thymosin and Neoplasm-Metastasis

β-thymosins, including thymosin β4 (Tβ4), Tβ10, and Tβ15, are a family of highly conserved 5 kDa peptides. They are involved not only in normal cell migration, but also in tumor metastasis. However, the molecular mechanisms of β-thymosins to regulate cell migration and other functions are not fully understood. Recently, this important area is under active investigation worldwide. Many new discoveries have been made from molecular biology and cell culture models as well as animal models and human diseases. This timely review provides the most updated information about functional roles and molecular mechanisms of β-thymosins in normal tissues and disease conditions.

Topics: Actins; Animals; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Neoplasm Metastasis; Neoplasms; Thymosin

Thymosin β4 (Tβ4), a 5 kDa protein, has been demonstrated to play an important role in a variety of biological activities, such as actin sequestering, cellular motility, migration, inflammation, and damage repair. Recently, several novel findings provided compelling evidence that Tβ4 played a key role in facilitating tumor metastasis and angiogenesis. It has been found that Tβ4 expressed increasingly in a number of metastatic tumors, which was associated with an increased expression of a known angiogenic factor, vascular endothelial growth factor. Thus, Tβ4 provided a potential target of opportunity for cancer management, especially for cancer metastasis therapy.

Topics: Animals; Cell Movement; Cell Survival; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Inflammation; Neoplasm Metastasis; Neoplastic Stem Cells; Neovascularization, Pathologic; Thymosin; Wound Healing

Thymosin beta 10 (Tbeta10) is a member of the beta-thymosin family, which has biological activities as an actin-sequestering protein involved in cell motility. Tbeta10 may be correlated with tumor biology such as cell proliferation, apoptosis, angiogenesis, and metastasis behavior. However, the molecular mechanisms of action of Tbeta10 in cancer are largely unknown. Tbeta10 is differentially expressed in embryogenesis and neuronal development. Its expression is also increased in many inflammatory conditions and tumorigenesis. This review briefly summarizes recent advances in Tbeta10 research including differential expression, functions, mechanisms, gene regulation, and therapeutic applications in cancer, wound healing, and other diseases.

Topics: Actins; Animals; Apoptosis; Cell Movement; Cell Proliferation; Gene Expression Regulation; Humans; Inflammation; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Neurogenesis; Neurons; Signal Transduction; Thymosin; Wound Healing

The beta-thymosins are a family of highly conserved polar 5 kDa peptides originally thought to be thymic hormones. About 10 years ago, thymosin beta(4) as well as other members of this ubiquitous peptide family were identified as the main intracellular G-actin sequestering peptides, being present in high concentrations in almost every cell. beta-Thymosins bind monomeric actin in a 1:1 complex and act as actin buffers, preventing polymerization into actin filaments but supplying a pool of actin monomers when the cell needs filaments. Changes in the expression of beta-thymosins appear to be related to the differentiation of cells. Increased expression of beta-thymosins or even the synthesis of a beta-thymosin normally not expressed might promote metastasis possibly by increasing mobility of the cells. Thymosin beta(4) is detected outside of cells in blood plasma or in wound fluid. Several biological effects are attributed to thymosin beta(4), oxidized thymosin beta(4), or to the fragment, acSDKP, possibly generated from thymosin beta(4). Among the effects are induction of metallo-proteinases, chemotaxis, angiogenesis and inhibition of inflammation as well as the inhibition of bone marrow stem cell proliferation. However, nothing is known about the molecular mechanisms mediating the effects attributed to extracellular beta-thymosins.

Topics: Actins; Amino Acid Sequence; Animals; Binding Sites; Cell Differentiation; Cell Movement; Humans; Molecular Sequence Data; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Oxidation-Reduction; RNA, Messenger; Thymosin; Wound Healing

Important contributions that stimulated studies in cancer immunotherapy included: (1) the discovery of tumour-associated antigens; (2) the observation that infection with bacille Calmette-Guérin (BCG) in animals was protective against tumour challenge; and (3) the observation that immunodepression due either to malignant disease or to treatment of the disease, was, in some instances, related to prognosis. Immunotherapy trials with microbial agents have involved attempts to obtain a local effect by injecting the agent into the tumour or into the region of the tumour and to obtain a "systemic" effect distant from the site of injection. Trials with active specific immunotherapy involving tumour cells or tumour cell extracts have frequently involved the combination of these specific agents with a nonspecific adjuvant such as BCG. Recent studies with thymosin and levamisole in patients with lung cancer and other types of malignant disease have shown prolonged survival in the groups receiving immunotherapy.

Topics: Animals; Antigens, Neoplasm; Antineoplastic Agents; BCG Vaccine; Humans; Hypersensitivity, Delayed; Immunotherapy; Leukemia, Lymphoid; Levamisole; Melanoma; Neoplasm Metastasis; Neoplasms; Neoplasms, Experimental; Skin Neoplasms; Thymosin

Topics: Adjuvants, Immunologic; Antigens, Neoplasm; Antineoplastic Agents; BCG Vaccine; Epitopes; Humans; Immunity, Cellular; Immunosuppression Therapy; Immunotherapy; Levamisole; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasms; Prognosis; Propionibacterium acnes; RNA; Thymosin; Transfer Factor

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents, Alkylating; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Combined Modality Therapy; Dacarbazine; Disease Progression; Disease-Free Survival; Drug Administration Schedule; Feasibility Studies; Female; Humans; Immunologic Factors; Interferon alpha-2; Interferon-alpha; Killer Cells, Natural; Lymphopenia; Male; Melanoma; Middle Aged; Neoplasm Metastasis; Recombinant Proteins; Safety; Survival Analysis; Thymalfasin; Thymosin; Treatment Outcome

