192343

NEWENTRY

Record to support submission of GeneRIFs for a gene not in Gene (human; man).;-;-

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INTRODUCTION: Acquisition of mesenchymal characteristics confers to breast cancer (BC) cells the capability of invading tissues different from primary tumor site, allowing cell migration and metastasis. Regulators of the mesenchymal-epithelial transition (MET) may represent targets for anticancer agents. Accruing evidence supports functional implications of choline phospholipid metabolism in oncogene-activated cell signaling and differentiation. We investigated the effects of D609, a xanthate inhibiting phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelin synthase (SMS), as a candidate regulator of cell differentiation and MET in the highly metastatic BC cell line MDA-MB-231. METHODS: PC-PLC expression and activity were investigated using confocal laser scanning microscopy (CLSM), immunoblotting and enzymatic assay on human MDA-MB-231 compared with MCF-7 and SKBr3 BC cells and a nontumoral immortalized counterpart (MCF-10A). The effects of D609 on PC-PLC and SMS activity, loss of mesenchymal markers and changes in migration and invasion potential were monitored in MDA-MB-231 cells by enzymatic assays, CLSM, immunoblotting and transwell chamber invasion combined with scanning electron microscopy examinations. Cell proliferation, formation and composition of lipid bodies and cell morphology were investigated in D609-treated BC cells by cell count, CLSM, flow-cytometry of BODIPY-stained cells, nuclear magnetic resonance and thin-layer chromatography. RESULTS: PC-PLC (but not phospholipase D) showed 2- to 6-fold activation in BC compared with nontumoral cells, the highest activity (up to 0.4 pmol/mug protein/min) being detected in the poorly-differentiated MDA-MB-231 cells. Exposure of the latter cells to D609 (50 mug/mL, 24-72 h) resulted into 60-80% PC-PLC inhibition, while SMS was transiently inhibited by a maximum of 21%. These features were associated with progressive decreases of mesenchymal traits such as vimentin and N-cadherin expression, reduced galectin-3 and milk fat globule EGF-factor 8 levels, beta-casein formation and decreased in vitro cell migration and invasion. Moreover, proliferation arrest, changes in cell morphology and formation of cytosolic lipid bodies typical of cell differentiation were induced by D609 in all investigated BC cells. CONCLUSIONS: These results support a critical involvement of PC-PLC in controlling molecular pathways responsible for maintaining a mesenchymal-like phenotype in metastatic BC cells and suggests PC-PLC deactivation as a means to promote BC cell differentiation and possibly enhance the effectiveness of antitumor treatments.

cancer progression is a complex series of events thought to incorporate the reversible developmental process of epithelial-to-mesenchymal transition (EMT). In vitro, the microRNA-200 family maintains the epithelial phenotype by posttranscriptionally inhibiting the E-cadherin repressors, ZEB1 and ZEB2. Here, we used in situ hybridization and immunohistochemistry to assess expression of miR-200 and EMT biomarkers in formalin-fixed paraffin-embedded human colorectal adenocarcinomas. In addition, laser capture microdissection and quantitative real-time polymerase chain reaction were employed to quantify levels of miR-200 in the normal epithelium, tumor core, invasive front, and stroma. We find that miR-200 is downregulated at the invasive front of colorectal adenocarcinomas that have destroyed and invaded beyond the basement membrane. However, regional lymph node metastases and vascular carcinoma deposits show strong expression of miR-200, suggesting this family of miRNAs is involved in the recapitulation of the primary tumor phenotype at metastatic sites. In contrast, adenomas and adenocarcinomas with intact basement membranes showed uniform miR-200 expression from the tumor core to the tumor-host interface. Taken together, these data support the involvement of EMT and mesenchymal-to-epithelial transition (MET) in the metastasis cascade and show that miR-200 is downregulated in the initial stages of stromal invasion but is restored at metastatic sites.

