transforming-growth-factor-beta and Kidney-Neoplasms

Molecular biomarkers that interact with the vascular and immune compartments play an important role in the progression of solid malignancies. CD105, which is a component of the transforming growth factor beta (TGF β) signaling cascade, has long been studied for its role in potentiating angiogenesis in numerous cancers. In renal cell carcinoma (RCC), the role of CD105 is more complicated due to its diverse expression profile on the tumor cells, tumor vasculature, and the components of the immune system. Since its discovery, its angiogenic role has overshadowed other potential functions, especially in cancers. In this review, we aim to summarize the recent evidence and findings of the multifunctional roles of CD105 in angiogenesis and immunomodulation in the context of the various subtypes of RCC, with a specific emphasis on the clear cell RCC subtype. Since CD105 is an established biomarker and tumor antigen, we also provide an update on the preclinical and clinical applications of CD105 as a therapeutic platform in RCC.

Topics: Carcinoma, Renal Cell; Endoglin; Humans; Kidney Neoplasms; Neovascularization, Pathologic; Transforming Growth Factor beta

Signal peptide, CUB, and EGF-like domain-containing proteins (SCUBE) are secretory cell surface glycoproteins that play key roles in the developmental process. SCUBE proteins participate in the progression of several diseases, including cancer, and are recognized for their oncogenic and tumor suppressor functions depending on the cellular context. SCUBE proteins promote cancer cell proliferation, angiogenesis, invasion, or metastasis, stemness or self-renewal, and drug resistance. The association of SCUBE with other proteins alters the expression of signaling pathways, including Hedgehog, Notch, TGF-β/Smad2/3, and β-catenin. Further, SCUBE proteins function as potential prognostic and diagnostic biomarkers for breast cancer, renal cell carcinoma, endometrial carcinoma, and nasopharyngeal carcinoma. This review presents key features of SCUBE family members, and their structure and functions, and highlights their contribution in the development and progression of cancer. A comprehensive understanding of the role of SCUBE family members offers novel strategies for cancer therapy.

Topics: beta Catenin; Biomarkers; Calcium-Binding Proteins; Epidermal Growth Factor; Humans; Kidney Neoplasms; Membrane Glycoproteins; Membrane Proteins; Protein Sorting Signals; Transforming Growth Factor beta

SUMOylation of proteins is an important regulatory element in modulating protein function and has been implicated in the pathogenesis of numerous human diseases such as cancers, neurodegenerative diseases, brain injuries, diabetes, and familial dilated cardiomyopathy. Growing evidence has pointed to a significant role of SUMO in kidney diseases such as DN, RCC, nephritis, AKI, hypertonic stress and nephrolithiasis. Recently, emerging studies in podocytes demonstrated that SUMO might have a protective role against podocyte apoptosis. However, the SUMO code responsible for beneficial outcome in the kidney remains to be decrypted. Our recent experiments have revealed that the expression of both SUMO and SUMOylated proteins is appreciably elevated in hypoxia-induced tubular epithelial cells (TECs) as well as in the unilateral ureteric obstruction (UUO) mouse model, suggesting a role of SUMO in TECs injury and renal fibrosis. In this review, we attempt to decipher the SUMO code in the development of kidney diseases by summarizing the defined function of SUMO and looking forward to the potential role of SUMO in kidney diseases, especially in the pathology of renal fibrosis and CKD, with the goal of developing strategies that maximize correct interpretation in clinical therapy and prognosis.

Topics: Acute Kidney Injury; Apoptosis; Carcinoma, Renal Cell; Diabetic Nephropathies; Fibrosis; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney Neoplasms; Nephritis; Nephrolithiasis; Podocytes; Protein Processing, Post-Translational; Smad Proteins; Small Ubiquitin-Related Modifier Proteins; Sumoylation; Transforming Growth Factor beta; Ureteral Obstruction

Wilms tumor is the most common kidney malignancy in children, especially in children aged less than 6 years. Although therapeutic approach has reached successful rates, there is still room for improvement. Considering the tumor microenvironment, cytokines represent important elements of interaction and communication between tumor cells, stroma, and immune cells. In this regard, the transforming growth factor beta (TGF-β) family members play significant functions in physiological and pathological conditions, particularly in cancer. By regulating cell growth, death, and immortalization, TGF-β signaling pathways exert tumor suppressor effects in normal and early tumor cells. Thus, it is not surprising that a high number of human tumors arise due to alterations in genes coding for various TGF-β signaling components. Understanding the ambiguous role of TGF-β in human cancer is of paramount importance for the development of new therapeutic strategies to specifically block the metastatic signaling pathway of TGF-β without affecting its tumor suppressive effect. In this context, this review attempt to summarize the involvement of TGF-β in Wilms tumor.

Topics: Animals; Child, Preschool; Humans; Kidney Neoplasms; Neoplasm Metastasis; Predictive Value of Tests; Prognosis; Signal Transduction; Transforming Growth Factor beta; Tumor Escape; Tumor Microenvironment; Wilms Tumor

The development of bone metastasis from renal cell carcinoma (RCC) signals a transition to a terminal state for the patient with previously isolated disease. These patients may suffer the morbidity of severe, persistent pain, pathologic fractures, and spinal compression from vertebral metastasis before they succumb to their cancer. Although recent advancements have been made in the understanding of breast and prostate bone metastasis, there has been less knowledge in the area of metastatic RCC to the skeleton. This particular cancer in bone remains relatively resistant to standard forms of treatment such as radiation and chemotherapy. A better understanding of the biology of RCC bone metastasis is critically needed in order to improve treatment. Bone-derived cell lines and an experimental animal model have been developed in order to explore the relevant mechanisms of how RCC cells survive within and destroy the bone. This review will focus on the growth factor signaling pathways most important for the RCC-stimulated osteoclast-mediated bone destruction, namely the epidermal growth factor receptor (EGF-R) and transforming growth factor-beta receptor (TGF-betaR) pathways. By inhibiting these receptors, growth of RCC within the bone is decreased which, directly or indirectly, decreases bone destruction.

Topics: Animals; Bone Neoplasms; Carcinoma, Renal Cell; Diphosphonates; Disease Models, Animal; ErbB Receptors; Humans; Interleukin-6; Kidney Neoplasms; Mice; Signal Transduction; Transforming Growth Factor beta

Genetic studies of families at high risk for developing malignant and benign renal neoplasms led to cloning of genes whose alteration results in tumor formation. These genes are either tumor suppressors (VHL, TSC) or oncogenes (MET). Their significance in understanding oncogenesis extends far beyond the familial syndromes. The identification of these genes and the elucidation of their biochemical functions are likely to facilitate (1) our understanding of the full clinical spectrum of the corresponding diseases, (2) genetic counseling, and (3) rational design of effective strategies to prevent and/or treat familial and sporadic forms of these neoplasms.

Topics: Animals; Carcinoma, Renal Cell; Cloning, Molecular; Elongin; Genes, Tumor Suppressor; Genotype; Humans; Kidney Diseases, Cystic; Kidney Neoplasms; Ligases; Middle Aged; Models, Genetic; Mutation; Pheochromocytoma; Proteins; Syndrome; Transcription Factors; Transforming Growth Factor beta; Tuberous Sclerosis; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; von Hippel-Lindau Disease; Von Hippel-Lindau Tumor Suppressor Protein

Topics: Animals; Epidermal Growth Factor; Fibroblast Growth Factors; Growth Substances; Humans; Kidney Neoplasms; Male; Prostatic Hyperplasia; Prostatic Neoplasms; Somatomedins; Transforming Growth Factor beta; Urinary Bladder Neoplasms; Urogenital Neoplasms

NIS793 is a human IgG2 monoclonal antibody that binds to transforming growth factor beta (TGF-β). This first-in-human study investigated NIS793 plus spartalizumab treatment in patients with advanced solid tumors.. Patients received NIS793 (0.3-1 mg/kg every 3 weeks (Q3W)) monotherapy; following evaluation of two dose levels, dose escalation continued with NIS793 plus spartalizumab (NIS793 0.3-30 mg/kg Q3W and spartalizumab 300 mg Q3W or NIS793 20-30 mg/kg every 2 weeks [Q2W] and spartalizumab 400 mg every 4 weeks (Q4W)). In dose expansion, patients with non-small cell lung cancer (NSCLC) resistant to prior anti-programmed death ligand 1 or patients with microsatellite stable colorectal cancer (MSS-CRC) were treated at the recommended dose for expansion (RDE).. Sixty patients were treated in dose escalation, 11 with NIS793 monotherapy and 49 with NIS793 plus spartalizumab, and 60 patients were treated in dose expansion (MSS-CRC: n=40; NSCLC: n=20). No dose-limiting toxicities were observed. The RDE was established as NIS793 30 mg/kg (2100 mg) and spartalizumab 300 mg Q3W. Overall 54 (49.5%) patients experienced ≥1 treatment-related adverse event, most commonly rash (n=16; 13.3%), pruritus (n=10; 8.3%), and fatigue (n=9; 7.5%). Three partial responses were reported: one in renal cell carcinoma (NIS793 30 mg/kg Q2W plus spartalizumab 400 mg Q4W), and two in the MSS-CRC expansion cohort. Biomarker data showed evidence of target engagement through increased TGF-β/NIS793 complexes and depleted active TGF-β in peripheral blood. Gene expression analyses in tumor biopsies demonstrated decreased TGF-β target genes and signatures and increased immune signatures.. In patients with advanced solid tumors, proof of mechanism of NIS793 is supported by evidence of target engagement and TGF-β pathway inhibition.. NCT02947165.

Topics: Adult; Antibodies, Monoclonal; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Humans; Kidney Neoplasms; Lung Neoplasms; Transforming Growth Factor beta

Purpose Interleukin-10 (IL-10) stimulates the expansion and cytotoxicity of tumor-infiltrating CD8+ T cells and inhibits inflammatory CD4+ T cells. Pegylation prolongs the serum concentration of IL-10 without changing the immunologic profile. This phase I study sought to determine the safety and antitumor activity of AM0010. Patients and Methods Patients with selected advanced solid tumors were treated with AM0010 in a dose-escalation study, which was followed by a renal cell cancer (RCC) dose-expansion cohort. AM0010 was self-administered subcutaneously at doses of 1 to 40 μg/kg once per day. Primary end points were safety and tolerability; clinical activity and immune activation were secondary end points. Results In the dose-escalation and -expansion cohorts, 33 and 18 patients, respectively, were treated with daily subcutaneous injection of AM0010. AM0010 was tolerated in a heavily pretreated patient population. Treatment-related adverse events (AEs) included anemia, fatigue, thrombocytopenia, fever, and injection site reactions. Grade 3 to 4 nonhematopoietic treatment-related AEs, including rash (n = 2) and transaminitis (n = 1), were observed in five of 33 patients. Grade 3 to 4 anemia or thrombocytopenia was observed in five patients. Most treatment-related AEs were transient or reversible. AM0010 led to systemic immune activation with elevated immune-stimulatory cytokines and reduced transforming growth factor beta in the serum. Partial responses were observed in one patient with uveal melanoma and four of 15 evaluable patients with RCC treated at 20 μg/kg (overall response rate, 27%). Prolonged stable disease of at least 4 months was observed in four patients, including one with colorectal cancer with disease stabilization for 20 months. Conclusion AM0010 has an acceptable toxicity profile with early evidence of antitumor activity, particularly in RCC. These data support the further evaluation of AM0010 both alone and in combination with other immune therapies and chemotherapies.

Topics: Adult; Aged; Aged, 80 and over; Anemia; Carcinoma, Renal Cell; Cytokines; Drug Eruptions; Exanthema; Fatigue; Female; Fever; Humans; Injections, Subcutaneous; Interferon-gamma; Interleukin-10; Interleukin-4; Interleukin-8; Kidney Neoplasms; Male; Melanoma; Middle Aged; Neoplasms; Polyethylene Glycols; Recombinant Proteins; Thrombocytopenia; Transforming Growth Factor beta; Uveal Neoplasms; Young Adult

In advanced cancers, transforming growth factor-beta (TGFβ) promotes tumor growth and metastases and suppresses host antitumor immunity. GC1008 is a human anti-TGFβ monoclonal antibody that neutralizes all isoforms of TGFβ. Here, the safety and activity of GC1008 was evaluated in patients with advanced malignant melanoma and renal cell carcinoma.. In this multi-center phase I trial, cohorts of patients with previously treated malignant melanoma or renal cell carcinoma received intravenous GC1008 at 0.1, 0.3, 1, 3, 10, or 15 mg/kg on days 0, 28, 42, and 56. Patients achieving at least stable disease were eligible to receive Extended Treatment consisting of 4 doses of GC1008 every 2 weeks for up to 2 additional courses. Pharmacokinetic and exploratory biomarker assessments were performed.. Twenty-nine patients, 28 with malignant melanoma and 1 with renal cell carcinoma, were enrolled and treated, 22 in the dose-escalation part and 7 in a safety cohort expansion. No dose-limiting toxicity was observed, and the maximum dose, 15 mg/kg, was determined to be safe. The development of reversible cutaneous keratoacanthomas/squamous-cell carcinomas (4 patients) and hyperkeratosis was the major adverse event observed. One malignant melanoma patient achieved a partial response, and six had stable disease with a median progression-free survival of 24 weeks for these 7 patients (range, 16.4-44.4 weeks).. GC1008 had no dose-limiting toxicity up to 15 mg/kg. In patients with advanced malignant melanoma and renal cell carcinoma, multiple doses of GC1008 demonstrated acceptable safety and preliminary evidence of antitumor activity, warranting further studies of single agent and combination treatments.. Clinicaltrials.gov NCT00356460.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Renal Cell; Female; Humans; Kidney Neoplasms; Male; Melanoma; Middle Aged; Neoplasm Staging; Positron-Emission Tomography; Tomography, X-Ray Computed; Transforming Growth Factor beta; Treatment Outcome

Recombinant interleukin-2 (rIL-2) in combination with recombinant interferon alpha (rIFN alpha) has been shown to mediate significant antitumoral effects in some patients with advanced renal cell cancer or malignant melanoma. The therapeutic effects may be partially modulated by secondarily induced cytokines, especially with regard to in vivo lymphocyte activation. To investigate possible negative effects on lymphocyte activation during immunotherapy, we designed a study on transcription of transforming growth factor beta 1 (TGF beta 1), a known inhibitor of lymphocyte function, in patients undergoing treatment with daily alternating administration of rIFN alpha and rIL-2. Here we present data on gene expression of TGF beta 1. Kinetic mRNA studies revealed an increase of TGF beta 1 mRNA in peripheral mononuclear cells 12 h after subcutaneous injection of rIFN alpha. The following intravenous rIL-2 administration significantly decreased the amounts of TGF beta 1-specific mRNA. In contrast to the effect of the first dose, subsequent application of rIFN alpha did not enhance TGF beta gene expression during rIFN alpha/IL-2 therapy. The diminished TGF beta 1 gene expression returned to pretreatment levels 1-7 days after the last rIL-2 administration. When concomitant with a decrease in TGF beta 1 transcripts. Our results indicate a complex regulatory effect on secondarily induced cytokines such as TGF beta 1 by immunotherapeutic approaches. The rIL-2-mediated down-regulation of increased TGF beta 1 steady-state mRNA levels following rIFN alpha may represent a positive immune regulatory effect on cytotoxic cells. Furthermore this effect may modulate proliferation of neoplastic tissues.

Topics: Autoradiography; Blotting, Northern; Carcinoma, Renal Cell; Cells, Cultured; Densitometry; Humans; Immunotherapy; Injections, Intravenous; Injections, Subcutaneous; Interferon Type I; Interleukin-2; Kidney Neoplasms; Leukocytes, Mononuclear; Melanoma; Recombinant Proteins; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta

CD105 (endoglin) is a transmembrane protein that functions as a TGF-beta coreceptor and is highly expressed on endothelial cells. Unsurprisingly, preclinical and clinical evidence strongly suggests that CD105 is an important contributor to tumor angiogenesis and tumor progression. Emerging evidence suggests that CD105 is also expressed by tumor cells themselves in certain cancers such as renal cell carcinoma (RCC). In human RCC tumor cells, CD105 expression is associated with stem cell-like properties and contributes to the malignant phenotype in vitro and in xenograft models. However, as a regulator of TGF-beta signaling, there is a striking lack of evidence for the role of tumor-expressed CD105 in the anti-tumor immune response and the tumor microenvironment. In this study, we report that tumor cell-expressed CD105 potentiates both the in vitro and in vivo tumorigenic potential of RCC in a syngeneic murine RCC tumor model. Importantly, we find that tumor cell-expressed CD105 sculpts the tumor microenvironment by enhancing the recruitment of immunosuppressive cell types and inhibiting the polyfunctionality of tumor-infiltrating CD4

Topics: Animals; Carcinoma, Renal Cell; CD8-Positive T-Lymphocytes; Endoglin; Endothelial Cells; Humans; Immunosuppression Therapy; Kidney Neoplasms; Mice; Neovascularization, Pathologic; Transforming Growth Factor beta; Tumor Microenvironment

Accumulating evidence shows HOOK1 disordered in human malignancies. However, the clinicopathological and biological significance of HOOK1 in renal cell carcinoma (RCC) remains rarely studied. In this study, the authors demonstrate that HOOK1 is downregulated in RCC samples with predicted poorer clinical prognosis. Mechanistically, HOOK1 inhibits tumor growth and metastasis via canonical TGF-β/ALK5/p-Smad3 and non-canonical TGF-β/MEK/ERK/c-Myc pathway. At the same time, HOOK1 inhibits RCC angiogenesis and sunitinib resistance by promoting degradation of TNFSF13B through the ubiquitin-proteasome pathway. In addition, HOOK1 is transcriptionally regulated by nuclear factor E2F3 in VHL dependent manner. Notably, an agonist of HOOK1, meletin, is screened and it shows antitumor activity more effectively when combined with sunitinib or nivolumab than it is used alone. The findings reveal a pivotal role of HOOK1 in anti-cancer treatment, and identify a novel therapeutic strategy for renal cell carcinoma.

Topics: B-Cell Activating Factor; Carcinoma, Renal Cell; Humans; Kidney Neoplasms; Sunitinib; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Transforming growth factor-β (TGF-β) plays a critical role in regulating cell growth and differentiation. Epithelial to mesenchymal transition (EMT) induced by TGF-β promotes cancer cell migration, invasion, and proliferation. Pirfenidone (5-methyl-1-phenyl-2(1 H)-pyridone, PFD), an approved drug for treating pulmonary and renal fibrosis, is a potent TGF-β inhibitor and found reduced incidence of lung cancer and alleviated renal function decline. However, whether PFD plays a role in controlling renal cancer progression is largely unknown. In the present study, we demonstrated that high TGF-β1 expression was negatively associated with ten-year overall survival of patients with renal cancer. Functionally, blockade of TGF-β signaling with PFD significantly suppressed the progression of renal cancer in a murine model. Mechanistically, we revealed that PFD significantly decreased the expression and secretion of TGF-β both in vitro and in vivo tumor mouse model, which further prevented TGF-β-induced EMT and thus cell proliferation, migration, and invasion. Importantly, the downregulation of TGF-β upon PFD treatment shaped the immunosuppressive tumor microenvironment by limiting the recruitment of tumor-infiltrating MDSCs. Therefore, our study demonstrated that PFD prevents renal cancer progression by inhibiting TGF-β production of cancer cells and downstream signaling pathway, which might be presented as a therapeutic adjuvant for renal cancer.

Topics: Animals; Carcinoma, Renal Cell; Epithelial-Mesenchymal Transition; Female; Fibrosis; Humans; Kidney Neoplasms; Male; Mice; Pyridones; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Microenvironment

The ETS transcription factor GABPA has long been thought of as an oncogenic factor and recently suggested as a target for cancer therapy due to its critical effect on telomerase activation, but the role of GABPA in clear cell renal cell carcinoma (ccRCC) is unclear. In addition, ccRCC is characterized by metabolic reprograming with aberrant accumulation of L-2-hydroxyglurate (L-2HG), an oncometabolite that has been shown to promote ccRCC development and progression by inducing DNA methylation, however, its downstream effectors remain poorly defined.. siRNAs and expression vectors were used to manipulate the expression of GABPA and other factors and to determine cellular/molecular and phenotypic alterations. RNA sequencing and ChIP assays were performed to identify GABPA target genes. A human ccRCC xenograft model in mice was used to evaluate the effect of GABPA overexpression on in vivo tumorigenesis and metastasis. ccRCC cells were incubated with L-2-HG to analyze GABPA expression and methylation. We carried out immunohistochemistry on patient specimens and TCGA dataset analyses to assess the effect of GABPA on ccRCC survival.. GABPA depletion, although inhibiting telomerase expression, robustly enhanced proliferation, invasion and stemness of ccRCC cells, whereas GABPA overexpression exhibited opposite effects, strongly inhibiting in vivo metastasis and carcinogenesis. TGFBR2 was identified as the GABPA target gene through which GABPA governed the TGFβ signaling to dictate ccRCC phenotypes. GABPA and TGFBR2 phenocopies each other in ccRCC cells. Higher GABPA or TGFBR2 expression predicted longer survival in patients with ccRCC. Incubation of ccRCC cells with L-2-HG mimics GABPA-knockdown-mediated phenotypic alterations. L-2-HG silenced the expression of GABPA in ccRCC cells by increasing its methylation.. GABPA acts as a tumor suppressor by stimulating TGFBR2 expression and TGFβ signaling, while L-2-HG epigenetically inhibits GABPA expression, disrupting the GABPA-TGFβ loop to drive ccRCC aggressiveness. These results exemplify how oncometabolites erase tumor suppressive function for cancer development/progression. Restoring GABPA expression using DNA methylation inhibitors or other approaches, rather than targeting it, may be a novel strategy for ccRCC therapy.