DTIC and interleukin-2 (IL-2), as single agents, have a limited anti-tumor activity in patients with metastatic melanoma. Experimentally, thymosin alpha 1 (TA1) may modulate the action of IL-2. We investigated the clinical and immunological effects of a combination with these three agents.. Forty-six patients with measurable metastatic melanoma were treated with DTIC 850 mg IV on day 1, TA1 2 mg s.c. on days 4 to 7, and IL-2 18 MU/m2/d by continuous intravenous infusion on days 8 to 12. Cycles were repeated every 3 weeks.. Objective responses were obtained in 15 (36%) of 42 evaluable patients (CI at 95%: 22%-50%). Two patients experienced complete responses, and stable disease was observed in five. The median time to progression was 5.5 months and median survival was 11 months. Side effects were predominantly caused by IL-2. Treatment was tolerated reasonably well, and there was no overlapping toxicity or interference between chemotherapy and biotherapy. Baseline sCD4 levels seem to correlate to tumor burden. Patients benefiting from treatment had lower sCD4 and higher sCD8 than did progressing patients.. The combination of DTIC + TA1 + IL-2 is active in the treatment of advanced melanoma, with acceptable toxicity. However, even more active regimens are needed, and the interactions between thymic hormones and cytokines should be further explored.

Topics: Adult; Aged; CD4 Antigens; CD8 Antigens; Combined Modality Therapy; Dacarbazine; Female; Humans; Interleukin-2; Male; Melanoma; Middle Aged; Neoplasm Metastasis; Remission Induction; Skin Neoplasms; Thymalfasin; Thymosin

Gastric cancer (GC) is histologically classified into intestinal-type gastric cancer (IGC) and diffuse-type gastric cancer (DGC), and the latter is poorly differentiated and highly metastatic. In this study, using quantitative real-time polymerase chain reaction, we described a complete protocol for in vivo CRISPR-Cas9-based knockout screening of essential genes for DGC metastasis. We functionally screened 30 candidate genes using our mouse DGC models lacking Smad4, p53, and E-cadherin. Pooled knockout mouse DGC cells were transplanted into a spleen of syngeneic immunocompetent mice to study clonal advantages in context of a complex process of liver metastasis. Tmsb4x (thymosin beta-4 X-linked), Hmox1, Ifitm3, Ldhb, and Itgb7 were identified as strong candidate genes that promote metastasis. In particular, Tmsb4x enhanced DGC metastasis and stomach organoid-generated tumor growth in in vivo transplantation models. Tmsb4x promoted tumor clonogenicity and anoikis resistance. In situ hybridization analysis showed that Tmsb4x is highly expressed in E-cadherin-negative mouse DGC models compared with mouse IGC and intestinal cancer models. E-cadherin deficiency also increased Tmsb4x expression in stomach organoids via Wnt signaling activation. Collectively, these results demonstrate that Tmsb4x promotes DGC metastasis. In addition, this experimental system will aid in the identification of novel target genes responsible for DGC metastasis.

Topics: Animals; Biomarkers, Tumor; CRISPR-Cas Systems; Disease Models, Animal; Gene Expression; Gene Knockout Techniques; Humans; Mice; Neoplasm Metastasis; Real-Time Polymerase Chain Reaction; Signal Transduction; Stomach Neoplasms; Thymosin

Cancer-initiating/sustaining stem cell subsets (CSCs) have the potential to regenerate cancer cell populations and are resistant to routine therapeutic strategies, thus attracting much attention in anticancer research. In this study, an innovative framework of endogenous microenvironment-renewal for addressing such a dilemma has been just developed. CSCs in three-dimensional multipotent spheroid-engineered biologics were prepared with 150 Gy radiation and inoculated into 15-mo-old BALB/c and C57BL/6 mice bearing diverse advanced tumors covering Mammary 4T1, liver Hepa, lung LL/2, and colon C26 tumors and distant metastases. Subsequently, the systematic microenvironment of tumor-bearing hosts was rapidly remodeled to resettle thymic cortex and medulla rudiment as an endogenous foxn1-thymosin reprogramming TCR-repertoire for resetting MHC-unrestricted multifunction renewal. Postrenewal Vγ4γδT-subsets would bind and lead migrating CSCs into apoptosis. Moreover, TCR repertoire multifunction renewal could reverse tumor metastases from tumoricidal resistance into eventual regression as a blockade of cancer-sustaining Bmi-1/Nanog-Oct4-Sox2 renewal loop with sequent multivalent depletion of both migrating/in situ CSCs and non-stem terminal cancer cell subsets. This study represents a promising start to set up a generalizable strategy of three-dimensional biologics evoking an endogenous integral microenvironment into pluripotent renewal versus advanced cancer.

Topics: Animals; Apoptosis; Biological Therapy; Cell Culture Techniques; Cell Differentiation; Cell Line, Tumor; Cell Self Renewal; Cellular Reprogramming Techniques; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Immunity, Cellular; Immunization; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Multipotent Stem Cells; Neoplasm Metastasis; Neoplasms, Experimental; Neoplastic Stem Cells; Radiation; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocytes; Thymosin; Thymus Gland; Tissue Engineering; Tumor Microenvironment

The genes for PFN1 and TMSB4 are both highly expressed in oral tissue and both encode actin monomer binding proteins thought to play a role in cell motility and possibly other crucial parts of tumor progression.. Oral brush cytology of epithelium from oral squamous cell carcinoma (OSCC) was used to measure PFN1 and TMSB4 mRNA in OSCC, while immunohistochemical analysis of tissue was used to check protein levels.. High but variable expression of mRNAs encoding these two proteins was observed suggesting they may contribute to tumor characteristics in a subset of OSCCs. Both proteins were highly expressed in normal appearing basal epithelium, in the cytoplasm, and perinuclear area, while expression was minimal in upper epithelial layers. In OSCCs, expression of these proteins varied. In tumors classified as later stage, based on size and/or lymph node involvement, PFN1 levels were lower in tumor epithelium. A control gene, KRT13, showed expression in normal differentiated basal and suprabasal oral mucosa epithelial cells and as reported was lost in OSCC cells.. Loss of PFN1 in tumor cells has been associated with lymph node invasion and metastasis in other tumor types, strengthening the argument that the protein has the potential to be a tumor suppressor in late-stage OSCC.

