Cancer-associated MDM2 W329G mutant attenuates ribosomal stress-mediated p53 responses to promote cell survival and glycolysis.

Although amplification/overexpression is the predominant mechanism for the oncogenic properties of MDM2, an increasing number of MDM2 somatic missense mutations were identified in cancer patients with the recent advances in sequencing technology. Here, we characterized an MDM2 cancer-associated mutant variant W329G identified from a patient sample that contains a wild-type p53 gene. Trp329 is one of residues that were reported to be critical to MDM2's binding to ribosomal protein L11 (RPL11). We found that the MDM2 W329G mutant was resistant to the inhibitory effect of RPL11 on MDM2-mediated p53 ubiquitination and degradation, in line with its defect on RPL11 binding. Using isogenic U2OS cells with or without endogenous MDM2 W329G mutation, we demonstrated that the expression of classic p53 targets induced by ribosomal stress signals was reduced in mutant cells. RNA-seq analysis revealed that upon 5-FU treatment, the p53 response was significantly impaired. Also, the 5-FU-mediated repression of genes in cell cycle progression and DNA replication was diminished in W329G mutant-containing cells. Physiologically, U2OS W329G cells were more resistant to cell growth inhibition induced by ribosomal stress and exhibited higher glycolytic rates upon 5-FU treatment. Together, our data indicated that cancer-associated MDM2 W329G mutant attenuates ribosomal stress-mediated p53 responses to promote cell survival and glycolysis.

Functional defects of cancer-associated MDC1 mutations in DNA damage repair.

Mediator of DNA damage checkpoint protein 1 (MDC1) serves as a docking platform to promote the localization of various DNA damage response (DDR) components to DNA double-strand break (DSB) sites. MDC1 is vital in controlling proper DDR and maintaining genomic stability. In cancers, genomic instability results from mutations in DNA repair genes and drives cancer development. The mutations of MDC1 in human cancers have not been systematically examined and little is known about the molecular phenotypes caused by these genetic changes. Here, we summarized cancer-associated mutations of MDC1 including insertion/deletion mutations as well as missense mutations in key functional domains of MDC1 from ICGC, TCGA and COSMIC databases. We analyzed 711 somatic mutations of MDC1 across 26 types of human cancers and examined the functional defects of these cancer-associated mutations of MDC1 in the context of DNA damage repair. 6 truncation mutations and 7 missense mutations of MDC1 were chosen for further study. 6 truncation mutations which abolish MDC1-γH2AX interaction abrogate its biological functions in DNA damage repair. 2 missense mutations in FHA domain impaired ATM (ataxia telangiectasia mutated) phosphorylation. 5 missense mutations in BRCT domain also abolished its interaction with γH2AX, resulting in defects in foci formation of MDC1, 53BP1 and BRCA1 as well as defects in G2/M checkpoints. We further used structural modeling to analyze the potential molecular mechanism by which the 7 missense mutations cause the DNA damage repair defects. Taken together, our results reveal these cancer-associated MDC1 mutations can result in functional defects in DNA damage response and may serve as biomarkers for cancer diagnostics in future.

Cancer-associated 53BP1 mutations induce DNA damage repair defects.

TP53 binding protein 1 (53BP1) plays an important role in DNA damage repair and maintaining genomic stability. However, the mutations of 53BP1 in human cancers have not been systematically examined. Here, we have analyzed 541 somatic mutations of 53BP1 across 34 types of human cancer from databases of The Cancer Genome Atlas, International Cancer Genome Consortium and Catalogue of Somatic Mutations in Cancer. Among these cancer-associated 53BP1 mutations, truncation mutations disrupt the nuclear localization of 53BP1 thus abolish its biological functions in DNA damage repair. Moreover, with biochemical analyses and structural modeling, we have examined the detailed molecular mechanism by which missense mutations in the key domains causes the DNA damage repair defects. Taken together, our results reveal the functional defects of a set of cancer-associated 53BP1 mutations.

Impairment of cytokinesis by cancer-associated DAPK3 mutations.

Death-associated protein kinase 3 (DAPK3), a member of the DAPK family, contributes to cytokinesis by phosphorylating myosin II regulatory light chain (MRLC). Missense mutations in DAPK3, T112M, D161N, and P216S, were observed in the lung, colon, and cervical cancers, respectively, but the effects of these mutations on cytokinesis remain unclear. Here, we show that cells expressing EGFP-DAPK3-T112M, -D161N, or -P216S exhibited reduced rates of cytokinesis, with an increased ratio of multinucleated cells. In addition, these cells exhibited reduced levels of phosphorylated MRLC at the contractile ring. Collectively, our data demonstrates that cancer-associated DAPK3 mutations impair cytokinesis by reducing phosphorylated MRLC.

Cancer-Associated Mutations but No Cancer: Insights into the Early Steps of Carcinogenesis and Implications for Early Cancer Detection.

Cancer is a disease of aging fueled by the accumulation of somatic mutations. While mutations in tumors are well characterized, little is known about the early mutational processes that initiate tumorigenesis. Recent advances in next-generation sequencing (NGS) have enabled the detection of mutations in normal tissue, revealing an unanticipated high level of age-related somatic mutations affecting most individuals and tissues. Surprisingly, many of these mutations are similar to mutations commonly found in tumors, suggesting an ongoing process of positive selection and clonal expansion akin to what occurs in cancer, but within normal tissue. Here we discuss some of the most important biological and clinical implications of these novel findings, with a special focus on their impact for cancer detection and prediction.

