Gene copy number alterations, tumor cell stemness, and the development of platinum chemotherapy resistance contribute to high-grade serous ovarian cancer (HGSOC) recurrence. Stem phenotypes involving Wnt-β-catenin, aldehyde dehydrogenase activities, intrinsic platinum resistance, and tumorsphere formation are here associated with spontaneous gains in Kras, Myc and FAK (KMF) genes in a new aggressive murine model of ovarian cancer. Adhesion-independent FAK signaling sustained KMF and human tumorsphere proliferation as well as resistance to cisplatin cytotoxicity. Platinum-resistant tumorspheres can acquire a dependence on FAK for growth. Accordingly, increased FAK tyrosine phosphorylation was observed within HGSOC patient tumors surviving neo-adjuvant chemotherapy. Combining a FAK inhibitor with platinum overcame chemoresistance and triggered cell apoptosis. FAK transcriptomic analyses across knockout and reconstituted cells identified 135 targets, elevated in HGSOC, that were regulated by FAK activity and β-catenin including Myc, pluripotency and DNA repair genes. These studies reveal an oncogenic FAK signaling role supporting chemoresistance.

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Ovarian cancer is the fifth-leading cause of cancer death among women. The dissemination of ovarian tumors and growth as spheroids accompanies late stage disease. In cell culture, ovarian tumor cell spheroids can exhibit elevated resistance to environmental stressors such as reactive oxygen species. Homeostatic balance of the antioxidant response is a protective mechanism that prevents anoikis, a form of programmed cell death. Signaling pathways activated by integrin receptors suppress anoikis. Rgnef (ARHGEF28/p190RhoGEF) is a guanine nucleotide exchange factor that is activated downstream of integrins. We find that Rgnef protein levels are elevated in late-stage serous ovarian cancer, high Rgnef mRNA levels are associated with decreased progression-free and overall survival, and genomic ARHGEF28 loss is associated with increased patient survival. Using transgenic and transplantable Rgnef knockout mouse models, we find that Rgnef is essential for supporting three-dimensional ovarian spheroid formation in vitro and tumor growth in mice. Using RNA-sequencing and bioinformatic analyses, we identify a conserved Rgnef-supported anti-oxidant gene signature including GPX4, Nqo1, and Gsta4; common targets of the NF-kB transcription factor. Antioxidant treatment enhanced growth of Rgnef-knockout spheroids and Rgnef re-expression facilitated NF-κB-dependent tumorsphere survival. These studies reveal a new role for Rgnef in ovarian cancer to facilitate NF-κB-mediated gene expression protecting cells from oxidative stress.

In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3α/β and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. Here we show that the axon guidance cue Netrin-1 promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, we demonstrate that Netrin-1 can substitute for blockade of Gsk3α/β and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, we reveal how Netrin-1 and the balance of its receptors Neo1 and Unc5B co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase to inactivate Gsk3α/β and stabilize β-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment.Animal studies. Teratoma assays were performed with 7-week-old severe combined immunodeficient (SCID) male mice (CB17/SCID, Charles River). Ntn1 βgeo reporter and Netrin-1 conditional-KO mESCs were derived from C57/bl6 mixed-background pregnant females at 8-15 weeks of age. Blastocyst injections were done using BALB/cANRj embryos. Embryos were flushed with M2 medium (Sigma) and grown overnight in KSOM (Sigma) or sequential blast (Origio) medium. Genotyping of Ntn1 fl/fl embryos was performed as previously described 44 . X-gal was detected in blastocysts using secondary antibodies coupled with biotin, the Vectastain ABC kit and DAB (Vector System). Teratoma-formation assays were performed by injecting 1 × 10 6 mESCs in the testes of 7-week-old SCID mice. After 3-4 weeks, the mice were euthanized and lesions were surgically removed and fixed in formalin or in 4% paraformaldehyde for sections. For blastocyst injections, Ntn1 fl/fl , untreated or treated with TAM for 48 h, were injected into BALB/cANRj blastocysts. The day before injection, frozen BALB/cANRj morulas from Quickblasto (Janvier) were thawed according to the manufacturer's instructions and incubated overnight in KSOM medium (Millipore) at 37 °C with 5% CO 2 . Between 5 and 15 cells were injected into expanded blastocysts in M2 medium (Sigma) using standard blastocyst injection techniques. Blastocysts were then allowed to recover for a period of 1-3 h prior to being implanted into pseudopregnant females. All animal procedures were performed in accordance with institutional guidelines (French ceccapp project 01369.01).Cell culture. The following cell lines were used in the study. Cgr8 ES cells (ECACC 07032901) were provided by the B. Pain laboratory (SBRI, Bron, France). E14Tg2a ES cells (ATCC CRL 1821) were provided by the M. E. Torres Padilla laboratory (IES, Munchen, Germany). Ntn1 βgeo reporter 27 and Netrin-1 conditional-KO (Ntn1 fl/fl ) 44 mESC...