Kenneth P. Nephew

MYC and HSF1 Cooperate to Drive Sensitivity to Polo-like Kinase 1 Inhibitor Volasertib in High-grade Serous Ovarian Cancer

Abstract Ovarian cancer is a deadly gynecologic disease with frequent recurrence. Current treatments for patients include platinum-based therapy regimens with PARP inhibitors specific for homologous recombination–deficient high-grade serous ovarian cancers (HGSOC). Despite initial effectiveness, patients inevitably develop disease progression as tumor cells acquire resistance. Toward the development of new therapeutic avenues, we describe a gene amplification involving both heat shock factor 1 (HSF1) and MYC, wherein these two genes are co-amplified in more than 30% of patients with HGSOC. We further found that HSF1 and MYC transcriptional activities were highly correlated with human HGSOC tumors and cell lines, suggesting that they may cooperate in the disease. CUT&RUN sequencing for HSF1 and MYC revealed overlapping HSF1 and MYC binding throughout the genome. Moreover, the binding peaks of both transcription factors in HGSOC cells were nearly identical, and a protein–protein interaction between HSF1 and MYC was detected, supporting molecular cooperation. Supporting a functional cooperation of these two transcription factors, the growth of HGSOC cells with the co-amplification was dependent on both HSF1 and MYC. To identify a therapeutic target that could take advantage of this unique HSF1 and MYC dependency, polo-like kinase 1 (PLK1) was correlated with HSF1 and MYC in HGSOC specimens. Targeting PLK1 with volasertib revealed a greater than 200-fold increased potency in HSF1–MYC co-amplified HGSOC cells compared with those with wild-type HSF1 and MYC copy numbers. Although the success of volasertib and other PLK1 inhibitors in clinical trials has been modest, the current study suggests that targeting PLK1 using a precision medicine approach based on HSF1–MYC co-amplification as a biomarker in HGSOC would improve therapy response and patient outcomes. Significance: We show that HSF1 and MYC genes are co-amplified in more than 30% of HGSOC and demonstrate that HSF1 and MYC functionally cooperate to drive the growth of HGSOC cells. This work provides the foundation for HSF1 and MYC co-amplification as a biomarker for treatment efficacy of the polo-like kinase 1 inhibitor volasertib in HGSOC.

EZH2-Mediated Downregulation of the Tumor Suppressor DAB2IP Maintains Ovarian Cancer Stem Cells

Abstract The majority of women diagnosed with epithelial ovarian cancer eventually develop recurrence, which rapidly evolves into chemoresistant disease. Persistence of ovarian cancer stem cells (OCSC) at the end of therapy may be responsible for emergence of resistant tumors. In this study, we demonstrate that in OCSC, the tumor suppressor disabled homolog 2–interacting protein (DAB2IP) is silenced by EZH2-mediated H3K27 trimethylation of the DAB2IP promoter. CRISPR/Cas9-mediated deletion of DAB2IP in epithelial ovarian cancer cell lines upregulated expression of stemness-related genes and induced conversion of non-CSC to CSC, while enforced expression of DAB2IP suppressed CSC properties. Transcriptomic analysis showed that overexpression of DAB2IP in ovarian cancer significantly altered stemness-associated genes and bioinformatic analysis revealed WNT signaling as a dominant pathway mediating the CSC inhibitory effect of DAB2IP. Specifically, DAB2IP inhibited WNT signaling via downregulation of WNT5B, an important stemness inducer. Reverse phase protein array further demonstrated activation of noncanonical WNT signaling via C-JUN as a downstream target of WNT5B, which was blocked by inhibiting RAC1, a prominent regulator of C-JUN activation. Coadministration of EZH2 inhibitor GSK126 and RAC1 inhibitor NSC23766 suppressed OCSC survival in vitro and inhibited tumor growth and increased platinum sensitivity in vivo. Overall, these data establish that DAB2IP suppresses the cancer stem cell phenotype via inhibition of WNT5B-induced activation of C-JUN and can be epigenetically silenced by EZH2 in OCSC. Targeting the EZH2/DAB2IP/C-JUN axis therefore presents a promising strategy to prevent ovarian cancer recurrence and has potential for clinical translation. Significance: These findings show that combining an epigenetic therapy with a noncanonical WNT signaling pathway inhibitor has the potential to eradicate ovarian cancer stem cells and to prevent ovarian cancer recurrence.

