Expression of Folate Pathway Genes in Stage III Colorectal Cancer Correlates with Recurrence Status Following Adjuvant Bolus 5-FU-Based Chemotherapy

Colorectal cancer is commonly treated with 5-fluorouracil and 5-formyltetrahydrofolate (leucovorin). Metabolic action of leucovorin requires several enzymatic steps that are dependent on expression of corresponding coding genes. To identify folate pathway genes with possible impact on leucovorin metabolism, a retrospective study was performed on 193 patients with stage III colorectal cancer. Relative expression of 22 genes putatively involved in leucovorin transport, polyglutamation and metabolism was determined in tumor and mucosa samples using quantitative real-time polymerase chain reaction. After surgery, patients received adjuvant 5-fluorouracil-based bolus chemotherapy with leucovorin during six months, and were followed for 3 to 5 years. Cox regression analysis showed that high tumoral expression of the genes SLC46A1/PCFT (proton-coupled folate transporter) and SLC19A1/RFC-1 (reduced folate carrier 1) correlated significantly (p < 0.001 and p < 0.01, respectively) with a decreased risk of recurrent disease, measured as disease-free survival (DFS). These two genes are involved in the transport of folates into the cells and each functions optimally at a different pH. We conclude that SLC46A1/PCFT and SLC19A1/RFC-1 are associated with DFS of patients with colorectal cancer and hypothesize that poor response to 5-fluorouracil plus leucovorin therapy in some patients may be linked to low expression of these genes. Such patients might need a more intensified therapeutic approach than those with high gene expression. Future prospective studies will determine if the expression of any of these genes can be used to predict response to leucovorin.     

Alcohol-associated folate disturbances result in altered methylation of folate-regulating genes

Folate plays a critical role in maintaining normal metabolic, energy, differentiation and growth status of all mammalian cells. The steady-state accumulation of folate seems to depend on the activity of two enzymes: folylpolyglutamate synthetase (FPGS), which adds glutamate residues, and gamma-glutamyl hydrolase (GGH), which removes them, enabling it to be transported across the biological membranes. Overexpression of GGH and downregulation of FPGS would be expected to decrease intracellular folate in its polyglutamylated form, thereby increasing efflux of folate and its related molecules, which might lead to resistance to drugs or folate deficiency. The study was sought to delineate the activity of GGH and expression FPGS in tissues involved in folate homeostasis during alcoholism and the epigenetic regulation of these enzymes and transporters regulating intracellular folate levels. We determined the activity of GGH and expression of FPGS in tissues after 3 months of ethanol feeding to rats at 1 g/kg body weight/day. The results showed that there was not any significant change in the activity of folate hydrolyzing enzyme GGH in ethanol-fed rats while there was significant down regulation in the expression of FPGS. Ethanol feeding decreased the total as well as polyglutamated folate levels. There was tissue-specific hyper/hypo methylation of folate transporter genes viz. PCFT and RFC by chronic ethanol feeding. Moreover, hypermethylation of FPGS gene was observed in intestine and kidney without any change in methylation levels of GGH in the ethanol-fed rats. In conclusion, the initial deconjugation of polyglutamylated folate by GGH was not impaired in ethanol-fed rats while the conversion of monoglutamylated folate to polyglutamylated form might be impaired. There was tissue-specific altered methylation of folate transporter genes by chronic ethanol feeding.     

sLea and sLex expression in colorectal cancer: implications for tumourigenesis and disease prognosis

The glycoconjugates expressed by cancer cells frequently contain sialylated oligosaccharide chains. Among these oligosaccharides the sialyl Lewis a (sLe(a)) and sialyl Lewis x (sLe(x)) antigens are found to be overexpressed in tumours of different origin. The current study assesses sLe(a) and sLe(x) expression in different colorectal specimens in order to establish the correlation of these biomarkers with both malignant transformation of colorectal mucosa and the progression of colorectal cancer (CRC). Healthy disease-free and inflammatory mucosa specimens showed no presence of the antigens. sLe(a) was expressed in 6.7% of the healthy tissue from CRC patients, in 20.8% of the adenomas, and in 33.3% and 42.6% of the transitional tissue and tumour tissue, respectively. sLe(x) expression was observed in 6.7% of the healthy tissue from CRC patients, in 27.0% of the adenomas, and in 25.6% and 74.8% of the transitional and the tumour tissue, respectively. The expression of the sLe(a) and sLe(x) antigens was correlated in adenomas, as well as in healthy and tumour tissue from CRC. Moreover, the high expression of sLe(x) in adenomas was correlated with a high degree of dysplasia (p=0.042). Finally, the survival analysis suggested that sLe(a) expression may be a prognostic factor for predicting disease-free survival in colorectal cancer (p=0.012).     

