JCI Insight

Evolutionary mode and timing of dissemination of high-grade serous carcinomas

Dissemination within the peritoneal cavity is a main determinant of poor patient outcomes from high-grade serous carcinomas (HGSCs). The dissemination process is poorly understood from a cancer evolutionary perspective. We reconstructed the evolutionary trajectories across a median of 5 tumor sites and regions from each of 23 patients based on deep whole-exome sequencing. Polyclonal cancer origin was detected in 1 patient. Ovarian tumors had more complex subclonal architectures than other intraperitoneal tumors in each patient, which indicated that tumors developed earlier in the ovaries. Three common modes of dissemination were identified, including monoclonal or polyclonal dissemination of monophyletic (linear) or polyphyletic (branched) subclones. Mutation profiles of initial or disseminated clones varied greatly among cancers, but recurrent mutations were found in 7 cancer-critical genes, including TP53, BRCA1, BRCA2, and DNMT3A, and in the PI3K/AKT1 pathway. Disseminated clones developed late in the evolutionary trajectory models of most cancers, in particular in cancers with DNA damage repair deficiency. Polyclonal dissemination was predicted to occur predominantly as a single and rapid wave, but chemotherapy exposure was associated with higher genomic diversity of disseminated clones. In conclusion, we described three common evolutionary dissemination modes across HGSCs and proposed factors associated with dissemination diversity.

APOBEC3A drives ovarian cancer metastasis by altering epithelial-mesenchymal transition

High-grade serous ovarian cancer (HGSOC) is the most prevalent and aggressive histological subtype of ovarian cancer and often presents with metastatic disease. The drivers of metastasis in HGSOC remain enigmatic. APOBEC3A (A3A), an enzyme that generates mutations across various cancers, has been proposed as a mediator of tumor heterogeneity and disease progression. However, the role of A3A in HGSOC has not been explored. We observed an association between high levels of APOBEC3-mediated mutagenesis and poor overall survival in primary HGSOC. We experimentally addressed this correlation by modeling A3A expression in HGSOC, and this resulted in increased metastatic behavior of HGSOC cells in culture and distant metastatic spread in vivo, which was dependent on catalytic activity of A3A. A3A activity in both primary and cultured HGSOC cells yielded consistent alterations in expression of epithelial-mesenchymal transition (EMT) genes resulting in hybrid EMT and mesenchymal signatures, providing a mechanism for their increased metastatic potential. Inhibition of key EMT factors TWIST1 and IL-6 resulted in mitigation of A3A-dependent metastatic phenotypes. Our findings define the prevalence of A3A mutagenesis in HGSOC and implicate A3A as a driver of HGSOC metastasis via EMT, underscoring its clinical relevance as a potential prognostic biomarker. Our study lays the groundwork for the development of targeted therapies aimed at mitigating the deleterious effect of A3A-driven EMT in HGSOC.

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.

4EBP1-mediated SLC7A11 protein synthesis restrains ferroptosis triggered by MEK inhibitors in advanced ovarian cancer

Loss of ferroptosis contributes to the development of human cancer, and restoration of ferroptosis has been demonstrated as a potential therapeutic strategy in cancer treatment. However, the mechanisms of how ferroptosis escape contributes to ovarian cancer (OV) development are not well elucidated. Here, we show that ferroptosis negative regulation signatures correlated with the tumorigenesis of OV and were associated with poor prognosis, suggesting that restoration of ferroptosis represents a potential therapeutic strategy in OV. High-throughput drug screening with a kinase inhibitor library identified MEK inhibitors as ferroptosis inducers in OV cells. We further demonstrated that MEK inhibitor-resistant OV cells were less vulnerable to trametinib-induced ferroptosis. Mechanistically, mTOR/eIF4E binding protein 1 (4EBP1) signaling promoted solute carrier family 7 member 11 (SLC7A11) protein synthesis, leading to ferroptosis inhibition in MEK inhibitor-resistant cells. Dual inhibition of MEK and mTOR/4EBP1 signaling restrained the protein synthesis of SLC7A11 via suppression of the mTOR/4EBP1 axis to reactivate ferroptosis in resistant cells. Together, these findings provide a promising therapeutic option for OV treatment through ferroptosis restoration by the combined inhibition of MEK and mTOR/4EBP1 pathways.

