Dormant origin firing promotes head-on transcription-replication conflicts at transcription termination sites in response to BRCA2 deficiency.

BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome.

Norepinephrine induces anoikis resistance in high-grade serous ovarian cancer precursor cells.

High-grade serous carcinoma (HGSC) is the most lethal gynecological malignancy in the United States. Late diagnosis and the emergence of chemoresistance have prompted studies into how the tumor microenvironment, and more recently tumor innervation, may be leveraged for HGSC prevention and interception. In addition to stess-induced sources, concentrations of the sympathetic neurotransmitter norepinephrine (NE) in the ovary increase during ovulation and after menopause. Importantly, NE exacerbates advanced HGSC progression. However, little is known about the role of NE in early disease pathogenesis. Here, we investigated the role of NE in instigating anchorage independence and micrometastasis of preneoplastic lesions from the fallopian tube epithelium (FTE) to the ovary, an essential step in HGSC onset. We found that in the presence of NE, FTE cell lines were able to survive in ultra-low-attachment (ULA) culture in a ß-adrenergic receptor-dependent (ß-AR-dependent) manner. Importantly, spheroid formation and cell viability conferred by treatment with physiological sources of NE were abrogated using the ß-AR blocker propranolol. We have also identified that NE-mediated anoikis resistance may be attributable to downregulation of colony-stimulating factor 2. These findings provide mechanistic insight and identify targets that may be regulated by ovary-derived NE in early HGSC.

Stromal mediated DNA damage promotes high grade serous ovarian cancer initiation.

The fundamental steps in high-grade serous ovarian cancer (HGSOC) initiation are unclear, thus providing critical barriers to the development of prevention or early detection strategies for this deadly disease. Increasing evidence demonstrates most HGSOC starts in the fallopian tube epithelium (FTE). Current models propose HGSOC initiates when FTE cells acquire increasing numbers of mutations allowing cells to evolve into serous tubal intraepithelial carcinoma (STIC) precursors and then to full blown cancer. Here we report that epigenetically altered mesenchymal stem cells (termed high risk MSC-hrMSCs) can be detected prior to the formation of ovarian cancer precursor lesions. These hrMSCs drive DNA damage in the form of DNA double strand breaks in FTE cells while also promoting the survival of FTE cells in the face of DNA damage. Indicating the hrMSC may actually drive cancer initiation, we find hrMSCs induce full malignant transformation of otherwise healthy, primary FTE resulting in metastatic cancer in vivo . Further supporting a role for hrMSCs in cancer initiation in humans, we demonstrate that hrMSCs are highly enriched in BRCA1/2 mutation carriers and increase with age. Combined these findings indicate that hrMSCs may incite ovarian cancer initiation. These findings have important implications for ovarian cancer detection and prevention.