J. Chhina

Bioenergetic Adaptations in Chemoresistant Ovarian Cancer Cells

Earlier investigations have revealed that tumor cells undergo metabolic reprogramming and mainly derive their cellular energy from aerobic glycolysis rather than oxidative phosphorylation even in the presence of oxygen. However, recent studies have shown that certain cancer cells display increased oxidative phosphorylation or high metabolically active phenotype. Cellular bioenergetic profiling of 13 established and 12 patient derived ovarian cancer cell lines revealed significant bioenergetics diversity. The bioenergetics phenotype of ovarian cancer cell lines correlated with functional phenotypes of doubling time and oxidative stress. Interestingly, chemosensitive cancer cell lines (A2780 and PEO1) displayed a glycolytic phenotype while their chemoresistant counterparts (C200 and PEO4) exhibited a high metabolically active phenotype with the ability to switch between oxidative phosphorylation or glycolysis. The chemosensitive cancer cells could not survive glucose deprivation, while the chemoresistant cells displayed adaptability. In the patient derived ovarian cancer cells, a similar correlation was observed between a high metabolically active phenotype and chemoresistance. Thus, ovarian cancer cells seem to display heterogeneity in using glycolysis or oxidative phosphorylation as an energy source. The flexibility in using different energy pathways may indicate a survival adaptation to achieve a higher ‘cellular fitness’ that may be also associated with chemoresistance.

Targeting of free fatty acid receptor 1 in EOC: A novel strategy to restrict the adipocyte-EOC dependence

OBJECTIVES Adipocyte derived free fatty acids (FFA) promote epithelial ovarian cancer (EOC) by acting as a fuel source to support the energy requirement of the cancer cells. FFA may also exert biological effects through signaling pathways. Recently, a family of FFA activated G-protein coupled receptors (FFAR/GPCRs) was identified. Our objective was to investigate the role of FFAR/GPCRs in EOC and assess their potential as therapeutic targets. METHODS The mRNA (RT-PCR) expression of FFAR/GPCR family members (FFAR1/GPR40; FFAR2/GPR43, FFAR3/GPR41, FFAR4/GPR120 and GPR84) was examined in: (1) a syngeneic mouse model of EOC fed high energy diet (60% fat) or regular diet (30% fat), (2) EOC cell lines exposed to free fatty acids and (3) specimens from 13 histologically normal ovaries and 28 high grade ovarian serous carcinomas. The GPR 40 antagonist, GW1100, was used to inhibit FFAR1/GPR40 and cell survival was assayed by MTT in various cell lines. RESULTS High Grade Serous carcinoma specimens expressed significantly increased GPR40 compared to normal ovaries (p=0.0020). Higher expression was noted in advanced stage disease. ID8 ovarian tumors from mice fed with high fat diet also showed higher GPR40 expression. Exposing EOC cells to FFAs, increased GPR40 expression. Treatment of EOC cell lines with GW100 resulted in growth inhibition and was associated with an alteration in their energy metabolism. CONCLUSION FFA-induced cancer cell growth may be partly mediated through FFAR1/GPR40. Targeting of FFAR1/GPR40 may be an attractive treatment strategy in EOC, and possibly offers a targeted treatment for a subset of EOC patients.

Abstract A81: Targeting of free fatty acid signaling in ovarian cancer may serve as a potential therapeutic approach.

