Abstract B093: Investigating early events of ovarian cancer transcoelomic metastasis identifies the small Rho-GTPase RHOV as an essential gene for cellular detachment and aggregate formation
Authors: Elhaw Amal T , Tang Priscilla , White Sierra , Javed Zaineb , Cheng Ya-Yun , Pan Weihua , Karthikeyan Mythreye , Hempel Nadine 
Published: March 2024

Abstract
A major mode of ovarian cancer (OVCA) metastasis is through transcoelomic spread, where cancer cells disseminate into the peritoneal fluid, form multicellular aggregates (MCA), and proceed to invade the peritoneal organs. MCAs can be detected in ascites and are associated with reduced patient survival rates. The formation of MCAs enables OVCA cells to survive and spread in anchorage independent (A-I) conditions more efficiently. While MCAs have been largely studied, the key molecular events promptly following OVCA detachment, and preceding MCA formation, are less known. In the present study, we focused on identifying early gene expression changes that are driven immediately post-matrix-detachment to identify essential early response genes that drive anoikis resistance and MCA formation. We applied bulk RNA-sequencing to determine the transcriptomic changes in OVCA cells shortly after matrix-detachment, using three serous OVCA cell lines: OVCA433, OVCAR3, and SKOV3. We discovered a significant transcript upregulation of the RHOV gene, expression of which peaked early within two hours of detachment, a pattern consistent across all three cell lines. RHOV, a member of the Rho-GTPase family that controls cellular morphology, migration, adhesion, and survival, was the only Rho-GTPase that showed differential upregulation during early detachment. Unlike other GTPases, RHOV is thought to be constitutively active once expressed, suggesting that RHOV’s rapid transcriptional increase in response to detachment, may be an important mechanism regulating its activity during OVCA metastasis. Overexpression of RHOV has been documented in lung and breast cancer, correlating with decreased patient survival and metastatic progression. Yet, its role in OVCA metastasis has not been studied. To explore the potential impact of elevated RHOV transcription during early detachment, we used CRISPR-Cas9 to create RHOV-knock out (KO) clones in OVCA cell lines. RHOV-KO cells are more vulnerable to anoikis and lose their ability to aggregate under A-I conditions. RHOV-KO cells displayed abrogated migration, and RHOV-KO MCAs were less capable of clearing mesothelial cells in a co-culture model simulating peritoneal invasion. Since both A-I survival and MCA formation are key to transcoelomic metastasis, we tested whether post-detachment RHOV expression is essential for peritoneal tumor formation in-vivo. Notably, we found that RHOV-KO significantly reduced intra-peritoneal tumor spread of SKOV3 cells, as observed through bioluminescence imaging. Current work is exploring how RHOV is rapidly expressed following matrix detachment and investigating how RHOV regulates cellular adhesion and extracellular matrix-deposition in this context by focusing on KRAS-GTPase signaling. Our research offers valuable insights into the oncogenic role of the understudied RHOV gene and provides a unique proof of concept: Interrupting early adaptations proceeding matrix detachment could impede the metastatic cascade in OVCA patients diagnosed in metastatic stages.
Citation Format: Amal T Elhaw, Priscilla Tang, Sierra White, Zaineb Javed, Ya-Yun Cheng, Weihua Pan, Mythreye Karthikeyan, Nadine Hempel. Investigating early events of ovarian cancer transcoelomic metastasis identifies the small Rho-GTPase RHOV as an essential gene for cellular detachment and aggregate formation [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B093.