Hou, Xiaonan

Abstract Ovarian cancer (OC) is the most lethal gynecologic malignancy and the fifth leading cause of cancer death in the United States. Metformin, an antidiabetic drug, has been shown to decrease the risk of mortality in cancer patients, particularly those with OC. Though the drug acts through inhibition of the PI3K/mTOR/AKT pathway, which is commonly altered in OC, knockout of key proteins in the pathway does not attenuate metformin’s antidiabetic effects. Further, intravenous metformin administration is less effective than oral, implying that some crucial interaction takes place in the gut. The gut is home to some 1013 bacterial cells, collectively known as the microbiota. These bacteria play important roles in fermentation of fiber, producing short-chain fatty acids (SCFA) that have positive health benefits, including prevention of metabolic syndrome, bowel disorders, and certain cancers. Mechanistically, SCFAs inhibit the PI3K/mTOR/AKT pathway, similar to metformin. Conditions such as obesity, metabolic syndrome, and diabetes significantly alter gut microbiota composition, possibly leading to a reduction in beneficial SCFAs. However, in type II diabetes, treatment with metformin leads to an increase in SCFA-producing bacteria, suggesting that metformin may positively influence the microbiota to improve patient metabolic profiles. In OC, metformin may play a similar role in promoting the presence of SCFA-producing bacteria. We hypothesized that metformin increases the abundance of SCFA-producing bacteria in the gut, with which it acts synergistically to decrease growth of OC cells in combination with OC chemotherapy. To investigate our hypothesis, we have employed the OC patient-derived xenograft (PDX) mouse model established at Mayo Clinic. Immunocompromised SCID mice received OC cells from Mayo Clinic patients and the tumors were allowed to grow to an appropriate size before treatment was started. Cage mates were split into one of four treatment arms: a control (no treatment) group, metformin only, carboplatin/paclitaxel (CP) chemotherapy only, or CP with metformin. Mice received 2mg/ml metformin in their water continuously and/or 51mg/kg carboplatin and 15mg/kg paclitaxel intravenously weekly. Stool was collected weekly prior to tumor injection, during tumor growth, and through the four weeks of treatment. DNA was extracted from stool and bacterial 16S rRNA amplicon sequencing was used to determine microbiota profiles. Mice with chemosensitive tumors treated with only CP chemotherapy had significant reductions in tumor growth along with significantly less microbial gut diversity than pretumor and pretreatment mice. Chemosensitive mice treated with metformin alone did not lose significant microbial diversity but did not have significant reductions in tumor growth as compared to the control mice. CP in combination with metformin both reduced tumor growth and protected against loss of microbiota diversity. Further, mice treated with only metformin had significant increases in abundance in the Firmicutes genera Ruminococcus and Oscillospira, SCFA producers that are associated with leanness and health. Mice treated with both metformin and CP had significant increases in another SCFA-producing Firmicute, Moryella. Finally, mice with chemoresistant tumors showed improved tumor growth inhibition with combination CP/metformin therapy, suggesting that metformin-associated changes in the gut microbiota may increase sensitivity to CP. These data support the hypothesis that metformin protects against detrimental loss of gut microbiota diversity induced by chemotherapy treatment, enriching beneficial SCFA producers. This work implies a role for the gut microbiota in OC treatment and provides support for metformin’s potential use as a beneficial addition to chemotherapy treatment. Citation Format: Dana M. Walsh, Ismail Mert, Chen Jun, Xiaonan Hou, Saravut John Weroha, Marina Walther-Antonio. Metformin alters the gut microbiota of ovarian cancer patients treated with carboplatin/paclitaxel chemotherapy and enhances sensitivity in resistant tumors. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A70.

Transforming growth factor beta (TGF-β) signaling has pleiotropic functions regulating cancer initiation, development, and metastasis, and also plays important roles in the interaction between stromal and cancer cells, making the pathway a potential therapeutic target. LY2157299 monohydrate (LY), an inhibitor of TGF-β receptor I (TGFBRI), was examined for its ability to inhibit ovarian cancer (OC) growth both in high-grade serous ovarian cancer (HGSOC) cell lines and xenograft models. Immunohistochemistry, qRT-PCR, and Western blot were performed to study the effect of LY treatment on expression of cancer- and fibroblast-derived genes. Results showed that exposure to TGF-β1 induced phosphorylation of SMAD2 and SMAD3 in all tested OC cell lines, but this induction was suppressed by pretreatment with LY. LY alone inhibited the proliferation, migration, and invasion of HGSOC cells in vitro. TGF-β1-induced fibroblast activation was blocked by LY. LY also delayed tumor growth and suppressed ascites formation in vivo. In addition, independent of tumor inhibition, LY reduces ascites formation in vivo. Using OVCAR8 xenograft specimens we confirmed the inhibitory effect of LY on TGF-β signaling and tumor stromal expression of collagen type XI chain 1 (COL11A1) and versican (VCAN). These observations suggest a role for anti-TGF-β signaling-directed therapy in ovarian cancer.

