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Ovarian carcinoma histotypes are distinct diseases with variable clinical outcomes and response to treatment. There is a need for new subtype‐specific treatment modalities, especially for women with widespread and chemo‐resistant disease. Stimulator of interferon genes (STING) is a part of the cGAS–STING pathway that mediates innate immune defence against infectious DNA‐containing pathogens and also detects tumour‐derived DNA and generates intrinsic antitumour immunity. The STING signalling pathway is suppressed by several mechanisms in a variety of malignant diseases and, in some cancers that may be a requirement for cellular transformation. The aim of this study was to use immunohistochemistry to evaluate STING protein expression across normal tissue, paratubal and ovarian cysts, and ovarian tumour histotypes including ovarian carcinomas. Herein, we show that the fallopian tube ciliated cells express STING protein, whereas the secretory cells are negative. STING expression differs among ovarian cancer histotypes; low‐grade serous ovarian carcinomas and serous borderline tumours have uniform high STING expression, while high‐grade serous and endometrioid carcinomas have heterogeneous expression, and clear cell and mucinous carcinomas show low expression. As low‐grade serous carcinomas are known to be genomically stable and typically lack a prominent host immune response, the consistently high STING expression is unexpected. High STING expression may reflect pathway activation or histogenesis and the mechanisms may be different in different ovarian carcinoma histotypes. Further studies are needed to determine whether the STING signalling pathway is active and whether these tumours would be candidates for therapeutic interventions that trigger innate immunity activation.

Background: Hepatocellular carcinoma (HCC) is the fourth leading cause of death from cancer worldwide, and for advanced HCC the prognosis is poor. Preliminary studies indicate mistletoe extracts may have anticancer activity for HCC. Methods: A prospective observational case series of advanced HCC patients that chose to take a mistletoe extract called viscum fraxini-2 (VF-2) alone for treatment. Time on treatment, imaging, and laboratory values were collected for descriptive analyses. Results: A total of 12 patients with advanced HCC enrolled onto the protocol, and 10 patients had data available for evaluation. The majority were male (10/12) with a median age of 64 (SD 11). Most patients had received sorafenib therapy (9/12) and had varying Child-Pugh classes (A-4, B-6, C-2). Treatment with VF-2 ranged from 1 to 36 weeks with a mean of 12.3 weeks (SD 12). Six patients received 8 weeks of treatment, and 3 patients received 12 or more weeks of treatment. For patients that received at least 4 weeks of treatment, the average AFP value stabilized during the first 4 weeks of treatment. Two patients experienced an AFP decrease of >30%, approximately 37 and 40% decreases at the nadir. One patient had stable disease of 9 months. Major side effects were fever, fatigue, rash, and local injection site reaction of swelling, redness, and tenderness. Conclusion: This case series of advanced HCC indicates that mistletoe extract VF-2 may have potential biological activity against HCC for selected patients. Research is needed to identify the active compound and predictive markers of response.

Background: We developed a fluorophore-conjugated peptide agent, SBK4, that detects a tumor-specific proteolyzed form of the cell adhesion molecule, PTPmu, found in the tumor microenvironment. We previously demonstrated its tissue specific distribution in high-grade brain tumors. To extend those studies to other aggressive solid tumor types, we assessed the tissue distribution of PTPmu/SBK4 in a set of matched gynecologic cancer patient derived xenografts (PDXs) and primary patient tumors, as well as a limited cohort of tumors from gynecological cancer patients. PDXs isolated from the tissues of cancer patients have been shown to yield experimentally manipulatable models that replicate the clinical characteristics of individual patients’ tumors. In this study, gynecological cancer PDXs and patient biopsies were examined to determine if tumor-specific proteolyzed PTPmu was present. Methods: We used the peptide agent SBK4 conjugated to the fluorophore Texas Red (TR) to label tumor tissue microarrays (TMAs) containing patient and/or PDX samples from several high-grade gynecologic cancer types, and quantified the level of staining with Image J. In one TMA, we were able to directly compare the patient and the matched PDX tissue on the same slide. Results: While normal tissue had very little SBK4-TR staining, both primary tumor tissue and PDXs have higher labeling with SBK4-TR. Matched PDXs and patient samples from high-grade endometrial and ovarian cancers demonstrated higher levels of PTPmu by staining with SBK4 than normal tissue. Conclusion: In this sample set, all PDXs and high-grade ovarian cancer samples had increased labeling by SBK4-TR compared with the normal controls. Our results indicate that proteolyzed PTPmu and its novel peptide detection agent, SBK4, allow for the visualization of tumor-specific changes in cell adhesion molecules by tissue-based staining, providing a rationale for further development as an imaging agent in aggressive solid tumors, including gynecological cancers.

Purpose: Acquired resistance to cisplatin is a major barrier to success in treatment of various cancers, and understanding mitotic mechanisms unique to cisplatin-resistant cancer cells can provide the basis for developing novel mitotic targeted therapies aimed at eradicating these cells. Experimental Design: Using cisplatin-resistant models derived from primary patient epithelial ovarian cancer (EOC) cells, we have explored the status of mitotic exit mechanisms in cisplatin-resistant cells. Results: We have uncovered an unexpected role of long-term cisplatin treatment in inducing mitotic exit vulnerability characterized by increased spindle checkpoint activity and functional dependency on Polo-like kinase 1 (PLK1) for mitotic exit in the presence of anaphase promoting complex/cyclosome (APC/C) dysfunction in a cisplatin-resistant state. Accordingly, PLK1 inhibition decreased the survival of cisplatin-resistant cells in vitro and in vivo and exacerbated spindle checkpoint response in these cells. APC/CCDC20 inhibition increased sensitivity to pharmacologic PLK1 inhibition, further confirming the existence of APC/C dysfunction in cisplatin-resistant cells. In addition, we uncovered that resistance to volasertib, PLK1 inhibitor, is due to maintenance of cells with low PLK1 expression. Accordingly, stable PLK1 downregulation in cisplatin-resistant cells induced tolerance to volasertib. Conclusions: We provide the first evidence of APC/C dysfunction in cisplatin-resistant state, suggesting that understanding APC/C functions in cisplatin-resistant state could provide a basis for developing novel mitotic exit–based therapies to eradicate cisplatin-resistant cancer cells. Our results also show that PLK1 downregulation could underlie emergence of resistance to PLK1-targeted therapies in cancers. Clin Cancer Res; 24(18); 4588–601. ©2018 AACR.

