deVere White Research Articles

Abstract 4468: Role of statins and PSA nadir after androgen-deprivation therapy in overall survival of patients with metastatic prostate cancer

Abstract Statins are known to reduce prostate cancer (CaP) aggressiveness in both localized and metastatic disease. Studies have indicated that the use of statin drugs to lower cholesterol levels was associated with decreased risk of localized CaP, less frequent high grade CaP and lower CaP volume, suggesting that statin use has a protective effect against localized CaP. However, less is known about a role for statins in metastatic CaP (mCaP). While it is known that hypercholesterolemia is associated with the development of castration resistant prostate cancer (CRPC) after androgen deprivation therapy (ADT) in patients with bone metastasis, it is not known whether the sequence of statin and ADT affects outcome. Charts of 162 patients with mCaP treated at the VA Northern California Health Care System (VANCHCS) with ADT and statins between 1992 and 2016 were analyzed. Overall survival (OS) was followed until 12/2016. Dates were noted from the medical records entered into the Computerized Patient Record System (CPRS). Time to event outcomes (with the exception of time to Nadir PSA, which was not censored) were analyzed using Cox proportional hazard models. PSA Nadir and time to PSA Nadir were analyzed using linear models, with outcomes log transformed. All models include age at PSA diagnosis and year of metastatic diagnosis as covariates. Of the 162 total patients, 110 were on ADT following biochemical recurrence (BCR) at the time of diagnosis with distant metastasis (ADT before mets, ABM) while 50 were diagnosed with a distant metastatic lesion at the time of ADT administration (mets before ADT, MBA). 46.3% of the patients did not receive statin treatment whereas 41.4% received statins prior to diagnosis with distant metastasis, and 12.3% started statins after metastasis diagnosis (29% prior to ADT treatment and 24.7% after starting on ADT). There was no effect of statins on OS among those who had a diagnosis of metastasis prior to initial ADT (MBA group). On the other hand, among those who developed metastases while on ADT (ABM group), statin use significantly increased OS (based on the time of metastasis diagnosis). However, within this group, the largest benefit was in the patients that received statins after starting ADT (HR = 0.51, p=0.022). The same group (subgroup within the ABM group who initiated statins after ADT) also took longer to reach PSA nadir following ADT compared to those who never received statins (R=1.79, p=0.031) and also took longer to be diagnosed with mCaP following ADT compared to those who had never been (HR=0.45, p=0.003). These advantages were not seen in patients in the ABM group who were already on statins at the time of ADT initiation, and no advantages of statins were seen in the MBA group. Our data indicate that treatment with statins initiated after the patient has undergone ADT is beneficial to patients who develop distant metastases while on ADT treatment. Citation Format: Salma Siddiqui, Blythe P. Durbin-Johnson, Stanley A. Yap, Ralph W. deVere White, Paramita M. Ghosh. Role of statins and PSA nadir after androgen-deprivation therapy in overall survival of patients with metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4468.

Abstract 4842: Overcoming EGFR and ERK-mediated resistance to enzalutamide in castration-resistant prostate cancer

Abstract The present project was undertaken to determine whether the activation of the EGFR family (EGFR/ErbB2/ErbB3/ErbB4) and its downstream signaling effector ERK1/2 plays a role in enzalutamide resistance and whether treatment with ErbB or ERK inhibitors overcome this resistance. C4-2B (enzalutamide sensitive) and 22Rv1 (partially enzalutamide resistant) cell lines cultured for prolonged periods in enzalutamide, developed resistance to this drug (C4-2B/MDVR and 22Rv1/MDVR, respectively). Whole genome comparison of enza-resistant to parental lines demonstrated that the ErbB signaling network was significantly upregulated in the enza-resistant lines. Comparison of various enza-sensitive and resistant lines demonstrated a strong correlation between phosphorylation at EGFR(Y1068) and enza resistance. Inhibition of EGFR, but not that of ErbB2 or ErbB3 prevented cell viability. The EGFR inhibitors erlotinib and dacomitinib, but not the ErbB2 inhibitor lapatinib, suppressed viability in combination with enzalutamide. We hypothesized that enza-resistant tumors expressing higher levels of EGFR would be more responsive to erlotinib. To test this hypothesis, we compared the effects of erlotinib and enzalutamide, individually or in combination, in organelles from PDX tumors expressing high or low EGFR levels. Significantly, tumors expressing high EGFR, but not those expressing low EGFR, were responsive to the combination. Finally, we investigated the cause for greater efficacy with erlotinib compared to lapatinib in PCa. Comparison of the effects of different ligands revealed that while both lapatinib and erlotinib inhibited EGFR phosphorylation, only erlotinib but not lapatinib was able to inhibit EGF-induced ERK phosphorylation. This indicated an important role for ERK in the mediation of EGFR ligand induced enzalutamide resistance. Continuous culture of enza-sensitive C4 cells in enzalutamide resulted in the development of ERK phosphorylation in these PTEN-null cells that normally do not express phosphorylated ERK, and the newly ERK phosphorylated cells also were more susceptible to erlotinib as well. In support of a role for ERK in mediating the effects of EGFR activation in enza-resistant cells, inhibition of EGFR but not ErbB2 or ErbB3 inhibited ERK phosphorylation, and the ERK inhibitor ulitertinib inhibited viability of enza-resistant lines. We conclude that enzalutamide treatment causes an increase in EGFR ligands that result in the phosphorylation of EGFR at Y1068, which further resulted in phosphorylation of ERK. EGFR inhibitors that prevent ERK phosphorylation were effective in suppressing viability of enza-resistant CRPC cells, whereas ErbB2 inhibitors that did not affect ERK phosphorylation were unable to affect cell viability. These results demonstrate why certain ErbB inhibitors failed to affect enza-resistant CRPC tumors but provide encouragement for others. Citation Format: Thomas M. Steele, Maitreyee K. Jathal, Salma Siddiqui, Sisi Qin, Clifford G. Tepper, Ralph W. deVere White, Manish Kohli, Liewei Wang, Allen C. Gao, Paramita M. Ghosh. Overcoming EGFR and ERK-mediated resistance to enzalutamide in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4842.

