P53 Gain-of-Function Mutants and Steroids in Ovarian Cancer Cell Metastasis

Abstract

High-grade serous ovarian cancer (HGSOC) is a heterogeneous disease for which there currentlyis no cure. Because p53 is mutated in >90% of all ovarian cancer, we studied specific gain-of-function (GOF) p53 mutants and steroid hormones for tumor morphology and metastasis in vivo. For this, we analyzed ALST (WT p53), SKOV3 (p53 null), TYK-NU (p53-R175H), OVCAR3 (p53-R248Q) and OVCA420 (p53-R273H) cell line xenografts in Foxn1-/- mice. ALST cells failed tometastasize, likely due to the known apoptotic effects of WT p53. SKOV3 and the p53-GOF celllines metastasized to the omentum and exhibited distinct morphologies: SKOV3, epithelial-like;TYK-Nu, vascular-like; OVCAR3, epithelial/mesenchymal; and OVCA420 epithelial exclusive. Despite different morphologies and p53 status, each tumor type contained large, Polyploid GiantCancer Cells (PGCCs) that are stem-like cells undergoing endoreplication. A specificphosphorylated, active form of β−catenin (pCTNNB1-S31/S37/T41; pCTNNB1) co-localizedselectively with GOF p53 and the mitotic stress regulatory kinase pMSK1-T581 in mitotic cells andPGCCs, indicating that in addition to GOF p53 mutants, pCTNNB1 and pMSK1 play a role intumor progression. To determine if ALST cells could be rendered metastatic, the p53 GOFmutants R175H and R273H were stably expressed in these cells. Remarkably, the ALST(WT/R273H) cells, but not the ALST (WT/R175H) cells, formed solid tumors on the ovary, visceralfat and uterus; but not on the omentum where OVCA420 (p53-R273H) cells formed tumors. TheALST (WT/R273H) tumors harbored discreet populations of pCTNNB1+ mitotic cells and PGCCsand exhibited distinct growth-promoting responses to estradiol while showing growth-inhibitoryeffects of DHT and nuclear AR in the tumor-associated stromal cells. Stably expressing p53-R175H or p53-R273H GOF mutants in SKOV3 (p53 null) cells enhanced tumor progression withthe R273H mutant being most aggressive. The parental SKOV3 tumors also contained pCTNNB1that was associated with mitotic cells and PGCCs. In the SKOV3 p53-R175H cells - and moreimpressively in the p53-R273H cells - pCTNNB1 co-localized with p53 in PGCCs. Whereas ESR1staining was diffuse in the ALST-R273H cells, it was nuclear in the SKOV3 cell lines. However,tumor promotion by estrogen and inhibition by DHT were observed in each SKOV3 cell line,suggesting that stromal cells may also contribute to the steroid dependent effects. Collectively, 1)elevated levels of specific phosphorylated forms of CTNNB1 and MSK1 identify mitotic cells andPGCCs within the tumors and new targets for therapeutic approaches; 2) knowing the status ofp53, and the presence and localization of pCTNNB1 and pMSK1 and steroid hormone receptorsin ovarian tumors suggest combinatorial approaches should be considered to combat the mostlethal gynecological cancer. NIH-CA181808; NIH-HD097321 (JSR).