4-(5-benzo(1-3)dioxol-5-yl-4-pyridin-2-yl-1h-imidazol-2-yl)benzamide and Breast-Neoplasms

The study examined the mechanisms of action of signal protein claudin 6 (CLDN6) on migration and invasion of breast cancer cell lines MCF-7 and SKBR-3. To this end, the signal proteins SMAD were blocked with their inhibitor SB431542, the genes CLDN6 and SNAIL were knocked down with short hairpin RNAs, and MMP2 and MMP9 were inhibited with TIMP-1. Expressions of MMP2 and MMP9 mRNAs were evaluated by reverse transcription PCR, Expressions of MMP-2, MMP-9, E-cadherin, N-cadherin, and vimentin were examined by Western blotting. Migration and invasion were analyzed by scratch test and Matrigel invasion assay. SB431542 inhibited expression of MMP2 and MMP9 in both cell lines. Single use of SB431542 inhibited expression of MMP-2/MMP-9 and corresponding mRNAs, but subsequent silencing of CLDN6 gene reversed this effect. TIMP-1 reversed down-regulation of E-cadherin, upregulation of N-cadherin and vimentin, facilitation of migration and invasion evoked by CLDN6 knocking down. Silencing of SNAIL gene inhibited migration and invasion, upregulated the expression of E-cadherin, and down-regulated expression of MMP2, MMP 9, N-cadherin, and vimentin. Thus, CLDN6 suppresses the epithelial-mesenchymal transition, migration, and invasion via blocking SMAD/Snail/MMP-2/9 signaling pathway in MCF-7 and SKBR-3 cancer cell lines.

Topics: Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; MCF-7 Cells; Tissue Inhibitor of Metalloproteinase-1; Vimentin

Tumor cells promote immune evasion through upregulation of programmed death-ligand 1 (PD-L1) that binds with programmed cell death protein 1 (PD1) on cytotoxic T cells and promote dysfunction. Though therapeutic efficacy of anti-PD1 antibody has remarkable effects on different type of cancers it is less effective in breast cancer (BC). Hence, more details understanding of PD-L1-mediated immune evasion is necessary. Here, we report BC cells secrete extracellular vesicles in form of exosomes carry PD-L1 and are highly immunosuppressive. Transforming growth factor beta (TGF-β) present in tumor microenvironment orchestrates BC cell secreted exosomal PD-L1 load. Circulating exosomal PD-L1 content is highly correlated with tumor TGF-β level. The later also found to be significantly associated with CD8+CD39+, CD8+PD1+ T-cell phenotype. Recombinant TGF-β1 dose dependently induces PD-L1 expression in Texos in vitro and blocking of TGF-β dimmed exosomal PD-L1 level. PD-L1 knocked down exosomes failed to suppress effector activity of activated CD8 T cells like tumor exosomes. While understanding its effect on T-cell receptor signaling, we found siPD-L1 exosomes failed to block phosphorylation of src family proteins, linker for activation of T cells and phosphoinositide phospholipase Cγ of CD8 T cells more than PD-L1 exosomes. In vivo inhibition of exosome release and TGF-β synergistically attenuates tumor burden by promoting Granzyme and interferon gamma release in tumor tissue depicting rejuvenation of exhausted T cells. Thus, we establish TGF-β as a promoter of exosomal PD-L1 and unveil a mechanism that tumor cells follow to promote CD8 T-cell dysfunction.

Topics: Aniline Compounds; Animals; B7-H1 Antigen; Benzamides; Benzylidene Compounds; Breast; Breast Neoplasms; Carcinoma, Ehrlich Tumor; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Dioxoles; Exosomes; Female; Gene Knockout Techniques; Granzymes; Healthy Volunteers; Humans; Interferon-gamma; Mice; Middle Aged; Phosphorylation; Primary Cell Culture; Receptor, Transforming Growth Factor-beta Type I; Receptors, Antigen, T-Cell; Recombinant Proteins; Signal Transduction; Transforming Growth Factor beta1; Tumor Escape; Tumor Microenvironment