Topics: Aged; Carcinoma, Squamous Cell; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Keratin-13; Lymphatic Metastasis; Male; Mouth Mucosa; Mouth Neoplasms; Neoplasm Metastasis; Neoplasm Staging; Profilins; RNA, Messenger; Thymosin

Thymosin beta 10 (TMSB10) has been demonstrated to be involved in the malignant process of many cancers. The purpose of this study was to determine the biological roles and clinical significance of TMSB10 in breast cancer and to identify whether TMSB10 might be used as a serum marker for the diagnosis of breast cancer.. TMSB10 expression was evaluated by immunohistochemical analysis (IHC) of 253 breast tumors and ELISA of serum from 80 patients with breast cancer. Statistical analysis was performed to explore the correlation between TMSB10 expression and clinicopathological features in breast cancer. Univariate and multivariate Cox regression analysis were performed to examine the association between TMSB10 expression and overall survival and metastatic status. In vitro and in vivo assays were performed to assess the biological roles of TMSB10 in breast cancer. Western blotting and luciferase assays were examined to identify the underlying pathway involved in the tumor-promoting role of TMSB10.. We found TMSB10 was upregulated in breast cancer cells and tissues. Univariate and multivariate analysis demonstrated that high TMSB10 expression significantly correlated with clinicopathological features, poor prognosis and distant metastases in patients with breast cancer. Overexpression of TMSB10 promotes, while silencing of TMSB10 inhibits, proliferation, invasion and migration of breast cancer cells in vitro and in vivo. Our results further reveal that TMSB10 promotes the proliferation, invasion and migration of breast cancer cells via AKT/FOXO signaling, which is antagonized by the AKT kinase inhibitor perifosine. Importantly, the expression of TMSB10 is significantly elevated in the serum of patients with breast cancer and is positively associated with clinical stages of breast cancer.. TMSB10 may hold promise as a minimally invasive serum cancer biomarker for the diagnosis of breast cancer and a potential therapeutic target which will facilitate the development of a novel therapeutic strategy against breast cancer.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Forkhead Transcription Factors; Humans; Kaplan-Meier Estimate; Neoplasm Metastasis; Prognosis; Proportional Hazards Models; Proto-Oncogene Proteins c-akt; Signal Transduction; Thymosin

Hepatoblastoma (HB) is the most common malignant hepatic tumor in children and complete surgical resection offers the highest possibility for cure in this disease. Tumor metastasis is the principle obstacle to the development of efficient treatments for patients with HB. The present study aimed to measure the expression levels of thymosin β4 (Tβ4) in liver samples from patients with HB and to investigate the involvement of Tβ4 in HB metastasis. The expression of Tβ4 was significantly higher in liver samples from patients with metastatic HB and in the HepG2 metastatic HB cell line, compared with that in adjacent healthy liver samples and in the L02 healthy hepatic cell line. By contrast, the expression levels of epithelial-cadherin (E-cadherin) and cytosolic accumulation of β-catenin, the two most prominent markers involved in epithelial-mesenchymal transition (EMT), were reduced in liver specimens from patients with metastatic HB compared with that of healthy adjacent control tissue. HepG2 cells were transfected with small interfering-RNA in order to downregulate Tβ4 gene expression. This resulted in a reduced cell migratory capacity compared with control cells. Tβ4 gene expression knockdown significantly inhibited transforming growth factor β1-mediated-EMT in vitro by upregulating the expression of E-cadherin. The results of the present study suggested that Tβ4 may promote HB metastasis via the induction of EMT, and that Tβ4 may therefore be a target for the development of novel treatments for patients with HB.

Topics: beta Catenin; Cadherins; Cell Proliferation; Child; Child, Preschool; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Hepatoblastoma; Humans; Infant; Male; Neoplasm Metastasis; Thymosin

Signaling by small guanosine triphosphatases (GTPase), Rap1/Rac1, is one of the major pathways controlling cancer cell migration and tumor metastasis. Thymosin beta-4 (Tβ4), an actin-sequestering protein, has been shown to increase migration of cancer cells. Episodes of hypoxia and re-oxygenation (H/R) are an important phenomenon in tumor microenvironment (TME). We investigated whether Tβ4 could play as an intermediary to crosstalk between Rac1- and Rap1- GTPase activation under hypoxia/reoxygenation (H/R) conditions. Inhibition of Tβ4 expression using transcription activator-like effector nucleases (TALEN) significantly decreased lung metastasis of B16F10 cells. Rac1 and Rap1 activity, as well as cancer cell migration, increased following induction of Tβ4 expression in normoxia- or H/R-experienced cells, but were barely detectable in Tβ4-depleted cells. Rap1-regulated Rac1 activity was decreased by a dominant negative Rap1 (Rap1N17), and increased by 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (CPT), a Rap1 activator. In contrast, a Rac1-specific inhibitor, NSC23766, and dominant negative Rac1 (Rac1N17) enhanced Tβ4 expression and aberrant Rap1 activity. While NSC23766 and Rac1N17 incompletely inhibited tumor metastasis in vivo, and H/R-experienced cancer cell migration in vitro, more efficient attenuation of cancer cell migration was accomplished by simultaneous inactivation of Rap1 and Rac1 with Rap1N17 and Rac1N17, respectively. These data suggest that a combination therapy targeting both Rap1 and Rac1 activity may be an effective method of inhibiting tumor metastasis.

