thymosin-beta(4) and Stomach-Neoplasms

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

Despite major advances in its diagnosis and treatment, gastric cancer (GC) remains a major life-threatening disease. Treatment of the disease is further aggravated by the lack of diagnostic biomarkers that can aid in the early detection of GC and promote its favorable prognosis. The present work aims to identify novel diagnostic biomarkers for GC.. The present work is a case-control study that focuses on proteomic analysis of serum from healthy volunteers and GC patients using ClinProt profiling technology based on mass spectrometry. A pattern of proteins/peptides with the ability to differentiate the studied populations was identified. Deregulated proteins/peptides differentially expressed in the serum of patients compared with healthy volunteers were identified by mass spectroscopy.. A pattern of proteins/peptides consisting of four protein/peptide peaks at m/z 1467, 1867, 2701, and 2094 was identified. These protein/peptide peaks were able to differentiate the studied populations with close to 100% sensitivity and specificity. Three of the deregulated proteins/peptides at m/z 1867, 2701, and 2094 were identified by mass spectroscopy (LTQ Orbitrap XL) as tubulin beta chain, thymosin beta-4-like protein 3, and cytochrome b-c₁ complex subunit 1, respectively.. The pattern of proteins/peptides identified in the present work shows great potential for GC diagnosis. Deregulated proteins of tubulin beta chain, thymosin beta-4-like protein 3, and cytochrome b-c₁ complex subunit 1 may be involved in the pathogenesis of GC and serve as potential serological diagnostic biomarkers.

Topics: Adenoma; Aged; Biomarkers, Tumor; Carrier Proteins; Case-Control Studies; Female; Gene Expression; Humans; Male; Mass Spectrometry; Middle Aged; Protein Isoforms; Sensitivity and Specificity; Stomach Neoplasms; Thymosin; Tubulin

Thymosin β4 (Tβ4), a small acidic actin binding peptide, is overexpressed in a side population of cancer stem cells and CD133-positive colorectal cancer stem cells. In order to understand the relationship between Tβ4 and CD133, we studied the expression patterns of Tβ4 and CD133 in ovarian cancers. The expression patterns of Tβ4 and CD133 were studied in normal ovaries, primary ovarian cancers, metastatic ovarian cancers, primary stomach cancers, and normal stomachs by Western blot and immunohistochemistry. Expression patterns and co-localization of Tβ4 and CD133 were examined by immunofluorescence and confocal laser-scanning microscopy. Tβ4 is overexpressed in primary ovarian cancers, but not in primary stomach cancers, when compared with normal controls. However, Tβ4 levels in metastatic stomach cancers to the ovary are significantly upregulated compared with levels in normal stomachs and primary stomach cancers. These results suggest that Tβ4 levels are related to tumorigenesis in ovarian cancers and metastasis in stomach cancers. The expression of Tβ4 in normal ovaries and normal stomachs was weak, but was co-localized with CD133 expression. Tβ4 expression was also co-localized with CD133 expression in primary ovarian carcinomas, metastatic ovarian cancers from stomach cancers and primary stomach cancers. These data suggest that Tβ4 expression is strongly related to CD133 expression and is a characteristic of stem cells or cancer stem cells.

Topics: AC133 Antigen; Antigens, CD; Biomarkers, Tumor; Female; Glycoproteins; Humans; Immunohistochemistry; Neoplastic Stem Cells; Ovarian Neoplasms; Peptides; Stomach Neoplasms; Thymosin; Up-Regulation

Thymosin beta-4 (Tβ4), actin-sequestering protein, plays important roles in many cellular functions including cancer cell migrations. Glycogen synthase kinase (GSK) in Wnt signaling pathway is a key molecule to control intercellular interaction. Here, we investigated whether GSK-3 activity is regulated by Tβ4 and it is associated with Tβ4-mediated migration in gastric cancer cells. Various expression level of Tβ4 was observed in human gastric tumor tissues. Migration in gastric cancer cells, SNU638 and SNU668, was dependent on a relative expression level of Tβ4. Cell migration was higher in SNU668 with a higher expression level of Tβ4 than that in SNU638 with a lower Tβ4. Although the level of phosphorylated(p)-GSK-3α (inactive), β-catenin, E-cadherin and E-cadherin:β-catenin complex was relatively higher, p-GSK-3β (inactive) was lower in SNU638 compared to those in SNU668 cells. LiCl, GSK-3α/β inhibitor, reduced lung metastasis of B16F10 mouse melanoma cells and SNU668 cell migration. Small interference (si)RNA of GSK-3α increased SNU638 cell migration in accordance with the reduction of E-cadherin:β-catenin complex formation through a decrease in β-catenin and E-cadherin. Expression level of GSK-3α/β, β-catenin and E-cadherin in SNU668 and SNU638 was reversed by Tβ4-siRNA and by the treatment with acetylated-serine-aspartic acid-lysine-proline (SDKP) tetrapeptide of Tβ4, respectively. E-cadherin expression in SNU638 cells was decreased by β-catenin-siRNA. PD98059, MEK inhibitor, or U0126, ERK inhibitor, reduced SNU668 cell migration accompanying an increase in p-GSK-3α, β-catenin and E-cadherin. Taken together, data indicated that the expression of GSK-3α, β-catenin and E-cadherin could be negatively regulated by Tβ4-induced ERK phosphorylation. It suggests that Tβ4 could be a novel regulator to control Wnt signaling pathways.

Topics: Animals; beta Catenin; Butadienes; Cadherins; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line, Tumor; Cell Movement; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Flavonoids; Glycogen Synthase Kinase 3; Humans; Lithium Chloride; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Nitriles; Peptides; Phosphorylation; RNA Interference; RNA, Small Interfering; Stomach Neoplasms; Thymosin; Wnt Signaling Pathway