Abstract 908: Overexpression of nicotinamide N-methyltransferase confers gemcitabine resistance in bladder cancer

Abstract Bladder cancer is among the top ten most common cancers diagnosed, with about ~380,000 new cases and ~150,000 deaths per year reported worldwide. Platinum-based chemotherapy in combination with gemcitabine, a nucleoside analog, is a widely used treatment option for advanced bladder cancer. It has been shown that only ~50% of the patients with advanced bladder cancer respond to platinum-based therapy. Cancer cells often become non-responsive to therapy that once proved efficacious and are now drug resistant. Drug resistance represents a significant, ongoing challenge in eradicating cancer. We have employed a patient-derived bladder cancer xenograft (PDX) platform to further investigate the molecular mechanisms that contribute to gemcitabine-induced drug resistance in advanced bladder cancer. Transcriptome profiling of passage 4 bladder cancer xenograft tumors from 2 gemcitabine sensitive PDX lines (BL0440 & BL0293) was performed using RNA sequencing (RNAseq) analysis, before and after a 21-day cisplatin/gemcitabine drug treatment regimen. Key regulatory pathways and genes contributing to drug resistant bladder cancer have been identified and validated by overexpression in a 5637 bladder cancer cell line. RNA sequencing data has indicated significant differences between the transcriptional profiles of drug-sensitive and drug-resistant tumors. PDXs retained morphology and shared 92-97% of genetic alterations of parental cancer cells. We identified 106 genes >1.5 fold up-regulated and 45 genes >1.5 fold down-regulated in the drug resistant tumors compared to their drug sensitive counterparts. Of the genes that were significantly upregulated, two methyltransferase enzymes were further validated: Nicotinamide N-methyltransferase (NNMT) and methionine methyltransferase 1A (MAT1A). These genes were found to be contributors to gemcitabine-mediated drug resistance. 5637 cells overexpressing NNMT yielded a 200-fold increase in gemcitabine resistance relative to parental strain. In conclusion, chemoresistance to gemcitabine is associated with differential expression of genes and alteration of downstream signaling pathways. Upregulation of NNMT & MAT1A can potentially be used as novel biomarkers for subpopulations of drug resistant bladder cancer for which improved therapeutics can be developed. Future direction will likely include studies to elucidate exact molecular mechanisms by which cancer cells utilize methyltransferases to no longer respond to gemcitabine therapy. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Kelly A. Martin, Aimy Sebastian, Nicholas R. Hum, Deepa K. Murugesh, Chong-xian Pan, Ai-Hong Ma, Ralph W. de Vere White, Gabriela G. Loots. Overexpression of nicotinamide N-methyltransferase confers gemcitabine resistance in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 908.

Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy.

Establishment of an in vivo small animal model of human tumor and human immune system interaction would enable preclinical investigations into the mechanisms underlying cancer immunotherapy. To this end, nonobese diabetic (NOD).Cg-PrkdcscidIL2rgtm1Wjl/Sz (null; NSG) mice were transplanted with human (h)CD34+ hematopoietic progenitor and stem cells, which leads to the development of human hematopoietic and immune systems [humanized NSG (HuNSG)]. HuNSG mice received human leukocyte antigen partially matched tumor implants from patient-derived xenografts [PDX; non–small cell lung cancer (NSCLC), sarcoma, bladder cancer, and triple-negative breast cancer (TNBC)] or from a TNBC cell line-derived xenograft (CDX). Tumor growth curves were similar in HuNSG compared with nonhuman immune-engrafted NSG mice. Treatment with pembrolizumab, which targets programmed cell death protein 1, produced significant growth inhibition in both CDX and PDX tumors in HuNSG but not in NSG mice. Finally, inhibition of tumor growth was dependent on hCD8+ T cells, as demonstrated by antibody-mediated depletion. Thus, tumor-bearing HuNSG mice may represent an important, new model for preclinical immunotherapy research.—Wang, M., Yao, L.-C., Cheng, M., Cai, D., Martinek, J., Pan, C.-X., Shi, W., Ma, A.-H., De Vere White, R. W., Airhart, S., Liu, E. T., Banchereau, J., Brehm, M. A., Greiner, D. L., Shultz, L. D., Palucka, K., Keck, J. G. Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy.

Abstract 97: RNA sequencing of bladder cancer patient-derived xenograft models identifies genes associated with chemoresistance

Abstract Background: Bladder cancer is among the top ten most common cancers, with about ~380,000 new cases and ~150,000 deaths per year worldwide. Platinum-based combination chemotherapy is commonly used to treat advanced bladder cancer. It has been shown that only ~50% of the patients with advanced bladder cancer respond to platinum-based therapy. Methods: We have utilized a patient-derived bladder cancer xenograft (PDX) platform to characterize the molecular mechanisms that contribute to resistance of gemcitabine-cisplatin combination therapy in advanced bladder cancer. We have also identified key regulatory pathways in our PDX models that can be targeted to treat chemotherapy resistant bladder cancer using RNAseq analysis. Transcriptome profiling of P0 (passage 0) bladder cancer xenograft tumors from 4 PDX lines (2 gemcitabine-cisplatin resistant lines and 2 drug sensitive lines) was performed by RNA-Seq analysis, before and after a 21-day cisplatin/gemcitabine drug treatment regimen. Results: The RNA-seq data has indicated significant differences between the transcriptional profiles of drug-sensitive and drug-resistant tumors. PDXs retained morphology and shared 92-97% of genetic alterations of parental cancer cells. We identified 333 genes >2 fold up or down regulated in the drug resistant tumors compared to the drug sensitive tumors. Significantly up-regulated genes in drug resistant tumors analyzed include metabolic enzymes ALDH2, ALDH3A1, ALDH4A1 and ALDH7A1, transporter proteins ABCA1, SLC1A4, SLC2A5, SLC30A1, SLC39A6, SLC7A5 and SLC9A3, Notch ligand JAG2, Growth hormone receptor GHR and transmembrane protein GPNMB. Consistent with the change of cell surface proteins such as GHR and GPNMB, the MAPK and the PI3K-AKT pathways were upregulated when PDXs became resistant to cisplatin treatment. Additional changes in gene expression based on RNA-seq data before and after drug treatment were also found. Conclusion: Chemoresistance to gemcitabine and cisplatin is associated with altered expression of several cell surface proteins and upregulation of the downstream signaling pathways. Targeting these cell surface proteins can possibly be harnessed to overcome chemoresistance. GPNMB is a type I transmembrane protein that has previously been shown to be up-regulated in many metastatic cancers including breast cancer. Potentially targeting GPNMB with glembatumumab vedotin, an antibody-drug-conjugate has shown promising results in treating several cancers including breast cancer and osteosarcoma. Further studies will elucidate whether targeting GPNMB is an effective strategy for the treatment of chemotherapy resistant bladder cancer. This study received funding by a developmental grant from the UCDCC. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM number: LLNL-688318 Citation Format: Kelly A. Martin, Nicholas R. Hum, Aimy Sebastian, Deepa K. Murugesh, Chong-Xian Pan, Ai-Hong Ma, Ralph de Vere White, Gaby Loots. RNA sequencing of bladder cancer patient-derived xenograft models identifies genes associated with chemoresistance [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 97. doi:10.1158/1538-7445.AM2017-97