Transforming growth factor-β (TGF-β) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-β signaling can lead to breast cancer. TGF-β elicits its effects on proliferation and differentiation via specific cell surface TGF-β type I and type II receptors (i.e., TβRI and TβRII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-β-induced heteromeric complex formation, activated TβRI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-β acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-β signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-β-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-β-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-β receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-β-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-β-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

Topics: Animals; Benzamides; Breast Neoplasms; Dioxoles; Epithelial-Mesenchymal Transition; Female; Humans; Mice; Phosphorylation; Pyrazoles; Signal Transduction; Smad2 Protein; Smad3 Protein; Transcription, Genetic; Transforming Growth Factor beta

Hepatocyte growth factor (HGF) and transforming growth factor β1 (TGFβ1) are biological stimuli of the micro-environment which affect bone metastasis phenotype through transcription factors, but their influence on the growth is scarcely known. In a xenograft model prepared with 1833 bone metastatic cells, derived from breast carcinoma cells, we evaluated mice survival and Twist and Snail expression and localization after competitive inhibition of HGF with NK4, or after blockade of TGFβ1-type I receptor (RI) with SB431542: in the latter condition HGF was also measured. To explain the in vivo data, in 1833 cells treated with SB431542 plus TGFβ1 we measured HGF formation and the transduction pathway involved. Altogether, HGF seemed relevant for bone-metastatic growth, being hampered by NK4 treatment, which decreased Twist more than Snail in the metastasis bulk. TGFβ1-RI blockade enhanced HGF in metastasis and adjacent bone marrow, while reducing prevalently Snail expression at the front and bulk of bone metastasis. The HGF accumulation in 1833 cells depended on an auxiliary signaling pathway, triggered by TGFβ1 under SB431542, which interfered in the transcription of HGF activator inhibitor type 1 (HAI-1) downstream of TGFβ-activated kinase 1 (TAK1): HGF stimulated Twist transactivation. In conclusion, the impairment of initial outgrowth with NK4 seemed therapeutically promising more than SB431542 chemotherapy; a functional correlation between Twist and Snail in bone metastasis seemed to be influenced by the biological stimuli of the micro-environment, and the targeting of these phenotype biomarkers might inhibit metastasis plasticity and colonization, even if it would be necessary to consider the changes of HGF levels in bone metastases undergoing TGFβ1-RI blockade.

Topics: Animals; Benzamides; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Dioxoles; Epithelial-Mesenchymal Transition; Female; Hepatocyte Growth Factor; Humans; Mice; Receptor, Transforming Growth Factor-beta Type I

The transforming growth factor-β (TGF-β) pathway plays a vital role in driving cancer cell epithelial-mesenchymal transition (EMT). Zonula occludens-1 (ZO-1), which is downregulated in response to TGF-β, is able to control endothelial cell-cell tension, cell migration, and barrier formation. However, the molecular mechanism of how TGF-β regulates ZO-1 expression remains unclear.. Breast cancer cells were treated with TGF-β to induce an EMT progress. Chromatin immunoprecipitation and dual-luciferase reporter assay were performed to investigate direct relationship between Snail and RNA binding motif protein 38 (RBM38). The RNA immunoprecipitation combined with RNA electrophoretic mobility shift assay and dual-luciferase reporter assay were conducted to testify direct relationship between RBM38 and ZO-1. The ZO-1 siRNA was transfected to breast cancer cells that overexpress RBM38 and the control, followed by transwell and Matrigel invasion assays to examine cell migratory and invasive ability.. Transforming growth factor-β induced a remarkable downregulation of RBM38 in breast cancer that was directly regulated by transcription repressor Snail targeting the E-box elements in promoter region of RBM38 gene. Additionally, RBM38 positively regulated ZO-1 transcript via directly binding to AU/U-rich elements in its mRNA 3'-UTR. Moreover, by magnifying RBM38 expression, cell migration and invasion mediated by knockdown of ZO-1 in breast cancer were reversed.. All the results clarified a linear regulation relationship among Snail, RBM38, and ZO-1, implicating RBM38 as a pivotal mediator in TGF-β-induced EMT in breast cancer.

