Marco A. Andonegui

The epigenetic origin of aneuploidy

Theodore Boveri, eminent German pathologist, observed aneuploidy in cancer cells more than a century ago and suggested that cancer cells derived from a single progenitor cell that acquires the potential for uncontrolled continuous proliferation. Currently, it is well known that aneuploidy is observed in virtually all cancers. Gain and loss of chromosomal material in neoplastic cells is considered to be a process of diversification that leads to survival of the fittest clones. According to Darwin’s theory of evolution, the environment determines the grounds upon which selection takes place and the genetic characteristics necessary for better adaptation. This concept can be applied to the carcinogenesis process, connecting the ability of cancer cells to adapt to different environments and to resist chemotherapy, genomic instability being the driving force of tumor development and progression. What causes this genome instability? Mutations have been recognized for a long time as the major source of genome instability in cancer cells. Nevertheless, an alternative hypothesis suggests that aneuploidy is a primary cause of genome instability rather than solely a simple consequence of the malignant transformation process. Whether genome instability results from mutations or from aneuploidy is not a matter of discussion in this review. It is most likely both phenomena are intimately related; however, we will focus on the mechanisms involved in aneuploidy formation and more specifically on the epigenetic origin of aneuploid cells. Epigenetic inheritance is defined as cellular information—other than the DNA sequence itself—that is heritable during cell division. DNA methylation and histone modifications comprise two of the main epigenetic modifications that are important for many physiological and pathological conditions, including cancer. Aberrant DNA methylation is the most common molecular cancer-cell lesion, even more frequent than gene mutations; tumor suppressor gene silencing by CpG island promoter hypermethylation is perhaps the most frequent epigenetic modification in cancer cells. Epigenetic characteristics of cells may be modified by several factors including environmental exposure, certain nutrient deficiencies, radiation, etc. Some of these alterations have been correlated with the formation of aneuploid cells in vivo. A growing body of evidence suggests that aneuploidy is produced and caused by chromosomal instability. We propose and support in this manuscript that not only genetics but also epigenetics, contribute in a major fashion to aneuploid cell formation.

Association between ERCC1 and XPA expression and polymorphisms and the response to cisplatin in testicular germ cell tumours

Background:Cisplatin cures over 80% of testicular germ cell tumours (TGCTs), and nucleotide-excision repair (NER) modifies the sensitivity to cisplatin. We explored the association between NER proteins and their polymorphisms with cisplatin sensitivity (CPS) and overall survival (OS) of patients with non-seminomatous (ns)-TGCTs.Methods:The expression of ERCC1 and XPA and the presence of γH2AX were evaluated in cancer cell lines and in fresh ns-TGCTs. The ERCC1 protein was also determined in ns-TGCTs. The differences between CPS and non-CPS cell lines and patients were analysed by Student’s t- or χ2-tests. The differences in OS were analysed using the log-rank test, and the hazard ratios (HRs) were calculated using the Cox model.Results:High ERCC1 expression was observed in the non-CPS cells, and both ERCC1 and γH2AX expressions were augmented after cisplatin treatment. Increased ERCC1 expression was also identified in non-CPS patients. Neither polymorphism was associated with either CPS or OS. The presence of ERCC1 was associated with non-CPS (P=0.05) and adjusted in the prognosis groups. The HR in ERCC1-negative and non-CPS patients was >14.43, and in ERCC1-positive and non-CPS patients the HR was >11.86 (P<0.001).Conclusions:High levels of ERCC1 were associated with non-CPS, suggesting that ERCC1 could be used as a potential indicator of the response to cisplatin and prognosis in ns-TGCTs.

MAD2γ, a novel MAD2 isoform, reduces mitotic arrest and is associated with resistance in testicular germ cell tumors

ABSTRACT 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.

Abstract LB-171: Repression of miR-125b-1 by epigenetic mechanisms in breast cancer cell lines