PD03-09 RISING INCIDENCE OF METASTATIC PROSTATE CANCER IN CALIFORNIA, 1988-2014

You have accessJournal of UrologyProstate Cancer: Epidemiology & Natural History I1 Apr 2017PD03-09 RISING INCIDENCE OF METASTATIC PROSTATE CANCER IN CALIFORNIA, 1988-2014 Marc Dall'Era, Ralph Devere White, Danielle Rodriguez, and Rosemary Cress Marc Dall'EraMarc Dall'Era More articles by this author , Ralph Devere WhiteRalph Devere White More articles by this author , Danielle RodriguezDanielle Rodriguez More articles by this author , and Rosemary CressRosemary Cress More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.215AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Early detection of prostate cancer with PSA based screening can reduce the risk of prostate cancer mortality by 21%. Screening for prostate cancer has dramatically declined in the United States since the United States Preventive Services Task Force (USPSTF) recommended against routine PSA based prostate cancer screening for all men in 2012. This led to dramatic reductions in the diagnosis of localized disease across all clinical risk groups. We sought to study trends in newly diagnosed metastatic prostate cancer incidence, specifically the impact of patient age and race. METHODS We analyzed new prostate cancer incidence by stage at diagnosis between 1988-2014 using data from the California Cancer Registry. We further stratified cases by age and four major race/ethnicity groups (non-Hispanic white (NHW), non-Hispanic black (NHB), Hispanic and non-Hispanic Asian/PI (API)). Incidence rates per 100,000 were age-adjusted to the 2000 US Standard Population. Joinpoint regression was used to detect changes in incidence and to calculate the average percent change (APC) over time. Joinpoint finds the best fit model with the smallest number of joinpoints and will not add an additional joinpoint if it does not make significant improvement on the model. RESULTS Adjusted rates of remote prostate cancer incidence for men of all races aged 65-74 and NHW men of all ages significantly increased over the most recent time period by 2.4% and 1%, respectively, p<0.05 (Figure 1 and 2). In contrast, incidence of remote prostate cancer continued to decline for NHB (-2.42%), Hispanic (-1.94%), and API (-1.66%) men. Localized disease incidence continues to decline significantly for all age and racial groups. CONCLUSIONS Incidence rates of newly metastatic prostate cancer have significantly increased for the first time in California for men aged 65-74 and white men. Although not possible to determine the etiology of these findings, decreased PSA screening and early detection of aggressive as well as indolent tumors is likely to contribute. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e60 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Marc Dall'Era More articles by this author Ralph Devere White More articles by this author Danielle Rodriguez More articles by this author Rosemary Cress More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

PD03-02 FREQUENCY OF DNA REPAIR GENE MUTATIONS IN LOCALIZED AND METASTATIC PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Epidemiology & Natural History I1 Apr 2017PD03-02 FREQUENCY OF DNA REPAIR GENE MUTATIONS IN LOCALIZED AND METASTATIC PROSTATE CANCER Allison Glass, Primo Lara, Ryan Hartmaier, Ralph deVere White, John McPherson, and Marc Dall'Era Allison GlassAllison Glass More articles by this author , Primo LaraPrimo Lara More articles by this author , Ryan HartmaierRyan Hartmaier More articles by this author , Ralph deVere WhiteRalph deVere White More articles by this author , John McPhersonJohn McPherson More articles by this author , and Marc Dall'EraMarc Dall'Era More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.208AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES DNA repair gene mutations are important molecular alterations in prostate cancer pathogenesis. Germline mutations in DNA repair genes, particularly BRCA2, were recently recognized as associated with metastatic prostate cancer. Prostate tumors with DNA repair defects may also be particularly sensitive to platinum based chemotherapy and PARP inhibitor therapy. We sought to characterize alterations in DNA repair pathway genes in both primary and metastatic prostate tumors. METHODS We studied the distribution of DNA repair gene mutations in 936 prostate cancers harvested from localized and metastatic tumors. Tumor DNA underwent hybrid capture for all coding exons of 395 cancer-related genes plus select introns from 19 or 31 genes frequently rearranged in cancer. Captured libraries were sequenced to a median exon coverage depth of >500x using Illumina sequencing and were analyzed for base substitutions/insertions, copy number alterations and rearrangements. We utilized two described lists of genes involved in DNA repair : our own in-house list of 74 (UCD) and a list of 20 DNA repair genes associated with cancer predisposition syndromes utilized in a recent publication by Pritchard et al. Nine genes were in common between the two lists yielding a total of 85 unique DNA repair genes. We further stratified the frequency of mutations by tissue site (prostate versus metastases). Only tissues represented by at least 10 samples in the set were included (868). Frequencies of DNA repair defects were compared across metastatic sites by Pearson's Chi-squared test. RESULTS We identified 228/936 unique samples with at least one likely functional mutation in a DNA repair gene (24.4%). Mutations were identified in 20.1% of prostate tumors (13% UCD, 18.4% Pritchard et al.) and in 18.8% of bone metastases. The highest rates of DNA repair mutations were found in visceral metastases including brain, pelvis and liver, which were significantly higher than either prostate tissue or bone sites (p=<0.01). The most commonly (=1% of samples) mutated genes in the DNA repair pathways are: BRCA2 (11.43%), ATM (5.77%), MSH6 (2.46%), MSH2 (2.14%), ATR (1.60%), MLH1 (1.28%), and BRCA1 (1.18%). CONCLUSIONS DNA repair gene mutations are more common in metastatic than localized prostate tumors. Visceral metastases appear enriched for these mutations compared with localized tumors or bone and lymph node metastases. Genomic profiling may identify prostate cancers potentially sensitive to platinum-based chemotherapy or PARP inhibition. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e56-e57 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Allison Glass More articles by this author Primo Lara More articles by this author Ryan Hartmaier More articles by this author Ralph deVere White More articles by this author John McPherson More articles by this author Marc Dall'Era More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract B22: Increased phosphorylation of eIF4E induces resistance to treatment with mTOR inhibitors together with AR antagonists in advanced prostate cancer