Topics: Animals; Carcinogenesis; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Epigenesis, Genetic; GA-Binding Protein Transcription Factor; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Mice; Receptor, Transforming Growth Factor-beta Type II; Telomerase; Transforming Growth Factor beta

WW domain-containing transcription regulator 1 (TAZ, or WWTR1) and Yes-associated protein 1 (YAP) are both important effectors of the Hippo pathway and exhibit different functions. However, few studies have explored their co-regulatory mechanisms in kidney renal clear cell carcinoma (KIRC). Here, we used bioinformatics approaches to evaluate the co-regulatory roles of TAZ/YAP and screen novel biomarkers in KIRC. GSE121689 and GSE146354 were downloaded from the GEO. The limma was applied to identify the differential expression genes (DEGs) and the Venn diagram was utilized to screen co-expressed DEGs. Co-expressed DEGs obtained the corresponding pathways through GO and KEGG analysis. The protein-protein interaction (PPI) network was constructed using STRING. The hub genes were selected applying MCODE and CytoHubba. GSEA was further applied to identify the hub gene-related signaling pathways. The expression, survival, receiver operating character (ROC), and immune infiltration of the hub genes were analyzed by HPA, UALCAN, GEPIA, pROC, and TIMER. A total of 51 DEGs were co-expressed in the two datasets. The KEGG results showed that the enriched pathways were concentrated in the TGF-β signaling pathway and endocytosis. In the PPI network, the hub genes (STAU2, AGO2, FMR1) were identified by the MCODE and CytoHubba. The GSEA results revealed that the hub genes were correlated with the signaling pathways of metabolism and immunomodulation. We found that STAU2 and FMR1 were weakly expressed in tumors and were negatively associated with the tumor stages. The overall survival (OS) and disease-free survival (DFS) rate of the high-expressed group of FMR1 was greater than that of the low-expressed group. The ROC result exhibited that FMR1 had certainly a predictive ability. The TIMER results indicated that FMR1 was positively correlated to immune cell infiltration. The abovementioned results indicated that TAZ/YAP was involved in the TGF-β signaling pathway and endocytosis. FMR1 possibly served as an immune-related novel prognostic gene in KIRC.

Topics: Biomarkers, Tumor; Carcinoma, Renal Cell; Computational Biology; Fragile X Mental Retardation Protein; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Kidney Neoplasms; Nerve Tissue Proteins; Prognosis; RNA-Binding Proteins; Transforming Growth Factor beta

DPF3, a component of the SWI/SNF chromatin remodeling complex, has been associated with clear cell renal cell carcinoma (ccRCC) in a genome-wide association study. However, the functional role of DPF3 in ccRCC development and progression remains unknown. In this study, we demonstrate that DPF3a, the short isoform of DPF3, promotes kidney cancer cell migration both in vitro and in vivo, consistent with the clinical observation that DPF3a is significantly upregulated in ccRCC patients with metastases. Mechanistically, DPF3a specifically interacts with SNIP1, via which it forms a complex with SMAD4 and p300 histone acetyltransferase (HAT), the major transcriptional regulators of TGF-β signaling pathway. Moreover, the binding of DPF3a releases the repressive effect of SNIP1 on p300 HAT activity, leading to the increase in local histone acetylation and the activation of cell movement related genes. Overall, our findings reveal a metastasis-promoting function of DPF3, and further establish the link between SWI/SNF components and ccRCC.

Topics: Carcinoma, Renal Cell; Chromatin; Chromatin Assembly and Disassembly; DNA-Binding Proteins; Genome-Wide Association Study; Humans; Kidney Neoplasms; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

Tumorigenic phenotype of M2 tumor-associated macrophages promote tumor progression in response to exosomes cues imposed by tumor cells. However, the effect and underlying mechanisms of clear cell renal cell carcinoma (ccRCC)-derived exosomes (ccRCC-exo) on instructing macrophages phenotype remains unclear.. Macrophages were cocultured with ccRCC-exo and then evaluate the polarization of macrophages and migration of ccRCC cells. The effect and mechanism of lncRNA AP000439.2 overexpressed or deleted exosomes on macrophages M2 polarization were examined. Xenograft tumor mice model was used for in vivo validation.. The ccRCC-exo significantly activated macrophages to M2 phenotype presented by increased expression of transforming growth factor-beta (TGF-β) and interleukin 10 (IL-10) at mRNA and protein levels, and these M2 macrophages in turn facilitating the migration of ccRCC cells. LncRNA AP000439.2 was highly enriched in the ccRCC-exo. Overexpression of exosomal AP000439.2 promoted M2 macrophage polarization whereas AP000439.2-deficient exosome had the opposite effects. Nuclear-localized AP000439.2 directly interacted with signal transducer and activator of transcription 3 (STAT3) proteins and phosphorylated STAT3 in macrophages. RNA-Seq results showed overexpression of AP000439.2 activated NF-κB signaling pathway. Silencing of STAT3 suppressed overexpression of AP000439.2-induced up-regulation of TGF-β and IL-10 expression, and p65 phosphorylation. AP000439.2-deleted exosome inhibited tumor growth in vivo.. Exosomes from ccRCC deliver AP000439.2 to promote M2 macrophage polarization via STAT3, thus enhancing ccRCC progression, indicating exosomal AP000439.2 might be a novel therapeutic target in ccRCC. Video Abstract.

Topics: Animals; Carcinoma, Renal Cell; Cell Line, Tumor; Exosomes; Humans; Interleukin-10; Kidney Neoplasms; Macrophage Activation; Macrophages; Mice; MicroRNAs; NF-kappa B; RNA, Long Noncoding; RNA, Messenger; STAT3 Transcription Factor; Transforming Growth Factor beta; Transforming Growth Factors

Mounting evidence points to alterations in mitochondrial metabolism in renal cell carcinoma (RCC). However, the mechanisms that regulate the TCA cycle in RCC remain uncharacterized. Here, we demonstrate that loss of TCA cycle enzyme expression is retained in RCC metastatic tissues. Moreover, proteomic analysis demonstrates that reduced TCA cycle enzyme expression is far more pronounced in RCC relative to other tumor types. Loss of TCA cycle enzyme expression is correlated with reduced expression of the transcription factor PGC-1α, which is also lost in RCC tissues. PGC-1α reexpression in RCC cells restores the expression of TCA cycle enzymes in vitro and in vivo and leads to enhanced glucose carbon incorporation into TCA cycle intermediates. Mechanistically, TGF-β signaling, in concert with histone deacetylase 7 (HDAC7), suppresses TCA cycle enzyme expression. Our studies show that pharmacologic inhibition of TGF-β restores the expression of TCA cycle enzymes and suppresses tumor growth in an orthotopic model of RCC. Taken together, this investigation reveals a potentially novel role for the TGF-β/HDAC7 axis in global suppression of TCA cycle enzymes in RCC and provides insight into the molecular basis of altered mitochondrial metabolism in this malignancy.

Topics: Animals; Citric Acid Cycle; Gene Expression Profiling; Histone Deacetylases; Humans; Kidney Neoplasms; Mice; Transfection; Transforming Growth Factor beta

Aberrant expression of transforming growth factor-β1 (TGF-β1) is associated with renal cell carcinoma (RCC) progression by inducing cancer metastasis. However, the downstream effector(s) in TGF-β signaling pathway is not fully characterized. In the present study, the elevation of secreted protein acidic and rich in cysteine (SPARC) as a TGF-β regulated gene in RCC was identified by applying differentially expressed gene analysis and microarray analysis, we further confirmed this result in several RCC cell lines. Clinically, the expression of these two genes is positively correlated in RCC patient specimens. Furthermore, elevated SPARC expression is found in all the subtypes of RCC and positively correlated with the RCC stage and grade. In contrast, SPARC expression is inversely correlated with overall and disease-free survival of patients with RCC, suggesting SPARC as a potent prognostic marker of RCC patient survival. Knocking down SPARC significantly inhibits RCC cell invasion and metastasis both in vitro and in vivo. Similarly, in vitro cell invasion can be diminished by using a specific monoclonal antibody. Mechanistically, SPARC activates protein kinase B (AKT) pathway leading to elevated expression of matrix metalloproteinase-2 that can facilitate RCC invasion. Altogether, our data support that SPARC is a critical role of TGF-β signaling network underlying RCC progression and a potential therapeutic target as well as a prognostic marker.

Topics: Animals; Carcinoma, Renal Cell; Cell Line, Tumor; Disease Models, Animal; Disease Progression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Male; Matrix Metalloproteinase 2; Mice, SCID; Neoplasm Invasiveness; Neoplasm Metastasis; Osteonectin; Snail Family Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Treatment Outcome

Recent studies of tumor microenvironments have revealed that clonal B cells reacting to tumor-derived antigens play an important role in anti-tumor immunity. We report a case of a 72-year-old Japanese man with a complaint of fever for 1 month. Computed tomography revealed a 48 mm mass in his right kidney. The patient underwent a right nephrectomy and histology revealed clear cell renal cell carcinoma (ccRCC) of Fuhrman Grade 4 with rhabdoid morphology. Focally, marked plasmacytoid cell infiltration was detected in the carcinoma. These plasmacytoid cells were immunohistochemically positive for immunoglobulin (Ig) G, and kappa light chain restriction was confirmed using mRNA in situ hybridization. Programmed death-ligand 1 (PD-L1) immunostaining and RNA in situ hybridization of transforming growth factor beta (TGF-β) revealed that both PD-L1 and TGF-β were highly expressed in the area with clonal plasmacytoid cell infiltration. The patient developed bone metastasis 3 months after surgery, and plasmacytoma was not detected during the observation period. We identified a potential link between an immunosuppressive microenvironment and clonal B cell proliferation. The latter posed a differential diagnosis problem between reactive and neoplastic clonal B cell proliferation vis-à-vis a plasmacytoma complicating carcinoma.

Topics: Aged; B-Lymphocytes; B7-H1 Antigen; Biomarkers, Tumor; Carcinoma, Renal Cell; Cell Proliferation; Diagnosis, Differential; Humans; Immunohistochemistry; In Situ Hybridization; Kidney Neoplasms; Male; Middle Aged; Neoplasm Grading; Plasmacytoma; Transforming Growth Factor beta; Tumor Microenvironment

IL-2 is a potent cytokine that activates natural killer cells and CD8+ cytotoxic T lymphocytes (CTLs) and has been approved for the treatment of metastatic renal cell carcinoma and metastatic melanoma. However, the medical use of IL-2 is restricted because of its narrow therapeutic window and potential side effects, including the expansion of regulatory T cells (Tregs).. In this study, the authors investigated the complementary effects of transforming growth factor-β2 (TGF-β2) anti-sense oligodeoxynucleotide (TASO) on the immunotherapeutic potential of IL-2 in a melanoma-bearing humanized mouse model.. The authors observed that the combination of TASO and IL-2 facilitated infiltration of CTLs into the tumor, thereby potentiating the tumor killing function of CTLs associated with increased granzyme B expression. In addition, TASO attenuated the increase in Tregs by IL-2 in the peripheral blood and spleen and also inhibited infiltration of Tregs into the tumor, which was partly due to decreased CCL22. Alteration of T-cell constituents at the periphery by TGF-β2 inhibition combined with IL-2 might be associated with the synergistic augmentation of serum pro-inflammatory cytokines (such as interferon γ and tumor necrosis factor α) and decreased ratio of Tregs to CTLs in tumor tissues, which consequently results in significant inhibition of tumor growth CONCLUSIONS: These results indicate that the application of TASO improves IL-2-mediated anti-tumor immunity, thus implying that blockade of TGF-β2 in combination with IL-2 may be a promising immunotherapeutic strategy for melanoma.

Topics: Animals; Carcinoma, Renal Cell; Immunotherapy; Interleukin-2; Kidney Neoplasms; Melanoma; Mice; Oligonucleotides, Antisense; Transforming Growth Factor beta; Transforming Growth Factor beta2; Transforming Growth Factors

Clear cell renal cell carcinoma (ccRCC) accounts for 60-70% of renal cell carcinoma (RCC) cases. It is an urgent mission to find more therapeutic targets for advanced ccRCC. Leucine-rich a-2-glycoprotein 1 (LRG1) is a secreted protein associated with a variety of malignancies. Our study focused on the expression and mechanism of LRG1 in ccRCC based on data from The Cancer Genome Atlas (TCGA) and provided primary verification including LRG1 expression detection, LRG1 gene methylation detection, and downstream signaling detection. We found that LRG1 was overexpressed in ccRCC kidney tissue samples, and the methylation level of LRG1 gene was significantly decreased in ccRCC. Moreover, the expression of LRG1 was negatively related to patient survival. Based on our previous study and the verification reported in this article, we propose that demethylation-induced overexpression of LRG1 is likely to accelerate ccRCC progression via the TGF-

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Renal Cell; Cytokines; Disease Progression; Female; Glycoproteins; Humans; Kidney Neoplasms; Male; Middle Aged; Signal Transduction; Transforming Growth Factor beta; Young Adult

Clear cell renal cell carcinoma (ccRCC) is the most common subtype among kidney cancer, which has poor prognosis. The aim of this study was to screen out novel prognostic biomarkers and therapeutic targets for immunotherapy, and some novel molecule drugs for ccRCC treatment. Immune scores ranged from -1109.36 to 2920.81 and stromal scores ranged from -1530.11 to 1955.39 were firstly calculated by applying ESTIMATE algorithm. Then 17 DEGs associated with immune score and stromal score were further identified. 6 candidate hub genes were screened out by performing overall survival (OS) and disease-free survival analyses based on TCGA-KIRC data, one of which including TGFBI was further regarded as hub gene associated with prognosis by calculating the R

Topics: Biomarkers, Tumor; Carcinoma, Renal Cell; Disease-Free Survival; DNA Methylation; Extracellular Matrix Proteins; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Kidney Neoplasms; Male; Prognosis; Transforming Growth Factor beta

Topics: Carcinoma, Renal Cell; Humans; Kidney Neoplasms; Neoplasm Recurrence, Local; Nephrectomy; Transforming Growth Factor beta

Renal cell carcinoma (RCC) is not sensitive to conventional radiotherapy and chemotherapy, and the effectiveness rate of molecular targeted therapy is low. Therefore, it is urgent to identify new treatment methods. Recently, adoptive T‑cell therapy has provided a new option for cancer treatment. Furthermore, low‑dose chemotherapy not only has no evident side effects and inhibitory effects on the human immune system, but can also enhance the immune activity of some effector cells. Therefore, it is surmised that the combination of different mechanisms of chemotherapy and immunotherapy could be a new treatment concept. In the present study, the effects of low‑dose chemotherapy combined with T cells in the treatment of renal cell carcinoma were explored using cytotoxicity assays, enzyme‑linked immunosorbent assay (ELISA), western blot analysis and flow cytometric analysis. The results revealed that low‑dose chemotherapy and T cells had synergistic effects on tumor cell elimination in vitro. The transforming growth factor (TGF)‑β signaling pathway may be involved in the inhibition of T‑cell functions. The targeted inhibition of TGF‑β signals may be a promising therapeutic strategy for the treatment of renal cancer. The present results provided a novel strategy for the combination of low‑dose chemotherapy and T cells to enhance the therapeutic efficacy of RCC treatment.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Renal Cell; Cell Line, Tumor; Chemotaxis, Leukocyte; Cisplatin; Combined Modality Therapy; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Female; Humans; Immunotherapy, Adoptive; Kidney Neoplasms; Mice; Mitomycin; Primary Cell Culture; Signal Transduction; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta

Kidney renal papillary renal cell carcinoma (KIRP) accounts for 10-15% of renal cell carcinoma (RCC). The need to find more therapeutic targets for KIRP is urgent because most targeted drugs have limited effects on advanced KIRP. Insulin-like growth factor (IGF) binding protein 5 (IGFBP5) is a secreted protein related to cell proliferation, cell adhesion, cell migration, the inflammatory response and fibrosis; these functions are independent of IGF. In our study, we determined the expression and functions of IGFBP5 with data from the database of The Cancer Genome Atlas (TCGA). We found that IGFBP5 is down regulated in KIRP kidney tissues compared to its expression in control tissues and that the expression of IGFBP5 is negatively related to patient survival. Bioinformatic analysis showed the probable processes and pathways involved in altered IGFBP5 expression, including blood vessel development, the cellular response to growth factor stimulus, the response to transforming growth factor

Topics: Carcinoma, Renal Cell; Cell Line, Tumor; Databases, Genetic; Disease-Free Survival; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Protein 5; Kidney Neoplasms; Neoplasm Proteins; Survival Rate; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Although transforming growth factor beta (TGF-β) is known to be involved in the pathogenesis and progression of many cancers, its role in renal cancer has not been fully investigated. In the present study, we examined the role of TGF-β in clear cell renal carcinoma (ccRCC) progression in vitro and in vivo. First, expression levels of TGF-β signaling pathway components were examined. Microarray and immunohistochemical analyses showed that the expression of c-Ski, a transcriptional corepressor of Smad-dependent TGF-β and bone morphogenetic protein (BMP) signaling, was higher in ccRCC tissues than in normal renal tissues. Next, a functional analysis of c-Ski effects was carried out. Bioluminescence imaging of renal orthotopic tumor models demonstrated that overexpression of c-Ski in human ccRCC cells promoted in vivo tumor formation. Enhancement of tumor formation was also reproduced by the introduction of a dominant-negative mutant TGF-β type II receptor into ccRCC cells. In contrast, introduction of the BMP signaling inhibitor Noggin failed to accelerate tumor formation, suggesting that the tumor-promoting effect of c-Ski depends on the inhibition of TGF-β signaling rather than of BMP signaling. Finally, the molecular mechanism of the tumor-suppressive role of TGF-β was assessed. Although TGF-β signaling did not affect tumor angiogenesis, apoptosis of ccRCC cells was induced by TGF-β. Taken together, these findings suggest that c-Ski suppresses TGF-β signaling in ccRCC cells, which, in turn, attenuates the tumor-suppressive effect of TGF-β.

Topics: Adult; Aged; Aged, 80 and over; Animals; Carcinoma, Renal Cell; Cell Line, Tumor; Disease Progression; DNA-Binding Proteins; Female; Gene Expression Profiling; Humans; Kidney Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Proto-Oncogene Proteins; Signal Transduction; Transforming Growth Factor beta; Transplantation, Heterologous

High-mobility group AT-hook 2 (HMGA2), a member of the high mobility group family, has been reported to correlate with cancer progression. However, there is no report concerning the correlation between HMGA2 and metastasis in renal cell carcinoma. In the present study, we found that HMGA2 was highly expressed in five renal cell carcinoma cell lines compared with that in the normal renal tubular epithelial HK2 cell line. Additionally, HMGA2 facilitated cell migration and invasion of renal cell carcinoma cells, as evidenced by wound healing and Transwell assays. Subsequently, our results revealed that the E‑cadherin level was upregulated, while N‑cadherin, Twist1 and Twist2 expression were downregulated in HMGA2-depleted ACHN cells. In contrast, overexpression of HMGA2 in 786‑O cells enhanced epithelial-mesenchymal transition (EMT). In addition, analysis of the database Cancer Browser further validated the positive correlation between HGMA2 and Twist1 or Twist2 in renal cell carcinoma. Meanwhile, Kaplan-Meier analysis indicated that low HMGA2 expression was closely associated with an increased overall survival in renal cell carcinoma patients. To confirm the underlying mechanism of HMGA2-regulated EMT, our results revealed that silencing of HMGA2 downregulated the mRNA and protein levels of TGF-β and Smad2, while HMGA2 overexpression had the opposite effect. Furthermore, TGF-β overexpression could partially reverse the anti-metastatic effect and mesenchymal-epithelial transition (MET) by HMGA2 loss, while TGF-β deficiency impeded the pro‑metastatic phenotype and high expression of EMT markers induced by HMGA2 overexpression. In summary, our results demonstrated that HMGA2 facilitated a metastatic phenotype and the EMT process in renal cell carcinoma cells in vitro through a TGF-β-dependent pathway. In addition, these data strongly suggest that HGMA2 may serve as a potential therapeutic target and prognostic biomarker against renal cell carcinoma in the future.

Topics: Carcinoma, Renal Cell; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; HMGA2 Protein; Humans; Kaplan-Meier Estimate; Kidney Neoplasms; Neoplasm Invasiveness; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Up-Regulation

Bone metastasis is common in renal cell carcinoma (RCC), and the lesions are mainly osteolytic. The mechanism of bone destruction in RCC bone metastasis is unknown.. We used a direct intrafemur injection of mice with bone-derived 786-O RCC cells (Bo-786) as an in vivo model to study if inhibition of osteoblast differentiation is involved in osteolytic bone lesions in RCC bone metastasis.. We showed that bone-derived Bo-786 cells induced osteolytic bone lesions in the femur of mice. We examined the effect of conditioned medium of Bo-786 cells (Bo-786 CM) on both primary mouse osteoblasts and MC3T3-E1 preosteoblasts and found that Bo-786 CM inhibited osteoblast differentiation. Secretome analysis of Bo-786 CM revealed that BIGH3 (Beta ig h3 protein), also known as TGFBI (transforming growth factor beta-induced protein), is highly expressed. We generated recombinant BIGH3 and found that BIGH3 inhibited osteoblast differentiation in vitro. In addition, CM from Bo-786 BIGH3 knockdown cells (786-BIGH3 KD) reduced the inhibition of osteoblast differentiation compared to CM from vector control. Intrafemural injection of mice with 786-BIGH3 KD cells showed a reduction in osteolytic bone lesions compared to vector control. Immunohistochemical staining of 18 bone metastasis specimens from human RCC showed strong BIGH3 expression in 11/18 (61%) and moderate BIGH3 expression in 7/18 (39%) of the specimens.. These results suggest that suppression of osteoblast differentiation by BIGH3 is one of the mechanisms that enhance osteolytic lesions in RCC bone metastasis, and raise the possibilty that treatments that increase bone formation may improve therapy outcomes.

Topics: Animals; Bone and Bones; Bone Neoplasms; Carcinoma, Renal Cell; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Extracellular Matrix Proteins; Gene Expression; Gene Knockout Techniques; Heterografts; Humans; Kidney Neoplasms; Mass Spectrometry; Mice; Osteoblasts; Osteolysis; Transforming Growth Factor beta; X-Ray Microtomography

TGF-β regulates both the tumor-forming and migratory abilities of various types of cancer cells. However, it is unclear how the loss of TGF-β signaling components affects these abilities in clear-cell renal cell carcinoma (ccRCC). In this study, we investigated the role of TGFBR3 (TGF-β type III receptor, also known as betaglycan) in ccRCC. Database analysis revealed decreased expression of TGFBR3 in ccRCC tissues, which correlated with poor prognosis in patients. Orthotopic inoculation experiments using immunocompromised mice indicated that low TGFBR3 expression in ccRCC cells enhanced primary tumor formation and lung metastasis. In the presence of TGFBR3, TGF-β2 decreased the aldehyde dehydrogenase (ALDH)-positive ccRCC cell population, in which renal cancer-initiating cells are enriched. Loss of TGFBR3 also enhanced cell migration in cell culture and induced expression of several mesenchymal markers in a TGF-β-independent manner. Increased lamellipodium formation by FAK-PI3K signaling was observed with TGFBR3 downregulation, and this contributed to TGF-β-independent cell migration in ccRCC cells. Taken together, our findings reveal that loss of TGFBR3 endows ccRCC cells with multiple metastatic abilities through TGF-β-dependent and independent pathways.