miR-125b-1 downregulates targets as ERBB2, BAK1 and ETS1. These targets are involved in cell proliferation, apoptosis and cell migration, respectively. Previous studies on tumor cells reveal that downregulation of miR-125b-1 is associated with poor prognosis in breast cancer patients. DNA methylation of the miR-125b-1 promoter can repress its expression, in addition, this promoter is embedded in an intermediate CpG island thus, DNA methylation and histone modifications could also affect its transcription. Repression by DNA methylation has been well characterized, but there is no information about the role of histone modifications in the regulation of miR-125b-1 promoter. We evaluated the enrichment of two histone modifications involved in gene repression, H3K9me3 and H3K27me3, on the miR-125b-1 promoter of two breast cancer cell lines, a luminal A, MCF 7, and a triple negative, MDA-MB-231, compared with a non-transformed breast cell line, MCF 10A. We found that breast cancer cell lines are enriched with H3K27me3 and H3K9me3 in MCF 7 and MDA-MB-231, respectively. Then, we focused on reactivating miR-125b-1 in MCF 7 using an EZH2 inhibitor. After the treatment with the EZH2 inhibitor, we evaluated the transcriptional levels of the pri-miR-125b-1 and the mature miR-125b by qRT-PCR. Our results suggest that transcripts, pri-miRNA and mature miRNA, increase their expression levels after the treatment in the MCF7 cell line, but not in the MDA-MB-231 and MCF 10A cell lines. Subsequently, we evaluated the BAK1 expression and protein levels to investigate whether the miR-125b-1 reactivation could affect some targets. We observed a 60% and 70% decrease in the expression and protein levels of after treatment with the EZH2 inhibitor. To determine if the H3K9me3 is involved on miR-125b-1 silencing in MDA-MB-231, we over-expressed KDM4B/JMJD2B to reactivate this miRNA. Then, we evaluated the transcript. A three-fold increase was observed compared. We conclude that the miR-125b-1 can be repressed by different epigenetic mechanisms depending on the breast cancer subtype; the miR-125b-1 reactivation by removing the repression histone modification marks affect the expression of BAK1, a pro-apoptotic target. Citation Format: Fernanda Cisneros-Soberanis, Marco Alonso Andonegui, Clementina Castro, Luis Alonso Herrera. Repression of miR-125b-1 by epigenetic mechanisms in breast cancer cell lines. [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 LB-171.

Abstract 2728: In silico identification of a MAD2-interacting motif in MAD2 spliced isoforms suggest a functional interaction with the spindle assemble checkpoint in cancer

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.

Abstract LB-84: The role of survivin and survivin variants in the mitotic arrest-induced cell death

Eukaryotic cells rely on a mitotic checkpoint in order to preserve their proper ploidy. This mechanism stops mitosis progression before anaphase until each kinetochore is adequately bound to mitotic microtubules, and tension is generated which splits up chromatids towards opposite poles of the mitotic spindle. The resulting paused state of the cell cycle is known as mitotic arrest, and has been long known to induce cell death. However, the mechanisms driving mitotic arrest-mediated cell death have not yet been elucidated. It has been hypothesized that an anti-apoptotic/pro-apoptotic protein interplay might define the mitotic arrest outcome. We are interested in defining the role of survivin and its splicing variants, survivin Δex3 and survivin 2b, in the mitotic arrest-induced cell death. Survivin is a multifaceted member of the IAP family of proteins with functions in the chromosome passenger complex that has been shown to be over expressed in nearly every human tumor type. Noteworthy, Survivin Δex3 retains IAP activity but Survivin 2b has been shown to act as a pro-apoptotic factor. In this study, we defined the mitotic index (MI) of HCT116 cells in response to different concentrations of taxol and vinblastine. The results show that vinblastine induces a wide MI oscillation in contrast to taxol, which produces a homogeneous behavior in the cell population. This difference in population behavior might be explained by the different mechanisms of action of taxol and vinblastine. From the maximum taxol-induced MI point on, the percentage of sub-2n cells increased at the expense of mitotic cells, as assessed by flow cytometry. These results suggest that, in our model, cells die by exposure to taxol in a mitotic arrest-dependent manner. We also determined, by RT-PCR, that survivin variants are endogenously expressed in HCT116 cells. Furthermore, in immunoblot experiments, we found that survivin protein level decreases throughout taxol treatment in contrast to Bub1 levels, a member of the spindle assembly checkpoint. These results suggest that the decreased survivin level might be related to mitotic arrest-dependent cell death. The analysis of the intracellular localization of survivin variants throughout the mitotic arrest showed that survivin is localized in mitotic chromosomes throughout mitosis (before anaphase) independently of the time of treatment with taxol and vinblastine. Noteworthy, we also found that survivin Δex3 localized to the cytosol, and its level decreases throughout mitosis while survivin 2b protein was diffusely localized in the cytosol and mitotic chromosomes, and its level and localization remained unchanged. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-84. doi:1538-7445.AM2012-LB-84