Abstract The anti-androgen bicalutamide is widely used in the treatment of advanced prostate cancer (CaP) in many countries, but its effect on castration resistant CaP (CRPC) is limited. We previously showed that resistance to bicalutamide results from increased activation of the mechanistic target of rapamycin (mTOR); indicating a role for mTOR inhibitors in CaP treatment. Interestingly, a clinical trial testing combinations of the mTOR inhibitor RAD001 with bicalutamide were initially effective (PSA decline&amp;gt;50%) in 62.5% CRPC patients, but many initial responders later developed resistance. Here we investigate causes for their difference in response. Evaluation of various CRPC cell lines identified resistant vs sensitive in vitro models, and revealed that increased eIF4E phosphorylation at S209 is associated with resistance to the combination. To recognize potential causes of resistance, we demonstrated using a human tumor xenograft mouse model, that bicalutamide use is associated with an increase in eIF4E(S209) phosphorylation. Investigations revealed that AR suppressed eIF4E phosphorylation, while the use of anti-androgens relieved this suppression, thereby triggering its increase. Additional investigation on the consequences of increased eIF4E phosphorylation in human prostatectomy samples revealed that increased eIF4E phosphorylation strongly correlated with the cell proliferation marker Ki67, indicating a role for eIF4E in tumor growth. Inhibition of eIF4E phosphorylation by siRNA suppression or treatment with CGP57380 (an inhibitor of MAPK interacting serine-threonine kinases Mnk1/2, the eIF4E upstream kinase,) sensitized CRPC cells to RAD001+bicalutamide, while eIF4E overexpression induced resistance. Our results demonstrate that bicalutamide treatment induced an increase in eIF4E phosphorylation by inhibition of AR which otherwise suppressed this phosphorylation. Increased eIF4E phosphorylation then induced resistance to the combination of anti-androgens with mTOR inhibitors by increasing proliferation. These results also indicate that resistance to RAD001+bicalutamide can be prevented by the concurrent use of a Mnk1/2 inhibitor. Citation Format: Leandro S. D'Abronzo, Michael Crapuchettes, Ryan Beggs, Ruth Vinall, Clifford Tepper, Salma Siddiqui, Maria Mudryj, Frank Melgoza, Blythe Durbin-Johnson, Ralph deVere White, Paramita Ghosh. Increased phosphorylation of eIF4E induces resistance to treatment with mTOR inhibitors together with AR antagonists in advanced prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B22.

Abstract IA29: Mice host selection for patient-derived xenograft (PDX) model development and other critical factors for success

Abstract IA29: Mice host selection for patient-derived xenograft (PDX) model development and other critical factors for success

Abstract 2106: Transcriptome analysis of patient-derived bladder cancer xenografts identifies genes associated with chemoresistance

Abstract Background: Bladder cancer is among the ten most common cancers, with about ∼380,000 new cases and ∼150,000 deaths per year worldwide. Platinum-based combination chemotherapy is commonly used to treat advanced bladder cancer. It has been shown that only ∼50% of the patients with advanced bladder cancer respond to platinum-based therapy. Methods: We employed a patient-derived bladder cancer xenograft (PDX) platform to characterize the molecular mechanisms contributing to resistance to gemcitabine-cisplatin combination therapy in advanced bladder cancer and to identify novel candidates that can be targeted to treat chemotherapy resistant bladder cancer. Transcriptome profiling of P0 (passage 0) bladder cancer xenograft tumors from 4 PDX lines (2 gemcitabine-cisplatin resistant lines and 2 drug sensitive lines) was performed by RNA-Seq analysis. Results: PDXs retained the morphology fidelity and shared 92-97% of genetic alterations of parental cancer cells. The RNA-seq data suggested the presence of significant differences between the transcription profiles of drug-sensitive and drug-resistant tumors. We identified 333 genes &amp;gt;2 fold up or down regulated in the drug resistant tumors compared to the drug sensitive tumors. Genes down-regulated in drug resistant tumors include tight junction protein CLDN3 and regulators of G-protein signaling RGS2 and RGS3. Significantly up-regulated genes include metabolic enzymes ALDH2, ALDH3A1, ALDH4A1 and ALDH7A1, transporter proteins ABCA1, SLC1A4, SLC2A5, SLC30A1, SLC39A6, SLC7A5 and SLC9A3, Notch ligand JAG2, Growth hormone receptor GHR and transmembrane glycoprotein GPNMB. Consistent with the change of cell surface proteins such as GHR and GPNMB, the MAPK and the PI3K-AKT pathways were upregulated when PDXs became resistant to cisplatin treatment. Conclusion: Chemoresistance to gemcitabine and cisplatin is associated with altered expression of several cell surface proteins and upregulation of the downstream signaling pathways. Targeting these cell surface proteins can possibly be harnessed to overcome chemoresistance. GPNMB, a type I transmembrane protein highly up-regulated in the drug resistant tumors, has previously been shown to be over-expressed in various cancers. Targeting GPNMB with an antibody-drug-conjugate, glembatumumab vedotin, has shown promising results in treating several cancers including breast cancer and osteosarcoma. Further studies will elucidate whether targeting GPNMB is an effective strategy for the treatment of chemotherapy resistant bladder cancer. Citation Format: Aimy Sebastian, Kelly A. Martin, Chong-xian Pan, Ai-hong Ma, Ralph W. deVere White, Gabriela G. Loots. Transcriptome analysis of patient-derived bladder cancer xenografts identifies genes associated with chemoresistance. [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 2106.

Abstract 4384: Targeting miR-124 inhibits growth of prostate cancer xenografts and sensitized prostate cancer cells to anti-androgen

