Huiying Piao

A comprehensive analysis of PAX8 expression in human epithelial tumors.

PAX8 is a paired-box gene important in embryogenesis of the thyroid, Müllerian, and renal/upper urinary tracts, and expression of PAX8 has been previously described in carcinomas from each of these sites. However, a large study including a wide variety of epithelial neoplasms from multiple organ sites other than the thyroid, kidney, or Müllerian system has not been performed. The goal of this study was to evaluate the utility of PAX8 immunostaining based on the evaluation of a wide range of epithelial tumors. PAX8 immunohistochemistry was performed on 1357 tumors (486 tumors in whole-tissue sections and 871 tumors in tissue microarrays, predominantly epithelial) from multiple organs. Only nuclear staining was scored as positive, and tumors were evaluated for the extent and intensity of staining. Western blot analysis with PAX8 was also performed on multiple tumor cell lines. Nuclear PAX8 staining was present in 91% (60 of 66) of thyroid tumors, 90% (158 of 176) of renal cell carcinomas (RCCs), 81% (13 of 16) of renal oncocytomas, 99% (164 of 165) of high-grade ovarian serous carcinomas, 71% (32 of 49) of nonserous ovarian epithelial neoplasms, 91% (10 of 11) of cervical epithelial lesions, and 98% (152 of 155) of endometrial adenocarcinomas. Of the remaining 719 evaluated tumors, only 30 cases (4%), including 12 thymic neoplasms, 3 bladder urothelial carcinomas, 4 lung squamous cell carcinomas, 2 esophageal adenocarcinomas, 1 pancreatic adenocarcinoma, 2 cholangiocarcinomas, 1 ovarian Sertoli-Leydig cell tumor, 1 ovarian sex cord stromal tumor, 3 testicular mixed germ cell tumors, and 1 acinic cell carcinoma, showed at least weak or focal PAX8 positivity. The unexpected finding was diffuse, moderate staining of PAX8 in a subset of thymomas and thymic carcinomas. The 689 remaining tumors, including but not limited to those from the prostate, colon, stomach, liver, adrenal gland, and head and neck, and small cell carcinomas from the lung, cervix, and ovary, were PAX8 negative. PAX8 specificity was confirmed by Western blot analysis, as expression was detected only in ovarian and RCC cell lines. These results show that PAX8 is a highly sensitive marker for thyroid, renal, Müllerian, and thymic tumors. Importantly, all lung adenocarcinomas, breast and adrenal neoplasms, and the majority of gastrointestinal tumors were negative for PAX8. Therefore, PAX8 is an excellent marker for confirming primary tumor site. In a subset of cases, additional markers, including but not limited to thyroid transcription factor-1, RCC, and Wilms tumor-1, may be needed to distinguish between the 3 most common PAX8-positive tumors.

Primary ex vivo cultures of human fallopian tube epithelium as a model for serous ovarian carcinogenesis

Recent studies suggest that some serous ovarian carcinomas (SOCs) arise from the fallopian tube (FT) epithelium rather than the ovarian surface epithelium. This hypothesis places emphasis on the FT secretory epithelial cell as a cell-of-origin. Herein, we report the development of a novel ex vivo primary human FT epithelium culture system that faithfully recapitulates the in vivo epithelium, as shown by morphological, ultrastructural and immunophenotypic analyses. Mass spectrometry-based proteomics reveal that these cultures secrete proteins previously identified as biomarkers for ovarian cancer. We also use this culture system to study the response of the FT epithelium to genotoxic stress and find that the secretory cells exhibit a distinct response to DNA damage when compared with neighboring ciliated cells. The secretory cells show a limited ability to resolve the damage over time, potentially leaving them more susceptible to accumulation of additional mutagenic injury. This divergent response is confirmed with in situ studies using tissue samples, further supporting the use of this ex vivo culture system to investigate FT epithelial pathobiology. We anticipate that this novel culture system will facilitate the study of SOC pathogenesis, and propose that similar culture systems could be developed for other organ site-specific epithelia.

An activated ErbB3/NRG1 autocrine loop supports in vivo proliferation in ovarian cancer cells.

Ovarian cancer is a leading cause of death from gynecologic malignancies. Treatment for advanced-stage disease remains limited and, to date, targeted therapies have been incompletely explored. By systematically suppressing each human tyrosine kinase in ovarian cancer cell lines by RNAi, we found that an autocrine signal-transducing loop involving NRG1 and activated ErbB3 operates in a subset of primary ovarian cancers and ovarian cancer cell lines. Perturbation of this circuit with ErbB3-directed RNAi decreased cell growth in three-dimensional culture and resulted in decreased disease progression and prolonged survival in a xenograft mouse model of ovarian cancer. Furthermore, a monoclonal ErbB3-directed antibody (MM-121) also significantly inhibited tumor growth in vivo. These findings identify ErbB3 as a potential therapeutic target in ovarian cancer.