Platinum-Induced Ubiquitination of Phosphorylated H2AX by RING1A Is Mediated by Replication Protein A in Ovarian Cancer

Abstract Platinum resistance is a common occurrence in high-grade serous ovarian cancer and a major cause of ovarian cancer deaths. Platinum agents form DNA cross-links, which activate nucleotide excision repair (NER), Fanconi anemia, and homologous recombination repair (HRR) pathways. Chromatin modifications occur in the vicinity of DNA damage and play an integral role in the DNA damage response (DDR). Chromatin modifiers, including polycomb repressive complex 1 (PRC1) members, and chromatin structure are frequently dysregulated in ovarian cancer and can potentially contribute to platinum resistance. However, the role of chromatin modifiers in the repair of platinum DNA damage in ovarian cancer is not well understood. We demonstrate that the PRC1 complex member RING1A mediates monoubiquitination of lysine 119 of phosphorylated H2AX (γH2AXub1) at sites of platinum DNA damage in ovarian cancer cells. After platinum treatment, our results reveal that NER and HRR both contribute to RING1A localization and γH2AX monoubiquitination. Importantly, replication protein A, involved in both NER and HRR, mediates RING1A localization to sites of damage. Furthermore, RING1A deficiency impairs the activation of the G2–M DNA damage checkpoint, reduces the ability of ovarian cancer cells to repair platinum DNA damage, and increases sensitivity to platinum. Implications: Elucidating the role of RING1A in the DDR to platinum agents will allow for the identification of therapeutic targets to improve the response of ovarian cancer to standard chemotherapy regimens.

Unintended Consequences of Antibiotic Therapy on the Microbiome Delivers a Gut Punch in Ovarian Cancer

Abstract While the early use of antibiotics during chemotherapy may be lifesaving, antibiotic therapy is associated with worse outcomes in patients with ovarian cancer during platinum chemotherapy. The study by Chambers and colleagues in this issue of Cancer Research provides mechanistic insights into how disrupting the gut microbiome with broad-spectrum antibiotics negatively influences the survival of patients with ovarian cancer and highlights the impact of the gut microbiome on tumor progression and response to therapy. Treatment of ovarian cancer models with a broad-spectrum antibiotic cocktail (ABX, vancomycin, neomycin sulfate, metronidazole, ampicillin) changed the gut microbiome and increased tumor growth and development of cisplatin resistance. Stem cells, reported to drive resistance to chemotherapy and disease recurrence in ovarian cancer, were enriched as a surprising consequence of ABX-induced microbiome disruption. Immune-competent and immune-deficient mice revealed that ABX treatment enhanced the cisplatin-induced stemness and provided evidence for immune surveillance of ovarian cancer stem cells through the gut microbiome. Two gut-derived metabolites, indole-3-propionic acid and indoxyl sulfate, suppressed by ABX treatment and reestablished with cecal microbial transplantation colonization of ABX-treated mice, were identified as potential effectors connecting the gut microbiome to ovarian cancer growth. This clinically relevant study opens new therapeutic opportunities for patients—one aimed at interventions to increase platinum sensitivity and another aimed at preventing the potential adverse effects of broad-spectrum antibiotic treatment. Both represent paradigm changes to standard care. See related article by Chambers et al., p. 4654