Clock gene expression levels and relationship with clinical and pathological features in colorectal cancer patients

The clock gene machinery controls cellular metabolism, proliferation, and key functions, such as DNA damage recognition and repair. Dysfunction of the circadian clock is involved in tumorigenesis, and altered expression of some clock genes has been found in cancer patients. The aim of this study was to evaluate the expression levels of core clock genes in colorectal cancer (CRC). Quantitative real-time polymerase chain reaction (qPCR) was used to examine ARNTL1, CLOCK, PER1, PER2, PER3, CRY1, CRY2, Timeless (TIM), TIPIN, and CSNK1? expression levels in the tumor tissue and matched apparently healthy mucosa of CRC patients. In the tumor tissue of CRC patients, compared to their matched healthy mucosa, expression levels of ARNTL1 (p=.002), PER1 (p=.002), PER2 (p=.011), PER3 (p=.003), and CRY2 (p=.012) were lower, whereas the expression level of TIM (p=.044) was higher. No significant difference was observed in the expression levels of CLOCK (p=.778), CRY1 (p=.600), CSNK1 (p=.903), and TIPIN (p=.136). As to the clinical and pathological features, a significant association was found between low CRY1 expression levels in tumor mucosa and age (p=.026), and female sex (p=.005), whereas high CRY1 expression levels in tumor mucosa were associated with cancer location in the distal colon (p?=?.015). Moreover, high TIM mRNA levels in the tumor mucosa were prevalent whenever proximal lymph nodes were involved (p= .013) and associated with TNM stages III-IV (p=.005) and microsatellite instability (p=.015). Significantly poorer survival rates were evidenced for CRC patients with lower expression in the tumor tissue of PER1 (p=.010), PER3 (p= .010), and CSNKIE (p=.024). In conclusion, abnormal expression levels of core clock genes in CRC tissue may be related to the process of tumorigenesis and exert an influence on host/tumor interactions.     

Circulating exosomal CPNE3 as a diagnostic and prognostic biomarker for colorectal cancer

Exosomal proteins are emerging as relevant diagnostic and prognostic biomarkers for cancer. This study was aimed at illustrating the clinical significance of exosomal Copine III (CPNE3) purified from the plasma of colorectal cancer (CRC) patients. The CPNE3 expression levels in CRC tissues were analyzed by real-time PCR, western blot, and immunohistochemistry. Plasma exosomes were isolated to examine the CPNE3 level using ELISA. Pearson’s correlation analysis was performed to investigate the CPNE3 levels between CRC tissues and matched plasma samples. Receiver operating characteristic curve analysis was developed to measure the diagnostic performance of exosomal CPNE3. The Kaplan–Meier method and Cox's proportional hazards model were utilized to determine statistical differences in survival times. CPNE3 showed increased expressions in the CRC tissues. A moderately significant correlation was found between CPNE3 expression in CRC tissues by immunohistochemistry and matched serum exosomal CPNE3 expression by ELISA (r = 0.645,(r = 0.645, p < 0.001). < 0.001). Exosomal CPNE3 yielded a sensitivity of 67.5% and a specificity of 84.4% in CRC at the cutoff value of 0.143 pg per 1ug1 ug exosome. Combined data from carcinoembryonic antigen and exosomal CPNE3 achieved 84.8% sensitivity and 81.2% specificity as a diagnostic tool. CRC patients with lower exosomal CPNE3 levels had substantially better disease-free survival (hazard ratio [HR], 2.9; 95% confidence interval [CI]: 1.3–6.4; p = 0.009) = 0.009) and overall survival (HR, 3.4; 95% CI: 1.2–9.9; p = 0.026) = 0.026) compared with those with higher exosomal CPNE3 levels. Exosomal CPNE3 show potential implications in CRC diagnosis and prognosis.     