Abstract Increasing evidence has shown that miR-124 is a tumor suppressor and plays an important role in pathogenesis of many types of human cancer. We previously found that miR-124 directly targets the androgen receptor (AR) and is significantly downregulated in clinical prostate cancer (CaP) samples. Moreover, overexpression of miR-124 induced apoptosis and inhibited cell proliferation and xenograft growth of CaP. In this study, we determined whether restoration of miR-124 may represent an adjunctively therapeutic option for CaP treatment. For this purpose, WST-1 and clonogenic assays were performed to evaluate the effects of miR-124 or miR-124 plus enzalutamide (one new androgen antagonist) on CaP cell proliferation. In animal experiment, male nude mice were inoculated s.c with CWR22 CaP cell suspensions. When tumor sizes reached ∼50 mm3, mice were treated with polyethylenimines (PEI)-complexed miR-124 (i.v., 10µg/day, thrice a week for 4 weeks) or PEI-miR-124 (i.v.) plus enzalutamide (p.o., 10mg/kg/day for 5 weeks). PEI can increase internalization and facilitate miRNA release into cytoplasm. After treatment, tumors were harvested for analyzing apoptosis and measuring miR-124 target levels. In in vitro experiment, 22Rv1 cells were separately treated with miR-124, enzalutamide or both. It was found that the combined treatment significantly increased growth inhibition of 22Rv1 cells. In addition, we established enzalutamide-resistant 22Rv1 subline (22Rv1-MDVR) by clone-selection in gradually increased doses (from 5μM to 40μM within four months). Treatment with miR-124 obviously inhibited clone formation of 22Rv1-MDVR cells. In mice receiving miR-124 or enzalutamide treatment, we observed tumor-inhibitory effects. However, the combination of miR-124 and enzalutamide exhibited more significant inhibition of tumor growth when compared to the single treatment. We examined the expression of AR and its variant 7 (AR-V7) that are targets of miR-124. Reduced levels of AR and AR-V7 were found in miR-124-treated tumors, indicating that PEI facilitates entry of miR-124 into the tumor cells. We also observed increased apoptosis in miR-124-treated tumors. Data from in vitro and in vivo studies show that miR-124 inhibited growth of CaP cells and sensitized CaP cells to anti-androgens, suggesting that miR-124 can be used as an adjunctively therapeutic agent for improved CaP treatment. Citation Format: Xu-Bao Shi, Aihong Ma, Lingru Xue, Ralph W. deVere White. Targeting miR-124 inhibits growth of prostate cancer xenografts and sensitized prostate cancer cells to anti-androgen. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4384. doi:10.1158/1538-7445.AM2014-4384

Abstract 327: Targeting autophagy overcomes enzalutamide resistance in castrate-resistant prostate cancer cells and improves therapeutic response in a xenograft model

Abstract Androgen deprivation or treatment with the androgen receptor signaling inhibitor (ARSI), Enzalutamide (MDV-3100, ENZA) or biclutamide induces autophagy in androgen-dependent and castration resistant CaP (CRPC) cell lines. This autophagic process is mediated by AMPK activation and the suppression of mTOR through Raptor phosphorylation (Serine 792). Using autophagy modulators such as clomipramine (CMI) or metformin in combination with ENZA drastically reduces the cell survival in the colony formation assay. Metformin in combination with ENZA causes AR degradation, which is inhibited by proteasome inhibitor MG132. Transcriptome deep sequencing of parental and ENZA resistant C4-2B cells was carried out to examine differential gene expression pattern that may be related to their ability to survive under constant high exposure to ARSI. In-vivo studies with mice orthotopically implanted with ENZA resistant cells demonstrates that the combination of ENZA and autophagy modulators, CMI or metformin significantly reduces tumor growth when compared to control groups (p&amp;lt;0.005). Blocking autophagy pharmacologically or genetically significantly impairs prostate cancer cell survival in- vitro and in-vivo, implying the therapeutics potential of autophagy inhibitors in the antiandrogen resistance setting.This work is supported in part by Stand Up To Cancer - Prostate Cancer Foundation–Prostate Dream Team Translational Cancer Research Grant. This research grant is made possible by the generous support of the Movember Foundation. Stand Up To Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research. Citation Format: Joy C. Yang, Hao G. Nguyen, Hsing-Jien Kung, Xu-Bao Shi, Derya Tilki, Ralph W. deVere White, Allen C. Gao, Christopher P. Evans. Targeting autophagy overcomes enzalutamide resistance in castrate-resistant prostate cancer cells and improves therapeutic response in a xenograft model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 327. doi:10.1158/1538-7445.AM2014-327

Abstract 2821: Dual-label microdosing approach to identify chemoresistance to carboplatin and gemcitabine combination chemotherapy

Abstract Gemcitabine is commonly given in combination with cisplatin or carboplatin (GC therapy) as the first-line chemotherapy regimen for several malignancies, including bladder cancer. Unfortunately, less than half of patients respond to the therapy, generating a critical need for a test that predicts tumor response to GC therapy. The goal of this project is to measure drug-DNA adducts after administration of a single non-toxic microdose of gemcitabine in combination with carboplatin and correlating the degree of modification to chemotherapy sensitivity. We have previously shown that resistance to carboplatin may be identified by using a [14C]carboplatin microdose (1% of therapeutic dose), and subsequent measurement of the [14C]-DNA adducts via accelerator mass spectrometry (AMS), an ultrasensitive method used for detecting rare isotopes. We postulate that adduct levels induced in tumors by GC microdoses are proportional to those formed during full dose chemotherapy. Furthermore, we hypothesize that there exists a threshold level of adducts above which tumors respond to GC therapy. We used bladder cancer cell lines and the nod scid gamma severe combined immunodeficient (NSG) mice containing patient-derived tumor xenografts (PDX) from bladder cancer patients to determine GC-DNA adduct levels using AMS. The NSG mouse is uniquely capable of accepting and propagating tumor tissue engrafted directly from patient biopsy samples. The resulting groups of mice each have identical tumors, but can be treated in a variety of ways with replicate experiments; characteristics that are impossible to achieve in normal clinical studies. We will report progress on developing protocols and preliminary GC microdosing data using the NSG-PDX mouse model of human bladder cancer. Citation Format: Maike Zimmermann, Tiffany M. Scharadin, Hongyong Zhang, Tzu-yin Lin, Ralph W. deVere White, Chong-xian Pan, Paul T. Henderson. Dual-label microdosing approach to identify chemoresistance to carboplatin and gemcitabine combination chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2821. doi:10.1158/1538-7445.AM2014-2821

Abstract LB-32: Patient-derived xenograft (PDX) paraclinical platform to guide precision medicine in urothelial cancer