Topics: 3' Untranslated Regions; Benzamides; Breast Neoplasms; Cell Movement; Dioxoles; Down-Regulation; E-Box Elements; Epithelial-Mesenchymal Transition; Female; Gene Expression; Humans; MCF-7 Cells; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA-Binding Proteins; RNA, Messenger; RNA, Small Interfering; Snail Family Transcription Factors; Transfection; Transforming Growth Factor beta; Zonula Occludens-1 Protein

To investigate the role of transmembrane prostate androgen-induced protein 1 (PMEPA1), an important gene downstream of transforming growth factor-β (TGF-β) signaling, in the process of breast cancer cell migration and epithelial-mesenchymal transition.. We treated MDA-MB-231 breast cancer cells with TGF-β and TGF-β inhibitor SB431542, and then detect the level of PMEPA1 using Western blotting. PMEPA1-specific siRNA was designed and its knockdown efficiency was tested by quantitative real-time PCR (qRT-PCR). After the expression of PMEPA1 in MDA-MB-231 cells was successfully silenced, the wound-healing assay and Transwell(TM) assay were used to investigate the effect of PMEPA1 silencing on the migration of MDA-MB-231 cells. Moreover, phalloidin was used to label the actin cytoskeleton of breast cancer cells to observe the effect of PMEPA1 silencing on cell morphology.. In breast cancer cells, PMEPA1 was upregulated by classical TGF-β/Smad signaling pathway. Silencing of PMEPA1 significantly inhibited the migration ability of MDA-MB-231 cells and promoted the process of mesenchymal-epithelial transition.. Over-expressed PMEPA1 can promote cell migration and maintain the mesenchymal-like morphology of breast cancer cells.

Topics: Benzamides; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Dioxoles; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Microscopy, Confocal; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction

Vasculogenic mimicry (VM) is a nonangiogenesis-dependent pathway that promotes tumor growth and disease progression. Nodal signaling has several vital roles in both embryo development and cancer progression. However, the effects of Nodal signaling on VM formation in breast cancer and its underlying mechanisms are ill-defined. We analyzed the relationship between Nodal signaling and VM formation in one hundred human breast cancer cases and the results showed that the expression of Nodal was significantly correlated with VM formation, tumor metastasis, differentiation grade, TNM stage and poor prognosis. Furthermore, up-regulation of Nodal expression promoted VM formation of breast cancer cells in vitro and in vivo. Knockdown of Nodal expression restrained VM formation. In addition, Nodal induced EMT and up-regulated the expression of Slug, Snail and c-Myc. We found that blocking the Smad2/3 pathway by administering SB431542 inhibited VM formation in breast cancer cell lines and xenografts. Taken together, Nodal signaling through the Smad2/3 pathway up-regulated Slug, Snail and c-Myc to induce EMT, thereby promoting VM formation. Our study suggests that the Nodal signaling pathway may serve as a therapeutic target to inhibit VM formation and improve prognosis in breast cancer patients.

Topics: Adult; Aged; Animals; Benzamides; Breast Neoplasms; Dioxoles; Epithelial-Mesenchymal Transition; Female; Humans; MCF-7 Cells; Mice; Mice, Inbred BALB C; Middle Aged; Neoplasm Invasiveness; Neovascularization, Pathologic; Nodal Protein; Signal Transduction; Smad2 Protein; Smad3 Protein

Some polypeptide N-acetyl-galactosaminyltransferases (GALNTs) are associated with cancer, but their function in organ-specific metastasis remains unclear. Here, we report that GALNT14 promotes breast cancer metastasis to the lung by enhancing the initiation of metastatic colonies as well as their subsequent growth into overt metastases. Our results suggest that GALNT14 augments the self-renewal properties of breast cancer cells (BCCs). Furthermore, GALNT14 overcomes the inhibitory effect of lung-derived bone morphogenetic proteins (BMPs) on self-renewal and therefore facilitates metastasis initiation within the lung microenvironment. In addition, GALNT14 supports continuous growth of BCCs in the lung by not only inducing macrophage infiltration but also exploiting macrophage-derived fibroblast growth factors (FGFs). Finally, we identify KRAS-PI3K-c-JUN signalling as an upstream pathway that accounts for the elevated expression of GALNT14 in lung-metastatic BCCs. Collectively, our findings uncover an unprecedented role for GALNT14 in the pulmonary metastasis of breast cancer and elucidate the underlying molecular mechanisms.