Folylpolyglutamate synthase and gamma-glutamyl hydrolase regulate leucovorin-enhanced 5-fluorouracil anticancer activity

Although 5-fluorouracil (5-FU) plus leucovorin (LV) is a standard chemotherapy regimen for colorectal cancer, the factors that determine the LV-mediated enhancement of the antitumor activity of 5-FU have remained unknown. We investigated the roles of folylpolyglutamate synthase (FPGS) and γ-glutamyl hydrolase (GGH), which are the main enzymes involved in folate metabolism, in the effect of LV. LV enhanced the anticancer activity of 5-FU and the level of reduced folate in human colon cancer cells. Small-interfering RNA (siRNA) transfected into DLD-1 cells to downregulate FPGS reduced the basal level of reduced folate, the folate level after LV treatment, and the enhancement of 5-fluoro-2′-deoxyuridine (FdUrd)-induced cytotoxicity elicited by LV. By contrast, the downregulation of GGH by siRNA increased cellular sensitivity to FdUrd combined with LV. These results suggest that FPGS and GGH expression levels in tumors are determinants of the efficacy of LV in enhancing the antitumor activity of 5-FU.     

Detection of p16 abnormalities in early-stage non-small cell lung cancer

We investigated the clinical significance of p16 abnormalities (mutations by sequencing and hypermethylation by methylation-specific PCR) in 52 radically resected stage I-II non-small cell lung cancers (NSCLCs). P16 abnormalities were detected in 20 (38%) patents (point mutations in 4 (8%) and promoter hypermethylation in 16 (31%) cases. No correlation was found between p16 abnormalities and various clinicopathologic factors, including sex, histological type of tumor and TNM (I vs. II) stage. The multivariate analysis of survival was performed using the Cox proportional hazard model. When the types of 16 inactivation were analyzed, p16 hypermethylation rather than point mutation was associated with poor Prognosis. The presented results prompt the conclusion that hypermethylation of p16 is the major mechanism for p16 gene inactivation in early stage NSCLC and could be a useful molecular marker for the prognosis.     

Molecular correlates and prognostic significance of SATB1 expression in colorectal cancer

ABSTRACT: BACKGROUND: Special AT-rich sequence-binding protein 1 (SATB1) is a global gene regulator that has been reported to confer malignant behavior and associate with poor prognosis in several cancer forms. SATB1 expression has been demonstrated to correlate with unfavourable tumour characteristics in rectal cancer, but its association with clinical outcome in colorectal cancer (CRC) remains unclear. In this study, we examined the prognostic impact of SATB1 expression in CRC, and its association with important molecular characteristics; i.e. beta-catenin overexpression, microsatellite instability (MSI) screening status, and SATB2 expression. METHODS: Immunohistochemical expression of SATB1 and beta-catenin was assessed in tissue microarrays with tumours from 529 incident CRC cases in the prospective population-based Malmo Diet and Cancer Study, previously analysed for SATB2 expression and MSI screening status. Spearman[ACUTE ACCENT]s Rho and Chi-Square tests were used to explore correlations between SATB1 expression, clinicopathological and investigative parameters. Kaplan Meier analysis and Cox proportional hazards modelling were used to explore the impact of SATB1 expression on cancer specific survival (CSS) and overall survival (OS). RESULTS: SATB1 was expressed in 222 (42%) CRC cases and negative, or sparsely expressed, in adjacent colorectal mucosa (n = 16). SATB1 expression was significantly associated with microsatellite stable tumours (p < 0.001), beta-catenin overexpression (p < 0.001) and SATB2 expression (p < 0.001). While not prognostic in the full cohort, SATB1 expression was significantly associated with poor prognosis in SATB2 negative tumours (HR = 2.63; 95% CI 1.46-4.71; pinteraction = 0.011 for CSS and HR = 2.31; 95% CI 1.32-4.04; pinteraction = 0.015 for OS), remaining significant in multivariable analysis. CONCLUSIONS: The results of this study demonstrate that SATB1 expression in CRC is significantly associated with beta-catenin overexpression, microsatellite stability and SATB2 expression. Furthermore, SATB1 expression is a factor of poor prognosis in SATB2 negative tumours. Altogether, these data indicate an important role for SATB1 in colorectal carcinogenesis and suggest prognostically antagonistic effects of SATB1 and SATB2. The mechanistic basis for these observations warrants further study.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1922643082772076.     