Epigenetic Attire in Ovarian Cancer: The Emperor's New Clothes

AbstractOvarian cancer is an aggressive epithelial tumor that remains a major cause of cancer morbidity and mortality in women. Epigenetic alterations including DNA methylation and histone modifications are being characterized in ovarian cancer and have been functionally linked to processes involved in tumor initiation, chemotherapy resistance, cancer stem cell survival, and tumor metastasis. The epigenetic traits of cancer cells and of associated tumor microenvironment components have been shown to promote an immunosuppressive tumor milieu. However, DNA methylation and histone modifications are reversible, and therapies targeting the epigenome have been implicated in potential reinvigoration of the antitumor immunity. In this review, we provide an overview specifically of DNA methylation and histone modifications as "clothes of the ovarian cancer genome" in relationship to their functional effects and highlight recent developments in the field. We also address the clinical implications of therapeutic strategies to remove or alter specific articles of genomic "clothing" and restore normal cellular function. As the clothes of the genome continue to be deciphered, we envision that the epigenome will become an important therapeutic target for cancer.

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.

Targeting Ovarian Cancer Stem Cells by Dual Inhibition of HOTAIR and DNA Methylation

Abstract Ovarian cancer is a chemoresponsive tumor with very high initial response rates to standard therapy consisting of platinum/paclitaxel. However, most women eventually develop recurrence, which rapidly evolves into chemoresistant disease. Persistence of ovarian cancer stem cells (OCSCs) at the end of therapy has been shown to contribute to resistant tumors. In this study, we demonstrate that the long noncoding RNA HOTAIR is overexpressed in HGSOC cell lines. Furthermore, HOTAIR expression was upregulated in OCSCs compared with non-CSC, ectopic overexpression of HOTAIR enriched the ALDH+ cell population and HOTAIR overexpression increased spheroid formation and colony-forming ability. Targeting HOTAIR using peptide nucleic acid-PNA3, which acts by disrupting the interaction between HOTAIR and EZH2, in combination with a DNMT inhibitor inhibited OCSC spheroid formation and decreased the percentage of ALDH+ cells. Disrupting HOTAIR-EZH2 with PNA3 in combination with the DNMTi on the ability of OCSCs to initiate tumors in vivo as xenografts was examined. HGSOC OVCAR3 cells were treated with PNA3 in vitro and then implanted in nude mice. Tumor growth, initiation, and stem cell frequency were inhibited. Collectively, these results demonstrate that blocking HOTAIR–EZH2 interaction combined with inhibiting DNA methylation is a potential approach to eradicate OCSCs and block disease recurrence.

Quality of Life and Adverse Events: Prognostic Relationships in Long-Term Ovarian Cancer Survival

Abstract Background There is a critical need to identify patient characteristics associated with long-term ovarian cancer survival. Methods Quality of life (QOL), measured by the Functional Assessment of Cancer Therapy-Ovarian-Trial Outcome Index (FACT-O-TOI), including physical, functional, and ovarian-specific subscales, was compared between long-term survivors (LTS) (8+ years) and short-term survivors (STS) (<5 years) of GOG 218 at baseline; before cycles 4, 7, 13, 21; and 6 months post-treatment using linear and longitudinal mixed models adjusted for covariates. Adverse events (AEs) were compared between survivor groups at each assessment using generalized linear models. All P values are 2-sided. Results QOL differed statistically significantly between STS (N = 1115) and LTS (N = 260) (P < .001). Baseline FACT-O-TOI and FACT-O-TOI change were independently associated with long-term survival (odds ratio = 1.05, 95% confidence interval = 1.03 to 1.06 and odds ratio = 1.06, 95% confidence interval = 1.05 to 1.07, respectively). A 7-point increase in baseline QOL was associated with a 38.0% increase in probability of LTS, and a 9-point increase in QOL change was associated with a 67.0% increase in odds for LTS. QOL decreased statistically significantly with increasing AE quartiles (cycle 4 quartiles: 0-5 vs 6-8 vs 9-11 vs ≥12 AEs, P = .01; cycle 21 quartiles: 0-2 vs 3 vs 4-5 vs ≥6 AEs, P = .001). Further, LTS reported statistically significantly better QOL compared with STS (P = .03 and P = .01, cycles 4 and 21, respectively), with similar findings across higher AE grades. Conclusions Baseline and longitudinal QOL change scores distinguished LTS vs STS and are robust prognosticators for long-term survival. Results have trial design and supportive care implications, providing meaningful prognostic value in this understudied population.