Topics: Animals; Carcinoma, Renal Cell; Cell Movement; Cells, Cultured; Down-Regulation; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; HEK293 Cells; Humans; Kidney Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Proteoglycans; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Topics: Carcinoma, Renal Cell; Cell Adhesion; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Transforming Growth Factor beta; Y-Box-Binding Protein 1

Renal cell carcinoma (RCC) is the most common malignancy in urogenital neoplasms worldwide. According to previous studies, valproic acid (VPA), an anticonvulsant drug, can suppress tumor metastasis and decrease the expression level of Mothers against decapentaplegic homolog 4 (SMAD4) and therefore may inhibit epithelial‑mesenchymal transition (EMT), which is responsible for cancer metastasis. However, the association between VPA, EMT and SMAD4 in RCC metastasis remains obscure. In the present study, it was demonstrated that in the RCC cell lines 786‑O and Caki‑1 treated with VPA, the neural (N)‑cadherin, vimentin and SMAD4 protein and mRNA levels were decreased, accompanied with an increase in expression of epithelial (E)‑cadherin. Silencing SMAD4 expression decreased the expression of EMT markers, including N‑cadherin and simultaneously upregulated E‑cadherin in RCC cell lines. SMAD4 overexpression counteracted the VPA‑mediated EMT‑inhibitory effect (P<0.05). The present study demonstrates that VPA inhibited EMT in RCC cells via altering SMAD4 expression. In addition, immunohistochemical staining demonstrated that transforming growth factor‑β (TGF‑β) and low expression of SMAD4 was associated with a lower Fuhrman grade and low expression of transcription intermediary factor 1‑γ was associated with a higher tumor Fuhrman grade (P<0.05), Therefore, based on the regulatory effect of SMAD4 on EMT‑associated transcription factors, SMAD4 which can form a SMAD3/SMAD4 complex induced by TGF‑β, could be a potential anticancer drug target inhibiting tumor invasion and metastasis in RCC.

Topics: Cadherins; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Intracellular Signaling Peptides and Proteins; Kidney Neoplasms; Middle Aged; RNA, Messenger; Smad4 Protein; Transforming Growth Factor beta; Up-Regulation; Valproic Acid; Vimentin

To investigate the expression pattern of a novel long non-coding ribonucleic acid activated by transforming growth factor β, long non-coding ribonucleic acid activated by transforming growth factor β, in renal cell carcinoma tissues among the patients with various clinicopathologic features and to detect the possible role of dysregulated long non-coding RNA-ATB in renal cell carcinoma.. The expression of long non-coding ribonucleic acid activated by transforming growth factor β in the renal cell carcinoma tissues and renal cancer cell lines was evaluated by quantitative real-time polymerase chain reaction. The association with clinicopathologic features was analyzed. The effects of long non-coding ribonucleic acid activated by transforming growth factor β on the renal cell carcinoma cell proliferation, apoptosis, migration, invasion and epithelial-to-mesenchymal transition were investigated by the loss-of-function approach.. The expression of long non-coding ribonucleic acid activated by transforming growth factor β was higher in the renal cell carcinoma tissues and renal cancer cells than in the adjacent non-tumor tissues and normal human proximal tubule epithelial cells HK-2. In addition, the long non-coding ribonucleic acid activated by transforming growth factor β expression was significantly higher in the renal cell carcinoma patients with metastasis. The elevated expression of long non-coding ribonucleic acid activated by transforming growth factor β was associated with tumor stages, histological grade, vascular invasion, lymph node metastasis and distant metastasis. Notably, we found that long non-coding ribonucleic acid activated by transforming growth factor β knockdown could (i) inhibit cell proliferation; (ii) trigger apoptosis; (iii) reduce epithelial-to-mesenchymal transition program; (iv) suppress cell migration and invasion.. Our data have shown that long non-coding ribonucleic acid activated by transforming growth factor β actively functions as a regulator of epithelial-to-mesenchymal transition during renal cell carcinoma metastasis, which suggests that long non-coding ribonucleic acid activated by transforming growth factor β may be a potential prognostic biomarker and therapeutic target for renal cell carcinoma.

Topics: Carcinoma, Renal Cell; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Prognosis; Real-Time Polymerase Chain Reaction; RNA, Long Noncoding; Transforming Growth Factor beta; Up-Regulation

The molecular mechanisms whereby transforming growth factor-β (TGF-β) promotes clear cell renal cell carcinoma (ccRCC) progression is elusive. The cell membrane bound TGF-β type I receptor (ALK5), was recently found to undergo proteolytic cleavage in aggressive prostate cancer cells, resulting in liberation and subsequent nuclear translocation of its intracellular domain (ICD), suggesting that ALK5-ICD might be a useful cancer biomarker. Herein, the possible correlation between ALK5 full length (ALK5-FL) and ALK5-ICD protein, phosphorylated Smad2/3 (pSmad2/3), and expression of TGF-β target gene PAI-1, was investigated in a clinical ccRCC material, in relation to tumor grade, stage, size and cancer specific survival. Expression of ALK5-FL, ALK5-ICD, pSmad2/3 and PAI-1 protein levels were significantly higher in higher stage and associated with adverse survival. ALK5-ICD, pSmad2/3 and PAI-1 correlated with higher grade, and ALK5-FL, pSmad2/3 and PAI-1 protein levels were significantly correlated with larger tumor size. Moreover, the functional role of the TGF-β - ALK5-ICD pathway were investigated in two ccRCC cell lines by treatment with ADAM/MMP2 inhibitor TAPI-2, which prevented TGF-β-induced ALK5-ICD generation, nuclear translocation, as well as cell invasion. The present study demonstrated that canonical TGF-β Smad2/3 pathway and generation of ALK5-ICD correlates with poor survival and invasion of ccRCC in vitro.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Renal Cell; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Kidney Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Plasminogen Activator Inhibitor 1; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

MicroRNAs (miRNAs) have been proven to be important oncogenes and tumor suppressors in wide range of cancers, including renal cell carcinoma (RCC). In our study, we evaluated miRNA-429 as potential diagnostic/prognostic biomarker in 172 clear cell RCC patients and as a potential regulator of epithelial-mesenchymal transition (EMT) in vitro. We demonstrated that miR-429 is down-regulated in tumor tissue samples (P < 0.0001) and is significantly associated with cancer metastasis (P < 0.0001), shorter disease-free (P = 0.0105), and overall survival (P = 0.0020). In addition, ectopic expression of miR-429 in 786-0 RCC cells followed by TGF-β treatment led to increase in the levels of E-cadherin expression (P < 0.0001) and suppression of cellular migration (P < 0.0001) in comparison to TGF-β-treated controls. Taken together, our findings suggest that miR-429 may serve as promising diagnostic and prognostic biomarker in RCC patients. We further suggest that miR-429 has a capacity to inhibit loss of E-cadherin in RCC cells undergoing EMT and consequently attenuate their motility.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Biomarkers, Tumor; Cadherins; Carcinoma, Renal Cell; Case-Control Studies; Cell Movement; Cell Proliferation; Combined Modality Therapy; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Lymphatic Metastasis; Male; MicroRNAs; Middle Aged; Neoplasm Grading; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Transforming Growth Factor beta; Tumor Cells, Cultured

Renal cell carcinoma (RCC) is an aggressive disease, with 35% chance of metastasis. The 'cancer stem cell' hypothesis suggests that a subset of cancer cells possess stem cell properties and is crucial in tumor initiation, metastasis and treatment resistance. We isolated RCC spheres and showed that they exhibit cancer stem cell/ tumor initiating cell-like properties including the formation of self-renewing spheres, high tumorigenicity and the ability to differentiate to cell types of the original tumor. Spheres showed increased expression of stem cell-related transcription factors and mesenchymal markers. miRNAs were differentially expressed between RCC spheres and their parental cells. Inhibition of miR-17 accelerated the formation of RCC spheres which shared molecular characteristics with the spontaneous RCC spheres. Target prediction pointed out TGFβ pathway activation as a possible mechanism to drive RCC sphere formation. We demonstrate that miR-17 overexpression interferes with the TGFβ-EMT axis and hinders RCC sphere formation; and validated TGFBR2 as a direct and biologically relevant target during this process. Thus, a single miRNA may have an impact on the formation of highly tumorigenic cancer spheres of kidney cancer.

Topics: Animals; Carcinoma, Renal Cell; Cell Growth Processes; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Humans; Kidney Neoplasms; Male; Mice; Mice, Inbred NOD; MicroRNAs; Neoplastic Stem Cells; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Spheroids, Cellular; Transfection; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney, and clear cell RCC (ccRCC) represents its most common histological subtype. To identify a therapeutic target for ccRCC, miRNA expression signatures from ccRCC clinical specimens were analyzed. miRNA microarray and real-time PCR analyses revealed that miR-629 expression was significantly upregulated in human ccRCC compared with adjacent noncancerous renal tissue. Functional inhibition of miR-629 by a hairpin miRNA inhibitor suppressed ccRCC cell motility and invasion. Mechanistically, miR-629 directly targeted tripartite motif-containing 33 (TRIM33), which inhibits the TGFβ/Smad signaling pathway. In clinical ccRCC specimens, downregulation of TRIM33 was observed with the association of both pathologic stages and grades. The miR-629 inhibitor significantly suppressed TGFβ-induced Smad activation by upregulating TRIM33 expression and subsequently inhibited the association of Smad2/3 and Smad4. Moreover, a miR-629 mimic enhanced the effect of TGFβ on the expression of epithelial-mesenchymal transition-related factors as well as on the motility and invasion in ccRCC cells. These findings identify miR-629 as a potent regulator of the TGFβ/Smad signaling pathway via TRIM33 in ccRCC.. This study suggests that miR-629 has biomarker potential through its ability to regulate TGFβ/Smad signaling and accelerate ccRCC cell motility and invasion.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis Regulatory Proteins; Carcinoma, Renal Cell; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Kidney Neoplasms; Male; MicroRNAs; Middle Aged; Mitochondrial Proteins; Neoplasm Metastasis; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

In this report, retrospectively, we analyzed fifteen histo-pathologically characterized FFPE based Wilms' Tumor (WT) samples following an integrative approach of copy number (CN) and loss of heterozygosity (LOH) imbalances. The isolated-DNA was tested on CN and somatic-mutation related Molecular-Inversion-Probe based-Oncoscan Array™ and was analyzed through Nexus-Express OncoScan-3.0 and 7.0 software. We identified gain of 3p13.0-q29, 4p16.3-14.0, 7, 12p13.33-q24.33, and losses of 1p36.11-q44, 11p15.5-q25, 21q 22.2-22.3 and 22q11.21-13.2 in six samples (W1-6) and validated them in nine more samples (W7-9, W12-15, W17-18). Some observed that discrete deletions (1p, 1q, 10p, 10q, 13q, 20p) were specific to our samples. Maximum-LOH was observed in Ch11 as reported in previous studies. However, LOH was also observed in different regions of Ch7 including some cancer genes. The identified LOH-regions (1q21.2-q21.3, 2p24.1-23.3, 2p24.3-24.3, 3p21.3-21.1, 4p16.3, 7p11.2-p11.1, 7q31.2-31.32, 7q34-q35 and Ch 8) in W1-W6 were also validated in W7-9, W12-15 and W18. In addition, previously reported LOH of 1p and 16q region was also observed in our cases. The proven and novel onco (OG)- and tumor-suppressor genes (TSGs) involved in the CNV regions affected the major pathways like Chromatin Modification, RAS, PI3K; RAS in 14/15 cases, NOTCH/TGF-β and Cell Cycle Apoptosis in 10/15 cases, APC in 9/15 cases and Transcriptional Regulation in 7/15 cases, PI3K and genome maintenance in 6/15 cases. This exhaustive profiling of OG and TG may help in prognosis and diagnosis of the disease after validation of all the relevant results, especially the novel ones, obtained in this research in a larger number of samples.

Topics: Apoptosis; Cell Cycle; Chromatin; Chromosome Inversion; Chromosomes, Human; Gene Dosage; Genes, Tumor Suppressor; Genome; Humans; Kidney Neoplasms; Loss of Heterozygosity; Microarray Analysis; Molecular Probes; Mutation; Phosphatidylinositol 3-Kinases; Retrospective Studies; Software; Transforming Growth Factor beta; Wilms Tumor

The study of kidney cancer pathogenesis and its treatment has been limited by the scarcity of genetically defined animal models. The FLCN gene that codes for the protein folliculin, mutated in Birt-Hogg-Dubé syndrome, presents a new target for mouse modeling of kidney cancer. Here we developed a kidney-specific knockout model by disrupting the mouse Flcn in the proximal tubules, thus avoiding homozygous embryonic lethality or neonatal mortality, and eliminating the requirement of loss of heterozygosity for tumorigenesis. This knockout develops renal cysts and early onset (6 months) of multiple histological subtypes of renal neoplasms featuring high tumor penetrance. Although the majority of the tumors were chromophobe renal cell carcinomas in affected mice under 1 year of age, papillary renal cell carcinomas predominated in the kidneys of older knockout mice. This renal neoplasia from cystic hyperplasia at 4 months to high-grade renal tumors by 16 months represented the progression of tumorigenesis. The mTOR and TGF-β signalings were upregulated in Flcn-deficient tumors, and these two activated pathways may synergetically cause renal tumorigenesis. Treatment of knockout mice with the mTOR inhibitor rapamycin for 10 months led to the suppression of tumor growth. Thus, our model recapitulates human Birt-Hogg-Dubé kidney tumorigenesis, provides a valuable tool for further study of Flcn-deficient renal tumorigenesis, and tests new drugs/approaches to their treatment.

Topics: Animals; Antibiotics, Antineoplastic; Carcinogenesis; Carcinoma, Renal Cell; Cysts; Disease Models, Animal; Hyperplasia; Kidney Neoplasms; Kidney Tubules, Proximal; Mice; Mice, Knockout; Proto-Oncogene Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transforming Growth Factor beta; Tumor Suppressor Proteins

Sunitinib is a multitargeting tyrosine kinase inhibitor used for metastatic renal cancer. There are no biomarkers that can predict sunitinib response. Such markers are needed to avoid administration of costly medication with side effects to patients who would not benefit from it. We compared global miRNA expression between patients with a short (≤12 months) versus prolonged (>12 months) progression-free survival (PFS) under sunitinib as first-line therapy for metastatic renal cell carcinoma. We identified a number of differentially expressed miRNAs and developed miRNA statistical models that can accurately distinguish between the two groups. We validated our models in the discovery set and an independent set of 57 patients. Target prediction and pathway analysis showed that these miRNAs are involved in vascular endothelial growth factor (VEGF), TGFβ, and mammalian target of rapamycin (mTOR)-mediated signaling and cell-cell communication. We tested the effect of these miRNAs on cellular proliferation and angiogenesis. We validated the negative correlation between miR-221 and its target, VEGFR2.miR-221 overexpression was associated with a poor PFS while its target, VEGFR2 was associated with longer survival. Gain of function experiments showed that miR-221 and miR-222 decreased angiogenesis and cellular proliferation in human umbilical vein endothelial cells (HUVEC) while increasing cellular proliferation in ACHN cells. miRNAs represent potential predictive markers for sunitinib response.

Topics: Angiogenesis Inhibitors; Animals; Biomarkers, Pharmacological; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Disease-Free Survival; Human Umbilical Vein Endothelial Cells; Humans; Indoles; Kidney Neoplasms; MicroRNAs; Middle Aged; Models, Statistical; Neovascularization, Pathologic; Prognosis; Pyrroles; Signal Transduction; Sunitinib; TOR Serine-Threonine Kinases; Transforming Growth Factor beta; Treatment Outcome; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a part of the ribonucleoprotein complex which regulates diverse biological events. While overexpression of hnRNP K has been shown to be related to tumorigenesis in several cancers, both the expression patterns and biological mechanisms of hnRNP K in renal cell carcinoma (RCC) cells remain unclear. In this study, we showed that hnRNP K protein was strongly expressed in selected RCC cell lines (ACHN, A498, Caki-1, 786-0), and knock-down of hnRNP K expression by siRNA induced cell growth inhibition and apoptosis. Based on immunohistochemical (IHC) analysis of hnRNP K expression in human clear cell RCC specimens, we demonstrated that there was a significant positive correlation between hnRNP K staining score and tumor aggressiveness (e.g., Fuhrman grade, metastasis). Particularly, the rate of cytoplasmic localization of hnRNP K in primary RCC with distant metastasis was significantly higher than that in RCC without metastasis. Additionally, our results indicated that the cytoplasmic distribution of hnRNP K induced by TGF-β stimulus mainly contributed to TGF-β-triggered tumor cell invasion in RCC cells. Dominant cytoplasmic expression of ectopic hnRNP K markedly suppressed the inhibition of invasion by knock-down of endogenous hnRNP K. The expression level of matrix metalloproteinase protein-2 was decreased by endogenous hnRNP K knock-down, and restored by ectopic hnRNP K. Therefore, hnRNP K may be a key molecule involved in cell motility in RCC cells, and molecular mechanism associated with the subcellular localization of hnRNP K may be a novel target in the treatment of metastatic RCC.

Topics: Apoptosis; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Cytoplasm; Down-Regulation; Gene Expression Regulation, Neoplastic; Heterogeneous-Nuclear Ribonucleoprotein K; Humans; Kidney Neoplasms; Mutation; Neoplasm Grading; Neoplasm Invasiveness; Neoplasm Metastasis; Protein Transport; Transforming Growth Factor beta

Dendritic cells (DCs) play an important role in anti-renal cell carcinoma (RCC) immunity. The aim of the study was to investigate effect of mimic RCC microenvironment on phenotype and function of DCs. We isolated conditioned media (CM) from supernatants of culturing RCC cells and adjacent non-RCC cells in patients. CD14+ monocytes were obtained from healthy donors. The monocytes derived DCs were treated by RCC CM and non-RCC CM. Maturation markers CD80, CD83, CD86, and HLA-DR on DCs were analyzed using flow cytometry, while the levels of IL-10, TGF-β, and IL12p70 in supernatants were examined by ELISA. The DCs migration treated with RCC CM and non-RCC CM was investigated using transwell assay. The DCs treated and allogenic T cells were co-cultured for detecting T-cell proliferation and change of phenotype on the T cells. Our results indicated that RCC CM inhibited the up-regulation of CD80,CD83, CD86, and HLA-DR in response to LPS in treated DCs and increased IL-10 and TGF-β secretion but reduced IL12p70 production. Moreover, the migration ability of DCs treated with RCC CM was also inhibited, compared to DCs treated with adjacent non-RCC CM. In addition, T-cell proliferation was suppressed in co-culture assay with DCs treated with RCC CM; proportion CD25+Foxp3+ regulatory T cells were induced to increase. This study suggests that RCC CM can inhibit maturation of DCs and impair its function; moreover, DCs treated with RCC CM induce regulatory T cells increase, thus could contribute RCC escape from antitumor immunity.

Topics: Antigens, CD; B7-1 Antigen; B7-2 Antigen; Carcinoma, Renal Cell; CD83 Antigen; Cell Movement; Cell Proliferation; Culture Media, Conditioned; Dendritic Cells; Female; HLA-DR Antigens; Humans; Immune Tolerance; Immunoglobulins; Interleukin-10; Kidney Neoplasms; Male; Membrane Glycoproteins; Phenotype; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment; Up-Regulation

TGF-beta can induce G1 arrest via many mechanisms including up-regulating p21, p27, and Rb. However, as the member of Rb family, whether RBL2 is induced by TGF-beta treatment remains exclusive.. The expression of RBL2 and miR-93 after TGF-beta treatment was determined by quantitative real-time PCR and western blot. The growth of renal cancer cells was determined by CCK-8 assays and cell cycle was determined by PI staining. The binding of miR-93 on RBL2 3'-UTR was determined by double luciferase system.. In renal cancer cells, TGF-beta treatment induced expression of RBL2 in a time- and concentration-dependent manner, and RBL2 mediated TGF-beta induced growth inhibition and cell cycle arrest in renal cancer cells. Furthermore, we found that miR-93 directly targeted RBL2 by binding to its 3'-UTR in renal cancer cells. Over-expression of miR-93 significantly reduced the expression of RBL2, whereas knock down of miR-93 up-regulated the expression of RBL2. More importantly, TGF-beta treatment inhibited miR-93 expression, which resulted in up-regulation of RBL2 after TGF-beta treatment.. TGF-beta induced RBL2 expression through down-regulating miR-93 in renal cancer cells. The newly identified TGF-beta/miR-93/RBL2 signal pathway reveals a new mechanism of TGF-beta induced growth arrest in renal cancer.

Topics: Blotting, Western; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; MicroRNAs; Real-Time Polymerase Chain Reaction; Retinoblastoma-Like Protein p130; Transfection; Transforming Growth Factor beta

Renal cell carcinoma (RCC) accounts for around 3% of cancers in the UK, and both incidence and mortality are increasing with the aging population. RCC can be divided into several subtypes: conventional RCC (the most common, comprising 75% of all cases), papillary RCC (15%) and chromophobe RCC (5%). Renal oncocytoma is a benign tumor and accounts for 5% of RCC. Cancer and epigenetics are closely associated, with DNA hypermethylation being widely accepted as a feature of many cancers. In this study the DNA methylation profiles of chromophobe RCC and renal oncocytomas were investigated by utilizing the Infinium HumanMethylation450 BeadChips. Cancer-specific hypermethylation was identified in 9.4% and 5.2% of loci in chromophobe RCC and renal oncocytoma samples, respectively, while the majority of the genome was hypomethylated. Thirty (hypermethylated) and 41 (hypomethylated) genes were identified as differentially methylated between chromophobe RCC and renal oncocytomas (p < 0.05). Pathway analysis identified some of the differentially hypermethylated genes to be involved in Wnt (EN2), MAPK (CACNG7) and TGFβ (AMH) signaling, Hippo pathway (NPHP4), and cell death and apoptosis (SPG20, NKX6-2, PAX3 and BAG2). In addition, we analyzed ccRCC and papillary RCC data available from The Cancer Genome Atlas portal to identify differentially methylated loci in chromophobe RCC and renal oncocytoma in relation to the other histological subtypes, providing insight into the pathology of RCC subtypes and classification of renal tumors.