Topics: Aminoquinolines; Animals; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Enzyme Activation; Gene Expression Regulation, Neoplastic; GTP Phosphohydrolases; HeLa Cells; Humans; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neuropeptides; Oxygen; Pyrimidines; rac1 GTP-Binding Protein; Shelterin Complex; Signal Transduction; Telomere-Binding Proteins; Thymosin; Tumor Microenvironment; Two-Hybrid System Techniques

Beta-thymosins constitute a group of small actin-sequestering peptides. These highly conserved peptides are involved in cytoskeleton dynamics and can influence different cell properties such as motility, substrate adhesion, shape and chemotaxis. As a marker for tumour metastasis, the mammalian thymosin beta15 is believed to have an important diagnostic relevance in cancer prognosis, although little is known about its physiological function. In order to study the role of thymosin beta15(avian) in embryogenesis, we cloned the chicken and quail orthologues of thymosin beta15 and used the chicken as a model for vertebrate development. Avian thymosin beta15, the first known non-mammalian thymosin beta15-like gene, encodes a peptide that possesses a cysteine at position one after the methionine which is a significant difference compared to its mammalian counterparts. Thymosin beta15(avian) expression starts at an early stage of development. The expression pattern changes rapidly with development and differs from that of the related thymosin beta4 gene. The most prominent expression domain is seen in developing muscles of limbs and trunk. Gain-of-function experiments revealed that thymosin beta15(avian) has a function in normal myotome development. Ectopic over-expression of thymosin beta15(avian) leads to premature elongation of myotome cells trespassing segment borders. We conclude that thymosin beta15(avian) has a still undescribed function in promoting myocyte elongation.

Topics: Amino Acid Sequence; Animals; Chick Embryo; Cloning, Molecular; Electroporation; Molecular Sequence Data; Muscle Cells; Muscle Development; Muscle, Skeletal; Neoplasm Metastasis; Retroviridae; Thymosin

Thymosin β10 (Tβ10) expression is associated with malignant phenotypes in many cancers. However, the role and mechanisms of Tβ10 in liver fluke-associated cholangiocarcinoma (CCA) are not fully understood. In this study, we investigated the expression of Tβ10 in CCA tumor tissues and cell lines as well as molecular mechanisms of Tβ10 in tumor metastasis of CCA cell lines.. Tβ10 expression was determined by real time RT-PCR or immunocytochemistry. Tβ10 silence or overexpression in CCA cells was achieved using gene delivery techniques. Cell migration was assessed using modified Boyden chamber and wound healing assay. The effect of silencing Tβ10 on CCA tumor metastasis was determined in nude mice. Phosphorylation of ERK1/2 and the expression of EGR1, Snail and matrix metalloproteinases (MMPs) were studied.. Ten pairs of CCA tissues (primary and metastatic tumors) and 5 CCA cell lines were studied. With real time RT-PCR and immunostaining analysis, Tβ10 was highly expressed in primary tumors of CCA; while it was relatively low in the metastatic tumors. Five CCA cell lines showed differential expression levels of Tβ10. Silence of Tβ10 significantly increased cell migration, invasion and wound healing of CCA cells in vitro; reversely, overexpression of Tβ10 reduced cell migration compared with control cells (P<0.05). In addition, silence of Tβ10 in CCA cells increased liver metastasis in a nude mouse model of CCA implantation into the spleen. Furthermore, silence of Tβ10 activated ERK1/2 and increased the expression of Snail and MMPs in CCA cell lines. Ras-GTPase inhibitor, FPT inhibitor III, effectively blocked Tβ10 silence-associated ERK1/2 activation, Snail expression and cell migration.. Low expression of Tβ10 is associated with metastatic phenotype of CCA in vitro and in vivo, which may be mediated by the activation of Ras, ERK1/2 and upregulation of Snail and MMPs. This study suggests a new molecular pathway of CCA pathogenesis and a novel strategy to treat or prevent CCA metastasis.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cholangiocarcinoma; Disease Models, Animal; Fasciola hepatica; Fascioliasis; Gene Expression; Gene Silencing; Humans; Mice; Mice, Nude; Neoplasm Metastasis; RNA Interference; Thymosin

Papillary thyroid carcinoma (PTC) is the most common well-differentiated thyroid cancer. Although the great majority of the cases exhibit an indolent clinical course, some of them develop local invasion with distant metastasis, and a few cases transform into undifferentiated/anaplastic thyroid carcinoma with a rapidly lethal course. To identify gene copy number alterations predictive of metastatic potential or aggressive transformation, array-based comparative genomic hybridization (CGH-array) was performed in 43 PTC cases. Formalin-fixed and paraffin-embedded samples from primary tumours of 16 cases without metastasis, 14 cases with only regional lymph node metastasis, and 13 cases with distant metastasis, recurrence or extrathyroid extension were analysed. The CGH-array and confirmatory quantitative real-time PCR results identified the deletion of the EIF4EBP3 and TRAK2 gene loci, while amplification of thymosin beta 10 (TB10) and Tre-2 oncogene regions were observed as general markers for PTC. Although there have been several studies implicating TB10 as a specific marker based on gene expression data, our study is the first to report on genomic amplification. Although no significant difference could be detected between the good and bad prognosis cases in the A-kinase anchor protein 13 (AKAP13) gene region, it was discriminative markers for metastasis. Amplification in the AKAP13 region was demonstrated in 42.9% and 15.4% of the cases with local or with distant metastasis, respectively, while no amplification was detected in non-metastatic cases. AKAP13 and TB10 regions may represent potential new genomic markers for PTC and cancer progression.