Abstract BACKGROUND: The overall efficiency in personalizing medicine by pairing targeted therapy with specific genomic alterations identified through omics technologies is disappointing as current computational biology methods have difficulty in distinguishing “driver” alterations from “passenger” ones. We have developed PDXs that share essentially the same genetic background as the parental human cancer, and use this platform to screen for most effective drugs and perform mechanistic study of drug resistance in order to guide design of precision medicine in urothelial cancer. MATERIALS AND METHODS: PDXs were developed by direct implantation of fresh clinical tumor specimens in immunocompromised NSG mice (The Jackson Laboratory, West Sacramento, CA). Whole exome, transcriptome and microRNA sequencing was performed, followed by computational biological analysis to identify druggable genetic alterations that were further validated with immunofluorescence (IF) and immunohistochemical (IHC) staining. Efficacy tests to determine response to treatment by targeted therapy, together with analyses to determine the mechanisms of resistance were performed in NSG mice carrying PDXs. RESULTS: The engraftment rate of PDX was 41 % (13/42). The fidelity between the PDXs and the parental patient cancers was confirmed with cell morphology, tissue microarray IHC/IF staining and mutation profiles. Cell morphology was maintained during passaging in both the subcutaneous and orthotopic PDXs. All PDXs had multiple druggable genetic alterations. Of the first 8 PDXs, 2 harbored PIK3CA mutations (H1047R at the kinase domain and D549Y at the helical domain); 5 had ERBB2 mRNA overexpression determined by transcriptome sequencing, and protein overexpression (3+ by IHC), and 5 had EphB4 overexpression. sEphB4-HSA is an experimental drug developed by Gill (co-author). In PDXs overexpressing EphB4, blockade of EphB4 with sEphB4-HSA significantly prolonged the progression-free survival (PFS) from 9.5 days in the control group to 16.7 days (p=3.17 X 10e-5). Western blot, IHC, and deep sequencing analyses in PDX tissue specimens collected before/during treatment, and after the development of resistance were used to determine the mechanism of resistance. CONCLUSION: PDXs can potentially serve as a para-clinical model for drug efficacy screening, study of resistance mechanisms, and drug development. Citation Format: Chong-xian Pan, Hongyong Zhang, Clifford Tepper, Paramita Ghosh, Sheri Kuslak-Meyer, Susie Airhart, Parkash Gill, David Gandara, Edison Liu, Ralph de Vere White. Patient-derived xenograft (PDX) paraclinical platform to guide precision medicine in urothelial cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-32. doi:10.1158/1538-7445.AM2014-LB-32

Abstract 905: Molecular dissection of platinum resistance through functional analysis

Abstract BACKGROUND: Platinum (Pt) agents (cisplatin, carboplatin and oxaliplatin) are active in many cancers including bladder cancer. Chemoresistance is the most common cause of treatment failure. This study is to determine the feasibility of using ultrasensitive accelerator mass spectrometry (AMS) to identify chemoresistance after cancer cells or patients are treated with one non-toxic microdose (1/100th of therapeutic dose) of Pt agents. The long-term goal is to identify chemoresistance before cancer patients receive toxic chemotherapy, and to determine the underlying resistance mechanisms to design personalized chemotherapy. METHODS: Cellular sensitivity to chemotherapeutic agents was determined by the MTT assay. Platinum-induced DNA adduct formation and repair of adducts was measured with AMS after cells were exposed 14C-labeled carboplatin and oxaliplatin. AMS quantifies the 14C label that is attached to genomic DNA when the 14C-labled drug forms adducts with DNA. Cell uptake and efflux was measured by liquid scintillation counting. Intracellular glutathione levels were measured by colorimetric analysis. RESULTS: Compared to the parental bladder cancer 5637 cells, chemoresistant 5637R cells are resistant to oxaliplatin (IC50: 2.45 µM versus 27.27 µM, p&amp;lt;0.0001), and cisplatin (0.59 µM versus 2.99 µM, p=0.049), carboplatin (24.34 µM versus 72.18 µM, p&amp;lt;0.0001), and gemcitabine (0.12 µM versus 1.44 µM, p=0.0015). Both 5637 and 5637R cells are still sensitive to other chemotherapeutic agents commonly used in treating bladder cancer, such as doxorubicin, methotrexate and vinblastine. Consistent with our hypothesis, chemoresistant 5637R cells have low oxaliplatin-induced DNA adduct levels than the parental 5637 cells (AUC of 943 versus 2,772 adducts per 109 nucleotide-hour for 5637, p=0.001). This low level of oxaliplatin-DNA adduct formation might be secondary to the pre-DNA damage mechanisms, such as decreased uptake (AUC of 4.42 versus 5.12 X 109 oxaliplatin molecules per cell for 5637, p=0.037) and increased intracellular inactivation of oxaliplatin by glutathione (53.91 versus 46.93 nmol/mg protein for 5637, p=0.003), plus increased repair of oxaliplatin-DNA adducts (3.48 versus 1.34 adducts per 108 nucleotides per hour for 5637, p=0.0004). We found the same correlation of low Pt-DNA adduct levels and chemoresistance in non-small cell lung (NSCLC) and breast cancer cell lines, and determined the same resistant mechanisms, such as cell uptake/efflux, intracellular inactivation and DNA repair. Carboplatin had partially different resistant mechanisms. CONCLUSION: Functional analysis of major resistant steps can identify some chemoresistance mechanisms that can potentially help design personalized chemotherapy to overcome resistance. This approach can be applied to several different cancer types. A Phase 0 microdosing clinical trial is currently going on in patients with NSCLC and bladder cancer. Citation Format: Amy W. Pan, Sisi Wang, Hongyong Zhang, Ruth Vinall, Tzu-yin Lin, Michael Malfatti, Maike Zimmermann, Tiffany Scharadin, Kenneth Turteltaub, Ralph de Vere White, Chong-xian Pan, Paul Henderson. Molecular dissection of platinum resistance through functional analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 905. doi:10.1158/1538-7445.AM2014-905

Abstract 5115: The Bcl-2 Homology Domain-3 Mimetic, Obatoclax, chemosensitizes muscle invasive bladder cancer cell lines to cisplatin treatment

