Theillet, C. | Search bioRxiv

bioRxiv2023

Replication poison treated BRCA1-deficient breast cancers are prone to MRE11 over-resection resulting in single strand DNA accumulation and mitotic catastrophe

BRCA1, BRCA2 and RAD51, key players of homologous recombination (HR) repair, are also involved in stalled DNA replication fork protection and repair. BRCA1-deficiency is encountered in 25% of Triple Negative Breast Cancer (TNBC). Here we investigated the sensitivity of BRCA1-deficient TNBC cell models to gemcitabine a frequently used replication poison that does not alter DNA structure. We show that BRCA1-deficient models, in contrast to their isogenic BRCA1-proficient counterparts, are superiorly sensitive to gemcitabine, accumulate massive levels of single strand DNA (ssDNA), in absence of RPA and RAD51 signals and elevated double strand break (DSB) numbers leading to cell death. Remarkably, ssDNA accumulation in gemcitabine-treated BRCA1-deficient cells was strongly diminished by the MRE11 inhibitor mirin, while it did not affect ssDNA levels resulting from PARP inhibitor olaparib treatment. The central role of MRE11 DNA resection strongly suggested that replication fork reversal may be important in response to replication poisoning by gemcitabine in BRCA1-deficient models. Furthermore, we demonstrate that gemcitabine-treated BRCA1-deficient cells showing massive ssDNA accumulation slipped into mitosis and produced mitotic bridges and micronuclei (MN) showing strong BrdU and {gamma}H2AX staining. Noticeably these BrdU-positive MN and DNA bridges triggered cGAS sensing. Our data, thus, strongly suggest that gemcitabine treatment could be beneficial in BRCA1-deficient TNBC both in terms of cancer cell death, but possibly as well in terms of antitumor immune response.

cancer biology↗

bioRxiv2020

MAGI1 inhibits the AMOTL2/p38 stress pathway and prevents luminal breast tumorigenesis

Alterations to cell polarization or to intercellular junctions are often associated with epithelial cancer progression, including breast cancers (BCa). We show here that the loss of the junctional scaffold protein MAGI1 is associated with bad prognosis in luminal BCa, and promotes tumorigenesis. E-cadherin and the actin binding scaffold AMOTL2 accumulate in MAGI1 deficient cells which are subjected to increased stiffness. These alterations are associated with low YAP activity, the terminal Hippo-pathway effector, but with an elevated ROCK and p38 Stress Activated Protein Kinase activities. Blocking ROCK prevented p38 activation, suggesting that MAGI1 limits p38 activity in part through releasing actin strength. Importantly, the increased tumorigenicity of MAGI1 deficient cells is rescued in the absence of AMOTL2 or after inhibition of p38, demonstrating that MAGI1 acts as a tumor-suppressor in luminal BCa by inhibiting an AMOTL2/p38 stress pathway.

cancer biology↗

bioRxiv2018

Distinct oncogenes drive distinct genome and epigenome alterations in human mammary epithelial cells

Gene expression differences, combined with distinct patterns of genomic rearrangements and epigenetic modifications, have laid the bases of molecular classification of breast cancer. Different molecular subtypes are thought to originate from different cell lineages in the mammary gland, but the early activation of an oncogene could also play a role. It is, however, difficult to discriminate the respective inputs of oncogene activation or cell type of origin in the natural history of the tumor. In this work, we have designed an experimental strategy aiming at determining whether activation of distinct oncogenic pathways in human mammary epithelial cells (HMEC) could lead to different patterns of genetic and epigenetic changes. We show that initial activation of CCNE1, WNT1 and RASv12, which activate distinct oncogenic pathways, in shp53 immortalized HMECs results in different and reproducible profiles of mRNA and miRNA expression, copy number alterations (CNA) and DNA methylation modifications. Noticeably, HMECs transformed by RAS bore very specific profiles of CNAs and DNA methylation, clearly distinct from those shown by CCNE1 and WNT1 transformed HMECs.\n\nGenes impacted by CNAs and CpG methylation in the RAS and the CCNE1/WNT1 clusters showed clear differences, illustrating the activation of distinct pathways. Our data show that early activation of distinct oncogenic pathways leads to active adaptive events resulting in specific sets of CNAs and DNA methylation changes. We, thus, propose that activation of different oncogenes could have a role in reshaping the genetic landscape of breast cancer subtypes.\n\nAuthor summaryGenetic and epigenetic changes are at the center of cancer development. Breast cancer molecular subtypes are defined on differences in genetic and epigenetic profiles and it is generally assumed these subtypes originate from different cell lineages in the mammary gland. We propose that founding oncogenic mutations could also have an impact. To address this question, we designed an experimental model, based on the ectopic expression of different oncogenes in human mammary epithelial cells (HMEC), and monitored genetic and DNA methylation changes occurring at different stages of cell transformation. We show that transformation of HMEC by distinct oncogenes resulted in clearly different and reproducible patterns of genetic and DNA methylation changes. Genes whose expression was modified by either CNAs or CpG methylation were consistent with the dominant pathways activated and reflected the phenotypes in the respective models. We propose that DNA methylation and CNA changes correspond to adaptive responses to the activation of the oncogenic pathways. Our data strongly suggest that early activation of distinct oncogenic insults will not only impinge on the phenotypic characteristics of the resulting tumors, but also have a strong impact on their genomic and epigenetic landscapes.

cancer biology↗