Norepinephrine induces anoikis resistance in high-grade serous ovarian cancer precursor cells

High-grade serous carcinoma (HGSC) is the most lethal gynecological malignancy in the United States. Late diagnosis and the emergence of chemoresistance have prompted studies into how the tumor microenvironment, and more recently tumor innervation, may be leveraged for HGSC prevention and interception. In addition to stess-induced sources, concentrations of the sympathetic neurotransmitter norepinephrine (NE) in the ovary increase during ovulation and after menopause. Importantly, NE exacerbates advanced HGSC progression. However, little is known about the role of NE in early disease pathogenesis. Here, we investigated the role of NE in instigating anchorage independence and micrometastasis of preneoplastic lesions from the fallopian tube epithelium (FTE) to the ovary, an essential step in HGSC onset. We found that in the presence of NE, FTE cell lines were able to survive in ultra-low-attachment (ULA) culture in a β-adrenergic receptor-dependent (β-AR-dependent) manner. Importantly, spheroid formation and cell viability conferred by treatment with physiological sources of NE were abrogated using the β-AR blocker propranolol. We have also identified that NE-mediated anoikis resistance may be attributable to downregulation of colony-stimulating factor 2. These findings provide mechanistic insight and identify targets that may be regulated by ovary-derived NE in early HGSC.

Cancer-associated mesothelial cell–derived ANGPTL4 and STC1 promote the early steps of ovarian cancer metastasis

Ovarian cancer (OvCa) preferentially metastasizes in association with mesothelial cell-lined surfaces. We sought to determine if mesothelial cells are required for OvCa metastasis and detect alterations in mesothelial cell gene expression and cytokine secretion upon interaction with OvCa cells. Using omental samples from patients with high-grade serous OvCa and mouse models with Wt1-driven GFP-expressing mesothelial cells, we validated the intratumoral localization of mesothelial cells during human and mouse OvCa omental metastasis. Removing mesothelial cells ex vivo from human and mouse omenta or in vivo using diphtheria toxin-mediated ablation in Msln-Cre mice significantly inhibited OvCa cell adhesion and colonization. Human ascites induced angiopoietin-like 4 (ANGPTL4) and stanniocalcin 1 (STC1) expression and secretion by mesothelial cells. Inhibition of STC1 or ANGPTL4 via RNAi obstructed OvCa cell-induced mesothelial cell to mesenchymal transition while inhibition of ANGPTL4 alone obstructed OvCa cell-induced mesothelial cell migration and glycolysis. Inhibition of mesothelial cell ANGPTL4 secretion via RNAi prevented mesothelial cell-induced monocyte migration, endothelial cell vessel formation, and OvCa cell adhesion, migration, and proliferation. In contrast, inhibition of mesothelial cell STC1 secretion via RNAi prevented mesothelial cell-induced endothelial cell vessel formation and OvCa cell adhesion, migration, proliferation, and invasion. Additionally, blocking ANPTL4 function with Abs reduced the ex vivo colonization of 3 different OvCa cell lines on human omental tissue explants and in vivo colonization of ID8p53-/-Brca2-/- cells on mouse omenta. These findings indicate that mesothelial cells are important to the initial stages of OvCa metastasis and that the crosstalk between mesothelial cells and the tumor microenvironment promotes OvCa metastasis through the secretion of ANGPTL4.

Characterization of the vaginal microbiome of postmenopausal patients receiving chemoradiation for locally advanced cervical cancer

The standard-of-care treatment of locally advanced cervical cancer includes pelvic radiation therapy with concurrent cisplatin-based chemotherapy and is associated with a 30%-50% failure rate. New prognostic and therapeutic targets are needed to improve clinical outcomes. The vaginal microbiome has been linked to the pathogenesis of cervical cancer, but little is known about the vaginal microbiome in locally advanced cervical cancer as it relates to chemoradiation. In this pilot study, we utilized 16S rRNA gene community profiling to characterize the vaginal microbiomes of 26 postmenopausal women with locally advanced cervical cancer receiving chemoradiation. Our analysis revealed diverse anaerobe-dominated communities whose taxonomic composition, diversity, or bacterial abundance did not change with treatment. We hypothesized that characteristics of the microbiome might correlate with treatment response. Pretreatment microbial diversity and bacterial abundance were not associated with disease recurrence. We observed a greater relative abundance of Fusobacterium in patients who later had cancer recurrence, suggesting that Fusobacterium could play a role in modifying treatment response. Taken together, this hypothesis-generating pilot study provides insight into the composition and dynamics of the vaginal microbiome, offering proof of concept for the future study of the microbiome and its relationship with treatment outcomes in locally advanced cervical cancer.

