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

Ovarian cancer is a heterogeneous disease that is categorized historically as a malignancy derived through the transformation of epithelial, sex-cord stromal or germ cells. Ninety percent of all cases are epithelial tumors, of which the most common serous subtype constitutes 80% of the cancer [1]. Surgery is the first line of treatment for ovarian cancer patients, while platinum- and taxane-based combination chemotherapy is given as a standard of care following surgery. However, the majority of ovarian cancer patients eventually experience a relapse due to failure of chemotherapy treatments, resulting in 30–40% 5-year survival rate, which has remained stagnant for the last three decades.

Platinum resistance is the major challenge in ovarian cancer treatment. The current classification strictly defines resistance to platinum as recurrence within 6 months of the last platinum administration [2]. Duration between the last administration of platinum treatment and cancer relapse dictates future line of treatment strategies with either platinum-based or another chemotherapeutics in the clinic. However, some argue that the empiric 6-month cut-off criterion for platinum-resistance may not be biologically relevant. More work needs to be done to identify mechanisms and molecular markers, which can be used clinically to design more effective targeted treatments for ovarian cancer patients [2].

Epithelial mesenchymal transition (EMT) has been associated with the acquisition of migratory and invasive like properties in cancer cells [3, 4]. During EMT, transcriptional and metabolic reprogramming triggers morphological and functional changes in cancer cells [5]. Loss of E-cadherin (CDH1) expression with the gain in N-cadherin and vimentin (VIM) expression associated with mesenchymal phenotype (enhanced migration, secretion of extracellular matrix [ECM] degrading proteases and ECM remodeling etc.) have been clinically associated with poor prognosis in many cancers [3, 6‐8]. In recent years, a good thrust of studies, including those in ovarian cancer, have demonstrated ‘incomplete or hybrid EMT’ or transitory cells within a ‘spectrum of EMT’ in tumors or cell lines where they retain both epithelial and mesenchymal features, and are able to migrate and invade like in a classical EMT process [6, 9]. Recent literature also suggests that EMT transformed cells can endorse on themselves the ability to evade host immunity by initiating several mechanisms. These include alterations in the antigen-processing machinery such as downregulation of MHC Class 1 molecule or transporters associated with antigen processing (TAP-1 and TAP-2) or enhancement in the expression of check point molecules such as PD-L1 on cancer cells to inhibit T cell function. In addition, genetic alterations which interfere with the interferon-gamma (IFN-γ) signaling pathway are also known to affect the antitumor responses in melanoma patients treated with immunotherapy [10].

An association between chemoresistance and the acquisition of EMT in ovarian cancer cells that attain cancer stem cells (CSC)-like phenotype has been demonstrated [11‐13]. These are a subpopulation of cells that possess the capacity of self-renewal and are responsible for drug resistance, tumor relapse and progression. They exploit therapy-induced selection pressure to give rise to resistant clones through plastic mechanisms such as EMT, which results in altered gene and protein expression and composition. Activation of EMT reprogramming and facilitation of cancer metastasis has been reported in multiple cancers through the induction of TGFβ and its inducible secreted extracellular matrix (ECM) associated proteins [14‐18]. The role of TGFBI is dependent on cellular context as its expression is elevated or suppressed in many cancers [19]. High expression of TGFBI interlinked with poor prognosis in patients has been noted in muscle invasive bladder cancer compared to non-muscle invasive bladder cancer tissues [15], pancreatic cancer [20], colorectal cancer [14], etc.

In addition to the tumor biology roles of TGFBI, pan-cancer analysis has indicated a prognostic role of TGFBI and associated that with various immune responses and functions (https://​papers.​ssrn.​com/​sol3/​cf_​dev/​AbsByAuth.​cfm?​per_​id=​4710390). In pancreatic cancer, cancer-associated fibroblasts express high levels of TGFBI which directly acts on tumor-specific CD8⁺ T cells and F4/80 macrophages in mice, reducing their proliferation and activation [20]. Targeting TGFBI in established lesions functionally reprogrammed F4/80 macrophages in tumor microenvironment [20]. In ovarian cancer, secreted TGFBI from tumor-associated macrophages in the ascites of ovarian cancer patients have been shown to promote migration of tumor cells [21, 22]. These observations suggest that tumor associated TGFBI is not only critical for driving cancer progression and metastasis but is also central in regulating cancer associated immune responses in host.

