The purpose of this study was to develop an integrated genomic-based approach to personalized treatment of patients with advanced-stage ovarian cancer. We have used gene expression profiles to ...identify patients likely to be resistant to primary platinum-based chemotherapy and also to identify alternate targeted therapeutic options for patients with de novo platinum-resistant disease.
A gene expression model that predicts response to platinum-based therapy was developed using a training set of 83 advanced-stage serous ovarian cancers and tested on a 36-sample external validation set. In parallel, expression signatures that define the status of oncogenic signaling pathways were evaluated in 119 primary ovarian cancers and 12 ovarian cancer cell lines. In an effort to increase chemotherapy sensitivity, pathways shown to be activated in platinum-resistant cancers were subject to targeted therapy in ovarian cancer cell lines.
Gene expression profiles identified patients with ovarian cancer likely to be resistant to primary platinum-based chemotherapy with greater than 80% accuracy. In patients with platinum-resistant disease, we identified expression signatures consistent with activation of Src and Rb/E2F pathways, components of which were successfully targeted to increase response in ovarian cancer cell lines.
We have defined a strategy for treatment of patients with advanced-stage ovarian cancer that uses therapeutic stratification based on predictions of response to chemotherapy, coupled with prediction of oncogenic pathway deregulation, as a method to direct the use of targeted agents.
Growth factors in tumor environments are regulators of cell survival and metastasis. Here, we reveal the dichotomy between TGF-β superfamily growth factors BMP and TGF-β/activin and their downstream ...SMAD effectors. Gene expression profiling uncovers SOX2 as a key contextual signaling node regulated in an opposing manner by BMP2, -4, and -9 and TGF-β and activin A to impact anchorage-independent cell survival. We find that SOX2 is repressed by BMPs, leading to a reduction in intraperitoneal tumor burden and improved survival of tumor-bearing mice. Repression of SOX2 is driven by SMAD1-dependent histone H3K27me3 recruitment and DNA methylation at SOX2’s promoter. Conversely, TGF-β, which is elevated in patient ascites, and activin A can promote SOX2 expression and anchorage-independent survival by SMAD3-dependent histone H3K4me3 recruitment. Our findings identify SOX2 as a contextual and contrastingly regulated node downstream of TGF-β members controlling anchorage-independent survival and metastasis in ovarian cancers.
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•SOX2 is a key node for anchorage-independent survival in cancer•SOX2 levels are differentially balanced by TGF-β/activin and BMPs in cancer•BMP9 is a robust intraperitoneal metastasis suppressor by lowering SOX2•SOX2 regulation is contextual and at the transcriptional level
Tumor cell survival upon loss of attachment is critical for metastasis. Shonibare et al. identify SOX2 as a contextual node regulated contrastingly by BMPs and TGF-β. Regulation occurs via distinct SMAD1- and SMAD3-dependent histone recruitment and DNA methylation mechanisms influencing anchorage-independent cell survival and intraperitoneal ovarian cancer metastasis.
Abstract Objective Detection of cell free tumor-specific DNA methylation has been proposed as a potentially useful noninvasive mechanism to detect malignancies, including ovarian cancer, and to ...monitor response to treatment. However, there are few easily implemented quantitative approaches available for DNA methylation analysis. Our objectives were to develop an absolute quantitative method for detection of DNA methylation using RASSF1A , a known target of promoter methylation in ovarian cancer, and test the ability to detect RASSF1A methylation in tumors and serum specimens of women with ovarian cancer. Methods Bisulfite modified DNAs were subjected to real time PCR using nondiscriminatory PCR primers and a probe with sequence containing a single CpG site, theoretically able to capture the methylation status of that CpG for every allele within a given specimen. Input DNA was normalized to ACTB levels detected simultaneously by assay multiplexing. Methylation levels were established by comparison to results obtained from universally methylated DNA. Results The assay was able to detect one methylated RASSF1A allele in 100,000 unmethylated alleles. RASSF1A was methylated in 54 of 106 (51%) invasive serous ovarian cancers analyzed and methylation status was concordant in 20/20 matched preoperative serum–tumor pairs. Serial serum specimens taken over the course of treatment for 8 of 9 patients showed fluctuations in RASSF1A methylation concomitant with disease status. Conclusions This novel assay provides a real-time PCR-based method for absolute quantitation of DNA methylation. Our results support feasibility of monitoring RASSF1A methylation from serum samples taken over the course of treatment from women with ovarian cancer.