Profiles of Genomic Instability in High-Grade Serous Ovarian Cancer Predict Treatment Outcome

Purpose: High-grade serous cancer (HGSC) is the most common cancer of the ovary and is characterized by chromosomal instability. Defects in homologous recombination repair (HRR) are associated with genomic instability in HGSC, and are exploited by therapy targeting DNA repair. Defective HRR causes uniparental deletions and loss of heterozygosity (LOH). Our purpose is to profile LOH in HGSC and correlate our findings to clinical outcome, and compare HGSC and high-grade breast cancers. Experimental Design: We examined LOH and copy number changes using single nucleotide polymorphism array data from three HGSC cohorts and compared results to a cohort of high-grade breast cancers. The LOH profiles in HGSC were matched to chemotherapy resistance and progression-free survival (PFS). Results: LOH-based clustering divided HGSC into two clusters. The major group displayed extensive LOH and was further divided into two subgroups. The second group contained remarkably less LOH. BRCA1 promoter methylation was associated with the major group. LOH clusters were reproducible when validated in two independent HGSC datasets. LOH burden in the major cluster of HGSC was similar to triple-negative, and distinct from other high-grade breast cancers. Our analysis revealed an LOH cluster with lower treatment resistance and a significant correlation between LOH burden and PFS. Conclusions: Separating HGSC by LOH-based clustering produces remarkably stable subgroups in three different cohorts. Patients in the various LOH clusters differed with respect to chemotherapy resistance, and the extent of LOH correlated with PFS. LOH burden may indicate vulnerability to treatment targeting DNA repair, such as PARP1 inhibitors. Clin Cancer Res; 18(20); 5806–15. ©2012 AACR.

An in-tumor genetic screen reveals that the BET bromodomain protein, BRD4, is a potential therapeutic target in ovarian carcinoma.

Significance The observations presented here demonstrate that inhibition of the BET bromodomain protein, BRD4, is a potential therapeutic approach to high-grade epithelial ovarian cancers that exhibit elevated MYCN expression. As BRD4 inhibitors enter clinical studies, these findings provide a rationale for stratification of patients in whom to test the effects of BRD4 inhibition. High-grade serous ovarian carcinoma (HGSOC) is the most common and aggressive form of epithelial ovarian cancer, for which few targeted therapies exist. To search for new therapeutic target proteins, we performed an in vivo shRNA screen using an established human HGSOC cell line growing either subcutaneously or intraperitoneally in immunocompromised mice. We identified genes previously implicated in ovarian cancer such as AURKA1, ERBB3, CDK2, and mTOR, as well as several novel candidates including BRD4, VRK1, and GALK2. We confirmed, using both genetic and pharmacologic approaches, that the activity of BRD4, an epigenetic transcription modulator, is necessary for proliferation/survival of both an established human ovarian cancer cell line (OVCAR8) and a subset of primary serous ovarian cancer cell strains (DFs). Among the DFs tested, the strains sensitive to BRD4 inhibition revealed elevated expression of either MYCN or c-MYC, with MYCN expression correlating closely with JQ1 sensitivity. Accordingly, primary human xenografts derived from high-MYCN or c-MYC strains exhibited sensitivity to BRD4 inhibition. These data suggest that BRD4 inhibition represents a new therapeutic approach for MYC-overexpressing HGSOCs.

Mesenchymal gene program-expressing ovarian cancer spheroids exhibit enhanced mesothelial clearance.

Metastatic dissemination of ovarian tumors involves the invasion of tumor cell clusters into the mesothelial cell lining of peritoneal cavity organs; however, the tumor-specific factors that allow ovarian cancer cells to spread are unclear. We used an in vitro assay that models the initial step of ovarian cancer metastasis, clearance of the mesothelial cell layer, to examine the clearance ability of a large panel of both established and primary ovarian tumor cells. Comparison of the gene and protein expression profiles of clearance-competent and clearance-incompetent cells revealed that mesenchymal genes are enriched in tumor populations that display strong clearance activity, while epithelial genes are enriched in those with weak or undetectable activity. Overexpression of transcription factors SNAI1, TWIST1, and ZEB1, which regulate the epithelial-to-mesenchymal transition (EMT), promoted mesothelial clearance in cell lines with weak activity, while knockdown of the EMT-regulatory transcription factors TWIST1 and ZEB1 attenuated mesothelial clearance in ovarian cancer cell lines with strong activity. These findings provide important insights into the mechanisms associated with metastatic progression of ovarian cancer and suggest that inhibiting pathways that drive mesenchymal programs may suppress tumor cell invasion of peritoneal tissues.

