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Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.

The deletion of exons 9 to 12 of BRCA1 (9–12 del BRCA1) is considered a founder mutation in the Mexican population. We evaluate the usefulness of the target detection of 9–12 del BRCA1 as the first molecular diagnostic strategy in patients with Hereditary Breast and Ovarian Cancer (HBOC). We performed the genetic assessment of 637 patients with suspected HBOC. The region corresponding to the breakpoints for the 9–12 del BRCA1 was amplified by polymerase chain reaction (PCR). An analysis of the clinical data of the carriers and non-carriers was done, searching for characteristics that correlated with the deletion. The 9–12 del BRCA1 was detected in 5% of patients with suspected HBOC (30/637). In patients diagnosed with ovarian cancer, 13 of 30 were 9–12 del BRCA1 carriers, which represents 43%. We found a significant association between the 9–12 del BRCA1 carriers with triple negative breast cancer and high-grade papillary serous ovarian cancer. We concluded that the detection of the 9–12 del BRCA1 is useful as a first molecular diagnostic strategy in the Mexican population. In particular, it shortens the gap in genetic assessment in patients with triple negative breast cancer and ovarian cancer.

Background: Prolonged mitotic arrest in response to anti-cancer chemotherapeutics, such as DNA-damaging agents, induces apoptosis, mitotic catastrophe, and senescence. Disruptions in mitotic checkpoints contribute resistance to DNA-damaging agents in cancer. MAD2 has been associated with checkpoint failure and chemotherapy response. In this study, a novel splice variant of MAD2, designated MAD2γ, was identified, and its association with the DNA damage response was investigated.Methods: Endogenous expression of MAD2γ and full-length MAD2 (MAD2α) was measured using RT-PCR in cancer cell lines, normal foreskin fibroblasts, and tumor samples collected from patients with testicular germ cell tumors (TGCTs). A plasmid expressing MAD2γ was transfected into HCT116 cells, and its intracellular localization and checkpoint function were evaluated according to immunofluorescence and mitotic index.Results: MAD2γ was expressed in several cancer cell lines and non-cancerous fibroblasts. Ectopically expressed MAD2γ localized to the nucleus and reduced the mitotic index, suggesting checkpoint impairment. In patients with TGCTs, the overexpression of endogenous MAD2γ, but not MAD2α, was associated with resistance to cisplatin-based chemotherapy. Likewise, cisplatin induced the overexpression of endogenous MAD2γ, but not MAD2α, in HCT116 cells.Conclusions: Overexpression of MAD2γ may play a role in checkpoint disruption and is associated with resistance to cisplatin-based chemotherapy in TGCTs.

Lynch syndrome (LS) is the main hereditary colorectal cancer syndrome. There have been few reports regarding the clinical and molecular characteristics of LS patients in Latin America; this is particularly true in the Mexican population, where no information is available. The present study aims to describe the clinical and molecular spectrum of variants in a cohort of patients diagnosed with LS in Mexico. We present a retrospective analysis of 412 patients with suspected LS, whose main site of cancer diagnosis was the colon (58.25%), followed by the endometrium (18.93%). Next-generation sequencing analysis, with an extensive multigene panel, showed that 27.1% (112/414) had a variant in one of the genes of the mismatch repair pathway (MMR); 30.4% (126/414) had a variant in non-MMR genes such as CHEK2, APC, MUTYH, BRCA1, and BRCA2; and 42.5% (176/414) had no genetic variants. Most of the variants were found in MLH1. Pathogenic variants (PVs) in MMR genes were identified in 65.7% (96/146) of the total PVs, and 34.24% (45/146) were in non-MMR genes. Molecular and clinical characterization of patients with LS in specific populations allowed personalized follow-up, with the option for targeted treatment with immune checkpoint inhibitors and the development of public health policies. Moreover, such characterization allows for family cascade testing and consequent prevention strategies.

Abnormal chromosome segregation plays a key role in cancer development. MAD2 is a component of the spindle assembly checkpoint (SAC), a cell cycle control mechanism that ensures an accurate segregation of chromosomes during mitosis. Changes in MAD2 expression have been associated with chemo-resistance both to spindle inhibitors and to DNA damaging agents. Also, a previous study has shown that the exogenous expression of MAD2β, a splicing variant of MAD2, was associated with resistance to Adriamycin and Vincristine in gastric cell lines. Additionally, we have previously identified that exogenous overexpression of MAD2γ upon paclitaxel-induced SAC activation in the colorectal cancer cell HCT116, reduces drug-induced mitotic arrest. These findings suggested a possible structural interaction of MAD2 isoforms with SAC components. To determine possible structural interactions between MAD2 isoforms and key SAC components (i.e. MAD1 and CDC20), we performed an in silico analysis of MAD2 isoforms, Interestingly, we found that alternative splicing of MAD2 generates a premature stop codon and a frameshift in exon 4 in MAD2γ and MAD2β. This change generates a new C-terminal region in MAD2γ and MAD2β isoforms that comprise 16 amino acids, which are not present in the major isoform (MAD2α). We aligned this region with the amino acid sequence of CDC20 from various species and identified a MAD2-interacting motif (MIM). This finding suggests that MAD2 isoforms may interact with the active conformation of MAD2 (C-MAD2). Since MAD2 isoforms and CDC20 may compete for the same region in MAD2, we propose a new model whereby MAD2 isoforms inhibits SAC by interfering with C-MAD2/CDC20 formation. This model helps to explain previous results where MAD2 isoforms over expression seem to have an opposite role in SAC signaling. Citation Format: Miguel Ramirez-Otero, Alejandro Lopez-Saavedra, Marco Andonegui, Jose Diaz-Chavez, Luis Alonso Herrera. In silico identification of a MAD2-interacting motif in MAD2 spliced isoforms suggest a functional interaction with the spindle assemble checkpoint in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2728.

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