Recent studies have revealed an association between adipocyte driven free fatty acids (FFA) and the aggressiveness of epithelial ovarian cancer (EOC). Adipocyte derived free fatty acids (FFA) seem to promote epithelial ovarian cancer (EOC) growth and progression by acting as mitochondrial fuel source to support the energy requirements of EOC cells. Apart from acting as a fuel source, FFA may also enhance tumor growth through other signaling pathways that can promote proliferation and regulate metabolism. Recently, a family of FFA activated G-protein coupled receptors (FFAR/GPCRs) was identified, including: FFAR1/GPR40, FFAR2/GPR42, FFAR3/GPR41, FFAR4/GPR120 and GPR84. Using RT-PCR, we have found that exposing EOC cells to adipocytes results in overexpression of FFAR1/GPR40 in multiple ovarian cancer cell lines (ID8, A2780, C200, OVCAR3 and SKOV3). In mRNA extracted from formalin-fixed paraffin embedded tumor specimens, approximately 80% of high-grade serous ovarian carcinomas exhibited increased FFAR1 expression. Additionally, analysis of TCGA database revealed that patients with tumors exhibiting increased mRNA expression of FFAR1/GPR40 had poor overall survival and progression free survival. We have also found that targeting FFAR1 using its specific antagonist GW1100 inhibited the proliferation of A2780, ID8, C200, OVCAR3 and SKOV3 cell lines and resulted in downstream inhibition of insulin secretion and the Akt signaling pathway. On the other hand, treatment of the same cells with the FFAR1 agonist ,CAY10587, had no effect on proliferation. Thus, we conclude that FFAR1 may play an important role in the regulation of FFA mediated EOC cell proliferation. Targeting of FFAR1/GPR40 may be an attractive strategy in EOC, particularly for EOC patients presenting with high adiposity. Citation Format: Adnan Munkarah, Suhail Hamid, Jasdeep Chhina, Ismail Mert, LaToya Jackson, Sharon Hensley-Alford, Dhananjay Chitale, Shailendra Giri, Ramandeep Rattan. Targeting of free fatty acid signaling in ovarian cancer may serve as a potential therapeutic approach. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A81.

Synergistic effect of MEK inhibitor and metformin combination in low grade serous ovarian cancer

OBJECTIVE Low-grade serous ovarian cancer (LGSOC) constitutes 5-8% of epithelial ovarian cancers and is refractory to chemotherapy. We and others have shown metformin to cause significant growth inhibition in high-grade ovarian cancer both in vitro and in vivo. Here, we aimed to analyze if metformin was effective in inhibiting proliferation of LGSOC alone and in combination with MEK inhibitor. METHODS Three LGSOC lines (VOA1056, VOA1312 and VOA5646) were treated with metformin, trametinib or 2-deoxyglucose (2DG) alone or in combination with metformin. Proliferation was measured by MTT assay over a period of four days. Protein expression was measured by western blotting. Seahorse Analyzer was used to measure effect of metformin on glycolysis and mitochondrial respiration. RESULTS All LGSOC cell lines showed significant inhibition with metformin in a dose- and time-dependent manner. Trametinib significantly inhibited the growth of Ras mutated LGSOC lines (VOA1312 and VOA1056), while VOA5646 cells without RAS mutation did not show any response. Metformin and trametinib combination showed synergistic inhibition of RAS mutated VOA1312 and VOA1056 cells, but not for non-Ras mutated VOA5646 cells. Metformin and trametinib increased phosphorylated AMPK expression in LGSOC lines with combination showing stronger expression. Trametinib decreased 42/44 mitogen activated kinase phosphorylation in all cell lines, while metformin and combination had no significant effect. 2-DG significantly inhibited glycolysis in all LGSOC lines and combination with metformin showed synergistic inhibitory effect. CONCLUSIONS Metformin alone or in combination with MEK and glycolytic inhibitors may be a potential therapy for LGSOC, a cancer that is indolent but chemo-resistant.