Background: The aim of this study was to evaluate whether molecular classification prognosticates treatment response in women with endometrial cancers and endometrial intraepithelial neoplasia (EIN) ...treated with levonorgestrel intrauterine system (LNG-IUS). Methods: Patients treated with LNG-IUS for endometrial cancer or EIN from 2013 to 2018 were evaluated. Using immunohistochemistry and single gene sequencing of POLE, patients were classified into four groups as per the Proactive Molecular Risk Classifier for Endometrial cancer (ProMisE): POLE-mutated, mismatch repair-deficient (MMRd), p53 wild type (p53wt), and p53-abnormal (p53abn). Groups were assessed relative to the primary outcome of progression or receipt of definitive treatment. Results: Fifty-eight subjects with endometrioid endometrial cancer or EIN treated with LNG-IUS were included. Of these, 22 subjects (37.9%) had endometrial cancer and 36 subjects (62.1%) had EIN. Per the ProMisE algorithm, 44 patients (75.9%) were classified as p53wt, 6 (10.3%) as MMRd, 4 (6.9%) as p53abn, and 4 (6.9%) as POLE-mutated. Of the 58 patients, 11 (19.0%) progressed or opted for definitive therapy. Median time to progression or definitive therapy was 7.5 months, with p53abn tumors having the shortest time to progression or definitive therapy. Conclusions: Molecular classification of endometrial cancer and EIN prior to management with LNG-IUS is feasible and may predict patients at risk of progression.
Over 21,000 women are diagnosed with ovarian cancer (OC) in the United States each year and over half that number succumb to this disease annually, often due to recurrent disease. A deeper ...understanding of the molecular events associated with recurrent disease is needed to identify potential targets. Using genome-scale DNA methylation and gene expression data for 16 matched primary-recurrent advanced stage serous epithelial OCs, we discovered that Claudin-1 (
1), a tight junction protein, shows a stronger correlation between expression and methylation in recurrent versus primary OC at multiple CpG sites (R= -0.47 to -0.64 versus R= -0.32 to -0.57, respectively). An independent dataset showed that this correlation is stronger in tumors from short-term (<3y) survivors than in tumors from long-term (>7y) survivors (R= -0.41 to -0.46 versus R= 0.06 to -0.19, respectively). The presence of this inverse correlation in short-term survivors and recurrent tumors suggests an important role for this relationship and potential predictive value for disease prognosis.
expression increased following pharmacologic inhibition of DNA methyltransferase activity (p< 0.001), thus validating the role of methylation in
gene inhibition.
knockdown enhanced chemosensitivity and suppressed cell proliferation, migration, and wound healing (p< 0.05). Stable
knockdown
resulted in reduced xenograft tumor growth but did not reach significance. Our results indicate that the relationship between
methylation and expression plays an important role in OC aggressiveness and recurrence.