Topics: A Kinase Anchor Proteins; Adolescent; Adult; Aged; Biomarkers, Tumor; Bone Neoplasms; Carcinoma, Papillary; Comparative Genomic Hybridization; DNA, Neoplasm; Female; Follow-Up Studies; Gene Amplification; Gene Dosage; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Middle Aged; Minor Histocompatibility Antigens; Neoplasm Grading; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Proto-Oncogene Proteins; Real-Time Polymerase Chain Reaction; Risk Factors; Thymosin; Thyroid Neoplasms; Young Adult

Thymosin β(4) (Tβ(4)) overexpression increases cell migration and tumor metastasis. Hence, understanding the mechanism of cancer cell migration induced by Tβ(4) may provide means to inhibit their metastasis. We demonstrated higher Rac1 activities and expression levels of IQGAP1 and ILK in highly migrated Tβ(4)-overexpressing SW480 cells. In addition, IQGAP1 formed a complex with ILK and knockdown of Tβ(4) simultaneously reduced ILK and IQGAP1 protein levels as well as their migration ability. These findings suggest that Tβ(4) increases migration of colon cancer cells via activating Rac1 by elevating IQGAP1/ILK complexes and IHC results illustrated a similar mechanism occurring in vivo.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Humans; Mice; Neoplasm Metastasis; Protein Serine-Threonine Kinases; rac1 GTP-Binding Protein; ras GTPase-Activating Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Thymosin; Tumor Cells, Cultured; Up-Regulation

Angiogenesis is induced by soluble factors such as vascular endothelial growth factor (VEGF) released from tumor cells in hypoxia. It enhances solid tumor growth and provides an ability to establish metastasis at peripheral sites by tumor cell migration. Thymosin beta-4 (TB4) is an actin-sequestering protein to control cytoskeletal reorganization. Here, we investigated whether angiogenesis and tumor metastasis are dependent on hypoxia conditioning-induced TB4 expression in B16F10 melanoma cells. TB4 expression in B16F10 cells was increased by hypoxia conditioning in a time-dependent manner. In addition, we found an increase of angiogenesis and HIF-1α expression in TB4-transgenic (Tg) mice as compared to wildtype mice. When wound healing assay was used to assess in vitro tumor cell migration, hypoxia conditioning for 1 h enhanced B16F10 cell migration. When TB4 expression in B16F10 cells was inhibited by the infection with small hairpin (sh) RNA of TB4 cloned in lentiviral vector, tumor cell migration was retarded. In addition, hypoxia conditioning-induced tumor cell migration was reduced by the infection of lentiviral shRNA of TB4. HIF-1α stabilization and the expression of VEGF isoform 165 and 121 in hypoxia were also reduced by the infection of lentiviral shRNA of TB4 in B16F10 cells. We also found an increase of tumor growth and lung metastasis count in TB4-Tg mice as compared to wildtype mice. Collectively, hypoxia conditioning induced tumor cell migration by TB4 expression-dependent HIF-1α stabilization. It suggests that TB4 could be a hypoxia responsive regulator to control tumor cell migration in angiogenesis and tumor metastasis.

Topics: Animals; Cell Hypoxia; Humans; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Transgenic; Neoplasm Metastasis; Neovascularization, Pathologic; Reverse Transcriptase Polymerase Chain Reaction; Thymosin; Tumor Cells, Cultured

Thymosin beta4 (Tbeta4) is a major actin-sequestering protein that has been implicated in the growth, survival, motility, and metastasis of certain tumors and is considered an indicator for malignant progression. Therefore, identifying compounds that can downregulate Tbeta4 expression is very important for the development of anti-cancer chemotherapies. In this study, we investigated the effects of elevated cAMP on Tbeta4 expression and the metastatic potential of murine B16 melanoma cells. In addition, we also dissected the mechanism underlying cAMP-mediated Tbeta4 suppression. We found that treatment with the cAMP-inducing compounds alpha-MSH (alpha-melanocyte stimulating hormone) and IBMX (3-isobutyl-1-methylxanthine) significantly suppressed Tbeta4 expression and regulated EMT-associated genes through the suppression of NF-kappaB activation in B16F10 cells. Along with decreased Tbeta4 expression, the in vitro invasiveness and anchorage-independent growth in a semi-solid agar of these cells were also inhibited. In animal experiments, the metastatic potential of the alpha-MSH- or IBMX-treated B16F10 melanoma cells was decreased compared to untreated control cells. Collectively, our data demonstrate that elevated intracellular cAMP significantly suppresses Tbeta4 expression and reduces MMP-9 activity, which leads to decreased metastatic potential. Moreover, suppression of NF-kappaB activation by alpha-MSH or IBMX is critical for inhibiting Tbeta4 expression.

Topics: Animals; Cell Proliferation; Cells, Cultured; Cyclic AMP; Gene Expression Regulation, Neoplastic; Matrix Metalloproteinase 9; Melanoma; Melanoma, Experimental; Mice; Neoplasm Metastasis; NF-kappa B; Thymosin

Thymosin beta15 (Tbeta15) is a small protein that comprises 44 amino acid residues. Tbeta15 is upregulated in malignant human prostate and breast tumors. The expression of Tbeta15 correlates with the metastatic potential of mouse lung cancer and human breast carcinoma cells. However, the correlation of Tbeta15 expression with human lung cancer remains unclear. Using immunohistochemistry and in situ hybridization, we analyzed the expression of Tbeta15 in tumors and tumor-adjacent tissues obtained from 76 patients with non-small cell lung cancer (NSCLC). The relationship between Tbeta15 expression and clinicopathological factors was investigated. Our findings showed that in NSCLC, Tbeta15 protein and mRNA were mainly expressed in the cytoplasm, and their expression correlated with stage (p=0.018 for both), differentiation (p=0.013 and 0.006, respectively), and lymph node metastasis (p=0.001 and 0.009, respectively). Tbeta15 expression was examined in PG-BE1 and PG-LH7 lung cancer cells. We found that both Tbeta15 protein and mRNA were highly expressed in BE1 cells as compared to LH7 cells. After transfecting the PG-LH7 cells with the pEGFP-Tbeta15 plasmid in order to increase Tbeta15 expression, the migration ability of the cells was enhanced. These findings suggest that increased Tbeta15 expression correlates with the progression and metastasis of NSCLC.