Abstract 11: Metformin exerts differential metabolic effects in ovarian cancer cell lines

Metformin is being actively repurposed for treatment of gynecologic malignancies including ovarian cancer, with various clinical trials underway. Metformin is known to alter the cancer cell metabolism, primarily by inhibiting oxidative phosphorylation and inducing glycolysis. Our aim was to investigate if metformin induces similar metabolic changes across ovarian cancer cells. Untargeted global metabolite assay, by ultra-high performance Liquid Chromatography and Gas Chromatography Mass Spectroscopy, was performed on A2780, C200, and SKOV3ip cell lines with and without metformin treatment (10mM for 48 hours). Per-metabolite comparisons were made across conditions. Interpretive analysis was performed using the KEGG molecular pathways (Kyoto Encyclopedia of Genes and Genomes) and the Ingenuity molecule library with a focus on energy pathways of glycolysis, oxidative phosphorylation and also other metabolic pathways. Additionally, glycolytic and mitochondrial respiration were measured using the Seahorse XFe Analyzer. Specific analysis of the glycolysis metabolites revealed that while glycolysis was increased by metformin in all the cells, the intracellular levels of glucose, lactose and pyruvate varied significantly across the cell lines and were differentially affected by metformin treatment. Metformin had little impact on the TCA cycle intermediates in the A2780 cells, which were significantly decreased in C200 and in contrast increased in SKOV3ip cells. Functional analysis showed the oxygen consumption rate to be significantly inhibited by metformin in all the three cell lines, while the increased fatty acid oxidation intermediates were observed across all the three cell lines albeit to a varying extent. Exploration of the global metabolite changes by metformin across the three cell lines revealed 57 common altered metabolites, of which 30 had consistent direction change, with 16 metabolite being up and 14 being downregulated by metformin treatment. Metabolite Set Enrichment analysis showed linolenic acid and methionine metabolism as most enriched in metabolites being increased by metformin, and RNA transcription and pyrimidine metabolism as most enriched in the metabolites downregulated by metformin treatment. Ingenuity analysis indicated cellular proliferation and signaling as the top common network pathway modulated by metformin. In conclusion metformin treatment had a significant and wide-spread effect on metabolism of ovarian cancer cell lines. While metformin resulted in certain consistent metabolic changes, it had cell line specific modulation on glycolysis and oxidative phosphorylation metabolites. These differential metabolic changes could indicate the degree of metformin response and suggest it to be context-dependent. Thus information about the cancer metabolism will aid in preclinical and clinical interpretation of metformin therapy in ovarian and other cancers. Citation Format: Adnan Munkarah, Laila Poisson, Seongho Kim, Jasdeep Chhina, Shailendra Giri, Ramandeep Rattan. Metformin exerts differential metabolic effects in ovarian cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 11.

Abstract POSTER-BIOL-1307: Bioenergetic adaptations in chemoresistant ovarian cancer cells

