Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C.

Pathogenic protein-truncating variants of RAD51C, which plays an integral role in promoting DNA damage repair, increase the risk of breast and ovarian cancer. A large number of RAD51C missense variants of uncertain significance (VUS) have been identified, but the effects of the majority of these variants on RAD51C function and cancer predisposition have not been established. Here, analysis of 173 missense variants by a homology-directed repair (HDR) assay in reconstituted RAD51C-/- cells identified 30 nonfunctional (deleterious) variants, including 18 in a hotspot within the ATP-binding region. The deleterious variants conferred sensitivity to cisplatin and olaparib and disrupted formation of RAD51C/XRCC3 and RAD51B/RAD51C/RAD51D/XRCC2 complexes. Computational analysis indicated the deleterious variant effects were consistent with structural effects on ATP-binding to RAD51C. A subset of the variants displayed similar effects on RAD51C activity in reconstituted human RAD51C-depleted cancer cells. Case-control association studies of deleterious variants in women with breast and ovarian cancer and noncancer controls showed associations with moderate breast cancer risk [OR, 3.92; 95% confidence interval (95% CI), 2.18-7.59] and high ovarian cancer risk (OR, 14.8; 95% CI, 7.71-30.36), similar to protein-truncating variants. This functional data supports the clinical classification of inactivating RAD51C missense variants as pathogenic or likely pathogenic, which may improve the clinical management of variant carriers. SIGNIFICANCE: Functional analysis of the impact of a large number of missense variants on RAD51C function provides insight into RAD51C activity and information for classification of the cancer relevance of RAD51C variants.

miR-181a modulates circadian rhythm in immortalized bone marrow and adipose derived stromal cells and promotes differentiation through the regulation of PER3.

miRNAs are important regulators of diverse cellular processes including proliferation, apoptosis, and differentiation. In the context of bone marrow derived stromal cell and adipose derived stromal cell differentiation, miRNAs are established regulators of both differentiation or stemness depending on their target. Furthermore, miRNA dysregulation can play a key role in various disease states. Here we show that miR-181a is regulated in a circadian manner and is induced during both immortalized bone marrow derived stromal cell (iBMSC) as well as primary patient adipose derived stromal cell (PASC) adipogenesis. Enhanced expression of miR-181a in iBMSCs  and PASCs produced a robust increase in adipogenesis through the direct targeting of the circadian factor period circadian regulator 3 (PER3). Furthermore, we show that knocking down endogenous miR-181a expression in iBMSC has a profound inhibitory effect on iBMSC adipogenesis through its regulation of PER3. Additionally, we found that miR-181a regulates the circadian dependency of the adipogenesis master regulator PPARγ. Taken together, our data identify a previously unknown functional link between miR-181a and the circadian machinery in immortalized bone marrow stromal cells and adipose derived stromal cells highlighting its importance in iBMSC and ASC adipogenesis and circadian biology.

CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors.

Effective targeting of cancer stem cells (CSCs) requires neutralization of self-renewal and chemoresistance, but these phenotypes are often regulated by distinct molecular mechanisms. Here we report the ability to target both of these phenotypes via CD55, an intrinsic cell surface complement inhibitor, which was identified in a comparative analysis between CSCs and non-CSCs in endometrioid cancer models. In this context, CD55 functions in a complement-independent manner and required lipid raft localization for CSC maintenance and cisplatin resistance. CD55 regulated self-renewal and core pluripotency genes via ROR2/JNK signaling and in parallel cisplatin resistance via lymphocyte-specific protein tyrosine kinase (LCK) signaling, which induced DNA repair genes. Targeting LCK signaling via saracatinib, an inhibitor currently undergoing clinical evaluation, sensitized chemoresistant cells to cisplatin. Collectively, our findings identify CD55 as a unique signaling node that drives self-renewal and therapeutic resistance through a bifurcating signaling axis and provides an opportunity to target both signaling pathways in endometrioid tumors.

Altered glutamine metabolism in platinum resistant ovarian cancer.