Abstract The primary objective of this study was to determine whether inhibition of anti-apoptotic Bcl-2 family protein activity causes decreased clonogenicity and/or increased apoptosis of muscle invasive bladder cancer (MI-BC) cell lines when used in combination with cisplatin, the standard of care treatment for MI-BC, and/or pre-miR-34a, a miRNA that targets several components of pathways know to promote tumor progression and chemoresistance. Obatoclax, a Bcl-2 homology domain-3 mimetic that has been demonstrated to prevent Bcl-2, Bcl-xL, and Mcl-1 from interacting with and inhibiting Bax and Bak, was used for this study. The impact of Obatoclax, in combination with cisplatin and/or pre-miR-34a, on clonogenic potential and apoptosis was assessed in three MI-BC cell lines (T24, TCCSuP, 5637) using clonogenic assay and annexin V flow cytometry respectively. The ability of Obatoclax to prevent interaction between Bcl-2, Bcl-xL, and Mcl-1, with Bax and Bak was assessed via immunoprecipitation and western blot. The impact of treatments on apoptosis/survival pathways was assessed via western blot. Obatoclax mediated a dose-dependent increase in apoptosis in all three MI-BC cell lines. Obatoclax was able to decrease binding of Bak to Bcl-xL, and cause decreased expression of Bcl-2 and Bcl-xL. Unexpectedly, Obatoclax also mediated a dose-dependent decrease in cdk4, cdk6, and cyclin D1. Combining Obatoclax with cisplatin or/and pre-miR-34a caused further inhibition of clonogenic potential of all three cell lines compared to treatment with single agents. Of the combination treatments, Obatoclax combined with cisplatin mediated the biggest increase in apoptosis levels. Treatment with Obatoclax, cisplatin, and pre-miR-34a actually decreased apoptosis levels. Western blot revealed that treatment with Obatoclax was unable to overcome the effect of pre-miR-34a on causing increased Bcl-2 and Bcl-xL expression. Our data demonstrate that Obatoclax chemosensitizes MI-BC cell lines to cisplatin and indicate that in vivo testing of this combination treatment is warranted. The data do not support further testing of pre-miR-34a in this setting. With the current 5-year survival rate for MI-BC being only 35%, clearly new and improved treatment options are needed for this disease. Citation Format: Thomas Steele, Dan Pham, Kathy Phan, Paramita Ghosh, Ralph deVere White, Ruth L. Vinall. The Bcl-2 Homology Domain-3 Mimetic, Obatoclax, chemosensitizes muscle invasive bladder cancer cell lines to cisplatin treatment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5115. doi:10.1158/1538-7445.AM2014-5115

Abstract 4270: ErbB inhibitors in bladder cancer cell lines: Effectiveness in cells that express high levels but low activity of the ErbB receptors.

Abstract Background: Various studies demonstrated increased expression of EGF-receptor (EGFR/HER1/ErbB1) and related receptors ErbB2/HER2/Neu, ErbB3/HER3 and ErbB4/HER4 in bladder cancer. Despite this, clinical trials using drugs inhibiting EGFR (erlotinib), ErbB2 (trastuzumab) or dual EGFR/ErbB2 inhibitors (lapatinib) were all deemed ineffective. Since ErbB receptors require dimerization with other members of this family, each of which regulate cell growth and survival, we hypothesized that all four receptors must be simultaneously suppressed to achieve lasting effects. Here we compare the effects of erlotinib, trastuzumab and lapatinib to a novel pan-ErbB inhibitor dacomitinib in four bladder cancer cell lines expressing high and low levels of these receptors. Methods: The bladder cancer cell lines T24, TCCSUP, J82 and HTB-2FT4 were used to evaluate the efficacy of erlotinib (Tarceva), trastuzumab (Herceptin), lapatinib (Tykerb, GW572016) and dacomitinib (PF00299804). Cell growth was estimated by MTT or crystal-violet staining. Protein expression was determined by western blotting and apoptosis was determined by flow cytometry after seventy-two hour incubation with the indicated treatment. Results: Comparison of the T24, TCCSUP, J82 and HTB-2FT4 bladder cancer cells with C4-2 (prostate) and MCF-7 (breast) cells showed that all but the HTB-2FT4 bladder cancer cells expressed high levels of EGFR and ErbB3 but not ErbB2, while HTB-2FT4 cells expressed only EGFR and TCCSUP cells had higher ErbB levels than T24. Interestingly, ErbB2 and ErbB3 are readily activated by EGF in T24 cells compared to TCCSUP. MTT demonstrated the efficacy of dacomitinib and erlotinib, primarily EGFR inhibitors, compared to lapatinib, primarily an ErbB2 inhibitor with anti-EGFR activity at higher concentrations, while the ErbB2 inhibitor, trastuzumab, had little effect. The trastuzumab and erlotinib combination performed worse than erlotinib alone in T24 cells; hence trastuzumab studies were not pursued further. Crystal-violet staining after treatment with erlotinib, lapatinib and dacomitinib indicated higher efficacy of dacomitinib after seven days in the T24 and TCCSUP cell lines. MTT and flow cytometry showed that lapatinib was ineffective alone but, in combination with dacomitinib, significantly decreased cell viability in TCCSUP cells. In contrast, combining erlotinib, lapatinib and dacomitinib proved most effective in T24 cells. Conclusions: We demonstrate that all ErbB receptors must be simultaneously inhibited to prevent proliferation of both high and low ErbB expressing bladder lines. Inhibition of ErbB2 alone had a deleterious effect, likely due to ErbB2 inhibition stimulating the activation of the other ErbB receptors. While high levels of ErbB kinases sensitized cells to the inhibitors, the drugs were more effective in cells that had lower activation of the receptors. Citation Format: Benjamin A. Mooso, Ralph W. deVere White, Paramita M. Ghosh. ErbB inhibitors in bladder cancer cell lines: Effectiveness in cells that express high levels but low activity of the ErbB receptors. [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 4270. doi:10.1158/1538-7445.AM2013-4270

Abstract 4190: miR-30 as a tumor suppressor connects EGF/Src signal to EMT and ERG.

Abstract Src tyrosine kinase (Src) is implicated in the development of bone metastasis and castration-resistance of prostate cancer. Src inhibitors are currently being tested in clinical trials for such diseases. Understanding the molecular and cellular actions of Src inhibitors holds the key to future improvement of this line of therapy. Here we describe the microRNA expression profiles modulated by two Src inhibitors and demonstrate that the miR-30 family members are the most prominently induced species. Consistent with its tumor suppressor role, miR-30 is downmodulated by oncogenic signals such as epidermal growth factor (EGF) and hepatocyte growth factor (HGF), and is generally underexpressed in prostate cancer specimens. A number of epithelial to mesenchymal transition (EMT)-associated genes are predicted targets of miR-30. Among these genes the Ets Related Gene (ERG) is the most frequently overexpressed-oncogene in prostate cancer activated by genomic fusion events between promoter upstream sequences of the TMPRSS2 and coding sequences of ERG. We showed by ERG 3’UTR-reporter and mutagenesis assays that ERG is a direct target of miR-30. Overexpression of miR-30 in prostate cancer cells suppresses EMT phenotypes and inhibits cell migration and invasion. It also inhibits the in vitro and in vivo growth of VCaP cells, which depends on TMPRSS2-ERG for proliferation. TMPRSS2-ERG is generally regulated by androgen at the transcriptional level. Our finding reveals a new post-transcriptional mechanism of TMPRSS2-ERG regulation by Src and growth signals via miR-30 providing a rationale for targeting ERG positive castration resistant tumors with Src inhibitors. Citation Format: Chiao-Jung Kao, Anthony Martiniez, Xu-Bao Shi, Albert Dobi, Ralph W. deVere White, Hsing-Jien Kung. miR-30 as a tumor suppressor connects EGF/Src signal to EMT and ERG. [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 4190. doi:10.1158/1538-7445.AM2013-4190

Abstract 3056: miR-125b suppresses p14ARF and modulates p53-dependent and p53-independent apoptosis in prostate cancer cells.

Abstract Increasing evidence has revealed that miR-125b functions as a potential oncogene in different types of human cancer. Our previous studies demonstrated that miR-125b directly targets p53, Puma and Bak1 in human prostate cancer (CaP). To further elucidate the role of miR-125b in modulating the p53 network, we utilized a bioinformatics approach to identify miR-125b-regulated, CaP-relevant tumor suppressor gene, and identified p14ARF gene as a miR-125b target. We first evaluated the effect of miR-125b on the regulation of p14ARF expression, and found that miR-125b treatment induced reduction of p14ARF by 80% in LNCaP CaP cells and 60% in 22Rv1 CaP cells. Conversely, anti-miR-125b increased the p14ARF level by 40% in LNCaP and 30% in 22Rv1. We then performed luciferase assay to validate that the miR-125b binding site in the 3’UTR of p14ARF mRNA is responsible for the regulation of p14ARF by miR-125b. Since p14ARF accelerates Mdm2 degradation, resulting in p53 up-regulation, we thus assessed the effect of miR-125b on the expression of Mdm2 and p53. Compared with miRNA control, miR-125b treatment induced a dramatic increase in Mdm2 and a significant reduction of p53 in CaP cell lines, as well as in miR-125b-overexpressed xenograft tumor. Additionally, miR-125b treatment resulted in a reduction of interaction between p14ARF and Mdm2. Next, we examined the effect of miR-125b on CaP cell proliferation. Both LNCaP and 22Rv1 cells were transfected with miR-125b and cell proliferation was determined by clone formation assay. When compared with the control, miR-125b treatment increased clonogenic survival of CaP cells, while anti-miR-125b caused a dramatic reduction in the number of colonies. We evaluated the effect of downregulation of miR-125b on apoptosis in p53-positive 22Rv1 CaP cells. Data from TUNEL assay demonstrated that anti-miR-125 caused 63% of cells to undergo apoptosis. Similarly, in p53-negative PC3 CaP cells, miR-125b treatment induced a 2-fold increase in survival, while anti-miR-125b caused 50% of cells to undergo apoptosis. In summary, we observed that overexpression of miR-125b negatively regulates the expression of p14ARF, resulting in promotion of cell proliferation and inhibition of apoptosis. Inactivation of miR-125b using anti-miR-125b affects apoptosis involving both p53-dependent and p53-independent pathways. Therefore, our data suggest that miR-125b has a great potential to be used as a therapeutic target for CaP treatment. Citation Format: Xu-Bao Shi, Sumaira Amir, Ai-Hong Ma, Lingru Xue, Ralph W. deVere White. miR-125b suppresses p14ARF and modulates p53-dependent and p53-independent apoptosis in prostate cancer cells. [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 3056. doi:10.1158/1538-7445.AM2013-3056

Abstract 1183: Phase 0 microdosing trial quantitating DNA adduct formation to identify platinum chemosensitivity: from bench to bedside.

Abstract Purpose: DNA adduct formation is a critical step in platinum(Pt)–induced cell death. The goal of this project is to measure carboplatin-DNA adducts after a single non-toxic microdose of [14C]carboplatin, use this information to predict efficacy before cancer patients receive toxic chemotherapy and assess the underlying chemoresistance mechanisms, thus enhancing personalized therapy. Methods: Six non-small cell lung cancer (NSCLC) and six bladder cancer cell lines were treated with 1 μM (microdose) or 100 μM (therapeutic dose) carboplatin, but with equal amounts of [14C]carboplatin. Carboplatin-DNA monoadduct levels were determined by measuring 14C content in genomic DNA with accelerator mass spectrometry (AMS). Cellular sensitivity to carboplatin (IC50 values) was correlated with drug-DNA adduct levels. To validate the in vitro findings, a pilot Phase 0 trial with microdosing [14C]carboplatin was initiated to determine the recommended Phase II dose (RP2D) of [14C]carboplatin for optimal measurement of DNA monoadducts and pharmacokinetics (PK) in lung and bladder cancer. PK and DNA monoadduct levels in peripheral blood mononuclear cells and tumor tissues were determined by liquid scintillation counting and AMS, respectively, validated with inductively coupled plasma mass spectrometry (ICP-MS), and correlated with tumor response to chemotherapy. Results: DNA monoadduct levels in cell lines induced by microdose and therapeutic concentrations of carboplatin were highly linearly proportional (r2 = 0.95, p &amp;lt; 0.001) as measured by AMS, and correlated to the total Pt-DNA adduct levels induced by therapeutic carboplatin (r2 = 0.80, p &amp;lt; 0.001) as measured by ICP-MS. DNA adduct levels were superior in predicting resistance to carboplatin when compared to the mRNA expression of ERCC1, a DNA repair gene commonly used as a resistant biomarker of Pt-based chemotherapy. Nine patients were recruited at the pilot Phase 0 trial. The Level I dose of [14C]carboplatin at 107 dpm/Kg with total carboplatin at 1% of therapeutic dose (area under curve of 6) was determined as the RP2D. No clinical toxicity was observed as a result of microdosing. The radiation exposure from 14C was less than a chest X-ray per patient. DNA monoadduct levels and PK parameters of carboplatin at the microdose and therapeutic doses were highly correlated (p&amp;lt;0.001), suggesting that this non-toxic microdosing approach can be used to predict DNA adduct levels and PK of therapeutic doses. Patients with the highest DNA monoadduct levels responded best to carboplatin-based chemotherapy, although more data is needed to achieve statistical significance in the clinical data. Conclusions: This novel microdosing approach predicts DNA monoadduct levels and PK of therapeutic carboplatin dosing; preliminary data suggest the feasibility of predicting chemosensitivity and the possibility of guiding personalized dosing. Citation Format: Chong-xian Pan, Sisi Wang, Miaoling He, Tao Li, Tzu-yin Lin, Hongyong Zhang, Michael Malfatti, David Gandara, Tianhong Li, Ken Yoneda, Joyce Lee, Marc Dall'Era, George Cimino, Ralph de Vere White, Kenneth Turteltaub, Paul Henderson. Phase 0 microdosing trial quantitating DNA adduct formation to identify platinum chemosensitivity: from bench to bedside. [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 1183. doi:10.1158/1538-7445.AM2013-1183