Daniela Matei

Epitranscriptomic Regulation of Platinum Resistance via the METTL3-ADAM23 Axis in Ovarian Cancer

N6-methyladenosine (m6A) has emerged as a pivotal regulator of post-transcriptional gene control, yet its contribution to chemotherapy resistance remains insufficiently defined. Here, we describe a previously unrecognized METTL3-ADAM23 epitranscriptomic regulatory relationship associated with platinum (Pt) resistance in ovarian cancer (OC). We show that cisplatin treatment increases global m6A levels and METTL3 expression, linking Pt exposure to activation of the m6A machinery. Functional perturbation studies demonstrate that METTL3 overexpression enhances cisplatin resistance, whereas METTL3 knockdown or pharmacologic inhibition with the selective METTL3 inhibitor STM2457 sensitizes OC cells to Pt treatment in vitro and improves Pt response in vivo. Transcriptomic profiling identifies ADAM23, a cell-adhesion-related tumor suppressor, as a METTL3-dependent, m6A-associated transcript, with altered mRNA expression observed across multiple experimental systems and several high-confidence predicted m6A sites within its transcript. Cisplatin-associated METTL3 upregulation correlates with reduced ADAM23 expression, suggesting a potential regulatory relationship that may contribute to chemoresistance. Together, these findings support a model in which METTL3-associated increases in m6A methylation are linked to Pt resistance, in part through modulation of ADAM23 expression, and highlight METTL3 as a potential therapeutic target in OC.

Metabolic Dependency on De Novo Pyrimidine Synthesis Is a Targetable Vulnerability in Platinum-Resistant Ovarian Cancer

Abstract Ovarian cancer is lethal because of near-universal development of resistance to platinum-based chemotherapy. Metabolic adaptations can play a pivotal role in therapy resistance. In this study, we aimed to identify key metabolic pathways that regulate platinum response and represent potential therapeutic targets. Transcriptomic and metabolomic analyses in cisplatin-sensitive and -resistant ovarian cancer cells identified enrichment of pyrimidine metabolism related to upregulated de novo pyrimidine synthesis. The 15N-glutamine flux analysis confirmed increased de novo pyrimidine synthesis in cisplatin-resistant cells. Targeting this pathway using brequinar (BRQ), an inhibitor of the key enzyme dihydroorotate dehydrogenase, decreased cell viability, delayed G2/M cell-cycle progression, and altered expression of genes related to mitochondrial electron transport in resistant cells. Under basal conditions, cisplatin-resistant cells had a lower oxygen consumption rate and spare respiratory capacity than cisplatin-sensitive cells. BRQ suppressed the oxygen consumption rate in both sensitive and resistant cells but only inhibited spare respiratory capacity in resistant cells. In cell line–derived and patient-derived xenograft models, BRQ attenuated the growth of cisplatin-resistant ovarian tumors and enhanced the inhibitory effects of carboplatin. Together, these results identify metabolic reprogramming in cisplatin-resistant ovarian cancer that induces an acquired dependency on de novo pyrimidine synthesis, which can be targeted to sensitize tumors to chemotherapy. Significance: De novo pyrimidine synthesis supports platinum resistance in ovarian cancer and can be targeted with DHODH inhibitors to suppress tumor growth, pointing to potential metabolic therapies for treating recurrent ovarian cancer.

Novel PROTACs targeting tissue transglutaminase (TG2) suppress tumorigenicity of ovarian cancer cells