Ovarian granulosa cell tumor characterization identifies FOXL2 as an immunotherapeutic target

Granulosa cell tumors (GCT) are rare ovarian malignancies. Due to the lack of effective treatment in late relapse, there is a clear unmet need for novel therapies. Forkhead Box L2 (FOXL2) is a protein mainly expressed in granulosa cells (GC) and therefore is a rational therapeutic target. Since we identified tumor infiltrating lymphocytes (TILs) as the main immune population within GCT, TILs from 11 GCT patients were expanded, and their phenotypes were interrogated to determine that T cells acquired late antigen-experienced phenotypes and lower levels of PD1 expression. Importantly, TILs maintained their functionality after ex vivo expansion as they vigorously reacted against autologous tumors (100% of patients) and against FOXL2 peptides (57.1% of patients). To validate the relevance of FOXL2 as a target for immune therapy, we developed a plasmid DNA vaccine (FoxL2-tetanus toxin; FoxL2-TT) by fusing Foxl2 cDNA with the immune-enhancing domain of TT. Mice immunization with FoxL2-TT controlled growth of FOXL2-expressing ovarian (BR5) and breast (4T1) cancers in a T cell-mediated manner. Combination of anti-PD-L1 with FoxL2-TT vaccination further reduced tumor progression and improved mouse survival without affecting the female reproductive system and pregnancy. Together, our results suggest that FOXL2 immune targeting can produce substantial long-term clinical benefits. Our study can serve as a foundation for trials testing immunotherapeutic approaches in patients with ovarian GCT.

CD73 restrains mutant β-catenin oncogenic activity in endometrial carcinomas

Approximately 30% of patients with endometrial carcinomas (ECs) with exon 3 CTNNB1 (β-catenin) mutations experience disease recurrence, whereas others with the same mutations remain recurrence-free. The molecular factors driving mutant β-catenin's oncogenic and clinical variability are unknown. Here we show that CD73 restrains the oncogenic activity of exon 3 β-catenin mutants, and CD73 loss is associated with recurrence. Using 7 patient-specific β-catenin mutants, together with genetic deletion or ectopic expression of CD73, we demonstrate that CD73 loss increases β-catenin-TCF/LEF transcriptional activity. In CD73-deficient cells, membrane levels of mutant β-catenin decreased, which corresponded with increased levels of nuclear and chromatin-bound mutant β-catenin. These results suggest that CD73 sequesters mutant β-catenin to the membrane to limit its oncogenic activity. Adenosine A1 receptor deletion phenocopied the effects of CD73 loss, implicating adenosine receptor signaling in this regulation. Ectopic CD73 expression suppressed the invasiveness and stemness capacity of β-catenin-mutant EC cells. TCGA analyses, GeoMx digital spatial profiling, and functional analyses showed that CD73 loss drives distinct Wnt-TCF/LEF-dependent gene expression programs linked to cancer cell stemness. These findings identify CD73 as a key regulator of mutant β-catenin, providing mechanistic insight into the variability of recurrence in CTNNB1-mutant EC.

Anoikis resistance and metastasis of ovarian cancer can be overcome by CDK8/19 mediator kinase inhibition

Anoikis resistance, or evasion of cell death triggered by matrix detachment, is a hallmark of cancer cell survival and metastasis. We showed that repeated exposure to suspension stress followed by recovery under attached conditions leads to development of anoikis resistance. The acquisition of anoikis resistance was associated with enhanced invasion, chemoresistance, and immune evasion in vitro and distant metastasis in vivo. This acquired anoikis resistance was not genetic, persisting for a finite duration without detachment stress, but was sensitive to CDK8/19 mediator kinase inhibition that could also reverse anoikis resistance. Transcriptomic analysis revealed that CDK8/19 kinase inhibition induces bidirectional transcriptional changes in both sensitive and resistant cells, disrupting the balanced reprogramming required for anoikis adaptation and resistance by reversing some resistance-associated pathways and enhancing others. Both anoikis resistance and in vivo metastatic growth of ovarian cancers are sensitive to CDK8/19 inhibition, thereby providing a therapeutic opportunity to both prevent and suppress ovarian cancer metastasis.