Large-scale chromosomal alterations, particularly chromosomal deletions in cancer genomes confer functional advantages to cancer cells via the loss of tumor suppressor genes (TSGs). However, due to the nature of these focal and arm-level deletions, essential house-keeping genes in the neighborhood of TSGs are potentially lost. We explore the emergence of metabolic adaptations and vulnerabilities that arise due to the collateral loss of essential metabolic genes. In our previous work, we showed that genomic loss in the locus containing SMAD4 and ME2 in pancreatic ductal adenocarcinomas forces these cells to rely on ME3 to compensate for the collateral loss of ME2; thereby revealing a highly selective metabolic target in these cells. Cancer cells can not only exploit such genetic redundancies but also rely on redundancies built into their complex metabolic network to compensate for the loss of metabolic function. Importantly, there is an unexplored landscape of these genomic loss events beyond well-characterized TSGs. To address these challenges, we have developed a platform to identify patient-specific metabolic vulnerabilities emerging due to distinct patterns of genomic loss events across tumors. Our platform presents opportunities for precision-based therapeutic intervention by targeting metabolic vulnerabilities in cancer patients. It uses genomic and clinical data available in cancer patient databases to obtain candidate metabolic genes that are lost to genomic deletions in an unbiased manner. To delineate metabolic redundances and tackle the complexity of genome-scale metabolic models, we employ an innovative multi-objective metabolic flux analysis approach. The utility of this platform is demonstrated via the discovery of a novel metabolic target in a cohort of ovarian cancer patients. The predicted collateral lethal target is validated in vitro using RNA interference and small-molecule inhibitors. Furthermore, we verify the metabolic mechanism of vulnerability predicted by the algorithm using deuterium tracing experiments. The target is also validated in vivo with mice containing ovarian tumors derived from cancer cells with and without the genomic deletion. Surprisingly, the collateral lethal metabolic target was also found to exist in a subset of aggressive endometrial cancers. Finally, we developed a multi-layer machine learning model to predict occurrence of the particular genomic deletion in ovarian and endometrial cancer patients with minimal molecular information to remove the need for whole-genome sequencing data. The model was trained and tested using the publicly-available molecular data from TCGA and AACR GENIE datasets. Citation Format: Abhinav Achreja, Tao Yu, Anjali Mittal, Srinadh Choppara, Noah Meurs, Olamide Animasahun, Jin Heon Jeon, Aradhana Mohan, Anusha Jayaraman, Reva Kulkarni, Justin Reinhold, Michele Cusato, Analisa Difeo, Xiongbin Lu, Deepak Nagrath. Genomic loss in cancers enable discovery of metabolic targets for precision cancer therapy via multiobjective flux analysis and machine learning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 236.

Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with limited and, very often, ineffective medical and surgical therapeutic options. The treatment of patients with advanced unresectable PDAC is restricted to systemic chemotherapy, a therapeutic intervention to which most eventually develop resistance. Recently, nab-paclitaxel (n-PTX) has been added to the arsenal of first-line therapies, and the combination of gemcitabine and n-PTX has modestly prolonged median overall survival. However, patients almost invariably succumb to the disease, and little is known about the mechanisms underlying n-PTX resistance. Using the conditionally reprogrammed (CR) cell approach, we established and verified continuously growing cell cultures from treatment-naïve patients with PDAC. To study the mechanisms of primary drug resistance, nab-paclitaxel–resistant (n-PTX-R) cells were generated from primary cultures and drug resistance was verified in vivo, both in zebrafish and in athymic nude mouse xenograft models. Molecular analyses identified the sustained induction of c-MYC in the n-PTX-R cells. Depletion of c-MYC restored n-PTX sensitivity, as did treatment with either the MEK inhibitor, trametinib, or a small-molecule activator of protein phosphatase 2a. Implications: The strategies we have devised, including the patient-derived primary cells and the unique, drug-resistant isogenic cells, are rapid and easily applied in vitro and in vivo platforms to better understand the mechanisms of drug resistance and for defining effective therapeutic options on a patient by patient basis.

Objective To evaluate the utility of amiodarone and its derivative dronedarone as novel drug repositioning candidates in EOC and to determine the potential pathways targeted by these drugs. Methods Drug-predict bioinformatics platform was used to assess the utility of amiodarone as a novel drug-repurposing candidate in EOC. EOC cells were treated with amiodarone and dronedarone. Cell death was assessed by Annexin V staining. Cell viability and cell survival were assessed by MTT and clonogenics assays respectively. c-MYC and mTOR/Akt axis were evaluated as potential targets. Effect on autophagy was determined by autophagy flux flow cytometry. Results "DrugPredict" bioinformatics platform ranked Class III antiarrhythmic drug amiodarone within the top 3.9% of potential EOC drug repositioning candidates which was comparable to carboplatin ranking in the top 3.7%. Amiodarone and dronedarone were the only Class III antiarrhythmic drugs that decreased the cellular survival of both cisplatin-sensitive and cisplatin-resistant primary EOC cells. Interestingly, both drugs induced degradation of c-MYC protein and decreased the expression of known transcriptional targets of c-MYC. Furthermore, stable overexpression of non-degradable c-MYC partially rescued the effects of amiodarone and dronedarone induced cell death. Dronedarone induced higher autophagy flux in EOC cells as compared to amiodarone with decreased phospho-AKT and phospho-4EBP1 protein expression, suggesting autophagy induction due to inhibition of AKT/mTOR axis with these drugs. Lastly, both drugs also inhibited the survival of EOC tumor-initiating cells (TICs). Conclusions We provide the first evidence of class III antiarrhythmic agents as novel c-MYC targeting drugs and autophagy inducers in EOC. Since c-MYC is amplified in >40% ovarian tumors, our results provide the basis for repositioning amiodarone and dronedarone as novel c-MYC targeting drugs in EOC with potential extension to other cancers.

// Andrew Wiechert 1, 2, * , Caner Saygin 1, * , Praveena S. Thiagarajan 1 , Vinay S. Rao 1 , James S. Hale 1 , Nikhil Gupta 3 , Masahiro Hitomi 1, 3, 4 , Anil Belur Nagaraj 5 , Analisa DiFeo 5 , Justin D. Lathia 1, 3, 4, 5 , Ofer Reizes 1, 3, 4, 5 1 Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA 2 Division of Gynecological Oncology, Women’s Health Institute, Cleveland Clinic, Cleveland, OH 44195, USA 3 Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195 4 Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine at Case Western Reserve University, Cleveland, OH 44195, USA 5 Department of Case Comprehensive Cancer Center, Cleveland, OH 44106, USA * These authors contributed equally to this work Correspondence to: Ofer Reizes, e-mail: reizeso@ccf.org Justin D. Lathia, e-mail: lathiaj@ccf.org Keywords: cancer stem cell, ovarian cancer, cisplatin, NANOG Received: December 01, 2015 Accepted: March 31, 2016 Published: April 20, 2016 ABSTRACT The mainstay of treatment for ovarian cancer is platinum-based cytotoxic chemotherapy. However, therapeutic resistance and recurrence is a common eventuality for nearly all ovarian cancer patients, resulting in poor median survival. Recurrence is postulated to be driven by a population of self-renewing, therapeutically resistant cancer stem cells (CSCs). A current limitation in CSC studies is the inability to interrogate their dynamic changes in real time. Here we utilized a GFP reporter driven by the NANOG-promoter to enrich and track ovarian CSCs. Using this approach, we identified a population of cells with CSC properties including enhanced expression of stem cell transcription factors, self-renewal, and tumor initiation. We also observed elevations in CSC properties in cisplatin-resistant ovarian cancer cells as compared to cisplatin-naive ovarian cancer cells. CD49f, a marker for CSCs in other solid tumors, enriched CSCs in cisplatin-resistant and -naive cells. NANOG-GFP enriched CSCs (GFP+ cells) were more resistant to cisplatin as compared to GFP-negative cells. Moreover, upon cisplatin treatment, the GFP signal intensity and NANOG expression increased in GFP-negative cells, indicating that cisplatin was able to induce the CSC state. Taken together, we describe a reporter-based strategy that allows for determination of the CSC state in real time and can be used to detect the induction of the CSC state upon cisplatin treatment. As cisplatin may provide an inductive stress for the stem cell state, future efforts should focus on combining cytotoxic chemotherapy with a CSC targeted therapy for greater clinical utility.

