Platinum-resistance in epithelial ovarian cancer: an interplay of epithelial-mesenchymal transition interlinked with reprogrammed metabolism.

BACKGROUND: Epithelial ovarian cancer is the most lethal gynaecological cancer worldwide. Chemotherapy resistance represents a significant clinical challenge and is the main reason for poor ovarian cancer prognosis. We identified novel expression of markers related to epithelial mesenchymal transitions (EMT) in a carboplatin resistant ovarian cancer cell line by proteomics. This was validated in the platinum resistant versus sensitive parental cell lines, as well as platinum resistant versus sensitive human ovarian cancer patient samples. The prognostic significance of the different proteomics-identified marker proteins in prognosis prediction on survival as well as their correlative association and influence on immune cell infiltration was determined by public domain data bases. METHODS: We explored the proteomic differences between carboplatin-sensitive OVCAR5 cells (parental) and their carboplatin-resistant counterpart, OVCAR5 CBPR cells. qPCR and western blots were performed to validate differentially expressed proteins at the mRNA and protein levels, respectively. Association of the identified proteins with epithelial-mesenchymal transition (EMT) prompted the investigation of cell motility. Cellular bioenergetics and proliferation were studied to delineate any biological adaptations that facilitate cancer progression. Expression of differentially expressed proteins was assessed in ovarian tumors obtained from platinum-sensitive (n = 15) versus platinum-resistant patients (n = 10), as well as matching tumors from patients at initial diagnosis and following relapse (n = 4). Kaplan-Meier plotter and Tumor Immune Estimation Resource (TIMER) databases were used to determine the prognostic significance and influence of the different proteomics-identified proteins on immune cell infiltration in the tumor microenvironment (TME). RESULTS: Our proteomics study identified 2422 proteins in both cell lines. Of these, 18 proteins were upregulated and 14 were downregulated by ≥ twofold (p < 0.05) in OVCAR5 CBPR cells. Gene ontology enrichment analysis amongst upregulated proteins revealed an overrepresentation of biological processes consistent with EMT in the resistant cell line. Enhanced mRNA and/or protein expression of the identified EMT modulators including ITGA2, TGFBI, AKR1B1, ITGAV, ITGA1, GFPT2, FLNA and G6PD were confirmed in OVCAR5 CBPR cells compared to parental OVCAR5 cell line. Consistent with the altered EMT profile, the OVCAR5 CBPR cells demonstrated enhanced migration and reduced proliferation, glycolysis, and oxidative phosphorylation. The upregulation of G6PD, AKR1B1, ITGAV, and TGFß1 in OVCAR5 CBPR cells was also identified in the tumors of platinum-resistant compared to platinum-sensitive high grade serous ovarian cancer (HGSOC) patients. Matching tumors of relapsed versus newly diagnosed HGSOC patients also showed enhanced expression of AKR1B1, ITGAV, TGFß1 and G6PD protein in relapsed tumors. Among the identified proteins, significant enhanced expression of GFPT2, FLNA, TGFBI (CDGG1), ITGA2 predicted unfavorable prognosis in ovarian cancer patients. Further analysis suggested that the expression of TGFBI to correlate positively with the expression of identified and validated proteins such as GFPT2, FLNA, G6PD, ITGAV, ITGA1 and ITGA2; and with the infiltration of CD8+ T cells, macrophages, neutrophils, and dendritic cells in the TME. CONCLUSIONS: Our research demonstrates proteomic-based discovery of novel EMT-related markers with an altered metabolic profile in platinum-resistant versus sensitive ovarian cancer cell lines. The study also confirms the expression of selected identified markers in the tumors of platinum-resistant versus sensitive, and in matching relapsed versus newly diagnosed HGSOC patients. The study provides insights into the metabolic adaptation of EMT-induced carboplatin resistant cells that confers on them reduced proliferation to provide effective migratory advantage; and the role of some of these identified proteins in ovarian cancer prognosis. These observations warrant further investigation of these novel target proteins in platinum-resistant patients.

Targeting XIAP and PPARγ in Granulosa Cell Tumors Alters Metabolic Signaling.