Topics: Adult; Aged; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Disease Progression; Female; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Metastasis; RNA, Messenger; Thymosin; Transfection; Up-Regulation

To investigate the differential expression levels of thymosin beta 10 (T beta 10) and the corresponding changes of actin filament organization in human tumor cell lines with different metastatic potential.. Four groups of nine human tumor cell lines with different metastatic potential were analyzed for the amount of T beta 10 mRNAs by Northern blot and for their peptide expression levels by immunohistochemistry. The filamentous actin (F-actin) was observed by staining of TRITC-phalloidin to detect changes in actin organization.. In comparison with non-/weakly metastatic counterparts, T beta 10 was upregulated in highly metastatic human lung cancer, malignant melanoma and breast cancer cell lines. Staining of TRITC-phalloidin revealed less actin bundles, a fuzzy network of shorter filaments and some F-actin aggregates in the highly metastatic tumor cells. Meanwhile, the actin filaments were robust and orderly arranged in the non-/weakly metastatic cancer cell lines.. T beta 10 levels correlate positively with the metastatic capacity in human tumors currently examined. The increasing metastatic potential of tumor cells is accompanied by a loss of F-actin, poorly arranged actin skeleton organizations and presence of F-actin aggregates. There is a consistent correlation between the elevated T beta 10 expression and the disrupted actin skeleton.

Topics: Actin Cytoskeleton; Blotting, Northern; Cell Line, Tumor; Humans; Immunohistochemistry; Neoplasm Metastasis; RNA, Messenger; Thymosin

To investigate the expression of thymosin beta10 (Tbeta10) and related changes of actin filament organization in human tumor cell lines with different metastatic potential.. Four groups of nine human tumor cell lines with different metastatic potential were analyzed for the expression of Tbeta10 mRNA detected by northern-blot and its peptide by immunohistochemical staining. The filamentous actin (F-actin) was stained with TRITC-phalloidin to detect changes in actin organization.. In comparison with the non and/or weakly metastatic counterparts, Tbeta10 was upregulated in highly metastatic human lung cancer, malignant melanoma and breast cancer cell lines. TRITC-phalloidin staining revealed less actin bundles and a fuzzy network of shorter filaments in the highly metastatic tumor cells, while in the non and/or weakly metastatic cancer cell lines, there were thick and orderly arranged actin filaments.. Tbeta10 levels correlate positively with the metastatic phenotype in human tumors currently examined. The increased metastatic potential of tumor cells is accompanied by the loss of F-actin and poorly organized actin skeleton. There is a consistent correlation between the elevated Tbeta10 expression and the disrupted actin skeleton.

Topics: Actins; Blotting, Northern; Cell Line, Tumor; Humans; Immunohistochemistry; Neoplasm Metastasis; Thymosin

Cell-matrix and cell-cell adhesive interactions play important roles in the normal organization and stabilization of the cell layer in epithelial tissue. Alterations in the expression and function of these adhesion systems that cause a switch to a migratory phenotype in tumor invasion and metastasis are critical for the malignant conversion of epithelial cells. Thymosin beta-4 (Tbeta-4) is the major actin-sequestering protein that has been shown to be upregulated in a wide variety of human carcinomas and has been implicated to be involved in altering the motility of certain tumors. We have recently demonstrated that the growth rate, colony formation in soft agar, and motility, all good indicators for malignant progression, of SW480 colon carcinoma cells are dramatically increased by enforced Tbeta-4 expression. To test the hypothesis that overexpression of this G-actin sequestering peptide also promotes tumor invasion, we examined not only the invasion capability of Tbeta-4-overexpressing SW480 cells, but also the expression levels of Tbeta-4 as well as several proteins that participate in different stages of tumor progression in matched samples of human primary colorectal adenocarcinoma and liver metastases from several patients. A marked increase on the invasiveness in Tbeta-4-overexpressing SW480 cells with increased levels and activity of matrix metalloproteinase-7 (MMP-7) was observed. Furthermore, the levels of Fas as well as the susceptibility to Fas ligand-mediated apoptosis in Tbeta-4-overexpressing cells were significantly decreased. Interestingly, the levels of Tbeta-4 mRNA, beta-catenin, c-Myc, and MMP-7 in metastatic liver lesions were relatively higher, whereas the levels of E-cadherin and Fas were significantly lower than those in the matched primary colorectal tumors. These results suggest that upregulation of Tbeta-4, by promoting the disruption of cell-cell adhesion and a consequential activation of the beta-catenin signaling, could be a key event in the acquisition of growth advantages as well as invasive phenotypes in human colorectal carcinomas.

Topics: Cell Line, Tumor; Colorectal Neoplasms; Gene Expression; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; RNA, Messenger; Thymosin

Early-stage non-small cell lung cancer (NSCLC) can be cured by surgical resection, but a substantial fraction of patients ultimately dies due to distant metastasis. In this study, we used subtractive hybridization to identify gene expression differences in stage I NSCLC tumors that either did or did not metastasize in the course of disease. Individual clones (n=225) were sequenced and quantitative RT-PCR verified overexpression in metastasizing samples. Several of the identified genes (eIF4A1, thymosin beta4 and a novel transcript named MALAT-1) were demonstrated to be significantly associated with metastasis in NSCLC patients (n=70). The genes' association with metastasis was stage- and histology specific. The Kaplan-Meier analyses identified MALAT-1 and thymosin beta4 as prognostic parameters for patient survival in stage I NSCLC. The novel MALAT-1 transcript is a noncoding RNA of more than 8000 nt expressed from chromosome 11q13. It is highly expressed in lung, pancreas and other healthy organs as well as in NSCLC. MALAT-1 expressed sequences are conserved across several species indicating its potentially important function. Taken together, these data contribute to the identification of early-stage NSCLC patients that are at high risk to develop metastasis. The identification of MALAT-1 emphasizes the potential role of noncoding RNAs in human cancer.