Ovarian cancer (OvC) is the deadliest of all female gynecologic malignancies, ranking as the fifth cause of cancer-related deaths among women. High-grade serous epithelial ovarian cancer (HGSOC) accounts for almost 90% of all forms of OvC, remaining as the most lethal subtype of ovarian malignancies. HGSOC is usually diagnosed at a later stage at which the disease has already progressed into metastatic, having a 5-year survival rate of only 17%. Treatment of HGSOC relies on aggressive approaches including surgery and platinum-based chemotherapies, with the majority of cases requiring combination of both. The molecular mechanism driving ovarian tumorigenesis is not fully understood, and its investigation is essential to provide better and novel therapeutic options for the treatment and cure of this disease. Targeted therapies have been vastly explored in the cancer research field due to their promise in reducing side effects by specifically targeting oncogenic pathways predominantly deregulated in human malignancies. DNA damage and repair (DDR) pathway aberrations and genomic instability are two main hallmarks of OvC. Extensive efforts in targeting this pathway have been devoted to introduce new drugs in the clinic that can target the DDR and homologous recombination (HR) pathways as therapeutic means for treatment of HGSOC. The use of PARP inhibitors (PARPi) to achieve synthetic lethality in OvC has been shown to have great promise in efficiently reducing tumor burden by specifically targeting tumor cells for cell death. However, PARPi’s antitumoral efficacy alone is limited by the status of the BRCA1/2 genes, narrowing down the patient population that can benefit from such therapies. Eventually, patients treated with PARPi alone relapse and develop resistant OvC, for which treatment options available are extremely limited. Our preliminary data show that first-in-class small-molecule activators of PP2A (SMAPs) in combination with PARPi can effectively reduce tumor burden in PDX models, promoting a synergistic effect in platinum-sensitive and -resistant OvC tumors, despite the BRCA gene status. Protein Phosphatase 2A (PP2A) and Protein Phosphatase 1 (PP1) account for 90% of all serine/threonine phosphatase activity in the cell and act as two main tumor suppressors. These phosphatases tightly regulate signaling pathways involved with cell cycle, growth, migration, metabolism, survival and many oncogenic pathways by counteracting kinases. HGSOC tumors express inactivated PP2A due to PPP2RA1 heterozygous loss, although lacking mutations, making it an ideal target for reactivation in these cancers. Moreover, we have shown that reactivation of PP2A via SMAPs leads to potent inhibition of several key DDR proteins. We have found that SMAPs treatment in BRCA1/2 WT tumors confers a “BRCAness” phenotype, which sensitizes tumors to DNA repair inhibitors such as PARPi. In sum, SMAPs can allow us to expand the patient population that can benefit from PARPi therapies and possibly overcome drug resistance. Citation Format: Rita A. Avelar, Amy Armstrong, Goutham Narla, Analisa DiFeo. Targeting Protein Phosphatase 2A in Combination with PARP inhibitors for the treatment of high-grade serous epithelial ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A34.

Effective practice of precision medicine in oncology relies on identifying and targeting unique characteristics of individual cancers. Coupling functional analysis with genomic screening will further enhance the selection of efficacious cancer therapies. Cancer is a heterogeneous disease that is defined by distinct capabilities, one of which is the evasion of apoptosis, controlled by the antiapoptotic protein members, Bcl-2, Bcl-xL, Mcl-1, and Bfl-1. These antiapoptotic proteins have become validated therapeutic targets, as manifested by the FDA-approved venetoclax, a selective Bcl-2 inhibitor, and several other molecules in clinical trials that target Mcl-1 and Bcl-xL. One of the translational challenges lies in predicting functional antiapoptotic dependence in a patient’s cancer and discriminating responders from nonresponders to Bcl-2 family targeted therapy. We have successfully characterized an updated array of Bcl-2 family antagonists, consisting of peptides and selective small-molecule BH3-mimetics via in vitro binding assays and functional analysis using model cell lymphoma cell lines. With this selective set of chemical tools, we employed the BH3 profiling assay across a wide spectrum of hematologic and solid tumor cell lines in order to functionally dissect survival dependence. To validate the results of BH3 profiling, we performed treatments with BH3-mimetics and analyzed their ability to induce apoptosis, which further demonstrated how cancers can be differentiated and targeted based on antiapoptotic dependence. The obtained data suggest that hematologic cancer cell lines mainly rely on Mcl-1 and Bcl-2 for survival and commonly undergo apoptosis in response to single-agent inhibitors of these proteins. Solid tumor cell lines are more dependent upon Bcl-xL and Mcl-1 and certain cell lines display increased involvement of Bfl-1. These cell lines undergo apoptosis when exposed to a combination of Bcl-xL/Mcl-1 inhibitors. Single agent-responsive solid tumor cell lines are less common but can be identified by BH3 profiling. This approach was successfully applied to primary patient samples to validate clinical utility. Further applications of dynamic BH3 profiling were explored to demonstrate how various FDA-approved therapies can alter antiapoptotic dependencies and aid in the design of rational combination therapies with BH3-mimetics. This work lays a translational foundation in the field of precision medicine by incorporating Bcl-2 protein family targeting into potential personalized cancer treatments. Citation Format: Karson J. Kump, Matthew Lieberman, Rita A. Avelar, Charles Foucar, Malathi Kandarpa, Antonio Di Cristofano, Russell J. Ryan, Sami N. Malek, Ryan A. Wilcox, Dale L. Bixby, Tycel J. Phillips, Moshe Talpaz, Analisa DiFeo, Zaneta Nikolovska-Coleska. Precision medicine strategy to elucidate and target Bcl-2 prosurvival proteins in a wide spectrum of cancers [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 10.

Host and Tumor Factor XII Drive Ovarian Cancer Maintenance and Progression Introduction . Epithelial ovarian cancer (EOC) is the leading cause of cancer death in women.Effective strategies to treat this disease are lacking due to the complexity of pathways involved and the multitude of cells that contribute to EOC biology. To this end, coagulation factor XII (FXII) and its receptor urokinase plasminogen activator receptor (uPAR) represent very promising therapeutic targets because i) uPAR is overexpressed in more than 90% of ovarian cancer patients, ii) FXII has been shown to be upregulated in the peritoneum of EOC patients and promotes EOC dissemination and iii) FXII-uPAR signal and upregulate key neutrophil functions, recently linked to tumor growth and metastasis. Through analysis of the TCGA ovarian cancer patient cohort, we have found that FXII and uPAR are co-expressed in ovarian tumors and their overexpression is associated with decreased overall survival (Fig 1A-B). Given that FXII and uPAR can be produced by both the EOC tumor and the host, the contribution of each of these compartments which has not been examined to date needs to be explored. Thus, we asked if the FXII-uPAR axis in neutrophils and EOC cells synergistically influences tumor biology. Methods - Results. We utilized a tissue microarray to determine FXII and uPAR expression. Both FXII and uPAR were co-expressed in all major histologic subtypes of EOC tumors but not in normal ovarian epithelium (Fig C-D). We adopted a composite expression score system and found significantly increased expression of FXII and uPAR in high grade tumors compared to low grade (grade 1-2) tumors, independently of tumor subtype and stage (Fig 1C-D). Given prior reports that neutrophils are enriched in the ovarian tumor microenvironment (TME) and our findings that FXII-uPAR are integral to neutrophil activation, tumors were also examined for their neutrophil content. Invariably, as tumor grade advanced, Neutrophil Elastase expression significantly increased (data not shown). We next asked if neutrophils contribute to EOC tumor progression or their recruitment merely correlates with advanced disease. We found that in the presence of neutrophils, EOC cells migrate significantly faster (p Next, in order to assess whether host FXII could contribute to EOC dissemination and progression we utilized WT, F12-/-, and uPAR deficient (Plaur-/-) mice for in vivo EOC tumor model. After orthotopic injection of ID8 EOC cells, WT mice exhibited faster rates of tumor development and ascitic fluid accumulation (13.4 ± 0.92 ml), relative to F12-/- (0.37 ± 0.26 ml) and Plaur-/- (0.5 ± 0.5 ml) mice (Fig 1G). Blinded examination of tumors showed significantly higher tumor burden in WT mice compared to F12-/-and Plaur-/- mice, with F12-/-mice exhibiting the highest degree of protection (Fig 1H). Conclusions . Our studies indicate three novel aspects: i) a direct crosstalk between ovarian cancer cells and neutrophils, that enhances tumor cell migration through FXII-uPAR signaling; ii) the presence of neutrophils in the TME induces EMT of cancer cells which is abolished when the FXII-uPAR interaction is inhibited and; iii) abrogation of the FXII-uPAR axis in EOC tumor cells and neutrophils, synergistically restricts the pro-invasive phenotype of ovarian tumor cells. Disclosures No relevant conflicts of interest to declare.

// Anil Belur Nagaraj 1 , Peronne Joseph 1 , Olga Kovalenko 1 , Sareena Singh 2 , Amy Armstrong 2 , Raymond Redline 3 , Kimberly Resnick 2 , Kristine Zanotti 2 , Steven Waggoner 2 and Analisa DiFeo 1 1 Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA 2 Department of Gynecology, Division of Gynecological Oncology, University Hospital Case Medical Center, Cleveland, OH, USA 3 Department of Pathology, University Hospital Case Medical Center, Cleveland, OH, USA Correspondence to: Analisa DiFeo, email: // Keywords : patient derived xenograft, platinum resistance, tumor spheres, cancer initiating cells, β-catenin Received : April 23, 2015 Accepted : June 01, 2015 Published : June 29, 2015 Abstract Resistance to platinum-based chemotherapy is the major barrier to treating epithelial ovarian cancer. To improve patient outcomes, it is critical to identify the underlying mechanisms that promote platinum resistance. Emerging evidence supports the concept that platinum-based therapies are able to eliminate the bulk of differentiated cancer cells, but are unable to eliminate cancer initiating cells (CIC). To date, the relevant pathways that regulate ovarian CICs remain elusive. Several correlative studies have shown that Wnt/β-catenin pathway activation is associated with poor outcomes in patients with high-grade serous ovarian cancer (HGSOC). However, the functional relevance of these findings remain to be delineated. We have uncovered that Wnt/β-catenin pathway activation is a critical driver of HGSOC chemotherapy resistance, and targeted inhibition of this pathway, which eliminates CICs, represents a novel and effective treatment for chemoresistant HGSOC. Here we show that Wnt/β-catenin signaling is activated in ovarian CICs, and targeted inhibition of β-catenin potently sensitized cells to cisplatin and decreased CIC tumor sphere formation. Furthermore, the Wnt/β-catenin specific inhibitor iCG-001 potently sensitized cells to cisplatin and decreased stem-cell frequency in platinum resistant cells. Taken together, our data is the first report providing evidence that the Wnt/β-catenin signaling pathway maintains stem-like properties and drug resistance of primary HGSOC PDX derived platinum resistant models, and therapeutic targeting of this pathway with iCG-001/PRI-724, which has been shown to be well tolerated in Phase I trials, may be an effective treatment option.

Neoadjuvant chemotherapy (NACT) has been recognized as a reliable therapeutic strategy in patients with unresectable advanced epithelial ovarian cancer (EOC). The molecular events leading to platinum (Pt) response in NACT settings have hitherto not been explored. In the present work, longitudinal changes of miRNA expression profile were investigated to identify miRNA families with prognostic role in high-grade serous EOC patients who received the NACT regimen.One hundred sixty-four matched tumor biopsies taken at initial laparoscopic evaluation and at interval-debulking surgery (IDS) after four courses of Pt-based therapy were selected from 82 stage IIIC-IV high-grade serous-EOC patients that were judged unsuitable for complete primary debulking and subjected the NACT protocol. miRNA profiling by microarray, real-time PCR and immuno-histochemical staining for Smad2 phosphorylation (P-Smad2) were used for data analysis.Analysis revealed that 369 miRNAs were differentially expressed in matched biopsies (referred to as DEMs). DEMs were not scattered across the genome, but clustered into families: miR-199, let-7, miR-30, miR-181 and miR-29. Multivariate analysis showed that miR-199a-3p, miR-199a-5p, miR-181a-5p and let-7g-5p associated with overall and progression-free survival (P0.05); miR-199a-3p, miR-199a-5p and miR-181a-5p associated with residual tumor volume and Pt-free interval (P0.05). Immuno-histochemical staining confirmed an enrichment of P-Smad2, a marker of transforming growth factor-β activation, in tumors from patients with shorter PFS and OS, and with high levels of expression of miR-181a-5p (P0.05). Kaplan-Meier curves plotting concomitant expression of P-Smad2 and miR-181a-5p show significant differences in PFS and OS compared with those depicting the expression of each biomarker alone (P0.001).This study describes several miRNA families with a prognostic role in the NACT setting. It also confirms that concomitant analysis of P-Smad2 and miR-181a-5p in surgical samples may be capable of identifying those ovarian cancer patients with poor outcome and little chance of response to Pt-based NACT.

Background Clinical and pathological parameters of patients with epithelial ovarian cancer (EOC) do not thoroughly predict patients' outcome. Despite the good outcome of stage I EOC compared with that of stages III and IV, the risk assessment and treatments are almost the same. However, only 20% of stage I EOC cases relapse and die, meaning that only a proportion of patients need intensive treatment and closer follow-up. Thus, the identification of cell mechanisms that could improve outcome prediction and rationalize therapeutic options is an urgent need in the clinical practice. Patients and methods We have gathered together 203 patients with stage I EOC diagnosis, from whom snap-frozen tumor biopsies were available at the time of primary surgery before any treatment. Patients, with a median follow-up of 7 years, were stratified into a training set and a validation set. Results and conclusions Integrated analysis of miRNA and gene expression profiles allowed to identify a prognostic cell pathway, composed of 16 miRNAs and 10 genes, wiring the cell cycle, ‘Activins/Inhibins’ and ‘Hedgehog’ signaling pathways. Once validated by an independent technique, all the elements of the circuit resulted associated with overall survival (OS) and progression-free survival (PFS), in both univariate and multivariate models. For each patient, the circuit expressions have been translated into an activation state index (integrated signature classifier, ISC), used to stratify patients into classes of risk. This prediction reaches the 89.7% of sensitivity and 96.6% of specificity for the detection of PFS events. The prognostic value was then confirmed in the external independent validation set in which the PFS events are predicted with 75% sensitivity and 94.7% specificity. Moreover, the ISC shows higher classification performance than conventional clinical classifiers. Thus, the identified circuit enhances the understanding of the molecular mechanisms lagging behind stage I EOC and the ISC improves our capabilities to assess, at the time of diagnosis, the patient risk of relapse.

Kruppel-like factor 6 splice variant 1 (KLF6-SV1) is an oncogenic splice variant of the KLF6 tumor suppressor gene that is specifically overexpressed in a number of human cancers. Previously, we have demonstrated that increased expression of KLF6-SV1 is associated with decreased survival in lung adenocarcinoma patient samples and that targeted reduction of KLF6-SV1 using siRNA induced apoptosis both alone and in combination with the chemotherapeutic drug cisplatin. Here, we demonstrate that chemoresistant lung cancer cells express increased levels of KLF6-SV1. Furthermore, targeted reduction of KLF6-SV1 using RNA interference restores chemotherapy sensitivity to lung cancer cells both in culture and in vivo through induction of apoptosis. Conversely, overexpression of KLF6-SV1 resulted in a marked reduction in chemotherapy sensitivity in a tumor xenograft model. Combined, these findings highlight a functional role for the KLF6-SV1 splice variant in the regulation of chemotherapy response in lung cancer and could provide novel insight into lung cancer therapy.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and is the fifth leading cause of cancer deaths in women. Developing adjuvant therapy to circumvent drug resistance represents an important aspect of current initiatives to improve survival in women with advanced EOC. A regulatory molecule that can act on multiple genes associated with a chemoresistant phenotype will be the ideal target for the development of therapeutics to overcome resistance and miRNAs constitute promising tools in this regard. In this review, we discuss the emerging role of miRNAs in regulating EOC phenotype with a focus on prognostic and therapeutic importance of miRNAs and the possibility of miRNA modulation as a tool to improve efficacy of chemotherapy in EOC.

Computation-based drug repositioning approaches that automatically search vast amounts of genomic-chemical-phenotypic data for tens of thousands of drugs and diseases can greatly speed up the traditional drug-discovery process. To date, systematic and comprehensive computation-based approaches to identify and validate drug repositioning candidates for High-grade serous ovarian cancer (HGSOC) have not been undertaken. Using a novel computational drug-repositioning platform (Drug-Predict) with HGSOC gene expression dataset from The Cancer Genome Atlas (TCGA) as the input, we have uncovered that Non-steroidal anti-inflammatory drugs (NSAIDs) could be potential candidates for Drug repositioning in HGSOC. Given that numerous epidemiological studies have shown that regular intake of NSAIDs in women is associated with decreased incidence of ovarian cancer, we assessed whether NSAIDs could have chemo-adjuvant applications in HGSOC and have identified Indomethacin as a novel chemo-adjuvant in HGSOC. Indomethacin decreased survival of primary HGSOC tumor cells and interestingly, cisplatin resistant ovarian tumor cells (derived from primary HGSOC PDX models) exhibited significantly higher cell death upon Indomethacin treatment suggesting that Indomethacin could exert chemo-adjuvant effects in HGSOC. Accordingly, Indomethacin treatment induced chemo-sensitivity in cisplatin resistant HGSOC tumor cells and combo treatment with Indomethacin and cisplatin exerted synergistic cell death as compared to individual drugs alone. Furthermore, Indomethacin decreased stem-like properties and induced chemo-sensitivity in ALDHhigh cisplatin-resistant tumor-initiating cells (TICs) thus suggesting that the chemo-adjuvant effect of the drug is mediated by decrease in TIC properties in HGSOC cells. Mechanistically, we found that Indomethacin inhibits Wnt/β-catenin signaling by degrading β-catenin and β-catenin modulation inversely affected Indomethacin functioning in HGSOC. Our study is the first report describing functional effects of an NSAID in patient-derived HGSOC models and demonstrates that combining novel computational predictions with experimental validation has potential in identifying viable drug candidates and moving them into patient trials efficiently and cost-effectively. Since NSAIDs are in routine clinical use in gynecological settings and have acceptable safety profile, this discovery provides with a potentially rapid and cost-effective translational opportunity for testing NSAIDs as chemo-adjuvants in patient trials in HGSOC. Citation Format: Anil Belur Nagaraj, QuanQiu Wang, Olga Kovalenko, Peronne Joseph, Yang Chen, Rong Xu and Analisa DiFeo. PHARMACOLOGIC ANALYSIS OF HIGH–GRADE SEROUS OVARIAN CANCER TCGA DATASET IDENTIFIES A NOVEL CHEMO–ADJUVANT ROLE FOR NON–STEROIDAL ANTI–INFLAMMATORY DRUGS (NSAIDS) [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-081.

e17054 Background: Racial disparities in outcomes exist between African-American (AA) and Caucasian (C) patients (pts) with high grade epithelial ovarian cancer (EOC). Not all disparity is explained by socio-economic and access related issues. Many argue that differences in tumor biology also play a role. Germline BRCA mutations confer a protective effect in these pts. Immunohistochemistry (IHC) for BRCA 1 is being developed to detect somatic BRCA1 mutations. Parp-inhibitors have been approved for use in pts with germline and/or somatic BRCA mutations. We describe the first comparison of the prognostic implications of BRCA1 somatic mutation status in tumor samples taken from AA and C EOC pts who underwent primary surgery followed by systemic chemotherapy. Methods: IHC staining of whole tumor sections from AA and C pts was performed using a commercially available monoclonal antibody against BRCA1 (Calbiochem). Staining was scored as: loss, retention, or equivocal in the presence of a positive control. Retention was described as wild-type and loss or equivocal as mutation. Standard statistical analyses were used to correlate race, BRCA1 status, and survival. Results: Tumor from 33 pts (16AA and 17C) was stained and scored. Most pts had Stage III (66.7%) disease and high grade serous subtype (82%). Stage, histology, and debulking status did not differ between groups. Overall, loss of BRCA 1 occurred 16/33 (48.5%) tumors. BRCA1 was mutated more frequently in tumors from AA pts (11/16) compared to C pts (5/17) (p=.024). BRCA 1 mutation conferred a protective effect, with a trend towards improved progression free survival: 9.5 vs 15.8 mos in AA (p=0.44) and 16.5 vs. 21 mos in C pts (p=0.97). No statistically significant difference in OS was seen. Conclusions: We demonstrate that IHC for BRCA 1 mutation is feasible in a standard hospital pathology laboratory. Race based differences in somatic BRCA 1 mutation exist; mutational differences are associated with a trend in improved PFS. A larger cohort is needed to elucidate the true effect of this status on outcome. Conceivably, knowledge of BRCA 1 tumor status could be used to improve outcomes in AA pts who may be able to achieve survival numbers comparable to C pts.

Endometrial cancer is the most commonly diagnosed malignancy of the female genital tract in the United States. The role of adjuvant radiation for the treatment of patients with early stage disease is still heavily debated, as no overall survival benefit has been demonstrated in large prospective trials. There is, however, evidence to suggest a decrease in recurrence risk with administration of adjuvant radiotherapy, especially in patients with high-risk features. In this review, we describe the relevant literature to date, define how we determine which patients are considered high or intermediate risk, and describe the findings related to the addition of chemotherapy to adjuvant brachytherapy.

Background Ovarian cancer is the most lethal gynecologic disease in the United States, with more women dying from this cancer than all gynecological cancers combined. Ovarian cancer has been termed the “silent killer” because some patients do not show clear symptoms at an early stage. Currently, there is a lack of approved and effective early diagnostic tools for ovarian cancer. There is also an apparent severe knowledge gap of ovarian cancer in general and of its indicative symptoms among both public and many health professionals. These factors have significantly contributed to the late stage diagnosis of most ovarian cancer patients (63% are diagnosed at Stage III or above), where the 5-year survival rate is less than 30%. The paucity of knowledge concerning ovarian cancer in the United States is unknown. Methods The present investigation examined current public awareness and knowledge about ovarian cancer. The study implemented design strategies to develop an unbiased survey with quality control measures, including the modern application of multiple statistical analyses. The survey assessed a reasonable proxy of the US population by crowdsourcing participants through the online task marketplace Amazon Mechanical Turk, at a highly condensed rate of cost and time compared to traditional recruitment methods. Conclusion Knowledge of ovarian cancer was compared to that of breast cancer using repeated measures, bias control and other quality control measures in the survey design. Analyses included multinomial logistic regression and categorical data analysis procedures such as correspondence analysis, among other statistics. We confirmed the relatively poor public knowledge of ovarian cancer among the US population. The simple, yet novel design should set an example for designing surveys to obtain quality data via Amazon Mechanical Turk with the associated analyses.

PP2A is a phosphatase that is functionally dysregulated and inactivated in over 50% of all breast cancers independent of histological type. While much attention has focused on oncogenic kinases as targets for cancer treatment, therapeutic targeting of phosphatases, the key negative regulators of these same signaling pathways, has remained largely unexplored. Starting with the observation that tricyclic neuroleptic drugs exert anticancer effects, our laboratory reverse engineered these drugs to generate a novel series of compounds that retain the anti-proliferative effects and favorable pharmacokinetic properties but are devoid of the undesirable central nervous system pharmacology of the parent drugs. It was subsequently demonstrated that these first generation novel derivatives exert anticancer effects in cell culture and xenograft models of cancer (without overt toxicity) by modulation of critical oncogenic signaling pathways. Recently, it was determined that the molecular basis of the actions of these novel derivatives is the activation of PP2A. Comprehensive profiling for activity in a panel of 240 cancer cell lines has shown that these first-in-class small molecule activators of PP2A (SMAPs) have significant anti-proliferative activity in breast cancer cell lines, and studies have been extended to disease relevant in vivo models (patient derived xenograft, GEMM, and traditional xenograft models). PP2A's ability to negatively regulate a diverse set of oncogenic drivers in breast cancer means SMAPs are able to exert their effects through multiple biological mechanisms, swiftly disrupting cellular energetics and metabolism, inducing apoptosis and inhibiting proliferation. This will be exhibited through a variety of biological assays evaluating cell growth, apoptosis and changes in cellular metabolism. Target engagement assessed via western blotting demonstrates inhibition of PP2A targets critical for mediating SMAP activity, notably c-MYC and AKT. As a result, this research demonstrates SMAPs have potent activity in a wide variety of breast cancer contexts where response to current therapies is modest and limited by the development of treatment resistance. Thus, SMAPs may be a viable treatment option for high-risk populations of patients who do not respond to the current standard of care. This research strives to demonstrate that therapeutic reactivation of PP2A may represent a novel approach for breast cancer treatment and that these molecules may favorably impact the lives of women suffering from breast cancer. Citation Format: Caroline C. Farrington, Xiaoyan Wang, Mahnaz Janghorban, Juan Liang, Analisa Difeo, Rosalie Sears, Goutham Narla. Pharmacological reactivation of the tumor suppressor protein phosphatase 2A as a novel approach for the treatment of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3857.

e17019Background: CIP2A is a newly elucidated oncogene that demonstrates high expression in many human cancers. CIP2A may not only serve as a drug target, but may play a role as a biomarker, predic...

e17098Background: Ovarian cancer (OC) is the most lethal gynecological cancer. Despite high rates of response to initial cytoreductive surgery and taxane-platinum combination therapy, vast majority...

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and is the fifth leading cause of cancer deaths in women. The most common and lethal form of EOC which makes up greater than 75% of cases is high-grade serous cancer (HGSOC). Platinum-based drugs are used as first-line chemotherapeutics for all women diagnosed with HGSOC. Unfortunately, ~20% do not respond to this treatment and up to 80% of those patients which do respond recur within 5 years at which point the cancer is particularly difficult to eradicate. Emerging evidence supports the concept that platinum based therapies are able to eliminate the bulk of differentiated cancer cells but are unable to eliminate tumor-initiating cells (TICs) which are rare populations of tumor cells characterized by their ability to self-renew indefinitely and thus drive the expansion of drug-resistant tumors. Hence, there is immediate need of novel TIC targeted therapeutic approaches that can overcome chemo-resistance in EOC. Thus, identifying critical drivers of the stem-like phenotype that can be pharmacologically modulated in ovarian TICs is required. To date, no TIC targeting drugs have successfully completed clinical trials in EOC. Using primary HGSOC cells derived from HGSOC patient derived xenograft (PDX) models, we have recently identified β-catenin as a critical driver of HGSOC TIC phenotype in-vitro. β-catenin knockdown decreased stem-cell frequency and induced chemo-sensitivity in-vitro in platinum resistant HGSOC PDX derived primary cells. ALDH positive Ovarian TIC subpopulation with high Wnt reporter activity was platinum resistant and exhibited increased expression of β-catenin regulated stem-cell markers clearly identifying β-catenin regulated stem-cell transcriptional axis as a driver of chemo-resistance in Ovarian TICs. Accordingly, the β-catenin stem-cell transcriptional axis specific inhibitor iCG-001 (PRI-724) decreased stem-cell frequency and induced chemo-sensitivity in platinum resistant ovarian TICs, suggesting that pharmacologic inhibition of β-catenin could be a potential novel approach aimed at eradicating TICs in EOC. Furthermore, in-vivo tumor-initiation assays showed a robust decrease in tumor-initiation capacity upon β-catenin knockdown in platinum resistant HGSOC cells, thus establishing β-catenin as a driver of TIC phenotype in HGSOC. In sum, we have identified that pharmacologic inhibition of β-catenin can be a novel TIC targeted therapy approach to overcome chemo-resistance and improve patient outcome in in EOC. Our results suggest that the β-catenin specific inhibitor PRI-724, that has shown promising results in phase I clinical trials in solid tumors, can be a potential TIC targeting drug in EOC. These results will form a solid foundation for clinical trials with PRI-724 in patients with chemo-recurrent EOC. Citation Format: Anil Belur Nagaraj, Peronne Joseph, Olga Kovalenko, Sareena Singh, Amy Armstrong, Analisa DiFeo. β-catenin as a TIC therapeutic target in epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A64.

Beyond their initial identification in prostate cancer, the tumor suppressor KLF6 and its alternatively spliced oncogenic isoform KLF6-SV1 have now been associated with a number of human cancers. Expression patterns of each have been linked to tumor stage, disease recurrence, chemotherapy response and overall survival. Most recently, inhibition of KLF6-SV1 has been shown to increase survival in a pre-clinical model of ovarian cancer. This chapter reviews the basic biology of KLF6 and KLF6-SV1 as it relates to cancer, summarizes the published studies to date, and highlights the scientific rationale for therapeutically targeting KLF6 and KLF6-SV1 as a novel treatment paradigm.

Kruppel-like factor 6 (KLF6) is a tumor suppressor gene that is functionally inactivated in human cancer by loss of heterozygosity, somatic mutation, decreased expression, and increased alternative splicing into an oncogenic splice variant, KLF6-SV1. Here we show that increased expression of KLF6-SV1 is associated with decreased survival in patients with lung adenocarcinoma. In addition, KLF6-SV1 is a novel antiapoptotic protein in lung cancer cell lines, and targeted reduction of KLF6-SV1 using siRNA induces apoptosis both alone and in combination with the chemotherapeutic drug cisplatin. Together, these findings highlight a critical role for KLF6-SV1 in lung cancer, and show a potential novel therapeutic strategy for the treatment of lung cancer. [Cancer Res 2008;68(4):965–70]

Torg-Winchester syndrome, which includes nodular arthropathy with osteolysis (OMIM 605156), is a condition associated with generalized osteoporosis. On the basis of usefulness of pamidronate in conditions with osteoporosis, we hypothesized that the drug will improve osteolysis and/or osteoporosis in this condition. After obtaining informed consent from the parents, two siblings affected with Torg-Winchester syndrome were administered intravenous pamidronate over a period of 3 years. The clinical status was monitored along with the bone mineral density (using radiographs and X-ray densitometry) to assess the effect of the drug. Clinically there was no improvement. Although the bone mineral density improved in axial skeleton, osteoporosis and osteolysis continued to worsen in the appendicular skeleton. We conclude that pamidronate does not improve peripheral osteolysis in multicentric osteolysis and nodular arthropathy caused by mutation in matrix metalloproteinase 2 gene.

e16594 Background: PP2A is a widely conserved phosphatase that functions as a major tumor suppressor. Inhibition of PP2A is a necessary prerequisite for the transformation of cancer cells. Dephosph...

cell lines, using the most potent and clinically relevant synergistic drug combinations identified by the screen. Results: Drug library screening revealed synergistic activity between B and specific classes of drugs, including taxanes, histone deacetylase (HDAC) inhibitors, and polo-like kinase (PLK) inhibitors. We proceeded to confirm results with docetaxel, panobinostat, volasertib, and olaparib in vitro. Favorable activity was observed using B with docetaxel, panobinostat, and volasertib but not with olaparib in eight ovarian cancer cell lines. Conclusions: An unbiased matrix screening platform enabled rapid identification of rational therapeutic combinations for the treatment of ovarian cancer. We identified three possible combinations to increase the anticancer activity of B. Future studies will evaluate the preclinical and clinical impact of these combinations.

Ovarian cancer is the most lethal gynecological cancer primarily due to advanced stage of disease at diagnosis. Effective therapeutic targets and prognostic biomarkers are still lacking due to insufficient knowledge of the pathways that regulate ovarian cancer progression. Here, we identify miR-181a as a mediator of disease dissemination through the induction of EMT and direct activation of the TGF-β signaling pathway via repression of its functional target, Smad7. High expression of miR-181a and phospho-Smad2 were associated with poor patient outcome and were enriched in recurrent compared to matched-primary tumors. Ectopic expression of miR-181a resulted in increased cellular survival, migration, drug resistance, and in vivo tumor burden, and dissemination. Conversely, targeting this miRNA using a decoy vector resulted in significant decreases in cell survival, migration, and MET in ovarian cancer cell lines. Combined, our findings identify miR-181a as a novel modulator of ovarian cancer dissemination through the induction of EMT and highlight its role as a potential biomarker and therapeutic target for aggressive late-stage ovarian cancer. Citation Format: Aditya Parikh, Christine Lee, Peronne Joseph, Sergio Marchini, Alessia Baccarini, Valentin Kolev, Chiara Romualdi, Robert Fruscio, Hardik Shah, Wang Feng, Gavriel Mullokandov, David Fishman, Maurizio D'Incalci, Jamal Rahaman, Tamara Kalir, Raymond Redline, Brian D. Brown, Goutham Narla, Analisa DiFeo. microRNA-181a plays a critical role in ovarian cancer progression through the regulation of epithelial-mesenchymal transition. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B47.

3014 Background: Improved biomarkers are needed to define recurrence risk in patients with completely resected skin melanomas. Standard prognostic indicators used in staging, including depth, ulceration, and mitotic rate, while useful, often fail to accurately predict recurrence for individual patients. The immune system may prevent recurrence in this population, but no evidence-based immune biomarkers are in clinical use. Biomarker development has been hindered by clinical standards necessitating that the entire specimen be formalin fixed and paraffin embedded (FFPE) for morphology evaluation, a process damaging to RNA. Methods: To define a biomarker for melanoma recurrence, mRNA copy number of immune-related genes from FFPE melanoma was measured using NanoString, a hybridization assay suited for analysis of partially degraded RNA. Genes predictive of non-recurrence were defined using receiver operating characteristic (ROC) curves in a training cohort and then validated in an independent patient cohort. Results: A panel of 21 genes predictive of non-recurrence were defined using ROC curves in a training cohort (N=44). This result was validated in an independent patient cohort (N=37, AUC=0.794). Protein levels of the most differentially expressed gene, CD2, also associated with non-recurrence (p

Breast cancer is a leading causes of cancer death in women worldwide. It is a heterogeneous and genetically complex disease has left a significant proportion of patients with inadequate treatment options. Since metastasis and drug resistance pose significant challengea in breast cancer treatment, elucidating the mechanisms of these processes is critical for understanding the key drivers of disease progression and for the development of targeted therapies. Accumulating evidence from our laboratory and other groups suggest that the tumor suppressor gene, KLF6, and its oncogenic splice variant, KLF6-SV1, play a role in breast cancer progression, dissemination, and chemoresistance. Here we demonstrate that in multiple independent clinical cohorts of over 1200 breast cancer patients with defined clinical outcome, high KLF6-SV1 mRNA levels in the primary tumor associated with poor survival and disease recurrence. Specifically, upregulated KLF6-SV1 expression in the primary tumors correlated with poor survival independent of disease stage and grade. Thus, we hypothesized that KLF6-SV1 is an early driver/molecular determinant of invasive breast cancer. In order to investigate the functional/biological relevance of KLF6-SV1 in breast cancer development and progression, we performed a series of experiments using retroviral-based overexpression in multiple non-tumorigenic, tumorigenic, and metastatic breast cancer cell lines. We demonstrated that overexpression of KLF6-SV1 increased mesenchymal marker gene expression, cellular survival, invasion, as well as the migratory potential of KLF6-SV1 transduced cells. Interestingly, KLF6-SV1 did not increase growth rate of these mammary epithelial cell lines, similar to the clinical data which showed an absence of a correlation between KLF6-SV1 expression and primary tumor size. In a 3D model KLF6-SV1 upregulation disrupted mammary acinar morphogenesis promoting complex multiacinar structures. To assay whether the KLF6-SV1-induced EMT phenotype conferred increased metastatic potential in vivo, we injected tumorigenic cells expressing high levels of KLF6-SV1 subcutaneously into immunodeficient mice. Strikingly, KLF6-SV1 overexpression alone drove the entire metastatic cascade resulting in dissemination to many organs including the liver, kidney, heart, lung, and spleen. Furthermore, KLF6-SV1 overexpression in a metastatic breast cancer cell line increased metastasis to the lungs and liver in an orthotopic model of the disease. This is consistent with our clinical data in which high KLF6-SV1 expression was correlated with decreased overall survival and metastasis free survival. Together these findings suggest a role for the KLF6-SV1 splice variant as a driver of breast cancer metastasis and validate its potential utility as a novel biomarker and therapeutic target for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5314. doi:1538-7445.AM2012-5314

Prostate cancer (PCa) is the second leading cause of cancer-related death among males in the United States. Many prostate tumors remain indolent and localized, while others result in metastatic and castrate-resistant disease. In order to uncover key molecular drivers that regulate the progression from indolent localized prostate cancer to lethal invasive and metastatic disease, the development of genetically relevant target driven mouse models is critical. Highly penetrant mouse models of this transition in which human disease relevant genes are the molecular drivers of disease progression are lacking. This transition is considered to be highly clinically relevant as our ability to distinguish between indolent disease and lethal metastatic forms of prostate cancer and has lead to either undertreatment of potentially poor behaving tumors or overtreatment of otherwise indolent disease. In addition, the paucity of biologically relevant models of advanced prostate cancer has hampered our ability to accurately and faithfully test novel therapeutic agents in relevant models of disease. To address this problem, we have developed a new murine model of PCa that combines transgenic overexpression of two genes known to be overexpressed in human prostate cancer (KLF6-SV1 and MYC). Overexpression of the c-Myc protein in the prostate of transgenic mice results in the development of both high-grade PIN and moderately differentiated prostate cancer. Interestingly, SV1 overexpression occurs late in disease progression with the highest protein expression occurring in locally invasive and metastatic human prostate cancers. In addition, SV1 overexpression in prostate cancer cell lines results in increased metastasis in vivo with no effect on local tumor growth. Combined, this data provides a strong experimental rationale for the characterization of the interactive and synergistic roles of these two disease relevant genes in the progression of moderately differentiated prostate cancer to poorly differentiated prostate cancer. Here we demonstrate for the first time that transgenic overexpression of MYC and SV1 results in invasive, metastatic prostate cancer with high penetrance in vivo. Pathological and molecular analyses identified a phenotype consistent with epithelial to mesenchymal transition (EMT), invasion and metastasis in this transgenic mouse model. Distant metastasis to several organs were observed and all double transgenic MYC and SV1 mice demonstrated evidence of capsular and stromal invasion by 36 weeks of age (n=14) as compared to none of the SV1 or MYC mice alone. To further understand the mechanism by which KLF6-SV1 and c-MYC regulate EMT, invasion, and metastasis, RWPE-1 cells, a non-tumorigenic prostate cell line that express low levels of KLF6-SV1 and c-MYC, were used to over-express c-MYC, KLF6-SV1 alone and in combination. Over-expression of these genes in RWPE-1, recapitulate our findings in vivo as assessed by a reduction in epithelial markers, such as E-cadherin and an increase in mesenchymal makers such as vimentin. These studies suggest that increased MYC and SV1 expression may drive a gene signature that is a prognostic indicator of aggressive and potentially metastatic human prostate cancer. In conclusion, this mouse model provides a novel tool for the functional evaluation of disease relevant genetic drivers of prostate cancer progression and should provide a roadmap for testing novel targeted molecular therapeutics to treat advanced prostate cancer. Citation Format: Sudeh Izadmehr, Christine H. Lee, Heriberto Fernandez, Raheleh Hatami, Alexander Kirschenbaum, Alice C. Levine, AnaLisa Difeo, Goutham Narl. Functional synergy between the KLF6-SV1 and c-MYC oncogenes in driving prostate cancer metastasis in a novel mouse model of the disease [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A57.