Ovarian granulosa cell tumors (GCTs) are hormonally active cancers characterized by indolent growth and late, invasive relapse. No therapies have yet proven to be efficacious. We previously reported that the inhibition of the antiapoptotic X-linked inhibitor of apoptosis protein (XIAP) removes transrepression of the pro-proliferative nuclear receptor, peroxisome proliferator-activated receptor (PPAR)-γ, in a GCT-derived cell line, KGN. Both PPARγ and XIAP are overexpressed in human GCT. The inhibition of XIAP with the restoration of PPARγ signaling using a SMAC-mimetic (Compound A (CmpdA)) and rosiglitazone (RGZ)/retinoic acid (RA), respectively, reduced cell proliferation and induced apoptosis in the KGN cells. Utilizing stable isotope labeling with amino acids in cell culture, we identified 32 differentially expressed proteins in the KGN cells following the CmpdA/RGZ/RA-treatment, 22 of which were upregulated by ≥1.5 fold. Of these, stearoyl-CoA desaturase (SCD; 4.5-fold induction) was examined for putative binding sites for PPARγ using in silico screening. Chromatin immunoprecipitation confirmed the direct binding of PPARγ on the promoter region of SCD, with increased binding in the CmpdA/RGZ/RA-treated KGN cells. Because PPARγ plays a pivotal role in lipid and glucose metabolism, the upregulation of proteins associated with metabolic processes such as SCD is consistent with the restoration of PPARγ activity.

Mutational Landscape of Ovarian Adult Granulosa Cell Tumors from Whole Exome and Targeted TERT Promoter Sequencing.

Adult granulosa cell tumor (aGCT), the most common malignant ovarian sex cord-stromal tumor, is characterized by the forkhead transcription factor FOXL2 p.C134W somatic mutation. Late recurrences are relatively common but the molecular mechanisms of relapse or aggressive behavior are not known. The mutational landscape of FOXL2 p.C134W mutation-positive tumors (n = 22) was determined using whole-exome sequencing (WES). An average of 64 coding and essential splice-site variants were identified per tumor. As the TERT promoter region is poorly covered by the WES, targeted sequencing identified the TERT -124C>T promoter mutation as the only recurrent mutation (∼40% of cases). Pathway analysis suggested an association with DNA replication/repair and the EGFR family canonical pathways. Copy number analysis confirmed that gains of chromosomes 12 and 14 occur in approximately 30% of aGCT and loss of chromosome 22 occurs in approximately 40% of cases. In summary, exome-wide analysis of the mutational landscape of aGCT revealed that, except for the TERT promoter mutation, recurrence and/or aggressive behavior is not defined by activation or loss of specific genes. IMPLICATIONS: This study found that although aGCTs are defined by the presence of a common FOXL2 gene mutation, recurrence and/or aggressive behavior cannot be attributed to subsequent mutation of specific gene(s) or pathways; however, there is a high frequency of the TERT -124C>T promoter mutation, which is associated with more aggressive disease.

Combined PPARγ Activation and XIAP Inhibition as a Potential Therapeutic Strategy for Ovarian Granulosa Cell Tumors.

Ovarian granulosa cell tumors (GCT) are characterized by indolent growth and late relapse. No therapeutic modalities aside from surgery have proven effective. We previously reported overexpression of the nuclear receptor, peroxisome proliferator-activated receptor-gamma (PPARγ), and constitutive activity of the NFκB and AP1 signaling pathways in GCT. PPARγ presents as a potential therapeutic target as it impedes proliferation and promotes terminal differentiation of granulosa cells. However, resistance to the actions of PPARγ is caused by NFκB transrepression in GCT-derived cell lines, KGN and COV434. We showed that abrogation of NFκB signaling in GCT cells enables PPARγ agonists to initiate apoptosis. In addition, we observed overexpression of an NFκB-induced gene, X-linked inhibitor of apoptosis protein (XIAP), in GCT and GCT-derived cells. XIAP is an attractive therapeutic target due to its role in inhibiting the apoptotic pathway. We investigated the antitumor effects of combined XIAP inhibition using Smac-mimetics and PPARγ activation using thiazolidinediones (TZD) in the GCT-derived cells. Transactivation assays revealed that NFκB transrepression of PPARγ can be relieved by NFκB or XIAP inhibition. Combined Smac-mimetic and TZD significantly induced apoptosis, reduced cell viability and proliferation in KGN cells in monolayer and 3D spheroid culture, and in GCT explant models. The Smac-mimetic and TZD cotreatment also delayed cell invasion, upregulated proapoptotic genes, and compromised cell metabolism in KGN cells. This study provides evidence that PPARγ and XIAP cotreatment has antineoplastic effects in GCT. As therapeutics that target these proteins are already in clinical or preclinical use, expedient translation to the clinic is possible.

Tumour microenvironment and metabolic plasticity in cancer and cancer stem cells: Perspectives on metabolic and immune regulatory signatures in chemoresistant ovarian cancer stem cells.

