Ovarian cancer has the highest mortality of all gynecologic malignancies. As such, there is a need to identify molecular mechanisms that underlie tumor metastasis in ovarian cancer. Increased expression of receptor tyrosine kinase, DDR2, has been associated with worse patient survival. Identifying downstream targets of DDR2 may allow specific modulation of ovarian cancer metastatic pathways. Additionally, stromal cells play a critical role in metastasis. The crosstalk between tumor and stromal cells can lead to tumor progression. We first identified that tumor cells co-cultured with DDR2-expressing fibroblasts had lower periostin expression when compared to tumor cells co-cultured with DDR2-depleted fibroblasts. We confirmed that DDR2 regulates POSTN expression in ovarian cancer-associated fibroblasts (CAFs). We found that mesothelial cell clearance and invasion by tumor cells were enhanced three-fold when DDR2 and POSTN-expressing CAFs were present compared to DDR2 and POSTN-depleted CAFs. Furthermore, DDR2-depleted and POSTN-overexpressing CAFs co-injected with ovarian tumor cells had increased tumor burden compared to mice injected with tumor cells and DDR2 and POSTN-depleted CAFs. Furthermore, we demonstrated that DDR2 regulates periostin expression through integrin B1 (ITGB1). Stromal DDR2 is highly correlated with stromal POSTN expression in ovarian cancer patient tumors. Thus, DDR2 expression in CAFs regulates the steps of ovarian cancer metastasis through periostin.

Uterine leiomyosarcoma (uLMS) is a rare and aggressive cancer with few effective therapeutics. The Notch signaling pathway is evolutionarily conserved with oncogenic properties, but it has not been well studied in uLMS. The purpose of our study was to determine expression of Notch family genes and proteins and to investigate the therapeutic effect of γ-secretase inhibitors (GSIs), indirect inhibitors of Notch signaling, in uLMS. We determined expression of Notch genes and proteins in benign uterine smooth muscle tissue, fibroids, and uLMS samples by immunostaining and in two uLMS cell lines, SK-UT-1B (uterine primary) and SK-LMS-1 (vulvar metastasis) by RT-PCR, Western blot and immunostaining. We exposed our cell lines to GSIs, DAPT and MK-0752, and measured expression of HES1, a downstream effector of Notch. Notch proteins were differentially expressed in uLMS. Expression of NOTCH3 and NOTCH4 was higher in uLMS samples than in benign uterine smooth muscle and fibroids. Expression of NOTCH4 was higher in SK-LMS-1 compared to SK-UT-1B. Exposure of SK-UT-1B and SK-LMS-1 to DAPT and MK-0752 decreased expression of HES1 and decreased uLMS cell viability in a dose- and time-dependent manner that was unique to each GSI. Our findings suggest that GSIs are potential therapeutics for uLMS, albeit with limited efficacy.

Introduction: Uterine leiomyosarcoma (uLMS) is a rare tumor with poor survival and few therapeutic options. The Notch pathway is evolutionarily conserved, is active in uterine processes, has oncogenic properties in many cancers, but has not been well studied in uLMS. Cancer stem cells (CSC) are subpopulations of cancer cells that are treatment resistant. In certain tumors, activation of canonical Notch signaling, via gamma secretase, supports CSC survival and the acquisition of chemoresistance. Our objective is to identify and characterize the subpopulation of uLMS cells that are viable after treatment with the half maximal inhibitory concentration (IC50) of MK-0752, a gamma secretase inhibitor (GSI) in phase I trials. We will determine Notch signaling activity and expression of markers associated with stem-like potential in GSI resistant uLMS cells. Methods: MTT assays were performed on SK-LMS-1 and SK-UT-1B, two uLMS cell lines, to identify the IC50 for MK-0752. Cells were exposed to MK-0752 at the IC50 or DMSO (control) for 24 hours and viable cells were collected. First, expression of stemness markers, CD133, cMYC and SOX2, in uLMS cells was compared to human uterine smooth muscle cells (hUT-SMC) at baseline. After treatment with MK-0752, the remaining subpopulations of resistant uLMS cells were evaluated for Notch signaling activity by expression of HES1. Stemness marker expression was also assessed. Gene expression was measured with qRT-PCR. Mean fold change (FC) gene expression was determined by FC experimental divided by FC control, relative to expression of 18S rRNA. P≤0.05 was considered significant. Results: In untreated uLMS cells, expression of CD133 and c-MYC was significantly higher in SK-LMS-1 (fibroblast morphology) vs hUT-SMC, while expression of CD133 and SOX2 was significantly higher in SK-UT-1B (epithelial morphology) vs hUT-SMC. After treatment with MK-0752 at IC50 (427.4 µM for SK-LMS-1 or 128.4µM for SK-UT-1B) expression of HES1 was decreased in SK-LMS-1 (0.35x, p≤0.05) and increased in SK-UT-1B (2.38x, p≤0.01) in treatment resistant, viable cells as compared to untreated cells. Expression of c-MYC was decreased (0.38x, p≤0.01) and CD133 was similar (1.64x, p>0.05) in GSI resistant SK-LMS-1 cells, while expression of CD133 and SOX2 was similar (1.43x and 1.30x, respectively, p>0.05) in GSI resistant SK-UT-1B cells compared to untreated cells. Conclusions: Cellular morphology, Notch signaling activity and expression of stemness markers differs in GSI resistant SK-LMS-1 compared to GSI resistant SK-UT-1B cells. The subpopulation of MK-0752 resistant SK-LMS-1 cells have reduced Notch activity and reduced expression of stemness marker, c-MYC, while the resistant SK-UT-1B cells have increased Notch activity. Further studies are required to identify additional factors associated with uLMS resistance to GSIs and the importance of this heterogeneity of uLMS in vivo. Citation Format: Yasmin Abedin, Emily Alpert, Erica Rego, Karla Larios, Emma Cheung, Sofia Gabrilovich, Qingshi Zhao, Mark H. Einstein, Nataki Douglas. Resistance to MK-0752 alters Notch activity and expression of stemness markers in uterine leiomyosarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6074.

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