Abstract Background: Endometrioid carcinoma (EC) and clear cell carcinoma (CC) histotypes of epithelial ovarian cancer are understudied compared with the more common high-grade serous carcinomas (HGSC). We therefore sought to characterize EC and CC transcriptomes in relation to HGSC. Methods: Following bioinformatics processing and gene abundance normalization, differential expression analysis of RNA sequence data collected on fresh-frozen tumors was completed with nonparametric statistical analysis methods (55 ECs, 19 CCs, 112 HGSCs). Association of gene expression with progression-free survival (PFS) was completed with Cox proportional hazards models. Eight additional multi-histotype expression array datasets (N = 852 patients) were used for replication. Results: In the discovery set, tumors generally clustered together by histotype. Thirty-two protein-coding genes were differentially expressed across histotype (P < 1 × 10−10) and showed similar associations in replication datasets, including MAP2K6, KIAA1324, CDH1, ENTPD5, LAMB1, and DRAM1. Nine genes associated with PFS (P < 0.0001) showed similar associations in replication datasets. In particular, we observed shorter PFS time for CC and EC patients with high gene expression for CCNB2, CORO2A, CSNK1G1, FRMD8, LIN54, LINC00664, PDK1, and PEX6, whereas, the converse was observed for HGSC patients. Conclusions: The results suggest important histotype differences that may aid in the development of treatment options, particularly those for patients with EC or CC. Impact: We present replicated findings on transcriptomic differences and how they relate to clinical outcome for two of the rarer ovarian cancer histotypes of EC and CC, along with comparison with the common histotype of HGSC. Cancer Epidemiol Biomarkers Prev; 27(9); 1101–9. ©2018 AACR.

Abstract E26 transformation-specific transcription factor ERG is aberrantly overexpressed in approximately 50% of all human prostate cancer due to TMPRSS2-ERG gene rearrangements. However, mice with prostate-specific transgenic expression of prostate cancer–associated ERG alone fail to develop prostate cancer, highlighting that ERG requires other lesions to drive prostate tumorigenesis. Forkhead box (FOXO) transcription factor FOXO1 is a tumor suppressor that is frequently inactivated in human prostate cancer. Here, we demonstrate that FOXO1, but not other FOXO proteins (FOXO3 and FOXO4), binds and inhibits the transcriptional activity of prostate cancer–associated ERG independently of FOXO1 transcriptional activity. Knockdown of endogenous FOXO1 increased invasion of TMPRSS2–ERG fusion–positive VCaP cells, an effect completely abolished by ERG knockdown. Patient specimen analysis demonstrated that FOXO1 and ERG protein expression inversely correlated in a subset of human prostate cancer. Although human ERG transgene expression or homozygous deletion of Foxo1 alone in the mouse prostate failed to promote tumorigenesis, concomitant ERG transgene expression and Foxo1 deletion resulted in upregulation of ERG target genes, increased cell proliferation, and formation of high-grade prostatic intraepithelial neoplasia. Overall, we provide biochemical and genetic evidence that aberrantly activated ERG cooperates with FOXO1 deficiency to promote prostate tumorigenesis and cell invasion. Our findings enhance understanding of prostate cancer etiology and suggest that the FOXO1–ERG signaling axis can be a potential target for treatment of prostate cancer. Cancer Res; 77(23); 6524–37. ©2017 AACR.

