thymosin-beta(4) and Colonic-Neoplasms

Aberrant expression of thymosin beta4 (Tbeta4) has recently been found to be associated with colorectal carcinoma (CRC) progression evidently due to an increase of the motility and invasion of tumor cells and the induction of a proangiogenic phenotype of endothelial cells. Both mechanisms depend upon matrix-degrading proteases, particularly plasmin and matrix metalloproteinases (MMPs) that are responsible for extensive tissue remodeling. Cleavage of ECM macromolecules weakens the structural integrity of tissues and exposes cryptic domains of extracellular components, which elicit biological responses distinct from intact molecules. Interestingly, signaling via integrins (alphaVbeta3, alpha5beta1) in CRC cells (HT29, CX1.1) is induced by Tbeta4 and VEGF-A only when they grow in 3D fibrin gels but not in 2D ones. The cells growing in 3D fibrin gels release upon Tbeta4 significant amounts of active MMPs (MMP-2, MMP-9, and MMP-7) that cause extensive proteolysis in their close vicinity. As evidenced by a variety of approaches (transfection experiments, coimmunoprecipitation, gene silencing with siRNA), we found that this involves interaction of Tbeta4 with Ku80, which has recently been described by us to mediate Tbeta4 intracellular activity.

Topics: Cell Movement; Cells; Colonic Neoplasms; Endothelial Cells; Extracellular Matrix; Humans; Integrins; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinases; Neoplasms; RNA, Small Interfering; Signal Transduction; Thymosin; Vascular Endothelial Growth Factor A

Thymosin beta 4 (Tβ4) is a ubiquitous peptide that plays pivotal roles in the cytoskeletal system and in cell differentiation. Recently, a role for Tβ4 has been proposed in experimental and human carcinogenesis, including gastrointestinal cancer. This study was aimed at evaluating the relationship between Tβ4 immunoreactivity and the initial steps of carcinogenesis.. In total, 60 intestinal biopsies, including 10 hyperplastic polyps, 10 sessile serrated adenomas/polyps, 15 colorectal adenomas with low-grade dysplasia, 15 adenomas with high-grade dysplasia, 15 adenocarcinomas and 10 samples of normal colon mucosa, were analyzed for Tβ4 expression by immunohistochemistry.. Weak cytoplasmic reactivity for Tβ4 was detected in the normal colon mucosa. No reactivity for Tβ4 was found in hyperplastic and sessile serrated polyps/adenomas. Tβ4 expression was observed in 10/15 colorectal adenocarcinomas. In adenomas with low-grade dysplasia, Tβ4 immunoreactivity was mainly detected in dysplastic glands but was absent in hyperplastic glands. Tβ4 immunoreactivity was characterized by spot-like perinuclear staining. In high-grade dysplastic polyps, immunostaining for Tβ4 appeared diffuse throughout the entire cytoplasm of dysplastic cells. Spot-like perinuclear reactivity was detected in adenocarcinoma tumor cells.. Our study shows for the first time that Tβ4 is expressed during different steps of colon carcinogenesis. The shift of Tβ4 immunolocalization from low-grade to high-grade dysplastic glands suggests a role for Tβ4 in colorectal carcinogenesis. However, the real meaning of Tβ4 reactivity in dysplastic intestinal epithelium remains unknown.

Topics: Adenoma; Biopsy; Cell Differentiation; Colon; Colonic Neoplasms; Colonic Polyps; Disease Progression; Female; Humans; Immunohistochemistry; Male; Neoplasm Proteins; Thymosin

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

Thymosin β(4) (Tβ(4)), overexpressed in various tumors, has been shown to be involved in cellular anti-oxidation. Reactive oxygen species (ROS) function as signaling molecules and play certain roles in tumor progression. To assess the anti-oxidative role of endogenous Tβ(4) in tumor cells, its expression in SW480 cells was knocked down by a shRNA, which induced significant increases of ROS. Interestingly, some cristae-lost and several electron-dense mitochondria appeared in cells with Tβ(4) knockdown that was accompanied by a marked decline of the membrane potential of these organelles. Strikingly, while the ATP and lactate levels in SW480 cells were notably elevated by Tβ(4) downregulation, this treatment significantly diminished the mitochondrial DNA copy number and protein levels of several subunits of the electron transport complexes. Finally, immunofluorescent staining results suggested the presence of Tβ(4) in mitochondria. To the best of our knowledge, this is the first report to demonstrate that Tβ(4) knockdown can disrupt the morphology and some crucial functions of mitochondria in human colorectal carcinoma (CRC) cells.