Tissue transglutaminase (TG2), a multifunctional enzyme involved in protein crosslinking through transamidation, fibronectin-integrin interactions and GTP hydrolysis, is upregulated in cancer. Due to its diverse functions, TG2 has been a challenging therapeutic target. Here, we investigate the use of PROteolysis TArgeting Chimeras (PROTACs) to degrade TG2 and inhibit its tumor-promoting functions in ovarian cancer models. We describe a novel family of VHL based PROTACs using a ligand that binds to the TG2 fibronectin interacting domain and a thiol ether PEG linker. Three structurally related PROTACs-P374, P404, and P405-induced significant proteasome dependent TG2 degradation at 24 h (p < 0.05), with stable effects at 48 h. These compounds also potently inhibited cell adhesion and migration (p < 0.005), outside-in signaling, and blocked TG2 enzymatic activity (p < 0.001). An unbiased evaluation using reverse phase protein array of P374-treated cells revealed 136 differentially expressed proteins, including protein networks related to cell adhesion and involved in extracellular matrix (ECM) interactions. P374 and P405 reduced omental colonization in vivo and P374 inhibited intraperitoneal tumor dissemination and growth. Visium HD based spatial profiling of human ovarian tumors identified TG2 as a highly enriched protein at the tumors invasive edge and the interface with the ECM. Together, our findings put forward novel TG2-targeting PROTACs which effectively degrade TG2, impair its functions, and block in-vivo tumor dissemination. These results highlight the potential development of TG2 degraders towards therapeutic targeting in ovarian cancer.

CRISPR screen reveals a simultaneous targeted mechanism to reduce cancer cell selenium and increase lipid oxidation to induce ferroptosis

Ferroptosis is a cell death mechanism distinguished by its dependence on iron-mediated lipid oxidation. Cancer cells highly resistant to conventional therapies often demonstrate lipid metabolic and redox vulnerabilities that sensitize them to cell death by ferroptosis. These include a unique dependency on the lipid antioxidant selenoenzyme, glutathione peroxidase 4 (GPx4), that acts as a ferroptosis inhibitor. Synthetic high-density lipoprotein-like nanoparticle (HDL NP) targets the high-affinity HDL receptor scavenger receptor class B type 1 (SR-B1) and regulates cell and cell membrane lipid metabolism. Recently, we reported that targeting cancer cell SR-B1 with HDL NP depleted cell GPx4, which is accompanied by increased cell membrane lipid peroxidation and cancer cell death. These data suggest that HDL NP may induce ferroptosis. Thus, we conducted an unbiased CRISPR-based positive selection screen and target validation studies in ovarian clear cell carcinoma (OCCC) cell lines to ascertain the mechanism through which HDL NP regulates GPx4 and kills cancer cells. The screen revealed two genes, acyl-CoA synthetase long chain family member 4 (ACSL4) and thioredoxin reductase 1 (TXNRD1), whose loss conferred resistance to HDL NP. Validation of ACSL4 supports that HDL NP induces ferroptosis as the predominant mechanism of cell death, while validation of TXNRD1 revealed that HDL NP reduces cellular selenium and selenoprotein production, most notably, GPx4. Accordingly, we define cancer cell metabolic targets that can be simultaneously actuated by a multifunctional, synthetic HDL NP ligand of SR-B1 to kill cancer cells by ferroptosis.

Chromatin Organization Governs Transcriptional Response and Plasticity of Cancer Stem Cells

Abstract Chromatin organization regulates transcription to influence cellular plasticity and cell fate. We explored whether chromatin nanoscale packing domains are involved in stemness and response to chemotherapy. Using an optical spectroscopic nanosensing technology we show that ovarian cancer‐derived cancer stem cells (CSCs) display upregulation of nanoscale chromatin packing domains compared to non‐CSCs. Cleavage under targets and tagmentation (CUT&amp;Tag) sequencing with antibodies for repressive H3K27me3 and active H3K4me3 and H3K27ac marks mapped chromatin regions associated with differentially expressed genes. More poised genes marked by both H3K4me3 and H3K27me3 were identified in CSCs vs. non‐CSCs, supporting increased transcriptional plasticity of CSCs. Pathways related to Wnt signaling and cytokine‐cytokine receptor interaction were repressed in non‐CSCs, while retinol metabolism and antioxidant response were activated in CSCs. Comparative transcriptomic analyses showed higher intercellular transcriptional heterogeneity at baseline in CSCs. In response to cisplatin, genes with low baseline expression levels underwent the highest upregulation in CSCs, demonstrating transcriptional plasticity under stress. Epigenome targeting drugs downregulated chromatin packing domains and promoted cellular differentiation. A disruptor of telomeric silencing 1‐like (Dot1L) inhibitor blocked transcriptional plasticity, reversing stemness. These findings support that CSCs harbor upregulated chromatin packing domains, contributing to transcriptional and cell plasticity that epigenome modifiers can target.

