Adaptations of key metabolic pathways in primary and metastatic epithelial ovarian cancer (253)

Abstract

Objectives: Patients with ovarian cancer (OC) often present with advanced-stage disease at diagnosis; however, the molecular mechanisms which drive the aggressive nature of OC metastases have not been well characterized. Metastasizing cancer cells undergo metabolic reprogramming to utilize available lipids in the metastatic niche of the omentum. This study aimed to understand the compensatory metabolic mechanisms that enable this OC metastasis. Our work focused on understanding the contributions of the pentose phosphate pathway (PPP)—a key mechanism responsible for redox homeostasis—in enabling this evolution. Methods: Using gene expression data of matched human primary and omental tumor samples (n=30), we performed targeted analysis of metabolic gene expression changes, which revealed upregulation of the PPP in omental metastases. We then performed in vitro and in vivo experiments using pre-clinical ovarian cancer models (SKOV3, IGROV, HEYA8). Given the canonical role of the PPP in maintaining redox homeostasis, we assessed oxidative stress in organoid cultures from omental metastases harvested at mouse necropsy and in cultures grown in omental conditioned media. Inhibition of the rate-limiting enzyme of the PPP, glucose-6-phosphate dehydrogenase (G6PD), was performed via shRNA knockdowns and treated with a pharmacological inhibitor. HeyA8 tumor-bearing mice were treated with the G6PD inhibitor, and the metastatic burden was evaluated using IVIS during treatment and in peritoneal organs following the sacrifice. Statistical significance was established using unpaired two-tailed Student’s t-tests. Results: Differential analysis of metabolic gene expression and pathway analysis revealed upregulation of the PPP in omental metastases compared to primary ovarian tumors (p<0.05, EnrichR Score = 800). Primary tumors and omental metastases were harvested from SKOV3, HeyA8, and IGROV1 tumor-bearing mice at necropsy and used to establish organoids. DCFH-DA, a fluorogenic probe for ROS, measured higher levels of oxidative stress in metastatic organoids than primary tumors (normalized F525= 11.3623, p<0.0005). SKOV3 organoids expressing HyPer, a ratiometric biosensor of intracellular H2O2, exhibited higher oxidative stress when grown in omental conditioned media versus basal organoid media (normalized F420/F500=1.27, p<0.05). Pharmacological and shRNA inhibition of G6PD sensitized cells to oxidative stress, reducing cell viability in vitro (p<0.05). Treatment with a G6PD inhibitor in vivo significantly decreased tumor growth and metastases compared to vehicle treatment (p<0.005). Objectives: Patients with ovarian cancer (OC) often present with advanced-stage disease at diagnosis; however, the molecular mechanisms which drive the aggressive nature of OC metastases have not been well characterized. Metastasizing cancer cells undergo metabolic reprogramming to utilize available lipids in the metastatic niche of the omentum. This study aimed to understand the compensatory metabolic mechanisms that enable this OC metastasis. Our work focused on understanding the contributions of the pentose phosphate pathway (PPP)—a key mechanism responsible for redox homeostasis—in enabling this evolution. Methods: Using gene expression data of matched human primary and omental tumor samples (n=30), we performed targeted analysis of metabolic gene expression changes, which revealed upregulation of the PPP in omental metastases. We then performed in vitro and in vivo experiments using pre-clinical ovarian cancer models (SKOV3, IGROV, HEYA8). Given the canonical role of the PPP in maintaining redox homeostasis, we assessed oxidative stress in organoid cultures from omental metastases harvested at mouse necropsy and in cultures grown in omental conditioned media. Inhibition of the rate-limiting enzyme of the PPP, glucose-6-phosphate dehydrogenase (G6PD), was performed via shRNA knockdowns and treated with a pharmacological inhibitor. HeyA8 tumor-bearing mice were treated with the G6PD inhibitor, and the metastatic burden was evaluated using IVIS during treatment and in peritoneal organs following the sacrifice. Statistical significance was established using unpaired two-tailed Student’s t-tests. Results: Differential analysis of metabolic gene expression and pathway analysis revealed upregulation of the PPP in omental metastases compared to primary ovarian tumors (p<0.05, EnrichR Score = 800). Primary tumors and omental metastases were harvested from SKOV3, HeyA8, and IGROV1 tumor-bearing mice at necropsy and used to establish organoids. DCFH-DA, a fluorogenic probe for ROS, measured higher levels of oxidative stress in metastatic organoids than primary tumors (normalized F525= 11.3623, p<0.0005). SKOV3 organoids expressing HyPer, a ratiometric biosensor of intracellular H2O2, exhibited higher oxidative stress when grown in omental conditioned media versus basal organoid media (normalized F420/F500=1.27, p<0.05). Pharmacological and shRNA inhibition of G6PD sensitized cells to oxidative stress, reducing cell viability in vitro (p<0.05). Treatment with a G6PD inhibitor in vivo significantly decreased tumor growth and metastases compared to vehicle treatment (p<0.005).