To understand the molecular basis of drug resistance in these cells, we analyzed the proteome in ovarian cancer cells and their platinum resistant counterpart in vitro. Our proteomic analyses revealed an enhanced expression of EMT and metabolic modulators in the carboplatin-resistant ovarian cancer cells. Given the increased expression of EMT modulators in our carboplatin-resistant ovarian cancer cells, we sought to explore this further by in vitro migration and bioenergetic assays. Evaluation of selected key proteomics-identified EMT-related proteins in platinum-sensitive, resistant, newly diagnosed, and relapsed ovarian tumor samples from ovarian cancer patients showed enhanced expression of some of these EMT-associated proteins in platinum-resistant vs sensitive and relapsed versus newly diagnosed patient’s tumors. Analysis with Kaplan–Meier plotter correlated high expression of some of the identified proteins as negative prognostic indicators. In addition, TIMER database analysis showed correlative positive expression of TGFBI with some of the identified proteins and their effect on infiltrating immune cells within the TME. Using these different platforms, our study provided a unified result depicting an induced EMT with an altered metabolic reprogramming in platinum-resistant ovarian cancer cells. This study has the potential to open avenues to design therapeutics aimed at targeting specific EMT- and metabolism-associated proteins to circumvent platinum resistance in ovarian cancer.

To understand the molecular changes between parental and carboplatin-resistant cells, we analyzed the proteomic changes in OVCAR5 parental and its carboplatin-resistant counterparts, OVCAR5 CBPR cell lines. Altogether, 2579 and 2576 proteins were identified in OVCAR5 parental and CBPR cell lines, respectively. Following filtering strategies described above, 2422 proteins were subjected to subsequent analyses. To analyze significant differences between OVCAR5 parental and CBPR cells, a selection criterion incorporating proteins which exhibited a fold change of > 2 at a statistically significant level (p < 0.05) were included in the study. Of these, 18 were upregulated (Table 1) and 14 downregulated (Table 2) by ≥ twofold at significant level (p < 0.05) in OVCAR5 CBPR cell lines. The peptide profile of up- and down-regulated proteins in OVCAR5 CBPR compared to OVCAR5 parental cell line is described in Additional file 2: Tables S3A and B.

Despite an initial good response to carboplatin, most ovarian cancer patients relapse due to an acquired or intrinsic resistance to platinum-based chemotherapy. In vitro studies have elucidated several cellular mechanisms driven by complex epigenetic and genetic changes to escape antitumor toxicities. Mechanisms of platinum resistance include, but not limited to, reduced accumulation of drugs due to impaired influx or potent efflux/secretion mechanisms, accumulation of reactive oxygen species (ROS) and simultaneous detoxification by antioxidants, elevated levels of DNA damage repair mechanisms, changes in membrane protein trafficking, aberrant protein and gene expressions altering drug transport and uptake, as well as pathways such as those hindering apoptosis and inducing EMT in response to chemotherapy treatment in surviving cells [29]. Platinum resistance has become the focus of cancer therapeutic development in recent years although no effective management has been shown to circumvent this phenomenon in resistant/refractory ovarian cancer patients. In this study, we sought to explore the proteome of ovarian cancer cell lines, the carboplatin sensitive OVCAR5 cells and their resistant counterparts, to identify proteins associated with carboplatin resistance and the associated functional consequences that may explain the resistance phenotype. We report an upregulation of several novel EMT modulators in the carboplatin-resistant cells including G6PD, AKR1B1, ITGAV, ITGA2, ITGA1, FLNA, GFPT2 and TGFBI. Functional analyses in vitro showed low proliferation and enhanced migratory features in the carboplatin-resistance cells, compared to parental chemo naïve cell line. An enhanced expression of EMT and metabolism regulators including G6PD and AKR1B1 and TGFβ1 observed in vitro in carboplatin resistant cell line was confirmed in platinum resistant and relapsed human ovarian tumor samples, compared to chemo naïve human tumors. In short, carboplatin-resistant cells acquired a plastic phenotype, and were less proliferative and more migratory. These slow-cycling resistant cells expressed drug resistance and CSC markers (ABCG2, CD44, CD105) and exhibited a low OXPHOS-glycolysis signature.

During cancer progression, tumor cells procure migratory feature imitating the phenotypic characteristics of EMT that occurs during embryogenesis and wound healing [30]. This feature of EMT is widely recognized as the central component of disseminated cancer cells that form distant metastasis [31]. Various mechanisms of enhanced migration in EMT-transformed cells have been elucidated, among those widely recognized is the loss of epithelial junction protein E-cadherin (CDH1) and replacement by other cadherins and intermediate filament protein vimentin (VIM) which facilitate mesenchymal transformation for single cell or collective migration [32]. In this study, we demonstrate decreased expression of E-cadherin and enhanced expression of vimentin, consistent with enhanced migration in CBPR OVCAR5 compared to their parental OVCAR5 cell line.