Topics: Animals; Benzamides; Bone Morphogenetic Protein Receptors; Breast Neoplasms; Cell Line, Tumor; Diamines; Dioxoles; Female; Gene Expression Regulation, Neoplastic; Glycosylation; Guanine Nucleotide Exchange Factors; Humans; Lung; Lung Neoplasms; Mice; Mice, Nude; Mutation; N-Acetylgalactosaminyltransferases; Neoplasms, Experimental; Polypeptide N-acetylgalactosaminyltransferase; Pyrazoles; Quinolines; Receptors, Notch; Receptors, Transforming Growth Factor beta; Thiazoles

Transforming growth factor-β (TGF-β) has both tumor suppressive and oncogenic activities. Autocrine TGF-β signaling supports tumor survival and growth in certain types of cancer, and the TGF-β signaling pathway is a potential therapeutic target for these types of cancer. TGF-β induces p21 expression, and p21 is considered as an oncogene as well as a tumor suppressor, due to its anti-apoptotic activity. Thus, we hypothesized that autocrine TGF-β signaling maintains the expression of p21 at levels that can support cell growth. To verify this hypothesis, we sought to examine p21 expression and cell growth in various cancer cells following the inhibition of autocrine TGF-β signaling using siRNAs targeting TGF-β signaling components and SB431542, a TGF-β receptor inhibitor. Results from the present study show that p21 expression and cell growth were reduced by knockdown of TGF-β signaling components using siRNA in MDA-MB231 and A549 cells. Cell growth was also reduced in p21 siRNA-transfected cells. Downregulation of p21 expression induced cellular senescence in MDA-MB231 cells but did not induce apoptosis in both cells. These data suggest that autocrine TGF-β signaling is required to sustain p21 levels for positive regulation of cell cycle. On the other hand, treatment with SB431542 up-regulated p21 expression while inhibiting cell growth. The TGF-β signaling pathway was not associated with the SB431542-mediated induction of p21 expression. Specificity protein 1 (Sp1) was downregulated by treatment with SB431542, and p21 expression was increased by Sp1 knockdown. These findings suggest that downregulation of Sp1 expression is responsible for SB43154-induced p21 expression.

Topics: Animals; Autocrine Communication; Benzamides; Breast Neoplasms; Caco-2 Cells; Cell Proliferation; Cellular Senescence; Chlorocebus aethiops; COS Cells; Cyclin-Dependent Kinase Inhibitor p21; Dioxoles; Female; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Male; MCF-7 Cells; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Sp1 Transcription Factor; Time Factors; Transfection; Transforming Growth Factor beta

The transforming growth factor beta (TGF-β) signalling pathway is known to control human breast cancer invasion and metastasis. We demonstrate that the zebrafish xenograft assay is a robust and dependable animal model for examining the role of pharmacological modulators and genetic perturbation of TGF-β signalling in human breast tumour cells.. We injected cancer cells into the embryonic circulation (duct of cuvier) and examined their invasion and metastasis into the avascular collagenous tail. Various aspects of the TGF-β signalling pathway were blocked by chemical inhibition, small interfering RNA (siRNA), or small hairpin RNA (shRNA). Analysis was conducted using fluorescent microscopy.. Breast cancer cells with different levels of malignancy, according to in vitro and in vivo mouse studies, demonstrated invasive and metastatic properties within the embryonic zebrafish model that nicely correlated with their differential tumourigenicity in mouse models. Interestingly, MCF10A M2 and M4 cells invaded into the caudal hematopoietic tissue and were visible as a cluster of cells, whereas MDA MB 231 cells invaded into the tail fin and were visible as individual cells. Pharmacological inhibition with TGF-β receptor kinase inhibitors or tumour specific Smad4 knockdown disturbed invasion and metastasis in the zebrafish xenograft model and closely mimicked the results we obtained with these cells in a mouse metastasis model. Inhibition of matrix metallo proteinases, which are induced by TGF-β in breast cancer cells, blocked invasion and metastasis of breast cancer cells.. The zebrafish-embryonic breast cancer xenograft model is applicable for the mechanistic understanding, screening and development of anti-TGF-β drugs for the treatment of metastatic breast cancer in a timely and cost-effective manner.