Cancer stem cells (CSCs) are a sub-population of tumour cells, which are responsible to drive tumour growth, metastasis and therapy resistance. It has recently been proposed that enhanced glucose metabolism and immune evasion by tumour cells are linked, and are modulated by the changing tumour microenvironment (TME) that creates a competition for nutrient consumption between tumour and different sub-types of cells attracted to the TME. To facilitate efficient nutrient distribution, oncogene-induced inflammatory milieu in the tumours facilitate adaptive metabolic changes in the surrounding non-malignant cells to secrete metabolites that are used as alternative nutrient sources by the tumours to sustain its increasing energy needs for growth and anabolic functions. This scenario also affects CSCs residing at the primary or metastatic niches. This review summarises recent advances in our understanding of the metabolic phenotypes of cancer cells and CSCs and how these processes are affected by the TME. We also discuss how the evolving TME modulates tumour cells and CSCs in cancer progression. Using previously described proteomic and genomic platforms, ovarian cancer cell lines and a mouse xenograft model we highlight the existence of metabolic and immune regulatory signatures in chemoresistant ovarian CSCs, and discuss how these processes may affect recurrence in ovarian tumours. We propose that progress in cancer control and eradication may depend not only on the elimination of highly chemoresistant CSCs, but also in designing novel strategies which would intervene with the tumour-promoting TME factors.

Measurement of Oxidative Stress: Mitochondrial Function Using the Seahorse System.

The Seahorse XFp Analyzer is a powerful tool for the assessment of various parameters of cellular respiration. Here we describe the process of the Seahorse Cell Phenotype Test using the Seahorse XFp Analyzer to characterize the metabolic phenotype of live cells. The Seahorse XFp Analyzer can also be coupled with other assays to measure cellular energetics. Given that mitochondrial dysfunction is implicated in preeclampsia, the Seahorse XFp Analyzer will serve as a useful tool for the understanding of pathological metabolism in this disorder.

Transcriptomic analysis of stage 1 versus advanced adult granulosa cell tumors.

Ovarian granulosa cell tumors (GCT) are hormonally-active neoplasms characterized, in the adult-subtype, by a mutation in the FOXL2 gene (C134W). They exhibit an indolent course with an unexplained propensity for late recurrence; ~80% of patients with aggressive, advanced stage tumors die from their disease; aside from surgery, therapeutic options are limited. To identify the molecular basis of advanced stage disease we have used whole transcriptome analysis of FOXL2 C134W mutation positive adult (a)GCT to identify genes that are differentially expressed between early (stage 1) and advanced (stage 3) aGCT. Transcriptome profiles for early (n = 6) and stage 3 (n = 6) aGCT, and for the aGCT-derived KGN, cell line identified 24 genes whose expression significantly differs between the early and stage 3 aGCT. Of these, 16 were more abundantly expressed in the stage 3 aGCT and 8 were higher in the stage 1 tumors. These changes were further examined for the genes which showed the greatest fold change: the cytokine CXCL14, microfibrillar-associated protein 5, insulin-like 3 and desmin. Gene Set Enrichment Analysis identified overexpression of genes on chromosome 7p15 which includes the homeobox A gene locus. The analysis therefore identifies a small number of genes with clearly discriminate patterns of expression arguing that the clinicopathological-derived distinction of the tumor stage is robust, whilst confirming the relative homogeneity of expression for many genes across the cohort and hence of aGCT. The expression profiles do however identify several overexpressed genes in both stage 1 and/or stage 3 aGCT which warrant further study as possible therapeutic targets.

Impact of FOXL2 mutations on signaling in ovarian granulosa cell tumors.

Granulosa cell tumors (GCT) are unique sex-cord stromal tumors which account for ∼ 8% of all ovarian malignancies. They exhibit morphological, biochemical and hormonal features similar to proliferating granulosa cells of the preovulatory follicle, including estrogen and inhibin synthesis. A somatic missense mutation in the forkhead box L2 (FOXL2) gene (C134W) is unique to adult GCT, and absent in other ovarian cancers. FOXL2 is a transcription factor that plays a critical role in ovarian function, in particular, proliferation and differentiation of granulosa cells. The molecular mechanisms underlying the pathogenicity of the mutant FOXL2 remain unresolved. Here we review the molecular alterations known to be associated with mutant FOXL2 and the potential signaling implications. Several studies suggest that dysregulated FOXL2 function may alter cell cycle progression and apoptosis. Further insights into the molecular mechanism of GCT pathophysiology may identify therapeutic targets for the treatment of these tumors.