Topics: Cell Line, Tumor; Colonic Neoplasms; Gene Knockdown Techniques; Glycolysis; Humans; Mitochondria; Reactive Oxygen Species; Thymosin

Thymosin β4 (Tβ4) is an actin-binding peptide overexpressed in several tumors, including colon carcinomas. The aim of this study was to investigate the role of Tβ4 in promoting the tumorigenic properties of colorectal cancer stem cells (CR-CSCs), which are responsible for tumor initiation and growth. We first found that CR-CSCs from different patients have higher Tβ4 levels than normal epithelial cells. Then, we used a lentiviral strategy to down-regulate Tβ4 expression in CR-CSCs and analyzed the effects of such modulation on proliferation, survival, and tumorigenic activity of CR-CSCs. Empty vector-transduced CR-CSCs were used as a control. Targeting of the Tβ4 produced CR-CSCs with a lower capacity to grow and migrate in culture and, interestingly, reduced tumor size and aggressiveness of CR-CSC-based xenografts in mice. Moreover, such loss in tumorigenic activity was accompanied by a significant increase of phosphatase and tensin homologue (PTEN) and a concomitant reduction of the integrin-linked kinase (ILK) expression, which resulted in a decreased activation of protein kinase B (Akt). Accordingly, exogenous expression of an active form of Akt rescued all the protumoral features lost after Tβ4 targeting in CR-CSCs. In conclusion, Tβ4 may have important implications for therapeutic intervention for treatment of human colon carcinoma.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cells, Cultured; Colonic Neoplasms; Down-Regulation; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Lentivirus; Mice; Mice, SCID; Neoplastic Stem Cells; Oncogene Protein v-akt; Protein Serine-Threonine Kinases; PTEN Phosphohydrolase; Thymosin

Thymosin β4 has multi-functional roles in cell physiology, but little is known about its mechanism(s) of action. We previously reported that thymosin β4 stimulated angiogenesis through the induction of vascular endothelial growth factor (VEGF). To identify the mechanism of VEGF induction by thymosin β4, we have used a luciferase assay system with VEGF in the 5' promoter region. We also analyzed the effect of thymosin β4 on VEGF mRNA stability and on the expression and stability of hypoxia-inducible factor (HIF)-1α. We found that thymosin β4 induces VEGF expression by an increase in the stability of HIF-1α protein. Analysis of the expression patterns of thymosin β4 and HIF-1α in colon cancer tissue microarray showed that thymosin β4 and HIF-1α co-localized in these biopsies. These data show that thymosin β4 induces the expression of VEGF indirectly by increasing the protein stability of HIF-1α.

Topics: Animals; Biopsy; Blotting, Western; Cell Line, Tumor; Colon; Colonic Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Luciferases; Male; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Promoter Regions, Genetic; Protein Stability; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thymosin; Tissue Array Analysis; Vascular Endothelial Growth Factor A

The epithelial-mesenchymal transition (EMT) is crucial for the invasion and metastasis of many epithelial tumors including colorectal carcinoma (CRC). In the present study, a scattering and fibroblastic morphology with reduced intercellular contacts was found in the SW480 colon cancer cells overexpressing the gene encoding thymosin beta4 (Tbeta4), which was accompanied by a loss of E-cadherin as well as a cytosolic accumulation of beta-catenin, two most prominent markers of EMT. Whereas E-cadherin downregulation was likely to be accounted by a ZEB1-mediated transcriptional repression, the accumulation of beta-catenin was a result of glycogen synthase kinase-3beta inactivation mediated by integrin-linked kinase (ILK) and/or its downstream effector, Akt. Intriguingly, ILK upregulation in Tbeta4-overexpressing SW480 cells seemed to be attributed mainly to a stabilization of this kinase by complexing with particularly interesting new Cys-His protein (PINCH) more efficiently. In the meantime, a strong correlation between the expression levels of Tbeta4, ILK and E-cadherin in CRC patients was also revealed by immunohistochemical analysis. Taken together, these data suggest a novel role of Tbeta4 in promoting CRC progression by inducing an EMT in tumor cells via upregulating ILK and consequentially its signal transduction.