OBJECTIVE: To explore the expression and significance of tumor specific growth factor (TSGF), carcinoembryonic antigen (CEA) and alpha fetoprotein (AFP) in cancer tissue and serum of patients with colon cancer. MATERIALS AND METHODS: Radical surgery for colon cancer was performed on 43 patients with laparoscope under conditions of general anesthesia. The Elisa method was used to detect the levels of serum TSGF, CEA and AFP before and after radical operation, and cancer tissue underwent TSGF, CEA and AFP immunohistochemistry staining after laparoscopic surgery. The decreased conditions of serum TSGF, CEA and AFP in patients with colon cancer at different levels of differentiation and clinical stagings were analyzed, and the relationships of expression rates between histological types, colon cancer morphology, lymph node metastasis and TSGF, CEA as well as AFP in cancer tissue were assessed. RESULTS: Compared with before radical surgery, the levels of serum TSGF, CEA and AFP decreased notably in patients after operations (p<0.01). The decreased degree of TSGF and CEA was the largest in patients with poorly differentiated cancer tissue (p<0.01), while that of AFP was noted in patients with moderately differentiated cancer tissue (p<0.01). The decreased degree of TSGF and AFP was the largest in patients at phase Dukes A (p<0.01), while that of CEA in patients at phase Dukes C (p<0.01). There were no significant differences among the positive expression rates of TSGF, CEA and AFP with different histological types and colon cancer morphologies (p>0.05). The positive expression rates of TSGF and CEA in patients with lymph node metastasis were significantly higher than those without lymph node metastasis (p<0.01). CONCLUSIONS: TSGF, CEA and AFP can be used to evaluate the effect of radical operation for colon cancer, and the changed levels of different markers are associated with tumor differentiation, clinical stating and presence or absence of lymph node metastasis.

cancer stem cells drive tumor formation and metastasis, but how they acquire metastatic traits is not well understood. Here, we show that all colorectal cancer stem cells (CR-CSCs) express CD44v6, which is required for their migration and generation of metastatic tumors. CD44v6 expression is low in primary tumors but demarcated clonogenic CR-CSC populations. Cytokines hepatocyte growth factor (HGF), osteopontin (OPN), and stromal-derived factor 1alpha (SDF-1), secreted from tumor associated cells, increase CD44v6 expression in CR-CSCs by activating the Wnt/beta-catenin pathway, which promotes migration and metastasis. CD44v6(-) progenitor cells do not give rise to metastatic lesions but, when treated with cytokines, acquire CD44v6 expression and metastatic capacity. Importantly, phosphatidylinositol 3-kinase (PI3K) inhibition selectively killed CD44v6 CR-CSCs and reduced metastatic growth. In patient cohorts, low levels of CD44v6 predict increased probability of survival. Thus, the metastatic process in colorectal cancer is initiated by CSCs through the expression of CD44v6, which is both a functional biomarker and therapeutic target.

BACKGROUND: Long interspersed nuclear element-1 (LINE-1 or L1), the most abundant and only autonomously active family of non-LTR retrotransposons in the human genome, expressed not only in the germ lines but also in somatic tissues. It contributes to genetic instability, aging, and age-related diseases, such as cancer. Our previous study identified in human gastric adenocarcinoma an upregulated transcript GCRG213, which shared 88% homology with human L1 sequence and contained a putative conserved apurinic/apyrimidinic endonucleas1 domain. METHODS: Immunohistochemistry was carried out by using a monoclonal mouse anti-human GCRG213 protein (GCRG213p) antibody produced in our laboratory, on tissue microarray constructed with specimens from 175 gastric adenocarcinoma patients. The correlation between GCRG213p expression and patient clinicopathological parameters was evaluated. GCRG213p expression in gastric cancer cell lines were studied using Western blotting analysis. L1 promoter methylation status of gastric cancer cells was tested using methylation-specific PCR. BLASTP was used at the NCBI Blast server to identify GCRG213p sequence to any alignments in the Protein Data Bank databases. RESULTS: Most primary gastric cancer, lymph node metastases and gastric intestinal metaplasia glands showed positive GCRG213p immunoreactivity. High GCRG213p immunostaining score in the primary gastric cancer was positively correlated with tumor differentiation (well differentiated, p = 0.001), Lauren's classification (intestinal type, p < 0.05) and a late age onset of gastric adenocarcinoma (>/=65 yrs; p < 0.05). GCRG213p expression has no association with other clinicopathological parameters, including survival. Western blotting analysis of GCRG213p expression in gastric cancer cells indicated that GCRG213p level was higher in gastric cancer cell lines than in human normal gastric epithelium immortalized cell line GES-1. Partial methylation of L1 in gastric cancer cells was confirmed by methylation-specific PCR. BLASTP program analysis revealed that GCRG213p peptide shared 83.0% alignment with the C-terminal region of L1 endonuclease (L1-EN). GCRG213p sequence possesses the important residues that compose the conserved features of L1-EN. CONCLUSIONS: GCRG213p could be a variant of L1-EN, a functional member of L1-EN family. Overexpression of GCRG213p is common in both primary gastric cancer and lymph node metastasis. These findings provide evidence of somatic L1 expression in gastric cancer, and its potential consequences in the form of tumor.

