No abstract

The presence of malignant ascites is a common feature of advanced stage ovarian cancer. During metastasis, as cells detach from the tumor and extravasate into the peritoneal fluid, ovarian cancer cells must adapt to survive the loss of anchorage support and evade anoikis. An important pro-survival adaptation in this context is the ability of tumor cells to increase their antioxidant capacity and restore cellular redox balance. We previously showed that the mitochondrial superoxide dismutase SOD2 is necessary for ovarian cancer cell anoikis resistance, anchorage-independent survival and spheroid formation, and intraperitoneal spread in vivo. We now demonstrate that the upregulation of SOD2 protein expression is an early event initiated in response to anchorage independence and occurs at the post-transcriptional level. SOD2 protein synthesis is rapidly induced in the cytosol within 2 hours of matrix detachment. Polyribosome profiling demonstrates an increase in the number of ribosomes bound to SOD2 mRNA, indicating an increase in SOD2 translation in response to anchorage-independence. Mechanistically, we find that anchorage-independence induces cytosolic accumulation of the RNA binding protein HuR/ELAVL1, leads to HuR binding to SOD2 mRNA, and that the presence of HuR is necessary for the increase in SOD2 mRNA association with the heavy polyribosome fraction and SOD2 protein synthesis. Cellular detachment activates the stress-response protein kinase p38 MAPK, which is necessary for HuR-SOD2 mRNA binding and the rapid increase in SOD2 protein expression. Moreover, HuR is necessary for optimal cell survival during early stages of anchorage independence. These findings uncover a novel post-transcriptional stress response mechanism by which tumor cells are able to rapidly increase their mitochondrial antioxidant capacity to adapt to stress associated with anchorage-independence.

Mitochondrial shape is integral for its proper function and is maintained by a dynamic balance between the events of fission and fusion. Hence, a disruption in the balance is detrimental and has been associated with multiple pathologies including tumorigenesis. We noticed significant heterogeneity in mitochondrial morphology and function in ovarian cancer, which remains the deadliest gynecologic malignancy to date. We discovered that heterogenous mitochondrial dynamics in ovarian cancer cells were associated with specific transcript variant signatures of the fission protein Drp1 (encoded by the gene DNM1L), the primary GTPase responsible for mitochondrial fission. While several Drp1 splice variants have been reported, few studies have linked expression and potential interplay of splice variants of Drp1 on mitochondrial dynamics and function with pathophysiology especially in ovarian cancer. We used 3’RACE, western blotting and LC-MS/MS proteomics analysis to establish the identity of the major Drp1 splice variants expressed in ovarian cancer. We found ovarian cancer cell lines as well as patient-ascites derived cells, predominantly express two Drp1 variants: a transcript including both exons 16 and 17 (16/17) and a transcript lacking exon 16 (-/17). We also validated our findings in TCGA ovarian cancer specimens by analyzing Drp1 splice variant transcripts following annotation of TCGA raw RNAseq data and Salmon expression analysis. Our TCGA analysis of these variants highlighted significant difference in overall survival of ovarian cancer patients. Samples with high Drp1(-/17) expression were associated with poorer overall survival compared to those predominantly expressing Drp1(16/17). Furthermore, carrying out gene set enrichment analysis (GSEA) on TCGA specimens split by high expression of these two variants showed enrichment of distinct gene expression signatures. Overexpression and splice variant specific siRNA knockdown studies demonstrated that Drp1 variants have unique localization and effects on mitochondrial morphology and function. Furthermore, metabolic profiling and 13C metabolic flux analysis highlighted variant specific alterations in mitochondrial metabolic pathways and the TCA cycle. Drp1(-/17) expression enhanced mitochondrial respiratory function and as previously shown, Drp1(-/17) associated with both mitochondria and microtubules, potentially implying a more regulated fission activity as a consequence of controlled subcellular localization. Additionally, Drp1(-/17) was enriched and associated with quiescent phenotype compared to more proliferative phenotype of Drp1(16/17). Hence, expression of distinct Drp1 splice variants may be a novel mechanism to regulate mitochondrial fission, and integral to ovarian cancer cell plasticity under different selection pressures during tumor progression. Citation Format: Zaineb Javed, Dong-Hui Shin, Weihua Pan, Amal Taher Elhaw, Priscilla Tang, Rebecca Phaeton, Mohamed Trebak, Vonn Walter, Nadine Hempel. Expression of ovarian cancer specific Drp1 splice variants regulate mitochondrial heterogeneity and cell plasticity during tumor progression [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 3781.

Ovarian cancer (OVCA) metastasis occurs through the process of transcoelomic spread, where cells disseminate into the peritoneal fluid and form multi-cellular aggregates (MCA) that mediate Anchorage-Independent (A-I) survival and invasion of the peritoneal organs. Isolation of MCAs from patient ascites has been extensively reported in the literature and found to correlate with poor patient survival and resistance to therapy. However, the mechanisms promoting OVCA MCA formation and survival in A-I are not fully understood. We previously demonstrated that MCAs upregulate their mitochondrial antioxidant defenses to maintain optimal survival in A-I, and that this is an early event following cellular detachment. To identify additional key molecular players promoting MCA formation and fitness in A-I, we employed sequential RNA seq analysis to compare the transcriptome of cells in attached, early A-I, and late A-I conditions using the serous cell line OVCA433. Analysis of early transcriptomic changes observed within 2hrs of A-I revealed that RHOV, a recently identified atypical member of the Rho GTPase family, is the top gene significantly upregulated in early A-I. Moreover, we found that RHOV expression rapidly declines in later A-I timepoints indicative of a tight temporal regulation of RHOV expression following cellular detachment. Next, we compared the expression of RHOV in primary ovarian tumor cells and matching malignant ascites cells from the publicly available dataset (GEO: GSE85296) and found increased RHOV expression specifically in the metastatic ascites of ovarian cancer patients when compared to the attached tumor. Previously, RHOV expression was found to regulate intercellular adhesion dynamics during specific stages of neural crest development, a physiological process mimicking epithelial to mesenchymal transition (EMT). RHOV has also been reported to be overexpressed in lung cancer where it was found to correlate with poor patient outcome and resistance to therapy. However, the role of RHOV in ovarian cancer metastasis remains unexplored. To test the consequences of increased RHOV transcription in A-I: its role in migration, aggregate formation, and anoikis resistance is being tested following silencing of RHOV expression using both CRISPR and siRNA. Current work is exploring how RHOV is rapidly transcribed following matrix detachment and how increased expression alters cellular signaling events in early A-I stages to promote optimal MCA fitness. This work aims to contribute new knowledge on the novel oncogenic role of an understudied member of the Rho family of GTPases, RHOV. Moreover, given that increased MCA fitness has been correlated with increases metastatic potential and resistance to therapy, we provide a unique proof-of-concept study to target key components of early MCA adaptations as a novel therapeutic strategy for prolonging survival rates in OVCA patients diagnosed in metastatic stages. Citation Format: Amal T. Elhaw, Yeon Soo Kim, Zaineb Javed, Priscilla Tang, Weihua Pan, Nadine Hempel. Orchestrated expression of the atypical Rho-GTPase, RHOV, in response to matrix detachment of ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A034.