While, normal cells primarily rely on mitochondrial oxidative phosporylation (OXPHOS), cancer cells are known to preferentially take up glucose to produce energy using aerobic glycolysis pathway, described as the ‘Warburg effect’. Recently, this view that all cancer cells are dependent on glycolysis is being challenged. We examined the bioenergetic characteristics of a panel of 10 human ovarian cancer cell lines and 2 immortalized ovarian surface epithelial cell lines, using the Seahorse XF Extracellular Flux analyzer to measure glycolysis and mitochondrial respiration in real time using the outputs of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) respectively. The mitochondrial bioenergetics was read by taking baseline OCR following sequential addition of oligomycin, FCCP and rotenone/antimycin, which inhibit mitochondrial ATP synthase, uncouple mitochondrial OXPHOS and induce maximal respiration respectively. Glycolytic profile (ECAR) was generated after keeping the cells glucose free followed by sequential addition of glucose to induce glycolysis, oligomycin and 2-deoxy glucose to inhibit glycolysis. The OCR profile showed ovarian cancer cells lines to have diverse mitochondrial bioenergetics and diverse ability to use glycolysis. The OCR:ECAR ratio showed varied bioenergetic organization, with some cells relying heavily on glycolysis or OXPHOS, but most using both pathways equally. A significant positive correlation (correlation coefficient 0.7705; p=0.003) was observed between mitochondrial respiration and glycolysis, confirming that glycolysis dependent cells have lower ATP-linked respiration rates. A similar diversity was observed in the mRNA expression of glycolytic (Glut1 and LDH) and mitochondrial (PGC-1α and CoxVb) genes. These findings highlight the actuality of extreme heterogeneity observed in cancer cells. A unique observation was the distinctive behavior of chemosensitive and resistant cell line pairs. Our panel contained a set of (i) cisplatin sensitive A2780 and resistant C200 cell lines and (ii) taxol sensitive PEO1 and resistant PEO4 cell lines. The resistant cells (C200 and PEO4) displayed higher ECAR and OCR profile compared to the sensitive cells (A2780 and PEO1), indicating an increased utilization of both energy pathways. The OCR:ECAR ratio suggested the sensitive cell lines to be glycolytic and the resistant cell lines to be highly metabolically active. This was further supported by increased mitochondrial function in the resistant cells, measured in terms of augmented fatty acid oxidation and mitochondrial potential in the resistant cells. On inhibition of glycolysis, the resistant cells were able to increase OX-PHOS and maintain their growth, whereas sensitive cells could not increase OX-PHOS and ceased growth. This led us to a novel hypothesis that chemo-resistant ovarian cancer cells exhibit greater plasticity than normal and sensitive cells, making them more adaptable to rearrange their metabolic phenotype according to microenvironment changes and stress, giving them a selective advantage to overcome adverse conditions. Thus, the metabolic diversity could be a means of selecting resilient chemo-resistant cells over a period of cytotoxic insults. Citation Format: J. Chhina, S. Dar, M. Deshpande, S. Giri, A. Munkarah, R. Rattan. Bioenergetic adaptations in chemoresistant ovarian cancer cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1307.

Abstract B77: Targeting ovarian cancer promoting effects of adipocytes by metformin

At the time of surgery, 80% of the ovarian cancers are found to be metastasized to the omentum, a large fat pad covering the abdominal organs. Omental metastasis is thought to be due to the promotion of homing, growth and migration of ovarian cancer cells by factors secreted by adipocytes, which include cytokines, adipokines and growth factors. These adipocytes also act as fuel depot providing for the energy needs of fast growing ovarian tumor cells. The objective of our study was to investigate if metformin can modulate or limit the adipocyte mediated tumor-promoting and migrating effects. For this, mouse preadipocyte cells were differentiated in the presence or absence of metformin and various parameters including lipid accumulation, AMPK activation and adipogenesis transcription factors were examined. Conditioned media from undifferentiated and differentiated adipocytes was used to evaluate ID-8 mouse ovarian cancer cell proliferation and migration. Conditioned media from differentiated but not from undifferentiated preadipocytes, increased ID-8 cell proliferation (p<0.05) and migration (p<0.050), which were attenuated in presence of metformin (p<0.05). Metformin treatment inhibited adipogenesis as evident by the reduction of neutral lipid accumulation. Metformin treatment increased AMP-activated protein kinase (AMPK) activity as evident from higher level of phosphorylation of AMPK and its immediate downstream target ACC (Acetyl Co Carboxylase). It also inhibited the key adipogenesis transcription factors (CEBPαa; CEBPβb, SREBP1) during the process of adipocyte diffrenetiation. A targeted Cancer Pathway Finder RT-PCR based gene array (Qiagen) revealed 21 genes that were upreguated and 2 were downregulated more than 2-fold in ID-8 cells in presence of adipocyte conditioned media, which were modulated by metformin. Together, metformin treatment inhibited the adipocyte mediated ovarian cancer cell proliferation and migration. It also blunted the expression of cancer associated genes that were induced by adipocyte influence in cancer cells. This study suggests that metformin could be a therapeutic option for ovarian cancer as it not only targets ovarian cancer but alternatively, also modulates the environmental milieu around it. Citation Format: Ramandeep Rattan, Calvin Tebbe, Jasdeep Chhina, Kalli Sarigiannis, Shailendra Giri, Adnan Munkarah. Targeting ovarian cancer promoting effects of adipocytes by metformin. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B77.