Nanoparticle Targeting in Chemo‐Resistant Ovarian Cancer Reveals Dual Axis of Therapeutic Vulnerability Involving Cholesterol Uptake and Cell Redox Balance

Abstract Platinum (Pt)‐based chemotherapy is the main treatment for ovarian cancer (OC); however, most patients develop Pt resistance (Pt‐R). This work shows that Pt‐R OC cells increase intracellular cholesterol through uptake via the HDL receptor, scavenger receptor type B‐1 (SR‐B1). SR‐B1 blockade using synthetic cholesterol‐poor HDL‐like nanoparticles (HDL NPs) diminished cholesterol uptake leading to cell death and inhibition of tumor growth. Reduced cholesterol accumulation in cancer cells induces lipid oxidative stress through the reduction of glutathione peroxidase 4 (GPx4) leading to ferroptosis. In turn, GPx4 depletion induces decreased cholesterol uptake through SR‐B1 and re‐sensitizes OC cells to Pt. Mechanistically, GPx4 knockdown causes lower expression of the histone acetyltransferase EP300, leading to reduced deposition of histone H3 lysine 27 acetylation (H3K27Ac) on the sterol regulatory element binding transcription factor 2 (SREBF2) promoter and suppressing expression of this key transcription factor involved in the regulation of cholesterol metabolism. SREBF2 downregulation leads to decreased SR‐B1 expression and diminished cholesterol uptake. Thus, chemoresistance and cancer cell survival under high ROS burden obligates high GPx4 and SR‐B1 expression through SREBF2. Targeting SR‐B1 to modulate cholesterol uptake inhibits this axis and causes ferroptosis in vitro and in vivo in Pt‐R OC.

Preclinical Evaluation of NTX-301, a Novel DNA Hypomethylating Agent in Ovarian Cancer

Abstract Purpose: DNA methylation causes silencing of tumor-suppressor and differentiation-associated genes, being linked to chemoresistance. Previous studies demonstrated that hypomethylating agents (HMA) resensitize ovarian cancer to chemotherapy. NTX-301 is a highly potent and orally bioavailable HMA, in early clinical development. Experimental Design: The antitumor effects of NTX-301 were studied in ovarian cancer models by using cell viability, stemness and ferroptosis assays, RNA sequencing, lipidomic analyses, and stimulated Raman spectroscopy. Results: Ovarian cancer cells (SKOV3, IC50 = 5.08 nmol/L; OVCAR5 IC50 = 3.66 nmol/L) were highly sensitive to NTX-301 compared with fallopian tube epithelial cells. NTX-301 downregulated expression of DNA methyltransferases 1–3 and induced transcriptomic reprogramming with 15,000 differentially expressed genes (DEG, P &amp;lt; 0.05). Among them, Gene Ontology enrichment analysis identified regulation of fatty acid biosynthesis and molecular functions related to aldehyde dehydrogenase (ALDH) and oxidoreductase, known features of cancer stem cells. Low-dose NTX-301 reduced the ALDH(+) cell population and expression of stemness-associated transcription factors. Stearoyl-coenzyme A desaturase 1 (SCD), which regulates production of unsaturated fatty acids (UFA), was among the top DEG downregulated by NTX-301. NTX-301 treatment decreased levels of UFA and increased oxidized lipids, and this was blunted by deferoxamine, indicating cell death via ferroptosis. NTX-301–induced ferroptosis was rescued by oleic acid. In vivo, monotherapy with NTX-301 significantly inhibited ovarian cancer and patient-derived xenograft growth (P &amp;lt; 0.05). Decreased SCD levels and increased oxidized lipids were detected in NTX-301–treated xenografts. Conclusions: NTX-301 is active in ovarian cancer models. Our findings point to a new mechanism by which epigenetic blockade disrupts lipid homeostasis and promotes cancer cell death.