Topics: Adenoma, Oxyphilic; Apoptosis; Carcinoma, Renal Cell; DNA Methylation; DNA, Neoplasm; Epigenesis, Genetic; Genome, Human; Humans; Kidney Neoplasms; MAP Kinase Signaling System; Transforming Growth Factor beta; Wnt Signaling Pathway

Clear cell renal cell carcinoma (ccRCC) is by far the most common type of kidney cancer and is characterized by loss of the tumor suppressor gene von Hippel-Lindau (VHL). ccRCC patients with metastatic disease has poor prognosis and today's therapy is insufficient. The cytokine Transforming Growth Factor-β (TGF-β) has been extensively studied in tumor biology and is believed to serve a variety of functions in tumor progression. We have previously shown that inhibition of NOTCH signaling causes a reduced migratory and invasive capacity of ccRCC cells, at least partly by a cross-talk with the TGF-β pathway. In the present study we aimed to further clarify the role of TGF-β signaling in ccRCC. We investigated the effects of TGF-β pathway modulation and showed that TGF-β inhibition attenuates the invasive capacity of ccRCC cells. By performing expression profiling we obtained a gene signature of the TGF-β induced response in ccRCC cells. The expression analyses revealed an extensive overlap between the TGF-β response and genes regulated by the hypoxia inducible factor (HIF). The link between the hypoxic and the TGF-β pathways was further corroborated by functional experiments, which demonstrated that TGF-β pathway activity was attenuated upon reintroduction of functional VHL in ccRCC.

Topics: Benzamides; Blotting, Western; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Dioxoles; Enzyme-Linked Immunosorbent Assay; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Oligonucleotide Array Sequence Analysis; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Von Hippel-Lindau Tumor Suppressor Protein

Micro RNAs (miRNAs) play an important role during renal development and show a tissue-specific enrichment in the kidney. Nephroblastomas, embryonal renal neoplasms of childhood, are considered to develop from nephrogenic rests (NRs) and resemble morphologically and genetically developing kidney. We therefore investigated the role of kidney-enriched miRNAs in the pathogenesis of nephroblastomas. miR-192, miR-215 and miR-194 had a significantly lower expression in nephroblastomas regardless of the subtype compared with mature kidney measured by quantitative real-time-PCR. miR-141 and miR-200c showed a significantly lower expression in blastema-type and mixed-type tumors. In comparison with NRs, a significantly lower expression of miR-192, miR-194 and miR-215 was identified in blastema-type, mixed-type and stroma-type nephroblastomas and of miR-141 and miR-200c in blastema-type tumors. Kidney parenchyma had a significantly higher expression of miR-192, miR-194, miR-215 and miR-200c compared with NRs. In this study, the activin receptor type 2B (ACVR2B), a member of the transforming growth factor (TGF)-β pathway, was identified as single common target gene for miR-192, miR-215, miR-194, miR-141 and miR-200c in silico for the first time. The interaction between all five miRNAs and ACVR2B was also verified by an in vitro assay. Additionally, a distinct protein expression of ACVR2B was detected in 53 of 55 nephroblastomas paralleled by an upregulation of ACVR2B messenger RNA demonstrated in 25 nephroblastomas of all subtypes. A differential regulation of ACVR2B by miRNAs in NRs and nephroblastomas appears to be an important step in the pathogenesis of nephroblastomas implicating for the first time the TGF-β pathway in this process.

Topics: Activin Receptors, Type II; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Kidney Neoplasms; MicroRNAs; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Up-Regulation; Wilms Tumor

Transforming growth factor-beta (TGF-beta) is a potent immunosuppressor that has been associated with tumor evasion from the host immune surveillance and, thus, tumor progression. We tested a novel immunotherapy for human renal cell cancer (RCC) using a technique that involves the adoptive transfer of autologous tumor-reactive, TGF-beta-insensitive CD8(+) T cells into human RCC-challenged immunodeficient mice to identify its potent antitumor responses.. The present study was conducted using a one-to-one adoptive transfer strategy to treat tumor-bearing severe combined immunodeficient (SCID/beige) mouse. The SCID/beige mice were humanized with peripheral blood mononuclear cells from patients with RCC (Hu-PBMC-SCID) before adoptive transfer. Autologous CD8(+) T cells were expanded ex vivo using autologous patient's dendritic cells pulsed with the tumor lysate and rendered TGF-beta insensitive by dominant-negative TGF-beta type II receptor. In addition, human RCC cell lines were generated using patients' tumor cells injected into SCID/beige mice.. Using flow cytometry analysis, we confirmed the expression of the tumor-reactive, TGF-beta-insensitive CD8(+) T cells were the effector CD8(+) cells (CD27(-)CDRA(+)). Adoptive transfer of autologous TGF-beta-insensitive CD8(+) T cells into tumor-bearing Hu-PBMC-SCID mice induced robust tumor-specific CTL responses in vitro, were associated with tumor apoptosis, suppressed lung metastasis, and prolonged survival times in vivo.. The one-to-one adoptive transfer strategy is an ideal in vivo murine model for studying the relationship between TGF-beta and immunosurveillance in RCC in vivo. Furthermore, this technique may offer the promise of a novel therapeutic option for the treatment of human patients with RCC.

Topics: Animals; Carcinoma, Renal Cell; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Humans; Immunotherapy, Adoptive; Interferon-gamma; Kidney Neoplasms; Mice; Mice, SCID; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Although dysregulation of transforming growth factor-beta (TGF-beta) signaling is implicated in renal carcinogenesis, its precise mechanism is unknown in renal cell carcinoma (RCC). In our study, we investigated Smad-mediated TGF-beta signaling pathway and its regulatory mechanisms in surgical samples from patients with RCC. We found that immunoreactivity for nuclear phosphorylated Smad2 was significantly decreased in RCC compared to normal renal tissues, thereby TGF-beta signaling was suggested to be attenuated in RCC tissues. In accordance with the result, transcriptional downregulation of Smad4 and post-transcriptional downregulation of TGF-beta type II receptor (TbetaR-II) were frequently found in RCC tissues compared to normal renal tissues. Next, to clarify the reason why the protein level of TbetaR-II was decreased in RCC, we investigated the activities of degradation and ubiquitination of TbetaR-II. We found that both proteasome-mediated degradation and ubiquitination of TbetaR-II were markedly enhanced in RCC tissues. Moreover, we found that the level of Smad-ubiquitination regulatory factor 2 (Smurf2), the E3 ligase for TbetaR-II, was increased in RCC tissues of the patients with higher clinical stages compared to the normal tissues and was inversely correlated with the level of TbetaR-II. Our results suggest that the low TbetaR-II protein level is due to augmented ubiquitin-dependent degradation via Smurf2 and might be involved in the attenuation of TGF-beta signaling pathway in RCC.

Topics: Carcinoma, Renal Cell; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Immunohistochemistry; Kidney Neoplasms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination

Loss of transforming growth factor-beta receptor III (TbetaRIII) correlates with loss of transforming growth factor-beta (TGF-beta) responsiveness and suggests a role for dysregulated TGF-beta signaling in clear cell renal cell carcinoma (ccRCC) progression and metastasis. Here we identify that for all stages of ccRCC TbetaRIII expression is downregulated in patient-matched tissue samples and cell lines. We find that this loss of expression is not due to methylation of the gene and we define GATA3 as the first transcriptional factor to positively regulate TbetaRIII expression in human cells. We localize GATA3's binding to a 10-bp region of the TbetaRIII proximal promoter. We demonstrate that GATA3 mRNA is downregulated in all stages, of ccRCC, mechanistically show that GATA3 is methylated in ccRCC patient tumor tissues as well as cell lines, and that inhibiting GATA3 expression in normal renal epithelial cells downregulates TbetaRIII mRNA and protein expression. These data support a sequential model whereby loss of GATA3 expression through epigenetic silencing decreases TbetaRIII expression during ccRCC progression.

Topics: Biomarkers, Tumor; Blotting, Western; Carcinoma, Renal Cell; Cell Line, Tumor; DNA Methylation; Electrophoretic Mobility Shift Assay; GATA3 Transcription Factor; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Immunoenzyme Techniques; Kidney Neoplasms; Luciferases; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Proteoglycans; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transfection; Transforming Growth Factor beta

Germline mutations in the FLCN gene are responsible for the development of fibrofolliculomas, lung cysts and renal neoplasia in Birt-Hogg-Dube' (BHD) syndrome. The encoded protein folliculin (FLCN) is conserved across species but contains no classic motifs or domains and its function remains unknown. Somatic mutations or loss of heterozygosity in the remaining wild type copy of the FLCN gene have been found in renal tumors from BHD patients suggesting that FLCN is a classic tumor suppressor gene.. To examine the tumor suppressor function of FLCN, wild-type or mutant FLCN (H255R) was stably expressed in a FLCN-null renal tumor cell line, UOK257, derived from a BHD patient. When these cells were injected into nude mice, tumor development was inversely dependent upon the level of wild-type FLCN expression. We identified genes that were differentially expressed in the cell lines with or without wild-type FLCN, many of which are involved in TGF-beta signaling, including TGF-beta2 (TGFB2), inhibin beta A chain (INHBA), thrombospondin 1 (THBS1), gremlin (GREM1), and SMAD3. In support of the in vitro data, TGFB2, INHBA, THBS1 and SMAD3 expression levels were significantly lower in BHD-associated renal tumors compared with normal kidney tissue. Although receptor mediated SMAD phosphorylation was not affected, basal and maximal TGF-beta-induced levels of TGFB2, INHBA and SMAD7 were dramatically reduced in FLCN-null cells compared with FLCN-restored cells. Secreted TGF-beta2 and activin A (homo-dimer of INHBA) protein levels were also lower in FLCN-null cells compared with FLCN-restored cells. Consistent with a growth suppressive function, activin A (but not TGF-beta2) completely suppressed anchorage-independent growth of FLCN-null UOK257 cells.. Our data demonstrate a role for FLCN in the regulation of key molecules in TGF-beta signaling and confirm deregulation of their expression in BHD-associated renal tumors. Thus, deregulation of genes involved in TGF-beta signaling by FLCN inactivation is likely to be an important step for tumorigenesis in BHD syndrome.

Topics: Animals; Cell Line, Tumor; Gene Expression Regulation; Genes, Tumor Suppressor; Genetic Vectors; Humans; Kidney Neoplasms; Lentivirus; Mice; Oligonucleotide Array Sequence Analysis; Proto-Oncogene Proteins; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Proteins

Dendritic cells (DCs) have been widely used as cancer vaccines. However, their functional abilities have often been suppressed by tumor-secreted immunosuppressants such as transforming growth factor-beta (TGF-beta). We developed a new strategy using a TGF-beta insensitive DC as cancer vaccine. The effect of this vaccine was tested in a murine pulmonary metastases model of renal carcinoma (Renca). Tumor lysate-pulsed DCs (TP-DCs) were infected with retrovirus containing gene of dominant negative TGF-beta type II receptor (TbetaRIIDN) and thus made TGF-beta insensitive. Vaccination of the mice bearing Renca pulmonary metastases with the TbetaRIIDN TP-DC induced powerful tumor-specific cytotoxic T lymphocyte (CTL) responses, suppressed pulmonary metastases, and prolonged survival times. These results suggest TGF-beta-insensitive TP-DC vaccine can be used to enhance the antitumor efficacy of DC vaccine.

Topics: Animals; Blotting, Western; Dendritic Cells; Female; Flow Cytometry; Kidney Neoplasms; Lung Neoplasms; Mice; Mice, Inbred BALB C; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta

To test, using a mouse renal cancer, Renca, whether adoptive transfer of tumor-reactive transforming growth factor (TGF)-beta-insensitive cytolytic T cells can inhibit tumor progression.. Cytolytic T cells were isolated from the spleen of male Balb/c mice repeatedly primed with irradiated Renca cells. They were expanded ex vivo and were rendered TGF-beta-insensitive by infecting with a retrovirus containing dominant negative TGF-beta type II receptor.. These tumor reactive TGF-beta-insensitive cytolytic T cells showed a specific and robust tumor killing activity against Renca cells, but not irrelevant cells, using an in vitro cytotoxic assay. Adoptive transfer of cytolytic T cells was performed in mice 10 days after they were challenged with Renca cells (5 x 10(5)) by tail vein injection. At 30 days after the adoptive transfer, the pulmonary tumor counts in mice who had received TGF-beta-insensitive cytolytic T cells (mean +/- standard deviation 130 +/- 140) was significantly less than those in mice that had received TGF-beta-sensitive cytolytic T cells (305 +/- 60) or in mice had received naive cytolytic T cells (375 +/- 50; P < .01). Kaplan-Meier survival analysis indicated that mice that had received adoptive transfer of TGF-beta-insensitive cytolytic T cells had a significantly greater rate of survival (75%) compared with mice that had received TGF-beta-sensitive cytolytic T cells (35%) or naive cytolytic T cells (15%), respectively (P < .05).. These results suggest that adoptive transfer of tumor-reactive TGF-beta-insensitive cytolytic T cells can warrant consideration for renal cell cancer immunotherapy.

Topics: Animals; Kidney Neoplasms; Mice; Mice, Inbred BALB C; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta

Alterations in transforming growth factor-beta (TGF-beta) signaling occur early during malignant transformation of renal epithelial cells and are associated with loss of type III TGF-beta receptor (TbetaRIII) expression. We evaluated the role of TbetaRIII in mediation of apoptosis using in vitro cell culture and in vivo animal models of clear cell renal cell carcinoma.. TbetaR3 expression was manipulated with adenoviral gene vector delivery system in vitro and in vivo. Induction of apoptosis and signaling through the Smad and mitogen-activated protein kinase (MAPK) pathways were examined at various time points after infection. To study viral oncolysis in vivo, human renal cell carcinoma cells were implanted s.c. in the flanks of nude mice and treated with intratumoral injections of adenovirus.. Restoring TbetaRIII expression in clear cell renal cell carcinoma resulted in a marked induction of apoptosis using in vitro cell culture and in vivo animal models. The expression of the cytoplasmic domain, but not the extracellular domain, of TbetaRIII mimicked the induction of apoptosis by full-length TbetaRIII in cell culture and the growth inhibition of tumors in athymic nude mice. TbetaRIII-associated apoptosis was not dependent on signaling through the canonical TGF-beta/Smad pathway but was mediated through p38 MAPK.. These findings indicate a novel mechanistic antitumor function for TbetaRIII and further support its role as an important tumor suppressor in clear cell renal cell carcinoma.

Topics: Animals; Apoptosis; Carcinoma, Renal Cell; Cell Line, Tumor; Humans; Kidney Neoplasms; Mice; p38 Mitogen-Activated Protein Kinases; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

We analyzed expression of candidate genes encoding cell surface or secreted proteins in normal kidney and kidney cancer. This screen identified a bone morphogenetic protein (BMP) antagonist, SOSTDC1 (sclerostin domain-containing-1) as down-regulated in kidney tumors. To confirm screening results, we probed cDNA dot blots with SOSTDC1. The SOSTDC1 message was decreased in 20/20 kidney tumors compared with normal kidney tissue. Immunohistochemistry confirmed significant decrease of SOSTDC1 protein in clear cell renal carcinomas relative to normal proximal renal tubule cells (p < 0.001). Expression of SOSTDC1 was not decreased in papillary and chromophobe kidney tumors. SOSTDC1 was abundantly expressed in podocytes, distal tubules, and transitional epithelia of the normal kidney. Transfection experiments demonstrated that SOSTDC1 is secreted and binds to neighboring cells and/or the extracellular matrix. SOSTDC1 suppresses both BMP-7-induced phosphorylation of R-Smads-1, -5, and -8 and Wnt-3a signaling. Restoration of SOSTDC1 in renal clear carcinoma cells profoundly suppresses proliferation. Collectively, these results demonstrate that SOSTDC1 is expressed in the human kidney and decreased in renal clear cell carcinoma. Because SOSTDC1 suppresses proliferation of renal carcinoma cells, restoration of SOSTDC1 signaling may represent a novel target in treatment of renal clear cell carcinoma.

Topics: Adaptor Proteins, Signal Transducing; Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Neoplasms; Mice; Phosphorylation; Proteins; RNA, Messenger; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Wnt Proteins; Wnt3 Protein; Wnt3A Protein

The mouse renal cell carcinoma line, Renca, is insensitive to transforming growth factor-beta (TGF-beta) in vitro. The present study was conducted to determine whether removal of TGF-beta from these tumor cells would inhibit tumor progression in vivo.. TGF-beta elimination was accomplished either by administration of neutralizing TGF-beta antibody into mice receiving intravenous injection of Renca tumor cells or infection of TGF-beta antisense expression vector into these tumor cells before subcutaneous injection into recipient mice.. Although a low dose of TGF-beta antibody (5 mg/kg every 3 days) was without any effect, a high dose of TGF-beta antibody (50 mg/kg every 3 days), administered to recipient mice, resulted in a significant reduction in lung metastasis and was accompanied by increased apoptosis in the tumor cells. When the tumor cells were transfected with a TGF-beta1 antisense expressing vector, a significant reduction occurred in the tumor incidence, as well as the tumor burden. However, in nude mice, cells with reduced TGF-beta1 production grew almost as well as did the unmodified Renca cells, suggesting that the host's immune system might play an antitumor role.. These results indicate that progression of Renca tumor can be inhibited by eliminating TGF-beta from the tumor cells. Our results also suggest that, although insensitive to TGF-beta under in vitro conditions, Renca tumors could be inhibited by TGF-beta removal through the systemic host environment.

Topics: Animals; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; In Vitro Techniques; Kidney Neoplasms; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Transfection; Transforming Growth Factor beta

To identify new target marker genes in renal cell carcinoma (RCC), we compared the gene expression profiles of clear cell RCC (cc-RCC) and normal kidney tissue using serial analysis of gene expression. Our results revealed that the transforming growth factor beta induced 68 kDa protein (TGF-betaI: beta ig-h3 (BIGH3), plasminogen activator inhibitor-1 (PAI-1) and transforming growth factor beta1 (TGF-beta1) genes are up-regulated in cc-RCC. To further assess the role of BIGH3 in RCC, we investigated the mRNA expression levels of BIGH3, TGFbeta1, PAI-1 and also of TGF-beta1 related genes in 53 RCC and 30 normal kidney tissues by quantitative real-time RT-PCR (QRT-PCR). We further determined the BIGH3 protein levels in 52 cc-RCC paraffin-embedded tissue samples by immunohistochemistry. BIGH3 mRNA was found to be highly overexpressed in cc-RCC compared with normal tissues with an average ratio of 27. The mRNA levels of TGF-beta1 and PAI-1 were also detected at significantly elevated levels in these cancers. Immunohistochemical analysis of BIGH3 also revealed strong staining in the majority of the cc-RCC samples. In addition, the up-regulation of BIGH3 and PAI-1 was found to correlate with the clinicopathological parameters associated with a poorer patient outcome, whereas TGF-beta1 expression was determined to be unrelated to cancer progression. Strong BIGH3 staining thus tended to be associated with a poor prognosis. BIGH3 was also induced in some RCC cell lines by TGF-beta1 stimulation and this correlated well with PAI-1 up-regulation, suggesting that these enhancements are regulated by a similar mechanism in these tumors.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Renal Cell; Extracellular Matrix Proteins; Female; Humans; Immunohistochemistry; Kidney Neoplasms; Male; Middle Aged; Plasminogen Activator Inhibitor 1; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

Mutations in the VHL gene are associated with highly vascular tumors of kidney, brain, retina, and adrenal gland. The inability of the mutant VHL protein to destabilize HIF-1 plays a crucial role in malignant angiogenesis. VHL is also associated with ECM assembly but the molecular mechanisms of this activity remain unclear. We used expression arrays and cell lines with different VHL status to identify ECM-associated genes controlled by VHL. One of them, adhesion-associated TGFBI, was repressed by VHL and overexpressed in renal, gastrointestinal, brain, and other tumors. Analyzing the mechanism of TGFBI up-regulation in clear cell carcinoma, we identified a novel VHL target, a Kruppel-like transcriptional factor 10 (KLF10). The TGFBI promoter, which we isolated and studied in Luc-reporter assay, was induced by KLF10 but not hypoxia. These data provide the molecular basis for the observed VHL effect on TGFBI and stimulate further research into the KLF10 and TGFBI roles in cancer.

Topics: Antineoplastic Agents; Carcinoma, Renal Cell; Cell Adhesion; Early Growth Response Transcription Factors; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Kruppel-Like Transcription Factors; Neoplasms; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Transcription, Genetic; Transforming Growth Factor beta; Up-Regulation; Von Hippel-Lindau Tumor Suppressor Protein

Bone metastases develop in approximately 30% of patients with RCC, and the mechanisms responsible for this phenomenon are unknown. We found that TGF-beta1 stimulation of RCC bone metastasis cells promotes tumor growth and bone destruction possibly by stimulating paracrine interactions between tumor cells and the bone.. Bone metastasis is a frequent complication and causes marked morbidity in patients with renal cell carcinoma (RCC). Surprisingly, the specific mechanisms of RCC interaction with bone have been scarcely studied despite the inability to prevent or effectively treat bone metastasis. Bone is a reservoir for various growth factors including the pleiotropic cytokine TGF-beta1. TGF-beta1 has been shown to have tumor-supportive effects on advanced cancers and evidence suggests its involvement in promoting the development of breast cancer bone metastasis. Here, we studied the potential role of TGF-beta1 in the growth of RCC bone metastasis (RBM).. To inhibit TGF-beta1 signaling, RBM cells stably expressing a dominant-negative (DN) TGF-betaRII cDNA were generated. The in vivo effect of TGF-beta1 on RBM tumor growth and osteolysis was determined by histological and radiographic analysis, respectively, of athymic nude mice after intratibial injection of parental, empty vector, or DN RBM cells. The in vitro effect of TGF-beta1 on RBM cell growth was determined after TGF-beta1 treatment by MTT assay.. TGF-beta1 and the TGF-beta receptors I and II (TGF-betaRI/II) were consistently expressed in both RBM tissues and cell lines. Inhibition of TGF-beta1 signaling in RBM cells significantly reduced tumor establishment and osteolysis observed in vivo after injection into the murine tibia, although no effect on tumor establishment was observed after injection of RBM cells subcutaneously or into the renal subcapsule. Treatment of five RBM cell lines with TGF-beta1 in vitro either had no effect (2/5) or resulted in a significant inhibition (3/5) of cell growth, suggesting that TGF-beta1 may promote RBM tumor growth indirectly in vivo.. TGF-beta1 stimulation of RBM cells plays a role in promoting tumor growth and subsequent osteolysis in vivo, likely through the initiation of tumor-promoting paracrine interactions between tumor cells and the bone microenvironment. These data suggest that inhibition of TGF-beta1 signaling may be useful in the treatment of RBM.

