thymosin-beta(4) and Colorectal-Neoplasms

Colorectal cancer (CRC) is a complicated tumor, involving several oncogenic signaling pathways, and with a molecular mechanism not fully understood yet. The implication of thymosin β4 (Tβ4) with tumor insurgence and in migration of CRC cells was evidenced in the past with different methodologies, while Tβ10 connection with CRC has been sporadically investigated. This study focused on the implication of both types of thymosin in CRC progression and invasion by analyzing the changes in their levels according to different zones of the tumor, and to Dukes stage and budding index.. Tβ4 and Tβ10 were analyzed in deep and superficial tumor samples, and normal mucosa from 18 patients. Concentrations of Tβ4 and Tβ10 have been measured by high-pressure liquid chromatography (HPLC) coupled to electrospray-ion trap mass spectrometry (ESI-IT-MS). MS data were compared by t-test and ANOVA statistical analysis. Identification of thymosin and their proteoforms has been performed by HPLC-high resolution-ESI-IT-MSMS.. Both Tβ4 and Tβ10, exhibited intra-tumoral quantitative differences, being upregulated in the deep part of the CRC. They exhibited, moreover, strong association with the Dukes stage and the budding grade, being more concentrated in patients at Dukes stage B and with budding index "2".. The results obtained in the present investigation encouraged the hypothesis that the two thymosin are involved in colorectal cancer progression, and in promoting cancer invasion. Thus, they are good candidates to be diagnostic/prognostic biomarkers and therapy targets.

Topics: Aged; Aged, 80 and over; Chromatography, High Pressure Liquid; Colorectal Neoplasms; Disease Progression; Female; Humans; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Prognosis; Signal Transduction; Spectrometry, Mass, Electrospray Ionization; Thymosin

DNA aneuploidy has been identified as a prognostic factor for epithelial malignancies. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for direct analysis of multiple proteins in tissue sections while maintaining the cellular and molecular integrity. We compared diploid and aneuploid colon cancer tissues against normal mucosa of the colon by means of IMS. DNA image cytometry determined the ploidy status of tissue samples that were subsequently subjected to MALDI-IMS. After obtaining protein profiles through direct analysis of tissue sections, a discovery and independent validation set were used to predict ploidy status by applying proteomic classification algorithms [Supervised Neural Network (SNN) and Receiver Operating Characteristic (ROC)]. Five peaks (m/z 2,395 and 4,977 for diploid vs. aneuploid comparison as well as m/z 3,376, 6,663, and 8,581 for normal mucosa vs. carcinoma comparison) were significant in both SNN and ROC analysis. Among these, m/z 4,977 was identified as thymosin beta 4 (Tβ-4). Tβ-4 was subsequently validated in clinical samples using a tissue microarray to predict overall survival in colon cancer patients.

Topics: Aged; Algorithms; Area Under Curve; Biomarkers, Tumor; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Male; Middle Aged; Ploidies; Prognosis; ROC Curve; Sensitivity and Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thymosin; Tissue Array Analysis

Topics: AC133 Antigen; Adult; Aged; Antigens, CD; Biomarkers, Tumor; Colorectal Neoplasms; Female; Glycoproteins; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Recurrence, Local; Peptides; Thymosin; Tissue Array Analysis; Up-Regulation

Thymosin beta-4 (Tβ4) is known to be involved in tumorigenesis. Overexpression of this polypeptide has been observed in a wide variety of cancers, including colorectal carcinoma (CRC). Accordingly, Tβ4 has been proposed to be a novel therapeutic target for CRC, especially in its metastatic form. Although in vitro tumor-suppressive effects of Tβ4 gene silencing mediated by small hairpin RNA (shRNA) have already been demonstrated, the in vivo efficacy of such an approach has not yet been reported. Herein, we demonstrated that infection with recombinant adenovirus expressing an shRNA targeting Tβ4 markedly reduced the growth of and robustly induced apoptosis in CT-26 mouse CRC cells in culture. Additionally, tumors grown in nude mice from the CT-26 cells whose Tβ4 expression already been downregulated by virus infection were also drastically reduced. Most importantly, significant growth arrest of tumors derived from the parental CT-26 cells was observed after multiple intratumoral injections of these viruses. Together, our results show for the first time that in vivo silencing of Tβ4 expression by its shRNA generated after adenoviral infection can suppress CRC growth. These results further demonstrate the feasibility of treating CRC by a Tβ4 knockdown gene therapeutic approach.