ADAR1 expression is associated with cervical cancer progression and negatively regulates NK cell activity

ADAR1 edits double-stranded RNAs (dsRNAs) by deaminating adenosines into inosines, preventing aberrant activation of innate immunity by endogenous dsRNAs, which may resemble viral structures. Several tumors exploit ADAR1 to evade immune surveillance; indeed, its deletion reduces tumor viability and reshapes infiltrating leukocytes. Here we investigated the role of ADAR1 in immune evasion mechanisms during cervical cancer (CC) progression. Patients' biopsy samples showed higher ADAR1 expression already in premalignant lesions (squamous intraepithelial lesions [SIL]) and a substantially reduced percentage of infiltrating CD7+ innate cells in in situ and invasive carcinomas compared with normal mucosa, with CD56+ NK cells showing phenotypic alterations that may have affected their functional responses. In CC-derived cell lines (SiHa, CaSki), ADAR1 silencing reduced cell proliferation, an effect further enhanced by exogenous IFN-β administration. It also induced proinflammatory gene expression, as demonstrated by RNA-Seq analysis, and conditioned supernatants collected from these cells activated several NK cell effector functions. NK cell infiltration and activation were also confirmed in organotypic 3D tissue models of SiHa cells knocked out for ADAR1. In conclusion, ADAR1 expression increased with CC progression and was accompanied by alterations in tumor-infiltrating NK cells, but its silencing in CC-derived cell lines potentiated antitumor NK cell activities. Thus, ADAR1 inhibition may represent a therapeutic perspective for CC and possibly other malignancies.

NFATC4 promotes quiescence and chemotherapy resistance in ovarian cancer

Development of chemotherapy resistance is a major problem in ovarian cancer. One understudied mechanism of chemoresistance is the induction of quiescence, a reversible nonproliferative state. Unfortunately, little is known about regulators of quiescence. Here, we identify the master transcription factor nuclear factor of activated T cells cytoplasmic 4 (NFATC4) as a regulator of quiescence in ovarian cancer. NFATC4 is enriched in ovarian cancer stem-like cells and correlates with decreased proliferation and poor prognosis. Treatment of cancer cells with cisplatin resulted in NFATC4 nuclear translocation and activation of the NFATC4 pathway, while inhibition of the pathway increased chemotherapy response. Induction of NFATC4 activity resulted in a marked decrease in proliferation, G0 cell cycle arrest, and chemotherapy resistance, both in vitro and in vivo. Finally, NFATC4 drove a quiescent phenotype in part via downregulation of MYC. Together, these data identify NFATC4 as a driver of quiescence and a potential new target to combat chemoresistance in ovarian cancer.

Loss of GATA2 promotes invasion and predicts cancer recurrence and survival in uterine serous carcinoma

BACKGROUNDA priori knowledge of recurrence risk in patients with nonmetastatic (International Federation of Gynecology and Obstetrics [FIGO] stage I) uterine serous carcinoma (USC) would enable a risk-stratified approach to the use of adjuvant chemotherapy. This would greatly reduce treatment-related morbidity and be predicted to improve survival.METHODSGATA2 expression was scored by IHC across a retrospective multiinstitutional cohort of 195 primary USCs. Associations between GATA2 levels and clinicopathologic metrics were evaluated using Student's t test, Fisher's exact test, Kaplan-Meier method, and Cox proportional hazard ratio. Invasion in patient-derived USC cells was assessed by Student's t test. RNA-Seq, anti-GATA2 ChIP-Seq, and confirmatory Western blotting enabled identification of GATA2 targets.RESULTSPatients with FIGO stage I GATA2hi USCs had 100% recurrence-free and 100% cancer-related survival, which was significantly better than patients with GATA2lo USCs. In patients for whom adjuvant chemotherapy was omitted, patients with GATA2hi USC had 100% recurrence-free 5-year survival compared with 60% recurrence-free survival in patients with GATA2lo USC. Depletion of GATA2 in patient-derived USC cells increased invasion in vitro.CONCLUSIONRoutine GATA2 IHC identifies 33% of patients with FIGO stage I USC who have a greatly reduced risk of posthysterectomy USC recurrence. Our results suggest that a GATA2-guided personalized medicine approach could be rapidly implemented in most hospital settings, would reduce treatment-related morbidity, and would likely improve outcomes in patients with USC.FUNDINGNIH grants R01 DK068634, P30 CA014520, S10 OD023526, K08 DK127244, T32 HL007899, the UW-Madison Department of Pathology and Laboratory Medicine, the UW-Madison Centennial Scholars Program, the Diane Lindstrom Foundation, the American Cancer Society, the V Foundation, The Hartwell Foundation, and the UMN Department of Obstetrics, Gynecology, and Women's Health.

