Within the ovarian follicle, immature oocytes are surrounded and supported by granulosa cells (GCs). Stimulation of GCs by FSH leads to their proliferation and differentiation, events that are necessary for fertility. FSH activates multiple signaling pathways to regulate genes necessary for follicular maturation. Herein, we investigated the role of Y-box-binding protein-1 (YB-1) within GCs. YB-1 is a nucleic acid binding protein that regulates transcription and translation. Our results show that FSH promotes an increase in the phosphorylation of YB-1 on Ser 102 within 15 min that is maintained at significantly increased levels until ϳ8 h post treatment. FSH-stimulated phosphorylation of YB-1(Ser 102 ) is prevented by pretreatment of GCs with the PKA-selective inhibitor PKA inhibitor (PKI), the MEK inhibitor PD98059, or the ribosomal S6 kinase-2 (RSK-2) inhibitor BI-D1870. Thus, phosphorylation of YB-1 on Ser 102 is PKA-, ERK-, and RSK-2-dependent. However, pretreatment of GCs with the protein phosphatase 1 (PP1) inhibitor tautomycin increased phosphorylation of YB-1(Ser 102 ) in the absence of FSH; FSH did not further increase YB-1(Ser 102 ) phosphorylation. This result suggests that the major effect of RSK-2 is to inhibit PP1 rather than to directly phosphorylate YB-1 on Ser 102 . YB-1 coimmunoprecipitated with PP1␤ catalytic subunit and RSK-2. Transduction of GCs with the dephospho-adenoviral-YB-1(S102A) mutant prevented the induction by FSH of Egfr, Cyp19a1, Inha, Lhcgr, Cyp11a1, Hsd17b1, and Pappa mRNAs and estradiol-17␤ production. Collectively, our results reveal that phosphorylation of YB-1 on Ser 102 via the ERK/ RSK-2 signaling pathway is necessary for FSH-mediated expression of target genes required for maturation of follicles to a preovulatory phenotype.In the female, fertility requires maturation of the ovarian follicle that contains the oocyte surrounded by granulosa cells (GCs) 2 and theca cells. Follicular maturation is a tightly regulated process that is initiated by FSH. FSH regulates at least 500 target genes within GCs whose expression drives development of the follicle, allowing it to respond to the surge of luteinizing hormone (LH) that promotes ovulation, oocyte maturation, and formation of the corpus luteum that serves to support the developing embryo after fertilization and implantation (for review, see Refs. 1 and 2). The mechanism by which FSH signals to regulate gene and protein expression in GCs has been extensively investigated. FSH binds to its G protein-coupled receptor (GPCR) expressed exclusively on GCs to activate adenylyl cyclase, raise intracellular cAMP levels, and activate PKA (3-6). PKA then either directly phosphorylates proteins that regulate transcription or indirectly activates signaling cascades whose targets regulate primarily transcription and translation. Direct PKA targets in GCs include cAMP-response element binding protein (CREB) (Ser 133 ) (7), histone H3 (Ser 10 ) (8), and ␤-catenin (Ser 552 and Ser 675 ) (9). Upon phosphorylation, these direct PKA target pro...

FSH promotes maturation of ovarian follicles. One pathway activated by FSH in granulosa cells (GCs) is phosphatidylinositol-3 kinase/AKT. The AKT target FOXO1 is reported to function primarily as a repressor of FSH genes, including Ccnd2 and Inha. Based on its broad functions in other tissues, we hypothesized that FOXO1 may regulate many more GC genes. We transduced GCs with empty adenovirus or constitutively active FOXO1 followed by treatment with FSH for 24 hours, and conducted RNA deep sequencing. Results show that FSH regulates 3,772 genes ≥ 2.0-fold; 60% of these genes are activated or repressed by FOXO1. Pathway Studio Analysis revealed enrichment of genes repressed by FOXO1 in metabolism, signaling, transport, development, and activated by FOXO1 in signaling, cytoskeletal functions, and apoptosis. Gene regulation was verified by q-PCR (eight genes) and ChIP analysis (two genes). We conclude that FOXO1 regulates the majority of FSH target genes in GCs.

Within the ovarian follicle, granulosa cells (GCs) surround and support immature oocytes. FSH promotes the differentiation and proliferation of GCs and is essential for fertility. We recently reported that ERK activation is necessary for FSH to induce key genes that define the preovulatory GC. This research focused on the phosphoregulation by FSH of ERK within GCs.