Topics: Carcinoma, Non-Small-Cell Lung; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization; Lung Neoplasms; Molecular Sequence Data; Neoplasm Metastasis; Neoplasm Staging; Predictive Value of Tests; Reference Values; Reverse Transcriptase Polymerase Chain Reaction; RNA, Untranslated; Survival Rate; Thymosin

Topics: Actins; Animals; Antineoplastic Agents; Cell Physiological Phenomena; Cell Transformation, Neoplastic; Humans; Male; Melanoma, Experimental; Neoplasm Metastasis; Neovascularization, Pathologic; Prostatic Neoplasms; Thymosin; Up-Regulation

Expression of the small peptide thymosin beta4 is associated with angiogenesis induction, accelerated wound healing, and the metastatic potential of tumor cells. However, little is known about the mechanism(s) by which thymosin beta4 promotes metastasis.. Northern blot analysis and immunohistochemistry were used to examine thymosin beta4 expression in mouse melanoma B16 cell lines and in B16-F10 cells derived from metastatic mouse lung tumors, respectively. B16-F10 cells infected with adenoviruses containing a thymosin beta4 expression vector or an empty vector were injected subcutaneously and intravenously into C57BL/6 mice to evaluate tumor growth and metastatic potential, respectively. In vitro assays were used to study cell migration, invasion, matrix metalloproteinase activity, cell proliferation, and angiogenic activity of adenovirus-infected B16-F10 cells. Statistical significance of all results was analyzed by two-tailed Student's t tests.. Thymosin beta4 mRNA was expressed in primary cultured B16-F10 cells derived from lung metastases and in B16-F10 cells that had formed lung tumors after being injected into mice but not in the B16-F1, B16-F10, or B16-BL6 cell lines. The mean tumor sizes in mice 20 days after injection with B16-F10 cells infected with thymosin beta4-expressing adenovirus and with control adenovirus were 21.7 mm (95% confidence interval [CI] = 17.7 to 25.7 mm) and 13.3 mm (95% CI = 11.1 to 15.3 mm), respectively (difference = 8.4 mm; P =.036). The mean numbers of metastatic lung nodules in mice (n = 20) 2 weeks after intravenous injection with thymosin beta4-expressing adenovirus and with control adenovirus were 46.7 (95% CI = 35.0 to 57.7) and 10.9 (95% CI = 6.2 to 15.6), respectively (difference = 35.8 metastatic lung nodules, P<.001). Thymosin beta4 overexpression was associated with a mean 2.3-fold increase (95% CI = 1.9- to 2.7-fold increase; P<.001) in B16-F10 cell migration and a mean 4.4-fold increase (95% CI = 3.3- to 5.5-fold increase; P<.001) in the number of blood vessels in solid tumors derived from injected B16-F10 cells but had no effect on cell invasion, proliferation, or matrix metalloproteinase activity. This induction of angiogenesis by thymosin beta4 was associated with induction of vascular endothelial growth factor expression.. Thymosin beta4 may stimulate tumor metastasis by activating cell migration and angiogenesis.

Topics: Adenoviridae; Animals; Cell Line, Tumor; Cell Movement; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Lung Neoplasms; Melanoma, Experimental; Mice; Neoplasm Metastasis; Neovascularization, Pathologic; Recombinant Proteins; Thymosin; Up-Regulation

Topics: Animals; Fibronectins; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Mice; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; ras Proteins; rho GTP-Binding Proteins; rhoC GTP-Binding Protein; Thymosin; Tumor Cells, Cultured; Up-Regulation

The most damaging change during cancer progression is the switch from a locally growing tumour to a metastatic killer. This switch is believed to involve numerous alterations that allow tumour cells to complete the complex series of events needed for metastasis. Relatively few genes have been implicated in these events. Here we use an in vivo selection scheme to select highly metastatic melanoma cells. By analysing these cells on DNA arrays, we define a pattern of gene expression that correlates with progression to a metastatic phenotype. In particular, we show enhanced expression of several genes involved in extracellular matrix assembly and of a second set of genes that regulate, either directly or indirectly, the actin-based cytoskeleton. One of these, the small GTPase RhoC, enhances metastasis when overexpressed, whereas a dominant-negative Rho inhibits metastasis. Analysis of the phenotype of cells expressing dominant-negative Rho or RhoC indicates that RhoC is important in tumour cell invasion. The genomic approach allows us to identify families of genes involved in a process, not just single genes, and can indicate which molecular and cellular events might be important in complex biological processes such as metastasis.

Topics: Animals; Fibronectins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Transfer Techniques; Humans; Lung Neoplasms; Melanoma; Mice; Mice, Inbred C57BL; Mice, Nude; Mutation; Neoplasm Metastasis; Neoplasm Transplantation; Oligonucleotide Array Sequence Analysis; ras Proteins; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; rhoC GTP-Binding Protein; Thymosin; Tumor Cells, Cultured

We previously isolated thymosin beta15 from highly metastatic Dunning rat prostatic carcinoma cells. Immunohistochemical study of human prostate cancer specimens revealed a general correlation between Gleason grade and thymosin beta15 expression, with high-grade (more malignant) tumors showing increased staining compared to low-grade tumors. To determine whether thymosin beta15 may be differentially expressed in cancer cells with different metastatic potential other than in the prostatic carcinoma cells, we examined thymosin beta15 mRNA levels in tumor cell lines from different species. We also examined thymosin beta15 protein levels in human breast cancer samples. Thymosin beta15 was upregulated in the highly metastatic mouse lung and human breast cancer cell lines in comparison to the nonmetastatic counterparts. Immunohistochemical staining showed the evidence of upregulation of thymosin beta15 in malignant human breast carcinomas as compared to benign breast tumors. The expression of thymosin beta15 was correlated with the metastatic potential of the mouse lung carcinoma and human breast carcinoma cells in addition to the prostatic carcinomas. Thymosin beta15 may be a useful marker to predict metastatic potential of certain human cancers.

Topics: Animals; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Mice; Neoplasm Metastasis; Prostatic Neoplasms; Rats; RNA, Messenger; RNA, Neoplasm; Thymosin; Tumor Cells, Cultured

Topics: Animals; Biomarkers, Tumor; Cell Movement; Humans; Male; Neoplasm Metastasis; Prostatic Neoplasms; Thymosin

The Dunning rat prostatic carcinoma is a model system where cell motility closely correlates with the metastatic phenotype. We have identified a novel gene, upregulated in the highly motile and metastatic Dunning cancer cell lines, that represents a new member of the thymosin-beta family, thymosin beta 15. Transfection of antisense thymosin beta 15 constructs into rat prostatic carcinoma cells demonstrates that this molecule positively regulates cell motility, a critical component of the metastatic pathway. Thymosin beta 15 levels are elevated in human prostate cancer and correlate positively with the Gleason tumor grade. Thymosin beta 15 may represent a potential new biochemical marker for human prostate cancer progression.