ZNFX1 Functions as a Master Regulator of Epigenetically Induced Pathogen Mimicry and Inflammasome Signaling in Cancer

Abstract DNA methyltransferase (DNMT) and PARP inhibitors induce a stimulator of IFN gene–dependent pathogen mimicry response (PMR) in ovarian and other cancers. In this study, we showed that combining DNMT and PARP inhibitors upregulates expression of the nucleic acid sensor NFX1-type zinc finger–containing 1 (ZNFX1) protein. ZNFX1 mediated the induction of PMR in mitochondria, serving as a gateway for stimulator of IFN gene–dependent IFN/inflammasome signaling. Loss of ZNFX1 in ovarian cancer cells promoted proliferation and spheroid formation in vitro and tumor growth in vivo. In patient ovarian cancer databases, expression of ZNFX1 was elevated in advanced stage disease, and ZNFX1 expression alone significantly correlated with an increase in overall survival in a phase III trial for patients with therapy-resistant ovarian cancer receiving bevacizumab in combination with chemotherapy. RNA sequencing revealed an association between inflammasome signaling through ZNFX1 and abnormal vasculogenesis. Together, this study identified that ZNFX1 is a tumor suppressor that controls PMR signaling through mitochondria and may serve as a biomarker to facilitate personalized therapy in patients with ovarian cancer. Significance: DNMT and PARP inhibitors induce a nucleic acid sensor, ZNFX1, that serves as a mitochondrial gateway to STING-dependent inflammasome signaling with tumor suppressor properties in ovarian cancer.

Ovarian tumor cells gain competitive advantage by actively reducing the cellular fitness of microenvironment cells

Cell competition and fitness comparison between cancer and tumor microenvironment (TME) cells determine oncogenic fate. Our previous study established a role for human Flower isoforms as fitness fingerprints, where the expression of Flower Win isoforms in tumor cells leads to growth advantage over TME cells expressing Lose isoforms. Here we demonstrate that the expression of Flower Lose and reduced microenvironment fitness is not a pre-existing condition but, rather, a cancer-induced phenomenon. Cancer cells actively reduce TME fitness by the exosome-mediated release of a cancer-specific long non-coding RNA, Tu-Stroma, which controls the splicing of the Flower gene in the TME cells and expression of Flower Lose isoform, which leads to reduced fitness status. This mechanism controls cancer growth, metastasis and host survival in ovarian cancer. Targeting Flower protein with humanized monoclonal antibody (mAb) in mice significantly reduces cancer growth and metastasis and improves survival. Pre-treatment with Flower mAb protects intraperitoneal organs from developing lesions despite the presence of aggressive tumor cells.

Claudin-4 Stabilizes the Genome via Nuclear and Cell-Cycle Remodeling to Support Ovarian Cancer Cell Survival

Abstract Alterations in the interplay between the nucleus and the cell cycle during cancer development lead to a state of genomic instability, often accompanied by observable morphologic aberrations. Tumor cells can regulate these aberrations to evade cell death, either by preventing or eliminating genomic instability. In epithelial ovarian cancer, overexpression of claudin-4 significantly contributes to therapy resistance through mechanisms associated with genomic instability regulation. However, the molecular mechanisms underlying claudin-4 overexpression in epithelial ovarian cancer remain poorly understood. In this study, we modified claudin-4 expression and employed a unique claudin mimic peptide to investigate claudin-4’s function. Our findings show that claudin-4 supports ovarian cancer cell survival by stabilizing the genome through nuclear and cell-cycle remodeling. Specifically, claudin-4 induced nuclear constriction by excluding lamin B1 and promoting perinuclear F-actin accumulation, thereby altering nuclear structure and dynamics. Similarly, cell-cycle modifications due to claudin-4 overexpression resulted in fewer cells entering the S-phase and reduced genomic instability in tumors. Importantly, disrupting claudin-4’s biological effects using claudin mimic peptide and forskolin increased the efficacy of PARP inhibitor treatment, correlating with alterations in the oxidative stress response. Our data indicate that claudin-4 protects tumor genome integrity by modulating the crosstalk between the nucleus and the cell cycle, leading to resistance to genomic instability formation and the effects of genomic instability–inducing agents. Significance: High-grade serous ovarian carcinoma is marked by chromosomal instability, which can serve to promote disease progression and allow cancer to evade therapeutic insults. The report highlights the role of claudin-4 in regulating genomic instability and proposes a novel therapeutic approach to exploit claudin-4–mediated regulation.