Our study also demonstrates reduced bioenergenetics in EMT transformed CBPR OVCAR5 cells, consistent with reduced proliferation in the resistant cell line compared to parental OVCAR5 cells. The link between EMT and reduced proliferation has been observed in many cellular model systems. Importantly, reduced proliferation in EMT-transformed disseminating cells at the invading margins of tumors has been noted. This presumably occurs to facilitate the re-establishment of migrating cells in their secondary niches to promote secondary growth and is enabled by the overexpression of cell cycle related cyclin-dependent kinase inhibitors p16(INK4a), p21(Cip1) or p27(Kip1) [33]. These observations are consistent with EMT programs described in cells under conditions that are not permissive for cellular growth, such as hypoxia and anoikis [34, 35]. It is also coherent with previous observations in chemotherapy surviving EMT transformed residual cells which had decreased bioenergetic demands, potentially to reduce their susceptibility to hostile cytotoxic microenvironment to enhance sustenance after therapeutic treatment [4]. Moreover, clinicopathological studies have demonstrated reduced proliferation in EMT-transformed circulating tumor cells (CTCs) and disseminated tumor cells during and after chemotherapy treatment [36]. In fact, slow-cycling phenotype in cancer cells has been recognized as a core mechanism for therapy resistance [37, 38]. Isolated populations of slow-growing, label-retaining colon cancer and breast cancer cells had enhanced survival when exposed to a third-generation platinum derivative, oxaliplatin, compared to non-labelled cells [38].

Altered expression of ECM components and integrins, have been widely documented in EMT-induced cancer cells preceding and through the metastatic process [39]. In ovarian cancer, the expression of several integrin complexes, including αvβ1, which binds fibronectin, and integrins α1β1 and α2β1, which interact with collagen, have been shown to contribute to cancer progression and chemoresistance [40]. Enhanced expression of ITGAV (αv subunit), ITGA1 (α1 subunit) and ITGA2 (α2 subunit) in OVCAR5 CBPR cells is indicative of ECM changes consistent with enhanced production of fibronectin and collagen observed in several malignancies and in most EMT-induced cancer models [41]. In addition, enhanced expression of filamin A (FLNA) may relate to cytoskeleton actin and tubulin reorganization as seen in different cancer models undergoing EMT [42].

In ovarian cancer, TGFβ1 induction has been shown to be a hallmark of EMT [43]. Cancer cells subjected to TGFβ1 treatment was found to become more motile and invasive [44]. TGFBI an ECM protein, with multiple functions in ovarian cancer is up regulated by TGFβ signaling pathway. Loss of TGFBI expression in HGSOC has been noted [19]. This occurs due to promoter hypermethylation of TGFBI promoter resulting in the silencing of the gene expression [19]. This promoter hypermethylation of TGFBI correlates with paclitaxel resistance in ovarian cancer [45]. In the same context, paclitaxel-resistant cells when treated with recombinant TGFBI protein showed increased paclitaxel sensitivity due to FAK-Rho dependent stabilization of microtubules [46]. These findings suggest that TGFBI plays an important role in chemoresistance. We thus investigated the expression of TGFβ1 and downstream targets following our observation of TGFBI overexpression in the mass spectrometry analysis of OVCAR5 CBPR cells. Enhanced expression of TGFβ1 protein as seen in our western blot analysis compliments the increased mRNA expression of TGFBI in OVCAR5 CBPR cells, suggesting an enhanced expression and functional activation of TGFβ1 in these cells. This is consistent with microarray studies that demonstrated significantly high expression of TGFBI in methotrexate, cisplatin, doxorubicin, vincristine, topotecan and paclitaxel-resistant ovarian cancer cell lines and reconciled those findings to changes in ECM remodeling in response to drug treatment [47].

Our proteomics findings and subsequent validation also demonstrated an enhanced expression of the ITGAV, ITGA1 and ITGA2 in OVCAR5 CBPR versus parental OVCAR5 cell lines. ITGAV expression has been shown to positively correlate with the molecular signature of mesenchymal cells and metastasis of cancer cells [7]. At the same token, TGFβ driven gemcitabine resistance and upregulated expression of ITGA1 has been noted in pancreatic ductal cell carcinoma, which promoted EMT and metastasis [48]. Similarly, overexpression of ITGA2 in esophageal squamous cell carcinoma cell lines promoted EMT and metastasis through FAK/AKT pathway [49] and also showed chemotherapy resistance in gastric cancer, while ITGA2 knockdown restored chemosensitivity by inducing apoptosis in chemoresistant cells [50]. These observations indicate a collective role of TGFBI, ITGAV, ITGA1 and ITGA2 in EMT-induced metastasis and chemotherapy resistance.

