Unraveling the molecular events driving BRCA1 -associated transformation in novel models of early müllerian cancer

Clinical and translational research studies over the last two decades have revealed that high-grade serous carcinomas (HGSCs) primarily originate from fallopian tube secretory epithelial cells (FTSECs). Germline mutations in BRCA1/2 are the strongest risk factors associated with HGSC development, but the earliest stages of neoplastic transformation, and the precise role in increasing cancer risk in FTSECs, remains poorly understood. To address this need, novel FTSEC models were generated by harvesting epithelial cells from fallopian tube fimbria by exfoliative cytology, a protocol that allowed for the use of tissue for both clinical and research applications. The study included 4 patients with high-risk germline BRCA1 mutations undergoing risk-reducing prophylactic salpingo-oophorectomy surgery, and 4 average risk patients undergoing salpingectomy for reasons unrelated to ovarian cancer risk. Immortalization and features of neoplastic transformation were induced by transducing cells with a cDNA encoding TERT , dominant negative p53 , mutant CDK4 R24C , and CMYC . Transduced cells bypassed replicative senescence and maintained expression of gynecologic epithelial keratins and FTSEC lineage marker PAX8. To explore if FTSECs with high-risk BRCA1 mutations were more prone to neoplastic transformation and DNA damage, colony formation assay, and camptothecin treatment with immunofluorescent analysis of gammaH2A.X, 53BP1, and Rad51, were assessed. We found that high-risk FTSECs displayed increased MYC -induced colony formation and DNA damage compared to average-risk FTSECs. Single cell RNA-sequencing of high-risk tubal brushings identified upregulated expression of genes encoding the electron transport chain, suggesting a metabolic alteration may characterize the precancer niche in BRCA1 -mutant fallopian tubes. Using Agilent's Seahorse assay, we found that oxidative phosphorylation is significantly decreased in high-risk FTSECs compared to FTSECs from average risk patients. Additionally, high-risk FTSECs exhibit decreased mitochondria fission. Decreased mitochondria fission coupled with a decrease in oxidative phosphorylation, suggests metabolic defects may be occurring due to the lack of mitochondria turnover and persistence of dysfunctional mitochondria. These observations uncover a new aspect of BRCA1 biology and give new insight into metabolic perturbations occurring in high-risk fallopian tube epithelia. This work provides new models to study early tumorigenesis in the fallopian tube of high-risk mutation carriers, and demonstrates the utility of these models to investigate mechanisms underlying elevated cancer susceptibility in high-risk mutation carriers. More importantly, they have the potential to serve as models for biomarker discovery to identify early-stage markers to aid in Stage I diagnoses in the general population.