The functional variant in promoter of OVCA1 was associated with the risk of gastric cancer in the northeast Chinese Han population.

We previously found allelic deletions on chromosomes 17 in primary gastric cancers (GC) using microsatellite markers for loss of heterozygosity (LOH). OVCA1 lies in one of these regions (17q21.33). The association between single nucleotide polymorphism (SNP) of OVCA1 gene and risk of gastric cancer is not yet clear. In this study, the peripheral blood of 505 gastric cancer patients and 544 healthy controls were genotyped for six SNPs (rs2273981, rs1131600, rs3752963, rs3803806, rs2236375, and rs1051322) of OVCA1, to evaluate the association of these SNPs with the risk of gastric cancer in the Han population in northeast China. The effect of rs2273981 located in the promoter region of OVCA1 on the transcription activity was determined using dual luciferase reporter assay. We found that the association between the AA + AG genotype of rs2273981 and the risk of gastric cancer was significant in smokers (AA + AG vs. GG, OR = 2.47, 95% CI = 1.04 - 5.87, P < 0.05). Stratified analysis of the clinicopathological parameters revealed that rs1131600 AG + GG genotype were significantly associated with increased gastric tumor volume (AG + GG vs. AA, OR = 1.81, 95% CI = 1.00 - 3.29, P < 0.05). The rs2236375 CT + TT genotype was also significantly associated with increased gastric tumor volume (CT + TT vs. CC, OR = 2.65, 95% CI = 1.38 - 5.10, P < 0.05). Additionally, by interacting with the transcription factor AP2A, the GG genotype the rs2273981 increased the transcription activity of OVCA1 compared with AA genotype, thus involved in gastric cancer development.

The tumor suppressor OVCA1 is a short half-life protein degraded by the ubiquitin-proteasome pathway.

Ovarian cancer gene 1 (OVCA1) is a tumor suppressor associated with ovarian cancer, which is involved in cell proliferation regulation, embryonic development and tumorigenesis. Loss of heterozygosity in the OVCA1 gene occurs in 50-86% of cases of ovarian cancer; however, the physiological and biochemical functions of OVCA1 are not yet clear. In the present study, the stability and degradation of OVCA1 were investigated in A2780, Hela and 293 cells. The results revealed that the OVCA1 protein was unstable by MG132 inhibiting proteasome mediated degradation, co-immunoprecipitation and half-life measurement experiments. The cellular protein levels of endogenous OVCA1 were too low to be detected by western blotting. In addition, carbobenzoxy-L-leucyl-L-leucyl-L-leucinal inhibited the degradation of OVCA1 in cells. The co-immunoprecipitation assay revealed that the OVCA1 protein interacted with ubiquitin to form a poly-ubiquitinated complex in cells. The half-life of OVCA1, measured by inhibiting protein synthesis with cycloheximide, was <2 h. The present study demonstrated that OVCA1 may be degraded by the ubiquitin-mediated proteasome pathway and may be considered a short half-life protein. In conclusion, the regulation of OVCA1 protein degradation via the ubiquitin-proteasome pathway may represent a novel direction in the development of ovarian cancer therapy.

Influence of DPH1 and DPH5 Protein Variants on the Synthesis of Diphthamide, the Target of ADPRibosylating Toxins.

The diphthamide on eukaryotic translation elongation factor 2 (eEF2) is the target of ADPribosylating toxins and -derivatives that serve as payloads in targeted tumor therapy. Diphthamide is generated by seven DPH proteins; cells deficient in these (DPHko) lack diphthamide and are toxin-resistant. We have established assays to address the functionality of DPH1 (OVCA1) and DPH5 variants listed in dbSNP and cosmic databases: plasmids encoding wildtype and mutant DPHs were transfected into DPHko cells. Supplementation of DPH1 and DPH5 restores diphthamide synthesis and toxin sensitivity in DPH1ko and DPH5ko cells, respectively. Consequently, the determination of the diphthamide status of cells expressing DPH variants differentiates active and compromised proteins. The DPH1 frameshift variant L96fs* (with Nterminal 96 amino acids, truncated thereafter) and two splice isoforms lacking 80 or 140 amino acids at their N-termini failed to restore DPH1ko deficiency. The DPH1 frameshift variant R312fs* retained some residual activity even though it lacks a large C-terminal portion. DPH1 missense variants R27W and S56F retained activity while S221P had reduced activity, indicated by a decreased capability to restore diphthamide synthesis. The DPH5 nonsense or frameshift variants E60*, W136fs* and R207* (containing intact N-termini with truncations after 60, 136 or 207 amino acids, respectively) were inactive: none compensated the deficiency of DPH5ko cells. In contrast, missense variants D57G, G87R, S123C and Q170H as well as the frequently occurring DPH5 isoform delA212 retained activity. Sensitivity to ADP-ribosylating toxins and tumor-targeted immunotoxins depends on diphthamide which, in turn, requires DPH functionality. Because of that, DPH variants (in particular those that are functionally compromised) may serve as a biomarker and correlate with the efficacy of immunotoxin-based therapies.

Comprehensive silencing of target-sharing microRNAs is a mechanism for SIRT1 overexpression in cancer.

Overexpression of SIRT1 is frequently observed in various types of cancers, suggesting its potential role in malignancies. However, the molecular basis of how SIRT1 is elevated in cancer is less understood. Here we show that cancer-related SIRT1 overexpression is due to evasion of Sirt1 mRNA from repression by a group of Sirt1-targeting microRNAs (miRNAs) that might be robustly silenced in cancer. Our comprehensive library-based screening and subsequent miRNA gene profiling revealed a housekeeping gene-like broad expression pattern and strong CpG island-association of the Sirt1-targeting miRNA genes. This suggests aberrant CpG DNA methylation as the mechanistic background for malignant SIRT1 elevation. Our work also provides an example where epigenetic mechanisms cause the group-wide regulation of miRNAs sharing a common key target.