Rad9 modulates the P21WAF1 pathway by direct association with p53

<p>Abstract</p> <p>Background</p> <p>Previous studies suggest that human <it>RAD9 </it>(hRad9), encoding a DNA damage checkpoint molecule, which is frequently amplified in epithelial tumor cells of breast, lung, head and neck cancer, participates in regulation of the tumor suppressor p53-dependent transactivation of pro-survival <it>P21</it>^{<it>WAF1</it>}. This study examined the exact mechanism of the hRad9 function, especially through the phosphorylation of the C-terminus, in the transcription regulation of <it>P21</it>^{<it>WAF1</it>}.</p> <p>Results</p> <p>The transfection of phosphorylation-defective <it>hRAD9 </it>mutants of C-terminus resulted in reduction of the p53-dependent <it>P21</it>^{<it>WAF1 </it>}transactivation; the knockdown of total hRad9 elicited an increased <it>P21</it>^{<it>WAF1 </it>}mRNA expression. Immunoprecipitation and a ChIP assay showed that hRad9 and p53 formed a complex and both were associated with two p53-consensus DNA-binding sequences in the 5' region of <it>P21</it>^{<it>WAF1 </it>}gene. The association was reduced in the experiment of phosphorylation-defective <it>hRAD9 </it>mutants.</p> <p>Conclusion</p> <p>The present study indicates the direct involvement of hRad9 in the p53-dependent <it>P21</it>^{<it>WAF1 </it>}transcriptional mechanism, presumably via the phosphorylation sites, and alterations of the hRad9 pathway might therefore contribute to the perturbation of checkpoint activation in cancer cells.</p

Vascular endothelial growth factor receptor-1 mRNA overexpression in peripheral blood as a useful prognostic marker in breast cancer

INTRODUCTION: Identification of useful markers associated with poor prognosis in breast cancer patients is critically needed. We previously showed that expression of vascular endothelial growth factor receptor-1 mRNA in peripheral blood may be useful to predict distant metastasis in gastric cancer patients. However, expression of vascular endothelial growth factor receptor-1 mRNA in peripheral blood of breast cancer patients has not yet been studied. METHODS: Real-time reverse transcriptase-PCR was used to analyze vascular endothelial growth factor receptor-1 mRNA expression status with respect to various clinical parameters in 515 patients with breast cancer and 25 controls. RESULTS: Expression of vascular endothelial growth factor receptor-1 mRNA in peripheral blood was higher in breast cancer patients than in controls. Increased vascular endothelial growth factor receptor-1 mRNA expression was associated with large tumor size, lymph node metastasis and clinical stage. Patients with high vascular endothelial growth factor receptor-1 mRNA expression also experienced a poorer survival rate than those with low expression levels, including those patients with triple-negative type and luminal-HER2(-) type disease. CONCLUSIONS: Expression of vascular endothelial growth factor receptor-1 mRNA in peripheral blood may be useful for prediction of poor prognosis in breast cancer, especially in patients with triple-negative type and luminal-HER2(-) type disease

Reduction of T-Box 15 gene expression in tumor tissue is a prognostic biomarker for patients with hepatocellular carcinoma

Genome-wide analysis is widely applied to detect molecular alterations during oncogenesis and tumor progression. We analyzed DNA methylation profiles of hepatocellular carcinoma (HCC), and investigated the clinical role of most heypermethylated of tumor, encodes T-box 15 (TBX15), which was originally involved in mesodermal differentiation. We conducted a genome-wide analysis of DNA methylation of tumor and non-tumor tissue of 15 patients with HCC, and revealed TBX15 was the most hypermethylated gene of tumor (Beta-value in tumor tissue = 0.52 compared with non-tumor tissue). Another validation set, which comprised 58 HCC with radical resection, was analyzed to investigate the relationships between tumor phenotype and TBX15 mRNA expression. TBX15 mRNA levels in tumor tissues were significantly lower compared with those of nontumor tissues (p < 0.0001). When we assigned a cutoff value = 0.5-fold, the overall survival 5-year survival rates of the low-expression group (n = 17) were significantly shorter compared with those of the high-expression group (n = 41) (43.3% vs. 86.2%, p = 0.001). Multivariate analysis identified low TBX15 expression as an independent prognostic factor for overall and disease-free survival. Therefore, genome-wide DNA methylation profiling indicates that hypermethylation and reduced expression of TBX15 in tumor tissue represents a potential biomarker for predicting poor survival of patients with HCC