In situ autoradiographic detection of folylpolyglutamate synthetase activity

The enzyme folylpolyglutamate synthetase (FPGS) catalyzes the conversion of folate (pteroylmonoglutamate) to the polyglutamate forms (pteroylpolyglutamates) that are required for folate retention by mammalian cells. A rapid in situ autoradiographic assay for FPGS was developed which is based on the folate cofactor requirement of thymidylate synthase. Chinese hamster AUX B1 mutant cells lack FPGS activity and are unable to accumulate folate. As a result, the conversion of [6-3H]deoxyuridine to thymidine via the thymidylate synthase reaction is impaired in AUX B1 cells and no detectable label is incorporated into DNA. In contrast, FPGS in wild-type Chinese hamster CHO cells causes folate retention and enables the incorporation of [6-3H]deoxyuridine into DNA. Incorporation may be detected by autoradiography of monolayer cultures or of colonies replica plated onto polyester discs. Introduction of Escherichia coli FPGS into AUX B1 cells restores the activity of the thymidylate synthase pathway and demonstrates that the E. coli FPGS enzyme can provide pteroylpolyglutamates which function in mammalian cells.     

Downregulation of exosome-encapsulated miR-548c-5p is associated with poor prognosis in colorectal cancer

The role of circulating exosomal microRNAs (miRNAs) in colorectal cancer (CRC) has drawn more and more attention during the past few years. Previously, we have identified several specific miRNAs in serum exosomes as potential CRC biomarkers. However, little is known about the association between exosome-encapsulated miR-548c-5p and outcomes of patients with CRC. In the current study, the expression of serum exosomal miR-548c-5p was investigated by quantitative real-time polymerase chain reaction. Its correlation with CRC prognosis was estimated by Kaplan-Meier survival and log-rank tests. Cox regression analysis based on uni- and multivariate analyses was performed to estimate the relationship of exosome-encapsulated miR-548c-5p with the clinicopathological factors of patients with CRC. Reduced levels of serum exosomal miR-548c-5p were more significant in CRC patients with liver metastasis and at later TNM stage (III/IV tumor stages). Serum exosomal miR-548c-5p could inhibit the proliferation of CRC cells, while the precise molecular mechanisms warranted further elucidation. In addition, decreased levels of serum exosomal miR-548c-5p were independently associated with shorter overall survival in CRC adjusted by age, sex, tumor grade vascular infiltration, TNM stage (III/IV tumor stages) and metastasis (hazard ratio = 3.40, 95% confidence interval 1.02-11.27; P = 0.046). The downregulation of exosomal miR-548c-5p in serum predicts poor prognosis in patients with CRC. Exosomal miR-548c-5p may be a critical biomarker for CRC diagnosis and prognosis.     

Clinical significance of preoperative neutrophil-lymphocyte versus platelet-lymphocyte ratio in patients with operable colorectal cancer

The objective of this study was to clarify whether the neutrophil-lymphocyte ratio (NLR) and the platelet-lymphocyte ratio (PLR) are significant prognostic markers in patients with resectable colorectal cancer (CRC). A total of 200 patients who underwent curative resection for CRC were enrolled. The NLR and PLR were positively correlated (p?相似文献   

The Prognostic Value of p16 Hypermethylation in Cancer: A Meta-Analysis

Background

The prognostic value of p16 promoter hypermethylation in cancers has been evaluated for several years while the results remain controversial. We thus performed a systematic review and meta-analysis of studies assessing the impact of p16 methylation on overall survival (OS) and disease-free survival (DFS) to clarify this issue.