Biology-driven therapy advances in high-grade serous ovarian cancer

Following a period of slow progress, the completion of genome sequencing and the paradigm shift relative to the cell of origin for high grade serous ovarian cancer (HGSOC) led to a new perspective on the biology and therapeutic solutions for this deadly cancer. Experimental models were revisited to address old questions, and improved tools were generated. Additional pathways emerging as drivers of ovarian tumorigenesis and key dependencies for therapeutic targeting, in particular, VEGF-driven angiogenesis and homologous recombination deficiency, were discovered. Molecular profiling of histological subtypes of ovarian cancer defined distinct genetic events for each entity, enabling the first attempts toward personalized treatment. Armed with this knowledge, HGSOC treatment was revised to include new agents. Among them, PARP inhibitors (PARPis) were shown to induce unprecedented improvement in clinical benefit for selected subsets of patients. Research on mechanisms of resistance to PARPis is beginning to discover vulnerabilities and point to new treatment possibilities. This Review highlights these advances, the remaining challenges, and unsolved problems in the field.

Long-Term Follow-Up and Overall Survival in NRG258, a Randomized Phase III Trial of Chemoradiation Versus Chemotherapy for Locally Advanced Endometrial Carcinoma

This randomized phase III trial aimed to determine whether treatment with cisplatin and volume-directed radiation followed by carboplatin and paclitaxel for four cycles (chemoradiotherapy [C-RT]) increased recurrence-free survival (RFS) and overall survival (OS) when compared with carboplatin and paclitaxel for six cycles (chemotherapy [CT]) in locally advanced endometrial cancer (UC). Previously reported results showed that C-RT did not improve RFS compared with CT. Here we report the final OS analysis. Patients with International Federation of Gynecology and Obstetrics (FIGO) 2009 stage III-IVA UC or stage I/II serous or clear cell UC and positive cytology were enrolled. The primary objective was RFS. Secondary objectives were OS, toxicity, and quality of life. Cumulative probabilities of OS were estimated using the Kaplan-Meier method. Subgroup analyses of treatment effect for FIGO stage, age, race, gross residual disease, histology, lymph-vascular space invasion, and body mass index were performed. In total, 813 patients were randomly assigned (407 C-RT and 406 CT). The median follow-up was 112 months. Median OS was not achieved in either arm. The stratified hazard ratio for death comparing C-RT versus CT was 1.05 (95% CI, 0.82 to 1.34, log-rank two-sided P value = .72). None of the factors analyzed predicted OS benefit from C-RT. Although C-RT reduced the rate of local recurrence compared with CT, it did not increase OS or RFS in stage III/IVA UC.

Clinical and Biological Activity of Chemoimmunotherapy in Advanced Endometrial Adenocarcinoma: A Phase II Trial of the Big Ten Cancer Research Consortium

Purpose: The objective of this study was to assess the efficacy and safety of pembrolizumab in combination with standard carboplatin/paclitaxel in patients with advanced endometrial cancer. Patients and Methods: This single-arm, open-label, multicenter phase II study enrolled patients with RECIST measurable advanced endometrial cancer. Patients could have received ≤ 1 prior platinum-based regimen and ≤ one non-platinum chemotherapy. The primary endpoint was objective response rate (ORR). Planned sample size of 46 subjects provided 80% power to detect 15% ORR improvement compared with historical control rate of 50%. Results: A total of 46 patients were enrolled and 43 were evaluable for ORR. Median age was 66 (range: 43–86). Thirty-four (73.9%) patients had recurrent and 12 (26.1%) primary metastatic endometrial cancer. Patients received carboplatin AUC6, paclitaxel 175 mg/m2, and pembrolizumab 200 mg i.v. every 3 weeks for up to six cycles. ORR was 74.4% (32/43), higher than historic controls (P = 0.001). Median progression-free survival (PFS) was 10.6 months (95% confidence interval, 8.3–13.9 months). The most common grade 1–2 treatment-related adverse event (TRAE) included anemia (56.5%), alopecia (47.8%), fatigue (47.8%), and neuropathy (13%), while the most common grade 3–4 TRAEs were lymphopenia, leukopenia, and anemia (19.6% each). High-dimensional spectral flow cytometry (CyTEK) identified enrichment in peripheral CD8+ and CD4+ T-cell populations at baseline in responders. The CD8+ T-cell compartment in responders exhibited greater expression levels of PD-1 and PD-L1 and higher abundance of effector memory CD8+ cells compared with nonresponders. Conclusions: Addition of pembrolizumab to carboplatin and paclitaxel for advanced endometrial cancer was tolerated and improved ORR compared with historical outcomes. Significance: The results of the study support that the combination of pembrolizumab with carboplatin and paclitaxel is well tolerated and active in patients with advanced endometrial cancer. The duration of response and the PFS were significantly longer in patients with mismatch repair deficient/microsatellite instability-high compared with mismatch repair proficient/microsatellite stable tumors. Responders to treatment tend to have enriched CD8+ T-cell and CD4+ T-cell populations among peripheral blood mononuclear cells at baseline.