PD-1 blockade restores helper activity of tumor-infiltrating, exhausted PD-1hiCD39+ CD4 T cells

Tumor antigen-specific CD4 T cells accumulate at tumor sites, evoking their involvement in antitumor effector functions in situ. Contrary to CD8 cytotoxic T lymphocyte exhaustion, that of CD4 T cells remains poorly appreciated. Here, using phenotypic, transcriptomic, and functional approaches, we characterized CD4 T cell exhaustion in patients with head and neck, cervical, and ovarian cancer. We identified a CD4 tumor-infiltrating lymphocyte (TIL) population, defined by high PD-1 and CD39 expression, which contained high proportions of cytokine-producing cells, although the quantity of cytokines produced by these cells was low, evoking an exhausted state. Terminal exhaustion of CD4 TILs was instated regardless of TIM-3 expression, suggesting divergence with CD8 T cell exhaustion. scRNA-Seq and further phenotypic analyses uncovered similarities with the CD8 T cell exhaustion program. In particular, PD-1hiCD39+ CD4 TILs expressed the exhaustion transcription factor TOX and the chemokine CXCL13 and were tumor antigen specific. In vitro, PD-1 blockade enhanced CD4 TIL activation, as evidenced by increased CD154 expression and cytokine secretion, leading to improved dendritic cell maturation and consequently higher tumor-specific CD8 T cell proliferation. Our data identify exhausted CD4 TILs as players in responsiveness to immune checkpoint blockade.

Phase I trial of ribociclib with platinum chemotherapy in ovarian cancer

BACKGROUNDNew therapeutic combinations to improve outcomes of patients with ovarian cancer are clearly needed. Preclinical studies with ribociclib (LEE-011), a CDK4/6 cell cycle checkpoint inhibitor, demonstrate a synergistic effect with platinum chemotherapy and efficacy as a maintenance therapy after chemotherapy. We tested the safety and initial efficacy of ribociclib in combination with platinum-based chemotherapy in recurrent ovarian cancer.METHODSThis phase I trial combined weekly carboplatin and paclitaxel chemotherapy with ribociclib, followed by ribociclib maintenance in patients with recurrent platinum-sensitive ovarian cancer. Primary objectives were safety and maximum tolerated dose (MTD) of ribociclib when given with platinum and taxane chemotherapy. Secondary endpoints were response rate (RR) and progression-free survival (PFS).RESULTSThirty-five patients were enrolled. Patients had a mean of 2.5 prior lines of chemotherapy, and 51% received prior maintenance therapy with poly(ADP-ribose) polymerase inhibitors and/or bevacizumab. The MTD was 400 mg. The most common adverse events included anemia (82.9%), neutropenia (82.9%), fatigue (82.9%), and nausea (77.1%). The overall RR was 79.3%, with a stable disease rate of 18%, resulting in a clinical benefit rate of 96.6%. Median PFS was 11.4 months. RR and PFS did not differ based on the number of lines of prior chemotherapy or prior maintenance therapy.CONCLUSIONThis work demonstrates that the combination of ribociclib with chemotherapy in ovarian cancer is feasible and safe. With a clinical benefit rate of 97%, this work provides encouraging evidence of clinical efficacy in patients with recurrent platinum-sensitive disease.TRIAL REGISTRATIONClinicalTrials.gov NCT03056833.FUNDINGThis investigator-initiated trial was supported by Novartis, which provided drugs and funds for trial execution.

CXCL13-producing CD4+ T cells accumulate in the early phase of tertiary lymphoid structures in ovarian cancer

Tertiary lymphoid structures (TLS) are transient ectopic lymphoid aggregates whose formation might be caused by chronic inflammation states, such as cancer. However, how TLS are induced in the tumor microenvironment (TME) and how they affect patient survival are not well understood. We investigated TLS distribution in relation to tumor infiltrating lymphocytes (TILs) and related gene expression in high-grade serous ovarian cancer (HGSC) specimens. CXCL13 gene expression correlated with TLS presence and the infiltration of T cells and B cells, and it was a favorable prognostic factor for patients with HGSC. Coexistence of CD8+ T cells and B cell lineages in the TME significantly improved the prognosis of HGSC and was correlated with the presence of TLS. CXCL13 expression was predominantly coincident with CD4+ T cells in TLS and CD8+ T cells in TILs, and it shifted from CD4+ T cells to CD21+ follicular DCs as TLS matured. In a mouse ovarian cancer model, recombinant CXCL13 induced TLS and enhanced survival by the infiltration of CD8+ T cells. These results suggest that TLS formation was associated with CXCL13-producing CD4+ T cells and that TLS facilitated the coordinated antitumor response of cellular and humoral immunity in ovarian cancer.