Topics: Animals; Benzamides; Breast Neoplasms; Chromones; Dioxoles; Dipeptides; Disease Models, Animal; Drug Screening Assays, Antitumor; Embryo, Nonmammalian; Enzyme Inhibitors; Female; Humans; Matrix Metalloproteinase Inhibitors; Morpholines; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA, Small Interfering; Signal Transduction; Smad2 Protein; Smad4 Protein; Small Molecule Libraries; Transforming Growth Factor beta; Xenograft Model Antitumor Assays; Zebrafish; Zebrafish Proteins

In solid tumors, a decreased oxygen and nutrient supply creates a hypoxic microenvironment in the central region. This hypoxic condition induces molecular responses of normal and cancer cells in the local area, including angiogenesis, metabolic changes, and metastasis. In addition, other cells including mesenchymal stem cells (MSCs) have been reported to be recruited into the hypoxic area of solid tumors. In our previous study, we found that hypoxic condition induces the secretion of growth factors and cytokines in MSCs, and here we demonstrate that elevated secretion of transforming growth factor-β1 (TGF-β1) by MSCs under hypoxia promotes the growth, motility, and invasive ability of breast cancer cells. It was found that TGF-β1 promoter activity was regulated by hypoxia, and the major hypoxia-regulated element was located between bp -1030 to -666 in front of the TGF-β1 promoter region. In ChIP assay, the results revealed that HIF-1 was bound to the hypoxia response element (HRE) of TGF-β1 promoter. Collectively, the results indicate that hypoxia microenvironment can enhance cancer cell growth through the paracrine effects of the MSCs by driving their TGF-β1 gene expression and secretion. Therefore, extra caution has to be exercised when considering hypoxia pretreatment of MSCs before cell transplantation into patients for therapeutic purposes, particularly in patients susceptible to tumor growth.

Topics: Benzamides; Bone Marrow Cells; Breast Neoplasms; Cell Hypoxia; Cell Movement; Cells, Cultured; Culture Media, Conditioned; Dioxoles; Epithelial-Mesenchymal Transition; Female; HEK293 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; MCF-7 Cells; Mesenchymal Stem Cells; Promoter Regions, Genetic; Protein Binding; Receptors, Transforming Growth Factor beta; Response Elements; Transforming Growth Factor beta1

Aberrant expression of mitotic checkpoint genes compromises mitotic checkpoint, leads to chromosome instability and tumorigenesis. However, the cell signals that control mitotic checkpoint gene expression have not been reported so far. In the present study we show that, in human breast cancer cells, chemical inhibition of Bone morphogenetic proteins (BMPs), but not Transforming Growth Factor-β (TGF-β), abrogates the mitotic arrest induced by nocodazole. Protein expression analysis reveals that inhibition of BMP signaling dramatically down regulates protein levels of mitotic checkpoint components BUB3, Hec1, TTK and MAD2, but inhibition of TGF-β has relatively minor effect on the expression of these proteins. Activation of BMP signaling specifically up regulates BUB3, and activation of Activin A signaling globally down regulates these proteins level. Furthermore, overexpressing MAD2, TTK, BUB3 or Hec1 significantly rescues the mitotic arrest defect caused by BMP inhibition. Our results demonstrated for the first time that TGF-β family cytokines are cellular signals regulating mitotic checkpoint and perturbations in intrinsic BMP signaling could lead to suppression of mitotic checkpoint signaling by downregulating key checkpoint proteins. The results suggest a possible mechanism by which dysregulation of TGF-β signaling causes mitotic checkpoint defects and drives tumorigenesis. The finding also provides a potential and more specific strategy for cancer prevention by targeting BMP and mitotic checkpoint connection.