C-C Chemokine receptor 5 knockout (Ccr5(-/-)) mice develop fewer experimental pulmonary metastases than wild-type (WT) mice. This phenomenon was explored by applying gene expression profiling to the lungs of mice with these metastases. Consequently, erythroid differentiation regulator 1 (Erdr1) was identified as upregulated in the WT mice. Though commonly associated with bone marrow stroma, Erdr1 was differentially expressed in WT pulmonary mesenchymal cells (PMC) and murine embryonic fibroblasts (MEF). Moreover, the Ccr5 ligand Ccl4 increased its expression by 3.36 +/- 0.14-fold. Ccr5 signaling was dependent on the mitogen-activated protein kinase kinase (Map2k) but not the phosphoinositide 3-kinase (Pi3k) pathway because treatment with U0126 inhibited upregulation of Erdr1, but treatment with LY294002 increased the expression by 3.44 +/- 0.92-fold (P < 0.05). The effect Erdr1 on B16-F10 melanoma metastasis was verified by the adoptive transfer of WT MEFs into Ccr5(-/-) mice. In this model, MEFs that had been transduced with Erdr1 short hairpin RNA (shRNA) lowered metastasis by 33% compared with control transduced MEFs. The relevance of ERDR1 on human disease was assessed by coculturing chronic lymphocytic leukemia (CLL) cells with M2-10B4 stromal cells that had been transfected with shRNA or control plasmids. After 96 hours of coculture, the cell counts were higher with control cell lines than with Erdr1 knockdown lines [odds ratio (OR), 1.88 +/- 0.27, 2.52 +/- 0.66, respectively]. This increase was associated with a decrease in apoptotic cells (OR, 0.69 +/- 0.18, 0.58 +/- 0.12, respectively). IMPLICATIONS: Therefore, ERDR1 is a stromal-derived factor that promotes cancer cell survival in vitro and in an experimental metastasis model.

Increased expression of cell adhesion molecule CD44v6, integrin-beta1, carbohydrate antigen 199 (CA199), and carcinoembryonic antigen (CEA) are closely associated with the progression and metastasis of numerous cancers. In this study, peripheral blood mononuclear cell (PBMC) and serum samples were collected from 37 pancreatic cancer patients and 12 healthy people. A novel triplex TaqMan real-time reverse transcription polymerase chain reaction assay was used to measure the expression levels of CD44v6 and integrin-beta1 gene in PBMCs, while chemiluminescence and enzyme-linked immunosorbent assay were used to measure the levels of CA199 and CEA expression in serum. The results showed that both the levels of CD44v6 and integrin-beta1 expression had significant correlation with clinical stage, lymph node, and liver metastasis of pancreatic cancer (P < 0.05). Age, tumor size, tumor differentiation, clinical stage, lymph nodes, and liver metastasis were significantly associated with the levels of CA199 and CEA expression (P < 0.05). The levels of CD44v6, integrin-beta1, CA199, and CEA expression in the patients prior cryosurgery and chemotherapy were significantly higher than those in the control group (P < 0.05), whereas no significant difference was found between the patients 1 month post cryosurgery and control group (P > 0.05). The expression levels of CD44v6, integrin-beta1, CA199, and CEA in the patients 1 month post cryosurgery were significantly lower than those in the patients prior cryosurgery (P < 0.05). Interestingly, no significant difference was found for the CD44v6, integrin-beta1, CA199, and CEA levels between the patients prior and post-chemotherapy (P > 0.05). The higher expression of CD44v6, integrin-beta1, CA199, and CEA are closely related to the progression and metastasis of pancreatic cancer and may play a important role in the curative evaluation of cryosurgery of pancreatic cancer.