AKR1B1 also functions as a rate-limiting enzyme that catalyzes the reduction of glucose in the polyol pathway [51]. AKR1B1 overexpression has been shown to strongly correlate with the molecular profile of mesenchymal-like cells in various cancer models [52]. In lung cancer, high expression of AKR1B1 resulted in enhanced glutathione (GSH) synthesis and resistance to EGFR inhibitors in cell lines and xenograft models [51]. Whether the same aspect of AKR1B1 exists in OVCAR5 CBPR cells remains to be evaluated. We also report for the first-time an association of GFPT2 with chemotherapy resistant ovarian cancer. GFPT2 has been associated with enhanced glycosylation of proteins concomitant with mesenchymal functions and morphology and is regulated by GSH [53]. GFPT2 has been reported to initiate EMT in serous ovarian cancer by activating the hexosamine synthetic pathway to enhance the nuclear localization of β-catenin [54]. These observations suggest that both AKR1B1 and GFPT2 may work in conjunction to sustain the levels of GSH in CBPR OVCAR5 cells for their sustenance against the ROS insult triggered by platinum treatment.

Our proteomics data also revealed expression of G6PD to be higher in the OVCAR5 CBPR cells and this was consistent in platinum-resistant human ovarian tumors compared to their chemo-sensitive counterparts. This enzyme drives the first step of pentose phosphate pathway and converts glucose to ribose-5-phosphate required for nucleotide synthesis. This is consistent with the upregulation of hypoxanthine guanine phosphoribosyl transferase (HPRT1), an enzyme critical for the maintenance of purine salvage pathway. The upregulation of these rate-limiting enzymes could divert glucose metabolism through glycolysis towards pentose phosphate pathway and purine salvage pathways, which may explain the reduction of the glycolytic pathway in the OVCAR5 CBPR cells. Whilst not investigated in this study, pentose phosphate pathway also produces NADPH, which is central to the detoxification of ROS. The survival benefit of G6PD overexpression is exemplified in G6PD-deficient mice with high levels of oxidative damage in the brain [55]. Like other platinum antineoplastic drugs, carboplatin induces ROS production upon DNA binding. In resistant cancer cells, elevated expression of G6PD, AKR1B1 and GFPT2 counteracts the effects of ROS production as a prerequisite for cell survival and therapy resistance [56]. A recent study utilizing ovarian cancer patient-derived spheroids also reported significant association between cisplatin resistance, elevated levels of G6PD and enhanced level of GSH-producing enzymes in resistant cells [57]. These observations suggest the importance of chemotherapy-induced ECM remodeling (potentially through TGFBI) and ROS counteracting measures for the survival of chemotherapy stressed cancer cells.

Using multi-omic and bioenergetics analyses, a recent study demonstrated that high-OXPHOS ovarian cancer cells with higher ROS content exhibited greater sensitivity to taxane and platinum treatment compared to low-OXPHOS cells [58]. Given the pharmacological role of platinum compounds in initiating ROS production, tumor metabolic reprogramming to survive under therapy-induced oxidative stress is anticipated. This can be achieved by reducing intracellular ROS production through low levels of mitochondrial respiration, elevated dependency on glycolytic phenotype, or enhanced capacity of antioxidant detoxification [58, 59]. Our profiling of cellular bioenergetics using Seahorse extracellular flux assay revealed that the carboplatin-resistant cells have a low-OXPHOS low-glycolysis phenotype. The low metabolism and activated pathways responsible for ROS scavenging likely work in synergy to confer resistance to carboplatin-induced oxidative stress. The carboplatin resistant OVCAR5 cells also possess the ability to switch between glycolysis and OXPHOS, which indicates plasticity to adapt to metabolic stresses. In addition, they exhibited a lower rate of other non-glycolytic means of ATP generation such as the TCA cycle and/or glycogenolysis. The slower rate of cell growth but higher migratory capacity of the less metabolically active OVCAR5 CBPR cells may suggest that these cells undergo migration at the expense of proliferation. The downregulation of PCK2 (required for the conversion of oxaloacetate to pyruvate that feeds into the anabolic gluconeogenic pathway) and ACACA (a rate-limiting enzyme in de novo fatty acid synthesis that catalyzes the conversion of acetyl-CoA to malonyl-CoA) in OVCAR5 CBPR cells may also indicate a lack of dependency of these cells on anabolic gluconeogenic and fatty acid synthesis pathways. Further work needs to be done to investigate if these cells get metabolically ‘switched-on’ to support migration and reduced proliferation as reported in other slow-cycling cells.