Topics: Actins; Amino Acid Sequence; Animals; Base Sequence; Biomarkers, Tumor; Carcinoma; Cell Movement; Cloning, Molecular; Gene Expression Regulation, Neoplastic; Humans; Hyperplasia; Male; Molecular Sequence Data; Neoplasm Metastasis; Organ Specificity; Prostate; Prostatic Neoplasms; Rats; Recombinant Fusion Proteins; RNA, Antisense; RNA, Messenger; RNA, Neoplasm; Thymosin; Tumor Cells, Cultured

Topics: Aged; Carcinoma, Renal Cell; Drug Evaluation; Humans; Hyperglycemia; Kidney Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Thymosin

Topics: Adjuvants, Immunologic; Animals; In Vitro Techniques; Lymphocyte Culture Test, Mixed; Mice; Neoplasm Metastasis; Neoplasms, Experimental; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Thymosin

In this report we describe the characterization of the immunomodulatory efficiency and therapeutic properties of thymosin fraction five (F5). We consistently observed the immunostimulation of T-cell activity in assays of allogeneic mixed lymphocyte response (MLR) and the development of cytotoxic effector cells in an allogeneic mixed lymphocyte tumor response-cell-mediated cytotoxicity assay (MLTR-CMC). No induction of suppressor cell activity was observed. Thymosin F5 also acted successfully as an adjuvant when admixed with irradiated tumor cells. We were unable to demonstrate either NK cell or macrophage activation by thymosin F5. Therapeutic protocols using thymosin F5 and directed against pre-existing experimental and spontaneous metastases, had a significant immunotherapeutic potential.

Topics: Animals; Cell Line; Fibrosarcoma; Immunotherapy; Leukemia, Experimental; Lymphocyte Culture Test, Mixed; Male; Melanoma; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasms, Radiation-Induced; T-Lymphocytes; Thymosin; Ultraviolet Rays

Thymosin, a product of the endocrine system, was used to further define the effects of immunomodulation of metastasis. Adult thymectomized C57BL/6 mice, 4 wk post-irradiation (400 R) had a decrease in the number of pulmonary metastasis (compared to controls) following tail vein injection of 5 X 10(4) B16 melanoma cells. Thymosin fraction 5 (fr. 5) administration (200 micrograms/mouse, 3 times weekly beginning 2 days post-thymectomy) returned the number of metastasis to the nonthymectomized values. Thymosin treatment of sham-operated, sham-operated irradiated, or thymectomized nonirradiated mice did not significantly elevate the number of metastases compared to the respective controls. Variant tumors which have an increase in metastasis following thymectomy and irradiation were also used. Thymosin administration reversed the effects of thymectomy in such variants, resulting in a decrease in metastasis. Metastases in thymosin-treated control mice were not significantly altered. A role for the thymus in metastasis via an endocrine product (thymosin) is suggested by these studies. Since thymosin did not increase metastasis in intact mice with tumors, further clinical trials with thymosin in cancer patients are not counterindicated by our results. These experiments confirm that thymosin fr. 5 is an important probe of the immunoendocrine events involved in tumor growth and metastasis.

Topics: Animals; Immunity; Melanoma; Mice; Neoplasm Metastasis; Thymectomy; Thymosin; Thymus Gland

One hundred and fifty-six patients were screened for the presence of urinary melanoma antigen and serum cytoplasmic antibody. It was found that 44% of symptomless Stage 1 patients tested five to 15 years after operation had detectable antigen (Ag) in their urine; the urines of 67% of Stage 2A (local recurrence) patients were positive for Ag; while in only 38% of those patients graded 2B (lymph-node involvement) were these tests positive. Urines of 83% of patients with generalized metastases (Stage 3) were positive. A sequential study was made of 23 patients seen and treated in 1976. Of this group, 14 reverted from a positive state to a negative one following excision of their tumour, while six were negative on first postoperative testing and subsequently became positive. Three out of the 23 remained persistently negative. T lymphocyte levels were assessed in 71 melanoma patients, and a stage-related fall was noticed. Thymosin (Hoffman LaRoche) on in vitro incubation significantly raised the levels of T lymphocytes.

Topics: Adult; Aged; Antigens, Neoplasm; Child, Preschool; Female; Humans; In Vitro Techniques; Male; Melanoma; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Skin Neoplasms; Stimulation, Chemical; T-Lymphocytes; Thymosin; Thymus Hormones

A Phase I clinical trial of thymosin administered in doses of 10 to 250 mg/M2 intramuscularly for seven days was undertaken in ten patients with disseminated malignancies and evidence of immunoincompetence. Toxicity was minimal; one patient experienced a mild urticarial rash which cleared spontaneously, two patients developed low grade fever and one patient experienced pain at the injection site. There was no evidence of systemic toxicity or parenchymal organ dysfunction. Thymosin administration was associated with an increase in the E-rosette forming capacity of the patient's lymphocytes and the development of new skin test reactivity to recall antigens in some of these patients. One objective tumor response was noted. These findings are preliminary but are encouraging for further utilization of thymosin as an immunostimulant in cancer patients with immunoincompetence.

Topics: Adenocarcinoma; Breast Neoplasms; Drug Eruptions; Dysgerminoma; Hodgkin Disease; Humans; Immune Adherence Reaction; Immunity, Cellular; Leiomyosarcoma; Lung Neoplasms; Lymphocytes; Melanoma; Neoplasm Metastasis; Neoplasms; Skin Tests; Thymosin; Thymus Hormones