Establishment of patient-derived tumor xenograft models of epithelial ovarian cancer for preclinical evaluation of novel therapeutics

Purpose: Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States, with high rates of recurrence and eventual resistance to cytotoxic chemotherapy. Model systems that allow for accurate and reproducible target discovery and validation are needed to support further drug development in this disease. Experimental Design: Clinically annotated patient-derived xenograft (PDX) models were generated from tumor cells isolated from the ascites or pleural fluid of patients undergoing clinical procedures. Models were characterized by IHC and by molecular analyses. Each PDX was luciferized to allow for reproducible in vivo assessment of intraperitoneal tumor burden by bioluminescence imaging (BLI). Plasma assays for CA125 and human LINE-1 were developed as secondary tests of in vivo disease burden. Results: Fourteen clinically annotated and molecularly characterized luciferized ovarian PDX models were generated. Luciferized PDX models retain fidelity to both the nonluciferized PDX and the original patient tumor, as demonstrated by IHC, array CGH, and targeted and whole-exome sequencing analyses. Models demonstrated diversity in specific genetic alterations and activation of PI3K signaling pathway members. Response of luciferized PDX models to standard-of-care therapy could be reproducibly monitored by BLI or plasma markers. Conclusions: We describe the establishment of a collection of 14 clinically annotated and molecularly characterized luciferized ovarian PDX models in which orthotopic tumor burden in the intraperitoneal space can be followed by standard and reproducible methods. This collection is well suited as a platform for proof-of-concept efficacy and biomarker studies and for validation of novel therapeutic strategies in ovarian cancer. Clin Cancer Res; 23(5); 1263–73. ©2016 AACR.

Elafin drives poor outcome in high-grade serous ovarian cancers and basal-like breast tumors.

High-grade serous ovarian carcinoma (HGSOC) and basal-like breast cancer (BLBC) share many features including TP53 mutations, genomic instability and poor prognosis. We recently reported that Elafin is overexpressed by HGSOC and is associated with poor overall survival. Here, we confirm that Elafin overexpression is associated with shorter survival in 1000 HGSOC patients. Elafin confers a proliferative advantage to tumor cells through the activation of the MAP kinase pathway. This mitogenic effect can be neutralized by RNA interference, specific antibodies and a MEK inhibitor. Elafin expression in patient-derived samples was also associated with chemoresistance and strongly correlates with bcl-xL expression. We extended these findings into the examination of 1100 primary breast tumors and six breast cancer cell lines. We observed that Elafin is overexpressed and secreted specifically by BLBC tumors and cell lines, leading to a similar mitogenic effect through activation of the MAP kinase pathway. Here too, Elafin overexpression is associated with poor overall survival, suggesting that it may serve as a biomarker and therapeutic target in this setting.