Methods

We searched Pubmed, Embase and ISI web of knowledge to identify studies on the prognostic impact of p16 hypermethylation in cancers. A total of 6589 patients from 45 eligible studies were included in the analysis. Pooled hazard ratios (HRs) with 95% confidence interval (95% CI) were calculated to estimate the effect using random-effects model.

Results

The analysis indicated that p16 hypermethylation had significant association with poor OS of non-small cell lung cancer (NSCLC) (HR 1.74, 95% CI: 1.36–2.22) and colorectal cancer (CRC) (HR 1.80; 95% CI 1.27–2.55). Moreover, the significant correlation was present between p16 hypermethylation and DFS of NSCLC (HR 2.04, 95% CI: 1.19–3.50) and head and neck cancer (HR 2.24, 95% CI: 1.35–3.73). Additionally, in the analysis of the studies following REMARK guidelines more rigorously, p16 hypermethylation had unfavorable impact on OS of NSCLC (HR 1.79, 95% CI: 1.35–2.39) and CRC (HR 1.96, 1.16–3.34), and on DFS of NSCLC (HR 2.12, 95% CI: 1.21–3.72) and head and neck cancer (HR 2.24, 95% CI: 1.35–3.73).

Conclusions

p16 hypermethylation might be a predictive factor of poor prognosis in some surgically treated cancers, particularly in NSCLC.     

脑胶质瘤组织中p16基因启动子区CpG岛甲基化检测及其临床意义研究

目的：研究脑胶质瘤中p16基因启动子区甲基化情况及其临床意义。方法：用甲基化特异性PCR技术检测42例脑胶质瘤组织和癌旁正常脑组织中p16基因启动子甲基化,并分析该基因启动子甲基化与临床病理特征之间的关系。结果：脑胶质瘤组织中p16基因异常甲基化率（38.27%）显著高于癌旁正常脑组织中p16基因的异常甲基化率（8.8%,P=0.000）。发生甲基化的肿瘤组织或者正常脑组织中p16基因mRNA和蛋白表达显著降低。此外,p16基因异常甲基化和肿瘤病理分级有相关性（P=0.007）,而与患者性别、年龄及肿瘤类型等临床特征无关（P=0.669,0.869和0.944）。结论：p16基因启动子区CpG岛高甲基化与p16表达下调相关,推测p16启动子区CpG岛高甲基化是导致p16基因在脑胶质瘤中表达下调的重要因素,有望成为脑胶质瘤早期辅助诊断的分子标志物之一。     

Epigenetic regulation of DACH1, a novel Wnt signaling component in colorectal cancer

Colorectal cancer (CRC) is one of the common malignant tumors worldwide. Both genetic and epigenetic changes are regarded as important factors of colorectal carcinogenesis. Loss of DACH1 expression was found in breast, prostate, and endometrial cancer. To analyze the regulation and function of DACH1 in CRC, 5 colorectal cancer cell lines, 8 cases of normal mucosa, 15 cases of polyps and 100 cases of primary CRC were employed in this study. In CRC cell lines, loss of DACH1 expression was correlated with promoter region hypermethylation, and re-expression of DACH1 was induced by 5-Aza-2'-deoxyazacytidine treatment. We found that DACH1 was frequently methylated in primary CRC and this methylation was associated with reduction in DACH1 expression. These results suggest that DACH1 expression is regulated by promoter region hypermethylation in CRC. DACH1 methylation was associated with late tumor stage, poor differentiation, and lymph node metastasis. Re-expression of DACH1 reduced TCF/LEF luciferase reporter activity and inhibited the expression of Wnt signaling downstream targets (c-Myc and cyclinD1). In xenografts of HCT116 cells in which DACH1 was re-expressed, tumor size was smaller than in controls. In addition, restoration of DACH1 expression induced G2/M phase arrest and sensitized HCT116 cells to docetaxel. DACH1 suppresses CRC growth by inhibiting Wnt signaling both in vitro and in vivo. Silencing of DACH1 expression caused resistance of CRC cells to docetaxel. In conclusion, DACH1 is frequently methylated in human CRC and methylation of DACH1 may serve as detective and prognostic marker in CRC.     