Topics: Animals; Bone Neoplasms; Carcinoma, Renal Cell; Cell Division; Cell Line, Tumor; DNA Primers; Enzyme-Linked Immunosorbent Assay; Humans; Kidney Neoplasms; Male; Mice; Mice, Nude; Osteolysis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Transforming Growth Factor beta

We investigated signal transducer and activator of transcription-5 (STAT5) activation status in renal cell carcinoma (RCC) and the role of transforming growth factor-beta (TGF-beta) in the process.. Twenty normal and RCC tissues were obtained from radical nephrectomy specimens for the assessment of expressions of phosphorylated STAT5 (p-STAT5) and TGF-beta1 (Western blot) and for localization and assessment of their relationship (immunohistochemical and immunofluorescence stains). By using four RCC cell lines and four primary cultured cells, the effect of TGF-beta1 and/or interleukin-2 (IL-2) on the expressions of p-STAT5 were analyzed.. In RCC samples, expression of p-STAT5 was significantly reduced while expression of TGF-beta was enhanced compared with normal kidney tissues (P < 0.001 and P = 0.003, respectively). P-STAT5 was observed almost exclusively in the nuclei of normal kidney tissues while TGF-beta was identified in the cytoplasm of cells of both tissues reflecting the Western results. In both RCC cell lines and cells from primary cultures, treatment with TGF-beta or antibody did not significantly alter STAT5 activation. However, TGF-beta significantly suppressed IL-2-induced STAT5 activation, whereas anti-TGF-beta antibodies enhanced IL-2-induced STAT5 further.. STAT5 activation is suppressed in RCC compared with normal renal parenchyma. It may be attributed to the RCC-derived TGF-beta which also interferes with IL-2-induced STAT5 pathway activation.

Topics: Adult; Blotting, Western; Carcinoma, Renal Cell; Down-Regulation; Humans; Immunohistochemistry; Interleukin-2; Kidney Neoplasms; Microscopy, Confocal; Middle Aged; Phosphorylation; STAT5 Transcription Factor; Transforming Growth Factor beta

Transforming growth factor beta (TGF-beta), which generally stimulates the growth of mesenchymally derived cells but inhibits the growth of epithelial cells, has been proposed as a possible target for cancer therapy. However, concerns have been raised that whereas inhibition of TGF-beta signaling could be efficacious for lesions in which TGF-beta promotes tumor development and/or progression, systemic pharmacologic blockade of this signaling pathway could also promote the growth of epithelial lesions.. We examined the effect of a TGF-beta inhibitor on mesenchymal (leiomyoma) and epithelial (renal cell carcinoma) tumors in Eker rats, which are genetically predisposed to develop these tumors with a high frequency.. Blockade of TGF-beta signaling with the ALK5/type I TGF-beta R kinase inhibitor, SB-525334, was efficacious for uterine leiomyoma; significantly decreasing tumor incidence and multiplicity, and reducing the size of these mesenchymal tumors. However, SB-525334 was also mitogenic and antiapoptotic for epithelial cells in the kidney and exacerbated the growth of epithelial lesions present in the kidneys of these animals.. Although pharmacologic inhibition of TGF-beta signaling with SB-525334 may be efficacious for mesenchymal tumors, inhibition of this signaling pathway seems to promote the development of epithelial tumors.

Topics: Activin Receptors, Type I; Animals; Apoptosis; Carcinoma, Renal Cell; Female; Imidazoles; Kidney Neoplasms; Leiomyoma; Mitosis; Protein Serine-Threonine Kinases; Quinoxalines; Rats; Rats, Inbred Strains; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Uterine Neoplasms

For a better understanding of the factors contributing to tumor progression in metastatic renal cell carcinoma and to identify possible targets for immunotherapeutic approaches, we characterized several primary cultures from renal cell carcinoma.. Cell cultures were tested for activity of telomerase, secretion of immunosuppressive cytokines and others. The induction of cytotoxic activity against the autologous tumor was tested in a cytotoxicity assay after coculture of immunological effector cells with antigen-pulsed dendritic cells. The data were tested for influence on survival.. We were able to establish primary cell cultures from 58 patients with renal cell carcinoma and their metastasis. 48/58 were positive for telomerase activity and all secreted IL-6, TGF-beta, VEGF and IL-8. High TGF-beta secretion, the activity of telomerase and the induction of a telomerase-specific immune response against telomerase peptides in telomerase-positive tumors had a significant impact on survival.. TGF-beta secretion, activity of telomerase in telomerase-positive tumors and the ability to generate a telomerase-specific immune response might serve as a prognostic marker for RCC. New approaches might focus on attacking the TGF-beta pathway and on induction of telomerase-specific immune cells.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Renal Cell; Cell Culture Techniques; Cytokines; Cytotoxicity, Immunologic; Female; Humans; Immunotherapy; Interleukin-6; Interleukin-8; Kaplan-Meier Estimate; Kidney Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Telomerase; Transforming Growth Factor beta; Treatment Outcome; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

The present study was carried out in order to examine molecular alterations of extracellular matrix (ECM), associated with cell-cell communication in conventional (clear-cell) renal cell carcinomas (cRCCs) influenced by persistent long-term, low-dose ionizing radiation (IR) exposure to patients living more than 19 years after the Chernobyl accident in Cesium 137 (137Cs)-contaminated areas of Ukraine. The ECM major components such as fibronectin, laminin, E-cadherin/beta-catenin complexes and p53 tumor suppressor gene protein, and transforming growth factor beta 1 (TGF-beta1) were immunohistochemically (IHC) evaluated in cRCCs from 59 Ukrainian patients, which represented 18 patients living in non-contaminated areas and 41 patients from 137Cs-contaminated areas. In contrast, a control group of 19 Spanish patients with analogue tumors were also investigated. For IHC evaluation, a tissue microarray technique was used. Decrease or loss and abnormal distribution of fibronectin, laminin, E-cadherin/beta-catenin complexes accompanied by elevated levels of p53 and TGF-beta1 were detected in the Ukrainian cRCCs from 137Cs-contaminated areas with statistically significant differences. Thus, our study suggests that chronic long-term, low-dose IR exposure might result in global remodeling of ECM components of the cRCCs with disruption in peri-epithelial stroma and epithelial basement membranes.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Cadherins; Carcinoma, Renal Cell; Chernobyl Nuclear Accident; Extracellular Matrix; Female; Fibronectins; Gene Expression; Humans; Immunohistochemistry; Kidney Neoplasms; Laminin; Male; Middle Aged; Neoplasm Staging; Radiation Effects; Time Factors; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Renal cell carcinoma (RCC) is the most prevalent cancer of the kidney. In human RCC cells, we recently showed that insulin-like growth factor I (IGF-I) has growth-promoting effects regulated by IGF-binding protein 3 (IGFBP-3). In this study, the analysis was expanded to include the interaction between the IGF and transforming growth factor-beta (TGF-beta) systems in the human RCC cells Caki-2 (from a primary tumor) and SK-RC-52 (from a metastasis). Functional effects such as cell proliferation, TGF-beta receptor (TbetaR) signaling, and IGFBP-3 levels were monitored after stimulation with various concentrations of IGF-I, TGF-beta, and IGFBP-3. In addition, human RCC tissues as well as experimental human RCC tumors were analyzed for cellular expression of phosphorylated Smad2 by immunohistochemistry. TGF-beta regulated the endogenous IGFBP-3 levels in these RCC cells as neutralizing anti-TGF-beta(1-3) antibodies strongly reduced the basal IGFBP-3 level. In addition, IGF-I increased the IGFBP-3 levels five- to eightfold with TGF-beta acting in synergy to enhance the IGFBP-3 levels 12- to 17-fold. Neutralizing TGF-beta(1-3) activity circumvented the growth inhibitory effects of IGFBP-3 seen in SK-RC-52, whereas it inhibited the growth-promoting effects of IGFBP-3 in Caki-2. Moreover, IGF-I interacted directly with TGF-beta activation of the TbetaR complex by enhancing phosphorylation and nuclear translocation of Smad2. This study demonstrates a direct interaction of the IGF and TGF-beta systems in human renal carcinoma cells. The observations that IGF-I enhances the TGF-beta signaling and that TGF-beta promotes IGFBP-3 production and thus influence the biological activity of IGF may be of importance for future therapeutic options.

Topics: Animals; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Kidney Neoplasms; Mice; Receptor Cross-Talk; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

The murine renal cell carcinoma (Renca) cells are insensitive to TGF-beta due to a lack of TGF-beta type II receptor (TbetaR-II). The objective of the present study is to determine the mechanism of this loss of sensitivity to TGF-beta in Renca cells. Renca cells were cultured and treated with 5-Aza-2'-Deoxycytidine (5-Aza), a specific inhibitor of methylation. Expression of TGF-beta type I receptor (TbetaRI) and TbetaRII was determined by RT-PCR and Western blot analysis before and after the treatment of Renca cells with 5-Aza. The expression of phosphorylated Smad2 (P-Smad2) was determined by Western blot analysis. TGF-beta levels in the conditioned medium were measured by ELISA. Renca cells did not express TbetaR-II prior to 5-Aza treatment. After 5-Aza treatment, these cells expressed TbetaR-II at both mRNA and protein levels, which corresponded to the restoration of sensitivity to TGF-beta by an increase in P-Smad2. Levels of TGF-beta1 were similar before and after 5-Aza treatment. Results of the present study indicated that, in Renca cells, the loss of sensitivity to TGF-beta is likely due to a promoter hypermethylation in the TbetaR-II gene.

Topics: Animals; Antimetabolites, Antineoplastic; Azacitidine; Carcinoma, Renal Cell; Cell Line, Tumor; Decitabine; DNA Methylation; Kidney Neoplasms; Mice; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

The major aim of this study was to investigate the association of the cytokine gene polymorphisms with the development of renal cell carcinoma (RCC). The study included 29 patients with RCC and 50 healthy controls. All genotyping (TNF-alpha, TGF-beta, IL-10, IL-6, IFN-gamma) experiments were performed using sequence-specific primers PCR (PCR-SSP). It was found that TNF-alpha -308 G/G and TGF-beta codon 10-25 T/T-G/C genotypes were significantly higher in frequency in the patients with RCC group compared with the healthy control group. Additionally, the frequency of TNF-alpha -308 G allele was significantly higher in the patients when compared to controls. On the other hand, the frequencies of TNF-alpha -308 G/A, IL-6 C/C and TGF-beta1 codon 10-25 C/C-G/G genotypes were significantly lower in the cancer group compared with the healthy control group. However, after correction for multiple comparisons (Bonferroni), these results did not remain significant. Nevertheless, these findings suggest that the TNF-alpha -308 G/G and TGF-beta codon 10-25 T/T-G/C genotypes may be potential risk factors for RCC, whereas TNF-alpha -308 G/A, IL-6 C/C and TGF-beta1 codon 10-25 C/C-G/G genotypes may be possible protective factors. The number of the cases has to be increased to investigate the independency of these polymorphisms involved in the oncogenesis of RCC.

Topics: Alleles; Carcinoma, Renal Cell; Codon; Cytokines; Female; Genotype; Humans; Interleukin-6; Kidney; Kidney Neoplasms; Male; Odds Ratio; Polymerase Chain Reaction; Polymorphism, Genetic; Risk; Risk Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

The competition between STAT1 and Smad3 for a limiting amount of the nuclear protein p300, a transcriptional coactivator, was suggested to be a mechanism for the antagonism between interferon-gamma (IFN-gamma) and transforming growth factor-beta1 (TGF-beta1). We investigated the effect of TGF-beta1 on IFN-gamma-induced HLA-DR production in cultured human glomerular endothelial cells (HGECs), and the involvement of p300 in this process.. Cell surface expression of HLA-DR and mRNA levels of HLA-DR and class II transactivator (CIITA), the master regulator of HLA-DR gene transcription, were measured by cellular ELISA and Northern blot, respectively. The levels of STAT1 and Smad3 protein were analyzed by Western blot. Nuclear binding activity of STAT1 was assessed by electrophoretic mobility shift assay.. IFN-gamma increased the cell surface expression of HLA-DR along with increases in the mRNA levels of CIITA and HLA-DR, while these stimulatory effects of IFN-gamma were down-regulated by TGF-beta1. IFN-gamma increased phosphorylation of STAT1 and this activation was not inhibited by TGF-beta1. IFN-gamma increased binding of p-STAT1 to p300, while TGF-beta1 increased binding of Smad3 to p300. TGF-beta1-induced Smad3 binding to p300 was inhibited by IFN-gamma, whereas IFN-gamma-induced p-STAT1 binding to p300 was not inhibited by TGF-beta1. IFN-gamma increased DNA binding activity of STAT1. Inhibition of interaction between STAT1 and p300 by addition of anti-p300 antibody to nuclear extract down-regulated DNA binding activity of STAT1. In contrast, TGF-beta1 did not inhibit IFN-gamma-induced STAT1 binding to DNA.. TGF-beta1 down-regulated IFN-gamma-induced CIITA and HLA-DR expression in HGECs. Though there was an antagonism between IFN-gamma and TGF-beta1, the competition for p300 between p-STAT1 and Smad3 was not the mechanism for it.

Topics: Blotting, Northern; Blotting, Western; Carcinoma, Renal Cell; Down-Regulation; E1A-Associated p300 Protein; Electrophoretic Mobility Shift Assay; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; HLA-DR Antigens; Humans; Immunohistochemistry; Interferon-gamma; Kidney Diseases; Kidney Glomerulus; Kidney Neoplasms; Nuclear Proteins; Smad3 Protein; STAT1 Transcription Factor; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Connective tissue growth factor (CTGF, CCN2) plays a fundamental role in the development of tissue fibrosis by stimulating matrix deposition and mediating many of the pro-fibrotic effects of transforming growth factor (TGF)-beta. CCN2 induction by TGF-beta in renal proximal tubule epithelial cells (PTECs) is likely to play an important role in the development of tubulointerstitial fibrosis. In this study, we investigated the induction of CCN2 by TGF-beta1 and the possible mechanisms of this induction in human PTECs.. Experiments were performed on primary and transformed (human kidney cell (HKC)-clone 8) human PTECs. Induction of CCN2 in response to TGF-beta1 was studied at the gene promoter level by reporter gene assay, mRNA by semi-quantitative RT-PCR and protein by immunoblotting. While chemical inhibitors were used to assess the role of Ras/MEK/ERK1,2 signalling, an HKC cell line over-expressing Smad7 was used to assess the role of Smad signalling in induction of CCN2 by TGF-beta1.. TGF-beta1 induced CCN2 promoter activity, mRNA and protein in human PTECs. TGF-beta1-dependent CCN2 promoter activity was reduced by inhibiting Ras and MEK activation. MEK inhibition also resulted in inhibition of the TGF-beta1-induced secreted CCN2 protein. There was no significant increase in CCN2 gene promoter activity or protein by TGF-beta1 in Smad7 over-expressing HKCs.. TGF-beta1 induces the expression of CCN2 in human PTECs. This induction is dependent on Ras/MEK/ERK and Smad signalling. Inhibiting TGF-beta induced CCN2 by targeting Smad and/or Ras/MEK/ERK1,2 signalling pathways could be of therapeutic value in renal fibrosis.

Topics: Connective Tissue Growth Factor; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Fibrosis; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Kidney; Kidney Neoplasms; Kidney Tubules, Proximal; MAP Kinase Signaling System; ras Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Wilms tumor is one of the most frequent neoplasias in children. Our previous microarray screening in a large series of Wilms tumors revealed several candidate genes that are deregulated in advanced tumors and are part of the retinoic acid signaling pathway. To investigate whether retinoic acid could be employed as a novel therapeutic agent in these tumors, we treated cultured Wilms tumor cells with different concentrations of all-trans retinoic acid (ATRA) and assessed gene expression changes by real-time RT-PCR as well as microarray analysis. Several genes like RARRES1, RARRES3, CTGF, CKS2, CCNA2, IGFBP3, UBE2C, CCL2 or ITM2B that were previously found to be deregulated in advanced tumors exhibited opposite expression changes after ATRA treatment. In addition to enhanced retinoid signaling, the transforming growth factor-beta (TGFbeta) pathway was strongly activated by ATRA treatment of Wilms tumor cells. Both the retinoic acid and the TGFbeta pathway mediate inhibition of cell growth. These findings represent the first molecular evidence of a potential benefit from ATRA treatment in Wilms tumors.

Topics: Antineoplastic Agents; Cell Proliferation; DNA-Binding Proteins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad Proteins; Trans-Activators; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured; Wilms Tumor

Transforming growth factor-beta1 (TGF-beta1) has a biphasic effect on the growth of renal epithelial cells. In transformed cells, TGF-beta1 appears to accelerate the proliferation of malignant cells. The diverse cellular functions of TGF-beta1 are regulated by three high-affinity serine/threonine kinase receptors, namely TbetaRI, TbetaRII and TbetaRIII. The renal serine protease tissue kallikrein acts on its endogenous protein substrate kininogen to form kinin peptides. The cellular actions of kinins are mediated through B1 and B2 G protein-coupled rhodopsin receptors. Both kinin peptides and TGF-beta1 are mitogenic, and therefore may play an important role in carcinogenesis. Experiments were designed to immunolabel tissue kallikrein, TGF-beta1, TbetaRII, TbetaRIII and kinin receptors using specific antibodies on serial sections of normal kidney and clear-cell renal carcinoma (CCRC) tissue, which included both the tumour and the adjacent renal parenchyma. The essential result was the localisation of tissue kallikrein, kinin B 1 and B 2 receptors and TGF-beta1 primarily on the cell membranes of CCRC cells. In the distal and proximal tubules of the renal parenchyma adjacent to the carcinoma (RPTAC), immunolabelling for tissue kallikrein was reduced, but the expression of kinin B1 and B2 receptors was enhanced. Immunolabelling for TbetaRII and TbetaRIII was more pronounced in the proximal tubules of the tissue adjacent to the carcinoma when compared to the normal kidney. The expression of tissue kallikrein, kinin receptors, and TbetaRII and TbetaRIII may be relevant to the parenchymal invasion and metastasis of clear-cell renal carcinoma.

Topics: Adenocarcinoma, Clear Cell; Humans; Kidney Neoplasms; Receptors, Bradykinin; Receptors, Transforming Growth Factor beta; Tissue Kallikreins; Transforming Growth Factor beta; Transforming Growth Factor beta1

We report the induction of gremlin, a bone morphogenetic protein antagonist, in cultured human mesangial cells exposed to high glucose and transforming growth factor beta (TGF-beta) levels in vitro and kidneys from diabetic rats in vivo.. Gremlin expression was assessed in human diabetic nephropathy by means of in situ hybridization, immunohistochemistry, and real-time polymerase chain reaction and correlated with clinical and pathological indices of disease.. Gremlin was not expressed in normal human adult kidneys. Conversely, abundant gremlin expression was observed in human diabetic nephropathy. Although some gremlin expression was observed in occasional glomeruli, gremlin expression was most prominent in areas of tubulointerstitial fibrosis, where it colocalized with TGF-beta expression. Gremlin messenger RNA levels correlated directly with renal dysfunction, determined by means of serum creatinine level, but not with proteinuria level. There was a strong correlation between gremlin expression and tubulointerstitial fibrosis score.. In aggregate, these results indicate that the developmental gene gremlin reemerges in the context of tubulointerstitial fibrosis in diabetic nephropathy and suggests a role for TFG-beta as an inducer of gremlin expression in this context.

Topics: Bone Morphogenetic Proteins; Cells, Cultured; Cytokines; Diabetic Nephropathies; Fibrosis; Gene Expression Regulation; Glomerular Mesangium; Glucose; Humans; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Kidney Neoplasms; Nephritis, Interstitial; Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

Allelic differences in gene promoter or codifying regions have been described to affect regulation of gene expression, consequently increasing or decreasing cytokine production and signal transduction responses to a given stimulus. This observation has been reported for interleukin (IL)-10 (-1082 A/G; -819/-592 CT/CA), transforming growth factor (TGF)-beta (codon 10 C/T, codon 25 G/C), tumor necrosis factor (TNF)-alpha (-308 G/A), TNF-beta (+252 A/G), interferon (IFN)-gamma (+874 T/A), IL-6 (-174 G/C), and IL-4R alpha (+1902 G/A). To evaluate the influence of these cytokine genotypes on the development of acute or chronic rejection, we correlated the genotypes of both kidney graft recipients and cadaver donors with the clinical outcome. Kidney recipients had 5 years follow-up, at least 2 HLA-DRB compatibilities, and a maximum of 25% anti-HLA pretransplantation sensitization. The clinical outcomes were grouped as follows: stable functioning graft (NR, n = 35); acute rejection episodes (AR, n = 31); and chronic rejection (CR, n = 31). The cytokine genotype polymorphisms were defined using PCR-SSP typing. A statistical analysis showed a significant prevalence of recipient IL-10 -819/-592 genotype among CR individuals; whereas among donors, the TGF-beta codon 10 CT genotype was significantly associated with the AR cohort and the IL-6 -174 CC genotype with CR. Other albeit not significant observations included a strong predisposition of recipient TGF-beta codon 10 CT genotype with CR, and TNF-beta 252 AA with AR. A low frequency of TNF-alpha -308 AA genotype also was observed among recipients and donors who showed poor allograft outcomes.

Topics: Cytokines; Genotype; Graft Rejection; Humans; Interleukin-10; Interleukin-6; Kidney Neoplasms; Transforming Growth Factor beta; Transplantation, Homologous; Treatment Outcome; Tumor Necrosis Factor-alpha

Topics: Adult; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Carcinoma, Renal Cell; Female; Humans; Immunohistochemistry; Kidney Neoplasms; Transforming Growth Factor beta

TGFbeta1 is one of several cytokines produced by proximal tubular and renal cancer cells. Previous studies have been mainly focused on determining plasma or serum TGFbeta levels, its effect on RCC cultures, and the expression of TGFbeta mRNA. Cancerous and autologous normal kidney samples were obtained from 24 patients treated by radical nephrectomy. TGFbeta1 expression was determined using a semi quantitative Western blot analysis and immunohistochemistry. Blot densities and immunohistochemical expression intensities in normal and neoplastic tissue were compared, and subsequently correlated to tumor stage, histological type and nuclear grade. All tissue samples examined expressed TGFbeta1; mean tumor to non-involved kidney spot density ratio correlated with advancing stage and higher nuclear grade. The overexpression of TGFbeta1 in certain RCCs may partially explain their resistance to the growth suppression action of TGFbeta. The correlation with tumor stage and grade indicates a possible role in the development of metastatic potential as well as in host's immune response modulation.