Complement activation at the interface between adipocytes and cancer cells drives tumor progression

The omentum is the primary site of metastasis for ovarian cancer (OC). Interactions between cancer cells and adipocytes drive an invasive and prometastatic phenotype. Here we studied cancer cell-adipocyte crosstalk by using a direct coculture model with immortalized human visceral nondiabetic pre-adipocytes (VNPADs) and OC cells. We demonstrated increased proliferation, invasiveness, and resistance to cisplatin of cocultured compared with monocultured OC cells. RNA sequencing of OC cells from coculture versus monoculture revealed significant transcriptomic changes, identifying over 200 differentially expressed genes common to OVCAR5 and OVCAR8 cell lines. Enriched pathways included PI3K/AKT and complement activation. Lipid transfer into OC cells from adipocytes induced upregulation of complement C3 and C5 proteins. Inhibiting C3 or C5 reversed the invasive phenotype and C3 knockdown reduced tumor progression in vivo. Increased C3 expression was observed in omental implants compared with primary ovarian tumors and C3 secretion was higher in OC ascites from high-BMI versus low-BMI patients. C3 upregulation in OC cells involved activation of the ATF4-mediated integrated stress response (ISR). Overall, adipocyte-cancer cell interactions promoted invasiveness and tumorigenesis via lipid transfer, activating the ISR, and upregulating complement proteins C3 and C5.

SOX9 drives a stem-like transcriptional state and platinum resistance in high-grade serous ovarian cancer

Chemotherapy resistance remains a formidable challenge to the treatment of high-grade serous ovarian cancer (HGSOC). The drug-tolerant cells may originate from a small population of inherently resistant cancer stem cells (CSCs) in primary tumors. In contrast, sufficient evidence suggests that drug tolerance can also be transiently acquired by nonstem cancer cells. Regardless of the route, key regulators of this plastic process are poorly understood. Here, we utilized multiomics, tumor microarrays, and epigenetic modulation to demonstrate that SOX9 is a key chemo-induced driver of chemoresistance in HGSOC. Epigenetic upregulation of SOX9 was sufficient to induce chemoresistance in multiple HGSOC lines. Moreover, this upregulation induced the formation of a stem-like subpopulation and significant chemoresistance in vivo. Mechanistically, SOX9 increased transcriptional divergence, reprogramming the transcriptional state of naive cells into a stem-like state. Supporting this, we identified a rare cluster of SOX9-expressing cells in primary tumors that were highly enriched for CSCs and chemoresistance-associated stress gene modules. Notably, single-cell analysis showed that chemo treatment results in rapid population-level induction of SOX9 that enriches for a stem-like transcriptional state. Altogether, these findings implicate SOX9 as a critical regulator of early steps of transcriptional reprogramming that lead to chemoresistance through a CSC-like state in HGSOC.