Topics: Adaptor Proteins, Signal Transducing; Animals; beta Catenin; Cadherins; Casein Kinase I; Colonic Neoplasms; Colorectal Neoplasms; DNA-Binding Proteins; Enzyme Inhibitors; Epithelial Cells; Fibroblasts; Glycogen Synthase Kinase 3; Homeodomain Proteins; Humans; Immunoenzyme Techniques; LIM Domain Proteins; Membrane Proteins; Mesoderm; Mice; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Antisense; RNA, Messenger; Signal Transduction; Thymosin; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Up-Regulation; Zinc Finger E-box-Binding Homeobox 1

Thymosin beta-4 (Tbeta-4), a small peptide originally isolated from calf thymus, modulates the formation of F-actin microfilaments by sequestering the monomeric G-actin. Recent studies have shown that overexpression of the Tbeta-4 gene occurs not only in many human carcinomas but also in the highly metastatic melanomas and fibrosarcomas. However, little is known about the specific growth advantages acquired by different tumors from this genetic abnormality. To address the above questions, Tbeta-4-overexpressing human colon carcinoma (SW480) cells were established by stable transfection and their phenotypic changes were monitored. We found that both the morphology and the cortical actin cytoskeleton of SW480 cells were altered by Tbeta-4 overexpression. Moreover, both cellular level and that distributed over the intercellular junctions of the E-cadherin were decreased in the Tbeta-4 overexpressers, which were accompanied by a twofold increase in their saturation densities. Meanwhile, these cells also exhibited an increased ability to form colonies in soft agar. Interestingly, a dramatic increase of growth rate was detected in the Tbeta-4 overexpressers, which might be attributed to an accelerated proliferation induced by c-Myc that was activated by nuclear beta-catenin. Finally, a motility increase of these cells was demonstrated by two independent migration assays, which was accompanied by an enhanced focal contact. Taken together, our data suggest that the drastic growth property and motility changes of the SW480 cells overexpressing Tbeta-4 gene are due mainly to a deregulated cell-cell adhesion arisen from the downregulation of E-cadherin, plus uncontrolled cell proliferation owing to the upregulation of beta-catenin, both resulted from a breakdown of actin microfilaments caused by the overexpression of this G-actin sequestering peptide.

Topics: beta Catenin; Cadherins; Carcinoma; Cell Division; Cell Movement; Colonic Neoplasms; Cytoskeletal Proteins; Disease Progression; Focal Adhesions; Humans; Proto-Oncogene Proteins c-myc; Thymosin; Trans-Activators; Transfection; Tumor Cells, Cultured; Tumor Stem Cell Assay

We constructed a "non-metastatic cell (SW837)--metastatic cell (PMCO1)" subtraction library and identified one cDNA that was strongly expressed in SW837 but weakly expressed in PMCO1. The nucleotide sequence of the cDNA revealed that it encoded thymosin beta-4. Four non-metastatic cell lines, which produced no experimental liver metastasis in nude mice, showed high expression of thymosin beta-4. However three of four metastatic cell lines showed weak expression of thymosin beta-4. Among surgical materials, thymosin beta-4 expression was high in tumors without metastasis in comparison with non-tumorous mucosa, but one case with liver metastasis showed decreased expression in both the primary and metastatic tumors. We suspect that down-regulation of thymosin beta-4 expression is correlated with the metastatic capacity of colorectal carcinomas.

Topics: Animals; Colonic Neoplasms; Colorectal Neoplasms; DNA, Neoplasm; Gene Expression; Gene Library; Humans; Mice; Mice, Nude; Neoplasm Transplantation; RNA, Messenger; Thymosin; Transplantation, Heterologous; Tumor Cells, Cultured