Topics: Adult; Aged; Blotting, Western; Carcinoma, Renal Cell; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Kidney; Kidney Neoplasms; Male; Middle Aged; Neoplasm Staging; Precipitin Tests; RNA, Messenger; Transforming Growth Factor beta

Rapamycin is an effective inhibitor of human renal cancer metastasis.. Human renal cell cancer (RCC) is common and is 10 to 100 times more frequent in patients with end-stage renal disease (ESRD) and candidates for renal transplantation. Treatment of metastatic RCC is largely ineffective and is further undermined by immunosuppressive therapy in transplant recipients. A treatment regimen that prevents transplant rejection while constraining RCC progression would be of high value.. We developed a human RCC pulmonary metastasis model using human RCC 786-O as the tumor challenge and the severe combined immunodeficient (SCID) beige mouse as the host. We explored the effect of rapamycin, cyclosporine, or rapamycin plus cyclosporine on the development of pulmonary metastases and survival. The effects of the drugs on tumor cell growth, apoptosis, and expression of vascular endothelial growth factor (VEGF-A) and transforming growth factor beta1 (TGF-beta1) were also investigated.. Rapamycin reduced, whereas cyclosporine increased, the number of pulmonary metastases. Rapamycin was effective in cyclosporine-treated mice, and rapamycin or rapamycin plus cyclosporine prolonged survival. Rapamycin growth arrested RCC 786-O at the G1 phase and reduced VEGF-A expression. Immunostaining of lung tissues for von Willebrand factor was minimal and circulating levels of VEGF-A and TGF-beta1 were lower in the rapamycin-treated mice compared to untreated or cyclosporine-treated mice.. Our findings support the idea that rapamycin may be of value for patients with RCC and that its antitumor efficacy is realized by cell cycle arrest and targeted reduction of VEGF-A and TGF-beta1. A regimen of rapamycin and cyclosporine, demonstrated to be effective in reducing acute rejection of renal allografts, may prevent RCC progression as well, and has the potential to prevent mortality due to RCC in patients with ESRD who have received renal allografts.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Cell Cycle; Cell Line, Tumor; Cyclosporine; Gene Expression; Humans; Immunosuppressive Agents; Kidney Neoplasms; Lung Neoplasms; Mice; Mice, SCID; Sirolimus; Survival Rate; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

The present study was carried out to clarify whether a histopathological analysis of vascular endothelial growth factor (VEGF), transforming growth factor-beta1 (TGF-beta1) and matrix metalloproteinase 2 (MMP-2) can help predict the outcome of renal cell carcinoma (RCC). We examined the expression of VEGF, TGF-beta1 and MMP-2 in a large series of RCC with a long follow-up, based on histopathological factors and survival.. Immunostaining for VEGF, TGF-beta1 and MMP-2 was performed on formalin-fixed, paraffin-embedded tissue sections from 84 patients with RCC who underwent nephrectomy at our institution between 1985 to 2000. The microvessel density (MVD) of tumor tissue was measured after it immunohistochemically stained with CD105 (Endoglin) monoclonal antibody.. A significant association was observed in the expression of VEGF and TGF-beta1 regarding the stage (P < 0.01, P < 0.01), nuclear grade (P < 0.01, P < 0.01) and MVD (P < 0.001, P < 0.001), respectively. However, no correlation was found among the results of MMP-2, nuclear grade and MVD. A multivariate analysis demonstrated both the nuclear grade and MVD to be independent prognostic factors.. Our results suggested that the expression of both VEGF and/or TGF-beta1 can be useful predictive prognostic factors RCC. In addition, a multivariate analysis demonstrated MVD to be an independent prognostic factor of RCC.

Topics: Carcinoma, Renal Cell; Confidence Intervals; Endothelial Growth Factors; Humans; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Kidney Neoplasms; Logistic Models; Lymphokines; Matrix Metalloproteinase 2; Middle Aged; Neovascularization, Pathologic; Survival Analysis; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

We evaluated the effects of transforming growth factor-beta1 (TGF-beta1) on growth activity, cytokine mRNA expression and cytokine protein production by renal cancer cells.. Exogenous continuous exposure of biologically active TGF-beta1 was performed on ACHN cells at various concentrations of 0.1 to 30 ng./ml. and the number of cells was counted each culture day. To determine the index of S-phase cells the bromodeoxyuridine pulse labeling method was used. Reverse transcriptase-polymerase chain reaction (PCR) was done and PCR products were quantified. Each supernatant cytokine level was measured using an enzyme-linked immunosorbent assay.. TGF-beta1 significantly inhibited the growth activities of ACHN cells compared with controls. Bromodeoxyuridine labeling indexes after treating ACHN cells with TGF-beta1 (10 ng./ml.) showed that it began to decrease gradually after 24 hours and after 72 hours it was inhibited to approximately 40% compared with controls. The mRNA of TGF-beta1, TGF-beta types 1 and 2 receptors, interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF) from ACHN cells was detected by reverse transcriptase-PCR assay. IL-6 and GM-CSF proteins were produced constitutively from ACHN cells but other cytokines were not. Adding TGF-beta1 to the cell culture medium did not influence mRNA expression, or the protein production of IL-6 or GM-CSF.. The inhibition of growth activities of ACHN cells by TGF-beta1 are mediated by its direct binding to specific receptors on ACHN cells followed by cell cycle inhibition, while TGF-beta1 seems to have no effect on the production of the cytokines studied.

Topics: Carcinoma, Renal Cell; Cell Division; Cytokines; Humans; Kidney Neoplasms; Protein Biosynthesis; RNA, Messenger; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Hypertrophic osteoarthropathy (HOA) is a paraneoplastic syndrome characterized by periosteal formation and arthritis and usually accompanied by clubbing ofthe digits. Many malignancies have been associated with HOA/clubbing, most being lung cancer and lung metastatic cancer. We herein present a 53-year-old man with lung metastasis from renal cell carcinoma (RCC). HOA occurred one year after the metastasis. Reviewing the literature, only five cases of RCC with HOA have been reported. If their clinical history was traceable, they consistently had disease progression. We reviewed the pathogenesis of HOA/clubbing and linked the prognosis of RCC to relevant cytokines. Therefore, HOA not only heralds a progression of disease but suggests a probable therapeutic choice by targeting some cytokines.

Topics: Carcinoma, Renal Cell; Disease Progression; Endothelial Growth Factors; Humans; Intercellular Signaling Peptides and Proteins; Kidney Neoplasms; Lymphokines; Male; Middle Aged; Osteoarthropathy, Secondary Hypertrophic; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

We have evaluated CD8+ and CD4+ T-cell responses against a new tumor-associated antigen, the receptor tyrosine kinase EphA2, which is broadly expressed in diverse cancer histologies and is frequently overexpressed in advanced stage/metastatic disease. We report herein that EphA2 is overexpressed in renal cell carcinoma (RCC) cell lines and clinical specimens of RCC, and find that the highest levels of EphA2 are consistently found in the most advanced stages of the disease. We identified and synthesized five putative HLA class I-binding and three class II-binding peptides derived from EphA2 that might serve as targets for immune reactivity. Each peptide induced specific, tumor-reactive CD8+ or CD4+T-cell responses as measured using IFN-gamma enzyme-linked immunospot assays. The EphA2 peptides elicited relatively weak responses from CD8+ T cells derived from normal healthy volunteers or from RCC patients with active disease. In marked contrast, immune reactivity to EphA2-derived epitopes was greatly enhanced in CD8+ T cells that had been isolated from patients who were rendered disease-free, after surgery. Furthermore, enzyme-linked immunospot analyses demonstrated prominent EphA2-restricted T-helper 1-type CD4+ T cell activity in patients with early stage disease, whereas T-helper 2-type and T regulatory-type responses predominated in patients with more advanced forms of RCC. These data suggest that the immune system of cancer patients actively monitors EphA2-derived epitopes, and that the magnitude and character of T-cell responses to EphA2 epitopes may convey much-needed predictive information about disease stage and outcome.

Topics: Adult; Aged; Amino Acid Sequence; Carcinoma, Renal Cell; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Epitope Mapping; Epitopes, T-Lymphocyte; Female; Humans; Interferon-gamma; Interleukin-10; Interleukin-5; Kidney Neoplasms; Lymphocyte Activation; Male; Middle Aged; Molecular Sequence Data; Neoplasm Staging; Receptor, EphA2; Transforming Growth Factor beta

Immunosuppressive therapy is a risk factor for the increased incidence and metastatic progression of malignancies in organ graft recipients. Transforming growth factor (TGF)-beta(1) has been associated with tumor invasion and metastasis, and we have implicated cyclosporine-associated TGF-beta(1) hyperexpression in tumor progression in mice.. BALB/c mice or severe combined immunodeficient-beige mice were treated with 2 or 4 mg/kg of tacrolimus, and the effect of treatment on mouse renal cancer cell pulmonary metastasis was investigated. We also determined whether tacrolimus induces TGF-beta(1) expression. Spleens from tacrolimus-treated mice were analyzed for level of expression of TGF-beta(1) mRNA with the use of competitive-quantitative polymerase chain reaction assay, and circulating levels of TGF-beta(1) protein were measured with the use of an enzyme-linked immunosorbent assay.. Treatment with tacrolimus resulted in a dose-dependent increase in the number of pulmonary metastases in the BALB/c mice (197+/-16 in untreated mice, 281+/-26 in mice treated with 2 mg/kg of tacrolimus, and 339+/-25 in mice treated with 4 mg/kg of tacrolimus; no treatment vs. 4 mg/kg tacrolimus, Bonferroni's P<0.001) and in the severe combined immunodeficient-beige mice (117+/-18 in untreated mice, 137+/-19 in mice treated with 2 mg/kg of tacrolimus, and 216+/-29 in mice treated with 4 mg/kg of tacrolimus; no treatment vs. 4 mg/kg tacrolimus, P<0.05). Treatment with 4 mg/kg but not 2 mg/kg of tacrolimus resulted in a significant increase in the levels of expression of TGF-beta(1) mRNA and circulating levels of TGF-beta(1) protein.. Tacrolimus has a dose-dependent effect on tumor progression and TGF-beta(1) expression, and tacrolimus-induced TGF-beta(1) overexpression may be a pathogenetic mechanism in tumor progression.

Topics: Animals; Carcinoma, Renal Cell; Disease Progression; Dose-Response Relationship, Drug; Immunosuppressive Agents; Kidney Neoplasms; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, SCID; Neoplasm Metastasis; Neoplasms; Spleen; Tacrolimus; Transforming Growth Factor beta; Transforming Growth Factor beta1

Renal cell carcinoma (RCC) is a major health issue. Whereas localized disease can be cured surgically, there is no effective therapy for metastatic disease. The development of an effective therapy will require an understanding of the pathways that are important in RCC carcinogenesis and progression. Using genomic profiling of patient-matched tissue, we have identified aberrations in the transforming growth factor beta (TGFbeta) signaling pathway in RCC. We observed loss of type III TGFbeta receptor (TBR3) expression in all RCC samples. This suggests that TBR3 loss is an early event in RCC carcinogenesis and plays a sentinel role in the acquisition of a tumorigenic phenotype. We also observed subsequent loss of type II TGFbeta receptor (TBR2) expression in metastatic RCCs. We propose that loss of TBR3 is necessary for RCC carcinogenesis, and that loss of TBR2 leads to acquisition of a metastatic phenotype. To this end, we have identified a human renal cell carcinoma line (UMRC6) that is representative of localized, nonmetastatic RCC, reflecting a loss of TBR3, but not TBR2 expression. Another cell line, UMRC3, is highly metastatic, having lost TBR3 and TBR2 expression. We demonstrate functional loss of TGFbeta responsiveness in these cell lines as observed through phenotypic and transcriptional responsiveness to exogenous TGFbeta. Restoring TBR2 and TBR3 expression in UMRC3 cells attenuates cell proliferation, completely restores TGFbeta-mediated transcriptional responses, and completely blocks anchorage independent-growth: while restoration of TBR2 partially restores TGFbeta-mediated signaling. Based on these data, we propose that dysregulation in TGFbeta signaling, through stepwise loss in receptor expression, plays a prominent role in RCC carcinogenesis and progression. In addition, these studies unequivocably demonstrate a link between loss of TBR3 and a human disease.

Topics: Carcinoma, Renal Cell; Cell Division; Disease Progression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Proteoglycans; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Bone morphogenetic proteins (BMPs) are members of a family of pleiotropic growth factors that play a critical role during renal development as well as maintaining kidney homeostasis. In the present study, we investigated the potential role of BMP receptors (BMPRs) in renal cell carcinoma (RCC) cells.. Immunohistochemistry was used to investigate the expression of BMPRs in human RCC tissues. As an in vitro model of RCC, three cell lines were used: 112, 117, and 181. Northern blot, immunoblot, and reverse transcription-PCR were used to study the expression of BMPRs in the cell lines. Finally, cells were transfected using LipofectAMINE.. Normal human kidney tissues express the three BMPRs: types RIA, RIB, and RII. In contrast, human RCC cells frequently exhibit a loss of expression of BMP-RII. In tissue culture, BMP-6 inhibits in a dose-dependent manner the proliferation of 112 cells but not of 117 and 181 cells. Assays for BMPRs demonstrated that 117 and 181 cells express low levels of BMP-RII RNA. When these two BMP-6 resistant cell lines were infected with the adenovirus containing the constitutively active form of BMP-RIA or -RIB in combination with a BMP-6-responsive luciferase reporter construct, luciferase activity increased. Finally, when these cell lines were transfected with BMP-RII, BMP-6-sensitivity was restored.. These results demonstrate that human RCC tissues frequently have decreased levels of expression of BMP-RII and that the human RCC cell lines 117 and 181 are resistant to the growth-inhibitory effect of BMP-6 because they have decreased levels of expression of BMP-RII.

Topics: Blotting, Northern; Bone Morphogenetic Protein 6; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Carcinoma, Renal Cell; Down-Regulation; Humans; Immunoblotting; Immunoenzyme Techniques; Kidney Neoplasms; Protein Serine-Threonine Kinases; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Tumor Cells, Cultured

The present study was performed to assess the expression of isoforms 1, 2 and 3 of transforming growth factor (TGF)-beta in skin nodular dermatofibrosis lesions, kidney, bladder and pancreas from a 10-year-old female German shepherd dog (GSD) affected by renal cystadenocarcinoma and nodular dermatofibrosis (RCND) compared with normal GSDs (n = 2). Formalin-fixed, paraffin-embedded tissues obtained from the dog affected by RCND, diagnosed by renal ultrasonography and histopathological examination were analysed by immunohistochemistry using polyclonal antibodies to TGF-beta1, 2 and 3, and evaluated semiquantitatively using an immunoreactivity score. Similar expression of TGF-beta2 and TGF-beta3 was observed in all tissue specimens in both the RCND-affected animal and normal dogs. In contrast, TGF-beta1 immunoreactivity was increased in the derma of the RCND canine. Comparable TGF-beta1 serum levels were found between the diseased and normal animals. The increased local cutaneous production of TGF-beta1 in the RCND dog, compared with the normal animals, suggests that this cytokine may play an important role in the induction of nodular dermatofibrosis associated with renal cystadenocarcinoma.

Topics: Animals; Case-Control Studies; Cystadenocarcinoma; Dog Diseases; Dogs; Female; Histiocytoma, Benign Fibrous; Immunohistochemistry; Kidney; Kidney Neoplasms; Pancreas; Skin; Skin Neoplasms; Transforming Growth Factor beta; Urinary Bladder

Blood vessel growth by angiogenesis plays an essential role in embryonic development, wound healing, and tumor growth. To understand the molecular cues underlying this process we have used the PCR-based subtractive hybridization method, representational difference analysis, to identify genes upregulated in endothelial cells (EC) forming tubes in 3D collagen gels, compared to migrating and proliferating cells in 2D cultures. We identified several previously characterized angiogenic markers, including the alpha(v) chain of the alpha(v)beta3 integrin and plasminogen activator inhibitor-1, suggesting overlap in gene expression between tube-forming cells in vitro and in vivo. We also found a 2- to 10-fold upregulation of (beta)ig-h3 (a collagen-binding extracellular matrix protein), NrCAM (a "neural" cell adhesion molecule), Annexin II (a tPA receptor), ESM-1 (an EC-specific molecule of unknown function), and Id2 (an inhibitory bHLH transcription factor). We identified a novel splice variant of the ESM-1 gene and also detected dramatically enhanced expression of ESM-1 and (beta)ig-h3 in several tumors. Antisense oligonucleotides to (beta)ig-h3 blocked both gene expression and tube formation in vitro, suggesting that (beta)ig-h3 may play a critical role in EC-matrix interactions. These data expand the suite of genes implicated in vascular remodeling and angiogenesis.

Topics: Alternative Splicing; Animals; Blotting, Northern; Cell Adhesion Molecules; Cell Division; Cells, Cultured; Cloning, Molecular; DNA, Complementary; Endothelium, Vascular; Extracellular Matrix Proteins; Humans; Kidney Neoplasms; Models, Biological; Neoplasm Proteins; Neovascularization, Pathologic; Neovascularization, Physiologic; Oligonucleotides, Antisense; Proteoglycans; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA; Tissue Distribution; Transforming Growth Factor beta

Up to now, clinical tumor-markers for renal cell carcinoma (RCC) have been lacking. Increased plasma levels of transforming growth factor-beta1 (TGF-beta1) were described as a tumor-marker and prognostic factor in RCC. The aim of this study was to test the clinical suitability of plasma TGF-beta1 as a tumor-marker for RCC. The concentrations of active and latent TGF-beta1 were determined in plasma of patients with localized (n = 39) and metastasised (n = 17) RCC. A newly developed, highly sensitive ELISA, which is specific for the isoform beta1, was used. Active TGF was directly measured in the EDTA plasma. To determine the amount of latent TGF-beta1, which is bound predominantly at beta2-macroglobulin, an optimized activation procedure was applied. Patients with localized RCC showed median concentrations of 16,700 ng/l (6,200-54,800 ng/l) for latent TGF-beta1. A total of 94 patients with various nonmalignant urological diseases were recruited as a control group. In comparison, this group had median concentrations of 19,900 ng/l (2,640-52,300 ng/l) for latent TGF-beta1. There was no significant difference (nonparametric Kruskal-Wallis ANOVA) between these groups. Patients with metastatic RCC showed median concentrations of 34,500 ng/l (6,800-48,960 ng/l) for latent TGF-beta1. In comparison to the localized RCC group, a statistically significant difference was found. Plasma levels after operative therapy (days 1, 5 and 10) and during follow-up without evidence of disease (2-6 months) showed no significant differences. Contrary to other study groups, our results suggest that TGF-beta1 is not a suitable tumor-marker for the diagnosis of localized RCC. In the face of higher TGF-beta1 plasma levels in metastatic disease, TGF-beta1 may be useful in the early detection of RCC recurrence or to control the success of immunochemotherapy.

Topics: Biomarkers, Tumor; Carcinoma, Renal Cell; Humans; Kidney Neoplasms; Male; Transforming Growth Factor beta; Transforming Growth Factor beta1

Captopril is known to inhibit the growth of renal cancer but the mechanism involved has been unclear. The current study elucidates the mechanism of captopril induced inhibition of the growth of the Renca mouse renal cancer cell line involving transforming growth factor-beta, which is known to be a growth inhibitory cytokine in epithelial cells and tissues.. Transforming growth factor-beta in conditioned medium was measured by bioassay. Levels of transforming growth factor-beta and transforming growth factor-beta type II receptor expression messenger RNA were determined by reverse transcriptase-polymerase chain reaction and flow cytometry. Cell viability was determined by bromodeoxyuridine (BrdU) incorporation and tetrazolium bromide assay.. Captopril (0.01 to 1 mM.) showed no significant effect on transforming growth factor-beta synthesis or transforming growth factor-beta messenger RNA in Renca cells. On the other hand, 1 mM. captopril significantly inhibited Renca cell growth. Reverse transcriptase-polymerase chain reaction and flow cytometry showed that 1 mM. captopril up-regulated type II receptor expression.. These findings suggest that captopril restores transforming growth factor-beta type II receptor expression and inhibits the growth of Renca cells by increasing their sensitivity to transforming growth factor-beta.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Captopril; Carcinoma, Renal Cell; Cell Division; Kidney Neoplasms; Mice; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

To assess the expression of the homeogene Pax-2 in adult renal cell carcinomas, we did a retrospective immunohistochemical analysis of 56 frozen tumor samples representing all major histologic subtypes of renal tumors. There were 33 conventional renal cell carcinomas (58.9%), 12 papillary renal cell carcinomas (21.4%), 4 chromophobe cell renal carcinomas, 4 urothelial cell renal carcinomas, and 3 oncocytomas. Forty-five tumors (62.5%) were localized, and 21 tumors had extrarenal involvement. Eight patients (14%) had metastatic disease at the end of the follow-up. We searched for relationships between Pax-2 expression and nuclear grading, TNM staging, Ki-67 proliferation index, expression of transforming growth factor-beta1 (TGF-beta 1), an in vitro down-regulator of Pax-2 expression, and finally cytogenetic abnormalities. All histologic subtypes expressed Pax-2 protein, except urothelial renal carcinomas. The highest expression was in papillary renal cell carcinomas. In this subtype, all tumors and 83.3% +/- 12.3% of tumor cells were immunoreactive for Pax-2. All but 2 conventional renal cell carcinomas expressed Pax-2, but with 26.3% +/- 29.6% of immunoreactive cells (P <.001). Pax-2 expression was not correlated with nuclear grading (P =.6), tumor size (P =.3), and TGF-beta 1 expression (P =.1). Nevertheless, Pax-2 expression correlated with the Ki-67 proliferation index only for the conventional histologic subtype (P =.03). In this histologic subtype, Pax-2 expression was higher in patients with metastatic disease than in those without (P =.02). Pax-2 expression was not associated with specific cytogenetic abnormalities like trisomy 7 (P =.1), 3p deletion (P =.5), and hyperdiploidy (P =.2). TGF-beta 1 expression, positive in 33 tumors (59%), was not correlated with either Pax-2 expression (P =.1) or current prognostic factors such as nuclear grading (P =.2). Interestingly, we also observed an expression of TGF-beta RI and TGF-beta RII in the tumors with high nuclear grading (P =.005). We conclude that Pax-2 protein is expressed in all major histologic subtypes of renal cell carcinomas. The pattern of expression differs between these subtypes. Pax-2 expression in conventional renal cell carcinomas is correlated with the proliferation index and is significantly higher in patients with metastatic disease. HUM PATHOL 32:282-287.