Abstract Background: Most ovarian cancer (OC) patients present with advanced disease and ultimately develop chemo-resistant relapses. Stroma plays a significant role in OC behavior including invasiveness, fibrosis and chemoresistance. TGF-β is important for crosstalk between stroma and cancer cells, and stromal activation in cancer has similarities to matrix remodeling in fibrosis. We have shown that TGF-β and fibrosis-inhibitors can suppress OC cell proliferation, migration and invasion. Such inhibitors are available for clinical use, and we have begun to test their efficacy in OC in vivo. Methods: Animal experiments were approved by Mayo Clinic IACUC. Intraperitoneal xenograft models were derived from OVCAR8 (5×106) cells (OV8-CDX) or 0.1 gm OC patient tumor tissue (PDX) in NSG mice. OV8-CDX and PDX PH003 (an aggressive, chemoresistant and ascites-prone model) (n=5-10 mice/arm) were treated with 2 agents and compared to untreated controls (C): 1) C vs. the fibrosis inhibitor Pirfenidone (P, 200 mg/kg, BID, provided by Genentech); 2) C vs. TGF-β receptor I inhibitor Galunisertib (G, 75 mg/kg, BID, provided by Eli Lily). Mice were pretreated for 2 days before xenografting and treated for 4 weeks with ultrasound (US) to measure tumor growth. At day (d) 28, we i) collected tumor (OV8-CDX); or ii) observed until progression (PH003, PH053). OV8-CDX tumors were used for target gene analysis. Results: P treatment delayed tumor growth in OV8-CDX relative to control (C): tumors established in 0/5 (P) vs. 1/5 (C), and 1/5 (P) vs. 4/5 (C) of mice at d14 and d28, respectively. The reduced tumor weight (g) at d28 was also confirmed (0.20±0.10 vs. 0.54±0.23, p=0.02). Similarly, P treatment in PH003 inhibited the tumor growth (tumor area (cm2) at d28: 0.74±0.43 vs. 1.04±0.51, p=0.28), ascites development (volume (ml): 1.14±0.54 vs. 3.37±0.62, p=0.05) and prolonged survival time (p=0.04). G treatment was also associated with delayed tumor establishment in OV8-CDX relative to C: tumors presented in 0/5 (G) vs. 1/5 (C), and 1/5 (G) vs. 4/5 (C) of mice at d14 and d28, respectively. Associated d28 tumor weight was significantly reduced (0.20±0.10 vs. 0.54±0.23, p=0.02) as well. For PH003, G treatment was associated with delayed tumor growth (tumor area at d28: 0.57±0.27 vs. 1.04±0.51, p=0.05), a marked reduction in ascites (volume: 1.25±0.56 vs. 3.37±0.62, p=0.05) and a prolonged survival time (p=0.04) We identified down-regulation of TGF-β signaling-related genes (e.g. TGFB1, TGFBR2) and fibrosis-related biomarkers (e.g. FAP, COL11A1) following drug treatment confirming inhibition of relevant pathways. Conclusion: We report that 2 currently available inhibitors targeting either fibrosis or TGF-β appear to have efficacy in vivo and delay tumor growth in OC PDX models. These results suggest promise for a strategy of targeting the stromal component of OC. Citation Format: Qing Zhang, Xiaonan Hou, Bradley J. Evans, John J. Weroha, William A. Cliby. Inhibitors of fibrosis and TGF-beta delay tumor xenograft growth and reduce ascites in ovarian cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 961. doi:10.1158/1538-7445.AM2017-961

Abstract Malignant ascites is one of the most common causes of morbidity in end stage ovarian cancer patients with negative impact on quality of life. Novel non-invasive palliative therapeutic options are lacking for such patients. The zinc metalloprotease, pregnancy-associated plasma protein-A (PAPP-A), plays a key role in the insulin-like growth factor (IGF) pathway, promoting ovarian cancer cellular transformation, growth and invasiveness. Furthermore, patient primary malignant ascites is known to contain high levels of PAPP-A by ELISA. Preliminary data shows that inhibition of PAPP-A through a neutralizing monoclonal PAPP-A antibody (mAb-PA) inhibits the accumulation and promote the regression of ascites in an ovarian cancer patient-derived xenograft (PDX) models. In the current study, we investigated whether mAb-PA can promote ascites regression in an additional ovarian PDX model with measurable ascites. Patient derived ascites xenograft (PDAX) models were defined as SCID mice that developed ascites after intraperitoneal heterotransplantation of patient solid tumor collected at the time of primary cytoreduction. Ascites from these models (n=51) was screened for human PAPP-A protein by ELISA and models were divided into two groups by the relative concentration of PAPPA-A: high (n=18) and low (n=33). PAPP-A High PDAX model PH438 was re-established intraperitoneal in 20 SCID mice by ascites injection (0.1 ml per mouse). When ascites area reached a threshold of >0.60 cm2 by ultrasound, mice were treated with 60mg/Kg of mAb-PA (n=10) or IgG2a control (n=10) on day one and three. On day four, a second ultrasound measurement was obtained and the mice were euthanized. Ascites burden was measured at necropsy. Personnel involved with the acquisition of ultrasounds measurements, subsequent ascites harvests, and post hoc analyses were blinded to the treatments. The ratio of mean ascites (mAb-PA/IgG2a in grams) collected at necropsy was 2.04/2.51, indicating that the mAb-PA arm had less ascites compared to control. These data were consistent with pre- and post-treatment ultrasound measurements of ascites burden; a statistically significant (p=0.0283 by paired t test) reduction of ascites burden of 52.3% was observed, compared to the starting baseline, indicating that mAb-PA treatment causes ascites regression. Ascites weight at necropsy was compared to the area of greatest fluid echogenicity by ultrasound and the Pearson correlation R value achieved was of 0.859 (p<0.0001). These PDAX data implicate PAPP-A attenuation as a potential strategy to treat malignant ascites in OC. Additional PDAX models are under examination to confirm these findings and support the translational development of PAPP-A as a new therapeutic target for women with refractory ascites. Citation Format: Valentina Zanfagnin, Laurie K. Bale, Marc A. Becker, Xiaonan Hou, Diogo Torres, Cheryl A. Conover, Saravut J. Weroha. Regression of malignant ascites via PAPP-A inhibition in ovarian cancer patient-derived xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4817. doi:10.1158/1538-7445.AM2017-4817