N6-Methyladenosine RNA Modifications Regulate the Response to Platinum Through Nicotinamide N-methyltransferase

Abstract Development of resistance to platinum (Pt) in ovarian cancer remains a major clinical challenge. Here we focused on identifying epitranscriptomic modifications linked to Pt resistance. Fat mass and obesity-associated protein (FTO) is a N6-methyladenosine (m6A) RNA demethylase that we recently described as a tumor suppressor in ovarian cancer. We hypothesized that FTO-induced removal of m6A marks regulates the cellular response of ovarian cancer cells to Pt and is linked to the development of resistance. To study the involvement of FTO in the cellular response to Pt, we used ovarian cancer cells in which FTO was knocked down via short hairpin RNA or overexpressed and Pt-resistant (Pt-R) models derived through repeated cycles of exposure to Pt. We found that FTO was significantly downregulated in Pt-R versus sensitive ovarian cancer cells. Forced expression of FTO, but not of mutant FTO, increased sensitivity to Pt in vitro and in vivo (P &amp;lt; 0.05). Increased numbers of γ-H2AX foci, measuring DNA double-strand breaks, and increased apoptosis were observed after exposure to Pt in FTO-overexpressing versus control cells. Through integrated RNA sequencing and MeRIP sequencing, we identified and validated the enzyme nicotinamide N-methyltransferase (NNMT), as a new FTO target linked to Pt response. NNMT was upregulated and demethylated in FTO-overexpressing cells. Treatment with an NNMT inhibitor or NNMT knockdown restored sensitivity to Pt in FTO-overexpressing cells. Our results support a new function for FTO-dependent m6A RNA modifications in regulating the response to Pt through NNMT, a newly identified RNA methylated gene target.

Methylomic Signatures of High Grade Serous Ovarian Cancer

High-grade serous ovarian cancer (HGSOC) harbours aberrant epigenetic features, including DNA methylation. In this study we delineate pathways and networks altered by DNA methylation and associated with HGSOC initiation and progression to a platinum-resistant state. By including tumours from patients who had been treated with the hypomethylating agent (HMA) guadecitabine, we also addressed the role of HMAs in treatment of HGSOC. Tumours from patients with primary (platinum-naïve) HGSOC (n = 20) were compared to patients with recurrent platinum-resistant HGSOC and enrolled in a recently completed clinical trial (NCT01696032). Human ovarian surface epithelial cells (HOSE; n = 5 samples) served as normal controls. Genome-wide methylation profiles were determined. DNA methyltransferase (DNMT) expression levels were examined by immunohistochemistry and correlated with clinical outcomes. Cancer-related and tumorigenesis networks were enriched among differentially methylated genes (DMGs) in primary OC vs. HOSE. When comparing platinum-resistant and primary tumours, 452 CpG island (CGI)-containing gene promoters acquired DNA methylation; of those loci, decreased (P < 0.01) methylation after HMA treatment was observed in 42% (n = 189 CGI). Stem cell pluripotency and cytokine networks were enriched in recurrent platinum-resistant OC tumours, while drug metabolism and transport-related networks were downregulated in tumours from HMA-treated patients compared to HOSE. Lower DNMT1 and 3B protein levels in pre-treatment tumours were associated with improved progression-free survival. The findings provide important insight into the DNA methylation landscape of HGSOC tumorigenesis, platinum resistance and epigenetic resensitization. Epigenetic reprogramming plays an important role in HGSOC aetiology and contributes to clinical outcomes.

Frizzled-7 Identifies Platinum-Tolerant Ovarian Cancer Cells Susceptible to Ferroptosis