Combined mutation of Apc, Kras and Tgfbr2 effectively drives metastasis of intestinal cancer.

金沢大学新学術創成研究機構ナノ生命科学研究所Embargo Period 12 month

Regulation of the MDM2-P53 pathway and tumor growth by PICT1 via nucleolar RPL11

PICT1 (also known as GLTSCR2) is considered a tumor suppressor because it stabilizes phosphatase and tensin homolog (PTEN), but individuals with oligodendrogliomas lacking chromosome 19q13, where PICT1 is located, have better prognoses than other oligodendroglioma patients. To clarify the function of PICT1, we generated Pict1-deficient mice and embryonic stem (ES) cells. Pict1 is a nucleolar protein essential for embryogenesis and ES cell survival. Even without DNA damage, Pict1 loss led to p53-dependent arrest of cell cycle phase G1 and apoptosis. Pict1-deficient cells accumulated p53, owing to impaired Mdm2 function. Pict1 binds Rpl11, and Rpl11 is released from nucleoli in the absence of Pict1. In Pict1-deficient cells, increased binding of Rpl11 to Mdm2 blocks Mdm2-mediated ubiquitination of p53. In human cancer, individuals whose tumors express less PICT1 have better prognoses. When PICT1 is depleted in tumor cells with intact P53 signaling, the cells grow more slowly and accumulate P53. Thus, PICT1 is a potent regulator of the MDM2-P53 pathway and promotes tumor progression by retaining RPL11 in the nucleolu

The Long Noncoding RNA CCAT2 Induces Chromosomal Instability Through BOP1-AURKB Signaling

BACKGROUND &amp; AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic.METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, gamma-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients.RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients.CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors

CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR-17-5p, and miR-20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk

Differentially expressed genes in endothelial differentiation

By screening differentially expressed genes in mouse embryonic stem (ES) cells by subtractive hybridization, we identified three conserved but uncharacterized genes encoding bromodomain containing 3 (BRD3), protein lysine methyltransferase (PLM), and kelch domain containing 2 (KLHDC2), which were downregulated during endothelial differentiation. An RNA blot study showed that these genes were markedly expressed in undifferentiated ES cells, whereas the expression was reduced upon endothelial differentiation; a study of mouse endothelium showed a significant reduction in the expression of BRD3. A study of human BRD3, located on chromosome 9 at q34, a region susceptible to genomic rearrangement, showed an altered expression in 4 of 12 patients with bladder cancer, compared with adjacent noncancerous tissues. Taken together with the result of siRNA inhibition showing the positive regulation of cell proliferation by BRD3, it is suggested that this molecule plays a role in allowing cells to enter the proliferative phase of the angiogenic process. © Mary Ann Liebert, Inc

Molecular mechanism of peritoneal dissemination in gastric cancer

Peritoneal dissemination (PD) is the most common cause of metastasis in gastric cancer (GC). Because there are no standard treatments for PD, it is associated with a poor prognosis. Although clinicians have performed intraperitoneal chemotherapy for GC with PD, the outcome remains unsatisfactory. Therefore, the development of novel treatments and diagnostic tools for PD is expected to improve the prognosis of GC patients with PD. Notably, it is essential to elucidate the molecular mechanisms involved in the development of PD in GC. In this review, the molecular mechanisms of PD (three steps: detachment from the primary tumor, adaptation to the microenvironment of the peritoneal cavity, and attachment to peritoneal mesothelial cells) and new topics in GC are highlighted