CRIMEtoYHU: a new web tool to develop yeast-based functional assays for characterizing cancer-associated missense variants.

Evaluation of the functional impact of cancer-associated missense variants is more difficult than for protein-truncating mutations and consequently standard guidelines for the interpretation of sequence variants have been recently proposed. A number of algorithms and software products were developed to predict the impact of cancer-associated missense mutations on protein structure and function. Importantly, direct assessment of the variants using high-throughput functional assays using simple genetic systems can help in speeding up the functional evaluation of newly identified cancer-associated variants. We developed the web tool CRIMEtoYHU (CTY) to help geneticists in the evaluation of the functional impact of cancer-associated missense variants. Humans and the yeast Saccharomyces cerevisiae share thousands of protein-coding genes although they have diverged for a billion years. Therefore, yeast humanization can be helpful in deciphering the functional consequences of human genetic variants found in cancer and give information on the pathogenicity of missense variants. To humanize specific positions within yeast genes, human and yeast genes have to share functional homology. If a mutation in a specific residue is associated with a particular phenotype in humans, a similar substitution in the yeast counterpart may reveal its effect at the organism level. CTY simultaneously finds yeast homologous genes, identifies the corresponding variants and determines the transferability of human variants to yeast counterparts by assigning a reliability score (RS) that may be predictive for the validity of a functional assay. CTY analyzes newly identified mutations or retrieves mutations reported in the COSMIC database, provides information about the functional conservation between yeast and human and shows the mutation distribution in human genes. CTY analyzes also newly found mutations and aborts when no yeast homologue is found. Then, on the basis of the protein domain localization and functional conservation between yeast and human, the selected variants are ranked by the RS. The RS is assigned by an algorithm that computes functional data, type of mutation, chemistry of amino acid substitution and the degree of mutation transferability between human and yeast protein. Mutations giving a positive RS are highly transferable to yeast and, therefore, yeast functional assays will be more predictable. To validate the web application, we have analyzed 8078 cancer-associated variants located in 31 genes that have a yeast homologue. More than 50% of variants are transferable to yeast. Incidentally, 88% of all transferable mutations have a reliability score >0. Moreover, we analyzed by CTY 72 functionally validated missense variants located in yeast genes at positions corresponding to the human cancer-associated variants. All these variants gave a positive RS. To further validate CTY, we analyzed 3949 protein variants (with positive RS) by the predictive algorithm PROVEAN. This analysis shows that yeast-based functional assays will be more predictable for the variants with positive RS. We believe that CTY could be an important resource for the cancer research community by providing information concerning the functional impact of specific mutations, as well as for the design of functional assays useful for decision support in precision medicine.

Novel mutations involving ßI-, ßIIA-, or ßIVB-tubulin isotypes with functional resemblance to ßIII-tubulin in breast cancer.

Tubulin is the target for very widely used anti-tumor drugs, including Vinca alkaloids, taxanes, and epothilones, which are an important component of chemotherapy in breast cancer and other malignancies. Paclitaxel and other tubulin-targeting drugs bind to the ß subunit of tubulin, which is a heterodimer of α and ß subunits. ß-Tubulin exists in the form of multiple isotypes, which are differentially expressed in normal and neoplastic cells and differ in their ability to bind to drugs. Among them, the ßIII isotype is overexpressed in many aggressive and metastatic cancers and may serve as a prognostic marker in certain types of cancer. The underpinning mechanisms accounting for the overexpression of this isotype in cancer cells are unclear. To better understand the role of ß-tubulin isotypes in cancer, we analyzed over 1000 clones from 90 breast cancer patients, sequencing their ß-tubulin isotypes, in search of novel mutations. We have elucidated two putative emerging molecular subgroups of invasive breast cancer, each of which involve mutations in the ßI-, ßIIA-, or ßIVB isotypes of tubulin that increase their structural, and possibly functional, resemblance to the ßIII isotype. A unifying feature of the first of the two subgroups is the mutation of the highly reactive C239 residue of ßI- or ßIVB-tubulin to L239, R239, Y239, or P239, culminating in probable conversion of these isotypes from ROS-sensitive to ROS-resistant species. In the second subgroup, ßI, ßIIA, and ßIVB have up to seven mutations to the corresponding residues in ßIII-tubulin. Given that ßIII-tubulin has emerged as a pro-survival factor, overexpression of this isotype may confer survival advantages to certain cancer cell types. In this mini-review, we bring attention to a novel mechanism by which cancer cells may undergo adaptive mutational changes involving alternate ß-tubulin isotypes to make them acquire some of the pro-survival properties of ßIII-tubulin. These "hybrid" tubulins, combining the sequences and/or properties of two wild-type tubulins (ßIII and either ßI, ßIIA, or ßIVB), are novel isotypes expressed solely in cancer cells and may contribute to the molecular understanding and stratification of invasive breast cancer and provide novel molecular targets for rational drug development.