Abstract Defining traits of platinum-tolerant cancer cells could expose new treatment vulnerabilities. Here, new markers associated with platinum-tolerant cells and tumors were identified using in vitro and in vivo ovarian cancer models treated repetitively with carboplatin and validated in human specimens. Platinum-tolerant cells and tumors were enriched in ALDH+ cells, formed more spheroids, and expressed increased levels of stemness-related transcription factors compared with parental cells. Additionally, platinum-tolerant cells and tumors exhibited expression of the Wnt receptor Frizzled-7 (FZD7). Knockdown of FZD7 improved sensitivity to platinum, decreased spheroid formation, and delayed tumor initiation. The molecular signature distinguishing FZD7+ from FZD7− cells included epithelial-to-mesenchymal (EMT), stemness, and oxidative phosphorylation-enriched gene sets. Overexpression of FZD7 activated the oncogenic factor Tp63, driving upregulation of glutathione metabolism pathways, including glutathione peroxidase 4 (GPX4), which protected cells from chemotherapy-induced oxidative stress. FZD7+ platinum-tolerant ovarian cancer cells were more sensitive and underwent ferroptosis after treatment with GPX4 inhibitors. FZD7, Tp63, and glutathione metabolism gene sets were strongly correlated in the ovarian cancer Tumor Cancer Genome Atlas (TCGA) database and in residual human ovarian cancer specimens after chemotherapy. These results support the existence of a platinum-tolerant cell population with partial cancer stem cell features, characterized by FZD7 expression and dependent on the FZD7–β-catenin–Tp63–GPX4 pathway for survival. The findings reveal a novel therapeutic vulnerability of platinum-tolerant cancer cells and provide new insight into a potential “persister cancer cell” phenotype. Significance: Frizzled-7 marks platinum-tolerant cancer cells harboring stemness features and altered glutathione metabolism that depend on GPX4 for survival and are highly susceptible to ferroptosis.

The Ratio of Toxic-to-Nontoxic miRNAs Predicts Platinum Sensitivity in Ovarian Cancer

Abstract Ovarian cancer remains one of the deadliest gynecologic malignancies affecting women, and development of resistance to platinum remains a major barrier to achieving a cure. Multiple mechanisms have been identified to confer platinum resistance. Numerous miRNAs have been linked to platinum sensitivity and resistance in ovarian cancer. miRNA activity occurs mainly when the guide strand of the miRNA, with its seed sequence at position 2–7/8, is loaded into the RNA-induced silencing complex (RISC) and targets complementary short seed matches in the 3′ untranslated region of mRNAs. Toxic 6mer seeds, which target genes critical for cancer cell survival, have been found in tumor-suppressive miRNAs. Many siRNAs and short hairpin RNAs (shRNA) can also kill cancer cells via toxic seeds, the most toxic of which carry G-rich 6mer seed sequences. We showed here that treatment of ovarian cancer cells with platinum led to increased RISC-bound miRNAs carrying toxic 6mer seeds and decreased miRNAs with nontoxic seeds. Platinum-tolerant cells did not exhibit this toxicity shift but retained sensitivity to cell death mediated by siRNAs carrying toxic 6mer seeds. Analysis of RISC-bound miRNAs in tumors from patients with ovarian cancer revealed that the ratio between miRNAs with toxic versus nontoxic seeds was predictive of treatment outcome. Application of the 6mer seed toxicity concept to cancer relevant miRNAs provides a new framework for understanding and predicting cancer therapy responses. Significance: These findings demonstrate that the balance of miRNAs that carry toxic and nontoxic 6mer seeds contributes to platinum resistance in ovarian cancer.

Ovarian cancer cell fate regulation by the dynamics between saturated and unsaturated fatty acids

Fatty acids are an important source of energy and a key component of phospholipids in membranes and organelles. Saturated fatty acids (SFAs) are converted into unsaturated fatty acids (UFAs) by stearoyl Co-A desaturase (SCD), an enzyme active in cancer. Here, we studied how the dynamics between SFAs and UFAs regulated by SCD impacts ovarian cancer cell survival and tumor progression. SCD depletion or inhibition caused lower levels of UFAs vs. SFAs and altered fatty acyl chain plasticity, as demonstrated by lipidomics and stimulated Raman scattering (SRS) microscopy. Further, increased levels of SFAs resulting from SCD knockdown triggered endoplasmic reticulum (ER) stress response with brisk activation of IRE1α/XBP1 and PERK/eIF2α/ATF4 axes. Disorganized ER membrane was visualized by electron microscopy and SRS imaging in ovarian cancer cells in which SCD was knocked down. The induction of long-term mild ER stress or short-time severe ER stress by the increased levels of SFAs and loss of UFAs led to cell death. However, ER stress and apoptosis could be readily rescued by supplementation with UFAs and reequilibration of SFA/UFA levels. The effects of SCD knockdown or inhibition observed in vitro translated into suppression of intraperitoneal tumor growth in ovarian cancer xenograft models. Furthermore, a combined intervention using an SCD inhibitor and an SFA-enriched diet initiated ER stress in tumors growing in vivo and potently blocked their dissemination. In all, our data support SCD as a key regulator of the cancer cell fate under metabolic stress and point to treatment strategies targeting the lipid balance.

