The Influence of Oncogenic RAS on Chemotherapy and Radiotherapy Resistance Through DNA Repair Pathways.

RAS oncogenes are chief tumorigenic drivers, and their mutation constitutes a universal predictor of poor outcome and treatment resistance. Despite more than 30 years of intensive research since the identification of the first RAS mutation, most attempts to therapeutically target RAS mutants have failed to reach the clinic. In fact, the first mutant RAS inhibitor, Sotorasib, was only approved by the FDA until 2021. However, since Sotorasib targets the KRAS G12C mutant with high specificity, relatively few patients will benefit from this therapy. On the other hand, indirect approaches to inhibit the RAS pathway have revealed very intricate cascades involving feedback loops impossible to overcome with currently available therapies. Some of these mechanisms play different roles along the multistep carcinogenic process. For instance, although mutant RAS increases replicative, metabolic and oxidative stress, adaptive responses alleviate these conditions to preserve cellular survival and avoid the onset of oncogene-induced senescence during tumorigenesis. The resulting rewiring of cellular mechanisms involves the DNA damage response and pathways associated with oxidative stress, which are co-opted by cancer cells to promote survival, proliferation, and chemo- and radioresistance. Nonetheless, these systems become so crucial to cancer cells that they can be exploited as specific tumor vulnerabilities. Here, we discuss key aspects of RAS biology and detail some of the mechanisms that mediate chemo- and radiotherapy resistance of mutant RAS cancers through the DNA repair pathways. We also discuss recent progress in therapeutic RAS targeting and propose future directions for the field.

Proteasome inhibition alters mitotic progression through the upregulation of centromeric α-Satellite RNAs.

Cell cycle progression requires control of the abundance of several proteins and RNAs over space and time to properly transit from one phase to the next and to ensure faithful genomic inheritance in daughter cells. The proteasome, the main protein degradation system of the cell, facilitates the establishment of a proteome specific to each phase of the cell cycle. Its activity also strongly influences transcription. Here, we detected the upregulation of repetitive RNAs upon proteasome inhibition in human cancer cells using RNA-seq. The effect of proteasome inhibition on centromeres was remarkable, especially on α-Satellite RNAs. We showed that α-Satellite RNAs fluctuate along the cell cycle and interact with members of the cohesin ring, suggesting that these transcripts may take part in the regulation of mitotic progression. Next, we forced exogenous overexpression and used gapmer oligonucleotide targeting to demonstrate that α-Sat RNAs have regulatory roles in mitosis. Finally, we explored the transcriptional regulation of α-Satellite DNA. Through in silico analyses, we detected the presence of CCAAT transcription factor-binding motifs within α-Satellite centromeric arrays. Using high-resolution three-dimensional immuno-FISH and ChIP-qPCR, we showed an association between the α-Satellite upregulation and the recruitment of the transcription factor NFY-A to the centromere upon MG132-induced proteasome inhibition. Together, our results show that the proteasome controls α-Satellite RNAs associated with the regulation of mitosis.

miR-125b-1 is repressed by histone modifications in breast cancer cell lines.

PURPOSE: Downregulation of miR-125b-1 is associated with poor prognosis in breast cancer patients. In this work we investigated the effect of histone modifications on the regulation of this gene promoter. METHODS AND RESULTS: We evaluated the enrichment of two histone modifications involved in gene repression, H3K9me3 and H3K27me3, on the miR-125b-1 promoter in two breast cancer cell lines, MCF7 (luminal A subtype) and MDA-MB-231 (triple-negative subtype), compared to the non-transformed breast cell line MCF10A. H3K27me3 and H3K9me3 were enriched in MCF7 and MDA-MB-231 cells, respectively. Next, we used an EZH2 inhibitor to examine the reactivation of miR-125b-1 in MCF7 cells and evaluated the transcriptional levels of pri-miR-125b-1 and mature miR-125b by qRT-PCR. pri-miRNA and mature miRNA transcripts were both increased after treatment of MCF7 cells with the EZH2 inhibitor, whereas no effect on miR-125b-1 expression levels was observed in MDA-MB-231 and MCF10A cells. We subsequently evaluated the effect of miR-125b-1 reactivation on the expression and protein levels of BAK1, a target of miR-125b. We observed 60 and 70 % decreases in the expression and protein levels of BAK1, respectively, compared to cells that were not treated with the EZH2 inhibitor. We over-expressed KDM4B/JMJD2B to reactivate this miRNA, resulting in a three-fold increase in miR-125b expression compared with the same cell line without KDM4B/JMJD2B over-expression. CONCLUSION: The miR-125b-1 is repressed by different epigenetic mechanisms depending on the breast cancer subtype and that miR-125b-1 reactivation specifically eliminates the effect of repressive histone modifications on the expression of an pro-apoptotic target.

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

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.

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.