Topics: Adenoma, Oxyphilic; Adult; Aged; Aged, 80 and over; Carcinoma, Papillary; Carcinoma, Renal Cell; Cell Division; Cell Nucleus; Chromosome Aberrations; Cryopreservation; Cytogenetic Analysis; DNA-Binding Proteins; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Kidney Neoplasms; Male; Middle Aged; Neoplasm Metastasis; PAX2 Transcription Factor; Retrospective Studies; Transcription Factors; Transforming Growth Factor beta

To analyze the role of the transforming growth factor (TGF)-beta pathway in renal tumors and to verify whether alterations in TGF-beta 1 pathway expression are associated with the grade of tumor differentiation and pathologic stage in renal cell carcinomas.. The expression of TGF-beta 1 and TGF-beta receptors (T beta RI and T beta RII), SMAD-2 and SMAD-4 was investigated by immunohistochemistry in normal peritumoral and tumoral tissue from 53 renal cell carcinomas (clear cell type). The gene expression of SMAD-2 and SMAD-4 was also studied by reverse transcription polymerase chain reaction (RT-PCR) in normal peritumoral and tumoral tissue from 6 of 56 primary tumors.. TGF-beta 1, T beta RI and T beta RII immunoreactivity was more frequent in tumoral than in normal peritumoral renal tissue (96.22%, 79.25% and 75.41% vs. 88.37%, 69.76% and 62.69%), whereas SMAD-2 and SMAD-4 immunoreactivity was more frequent in normal peritumoral than in tumoral tissue (23.25% and 30.23% vs. 15.09% and 7.54%). In tumor areas, immunohistochemical scores were lower for T beta RII than for T beta RI and TGF-beta 1 and higher than SMAD-4 and SMAD-2 scores. TGF-beta 1, T beta RI, T beta RII and SMAD-4 histologic scores correlated with neither the histologic grade of malignancy nor TNM clinical stage, whereas SMAD-2 protein levels were significantly lower in grade 3 than in grade 1 tumors. In the samples of normal kidney and carcinoma studied, RT-PCR detected the correct transcripts for SMAD-2 and SMAD-4, indicating that the RNA of the samples analyzed contained RNA sequences coding for these genes.. Our data support the concept that the reduction of T beta RII and SMAD proteins in renal cell carcinomas is involved in tumor development and suggest an altered TGF-beta/SMAD signaling pathway in kidney neoplasia.

Topics: Carcinoma, Renal Cell; DNA-Binding Proteins; Humans; Immunohistochemistry; Kidney; Kidney Neoplasms; Polymerase Chain Reaction; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) plays central roles in embryonic development, organogenesis, and physiologic connective tissue remodeling during wound healing and tissue repair as well as in carcinogenesis. Estrogens have key roles in a variety of biological events, such as the development and maintenance of female reproductive organs and bone and lipid metabolism. Previous studies demonstrated that estrogens suppress TGF-beta-induced gene expression, such as type IV collagen in kidney mesangial cells. However, the molecular mechanisms that mediate this antagonistic effect are unknown. To elucidate the mechanisms of cross-talk between TGF-beta and estrogen receptor (ER) signaling pathways, we reconstituted TGF-beta and ER signaling in human kidney carcinoma cells. Here we demonstrate that TGF-beta-induced activation of Sma and MAD-related protein 3 (Smad3) activity, one of the major intracellular transducers of TGF-beta signaling, was suppressed by ER, whereas ER-mediated transcriptional activation was enhanced by TGF-beta signaling. We provide evidence that this two-way cross-talk between the estrogen and TGF-beta signaling pathways was the result of direct physical interactions between Smad3 and ER. These findings have implications for a variety of disease states, such as the pathophysiology of kidney function, atherosclerosis, and breast cancer.

Topics: Blotting, Northern; DNA-Binding Proteins; Enzyme Activation; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens; Humans; Immunoblotting; Kidney Neoplasms; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Receptors, Estrogen; Recombinant Proteins; Signal Transduction; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Wilms tumor is one of the most common solid tumors in children. A transforming growth factor-alpha (TGF-alpha)/epidermal growth factor receptor (EGF-R) autocrine loop plays an important role in tumor growth. Abnormal expression of TGF-alpha, EGF-R and c-erb B-2 has been demonstrated in several human malignancies.. The immunohistochemical expression of TGF-alpha, EGF-R, and c-erb B-2 was studied in paraffin material of 62 clinical Wilms tumors. Patients had a mean follow-up of 5.7 years.. Generally, TGF-alpha, EGF-R, and c-erb B-2 were expressed in tissue of the normal kidney and at variable levels in the three cell types of Wilms tumor, i.e., blastemal, epithelial, and stromal cells. Immunoreactive blastema cells were found in 48%, 44%, and 34% of tumors for TGF-alpha, EGF-R, and c-erb B-2, respectively. It was found that TGF-alpha, EGF-R, and c-erb B-2 blastemal and epithelial expression gradually increased from T1 to T3. The blastemal expression of TGF-alpha was statistically significantly correlated with clinicopathologic stages. Both univariate and multivariate analysis showed that blastemal TGF-alpha expression was indicative for clinical progression, but neither blastemal TGF-alpha, nor EGF-R or c-erb B-2 expression correlated with patients survival. Epithelial staining was of no prognostic value. The simultaneous expression of TGF-alpha/EGF-R was indicative for clinical progression at univariate level.. Increased expression of TGF-alpha in the blastemal part of Wilms tumor correlated with tumor classification and clinical progression. These findings suggest that significant expression of TGF-alpha and EGF-R may play a role in promoting transformation and/or proliferation of Wilms tumor, perhaps by an autocrine mechanism. Therefore, their expression may be of value in identifying patients at high risk of tumor recurrence.

Topics: Biomarkers, Tumor; Child; Child, Preschool; Disease Progression; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Kidney Neoplasms; Male; Neoplasm Recurrence, Local; Prognosis; Receptor, ErbB-2; Risk Factors; Transforming Growth Factor beta; Wilms Tumor

Invasion of tumor cells into the extracellular matrix is an essential step in the formation of metastases in renal cancer. Cell adhesion molecules such as beta(1)-integrins, which bind to the RGD sequence (arginine-glycine-asparagine) and CD44 are involved in this process. We examined the invasion of a renal carcinoma cell line (CCF-RC1) into the extracellular matrix compounds fibronectin, collagen IV and laminin and the effect of TGFbeta and IFNgamma on this process. The inhibitory effect of an antibody against the beta(1)-subunit of integrins (CD29), as well as a pentapeptide including the RGD sequence, was also evaluated. A micro-chemotaxis chamber, including a polycarbonate membrane with a pore diameter of 8 microm, was used for quantification of cell migration. The addition of the extracellular matrix compounds fibronectin, laminin and collagen IV resulted in a 5-10-fold increase in invasion. This increased invasion depends strongly on the presence of beta(1)-integrins, shown by the use of an antibody against CD29 or a RGD including peptide which inhibit the cell migration by approximately 88%. CD44 is less involved in collagen IV dependent migration and almost no influence of CD44 was observed on a fibronectin and laminin dependent migration. TNFalpha and IFNgamma did not significantly influence the expression of CD29 or CD44, and no alteration in tumor cell migration was observed. These results show that the invasion of renal cancer cells is differentially regulated by compounds of the extracellular matrix, whereby fibronectin seems to be the most critical factor. The molecular interactions in this process are strongly dependent on beta(1)-integrins and the corresponding amino acid sequence RGD.

Topics: Carcinoma, Renal Cell; Cell Movement; Chemotaxis; Collagen; Dose-Response Relationship, Drug; Extracellular Matrix; Fibronectins; Humans; Hyaluronan Receptors; Integrin beta1; Interferon-gamma; Kidney Neoplasms; Laminin; Neoplasm Invasiveness; Oligopeptides; Transforming Growth Factor beta; Tumor Cells, Cultured

We previously reported elevated levels of TGF-beta1 in patients with renal carcinoma. Certain aspects led us to ask whether they might be caused by chronic damage to the kidney(s). Here we report on an extended set of patients with various renal diseases, lung cancer, humoral immunodeficiency and controls. For latent TGF-beta1 in plasma, we find that the control, immunodeficiency, lung cancer and kidney transplant groups do not differ significantly (means, 7.0-8.8 ng/ml). Also, acute short-term renal stress (extracorporal lithotrypsy) does not lead to an increase of TGF-beta1. However, the pyelonephritis patients present with levels of 19.0 ng/ml, chronic extracorporal dialysis patients with 15.5 ng/ml, and renal cell carcinoma patients with 22.8 ng/ml. For active TGF-beta1 these findings are exactly recovered. For serum levels, only the renal carcinoma group presents with significantly elevated levels of TGF-beta1. Kidney transplantation seems to normalize TGF-beta1 levels, while in the kidney cancer patients surgery has an effect only in part of the group. We conclude that elevated plasma TGF-beta1 levels are common in at least two chronic renal disease conditions, and that it normalizes with restoration of renal function. It is tempting to speculate that chronic elevation of TGF-beta1 in these patients may be critically involved in these conditions predisposing to renal cancer.

Topics: Carcinoma, Renal Cell; Common Variable Immunodeficiency; Humans; Kidney Diseases; Kidney Failure, Chronic; Kidney Neoplasms; Kidney Transplantation; Lithotripsy; Lung Neoplasms; Pyelonephritis; Transforming Growth Factor beta

The von Hippel-Lindau (VHL) tumor suppressor gene is mutated in patients with VHL disease and in the majority of patients with sporadic clear cell renal carcinoma (RCC). Overexpression of transforming growth factor (TGF) beta1 has been observed in patients with several cancers, including RCCs, with serum and urine levels correlating inversely with prognosis. We have demonstrated that the VHL tumor suppressor gene product represses TGF-beta1 mRNA and protein levels (approximately 3-4-fold) in 786-O RCC cells by decreasing the TGF-beta1 mRNA half-life. Exogenously added TGF-beta1 did not suppress the growth of 786-O cells in vitro, nor did the addition of neutralizing antibody (Ab) against TGF-beta have any effect. Indeed, 786-O cells were found to express no TGF-beta type II receptor protein, thus allowing them to escape from the negative growth control of TGF-beta1. In contrast to the in vitro data, neutralizing Ab to TGF-beta inhibited tumorigenesis and, in some cases, regressed established 786-O tumors in athymic mice. Immunohistochemistry for von Willebrand's factor revealed a 3-4-fold lower tumor microvessel count in the mice treated with TGF-beta Ab compared to controls, suggesting that the Ab was inhibiting angiogenesis. Our findings indicate that TGF-beta1 is a novel target for the VHL tumor suppressor and that antagonizing its paracrine action may provide novel avenues for treatment of RCCs as well as other tumors that secrete TGF-beta1.

Topics: Adenocarcinoma, Clear Cell; Animals; Antibodies, Monoclonal; Gene Deletion; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Half-Life; Humans; Kidney Neoplasms; Ligases; Mice; Mice, Mutant Strains; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Neovascularization, Pathologic; Proteins; Recombinant Fusion Proteins; RNA Processing, Post-Transcriptional; RNA, Messenger; RNA, Neoplasm; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Von Hippel-Lindau Tumor Suppressor Protein; von Willebrand Factor

We investigated the effects of constant and intermittently increased glucose concentrations on human proximal tubule cells and cortical fibroblasts in primary culture.. Cells were grown to confluence and then exposed for 4 days to 6.1 mmol/l D-glucose (normal), 25 mmol/l D-glucose (high), or 6.1 mmol/l alternating with 25 mmol/l D-glucose on a daily basis.. In proximal tubular cells, exposure to high glucose caused an 11 % increase in thymidine uptake (p < 0.05), a 230 % increase in secretion of transforming growth factor beta 1 (TGF-beta1; p < 0.05) and a 393 % increase in platelet derived growth factor. Intermittent exposure to high glucose caused thymidine uptake to further increase by 42 % (p < 0.01) and TGF-beta1 secretion by 352 % (p < 0.01) but no additional increase in platelet-derived growth factor secretion was observed. Cellular protein content increased by 27 % (p < 0.05) and collagen synthesis by 29 % (p < 0.05), changes that were not observed in cells constantly exposed to high glucose. In cortical fibroblasts constant exposure to high glucose caused a 35 % increase in thymidine uptake (p < 0.01). Intermittently high glucose increased thymidine incorporation a further 58 % (p < 0.001), collagen synthesis by 65 % (p < 0.01) and insulin-like growth factor binding protein 3 secretion by 216 % (p < 0.01).. In cultured human tubulointerstitial cells, increased glucose concentrations change cell growth, collagen synthesis and cytokine secretion. These effects are enhanced following intermittent exposure to high glucose, indicating that short lived excursions in glycaemic control have important pathological effects on the human tubulointerstitium.

Topics: Aged; Analysis of Variance; Cell Division; Cells, Cultured; Collagen; Epithelial Cells; Female; Glucose; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Kidney Cortex; Kidney Neoplasms; Kidney Tubules; L-Lactate Dehydrogenase; Male; Platelet-Derived Growth Factor; Time Factors; Transforming Growth Factor beta

We present a case of a 25-year-old woman with a renin-secreting juxtaglomerular cell tumor, retroperitoneal fibrosis associated with glomerular hypertrophy, glomerulonephritis, and marked tubulointerstitial alterations. Myofibroblasts, as shown by positive immunostaining for alpha-smooth muscle actin, were found along with transforming growth factor-beta (TGF-beta) in the interstitium of the tumor-free kidney. Regarding the pathogenesis of renal fibrosis and glomerular hypertrophy, this case may provide evidence not only experimentally but also clinically that the renin-angiotensin system plays an important role because angiotensin II is known to induce renal fibrosis associated with increased TGF-beta and the appearance of myofibroblasts.

Topics: Actins; Adenocarcinoma; Adult; Female; Glomerulonephritis; Humans; Kidney Neoplasms; Renin; Renin-Angiotensin System; Retroperitoneal Fibrosis; Transforming Growth Factor beta

Denys-Drash syndrome (DDS) and Frasier syndrome (FS) are rare diseases caused by the mutations of Wilms tumor gene, WT1. The common denominator in these syndromes is a nephropathy which is manifested by early-onset proteinuria, nephrotic syndrome and end stage renal failure. Although these syndromes are genetic models of nephropathy and the mutations of WT1 gene are characterized in these patients the mechanism how mutations of WT1 gene affect the embryonic kidney adversely has not been elucidated. Recently, there was a report that FS is caused by mutations in the donor splice site of WT1. These mutations predicted loss of +KTS isoform, which is one of the four splicing variants of WT1. In this study, two +KTS deletion mutants of WT1 were made as well as a WT1 mutant mimicking a mutation found in a patient who had diffuse mesangial sclerosis, end stage renal failure and Wilms tumor. Mutant embryonic kidney cell lines were established by transfection of 293 embryonic kidney cells with WT1 mutants. We investigated the transcription regulation of mutant WT1 among these cell lines using the reporter vectors containing PDGF-A and TGF-beta promoter sequence. Our results showed that the promoter activity of PDGF-A and TGF-beta, which are related to the progression of glomerular diseases, was modestly increased in the mutant cell mimicking the patent, while those activities were markedly increased in other two deletion mutant cell lines. This study demonstrated that +KTS WT1 mutation found in DDS affected the cytokine expression adversely in vitro. From these results, we suggest that the alteration of +KTS WT1 expression be responsible for the rapid progression of renal diseases in DDS and FS.

Topics: Child, Preschool; Chromosome Deletion; Humans; Kidney Diseases; Kidney Neoplasms; Male; Mutation; Platelet-Derived Growth Factor; Syndrome; Transcription, Genetic; Transforming Growth Factor beta; Wilms Tumor

The most worrying problem with renal cell carcinoma (RCC) seems to be the prediction of metastases by means of tumor-specific markers. Therefore, much effort is committed to the development of new markers.. The level of latent transforming growth factor beta1 (TGF-beta1) was measured in plasma samples by ELISA. These samples were collected from patients with RCC before they underwent radical nephrectomy, from patients 1 h after extracorporeal lithotripsy, from patients with pyelonephritis, and from healthy controls.. In all cases of RCC the levels of latent TGF-beta1 in plasma were much higher (n = 20, 41.0 +/- 13.9 ng/ml, range 19.3-78.1 ng/ml) than in healthy controls (n = 20, 3.8 +/- 2.9 ng/ml, range 0.6-9.9 ng/ml, p < 0.0001). The TGF-beta1 levels in plasma after extracorporeal lithotripsy (n = 20, 7.4 +/- 4. 64 ng/ml, range 2.9-21.7 ng/ml, p < 0.01) and in patients suffering from pyelonephritis (n = 20, 18.93 +/- 14.2 ng/ml, range 4.2-46.7 ng/ml, p < 0.001) were also higher than in healthy controls.. We conclude that increased levels of latent TGF-beta1 are common in the plasma of RCC patients. The TGF-beta1 plasma level in RCC was found to be significantly higher than in cases of inflammation. Thus, TGF-beta1 is a possible tumor-prognostic marker in RCC.

Topics: Biomarkers, Tumor; Carcinoma, Renal Cell; Enzyme-Linked Immunosorbent Assay; Humans; Kidney Neoplasms; Lithotripsy; Pyelonephritis; Transforming Growth Factor beta

Renal cell carcinoma (RCC) is a solid tumour of the kidney and is the most common renal neoplasm. Despite the presence of tumour infiltrating lymphocytes (TIL) in RCC, these tumours continue to progress in vivo suggesting a poor host immune response to the tumour, and the suppression of TIL effector function. Cytokines are key molecules that modulate the function of T cells. The possibility is investigated that the local production of cytokines in RCC contributes to immunosuppression of TIL. The expression of pro-inflammatory (IFN-gamma/IL-2) and immunosuppressive (IL-10/TGF-beta) cytokine mRNA transcripts was determined in RCC, normal kidney and peripheral blood of RCC patients using a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) with cytokine-specific primers. Following Southern blot hybridization of the PCR products with internal radiolabelled oligonucleotide probes, cytokine transcript levels were measured by densitometry and expressed relative to the glyceraldehyde-3-phosphate dehydrogenase densitometry score. With the exception of IL-10, there were no differences in expression of cytokine mRNA transcripts between the peripheral blood of patients and normal healthy individuals. It was found that TGF-beta transcripts were well represented in normal kidney and RCC. In contrast, the expression of IFN-gamma transcripts, while low in the majority of samples, was significantly increased in RCC when compared to normal kidney (P=0.05). The IL-2 and IL-10 transcripts showed a more variable expression in normal kidney and RCC, with no significant differences in expression between the sample groups. The data demonstrating pro-inflammatory and immunosuppressive cytokine expression in RCC do not support a prominent immunosuppressive cytokine profile in these tumours.

Topics: Adult; Aged; Carcinoma, Renal Cell; Cytokines; Female; Gene Expression; Humans; Interferon-gamma; Interleukin-10; Interleukin-2; Kidney; Kidney Neoplasms; Leukocytes, Mononuclear; Male; Middle Aged; RNA, Messenger; Transforming Growth Factor beta

Wilms' tumor, or nephroblastoma, arises from metanephric blastema and caricatures renal organogenesis. An alteration in at least one of the genes involved in control of renal differentiation is therefore a likely event in tumorigenesis, and indeed some of the genes involved in renal development, for example, hepatocyte growth factor (HGF) and its receptor c-met, the transcription factor Wilms' tumor gene (WT1), and transforming growth factor-beta family member bone morphogenetic protein (BMP)-7, have also been implicated in various models of tumorigenesis. In a comparison of mRNA expression patterns for these genes in normal rat embryonic or fetal kidney and nephroblastoma, we found that the patterns for HGF, met, and WT1 detected by in situ hybridization or ribonuclease protection assay (RPA) in the nephroblastomas were similar to those of normal developing kidney. BMP-7 expression, on the other hand, was lower in most tumors examined both by in situ hybridization and RPA than in normal tissues. This deficiency in a defined inductive factor that has been shown to function in renal tubulogenesis may play a role in tumorigenesis by allowing the accumulation of blastemal populations typical of nephroblastomas.

Topics: Animals; Base Sequence; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; DNA Primers; DNA-Binding Proteins; Female; Hepatocyte Growth Factor; In Situ Hybridization; Kidney; Kidney Neoplasms; Male; Proto-Oncogene Proteins c-met; Rats; Ribonucleases; RNA, Messenger; Transcription Factors; Transforming Growth Factor beta; Wilms Tumor; WT1 Proteins

We investigated the immunomodulatory capacity of primary cultures of renal cell carcinomas (RCC) by assessing production of cytokines and modulation of mitogen-induced T lymphocyte blast transformation. The results clearly show that immunomodulatory capacity is a common feature of RCC and that in vitro these tumors can produce interleukin-10 (IL-10) up to 20 ng/ml, IL-6 up to 35 micrograms/ml (> 250 kU/ml in the B9 system), IL-11 up to 15 micrograms/ml, and transforming growth factor-beta 1 (TGF-beta 1) up to 22 ng/ml. Furthermore, these tumors have the capacity to modulate T cell blast transformation over two orders of magnitude in each direction. The correlations of the immunologic properties of tumor cell cultures with the conventional classification of tumors (histology, cytology, staging, grading, presence of metastases, and secondary tumors) are analyzed. The significance of these findings for modulation of local immunity by RCC as well as for patient outcome is discussed.

Topics: Adjuvants, Immunologic; Carcinoma, Renal Cell; Concanavalin A; Humans; Interleukin-10; Interleukin-11; Interleukin-6; Interleukins; Kidney Neoplasms; Lymphocyte Activation; Mitogens; Reference Values; T-Lymphocytes; Transforming Growth Factor beta; Tumor Cells, Cultured

A major problem of renal cell carcinoma is the prediction of metastases via tumor prognostic markers. Therefore, much effort has been committed to the development of new prognostic markers.. The level of transforming growth factor-beta1 was measured by enzyme-linked immunosorbent assay in serum samples collected from patients with renal cell carcinoma before radical nephrectomy.. In serum samples from 21 patients with renal cell carcinoma and 21 healthy controls mean transforming growth factor-beta1 levels were 177 +/- 54.1 versus 65.6 +/- 15.8 ng./ml., respectively. This difference was statistically significant (Mann-Whitney U test p <0.001).. Increased levels of transforming growth factor-beta1 are common in serum of patients with renal cell carcinoma.