Targeting Ovarian Cancer Stem Cells by Dual Inhibition of the Long Noncoding RNA HOTAIR and Lysine Methyltransferase EZH2

Abstract The persistence of cancer stem cells (CSC) is believed to contribute to resistance to platinum-based chemotherapy and disease relapse in ovarian cancer, the fifth leading cause of cancer-related death among US women. HOXC transcript antisense RNA (HOTAIR) is a long, noncoding RNA (lncRNA) overexpressed in high-grade serous ovarian cancer and linked to chemoresistance. However, HOTAIR impacts chromatin dynamics in ovarian CSCs. Oncogenic lncRNA’s contributions to drug-resistant disease are incompletely understood. Here, we generated HOTAIR knockout (KO) high-grade serous ovarian cancer cell lines using paired CRISPR guide RNA design to investigate the function of HOTAIR. We show the loss of HOTAIR function resensitized ovarian cancer cells to platinum treatment and decreased the population of ovarian CSCs. Furthermore, HOTAIR KO inhibited the development of stemness-related phenotypes, including spheroid formation ability and expression of key stemness-associated genes ALDH1A1, NOTCH3, SOX9, and PROM1. HOTAIR KO altered the cellular transcriptome and chromatin accessibility landscape of multiple oncogenic-associated genes and pathways, including the NF-kB pathway. HOTAIR functions as an oncogene by recruiting enhancer of zeste homolog 2 (EZH2) to catalyze H3K27 trimethylation to suppress downstream tumor suppressor genes, and it was of interest to inhibit both HOTAIR and EZH2. In vivo, combining a HOTAIR inhibitor with an EZH2 inhibitor and platinum chemotherapy decreased tumor formation and increased survival. These results suggest a key role for HOTAIR in ovarian CSCs and malignant potential. Targeting HOTAIR in combination with epigenetic therapies may represent a therapeutic strategy to ameliorate ovarian cancer progression and resistance to platinum-based chemotherapy.

Not All STICs Are Equal: Unraveling the Implications of Precursor Heterogeneity

Serous tubal intraepithelial carcinomas (STIC) are precursors of high-grade serous carcinoma (HGSC), the deadliest subtype of ovarian carcinoma. To establish clinically actionable strategies against these lesions, a better understanding of the mutational, transcriptional, and genetic/epigenetic alterations, as well as interactions among epithelial, immune, and stromal cells, is essential. In this issue of Cancer Research, Shih and colleagues conducted the first integrated spatial multiomics analysis of ovarian precancerous lesions, revealing substantial heterogeneity within the fallopian tube epithelium that may influence cancer susceptibility. They described four molecular subclasses of STICs according to their epithelial transcriptomic profiles: proliferative, immunoreactive, mixed, and dormant (PIMD) subtypes. Molecular links of this “PIMD” STIC subclassification to tumor progression were proposed, uncovering early events in ovarian tumorigenesis and potential genetic drivers of STIC heterogeneity. Furthermore, the STIC subtypes showed distinct histologic and molecular characteristics that warrant further investigation to develop a deeper understanding of the molecular and cellular processes driving the evolution of STIC heterogeneity, which may facilitate the development of early diagnostic approaches for HGSC. Collectively, the findings that not all STICs are equal open new avenues for further clinicopathologic, translational, and basic research to improve risk classification and early intervention in HGSC. See related article by Chang et al., p. 1739