The MLH1 c.1852_1853delinsGC (p.K618A) Variant in Colorectal Cancer: Genetic Association Study in 18,723 Individuals

Colorectal cancer is one of the most frequent neoplasms and an important cause of mortality in the developed world. Mendelian syndromes account for about 5% of the total burden of CRC, being Lynch syndrome and familial adenomatous polyposis the most common forms. Lynch syndrome tumors develop mainly as a consequence of defective DNA mismatch repair associated with germline mutations in MLH1, MSH2, MSH6 and PMS2. A significant proportion of variants identified by screening these genes correspond to missense or noncoding changes without a clear pathogenic consequence, and they are designated as "variants of uncertain significance'', being the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene a clear example. The implication of this variant as a low-penetrance risk variant for CRC was assessed in the present study by performing a case-control study within a large cohort from the COGENT consortium-COST Action BM1206 including 18,723 individuals (8,055 colorectal cancer cases and 10,668 controls) and a case-only genotype-phenotype correlation with several clinical and pathological characteristics restricted to the Epicolon cohort. Our results showed no involvement of this variant as a low-penetrance variant for colorectal cancer genetic susceptibility and no association with any clinical and pathological characteristics including family history for this neoplasm or Lynch syndrome.

Involvement of ANXA5 and ILKAP in Susceptibility to Malignant Melanoma

Single nucleotide-polymorphisms (SNPs) are a source of diversity among human population, which may be responsible for the different individual susceptibility to diseases and/or response to drugs, among other phenotypic traits. Several low penetrance susceptibility genes associated with malignant melanoma (MM) have been described, including genes related to pigmentation, DNA damage repair and oxidative stress pathways. In the present work, we conducted a candidate gene association study based on proteins and genes whose expression we had detected altered in melanoma cell lines as compared to normal melanocytes. The result was the selection of 88 loci and 384 SNPs, of which 314 fulfilled our quality criteria for a case-control association study. The SNP rs6854854 in ANXA5 was statistically significant after conservative Bonferroni correction when 464 melanoma patients and 400 controls were analyzed in a discovery Phase I. However, this finding could not be replicated in the validation phase, perhaps because the minor allele frequency of SNP rs6854854 varies depending on the geographical region considered. Additionally, a second SNP (rs6431588) located on ILKAP was found to be associated with melanoma after considering a combined set of 1,883 MM cases and 1,358 disease-free controls. The OR was 1.29 (95% CI 1.12-1.48; p-value= 4x10(-4)). Both SNPs, rs6854854 in ANXA5 and rs6431588 in ILKAP, show population structure, which, assuming that the Spanish population is not significantly structured, suggests a role of these loci on a specific genetic adaptation to different environmental conditions. Furthermore, the biological relevance of these genes in MM is supported by in vitro experiments, which show a decrease in the transcription levels of ANXA5 and ILKAP in melanoma cells compared to normal melanocytes.

A High Degree of LINE-1 Hypomethylation Is a Unique Feature of Early-Onset Colorectal Cancer

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Leukemia-Associated Mutations in Nucleophosmin Alter Recognition by CRM1: Molecular Basis of Aberrant Transport

Nucleophosmin (NPM) is a nucleocytoplasmic shuttling protein, normally enriched in nucleoli, that performs several activities related to cell growth. NPM mutations are characteristic of a subtype of acute myeloid leukemia (AML), where mutant NPM seems to play an oncogenic role. AML-associated NPM mutants exhibit altered subcellular traffic, being aberrantly located in the cytoplasm of leukoblasts. Exacerbated export of AML variants of NPM is mediated by the nuclear export receptor CRM1, and due, in part, to a mutationally acquired novel nuclear export signal (NES). To gain insight on the molecular basis of NPM transport in physiological and pathological conditions, we have evaluated the export efficiency of NPM in cells, and present new data indicating that, in normal conditions, wild type NPM is weakly exported by CRM1. On the other hand, we have found that AML-associated NPM mutants efficiently form complexes with CRM1HA (a mutant CRM1 with higher affinity for NESs), and we have quantitatively analyzed CRM1HA interaction with the NES motifs of these mutants, using fluorescence anisotropy and isothermal titration calorimetry. We have observed that the affinity of CRM1HA for these NESs is similar, which may help to explain the transport properties of the mutants. We also describe NPM recognition by the import machinery. Our combined cellular and biophysical studies shed further light on the determinants of NPM traffic, and how it is dramatically altered by AML-related mutations.

Structure-function analysis of USP1: insights into the role of Ser313 phosphorylation site and the effect of cancer-associated mutations on autocleavage

Background: In complex with its cofactor UAF1, the USP1 deubiquitinase plays an important role in cellular processes related to cancer, including the response to DNA damage. The USP1/UAF1 complex is emerging as a novel target in cancer therapy, but several aspects of its function and regulation remain to be further clarified. These include the role of the serine 313 phosphorylation site, the relative contribution of different USP1 sequence motifs to UAF1 binding, and the potential effect of cancer-associated mutations on USP1 regulation by autocleavage. Methods: We have generated a large set of USP1 structural variants, including a catalytically inactive form (C90S), non-phosphorylatable (S313A) and phosphomimetic (S313D) mutants, deletion mutants lacking potential UAF1 binding sites, a mutant (GG/AA) unable to undergo autocleavage at the well-characterized G670/G671 diglycine motif, and four USP1 mutants identified in tumor samples that cluster around this cleavage site (G667A, L669P, K673T and A676T). Using cell-based assays, we have determined the ability of these mutants to bind UAF1, to reverse DNA damage-induced monoubiquitination of PCNA, and to undergo autocleavage. Results: A non-phosphorylatable S313A mutant of USP1 retained the ability to bind UAF1 and to reverse PCNA ubiquitination in cell-based assays. Regardless of the presence of a phosphomimetic S313D mutation, deletion of USP1 fragment 420-520 disrupted UAF1 binding, as determined using a nuclear relocation assay. The UAF1 binding site in a second UAF1-interacting DUB, USP46, was mapped to a region homologous to USP1(420-520). Regarding USP1 autocleavage, co-expression of the C90S and GG/AA mutants did not result in cleavage, while the cancer-associated mutation L669P was found to reduce cleavage efficiency. Conclusions: USP1 phosphorylation at S313 is not critical for PCNA deubiquitination, neither for binding to UAF1 in a cellular environment. In this context, USP1 amino acid motif 420-520 is necessary and sufficient for UAF1 binding. This motif, and a homologous amino acid segment that mediates USP46 binding to UAF1, map to the Fingers sub-domain of these DUBs. On the other hand, our results support the view that USP1 autocleavage may occur in cis, and can be altered by a cancer-associated mutation.