Topics: Actins; Adenoviridae; Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Transformation, Neoplastic; Colorectal Neoplasms; Disease Models, Animal; Gene Knockout Techniques; Genetic Vectors; Humans; Mice; RNA Interference; RNA, Messenger; RNA, Small Interfering; Thymosin; Transduction, Genetic; Tumor Burden; Xenograft Model Antitumor Assays

Thymosin β4 (Tβ4) is a 43-amino-acid peptide involved in many biological processes. However, the precise molecular signaling mechanism(s) of Tβ4 in cell invasion and migration remain unclear. In this study, we show that Tβ4 was significantly overexpressed in colorectal cancer tissues compared to adjacent normal tissues and high levels of Tβ4 were correlated with stage of colorectal cancer, and that Tβ4 expression was associated with morphogenesis and EMT. Tβ4-upregulated cancer cells showed increased adhesion, invasion and migration activity, whereas Tβ4-downregulated cells showed decreased activities. We also demonstrated that Tβ4 interacts with ILK, which promoted the phosphorylation and activation of AKT, the phosphorylation and inactivation of GSK3β, the expression and nuclear localization of β-catenin, and integrin receptor activation. These results suggest that Tβ4 is an important regulator of the ILK/AKT/β-catenin/Integrin signaling cascade to induce cell invasion and migration in colorectal cancer cells, and is a potential target for cancer treatment.

Topics: Aged; beta Catenin; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Integrins; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Thymosin; Tumor Microenvironment

Thymosin β 4 (Tβ(4)) is a ubiquitous peptide that plays pivotal roles in the cytoskeletal system and in cell differentiation during embryogenesis. Recently, a role for Tβ(4) has been proposed in experimental and human carcinogenesis. This study was aimed at evaluating the correlation between Tβ(4) immunoractivity and colorectal cancer, with particular attemption to tumor cells undergoing epithelial-mesenchymal transition.. 86 intestinal biopsies were retrospectively analyzed including 76 colorectal adenocarcinomas with evident features of epithelial-mesenchymal transition, and 10 samples of normal colorectal mucosa. Paraffin sections were immunostained for Tβ(4) and for E-cadherin. Total RNA was isolated from frozen specimens obtained, at surgery, from the normal colon mucosa, the deeper regions and the superficial tumor regions in four cases of colon cancer. Tβ(4) immunoreactivity was detected in the vast majority (59/76) of colon carcinomas, showing a patchy distribution, with well differentiated areas significantly more reactive than the less differentiated tumor zones. We also noted a zonal pattern in the majority of tumors, characterized by a progressive increase in immunostaining for Tβ(4) from the superficial toward the deepest tumor regions. The strongest expression for Tβ(4) was frequently detected in invading tumor cells with features of epithelial-mesenchymal transition. The increase in reactivity for Tβ(4) matched with a progressive decrease in E-cadherin expression in invading cancer cells. At mRNA level, the differences in Tβ(4) expression between the surrounding colon mucosa and the tumors samples were not significant.. Our data show that Tβ(4) is expressed in the majority of colon cancers, with preferential immunoreactivity in deep tumor regions. The preferential expression of the peptide and the increase in intensity of the immunostaining at the invasion front suggests a possible link between the peptide and the process of epithelial mesenchymal transition, suggesting a role for Tβ(4) in colorectal cancer invasion and metastasis.

Topics: Adenocarcinoma; Cadherins; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Cohort Studies; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Immunohistochemistry; Thymosin

Thymosin β4 (Tβ4) is highly expressed in saliva of human newborns but not in adults. Here preliminary immunohistochemical analyses on different human tissues are reported. Immunoreactivity for Tβ4 in human salivary glands show high quantities of Tβ4 before birth, followed by downregulation of expression in adulthood. In contrast, Tβ4 is detected in tumors of salivary glands, suggesting that tumor cells might utilize fetal programs, including Tβ4 synthesis. Immunohistochemical analyses in the gastrointestinal tract showed strong reactivity for Tβ4 in enterocytes during development, but weak immunostaining in mature enterocytes. In colorectal cancer, the association of a high expression of Tβ4 with epithelial-mesenchymal transition was observed. On the basis of these data, the process of epithelial-mesenchymal transition could represent the unifying process that explains the role of Tβ4 during fetal development and in cancer progression.

Topics: Colorectal Neoplasms; Enterocytes; Epithelial-Mesenchymal Transition; Gastrointestinal Tract; Humans; Liver; Salivary Glands; Thymosin

Thymosin β4 has been reported to play the key roles in tumor growth, metastasis, and angiogenesis. Although the importance of thymosin β4 in angiogenesis and metastasis is known, few studies to show the expression patterns of thymosin β4 in human tissues including tumors have been conducted. The comparisons of the expression of thymosin β4 between the normal and tumor tissues are also needed to study the role of thymosin β4 in tumor formation. Using tissue microarray analysis, we compared the expression patterns of thymosin β4 in the normal human tissues and in the tumors to screen certain tumors and upregulated the expression of thymosin β4 by tumorigenesis. Thymosin β4 was highly expressed in the hepatic cells in the normal adult liver, duct, and acinar cells in pancreas, and muscle cells in the heart and also expressed highly in certain tumor cells, including osteosarcoma, colon adenocarcinoma, esophageal squamous cell carcinoma, kidney and urinary bladder transitional carcinoma, lung cancer, and liver cancer. Comparing the thymosin β4 expression between normal and tumors, thymosin β4 was upregulated specifically in osteosarcoma, colorectal carcinoma, and esophageal cancer. To confirm the over-expression of thymosin β4 in these tumors, we analyzed expression of thymosin β4 with each additional microarray of osteosarcoma, colorectal carcinoma, and esophageal cancer. The significant increased expression of thymosin β4 was observed in osteosarcoma and in colorectal cancer. These results suggest that the expression of thymosin β4 is highly related with tumorigenesis of certain tumors including the osteosarcoma and colorectal cancers.