Abstract 1202: Patient derived xenograft model platform of high grade serous ovarian cancer supporting discovery of targeted therapies and biomarkers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA High grade serous ovarian carcinoma (HGSOC) is the most lethal gynecologic malignancy in the United States. It is the most common form of ovarian cancer but early detection and effective therapeutics remain elusive and are a major unmet medical need. The current standard of care for ovarian cancer is platinum based therapy, without consideration of histological subtypes. While many patients exhibit an initial response to this standard therapy, few achieve long term remissions or cures. A major limitation in advancing therapeutic development has been a lack of experimental animal models that accurately represent the spectrum of HGSOC, are amenable to assess efficacy of novel therapeutics, are predictive of patient responses in the clinic and can identify patient subsets that will benefit from specific targeted therapeutics. To address this gap, we generated a platform of clinically relevant, early passage, orthotopic HGSOC patient-derived xenograft (PDX) models. These models were established by implanting mice intraperitoneally with fresh human ovarian cancer cells purified from operative or paracentesis samples under an IRB-approved protocol. Implanted mice were sacrificed after showing signs of abdominal distension and ascites development. Fresh mouse ascites-derived ovarian cancer cells were luciferized ex vivo by sophisticated lentivirus based methodologies to allow for non-invasive methods of tumor burden measurement. Luciferized PDX models were further expanded, banked and utilized for drug efficacy and biomarker evaluation studies. Surrogate biomarkers such as plasma CA-125 measured by BioScale AMMP method and circulating human cfDNA by LINE-1 qPCR have been validated and qualified in these models to support secondary methods of tumor burden evaluation. A diverse library of clinically-annotated, HGSOC PDX models has been established that encompass both treatment naive and treatment refractory HGSOC. Consistent with clinical disease, these orthotopic PDX models exhibit diffusely disseminated peritoneal disease with tissue infiltration to the omentum, ovaries, pancreas and spleen; along with distended abdomens full with ascites. Immunohistochemistry for ovarian specific markers including PAX8 and CK7 were analyzed in these tissues and shown to maintain fidelity to patient material through serial passages in mice. In vivo drug sensitivity studies with platinum and platinum/taxane doublet regimens recapitulate response as expected in HGSOC and, in a model of chemotherapy-resistant disease, demonstrate expected resistance as predicted by the clinical history. A clinically relevant HGSOC PDX model platform, amenable for testing efficacy of novel therapeutics and delineating responder ID has been established. Data will be presented with ongoing additional characterization of this platform. Citation Format: Sangeetha S. Palakurthi, Joyce F. Liu, Qing Zeng, Shan Zhou, Elena Ivanova, Cloud Paweletz, John Murgo, Justin Evangelista, Jennifer Curtis, Huiying Piao, Prafulla Gokhale, Jessie M. English, Paul Kirschmeier, Kwok-Kin Wong, Ursula A. Matulonis, Ronny Drapkin. Patient derived xenograft model platform of high grade serous ovarian cancer supporting discovery of targeted therapies and biomarkers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1202. doi:10.1158/1538-7445.AM2014-1202

Abstract 2837: Hitting the target: Dynamic BH3 profiling, a novel functional assay to predict chemotherapy response

Background: Most chemotherapeutic agents kill via the mitochondrial pathway of apoptosis, but unfortunately there is a lack of effective predictive biomarkers to assess the optimal treatment for each patient. When effective death signaling is initiated by a targeted therapy, an increase in mitochondrial apoptotic sensitivity (or ‘priming for death’) can be observed within hours. We developed a new technique, Dynamic BH3 Profiling, that measures changes in priming induced by chemotherapy in cancer cells, without the requirement for prolonged ex vivo culture, and we assessed if it could be used as predictive assay to personalize cancer therapy in patients. Hypothesis : Early apoptotic signaling detected via Dynamic BH3 Profiling can predict later cytotoxic cellular response. Results: Our first test was whether dynamic BH3 profiling performed following only 16 hours of drug exposure could predict cytotoxicity at a much later (72-96 hours) time point. We initiated our studies on a diverse panel of human cancer cell lines treated with a wide range of kinase inhibitors. These cell lines included solid tumors: NSCLC, breast cancer, melanoma and colon carcinoma; and hematological malignancies: multiple myeloma, CML, diffuse large B cell lymphoma and AML. We used different treatments (primary targets): Gefitinib (EGFR inh), Imatinib (BCR-ABL inh), Lapatinib (HER2 inh), TAE-684 (ALK inh), MK-2206 (Akt inh), PLX-4032 (BRaf V600E inh), AZD6244 (MEK inh), BEZ235 (PI3K/mTOR inh) and others. We observed a significant correlation between the increase in priming following short-term exposure to the agents and cell death at 72-96 hours, demonstrating the predicting capacity of dynamic BH3 profiling in different cancer cells. We then tested if dynamic BH3 profiling can predict clinical response in cancer patients. For that purpose, we used samples from stable phase CML and ovarian adenocarcinoma patients of known clinical outcome to imatinib or carboplatin treatment respectively. In both cases we found that the induction of mitochondrial priming caused by short term (16 hr) ex vivo exposure to treatment predicted clinical response. Moreover, the ROC curve analysis demonstrated that Dynamic BH3 profiling is an excellent binary predictor of response in vitro and in the clinic. Conclusions: Our cell line and clinical experiments demonstrate the potential for Dynamic BH3 profiling to be used as a powerful real-time tool to predict chemotherapy response across many cancers and many agents, including combinations of agents. It is an excellent binary predictor in multiple cell lines and different types of primary samples. and could be used to recognize the best agent from a list of possible therapies for an individual tumor and improve cancer therapy. Citation Format: Joan Montero, Kris A. Sarosiek, Joe D. DeAngelo, Huiying Piao, Neil Horowitz, Ross Berkowitz, Ursula Matulonis, Ronny Drapkin, Anthony Letai. Hitting the target: Dynamic BH3 profiling, a novel functional assay to predict chemotherapy response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2837. doi:10.1158/1538-7445.AM2014-2837