Heterozygous colon cancer-associated mutations of SAMHD1 have functional significance.
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ABSTRACT:
SUBMITTER: Rentoft M
PROVIDER: S-EPMC4855590 | biostudies-other | 2016 Apr
REPOSITORIES: biostudies-other
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Project description:Multiple genome-wide association studies have identified several susceptibility variants for colon cancer at 8q24. However, the functional roles of these variants have yet to be elucidated. Here, we evaluated the potential role of these markers in tumor progression and examined association with commonly observed structural abnormalities in this region, c-MYC amplification and chromosome fragility at FRA8C and FRA8D. We first replicated the previously reported association by testing 1,178 cases and 1,009 clinic-based controls with eight markers localized to three specific regions at 8q24. We observed significant associations with colon cancer risk with markers rs13254738 (ordinal odds ratio, 0.82; 95% confidence interval, 0.072-0.94; P(trend) = 0.0037) and rs6983267 (ordinal odds ratio, 1.17; 95% confidence interval, 1.03-1.32, P(trend) = 0.013). Survival analysis was done using a separate set of 460 cases to evaluate the clinical significance of these markers. Overall, univariate analysis did not detect survival differences for any of the markers. We also tested a subset of the 460 cases (n = 380) for structural abnormalities at or near the c-MYC locus using fluorescence in situ hybridization analysis. Furthermore, we evaluated a small number of cases homozygous for the rs6983267 alleles to test for differences in fragile site induction. None of the 8q markers correlated with amplification at the c-MYC locus as detected by fluorescence in situ hybridization, and no clear pattern of breakage was observed at the FRA8C and FRA8D sites. In this study, we confirm the association for several single nucleotide polymorphisms at 8q24 in colon cancer but have not detected any structural role relating to c-MYC amplification or chromosomal fragility. Finally, these risk alleles do not seem to be associated with survival.
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Project description:The expression of sterile α motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1) and its mutation play a key role in the prognosis of colon cancer. The aim of the present study was to investigate the mechanism and the role of SAMHD1 in colon cancer. Microarray data from 187 patients with colon cancer and 45 adjacent normal tissue obtained from the Gene Expression Omnibus (GEO) were analyzed. A protein-protein interaction (PPI) network was constructed to identify key genes associated with colon cancer prognosis. Cox proportional hazard regression and survival analyses were performed to identify the potential for SAMHD1 to serve as a prognostic biomarker. Immunohistochemistry (IHC) and immunofluorescence (IF) were performed to assess the expression levels and distribution of SAMHD1 in tissues and cells. Western blotting (WB) and Cell Counting Kit-8 (CCK-8) assays were used to identify the proliferation and apoptotic effects of SAMHD1 on HT-29 (Cas9-SAMHD1) cell lines. A total of 6,905 consistently differentially expressed genes were identified in the GEO database. Through the PPI network, SAMHD1 was found to be associated with Kirsten rat sarcoma virus (KRAS). SAMHD1 expression was negatively associated with KRAS. Proportional hazards regression and survival analyses demonstrated that low expression of SAMHD1 was associated with increased patient mortality. IHC and IF results demonstrated that SAMHD1 expression in patients with colon cancer was decreased compared with controls (both P<0.05). CCK-8 and WB results showed that proliferation was significantly promoted, and the expression levels of apoptosis-related proteins were significantly inhibited in the D137N and D311A groups as a result of a mutation in the deoxynucleoside triphosphohydrolase (dNTPase) site (both P<0.05 vs. wild-type). Proliferation was inhibited and apoptosis-related protein expression levels were promoted in the wild-type (WT) and D137N groups following 20 µg/ml 5-fluorouracil (5-FU) treatment (both P<0.05). WB and CCK-8 results showed cell proliferation was promoted and cell apoptosis-related protein expression was inhibited in the D137N group following treatment with 20 µg/ml 5-FU (all P<0.05) compared with the WT group. In conclusion, SAMHD1 expression was low in colon cancer. The dNTPase function of SAMHD1 may inhibit colon cancer cell proliferation and may enhance apoptosis. In addition, first-line chemotherapy with 5-FU has a time-dependent effect, which may provide novel options for clinical treatment of colon cancer.
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Project description:BackgroundTo investigate whether one or more SAMHD1 gene mutations are associated with cerebrovascular disease in the general population using a Chinese stroke cohort.MethodsPatients with a Chinese Han background (N = 300) diagnosed with either cerebral large-artery atherosclerosis (LAA, n = 100), cerebral small vessel disease (SVD, n = 100), or other stroke-free neurological disorders (control, n = 100) were recruited. Genomic DNA from the whole blood of each patient was isolated, and direct sequencing of the SAMHD1 gene was performed. Both wild type and mutant SAMHD1 proteins identified from the patients were expressed in E. coli and purified; then their dNTPase activities and ability to form stable tetramers were analysed in vitro.ResultsThree heterozygous mutations, including two missense mutations c.64C>T (P22S) and c.841G>A (p.E281K) and one splice site mutation c.696+2T>A, were identified in the LAA group with a prevalence of 3%. No mutations were found in the patients with SVD or the controls (p = 0.05). The mutant SAMHD1 proteins were functionally impaired in terms of their catalytic activity as a dNTPase and ability to assemble stable tetramers.ConclusionsHeterozygous SAMHD1 gene mutations might cause genetic predispositions that interact with other risk factors, resulting in increased vulnerability to stroke.
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2015-01-30 | E-GEOD-56973 | biostudies-arrayexpress
Project description:Colorectal cancer is one of the most commonly diagnosed malignancies in the Western world and is associated with elevated expression and activity of epidermal growth factor receptors (EGF-R). The metalloproteinase ADAM17 is involved in EGF-R activation by processing EGF-R ligands from membrane-bound pro-ligands. Underlining the link between colon cancer and ADAM17, genetic intestinal cancer models in ADAM17-deficient mice show a reduced tumor burden. In this study, we characterize point mutations within the ADAM17 gene found in the tissue of colon cancer patients. In order to shed light on the role of ADAM17 in cancer development, as well as into the mechanisms that regulate maturation and cellular trafficking of ADAM17, we here perform overexpression studies of four ADAM17 variants located in the pro-, membrane-proximal- and cytoplasmic-domain of the ADAM17 protein in ADAM10/17-deficient HEK cells. Interestingly, we found a cancer-associated point mutation within the pro-domain of ADAM17 (R177C) to be most impaired in its proteolytic activity and trafficking to the cell membrane. By comparing this variant to an ADAM17 construct lacking the entire pro-domain, we discovered similar functional limitations and propose a crucial role of the pro-domain for ADAM17 maturation, cellular trafficking and thus proteolytic activity.
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2015-01-30 | E-GEOD-56972 | biostudies-arrayexpress
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