Metabolic reprogramming from glycolysis to fatty acid uptake and beta-oxidation in platinum-resistant cancer cells

AbstractIncreased glycolysis is considered as a hallmark of cancer. Yet, cancer cell metabolic reprograming during therapeutic resistance development is under-studied. Here, through high-throughput stimulated Raman scattering imaging and single cell analysis, we find that cisplatin-resistant cells exhibit increased fatty acids (FA) uptake, accompanied by decreased glucose uptake and lipogenesis, indicating reprogramming from glucose to FA dependent anabolic and energy metabolism. A metabolic index incorporating glucose derived anabolism and FA uptake correlates linearly to the level of cisplatin resistance in ovarian cancer (OC) cell lines and primary cells. The increased FA uptake facilitates cancer cell survival under cisplatin-induced oxidative stress by enhancing beta-oxidation. Consequently, blocking beta-oxidation by a small molecule inhibitor combined with cisplatin or carboplatin synergistically suppresses OC proliferation in vitro and growth of patient-derived xenografts in vivo. Collectively, these findings support a rapid detection method of cisplatin-resistance at single cell level and a strategy for treating cisplatin-resistant tumors.

Tissue transglutaminase activates integrin-linked kinase and β-catenin in ovarian cancer

Ovarian cancer (OC) is the most lethal gynecological cancer. OC cells have high proliferative capacity, are invasive, resist apoptosis, and tumors often display rearrangement of extracellular matrix (ECM) components, contributing to accelerated tumor progression. The multifunctional protein tissue transglutaminase (TG2) is known to be secreted in the tumor microenvironment, where it interacts with fibronectin (FN) and the cell surface receptor integrin β1. However, the mechanistic role of TG2 in cancer cell proliferation is unknown. Here, we demonstrate that TG2 directly interacts with and facilitates the phosphorylation and activation of the integrin effector protein integrin-linked kinase (ILK) at Ser246. We show that TG2 and p-Ser246-ILK form a complex that is detectable in patient-derived OC primary cells grown on FN-coated slides. In addition, we show that coexpression of TGM2 and ILK correlates with poor clinical outcome. Mechanistically, we demonstrate that TG2-mediated ILK activation causes phosphorylation of glycogen synthase kinase-3α/β, allowing β-catenin nuclear translocation and transcriptional activity. Furthermore, inhibition of TG2 and ILK using small molecules, neutralizing antibodies, or shRNA-mediated knockdown blocks cell adhesion to the FN matrix, as well as the Wnt receptor response to the Wnt-3A ligand, and ultimately, cell adhesion, growth, and migration. In conclusion, we demonstrate that TG2 directly interacts with and activates ILK in OC cells and tumors and define a new mechanism that links ECM cues with β-catenin signaling in OC. These results suggest a central role of TG2-FN-integrin clusters in ECM rearrangement and indicate that downstream effector ILK may represent a potential new therapeutic target in OC.

SGI-110 in Combination With Carboplatin in Ovarian Cancer

A 2-part, Phase 2 controlled, open-label, randomized study in participants with platinum-resistant recurrent ovarian cancer. In Part 1, participants received SGI-110 and carboplatin. The optimum dose of SGI-110 (guadecitabine) was identified in Part 1 based on safety and efficacy. In Part 2, participants were randomized to receive the dose identified in Part 1 plus carboplatin or one of four treatment of choice at the discretion of the investigator. The treatment of choice consisted of topotecan, pegylated liposomal doxorubicin, paclitaxel or gemcitabine.