Topics: Adult; Cell Line, Tumor; Colorectal Neoplasms; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Organ Specificity; Osteosarcoma; Thymosin; Tissue Array Analysis; Up-Regulation

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

Overexpression of thymosin beta-4 has been linked to malignant progression but the localization of this polypeptide within tumors is incompletely known. We therefore examined breast cancers for thymosin beta-4 using immunofluorescence. Reactive cells were identified with monoclonal cell marker antibodies. A very heterogeneous staining pattern for thymosin beta-4 was observed. Thus, while leukocytes and macrophages showed intense reactivity for this polypeptide, cancer cells, and endothelial cells showed a much more variable reactivity. A similar heterogeneous staining was observed also in colorectal carcinomas. The degree of staining of breast cancer cells for thymosin beta-4 correlated neither to histological grade nor to endothelial cell staining. However, there was a tendency toward correlation (P = 0.07) between staining of endothelial cells and histological grade. Treatment of cultured breast cancer cells (SK-BR-3) with 1-4 microg thymosin beta-4/mL significantly increased cell numbers, as determined by MTT-assays. These data reveal an unexpected cellular heterogeneity of thymosin beta-4 expression in breast and colonic carcinomas and suggest that local release of this polypeptide in the tumor microenvironment may modulate tumor behavior.

Topics: Breast Neoplasms; Cell Nucleus; Colorectal Neoplasms; Cytoplasm; Female; Fluorescent Antibody Technique; Humans; Keratins; Leukocytes; Thymosin

For hMLH1, a key enzyme of DNA mismatch repair and frequently mutated in human cancers, several additional functions have been suggested. We now identified Thymosin beta4 (Tbeta4), an actin-binding and cell motility regulating protein, by bacterial two-hybrid screening. Interaction was confirmed by coimmunoprecipitation. Tbeta4 was weakly expressed in the hMLH1-deficient cell lines 293T and HCT-116. Reconstitution of hMLH1 resulted in strong expression of Tbeta4. Confocal laser microscopy revealed nuclear colocalization of both proteins. Reconstitution with hMLH1 mutants lacking a functional nuclear localization sequence resulted in cytoplasmatic retention of both proteins. After Tbeta4- or hMLH1-siRNA treatment, cell migration of hMLH1-proficient cells was markedly decreased. Our results show that hMLH1 interacts with Tbeta4 and regulates its expression and nuclear transport. Moreover, loss of hMLH1 causes Tbeta4 deprivation and results in reduced migratory activity in vitro. These data give insight into novel functions of hMLH1 and probably disease related dysregulated mechanisms.

Topics: Active Transport, Cell Nucleus; Adaptor Proteins, Signal Transducing; Cell Line; Colorectal Neoplasms; DNA Repair; Gene Expression Regulation; Humans; Kidney; MutL Protein Homolog 1; Nuclear Proteins; 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

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for their anti-inflammatory effects and have been shown to have chemopreventive effects as well. NSAIDs inhibit cyclooxygenase (COX) activity to exert their anti-inflammatory effects, but it is not clear whether their antitumorigenic ability is through COX inhibition. Using subtractive hybridization, we previously identified a novel member of the transforming growth factor-beta superfamily that has antitumorigenic activity from indomethacin-treated HCT-116 human colorectal cancer cells. On further investigation of this library, we now report the identification of a new cDNA corresponding to the thymosin beta-4 gene. Thymosin beta-4 is a small peptide that is known for its actin-sequestering function, and it is associated with the induction of angiogenesis, accelerated wound healing, and metastatic potential of tumor cells. However, only selective NSAIDs induce thymosin beta-4 expression in a time- and concentration-dependent manner. For example, indomethacin and SC-560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole] induce thymosin beta-4 expression whereas sulindac sulfide does not. We show that selective NSAIDs induce actin cytoskeletal reorganization, a precursory step to many dynamic processes regulating growth and motility including tumorigenesis. This is the first report to link thymosin beta-4 induction with NSAIDs. These data suggest that NSAIDs alter the expression of a diverse number of genes and provide new insights into the chemopreventive and biological activity of these drugs.

Topics: Actins; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Northern; Cell Line, Tumor; Chromatography, High Pressure Liquid; Colorectal Neoplasms; Cytokines; Cytoskeleton; Dinoprostone; Dose-Response Relationship, Drug; Fluorescent Antibody Technique; Growth Differentiation Factor 15; Humans; Indicators and Reagents; Indomethacin; Prostaglandin-Endoperoxide Synthases; Pyrazoles; RNA, Neoplasm; Thymosin

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