Correlation between PTEN and P62 gene expression in rat colorectal cancer cell

ObjectiveAutophagy is a cellular pathway that regulates the transportation and degradation of cytoplasmic macromolecules and organelles towards lysosome, which is often related to the tumorigenesis and tumor suppression. Here, we investigate the regulating effect of PTEN gene on autophagy-related protein P62 in rat colorectal cancer (CRC) cells and explore the application value of PTEN gene in clinic.MethodsRat colorectal cancer was induced by intraperitoneal injection of 1,2-dimethyl hydrazine in male ACI rats. A total of 20 rats were randomly selected from those successfully induced with CRC as the experimental group, while 10 healthy rats as control. The rat CRC cells were isolated and cultured. After transfecting the rat CRC cells with pEGFP-N1-PTEN plasmid, RT-PCR was adopted to examine that gene expression of p62 and PTEN, while Western blotting was used to detect the protein expression of p62 and PTEN. Also, the proliferation of CRC cells was measured by MTT assay.ResultsThe expression of PTEN gene in the experimental group was significantly inhibited as compared with the control group, while the expression of P62 gene was significantly increased (p < 0.05). Western blotting demonstrated that the PTEN protein in the experimental group was lower, while the expression of P62 protein was higher. When the CRC cells were transfected with pEGFP-N1-PTEN plasmid, the PTEN expressions were elevated, while p62 was down-regulated. Also, the proliferation of CRC cells was inhibited.ConclusionThe expression of PTEN gene is negatively correlated with the expression of P62 gene in rat CRC cells. And the expression of PTEN gene can inhibit the occurrence and development of colorectal cancer, thus providing theoretical basis for future clinical treatment.     

p16基因甲基化状态与散发性大肠癌的相关性研究

为探讨p1 6基因甲基化状态与散发性大肠癌发生发展的关系 ,用甲基化特异性的聚合酶链反应 (methylati omspecificPCR ,MSP)结合测序检测散发性大肠癌及相应癌旁组织p1 6基因甲基化状态。研究发现p1 6基因在散发性大肠癌中甲基化率为 2 8 9% (1 3 4 5 ) ,有 8例癌及癌旁组织都发生了甲基化 ;有淋巴结及远处转移的甲基化率为5 0 % (8 1 6 ) ,高于无转移的甲基化率 2 0 8(5 2 4 ) (P <0 0 5 )。p1 6基因高甲基化是散发性大肠癌中常见的分子改变之一 ,大肠癌中p1 6基因高甲基化可能发生在癌变早期并与大肠癌的恶性进展有相关性     

Aberrant O‐glycosylation contributes to tumorigenesis in human colorectal cancer

Aberrant O‐glycosylation is frequently observed in colorectal cancer (CRC) patients, but it is unclear if it contributes intrinsically to tumorigenesis. Here, we investigated the biological consequences of aberrant O‐glycosylation in CRC. We first detected the expression profile of Tn antigen in a serial of human CRC tissues and then explored the genetic and biosynthetic mechanisms. Moreover, we used a human CRC cell line (LS174T), which express Tn antigen, to assess whether aberrant O‐glycosylation can directly promote oncogenic properties. It showed that Tn antigen was detected in around 86% human primary and metastatic CRC tissues. Bio‐functional investigations showed that T‐synthase and Cosmc were both impaired in cancer tissues. A further analysis detected an occurrence of hypermethylation of Cosmc gene, which possibly caused its loss‐of‐function and a consequent inactive T‐synthase. Transfection of LS174T cells with WT Cosmc restored mature O‐glycosylation, which subsequently down‐regulated cancer cell proliferation, migration and apoptotic‐resistant ability. Significantly, the expression of MUC2, a heavily O‐glycosylated glycoprotein that plays an essential role in intestinal function, was uniformly reduced in human CRC tissues as well as in LS174T cells. These data suggest that aberrant O‐glycosylation contributes to the development of CRC through direct induction of oncogenic properties in cancer cells.