Topics: Activins; Benzamides; Bone Morphogenetic Proteins; Breast Neoplasms; Calcium-Binding Proteins; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cell Transformation, Neoplastic; Cytoskeletal Proteins; Dioxoles; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Mad2 Proteins; Nocodazole; Nuclear Proteins; Plasmids; Poly-ADP-Ribose Binding Proteins; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Repressor Proteins; Signal Transduction; Transfection; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stages. In contrast to the mechanisms by which TGF-β induces growth arrest, the pathways that mediate tumor invasion are not well understood. Here, we describe a TGF-β-dependent invasion assay system consisting of spheroids of MCF10A1 normal breast epithelial cells (M1) and RAS-transformed (pre-)malignant derivatives (M2 and M4) embedded in collagen gels. Both basal and TGF-β-induced invasion of these cell lines was found to correlate with their tumorigenic potential; M4 showing the most aggressive behavior and M1 showing the least. Basal invasion was strongly inhibited by the TGF-β receptor kinase inhibitor SB-431542, indicating the involvement of autocrine TGF-β or TGF-β-like activity. TGF-β-induced invasion in premalignant M2 and highly malignant M4 cells was also inhibited upon specific knockdown of Smad3 or Smad4. Interestingly, both a broad spectrum matrix metalloproteinase (MMP) inhibitor and a selective MMP2 and MMP9 inhibitor mitigated TGF-β-induced invasion of M4 cells, while leaving basal invasion intact. In line with this, TGF-β was found to strongly induce MMP2 and MMP9 expression in a Smad3- and Smad4-dependent manner. This collagen-embedded spheroid system therefore offers a valuable screening model for TGF-β/Smad- and MMP2- and MMP9-dependent breast cancer invasion.

Topics: Benzamides; Breast Neoplasms; Cell Line, Tumor; Dioxoles; Female; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Smad Proteins; Spheroids, Cellular; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

The transcriptional repressors Snail and Slug are situated at the core of several signaling pathways proposed to mediate epithelial to mesenchymal transition or EMT, which has been implicated in tumor metastasis. EMT involves an alteration from an organized, epithelial cell structure to a mesenchymal, invasive and migratory phenotype. In order to obtain a global view of the impact of Snail and Slug expression, we performed a microarray experiment using the MCF-7 breast cancer cell line, which does not express detectable levels of Snail or Slug. MCF-7 cells were infected with Snail, Slug or control adenovirus, and RNA samples isolated at various time points were analyzed across all transcripts. Our analyses indicated that Snail and Slug regulate many genes in common, but also have distinct sets of gene targets. Gene set enrichment analyses indicated that Snail and Slug directed the transcriptome of MCF-7 cells from a luminal towards a more complex pattern that includes many features of the claudin-low breast cancer signature. Of particular interest, genes involved in the TGF-beta signaling pathway are upregulated, while genes responsible for a differentiated morphology are downregulated following Snail or Slug expression. Further we noticed increased histone acetylation at the promoter region of the transforming growth factor beta-receptor II (TGFBR2) gene following Snail or Slug expression. Inhibition of the TGF-beta signaling pathway using selective small-molecule inhibitors following Snail or Slug addition resulted in decreased cell migration with no impact on the repression of cell junction molecules by Snail and Slug. We propose that there are two regulatory modules embedded within EMT: one that involves repression of cell junction molecules, and the other involving cell migration via TGF-beta and/or other pathways.

Topics: Acetylation; Benzamides; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Movement; Dioxoles; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Genetic Loci; Histones; Humans; Intercellular Junctions; Mammary Glands, Human; Phenotype; Protein Serine-Threonine Kinases; Pyrazoles; Pyrroles; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Snail Family Transcription Factors; Substrate Specificity; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta

During the course of breast cancer progression, normally dormant tumour-promoting effects of transforming growth factor beta (TGFbeta), including migration, invasion, and metastasis are unmasked. In an effort to identify mechanisms that regulate the pro-migratory TGFbeta 'switch' in mammary epithelial cells in vitro, we found that TGFbeta stimulates the phosphorylation of Smad1 and Smad5, which are typically associated with bone morphogenetic protein signalling. Mechanistically, this phosphorylation event requires the kinase activity and, unexpectedly, the L45 loop motif of the type I TGFbeta receptor, ALK5, as evidenced by studies using short hairpin RNA-resistant ALK5 mutants in ALK5-depleted cells and in vitro kinase assays. Functionally, Smad1/5 co-depletion studies demonstrate that this phosphorylation event is essential to the initiation and promotion of TGFbeta-stimulated migration. Moreover, this phosphorylation event is preferentially detected in permissive environments such as those created by tumorigenic cells or oncogene activation. Taken together, our data provide evidence that TGFbeta-stimulated Smad1/5 phosphorylation, which occurs through a non-canonical mechanism that challenges the notion of selective Smad phosphorylation by ALK5, mediates the pro-migratory TGFbeta switch in mammary epithelial cells.