Abstract Purpose: The treatment of ovarian cancer poses a challenge since 70% of patients will relapse with incurable platinum resistant disease. Although ovarian cancers lack frequently-occurring driver mutations or amplifications, the study presented herein examines the concept of individualized therapy based on mate pair sequencing and protein expression in ovarian cancer patient-derived xenografts. Experimental Procedure: Macrodissection of patient tumor was followed by genomic DNA isolation and next-generation sequencing using an Illumina Mate Pair protocol to identify structural genomic changes and copy number variations. Secondary analyses were carried out to select potential therapeutic targets among those. The tumors were also propagated intraperitoneally in immunocompromised mice and treated with standard chemotherapy or/and targeted therapy chosen based on genomic analyses. Results: Mate pair analysis revealed that, in general, copy number changes (amplifications, gains and losses) were common in ovarian tumors and included genes potentially targetable. Tumors from early generation patient- derived xenografts showed a landscape of genomic aberrations identical to that of original patient tumor. One PDX model in which high expression of HER2 and copy number gains at RICTOR and AKT genetic loci were revealed, was treated with either chemotherapy and Pertuzumab/trastuzumab (HER2 inhibitors), MK-8669 (mTOR inhibitor) or MK-2206 (AKT inhibitor) or chemotherapy alone. Tumor size was measured by serial abdominal ultrasound and after 28 days, the final tumor size ratio compared to baseline was 0.23, 0.36, 0.55 and 0.56, respectively, indicating that the best response was seen in the combined chemotherapy and Pertuzumab/trastuzumab cohort and that was better than in the chemotherapy only cohort. Conclusions: Patient-derived xenografts models in conjunction with genomic and protein analyses of patient tumors are valuable for testing of combination therapies including specific targeting drugs. This approach also provides actionable information for more tailored treatment of ovarian cancer patients. Citation Format: Konstantinos Leventakos, Faye R. Harris, Lin Yang, Xiaonan Hou, Saravut Weroha, Irina V. Kovtun. Testing personalized therapies for ovarian cancer using mate pair genomic analysis and patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4814. doi:10.1158/1538-7445.AM2017-4814