Ovarian cancer is characterized by an increase in cellular energy metabolism, which is predominantly satisfied by glucose and glutamine. Targeting metabolic pathways is an attractive approach to enhance the therapeutic effectiveness and to potentially overcome drug resistance in ovarian cancer. In platinum-sensitive ovarian cancer cell lines the metabolism of both, glucose and glutamine was initially up-regulated in response to platinum treatment. In contrast, platinum-resistant cells revealed a significant dependency on the presence of glutamine, with an upregulated expression of glutamine transporter ASCT2 and glutaminase. This resulted in a higher oxygen consumption rate compared to platinum-sensitive cell lines reflecting the increased dependency of glutamine utilization through the tricarboxylic acid cycle. The important role of glutamine metabolism was confirmed by stable overexpression of glutaminase, which conferred platinum resistance. Conversely, shRNA knockdown of glutaminase in platinum resistant cells resulted in re-sensitization to platinum treatment. Importantly, combining the glutaminase inhibitor BPTES with platinum synergistically inhibited platinum sensitive and resistant ovarian cancers in vitro. Apoptotic induction was significantly increased using platinum together with BPTES compared to either treatment alone. Our findings suggest that targeting glutamine metabolism together with platinum based chemotherapy offers a potential treatment strategy particularly in drug resistant ovarian cancer.

Cisplatin induces stemness in ovarian cancer.

The mainstay of treatment for ovarian cancer is platinum-based cytotoxic chemotherapy. However, therapeutic resistance and recurrence is a common eventuality for nearly all ovarian cancer patients, resulting in poor median survival. Recurrence is postulated to be driven by a population of self-renewing, therapeutically resistant cancer stem cells (CSCs). A current limitation in CSC studies is the inability to interrogate their dynamic changes in real time. Here we utilized a GFP reporter driven by the NANOG-promoter to enrich and track ovarian CSCs. Using this approach, we identified a population of cells with CSC properties including enhanced expression of stem cell transcription factors, self-renewal, and tumor initiation. We also observed elevations in CSC properties in cisplatin-resistant ovarian cancer cells as compared to cisplatin-naïve ovarian cancer cells. CD49f, a marker for CSCs in other solid tumors, enriched CSCs in cisplatin-resistant and -naïve cells. NANOG-GFP enriched CSCs (GFP+ cells) were more resistant to cisplatin as compared to GFP-negative cells. Moreover, upon cisplatin treatment, the GFP signal intensity and NANOG expression increased in GFP-negative cells, indicating that cisplatin was able to induce the CSC state. Taken together, we describe a reporter-based strategy that allows for determination of the CSC state in real time and can be used to detect the induction of the CSC state upon cisplatin treatment. As cisplatin may provide an inductive stress for the stem cell state, future efforts should focus on combining cytotoxic chemotherapy with a CSC targeted therapy for greater clinical utility.

Critical role of Wnt/ß-catenin signaling in driving epithelial ovarian cancer platinum resistance.

Resistance to platinum-based chemotherapy is the major barrier to treating epithelial ovarian cancer. To improve patient outcomes, it is critical to identify the underlying mechanisms that promote platinum resistance. Emerging evidence supports the concept that platinum-based therapies are able to eliminate the bulk of differentiated cancer cells, but are unable to eliminate cancer initiating cells (CIC). To date, the relevant pathways that regulate ovarian CICs remain elusive. Several correlative studies have shown that Wnt/ß-catenin pathway activation is associated with poor outcomes in patients with high-grade serous ovarian cancer (HGSOC). However, the functional relevance of these findings remain to be delineated. We have uncovered that Wnt/ß-catenin pathway activation is a critical driver of HGSOC chemotherapy resistance, and targeted inhibition of this pathway, which eliminates CICs, represents a novel and effective treatment for chemoresistant HGSOC. Here we show that Wnt/ß-catenin signaling is activated in ovarian CICs, and targeted inhibition of ß-catenin potently sensitized cells to cisplatin and decreased CIC tumor sphere formation. Furthermore, the Wnt/ß-catenin specific inhibitor iCG-001 potently sensitized cells to cisplatin and decreased stem-cell frequency in platinum resistant cells. Taken together, our data is the first report providing evidence that the Wnt/ß-catenin signaling pathway maintains stem-like properties and drug resistance of primary HGSOC PDX derived platinum resistant models, and therapeutic targeting of this pathway with iCG-001/PRI-724, which has been shown to be well tolerated in Phase I trials, may be an effective treatment option.

miRNAs as prognostic and therapeutic tools in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and is the fifth leading cause of cancer deaths in women. Developing adjuvant therapy to circumvent drug resistance represents an important aspect of current initiatives to improve survival in women with advanced EOC. A regulatory molecule that can act on multiple genes associated with a chemoresistant phenotype will be the ideal target for the development of therapeutics to overcome resistance and miRNAs constitute promising tools in this regard. In this review, we discuss the emerging role of miRNAs in regulating EOC phenotype with a focus on prognostic and therapeutic importance of miRNAs and the possibility of miRNA modulation as a tool to improve efficacy of chemotherapy in EOC.