Background Epithelial ovarian cancer (OC) is the most lethal gynecological malignancy and patients present with significant metastatic burden, particularly to the adipose-rich microenvironment of the ...omentum. Recent evidence has highlighted the importance of metabolic adaptations in enabling this metastasis, leading to significant interest in evolving the arsenal of tools used to study OC metabolism. In this study, we demonstrate the capability of genetically encoded fluorescent biosensors to study OC, with a focus on 3D organoid models that better recapitulate in vivo tumor microenvironments. Materials and methods Plasmids encoding the metabolic biosensors HyPer, iNap, Peredox, and Perceval were transfected into 15 ovarian cancer cell lines to assay oxidative stress, NADPH/NADP+, NADH/NAD+, and ATP/ADP, respectively. Fluorescence readings were used to assay dynamic metabolic responses to omental conditioned media (OCM) and 100 muM carboplatin treatment. SKOV3 cells expressing HyPer were imaged as 2D monolayers, 3D organoids, and as in vivo metastases via an intravital omental window. We further established organoids from ascites collected from Stage III/IV OC patients with carboplatin-resistant or carboplatin-sensitive tumors (n = 8 total). These patient-derived organoids (PDOs) were engineered to express HyPer, and metabolic readings of oxidative stress were performed during treatment with 100 muM carboplatin. Results Exposure to OCM or carboplatin induced heterogenous metabolic changes in 15 OC cell lines, as measured using metabolic sensors. Oxidative stress of in vivo omental metastases, measured via intravital imaging of metastasizing SKOV3-HyPer cells, was more closely recapitulated by SKOV3-HyPer organoids than by 2D monolayers. Finally, carboplatin treatment of HyPer-expressing PDOs induced higher oxidative stress in organoids derived from carboplatin-resistant patients than from those derived from carboplatin-sensitive patients. Conclusions Our study showed that biosensors provide a useful method of studying dynamic metabolic changes in preclinical models of OC, including 3D organoids and intravital imaging. As 3D models of OC continue to evolve, the repertoire of biosensors will likely serve as valuable tools to probe the metabolic changes of clinical importance in OC.
In pathologies including cancer, aberrant Transforming Growth Factor-β (TGF-β) signaling exerts profound tumor intrinsic and extrinsic consequences. Intense clinical endeavors are underway to target ...this pathway. Central to the success of these interventions is pinpointing factors that decisively modulate the TGF-β responses. Betaglycan/type III TGF-β receptor (TβRIII), is an established co-receptor for the TGF-β superfamily known to bind directly to TGF-βs 1-3 and inhibin A/B. Betaglycan can be membrane-bound and also undergo ectodomain cleavage to produce soluble-betaglycan that can sequester its ligands. Its extracellular domain undergoes heparan sulfate and chondroitin sulfate glycosaminoglycan modifications, transforming betaglycan into a proteoglycan. We report the unexpected discovery that the heparan sulfate glycosaminoglycan chains on betaglycan are critical for the ectodomain shedding. In the absence of such glycosaminoglycan chains betaglycan is not shed, a feature indispensable for the ability of betaglycan to suppress TGF-β signaling and the cells' responses to exogenous TGF-β ligands. Using unbiased transcriptomics, we identified TIMP3 as a key inhibitor of betaglycan shedding thereby influencing TGF-β signaling. Our results bear significant clinical relevance as modified betaglycan is present in the ascites of patients with ovarian cancer and can serve as a marker for predicting patient outcomes and TGF-β signaling responses. These studies are the first to demonstrate a unique reliance on the glycosaminoglycan chains of betaglycan for shedding and influence on TGF-β signaling responses. Dysregulated shedding of TGF-β receptors plays a vital role in determining the response and availability of TGF-βs', which is crucial for prognostic predictions and understanding of TGF-β signaling dynamics.
Despite advances in surgery and targeted therapies, the prognosis for women with high-grade serous ovarian cancer remains poor. Moreover, unlike other cancers, immunotherapy has minimally impacted ...outcomes in patients with ovarian cancer. Progress in this regard has been hindered by the lack of relevant syngeneic ovarian cancer models to study tumor immunity and evaluate immunotherapies. To address this problem, we developed a luciferase labeled murine model of high-grade serous ovarian cancer, STOSE.M1 luc. We defined its growth characteristics, immune cell repertoire, and response to anti PD-L1 immunotherapy. As with human ovarian cancer, we demonstrated that this model is poorly sensitive to immune checkpoint modulators. By developing the STOSE.M1 luc model, it will be possible to probe the mechanisms underlying resistance to immunotherapies and evaluate new therapeutic approaches to treat ovarian cancer.
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