Protein kinase A (PKA) has recently been shown to mimic the actions of follicle-stimulating hormone (FSH) by activating signaling pathways that promote granulosa cell (GC) differentiation, such as phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK). We sought to elucidate the mechanism by which PKA, a Ser/Thr kinase, intersected the PI3K/AKT and MAPK/ERK pathways that are canonically activated by receptor tyrosine kinases (RTKs). Our results show that for both of these pathways, the RTK is active in the absence of FSH yet signaling down the pathways to commence transcriptional responses requires FSH-stimulated PKA activation. For both pathways, PKA initiates signaling by regulating the activity of a protein phosphatase (PP). For the PI3K/AKT pathway, PKA activates the Ser/Thr PP1 complexed with the insulinlike growth factor 1 receptor (IGF-1R) and insulin receptor substrate 1 (IRS1) to dephosphorylate Ser residues on IRS1, authorizing phosphorylation of IRS1 by the IGF-1R to activate PI3K. Treatment of GCs with FSH and exogenous IGF-1 initiates synergistic IRS1 Tyr phosphorylation and resulting gene activation. The mechanism by which PKA activates PI3K is conserved in preovulatory GCs, MCF7 breast cancer cells, and FRTL thyroid cells. For the MAPK/ERK pathway, PKA promotes inactivation of the MAPK phosphatase (MKP) dual specificity phosphatase (DUSP) MKP3/DUSP6 to permit MEK-phosphorylated ERK to accumulate downstream of the epidermal growth factor receptor. Thus, for the two central signaling pathways that regulate gene expression in GCs, FSH via PKA intersects canonical RTK-regulated signaling by modulating the activity of PPs.

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Triple negative breast cancer (TNBC) is a collection of heterogeneous diseases with limited therapeutic options primarily involving cytotoxic chemotherapy. The distinct molecular profile and increased chromosomal instability (CIN) of TNBC make it a difficult disease to treat. While many patients initially respond to treatment, resistance is common, resulting in poor patient outcomes. Thus identifying vulnerabilities in this disease is necessary to improve TNBC patient outcomes. To identify potential therapeutic targets in this disease, we focused on Src Family Kinases (SFKs). The SFK family is comprised of 9 non-receptor tyrosine kinases that interact with upstream signaling partners to regulate cell phenotypes such as adhesion, motility, survival and mitosis. Five SFKs are overexpressed in breast cancer, including Src, the founding member of the family. Within the basal breast cancer subtype, one of the most highly overexpressed SFKs is YES1. High YES1 expression is also associated with poorer outcomes in TNBC patients when compared to tumors with low YES1 expression. We have found that TNBC cells are reliant on sustained YES1 expression for viability, growth, cell cycle progression, and maintenance of genomic stability. Transiently silencing YES1 causes a significant decrease in cell growth and increase in apoptosis. Furthermore, loss of YES1 expression induces features of whole chromosomal instability (w-CIN), including micronucleation, multinucleation, and dysmorphic nuclei. An increase in γ-H2AX positive staining was also noted in cells with decreased YES1 expression, indicating an accumulation of double strand DNA breaks (DSB) that are indicative of structural chromosomal instability (s-CIN). These findings were recapitulated using several pan-SFK inhibitors, including FDA approved agents dasatinib and saracatinib. Loss of YES1 function also results in perturbed cell cycle progression, particularly a G2/M delay. RNA-sequencing and Reverse Phase Protein Array (RPPA) revealed alterations in mitotic, replication stress, and DNA repair pathways following the loss of YES1. These data demonstrate that YES1 is a previously understudied signaling component of mitotic- and DNA damage-regulating pathways in TNBC. More broadly, they suggest that YES1 may be a therapeutically targetable vulnerability that could be paired with other DNA damaging or mitotic inhibitors to improve treatment efficacy in TNBC. Current SFK-targeted agents globally impact all SFKs. The data presented here support efforts to identify a YES1 selective inhibitor that should be more specific for treating TNBC with less toxicity that pan-SFK drugs. Citation Format: Katrina Piemonte, Elyse Donaubauer, Ruth Keri. The SRC family kinase, YES1, controls chromosomal stability and promotes growth of triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-06.

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