The expression levels of carboplatin resistance-associated proteins, AKR1B1, ITGAV, TGFβ1 and G6PD, in platinum resistant and relapsed human ovarian cancer samples are consistent with our observations in vitro data on control and carboplatin-resistant cells. However, no prognostic role of AKRIBI, ITGAV and G6PD was evident in TCGA data and GSE (from Gene Expression Omnibus) datasets which included 738 high grade ovarian cancer patients that had undergone chemotherapy treatments. High expression of TGFBI, GFPT2, FLNA and ITGA2 on the other hand, were bad prognostic indicators in this patient cohort. Interestingly, the positive expression profiling of TGFBI with important ECM remodeling proteins such as GFPT2, FLNA, G6PD, ITGAV, ITGA1 and ITGA2 shown by TIMER datasets suggest a potential role of these proteins in remodeling ECM in response to carboplatin treatment. Consistent with our findings, a recent study has shown increased migratory, amino acid metabolism, protein catabolism and IFN1 signaling perturbation in platinum resistant ovarian cancer cell lines [60].

EMT in cancer cells has been associated with mitigating the immune system escape mechanisms in host [61]. In breast cancer, induction of EMT by overexpression of transcription factor SNAIL can render breast cancer cells resistant to the cytotoxic effect of CD8⁺T cells through the induction of autophagy; and targeting an autophagy inducer (BECN1) restored CD8⁺T mediated tumor cell lysis [62]. A recent pan cancer study indicated TGFBI as a prognostic marker in various cancers due to its involvement in various immune responses [63]. This is consistent with our analysis which showed a statistically significant positive association of infiltration of CD8⁺ T cells, CD4⁺ T cells, macrophages, neutrophils, and dendritic cells with TGFB1 expression. In addition, the enhanced expression of FLNA and ITGAV significantly associated with CD8⁺T cells, macrophages, and dendritic cells. Infiltration of dendritic cells was positively regulated by all the genes, except FLNA. As most of the ECM components in the study facilitated the infiltration of dendritic cells, may indicate essential role of remodeled ECM components in facilitating dendritic cell function which contributes in a significant way to antitumor response. However, approaches to use immunotherapy have not shown success in primary HGSOC or platinum resistant HGSOC patients [64, 65]. Potential reasons for the lack of immunotherapy response in HGSOC patients may include abundance of immunosuppressive factors in ovarian TME such as infiltration of a variety of immunosuppressive cells such as myeloid-derived suppressor cells (MDSC), cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAM) and Tregs. In addition, immunoregulatory enzymes (arginase, COX2, INOS) and immunosuppressive substances produced by these cells such as IL-10, TGFβ, vascular endothelial growth factor, PGE2, or PD-L1 inhibit innate and adaptive immunities and dendritic cell maturation. In addition, increased expression of checkpoint inhibitors such as PD-L1, CD47, CD73, in chemo naïve ovarian cancer cells and chemotherapy treated ovarian cancer cells have been noted [66, 67]. In addition, the density of intraepithelial CD8⁺T cells was shown to inversely correlate with the expression of PD-L1 on tumor cells, suggesting that the expression of PD-L1 on tumor cells may result in the exclusion of CD8 + T-cell in the tumors [66]. Some of these factors may indicate limited activity of immune check point inhibitors in treating chemonaive and platinum resistant ovarian cancer patients [65].

In summary, this study demonstrates that carboplatin-resistant cells acquired an EMT phenotype, which was less proliferative but more migratory. Altered expression of metabolic proteins also suggest a potential metabolic switch towards pentose phosphate pathway, polyol and hexosamine biosynthesis and purine salvage pathways with less dependency on anabolic gluconeogenesis and fatty acid biosynthesis. Enhanced expression of AKR1B1, GFPT2 and G6PD may sustain adequate level of GSH crucial for the survival of platinum—treated EMT transformed cells. On the other hand, upregulation of TGFBI in conjunction with ECM related proteins such as ITGAV, ITGA1, ITGA2, FLNA, etc. may facilitate ECM remodeling advantageous for chemoresistance and sustenance in the EMT transformed TME. Taken together, our findings provide molecular and functional evidence of EMT, altered metabolic and redox metabolism which supports carboplatin chemoresistance in ovarian cancer. The findings of this study have been depicted in Fig. 13.