Metformin is a widely used agent for the treatment of diabetes and infertility, however, it has been found to have anti-cancer effects in a variety of malignancies including high grade serous ovarian cancer (HGSC). Studies describing the mechanisms by which metformin affects HGSC are ongoing, but detailed analysis of its effect on the cellular metabolism of both HGSC cells and their precursor, normal fallopian tube secretory epithelial cells (FTSECs), is lacking. We addressed the effects of metformin and the more potent biguanide, phenformin, on HGSC cell lines and normal immortalized FTSECs. Cell proliferation assays identified that FTSECs and a subset of HGSC cell lines are relatively resistant to the anti-proliferative effects of metformin. Bioenergetic and metabolomic analyses were used to metabolically differentiate the metformin-sensitive and metformin-resistant cell lines. Bioenergetically, biguanides elicited a significant decrease in mitochondrial respiration in all HGSC cells and FTSECs. However, biguanides had a greater effect on mitochondrial respiration in metformin sensitive cells. Metabolomic analysis revealed that metformin and phenformin generally induce similar changes in metabolic profiles. Biguanide treatment led to a significant increase in NADH in FTSECs and HGSC cells. Interestingly, biguanide treatment induced changes in the levels of mitochondrial shuttle metabolites, glycerol-3-phopshate (G3P) and aspartate, specifically in HGSC cell lines and not in FTSECs. Greater alterations in G3P or aspartate levels were also found in metformin sensitive cells relative to metformin resistant cells. These data identify bioenergetic and HGSC-specific metabolic effects that correlate with metformin sensitivity and novel metabolic avenues for possible therapeutic intervention.

Glucose is considered the primary energy source for all cells, and some cancers are addicted to glucose. Here, we investigated the functional consequences of chronic glucose deprivation in serous ovarian cancer cells. We found that cells resistant to glucose starvation (glucose-restricted cells) demonstrated increased metabolic plasticity that was dependent on NNMT (Nicotinamide N-methyltransferase) expression. We further show that ZEB1 induced NNMT, rendered cells resistant to glucose deprivation and recapitulated metabolic adaptations and mesenchymal gene expression observed in glucose-restricted cells. NNMT depletion reversed metabolic plasticity in glucose-restricted cells and prevented de novo formation of glucose-restricted colonies. In addition to its role in glucose independence, we found that NNMT was required for other ZEB1-induced phenotypes, such as increased migration. NNMT protein levels were also elevated in metastatic and recurrent tumors compared to matched primary carcinomas, while normal ovary and fallopian tube tissue had no detectable NNMT expression. Our studies define a novel ZEB1/NNMT signaling axis, which elicits mesenchymal gene expression, as well as phenotypic and metabolic plasticity in ovarian cancer cells upon chronic glucose starvation. Understanding the causes of cancer cell plasticity is crucial for the development of therapeutic strategies to counter intratumoral heterogeneity, acquired drug resistance and recurrence in high-grade serous ovarian cancer (HGSC).

Sphingosine kinase 1 (SPHK1), the enzyme that produces sphingosine 1 phosphate (S1P), is known to be highly expressed in many cancers. However, the role of SPHK1 in cells of the tumor stroma remains unclear. Here, we show that SPHK1 is highly expressed in the tumor stroma of high-grade serous ovarian cancer (HGSC), and is required for the differentiation and tumor promoting function of cancer-associated fibroblasts (CAFs). Knockout or pharmacological inhibition of SPHK1 in ovarian fibroblasts attenuated TGF-β-induced expression of CAF markers, and reduced their ability to promote ovarian cancer cell migration and invasion in a coculture system. Mechanistically, we determined that SPHK1 mediates TGF-β signaling via the transactivation of S1P receptors (S1PR2 and S1PR3), leading to p38 MAPK phosphorylation. The importance of stromal SPHK1 in tumorigenesis was confirmed in vivo, by demonstrating a significant reduction of tumor growth and metastasis in SPHK1 knockout mice. Collectively, these findings demonstrate the potential of SPHK1 inhibition as a novel stroma-targeted therapy in HGSC.

Background

Succinate dehydrogenase (SDH) is a mitochondrial metabolic enzyme complex involved in both the electron transport chain and the citric acid cycle. SDH mutations resulting in enzymatic dysfunction have been found to be a predisposing factor in various hereditary cancers. Therefore, SDH has been implicated as a tumor suppressor.

Results

We identified that dysregulation of SDH components also occurs in serous ovarian cancer, particularly the SDH subunit SDHB. Targeted knockdown of Sdhb in mouse ovarian cancer cells resulted in enhanced proliferation and an epithelial-to-mesenchymal transition (EMT). Bioinformatics analysis revealed that decreased SDHB expression leads to a transcriptional upregulation of genes involved in metabolic networks affecting histone methylation. We confirmed that Sdhb knockdown leads to a hypermethylated epigenome that is sufficient to promote EMT. Metabolically, the loss of Sdhb resulted in reprogrammed carbon source utilization and mitochondrial dysfunction. This altered metabolic state of Sdhb knockdown cells rendered them hypersensitive to energy stress.

Conclusions

These data illustrate how SDH dysfunction alters the epigenetic and metabolic landscape in ovarian cancer. By analyzing the involvement of this enzyme in transcriptional and metabolic networks, we find a metabolic Achilles' heel that can be exploited therapeutically. Analyses of this type provide an understanding how specific perturbations in cancer metabolism may lead to novel anticancer strategies.