Abstract Mutations in the EGFR gene or transcript fusion involving EML4-ALK result in oncogenic activation of these oncogenes driving lung adenocarcinoma growth in tumors carrying such an alteration. For lung adenocarcinoma with these genetic changes, small-molecule tyrosine kinase inhibitors have been highly effective at reducing tumor burden and improve the outcome of the patients. Unfortunately, drug resistance eventually develops in these tumors and challenges remain in controlling lung cancer recurrence after targeted therapy. To overcome recurrence, we initiated a prove-of-principle study to evaluate the feasibility of an integrated strategy utilizing genomic profiles of the tumor and patient-specific tumor xenografts derived (PDX) from biopsies for ex vivo evaluation of antitumor drugs to help guide personalized treatment of lung cancer in patients who have developed resistance to targeted therapy drugs. Our study has three main objectives. 1) Use tumor biopsies obtained at the time of recurrence for oncogene mutation and RNAseq analyses to identify molecular changes in oncogenes and gene pathways that can be potentially targeted. 2) Evaluate drug efficacy and optimize personalized therapy for each patient using PDX models. 3) Assess clinical feasibility and our experience using integrated approaches for lung cancer patients who failed targeted therapy. This study was approved by the Mayo Clinic Institutional Review Board (IRB) and utilizes both clinical and research biopsies. A dedicated nurse study coordinator reviews clinical patient list and history on weekly basis and informs the study team of each potential candidate patient. A total of 30 patients having ALK positive lung cancers have been identified and followed up from nearly 500 potentially ALK positive cases between April 2014 to Sept. 2015. Most patients were never smokers but six were former smokers and two were current smokers. Age at diagnoses ranged from 27-78 years (median = 61). Seven patients (23%) were 45 years or younger at the time of diagnosis. A total of 22 patients are currently being treated with crizotinib while eight are on ceritinib or alectinib. For each biopsy, tumor tissues are obtained using a 20 gauge needle, transferred on ice in preserving media and implanted within one hour into 6-8 week old NOD/SCID mice. Many patients have been on treatment for 2-3 years with stable disease so they do not require biopsy. Using a similar strategy, we also obtained biopsies from patients with EGFR gene mutations in their tumors and have progressed while on targeted therapy. A total of seven cases have been implanted. We will report our experiences in patient selection, clinical follow up, patient consent, PDX development, time from biopsy to tumor establishment, and the results of molecular analyses. Our study enabled us to gain new insights regarding the molecular changes associated with recurring tumors after they have failed targeted therapy as well as clinical experiences on how to utilize state-of-the art approaches and comprehensive genomic information to further improve cancer patient care upon disease progression. Acknowledgements: This work is supported in part by the Hillsberg Award from the National Foundation for Cancer Research and by the Biomarker Discovery Program at the Mayo Clinic Center for Individualized Medicine. Citation Format: Jin Jen, Aaron Mansfield, Patrick W. Eiken, Shawn Stoddard, Karlyn Pierson, Xiaonan Hou, Hong Zheng Ren, Julian Molina, Joanne Yi, Ping Yang. Integrated Approaches to Treating Lung Adenocarcinoma Resistant to Targeted Therapy. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B34.

Abstract Over the recent years significant knowledge has been accumulated on which drugs can be therapeutically beneficial for a given patient based on mutational profiling of their tumors using selected gene panels. Much less, however, is known under which conditions targeting of genes which are amplified could be beneficial. Apart from successfully targeting HER2-positive cancers, many of which frequently carry amplified ERBB2 gene, no other targeted therapy based on DNA amplifications or gains of the genes is offered to the patients due to lack of clinical response data. Using mate pair sequencing we have profiled 60 high grade serous ovarian tumors to compare genomic rearrangements and copy number changes. The analyses revealed that these tumors carry highly rearranged genome. Copy number changes amplifications, gains and losses were very common and included genes potentially therapeutically targetable. In contrast, only a few of targetable fusion genes were identified in a set of 60 cases. The tumors were propagated in immunocompromised mice (mouse avatars), sequenced and showed identical to original patient tumor landscape of rearrangements. Using these mouse avatar models we tested whether a therapy inhibiting selected based on genomic amplification targets provided a benefit compared to standard chemotherapy. Mice which harbored tumors showing amplifications of CDKs (CDK2, 4 and 9) and ERBB3 were treated with flavopiridol and pertuzumab respectively, and response was compared to that of standard for ovarian cancer chemotherapy (carboplatin and paclitaxel combination). We observed that application of either agent, significantly inhibited tumor growth in treated mice as compared to untreated. However, neither drug alone caused tumor regression. The treatment regimen then was modified to administer chemotherapy and targeted therapy together. A mouse avatars in which genomic analyses revealed amplification at loci involving AKT3 and RICTOR genes was treated with chemotherapy alone and in combination with MK2206 (AKT inhibitor) or MK8669 (mTOR inhibitor). The high level of both target proteins was confirmed by immunoblotting. Significant regression of tumor was observed after 6 weeks of treatment. Addition of either targeted drug provided an extra benefit in killing tumor cells compared to chemotherapy alone. The mice are followed to study tumor recurrence. These data indicated that genomic information obtained using next generation sequencing can aid therapy decisions by tailoring treatments to individual tumor makeup. Avatar models in conjunction with genomic analyses of the original tumors are invaluable for testing specific targeted therapies selected based on amplification/gains of putative genes-targets and for studying mechanisms of tumor recurrence and therapy resistance. Citation Format: Faye R. Harris, Lin Yang, Xiaonan Hou, Konstantinos Leventakos, Saravut J. Weroha, George Vasmatzis, Irina V. Kovtun. Targeting genes which are amplified in ovarian cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A34.