Topics: Activins; Animals; Benzamides; Bone Morphogenetic Proteins; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Dioxoles; Humans; Mice; Phosphorylation; Protein Binding; Protein Isoforms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad1 Protein; Smad5 Protein; Transforming Growth Factor beta

The type III transforming growth factor-beta receptor (TbetaRIII or betaglycan) is a ubiquitously expressed transforming growth factor-beta (TGF-beta) superfamily coreceptor with essential roles in embryonic development. Recent studies have defined a role for TbetaRIII in the pathogenesis of human cancers, with frequent loss of TbetaRIII expression at the message and protein level. Mechanisms for the loss of TbetaRIII expression remain to be fully defined. Advanced human cancers often have elevated circulating levels of TGF-beta1. Here, we define a specific role for TGF-beta1 in negatively regulating TbetaRIII at the message level in breast and ovarian cancer models. TGF-beta1 decreased TbetaRIII message and protein levels in ovarian (Ovca420) and breast cancer (MDA-MB-231) cell lines in both a dose- and time-dependent manner. TGF-beta1-mediated TbetaRIII repression is mediated by the type I TGF-beta receptor/Smad2/3 pathway as the activin receptor-like kinase 5 (ALK5) inhibitor, SB431542, abrogated this effect, while the expression of constitutively active ALK5 was sufficient to repress TbetaRIII expression. Mechanistically, TGF-beta1 does not affect TbetaRIII messenger RNA (mRNA) stability, but instead directly regulates the TbetaRIII promoter. We define alternative promoters for the TGFBR3 gene, a distal and proximal promoter. Although both promoters are active, only the proximal promoter was responsive and negatively regulated by TGF-beta1 and constitutively active ALK5. Taken together, these studies define TGF-beta1-mediated downregulation of TbetaRIII mRNA expression through effects on the ALK5/Smad2/3 pathway on the TGFBR3 gene proximal promoter as a potential mechanism for decreased TbetaRIII expression in human cancers.

Topics: Benzamides; Breast Neoplasms; Cell Line, Tumor; Dioxoles; DNA Primers; DNA, Complementary; Female; Gene Expression Regulation; Genes, Reporter; Humans; Ovarian Neoplasms; Promoter Regions, Genetic; Protein Kinase Inhibitors; Proteoglycans; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta1

The anticancer agent prodigiosin has been shown to act as an efficient immunosuppressant, eliciting cell cycle arrest at non-cytotoxic concentrations, and potent proapoptotic and antimetastatic effects at higher concentrations. Gene expression profiling of MCF-7 cells after treatment with a non-cytotoxic concentration of prodigiosin showed that expression of the p21WAF1/CIP1 gene, a negative cell cycle regulator was induced. In this study, we show that prodigiosin induces p21 expression leading to cell cycle blockade. Subsequently, we attempted to elucidate the molecular mechanisms involved in prodigiosin-mediated p21 gene expression. We demonstrate that prodigiosin induces p21 in a p53-independent manner as prodigiosin induced p21 in cells with both mutated and dominant negative p53. Conversely, the transforming growth factor-beta (TGF-beta) pathway has been found to be necessary for p21 induction. Prodigiosin-mediated p21 expression was blocked by SB431542, a TGF-beta receptor inhibitor. Nevertheless, this pathway alone is not enough to induce p21 expression. The TGF-beta family member (nonsteroidal anti-inflammatory drug)-activated gene 1/growth differentiation factor 15 (NAG-1) may activate this pathway, as it has previously been suggested to signal through the TGF-beta pathway and is overexpressed in response to prodigiosin treatment. We show that NAG-1 colocalizes with TGF-beta receptor type I, suggesting a possible interaction between them. Taken together, these results suggest the TGF-beta pathway is required for induction of p21 expression after prodigiosin treatment of MCF-7 cells.

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Dioxoles; Female; Humans; Immunohistochemistry; Prodigiosin; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Protein p53