Topics: Carcinoma, Renal Cell; Humans; Kidney Neoplasms; Transforming Growth Factor beta

The aim of the present study was to analyze the contribution of different stimulatory and inhibitory growth factors to the deregulated proliferation of human RCCs.. The expression of different growth factors and their corresponding receptors were analyzed by Northern blot, FACS, ELISA and immunocytochemistry in 13 permanent human RCC cell lines of the clear cell type. Moreover, the functional intactness of growth factor-related signal transduction pathways was investigated.. All RCC cell lines expressed EGF-receptor mRNA and protein and 10 cell lines secreted TGF-alpha. Exogeneously added TGF-alpha resulted in a significant (p < 0.05) stimulation of growth in 6 RCC cell lines and a significant (p < 0.05) inhibition of proliferation in 3 cell lines. PDGF B and the corresponding type beta receptor were expressed in a single cell line. mRNA expression of PDGF A and PDGF-alpha-receptor as well as IGF-1 and its receptor could not be detected in any cell line. Eleven RCC cell lines expressed TGF-beta 1 mRNA and in all cell lines TGF-beta 1 secretion into the supernatant could be demonstrated. Whereas all cell lines exhibited TGF-beta type II-receptor mRNA, type I-receptor mRNA could be detected only in 3 cell lines. TGF-beta type III-receptor was observed in 1 cell line. Exogeneously added TGF-beta1 resulted in a significant (p < 0.05) inhibition of proliferation in 7 RCC cell lines.. Clear cell RCCs exhibit a complex and heterogeneous expression pattern for various growth factors and their receptors. Growth factor secretion and intact signal transduction pathways in most clear cell RCCs facilitate an intricate modulation of RCC growth by autocrine and paracrine interactions between tumor cells and host tissue.

Topics: Carcinoma, Renal Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Kidney Neoplasms; Platelet-Derived Growth Factor; Receptor, IGF Type 1; Receptors, Platelet-Derived Growth Factor; RNA; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

The balance of growth regulation in tumors can be profoundly disturbed by defects in the transforming growth factor-beta 1 (TGF-beta 1) system. Thus far, investigations into the secretion of TGF-beta 1, the expression of its type I, II, and III receptors, as well as the functional intactness of the TGF-beta 1 signal transduction pathways in human renal cell carcinomas (RCC) have been lacking. The objective of the present study, therefore, was to elucidate the role of the TGF-beta 1 system in RCC. We were able to determine the status of this system in 20 primary RCC and in 30 newly established human RCC cell lines of all major histologic types. All primary RCC showed expression of TGF-beta 1 and its type I and II receptors by immunohistochemistry, irrespective of histologic type. In vitro, all RCC cell lines secreted TGF-beta 1 protein as a biologically inactive complex, which cannot interact with cell-surface receptors. Type I ALK-5-receptor mRNA and protein were detected in 29 RCC cell lines, whereas type I ALK-2-receptor mRNA was found in all cell lines. Type II-receptor mRNA and protein could be demonstrated in all cell lines analyzed, and type III-receptor mRNA was observed in five RCC cell lines. Exogenously added, biologically active TGF-beta 1 (1 ng/ml) resulted in significant (p < 0.05) inhibition of proliferation in 14 of 30 RCC cell lines. Sixteen of thirty RCC cell lines, however, proved to be TGF-beta 1-resistant. This resistance could not be explained by mutations in two "hot spot" regions of the type II-receptor gene (bp 622 to 795 and bp 1868 to 2019), as was demonstrated by DNA sequencing in the TGF-beta 1-resistant RCC cell lines. In conclusion, our observations are the first to provide evidence of an "escape" from the negative growth control of TGF-beta 1 by a significant proportion of RCC, suggesting that the acquisition of TGF-beta 1 resistance is an important progression factor for human RCC.

Topics: Biomarkers, Tumor; Carcinoma, Renal Cell; Cell Division; Disease Progression; DNA Mutational Analysis; Drug Resistance, Neoplasm; Humans; Kidney Neoplasms; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

The levels of active and latent transforming growth factor beta 1 (aTGF-beta 1, ITGF-beta 1, respectively) in plasma samples were measured by enzyme-linked immunoabsorbent assay (ELISA). Samples were collected from patients suffering from renal cell carcinoma (RCC) before they underwent tumour resection. In all cases tested, the levels of latent TGF-beta 1 were much higher (n = 23, 41.0 +/- 13.9, range 19.3-78.1 ng/ml) than in healthy controls (n = 21, 3.8 +/- 2.9, range 0.6-9.9, P < < 0.0001), while active TGF-beta 1 did not differ that impressively (n = 38, 1.2 +/- 1.3, range 0.0-4.5 for RCC, n = 21, 0.1 +/- 0.2, range 0.0-1.1 in controls, P < 0.001). As for TGF-beta 1 production by proximal tubulus cells has been shown, it was speculated that these high TGF-beta 1 levels might be due to secretion by the tumour, which usually originates from proximal tubuli. Indeed, production of TGF-beta 1 was found in culture supernatants, and it was possible to show TGF-beta 1 mRNA expression in tumour samples. This TGF-beta 1 was predominantly secreted in the latent form. This is in contrast to data from other authors, who found TGF-beta 1 to be secreted mainly in the active form, but need not mean that it is inactive locally as the low pH encountered within tumours is in the range necessary for its activation. It is concluded that elevated latent TGF-beta 1 is common in RCC, is at least partially produced by the tumour and might be a cause for local immunosuppressive effects within the tumour.

Topics: Carcinoma, Renal Cell; Enzyme-Linked Immunosorbent Assay; Humans; Kidney Neoplasms; Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

The biological activity of transforming growth factor-beta 1 (TGF-beta) is governed by dissociation from its latent complex. Immunohistochemical discrimination of active and latent TGF-beta could provide insight into TGF-beta activation in physiological and pathological processes. However, evaluation of immunoreactivity specificity in situ has been hindered by the lack of tissue in which TGF-beta status is known. To provide in situ analysis of antibodies to differentiate between these functional forms, we used xenografts of human tumor cells modified by transfection to overexpress latent TGF-beta or constitutively active TGF-beta. This comparison revealed that, whereas most antibodies did not differentiate between TGF-beta activation status, the immunoreactivity of some antibodies was activation dependent. Two widely used peptide antibodies to the amino-terminus of TGF-beta, LC(1-30) and CC(1-30) showed marked preferential immunoreactivity with active TGF-beta versus latent TGF-beta in cryosections. However, in formalin-fixed, paraffin-embedded tissue, discrimination of active TGF-beta by CC(1-30) was lost and immunoreactivity was distinctly extracellular, as previously reported for this antibody. Similar processing-dependent extracellular localization was found with a neutralizing antibody raised to recombinant TGF-beta. Antigen retrieval recovered cell-associated immunoreactivity of both antibodies. Two antibodies to peptides 78-109 showed mild to moderate preferential immunoreactivity with active TGF-beta only in paraffin sections. LC(1-30) was the only antibody tested that discriminated active from latent TGF-beta in both frozen and paraffin-embedded tissue. Thus, in situ discrimination of active versus latent TGF-beta depends on both the antibody and tissue preparation. We propose that tissues engineered to express a specific form of a given protein provide a physiological setting in which to evaluate antibody reactivity with specific functional forms of a protein.

Topics: Antibodies, Monoclonal; Humans; Immunohistochemistry; Kidney Neoplasms; Mutagenesis, Site-Directed; Sarcoma; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

We have here studied the composition and regulation of stromal extracellular matrix components in an experimental tumor model. Nude mice were inoculated with WCCS-1 cells, a human Wilms' tumor cell line. In the formed tumors the stroma was found to contain mesenchymal extracellular matrix proteins such as tenascin-C, fibulins-1 and 2 and fibronectin, but no nidogen. Nidogen was confined to basement membranes of tumor blood vessels. Since glucocorticoids have been shown to downregulate tenascin-C expression in vitro, we tested whether dexamethasone can influence biosynthesis of extracellular matrix components during tumor formation in vivo. A downregulation of tenascin-C mRNA and an upregulation of fibronectin mRNA expression by dexamethasone was noted. Transforming growth factor-beta 1 mRNA levels were unaffected by the dexamethasone treatment. Glucocorticoids can thus downregulate tenascin-C synthesis although local stimulatory growth factors are present. The competition between a negative and a positive extrinsic factor on synthesis of stromal extracellular matrix components was studied in a fibroblast/preadipocyte cell line. Transforming growth factor-beta 1 stimulated tenascin-C synthesis but did not affect fibronectin or fibulin-2 synthesis. Dexamethasone at high concentrations could completely suppress the effect of transforming growth factor-beta 1 on tenascin-C mRNA expression. Transforming growth factor-beta 1 could in turn overcome the downregulation of tenascin-C mRNA expression caused by a lower concentration of dexamethasone. We therefore suggest that the limited expression of tenascin-C in part is due to a continuous suppression by physiological levels of glucocorticoids, which can be overcome by local stimulatory growth factors when present in sufficient amounts.

Topics: Animals; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Glucocorticoids; Humans; Kidney Neoplasms; Mice; Mice, Nude; Neoplasms, Experimental; RNA, Messenger; Stromal Cells; Transforming Growth Factor beta; Tumor Cells, Cultured; Wilms Tumor

Continuous local delivery of interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) via gene transfer appears to be more effective than systemic therapy in preventing the growth of human renal cell carcinoma (RCC) in vitro and in vivo. To understand further if cytokine-gene transfection of RCC could alter certain cellular properties that are associated with the invasive and metastatic potentials of tumor, the authors characterized six cell lines that produce IL-2 and/or IFN-alpha in their expression of intercellular adhesion molecule-1 (ICAM-1) and CD44; binding affinity to extracellular matrix (ECM) components (fibronectin, laminin, type IV collagen, and vitronectin); and preference in forming homotypic aggregation and mRNA levels of c-myc, epidermal growth factor receptor (EGF-R), tumor transforming growth factor-beta (TGF-beta) and type IV collagenase. These six lines were compared with control vector transfected parental R11 line.. The expression of ICAM-1 and CD44 was determined by fluorescence-activated cell sorter (FACS) analysis, the tumor cell binding affinity to ECM components was measured by cell attachment assay, the degree of homotypic aggregation was quantified by cell aggregation assay, and the mRNA levels of c-myc, EGF-R, TGF-beta, and collagenase were analyzed by a quantitative polymerase chain reaction analysis.. Both IL-2-gene- and IFN-alpha-gene-modified R11 exhibited enhanced expression of ICAM-1, suppression of CD44, and decreased binding affinity to ECM components, when compared with the R11-control vector. All cytokine-producing tumor lines showed a decreased preference to form homotypic aggregation. Interferon-alpha gene transfer downregulated c-myc, EGF-R, and type IV collagenase mRNA expression, whereas only the higher producers of IL-2 downregulated TGF-beta mRNA expression. Exogenous IL-2 and/or IFN-alpha treatment of a IFN-alpha-resistant RCC enhanced both HLA class I antigen and ICAM-1 expression and suppressed CD44 expression, but had no effect on tumor growth rate.. The local production of high concentrations of IL-2 and IFN-a at the tumor site may directly alter tumor properties associated with invasive and metastatic phenotypes of RCC. Interleukin-2 and/or IFN-alpha gene therapy may be an effective strategy for treatment of patients with advanced renal cancer.

Topics: Base Sequence; Carcinoma, Renal Cell; Carrier Proteins; Cell Adhesion; Cell Adhesion Molecules; Cell Membrane; Collagenases; Down-Regulation; Drug Resistance; ErbB Receptors; Extracellular Matrix; Gene Transfer Techniques; Humans; Hyaluronan Receptors; Interferon-alpha; Interleukin-2; Kidney Neoplasms; Matrix Metalloproteinase 9; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Metastasis; Polymerase Chain Reaction; Proto-Oncogene Proteins c-myc; Receptors, Cell Surface; Receptors, Lymphocyte Homing; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

An orthotopic metastatic human renal cell carcinoma model in nude mice was established to evaluate the mechanism of metastasis as a preliminary phase in the development of a treatment to prevent cancer metastasis. The effect of host fibroblasts from different organs on the production of type IV collagenase by human renal cell carcinoma and the factors which influenced the enzyme production and secretion by fibroblasts were also investigated. KG-2 cells were established from human renal cell carcinoma, and produced tumors following implantation to both the subrenal capsular space (orthotopic site) and subcutis (ectopic site). Histologically, the tumors in the subcuit (SC tumors) were well encapsulated with a thick fibrous capsule and did not produce metastasis or invasion, whereas those in the subrenal capsular space (SRC tumors) lacked a fibrous capsule and produced metastasis at the lung or regional lymph nodes. The production of type IV collagenase in conditioned media from metastatic SRC tumors and lung metastatic lesions was larger than that from non-metastatic SC tumors. The conditioned media separated from mouse kidney or lung fibroblasts stimulated the production of type IV collagenase by KG-2 cells, whereas, that separated from mouse skin fibroblasts decreased the enzyme production. The production of type IV collagenase by KG-2 cells was stimulated by cocultured KG-2 cells and fibroblasts from the kidney or lung, whereas it was suppressed by cocultured KG-2 cells and skin fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carcinoma, Renal Cell; Collagenases; Fibroblasts; Humans; Kidney Neoplasms; Matrix Metalloproteinase 9; Mice; Mice, Nude; Subrenal Capsule Assay; Transforming Growth Factor beta

We have recently established a human renal cell carcinoma KG-2 line that is tumorigenic in the subcutis (ectopic) and kidney (orthotopic) of nude mice but spontaneously metastasizes to the lung only after orthotopic implantation. KG-2 cells growing in the kidney (orthotopic) and lung metastases secreted higher levels of gelatinase than did cells growing in the subcutis (ectopic). We examined whether organ-specific fibroblasts play a role in the regulation of gelatinase production and invasion by renal carcinoma cells. The gelatinase level in the culture supernatants of KG-2 cells was increased by their cultivation with mouse kidney or lung fibroblasts. In contrast, cocultivation of KG-2 cells with mouse skin fibroblasts resulted in a significant reduction of gelatinase activity. Similar results were obtained by culturing KG-2 cells in the media conditioned by the different mouse fibroblasts. We, therefore, investigated effects on KG-2 cells of cytokines and growth factors known to be produced by fibroblasts of various origins. Of ten cytokines and growth factors tested, basic fibroblast growth factor, hepatocyte growth factor, and transforming growth factor-beta 1 (TGF-beta 1) stimulated gelatinase expression by the cultured KG-2 cells. Parallel immunohistochemical analyses revealed that mouse kidney and lung fibroblasts produced higher levels of TGF-beta 1 than did skin fibroblasts. These results indicate that gelatinase production by KG-2 renal cell carcinoma cells is influenced by the organ microenvironment. Specifically, organ-specific fibroblasts regulate the production of degradative enzymes by KG-2 cells and, hence, profoundly influence their invasive and metastatic capacity.

Topics: Animals; Carcinoma, Renal Cell; Fibroblasts; Gelatinases; Humans; Kidney Neoplasms; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Organ Specificity; Skin Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured

We investigated the influence of epidermal growth factor (EGF) and transforming growth factor (TGF)-beta 1 on the in vitro invasion and type IV collagenolytic activity of two new cell lines of renal cell carcinoma (SRCC-1P and SRCC-1M). When cells were treated with EGF or with TGF-beta 1, EGF increased the number of cells penetrating through the reconstituted basement membrane in SRCC-1P. In contrast, TGF-beta 1 suppressed the number of cells penetrating through the membrane in SRCC-1M. In accordance with the invasiveness, EGF enhanced the activity of type IV collagenolysis in SRCC-1P, and TGF-beta 1 suppressed it in SRCC-1M. The growth factors did not affect DNA synthesis of the cells as evaluated by 3H-thymidine incorporation. These results suggest that EGF and TGF-beta 1 can influence the in vitro invasive process of renal cell carcinoma cells through their actions on proteolysis such as type IV collagenolysis.

Topics: Carcinoma, Renal Cell; Collagen; DNA, Neoplasm; Epidermal Growth Factor; Humans; Kidney Neoplasms; Neoplasm Invasiveness; Transforming Growth Factor beta; Tumor Cells, Cultured

The proto-oncogene C-jun acts as a transcriptional activator or repressor for numerous cellular genes, and the overexpression of these genes may cause malignant transformation. JunB inhibits c-jun's transforming activities. We investigated the expression of jun genes in renal cell cancer (RCC) and their regulation by cytokines and transforming growth factor beta 1 (TGF-b1). The constitutive expression of c-jun was detected in 39 of 43 fresh frozen RCC, 5 of 10 normal kidneys, and the expression of junB detected in 28 of 34 RCC, 5 of 6 normal kidneys. C-jun was also found expressed in all 10 RCC tumor lines examined and junB was expressed at low levels in 6 of 10 renal tumor lines. TGF-b1 and tumor necrosis factor alpha (TNF-a) have been shown to alter the expression of jun genes in other tissue types. Additionally, TGF-b1, TNF-a, and gamma interferon (g-IFN) were shown to inhibit the growth of RCC. We found that TGF-b1 highly augmented the expression of junB (mean of 34 folds, p less than .05), but did not significantly alter the expression of c-jun, the transforming gene. In contrast, TNF-a significantly enhanced the expression of both c-jun (mean fold enhancement of 2.1, p less than .05) and junB (2.2 folds, p less than .05). Interleukin-2 (IL-2), interleukin-4 (IL-4) and g-IFN did not significantly alter jun expression. The findings presented suggest that c-jun may have a role in inducing malignant transformation in RCC and a novel mechanism by which TGF-b1 may exert its anti-tumor effects, via the activation of junB. Additionally, although TGF-b1, TNF-a, and g-IFN all have anti-proliferative actions on RCC in vitro, they were found to have different effects in altering jun expressions.

Topics: Carcinoma, Renal Cell; Gene Expression Regulation, Neoplastic; Genes, jun; Humans; Interferon-gamma; Interleukin-1; Interleukin-4; Kidney Neoplasms; Proto-Oncogene Mas; Proto-Oncogene Proteins c-jun; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Suramin is a polyanionic compound used clinically for the treatment of trypanosomiasis, which is known to inhibit the action of many protein factors in vitro. Transforming growth factor-beta (TGF-beta) is a multifunctional regulatory protein which inhibits the growth of renal cell carcinoma in culture. While suramin at 50-500 micrograms/ml had no significant effect on the growth of renal cell carcinoma in culture in our experiments, it did partially reverse the growth inhibition induced by TGF-beta in the two cell lines tested. This effect apparently is caused by suramin's direct interference with 125I-labeled TGF-beta's ability to bind to the cell, and not by any effect of suramin on the TGF-beta receptor. Furthermore, suramin dissociates TGF-beta bound to the cell with a t1/2 of less than 30 min. These results are consistent with those previously reported regarding suramin's interaction with other protein growth factors, and suggest that suramin may interact with the TGF-beta protein itself to inactivate it.

Topics: Carcinoma, Renal Cell; Cell Division; Dose-Response Relationship, Drug; Humans; Kidney Neoplasms; Suramin; Transforming Growth Factor beta; Tumor Cells, Cultured

The growth and differentiation of the epithelium in many tissues is mediated by interactions with the adjacent mesenchyme, but the mechanisms responsible remain undefined. To identify the factors involved in the growth and branching morphogenesis of ureteric bud, which is the collecting duct anlagen, buds from 13-gestation-day rat embryos were separated from the metanephrogenic mesenchyme and explanted to culture dishes coated with gelled type I collagen in a defined medium. Under these conditions buds attached to the substrate and grew out without indication of cell senescence. When buds were instead suspended in gelled type I collagen, branching morphogenesis was observed despite the absence of mesenchyme although it was not as extensive as in vivo. Since growth occurred much more slowly in culture than expected, culture conditions were varied in attempts to accelerate the process. Despite extensive screening of matrices and growth factors, only epidermal and endothelial cell growth factors stimulated growth to a significant extent. Transforming growth factor-beta, on the other hand, was a potent inhibitor of growth. Homogenates from tumors that caricature metanephrogenic mesenchyme were highly mitogenic for bud cells and, thus, will be a source of material for characterizing regulatory factors involved in renal growth. These studies show that growth and branching morphogenesis of the ureteric bud can occur without direct cell-cell interactions with the metanephrogenic mesenchyme and that matrices and factors secreted by the mesenchyme may mediated these activities in vivo.

Topics: Animals; Cell Differentiation; Collagen; Culture Media, Serum-Free; Endothelial Growth Factors; Epidermal Growth Factor; Female; Kidney Neoplasms; Kidney Tubules, Collecting; Male; Mesoderm; Morphogenesis; Rats; Rats, Inbred F344; Stem Cells; Transforming Growth Factor beta; Tumor Cells, Cultured

Renal cell carcinomas (RCCs) frequently metastasize to distant organs in their clinical course. However, the mechanism of the metastasis had not been fully elucidated. In vitro invasion assay has been reported to be a rapid method for the evaluation of the invasive potential of various malignant cells. In vitro invasive potential of RCC has not been investigated by this method. Thus, in the present study, we first attempted to characterize the in vitro invasive potential of four human RCC cell lines which had been established in our institute. Secondly, we investigated the influence of two growth factors (EGF, TGF-beta 1) on the invasive potential of these cell lines when the two factors were applied as chemoattractants. SMKT-R-3 and R-4 cell lines showed more cell penetration through Matrigel than SMKT-R-1 and R-2 cell lines, suggesting that the former cell lines have higher invasive potential. While invasive potential varied in each cell line, it was enhanced by EGF in all cell lines. However, TGF-beta 1 suppressed the invasive potential of all four cell lines. These results suggest that two factors have different actions on the invasion of RCCs.

Topics: Carcinoma, Renal Cell; Cell Division; Epidermal Growth Factor; Humans; Kidney Neoplasms; Neoplasm Invasiveness; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factors (TGFs)-alpha and -beta are regulatory polypeptides that reversibly confer a transformed phenotype upon normal cultured fibroblasts. TGF-alpha is synthesized primarily by malignant cells and shares many properties with the tissue mitogen, epidermal growth factor (EGF). The expression of TGF-beta mRNA has been demonstrated in a variety of normal and malignant cell types, some of which secrete the mature protein in an inactive form. To investigate the role of TGFs in human renal cell carcinoma (RCC), we used two renal tumor-derived cell lines and one established RCC cell line for analysis of TGF-alpha and TGF-beta mRNA production and for evaluation of TGF-beta protein secretion. By northern blot hybridization, all three RCC cell lines expressed TGF-alpha and -beta mRNA. In addition, TGF-beta activity was found in the conditioned medium from these cells. The secreted TGF-beta protein, however, displayed biological activity only after activation by acid-treatment. These data demonstrate the constitutive expression of TGF-alpha and -beta mRNA by RCC cell lines and, also, the secretion by this tumor of endogenous TGF-beta protein in a latent form.

Topics: Carcinoma, Renal Cell; Cell Line; Gene Expression; Humans; Kidney Neoplasms; Radioligand Assay; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured