Monjri M. Shah

Smoothened Antagonists Reverse Taxane Resistance in Ovarian Cancer

The hedgehog pathway has been implicated in the formation and maintenance of a variety of malignancies, including ovarian cancer; however, it is unknown whether hedgehog signaling is involved in ovarian cancer chemoresistance. The goal of this study was to determine the effects of antagonizing the hedgehog receptor, Smoothened (Smo), on chemotherapy response in ovarian cancer. Expression of hedgehog pathway members was assessed in three pairs of parental and chemotherapy-resistant ovarian cancer cell lines (A2780ip2/A2780cp20, SKOV3ip1/SKOV3TRip2, HeyA8/HeyA8MDR) using quantitative PCR and Western blot analysis. Cell lines were exposed to increasing concentrations of two different Smo antagonists (cyclopamine, LDE225) alone and in combination with carboplatin or paclitaxel. Selective knockdown of Smo, Gli1, or Gli2 was achieved using siRNA constructs. Cell viability was assessed by MTT assay. A2780cp20 and SKOV3TRip2 orthotopic xenografts were treated with vehicle, LDE225, paclitaxel, or combination therapy. Chemoresistant cell lines showed higher expression (>2-fold, P < 0.05) of hedgehog signaling components compared with their respective parental lines. Smo antagonists sensitized chemotherapy-resistant cell lines to paclitaxel, but not to carboplatin. LDE225 treatment also increased sensitivity of ALDH-positive cells to paclitaxel. A2780cp20 and SKOV3TRip2 xenografts treated with combined LDE225 and paclitaxel had significantly less tumor burden than those treated with vehicle or either agent alone. Increased taxane sensitivity seems to be mediated by a decrease in P-glycoprotein (MDR1) expression. Selective knockdown of Smo, Gli1, or Gli2 all increased taxane sensitivity. Smo antagonists reverse taxane resistance in chemoresistant ovarian cancer models, suggesting combined anti-hedgehog and chemotherapies could provide a useful therapeutic strategy for ovarian cancer. Mol Cancer Ther; 11(7); 1587–97. ©2012 AACR.

Ovarian cancer stem cells: Are they real and why are they important?

The cancer stem cell hypothesis has been put forward as a paradigm to describe varying levels of aggressiveness in heterogeneous tumors. Specifically, many subpopulations have been clearly demonstrated to possess increased tumorigenicity in mice, broad differentiating capacity, and resistance to therapy. However, the extent to which these experimental findings are potentially clinically significant is still not clear. This review will describe the principles of this emerging hypothesis, ways in which it may be appropriate in ovarian cancer based on the clinical course of the disease, and how we might exploit it to improve outcomes in ovarian cancer patients.

Endoglin (CD105) Contributes to Platinum Resistance and Is A Target for Tumor-Specific Therapy in Epithelial Ovarian Cancer

Purpose: Endoglin (CD105) is a membranous protein overexpressed in tumor-associated endothelial cells, chemoresistant populations of ovarian cancer cells, and potentially stem cells. Our objective was to evaluate the effects and mechanisms of targeting endoglin in ovarian cancer. Experimental Design: Global and membranous endoglin expression was evaluated in multiple ovarian cancer lines. In vitro, the effects of siRNA-mediated endoglin knockdown with and without chemotherapy were evaluated by MTT assay, cell-cycle analysis, alkaline comet assay, γ-H2AX foci formation, and quantitative PCR. In an orthotopic mouse model, endoglin was targeted with chitosan-encapsulated siRNA with and without carboplatin. Results: Endoglin expression was surprisingly predominantly cytoplasmic, with a small population of surface-positive cells. Endoglin inhibition decreased cell viability, increased apoptosis, induced double-stranded DNA damage, and increased cisplatin sensitivity. Targeting endoglin downregulates expression of numerous DNA repair genes, including BARD1, H2AFX, NBN, NTHL1, and SIRT1. BARD1 was also associated with platinum resistance, and was induced by platinum exposure. In vivo, antiendoglin treatment decreased tumor weight in both ES2 and HeyA8MDR models when compared with control (35%–41% reduction, P < 0.05). Endoglin inhibition with carboplatin was associated with even greater inhibitory effect when compared with control (58%–62% reduction, P < 0.001). Conclusions: Endoglin downregulation promotes apoptosis, induces significant DNA damage through modulation of numerous DNA repair genes, and improves platinum sensitivity both in vivo and in vitro. Antiendoglin therapy would allow dual treatment of both tumor angiogenesis and a subset of aggressive tumor cells expressing endoglin and is being actively pursued as therapy in ovarian cancer. Clin Cancer Res; 19(1); 170–82. ©2012 AACR.

Reasons for failure to deliver National Comprehensive Cancer Network (NCCN)-adherent care in the treatment of epithelial ovarian cancer at an NCCN cancer center.

OBJECTIVE The National Comprehensive Cancer Network (NCCN) has established guidelines for treating epithelial ovarian cancer (EOC) which includes cytoreductive surgery and platinum and taxane-based chemotherapy (CT). The objective of this study was to determine the reasons for failure to deliver NCCN-adherent care at an NCCN cancer center serving a diverse racial and socioeconomic population. METHODS Medical records of women with EOC diagnosed between 2004 and 2009 were reviewed for demographic, clinical, tumor, treatment, and survival data. Independent reviewers determined if their treatment met criteria for being NCCN-adherent. Progression-free survival (PFS) and overall survival (OS) were calculated with Kaplan-Meier estimates and compared with the log-rank test. RESULTS 367 patients were identified. 79 (21.5%) did not receive NCCN-adherent care. Non-adherent CT in 75 patients was the most common reason for failure to receive NCCN-adherent care. 39 patients did not complete CT due to treatment toxicities or disease progression. 12 patients received single agent CT only and 4 received no CT due to comorbidities. 2 patients declined CT. 18 patients died in the postoperative period without receiving CT. 8 patients did not undergo cytoreduction due to disease progression or comorbidities. PFS and OS were improved in the NCCN-adherent cohort (PFS: 5.7 vs. 18.3 months, p<.005) (OS: 11.4 vs. 49.5 months, p<.005). CONCLUSIONS The vast majority of patients at an NCCN cancer center received NCCN-adherent treatment. Reasons for failure to receive NCCN-adherent care were variable, but most did not receive chemotherapy in accordance with guidelines due to comorbidities or disease progression.

Diabetes mellitus and ovarian cancer: More complex than just increasing risk ☆

OBJECTIVE Diabetes mellitus (DM) is a risk factor for endometrial cancer and is associated with poorer outcomes in breast and colon cancers. This association is less clear in epithelial ovarian cancer (EOC). We sought to examine the effect of DM on progression-free (PFS) and overall survival (OS) in women with EOC. METHODS A retrospective cohort study of EOC patients diagnosed between 2004 and 2009 at a single institution was performed. Demographic, pathologic and DM diagnosis data were abstracted. Pearson chi-square test and t test were used to compare variables. The Kaplan-Meier method and the log rank test were used to compare PFS and OS between non-diabetic (ND) and DM patients. RESULTS 62 (17%) of 367 patients had a diagnosis of DM. No differences in age, histology, debulking status, or administration of intraperitoneal chemotherapy between ND and DM patients were present, although there were more stage I and IV patients in the ND group (p=0.04). BMI was significantly different between the two groups (ND vs. DM, 27.5 vs. 30.7kg/m(2), p<0.001). While there were no differences in survival based on BMI, diabetic patients had a poorer PFS (10.3 vs. 16.3months, p=0.024) and OS (26.1 vs. 42.2months, p=0.005) compared to ND patients. Metformin use among diabetic patients did not appear to affect PFS or OS. CONCLUSIONS EOC patients with DM have poorer survival than patients without diabetes; this association is independent of obesity. Metformin use did not affect outcomes. The pathophysiology of this observation requires more inquiry.

Does a standardized preoperative algorithm of clinical data improve outcomes in patients with ovarian cancer? A quality improvement project.

Objective To evaluate the potential impact of a standardized preoperative algorithm on outcomes of patients with suspected ovarian cancer. Methods From January 1 to December 31, 2013, patients with suspected ovarian cancer were triaged to primary debulking surgery or neoadjuvant chemotherapy/interval debulking surgery (NACT/IDS) based on a comprehensive review of preoperative clinical data as part of a quality improvement project. Demographics, surgical, and postoperative data were collected. Results A total of 110 patients with newly diagnosed ovarian cancer were identified: 68 (62%) underwent PDS with an 85% optimal debulking rate. The 30-day readmission rate was 14.7% with a 2.9% 60-day mortality rate. Forty-two patients (38%) underwent NACT. Two patients (4.8%) died before receiving NACT. Thirty-five patients have undergone IDS with an 89% optimal debulking rate. The 30-day readmission rate was 8.5% with a 5.7% 60-day mortality rate after IDS. Conclusions Although it is difficult to predict which patients will undergo optimal debulking at the time of PDS, surgical morbidity and mortality can be decreased by using NACT in select patients. The initiation of a quality improvement project has contributed to an improvement in patient outcomes at our institution.

Abstract A58: An ovarian patient-derived xenograft model to identify the chemoresistant population

Introduction: A cornerstone of preclinical ovarian cancer research over the last 30 years has been the use of cell lines both in vitro and in vivo . This resource has underperformed in its ability to identify effective novel therapeutics and evaluate the heterogeneity of an ovarian neoplasm. Developing a patient-derived xenograft (PDX) allows for a comprehensive study of the heterogeneous tumor and potentially the identification of cellular populations responsible for chemoresistance and recurrence, but has not been fully characterized for ovarian cancer. We have developed an ovarian PDX model to demonstrate many important similarities and differences between these and primary patient tumors, and begun to explore its utility in identifying mediators of chemoresistance and personalizing therapy. Methods: Tumors removed from patients undergoing primary tumor reductive surgery were implanted into the subcutaneous flanks of SCID mice. Once a tumor developed, it was expanded into additional mice to propagate a PDX line. Tumors were analyzed for heterogeneity by examination of the tumor initiating cell populations, its stromal content with HLA immunohistochemistry, and proliferation with Ki-67. Cohorts of PDX lines were treated with maximum-tolerated dose of combined carboplatin and paclitaxel or vehicle and response was compared between the PDX and the patient. Chemoresistant PDX lines were generated by treatment with MTD carboplatin and paclitaxel until no evidence of disease (NED), observation until recurrence, and retreatment until fully resistant. RNAseq was conducted comparing treated PDX lines to the untreated PDX lines (n=6 pair) to identify important mediators of chemotherapy resistance. Finally, PDX lines were characterized for defects in homologous recombination (HR) repair with an ex vivo assay, correlating HR status with response to PARP inhibition in vivo . Results: Refinement of xenografting procedures yields an 85% success rate in establishing a PDX. PDX tumors maintain the patient9s histology, but the stromal component is replaced by murine cells in the first generation. The xenografts retain primary tumor heterogeneity, at least in expression of putative tumor initiating cells (TIC) (ALDH: 17% vs 19%, CD44: 2.4% vs 5%, CD133 10% vs 3%). However, treatment with chemotherapy significantly increases these populations (ALDH1 increased to 36.1% from 16.2% (p=0.002); CD133 increased to 33.8% from 16.2% (p=0.026), suggesting a role in chemotherapy resistance. The treated PDX tumors showed a decrease in Ki67 staining (from 64% to 34%, p=0.001), suggesting dormancy is induced in the surviving population. The PDX tumors show similar response to chemotherapy as patient tumors, in that PDX tumors from patients with a partial response respond more slowly (if at all) than those from patients achieving a complete response (mean change in tumor volume -5.11% vs -63.73%, p=0.029). RNAseq comparing treated and untreated PDX lines indicate only 54 common genes with a significant change in mRNA (p ex vivo assay show regression of tumor or stable disease with single-agent PARP inhibitor therapy. Conclusions: The ovarian PDX model recapitulates patient tumor heterogeneity and mirrors response to chemotherapy. This model may prove superior in predicting response to therapy in patients, and allows a better model to study complex tumor biology that is impacted by tumor heterogeneity, such as survival of small populations with chemotherapy. Citation Format: Zachary C. Dobbin, Ashwini K. Katre, Monjri M. Shah, Britt K. Erickson, Hauping Chen, Ronald D. Alvarez, Michael G. Conner, Dongquan Chen, Charles N. Landen. An ovarian patient-derived xenograft model to identify the chemoresistant population. [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 A58.

Abstract 5475: Endoglin (CD105) is a target for ovarian cancer cell-specific therapy through induction of DNA damage.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Endoglin (ENG, CD105) is a protein markedly overexpressed in tumor-associated endothelial cells, and a target for anti-angiogenic therapy. Recently we have noted it to be overexpressed in chemoresistant ovarian cancer cells as well. Our objective was to evaluate the effects and mechanisms of targeting endoglin specifically in ovarian cancer cells. Methods: Endoglin expression was assessed in multiple ovarian cancer lines by Western blot, immunohistochemistry, and flow cytometry. Anti-endoglin siRNAs were used to downregulate expression in ES2 and HeyA8MDR. In vitro, the effects of endoglin-knockdown individually and with chemotherapy were evaluated by MTT assay, cell-cycle analysis, alkaline comet assay, γ-H2AX foci formation and qPCR array for mediators of DNA damage and repair. In an orthotopic murine model, mice inoculated with ES2 or HeyA8MDR cell lines were administered chitosan-encapsulated anti-ENG siRNA or control siRNA with and without carboplatin. PCNA, γH2AX, and 53BP1 IHC and the TUNEL assay were performed to examine biologic effects of endoglin knockdown. Results: Endoglin is expressed to varying degrees by multiple ovarian cancer cell lines. Expression was on the cell surface, consistent with its recognized role as a cofactor with TGF-beta receptor, in only 5% of cells, with most other cells having cytoplasmic expression. In ES2 and HeyA8MDR cell lines, endoglin inhibition decreased cell viability, increased apoptosis, induced double-stranded DNA damage, and increased cisplatin sensitivity. Endoglin downregulation led to a decrease in expression of several DNA repair genes, including NBN, NTHL1, BARD1, H2AFX and SIRT1, and an increase in expression of DDIT3 and PPP1R15A. BARD1-specific downregulation, in turn, led to a significant decrease in BRCA1 expression, likely through ubiqutinylation. In vivo, anti-endoglin treatment decreased tumor weight in both ES2 and HeyA8MDR models when compared to control (35-41% reduction, p<0.05). Combination treatment with anti-Endoglin siRNA and carboplatin was associated with even greater inhibitory effect compared to control (58-62% reduction, p<0.001). Conclusions: Targeting endoglin improves platinum sensitivity of ovarian cancer cells both in vivo and in vitro. Endoglin downregulation induces DNA damage at multiple levels, one of which is through decreased expression of BARD1 and BRCA1. Anti-endoglin therapies should be pursued further as their development would allow dual treatment of both tumor angiogenesis and an aggressive subset of chemoresistant tumor cells. Citation Format: Angela J. Ziebarth, Somaria Nowsheen, Adam D. Steg, Monjri M. Shah, Ashwini A. Katre, Zachary C. Dobbin, Anil K. Sood, Michael G. Conner, Eddy S. Yang, Charles N. Landen. Endoglin (CD105) is a target for ovarian cancer cell-specific therapy through induction of DNA damage. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5475. doi:10.1158/1538-7445.AM2013-5475 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.