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The mutation status and absolute IC50 of bladder cancer cells

Analysis of survival curves, tumor volumes and body weights

Purpose: Activation of the PI3K pathway occurs in over 40% of bladder urothelial cancers. The aim of this study is to determine the therapeutic potential, the underlying action, and the resistance mechanisms of drugs targeting the PI3K pathway.Experimental Design: Urothelial cancer cell lines and patient-derived xenografts (PDXs) were analyzed for alterations of the PI3K pathway and for their sensitivity to the small-molecule inhibitor pictilisib alone and in combination with cisplatin and/or gemcitabine. Potential predictive biomarkers for pictilisib were evaluated, and RNA sequencing was performed to explore drug resistance mechanisms.Results: The bladder cancer cell line TCCSUP, which harbors a PIK3CA E545K mutation, was sensitive to pictilisib compared to cell lines with wild-type PIK3CA. Pictilisib exhibited stronger antitumor activity in bladder cancer PDX models with PI3KCA H1047R mutation or amplification than the control PDX model. Pictilisib synergized with cisplatin and/or gemcitabine in vitro, significantly delayed tumor growth, and prolonged survival compared with single-drug treatment in the PDX models. The phosphorylation of ribosomal protein S6 correlated with response to pictilisib both in vitro and in vivo, and could potentially serve as a biomarker to predict response to pictilisib. Pictilisib activated the compensatory MEK/ERK pathway that likely contributed to pictilisib resistance, which was reversed by cotreatment with the RAF inhibitor sorafenib. RNA sequencing of tumors resistant to treatment suggested that LSP1 downregulation correlated with drug resistance.Conclusions: These preclinical results provide new insights into the therapeutic potential of targeting the PI3K pathway for the treatment of bladder cancer. Clin Cancer Res; 23(21); 6580–91. ©2017 AACR.

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Immunostaining of AR-V7 in miR-124-treated 22Rv1-EnzR cells.

miR-124 targets the androgen receptor (AR) transcript, acting as a tumor suppressor to broadly limit the growth of prostate cancer. In this study, we unraveled the mechanisms through which miR-124 acts in this setting. miR-124 inhibited proliferation of prostate cancer cells in vitro and sensitized them to inhibitors of androgen receptor signaling. Notably, miR-124 could restore the apoptotic response of cells resistant to enzalutamide, a drug approved for the treatment of castration-resistant prostate cancer. We used xenograft models to examine the effects of miR-124 in vivo when complexed with polyethylenimine-derived nanoparticles. Intravenous delivery of miR-124 was sufficient to inhibit tumor growth and to increase tumor cell apoptosis in combination with enzalutamide. Mechanistic investigations revealed that miR-124 directly downregulated AR splice variants AR-V4 and V7 along with EZH2 and Src, oncogenic targets that have been reported to contribute to prostate cancer progression and treatment resistance. Taken together, our results offer a preclinical rationale to evaluate miR-124 for cancer treatment. Cancer Res; 75(24); 5309–17. ©2015 AACR.

Legend for Supplementary Figures S1-S5.

Description of additional methods and procedures used in the study. Also includes Supplementary Reference.

Patients with advanced prostate cancer (PCa) are initially susceptible to androgen withdrawal (AW), but ultimately develop resistance to this therapy (castration-resistant PCa, CRPC). Here, we show that AW can promote CRPC development by increasing the levels of the receptor tyrosine kinase ErbB3 in androgen-dependent PCa, resulting in AW-resistant cell cycle progression and increased androgen receptor (AR) transcriptional activity. CRPC cell lines and human PCa tissue overexpressed ErbB3, whereas downregulation of ErbB3 prevented CRPC cell growth. Investigation of the mechanism by which AW augments ErbB3, using normal prostate-derived pRNS-1-1 cells, and androgen-dependent PCa lines LNCaP, PC346C, and CWR22 mouse xenografts, revealed that the AR suppresses ErbB3 protein levels, whereas AW relieves this suppression, showing for the first time the negative regulation of ErbB3 by AR. We show that AR activation promotes ErbB3 degradation in androgen-dependent cells, and that this effect is mediated by AR-dependent transcriptional upregulation of neuregulin receptor degradation protein-1 (Nrdp1), an E3 ubiquitin ligase that targets ErbB3 for degradation but whose role in PCa has not been previously examined. Therefore, AW decreases Nrdp1 expression, promoting ErbB3 protein accumulation, and leading to AR-independent proliferation. However, in CRPC sublines of LNCaP and CWR22, which strongly overexpress the AR, ErbB3 levels remain elevated due to constitutive suppression of Nrdp1, which prevents AR regulation of Nrdp1. Our observations point to a model of CRPC development in which progression of PCa to castration resistance is associated with the inability of AR to transcriptionally regulate Nrdp1, and predict that inhibition of ErbB3 during AW may impair CRPC development. Cancer Res; 70(14); 5994–6003. ©2010 AACR.

Supplementary Table 1, Figures 1-4, Methods from Nrdp1-Mediated Regulation of ErbB3 Expression by the Androgen Receptor in Androgen-Dependent but not Castrate-Resistant Prostate Cancer Cells

Development of candidate cancer treatments is a resource-intensive process, with the research community continuing to investigate options beyond static genomic characterization. Toward this goal, we have established the genomic landscapes of 536 patient-derived xenograft (PDX) models across 25 cancer types, together with mutation, copy number, fusion, transcriptomic profiles, and NCI-MATCH arms. Compared with human tumors, PDXs typically have higher purity and fit to investigate dynamic driver events and molecular properties via multiple time points from same case PDXs. Here, we report on dynamic genomic landscapes and pharmacogenomic associations, including associations between activating oncogenic events and drugs, correlations between whole-genome duplications and subclone events, and the potential PDX models for NCI-MATCH trials. Lastly, we provide a web portal having comprehensive pan-cancer PDX genomic profiles and source code to facilitate identification of more druggable events and further insights into PDXs’ recapitulation of human tumors., Patient-derived xenograft models (PDX) have been extensively used to study the molecular and clinical features of cancers. Here the authors present a cohort of 536 PDX models from 25 cancers, as well as their genomic and evolutionary profiles and their suitability for clinical trials.

Background Emerging evidence has suggested that DNA repair gene alterations may be important in prostate cancer pathogenesis. In the current study, the authors sought to characterize alterations in DNA repair pathway genes in both primary and metastatic prostate tumors with attention to tissue distribution as well as specific genomic alterations. Methods The authors studied the distribution and type of alterations in 24 genes that are considered important for DNA repair in 944 prostate cancers harvested from localized and metastatic tumors. Tumor DNA underwent hybrid capture for all coding exons of 287 or 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 >×500. Specific genomic alterations were characterized and the frequencies of mutations by tissue site (prostate vs metastases) were compared using logistic regression. Results A total of 152 patients from the cohort of 944 men (16%) harbored a germline or somatic mutation in ≥1 DNA repair genes. The most frequently mutated genes were BRCA2 (11.4%) and ATM (5.8%), followed by MSH6 (2.5%) and MSH2 (2.1%). Mutations were identified in approximately 20.1% of primary prostate tumors compared with 18.8% of bone metastases. When stratified by tissue site, the highest rates of DNA repair mutations were found in solid organ metastases, including brain and visceral metastases, compared with prostate. Conclusions DNA repair gene mutations are more common in metastatic than localized prostate tumors. Visceral and other solid organ metastases appear enriched for these mutations compared with localized tumors or bone and lymph node metastases.

Our group and others have previously shown that genistein combined polysaccharide (GCP), an aglycone isoflavone-rich extract with high bioavailability and low toxicity, can inhibit prostate cancer (CaP) cell growth and survival as well as androgen receptor (AR) activity. We now elucidate the mechanism by which this may occur using LNCaP and PC-346C CaP cell lines, GCP can inhibit intracrine androgen synthesis in CaP cells. UPLC-MS/MS and qPCR analyses demonstrated that GCP can mediate a ~3-fold decrease in testosterone levels (p &lt, 0.001) and cause decreased expression of intracrine androgen synthesis pathway enzymes (~2.5-fold decrease of 3&beta, HSD (p &lt, 0.001), 17&beta, 0.001), CYP17A (p &lt, 0.01), SRB1 (p &lt, 0.0001), and StAR (p &lt, 0.01)), respectively. Reverse-phase HPLC fractionation and bioassay identified three active GCP fractions. Subsequent NMR and LC-MS analysis of the fraction with the highest level of activity, fraction 40, identified genistein as the primary active component of GCP responsible for its anti-proliferative, pro-apoptotic, and anti-AR activity. GCP, fraction 40, and genistein all mediated at least a ~2-fold change in these biological activities relative to vehicle control (p &lt, 0.001). Genistein caused similar decreases in the expression of 17&beta, HSD and CYP17A (2.5-fold (p &lt, 0.001) and 1.5-fold decrease (p &lt, 0.01), respectively) compared to GCP, however it did not cause altered expression of the other intracrine androgen synthesis pathway enzymes, 3&beta, HSD, SRB1, and StAR. Our combined data indicate that GCP and/or genistein may have clinical utility and that further pre-clinical studies are warranted.

Noncoding RNAs (ncRNAs) produced in live cells may better reflect intracellular ncRNAs for research and therapy. Attempts were made to produce biologic ncRNAs, but at low yield or success rate. Here we first report a new ncRNA bioengineering technology using more stable ncRNA carrier (nCAR) containing a pre-miR-34a derivative identified by rational design and experimental validation. This approach offered a remarkable higher level expression (40%–80% of total RNAs) of recombinant ncRNAs in bacteria and gave an 80% success rate (33 of 42 ncRNAs). New FPLC and spin-column based methods were also developed for large- and small-scale purification of milligrams and micrograms of recombinant ncRNAs from half liter and milliliters of bacterial culture, respectively. We then used two bioengineered nCAR/miRNAs to demonstrate the selective release of target miRNAs into human cells, which were revealed to be Dicer dependent (miR-34a-5p) or independent (miR-124a-3p), and subsequent changes of miRNome and transcriptome profiles. miRNA enrichment analyses of altered transcriptome confirmed the specificity of nCAR/miRNAs in target gene regulation. Furthermore, nCAR assembled miR-34a-5p and miR-124-3p were active in suppressing human lung carcinoma cell proliferation through modulation of target gene expression (e.g., cMET and CDK6 for miR-34a-5p; STAT3 and ABCC4 for miR-124-3p). In addition, bioengineered miRNA molecules were effective in controlling metastatic lung xenograft progression, as demonstrated by live animal and ex vivo lung tissue bioluminescent imaging as well as histopathological examination. This novel ncRNA bioengineering platform can be easily adapted to produce various ncRNA molecules, and biologic ncRNAs hold the promise as new cancer therapeutics.

Cisplatin-based therapy is highly toxic, but moderately effective in most cancers. Concurrent inhibition of cyclooxygenase-2 (COX-2) and soluble epoxide hydrolase (sEH) results in antitumor activity and has organ-protective effects. The goal of this study was to determine the antitumor activity of PTUPB, an orally bioavailable COX-2/sEH dual inhibitor, in combination with cisplatin and gemcitabine (GC) therapy. NSG mice bearing bladder cancer patient-derived xenografts were treated with vehicle, PTUPB, cisplatin, GC, or combinations thereof. Mouse experiments were performed with two different PDX models. PTUPB potentiated cisplatin and GC therapy, resulting in significantly reduced tumor growth and prolonged survival. PTUPB plus cisplatin was no more toxic than cisplatin single-agent treatment as assessed by body weight, histochemical staining of major organs, blood counts, and chemistry. The combination of PTUPB and cisplatin increased apoptosis and decreased phosphorylation in the MAPK/ERK and PI3K/AKT/mTOR pathways compared with controls. PTUPB treatment did not alter platinum–DNA adduct levels, which is the most critical step in platinum-induced cell death. The in vitro study using the combination index method showed modest synergy between PTUPB and platinum agents only in 5637 cell line among several cell lines examined. However, PTUPB is very active in vivo by inhibiting angiogenesis. In conclusion, PTUPB potentiated the antitumor activity of cisplatin-based treatment without increasing toxicity in vivo and has potential for further development as a combination chemotherapy partner. Mol Cancer Ther; 17(2); 474–83. ©2017 AACR.

Precision cancer medicine seeks to target the underlying genetic alterations of cancer; however, it has been challenging to use genetic profiles of individual patients in identifying the most appropriate anti-cancer drugs. This spurred the development of patient avatars; for example, patient-derived xenografts (PDXs) established in mice and used for drug exposure studies. However, PDXs are associated with high cost, long development time and low efficiency of engraftment. Herein we explored the use of microfluidic devices or microchambers as simple and low-cost means of maintaining bladder cancer cells over extended periods of times in order to study patterns of drug responsiveness and resistance. When placed into 75 µm tall microfluidic chambers, cancer cells grew as ellipsoids reaching millimeter-scale dimeters over the course of 30 days in culture. We cultured three PDX and three clinical patient specimens with 100% success rate. The turn-around time for a typical efficacy study using microchambers was less than 10 days. Importantly, PDX-derived ellipsoids in microchambers retained patterns of drug responsiveness and resistance observed in PDX mice and also exhibited in vivo-like heterogeneity of tumor responses. Overall, this study establishes microfluidic cultures of difficult-to-maintain primary cancer cells as a useful tool for precision cancer medicine.

Metastasis is a major cause of mortality for cancer patients and remains as the greatest challenge in cancer therapy. Driven by multiple factors, metastasis may not be controlled by the inhibition of single target. This study was aimed at assessing the hypothesis that drugs could be rationally combined to co-target critical DNA, RNA and protein molecules to achieve "saturation attack" against metastasis. Independent actions of the model drugs DNA-intercalating doxorubicin, RNA-interfering miR-34a and protein-inhibiting sorafenib on DNA replication, RNA translation and protein kinase signaling in highly metastatic, human osteosarcoma 143B cells were demonstrated by the increase of γH2A.X foci formation, reduction of c-MET expression and inhibition of Erk1/2 phosphorylation, respectively, and optimal effects were found for triple-drug combination. Consequently, triple-drug treatment showed a strong synergism in suppressing 143B cell proliferation and the greatest effects in reducing cell invasion. Compared to single- and dual-drug treatment, triple-drug therapy suppressed pulmonary metastases and orthotopic osteosarcoma progression to significantly greater degrees in orthotopic osteosarcoma xenograft/spontaneous metastases mouse models, while none showed significant toxicity. In addition, triple-drug therapy improved the overall survival to the greatest extent in experimental metastases mouse models. These findings demonstrate co-targeting of DNA, RNA and protein molecules as a novel therapeutic strategy for the treatment of metastasis.

Introduction: We identify the impact of neoadjuvant chemotherapy before open radical cystectomy on perioperative outcomes and identify actionable areas for improvement.Methods: The impact of neoadjuvant chemotherapy on perioperative outcomes after radical cystectomy for muscle invasive bladder cancer from 2003 to 2014 was assessed using an institutional database. Individual outcomes (venous thromboembolism, surgical site infection, cardiac event) and a composite score using the Clavien-Dindo classification were identified. Univariable and multivariable logistic regression models were used to identify predictors of perioperative complication and 30-day readmission rates.Results: A total of 241 patients were included in the study, of whom 175 underwent radical cystectomy alone (72.6%) and 66 were treated with neoadjuvant chemotherapy plus radical cystectomy (27.4%). The 30-day readmission rate for the neoadjuvant chemotherapy cohort was 30.5% compared to 17.2% for radical cystectomy alone. Multivari...

BACKGROUND The mammalian target of rapamycin (mTOR) pathway is up-regulated in castration-resistant prostate cancer (CRPC). Nevertheless, inhibition of mTOR is ineffective in inducing apoptosis in prostate cancer cells, likely because of the compensatory up-regulation of the androgen receptor (AR) pathway. METHODS Patients who were eligible for this study had to have progressive CRPC with serum testosterone levels

miR-124 targets the androgen receptor (AR) transcript, acting as a tumor suppressor to broadly limit the growth of prostate cancer. In this study, we unraveled the mechanisms through which miR-124 acts in this setting. miR-124 inhibited proliferation of prostate cancer cells in vitro and sensitized them to inhibitors of androgen receptor signaling. Notably, miR-124 could restore the apoptotic response of cells resistant to enzalutamide, a drug approved for the treatment of castration-resistant prostate cancer. We used xenograft models to examine the effects of miR-124 in vivo when complexed with polyethylenimine-derived nanoparticles. Intravenous delivery of miR-124 was sufficient to inhibit tumor growth and to increase tumor cell apoptosis in combination with enzalutamide. Mechanistic investigations revealed that miR-124 directly downregulated AR splice variants AR-V4 and V7 along with EZH2 and Src, oncogenic targets that have been reported to contribute to prostate cancer progression and treatment resistance. Taken together, our results offer a preclinical rationale to evaluate miR-124 for cancer treatment. Cancer Res; 75(24); 5309–17. ©2015 AACR.

© 2018 American Urological Association Education and Research, Inc. Introduction: Radical cystectomy with neoadjuvant chemotherapy is the standard of care for patients with localized muscle invasive urothelial carcinoma of the bladder. One of the strongest predictors of survival in these patients is pathological response to initial treatment. Our objective was to determine whether we could stratify the need for radical cystectomy based on pathological response to neoadjuvant chemotherapy. Methods: We present a cohort of patients with muscle invasive urothelial carcinoma of the bladder to whom surveillance and bladder preservation were offered if complete response was achieved following neoadjuvant chemotherapy. Descriptive statistics and survival analysis were performed to assess overall, cancer specific and metastasis-free survival. Patients were stratified based on pathological response to neoadjuvant chemotherapy. Results: A total of 60 patients were included in the cohort, of whom 32 (55%) had absence of residual disease on post-neoadjuvant chemotherapy transurethral resection and 27 (45%) had persistent disease. Of patients undergoing surveillance 52% maintained the bladder without evidence of recurrence. By comparison, of those with recurrence only 20% preserved the bladder and were without evidence of disease. Conclusions: Long-term followup shows a subset of patients achieving good outcomes while preserving the bladder. However, we also observed an inability to reliably identify this subset of patients given current clinical and pathological markers. Until we are able to achieve that goal, the safest oncologic approach remains neoadjuvant chemotherapy followed by radical cystectomy.

Photodynamic therapy is a promising and effective non-invasive therapeutic approach for the treatment of bladder cancers. Therapies targeting HSP90 have the advantage of tumor cell selectivity and have shown great preclinical efficacy. In this study, we evaluated a novel multifunctional nanoporphyrin platform loaded with an HSP90 inhibitor 17AAG (NP-AAG) for use as a multi-modality therapy against bladder cancer. NP-AAG was efficiently accumulated and retained at bladder cancer patient-derived xenograft (PDX) over 7 days. PDX tumors could be synergistically eradicated with a single intravenous injection of NP-AAG followed by multiple light treatments within 7 days. NP-AAG mediated treatment could not only specifically deliver 17AAG and produce heat and reactive oxygen species, but also more effectively inhibit essential bladder cancer essential signaling molecules like Akt, Src, and Erk, as well as HIF-1α induced by photo-therapy. This multifunctional nanoplatform has high clinical relevance and could dramatically improve management for bladder cancers with minimal toxicity.

Objectives To demonstrate patterns of uptake and impact on recurrence of intravesical chemotherapy (IC) immediately following transurethral resection of bladder tumor (TURBT) for low-grade non-muscle-invasive bladder cancer (NMIBC) at a population level. Methods Incident cases of low-grade (LG) Ta or T1 NMIBC from 2005 to 2012 were identified from the California Cancer Registry. We determined rates of IC utilization following TURBT. Multivariable logistic regression models were utilized to assess predictors of IC utilization. Multivariable Cox proportional hazards regression was used to assess the association of IC utilization with recurrence-free survival, bladder cancer-specific survival, and overall survival. Results Ten thousand thirty-one patients with LG NMIBC diagnosed in California between 2005 and 2012. The overall rate of IC utilization was 5.1%, and increased from 1.7% (2005–2006) to 9.6% (2011–2012). More recent year of diagnosis (Odds ratio 1.74, confidence interval 1.60–1.90 for 2-year increments) was associated with an increased likelihood of undergoing immediate postoperative IC. The cumulative incidence of recurrence at 24 months for patients who received IC was 25.2% compared to 30.2% among those who did not receive IC. Use of IC was significantly associated with improved recurrence-free survival (Hazards ratio 0.82, confidence interval 0.70–0.97). Conclusion Utilization of IC for LG NMIBC remains dismally low, with less than 10% of patients receiving this standard of care. Low utilization is associated with increased rates of recurrence. We demonstrate a major shortcoming in quality of care with potential widespread impact on outcomes and cost of care.

© 2015 Elsevier Inc. New approaches to optimization of cancer drug development in the laboratory and the clinic will be required to fully achieve the goal of individualized, precision cancer therapy. Improved preclinical models that more closely reflect the now recognized genomic complexity of human cancers are needed. Here we describe a collaborative research project that integrates core resources of The Jackson Laboratory Basic Science Cancer Center with genomics and clinical research facilities at the UC Davis Comprehensive Cancer Center to establish a clinically and genomically annotated patient-derived xenograft (PDX) platform designed to enhance new drug development and strategies for targeted therapies. Advanced stage non-small-cell lung cancer (NSCLC) was selected for initial studies because of emergence of a number of "druggable" molecular targets, and recent recognition of substantial inter- and intrapatient tumor heterogeneity. Additionally, clonal evolution after targeted therapy interventions make this tumor type ideal for investigation of this platform. Using the immunodeficient NOD scid gamma mouse, > 200 NSCLC tumor biopsies have been xenotransplanted. During the annotation process, patient tumors and subsequent PDXs are compared at multiple levels, including histomorphology, clinically applicable molecular biomarkers, global gene expression patterns, gene copy number variations, and DNA/chromosomal alterations. NSCLC PDXs are grouped into panels of interest according to oncogene subtype and/or histologic subtype. Multiregimen drug testing, paired with next-generation sequencing before and after therapy and timed tumor pharmacodynamics enables determination of efficacy, signaling pathway alterations, and mechanisms of sensitivity-resistance in individual models. This approach should facilitate derivation of new therapeutic strategies and the transition to individualized therapy.

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.

Advances in nutrition during the past century have helped untold numbers of people around the world enjoy healthier and longer lives and be more productive members of society. These advances include the identification of numerous essential nutrients, the identification of common disease states that can arise as a consequence of deficiencies of these essential nutrients, the use of food fortification to correct common deficiencies in the diet, and improvements in agricultural practices and food processing that have resulted in marked advances in food safety and quality. However, many challenges still remain. To a significant extent, these challenges reflect expectations of what constitutes a good diet and what the result of following food guidelines will produce. Moving forward in time in an era of limited economic resources and expanding populations, a critical focus is required to direct attention to the most pressing challenges with the greatest need and opportunity for return on investment. Balancing the desire for quick and effective solutions with the slow, steady, and incremental nature of nutrition research is a struggle confronting academia, industry, and government. To address these challenges, a group of distinguished nutrition scientists gathered for a panel symposium in celebration of the 10th anniversary of the Kosuna Distinguished Lecture in Nutrition at the University of California, Davis. Eight of the panelists were previous Kosuna Distinguished Lecturers. The symposium discussion revolved around 2 questions that were posed to the panel members prior to the meeting: (1) What will be the hottest areas of nutrition research in 2020 and (2) If one were just starting a career in nutrition, what would be a reasonable focus for one s work? A distillation of the discussion follows, organized from the most global to the most individual topics, with some concluding thoughts on the nature of nutrition research.

In a prospective study of 31,925 men with 18 years of follow up, higher ejaculation frequency (EF) throughout adulthood was associated with lower rates of prostate cancer. To further explore this association, we evaluated whole transcriptome gene expression in prostate tissue from study participants who developed prostate cancer between 1992 and 2004 (N=157 tumor tissue, N=85 adjacent normal). We tested for trends in gene expression according to level of EF as self-reported in 1992 for ages 20-29; 40-49; and the year prior to the questionnaire, 1991. There were no associations between EF and gene expression in areas of tumor after accounting for multiple testing. In contrast, in the adjacent normal tissue, 409 genes and six pathways were differentially expressed at a false discovery rate ≤0.2 across categories of EF in 1991. These results suggest that ejaculation affects the expression of genes in normal prostate tissue. The identified genes and pathways provide potential biological links between EF and prostate tumorigenesis.

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.

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.

BACKGROUND Prostate cancer mortality in the United States has declined by nearly 40% over the last 25 years. However, to the authors' knowledge, the contribution of prostate-specific antigen (PSA) screening for the early detection of prostate cancer remains unclear and controversial. In the current study, the authors attempted to determine whether improvements in survival over time among patients with metastatic prostate cancer have contributed to the decline in mortality. METHODS Men aged ≥ 45 years who presented with de novo metastatic prostate cancer from 1988 to 2009 were identified within the California Cancer Registry. Overall survival and disease-specific survival were estimated using the Kaplan-Meier method. A multivariate analysis with Cox proportional hazards modeling was performed to adjust for different distributions of variables between groups. RESULTS A total of 19,336 men presented with de novo metastatic prostate cancer during the study period. On multivariate analysis, overall survival was found to be better for men diagnosed from 1988 through 1992 and 1993 through 1998 than for men diagnosed in the most recent era (hazards ratio, 0.78; 95% confidence interval, 0.72-0.85 [P

© 2016 American Urological Association Education and Research, Inc. Purpose Bladder cancer is a common malignancy often diagnosed in older adults. Previous studies have reported racial/ethnic disparities in bladder cancer survival outcomes but have not focused on younger patients. We identified whether factors influencing cause specific survival in adolescents and young adults (ages 15 to 39) differed from older adults, and defined prognostic factors specifically in adolescents and young adults using the California Cancer Registry. Materials and Methods Patients diagnosed with bladder cancer between 1988 through 2012 were included in the study. The primary outcome measure was cause specific survival. A multivariable Cox proportional hazards regression model was used to evaluate predictors of cause specific survival in patients of all ages and in adolescents/young adults. Interactions of age and other variables between younger and older adult patients were assessed. Results Of 104,974 patients with bladder cancer we identified 1,688 adolescent and young adult patients (1.6%). Compared to older patients these patients had a 58% reduced risk of bladder cancer death (HR 0.42, p

© 2016 Elsevier Ltd The overall prognosis of bladder cancer has not been improved over the last 30 years and therefore, there is a great medical need to develop novel diagnosis and therapy approaches for bladder cancer. We developed a multifunctional nanoporphyrin platform that was coated with a bladder cancer-specific ligand named PLZ4. PLZ4-nanoporphyrin (PNP) integrates photodynamic diagnosis, image-guided photodynamic therapy, photothermal therapy and targeted chemotherapy in a single procedure. PNPs are spherical, relatively small (around 23 nm), and have the ability to preferably emit fluorescence/heat/reactive oxygen species upon illumination with near infrared light. Doxorubicin (DOX) loaded PNPs possess slower drug release and dramatically longer systemic circulation time compared to free DOX. The fluorescence signal of PNPs efficiently and selectively increased in bladder cancer cells but not normal urothelial cells in vitro and in an orthotopic patient derived bladder cancer xenograft (PDX) models, indicating their great potential for photodynamic diagnosis. Photodynamic therapy with PNPs was significantly more potent than 5-aminolevulinic acid, and eliminated orthotopic PDX bladder cancers after intravesical treatment. Image-guided photodynamic and photothermal therapies synergized with targeted chemotherapy of DOX and significantly prolonged overall survival of mice carrying PDXs. In conclusion, this uniquely engineered targeting PNP selectively targeted tumor cells for photodynamic diagnosis, and served as effective triple-modality (photodynamic/photothermal/chemo) therapeutic agents against bladder cancers. This platform can be easily adapted to individualized medicine in a clinical setting and has tremendous potential to improve the management of bladder cancer in the clinic.

The identification and development of biomarkers which predict response of muscle invasive bladder cancer (MIBC) patients to neoadjuvant chemotherapy would likely increase usage of this treatment option and thereby improve patient survival rates. MiRNA array and qRT-PCR validation was used to identify miRNA which are associated with response to neoadjuvant chemotherapy. RNA was extracted from a total of 41 archival, fully annotated, MIBC patient diagnostic biopsies (20 chemo-responders and 21 non-responders (response is defined as > 5 year survival rate and being pT0 post-chemotherapy)). Microarray and qPCR identified let-7c as being differentially expressed in chemo-responder versus non-responder patients. Patients with higher let-7c expression levels had significantly higher odds of responding to chemotherapy (p = 0.023, OR 2.493, 95% CI 1.121, 5.546), and assessment of let-7c levels allowed for prediction of patient response (AUC 0.72, positive predictive value 59%). Decreased let-7c was associated with MIBC incidence (p < 0.001), and significantly correlated with other related miRNA including those that were not differentially expressed between responders and non-responders. The combined data indicate let-7c plays a role in mediating chemoresistance to neoadjuvant chemotherapy in MIBC patients, and is a modest, yet clinically meaningful, predictor of patient response.

Objective—To determine expression of microRNA (miRNA) in urinary bladder samples obtained from dogs with grossly normal urinary bladders, inflammatory bladder disease, or transitional cell carcinoma (TCC) and in cells of established canine TCC cell lines. Sample—Samples of grossly normal bladders (n = 4) and bladders from dogs with inflammatory bladder disease (13) or TCC (18), and cells of 5 established canine TCC cell lines. Procedures—Expression of 5 miRNAs (miR-34a, let-7c, miR-16, miR-103b, and miR-106b) that target p53, Rb, or Bcl-2 protein pathways was determined for bladder samples and cells via quantitative real-time PCR assay. Effects of cisplatin (5μM) on proliferation and miRNA expression of cells were determined. Results—Expression of miR-34a and miR-106b was significantly higher in TCC samples than it was in samples of grossly normal bladders. Expression of miR-34a, miR-16, miR-103b, and miR-106b was higher in TCC samples than it was in bladder samples from dogs with inflammatory bladder disease. Cells of established canine TCC cell lines that had the lowest growth after cisplatin treatment had increased miR-34a expression after such treatment. Conclusions and Clinical Relevance—Findings of this study indicated results of miRNA expression assays can be used to distinguish between samples of grossly normal bladders and bladders of dogs with inflammatory bladder disease or TCC. This finding may have clinical relevance because currently available diagnostic tests cannot be used to differentiate these tissues, and inflammatory bladder disease and TCC are both prevalent in dogs. Validation of miRNA expression assays as diagnostic tests may be warranted.

As prostate cancer (CaP) is regulated by androgen receptor (AR) activity, metastatic CaP is treated with androgen deprivation therapy (ADT). Despite initial response, patients on ADT eventually progress to castration-resistant CaP (CRPC), which is currently incurable. We previously showed that cleavage of the 280 kDa structural protein Filamin A (FlnA) to a 90 kDa fragment, and nuclear localization of the cleaved product, sensitized CRPC cells to ADT. Hence, treatment promoting FlnA nuclear localization would enhance androgen responsiveness. Here, we show that FlnA nuclear localization induced apoptosis in CRPC cells during ADT, identifying it as a treatment tool in advanced CaP. Significantly, the natural product genistein combined polysaccharide (GCP) had a similar effect. Investigation of the mechanism of GCP-induced apoptosis showed that GCP induced FlnA cleavage and nuclear localization and that apoptosis resulting from GCP treatment was mediated by FlnA nuclear localization. Two main components of GCP are genistein and daidzein: the ability of GCP to induce G2 arrest was due to genistein whereas sensitivity to ADT stemmed from daidzein; hence, both were needed to mediate GCP's effects. FlnA cleavage is regulated by its phosphorylation; we show that ADT enhanced FlnA phosphorylation, which prevented its cleavage, whereas GCP inhibited FlnA phosphorylation, thereby sensitizing CaP cells to ADT. In a mouse model of CaP recurrence, GCP, but not vehicle, impeded relapse following castration, indicating that GCP, when administered with ADT, interrupted the development of CRPC. These results demonstrate the efficacy of GCP in promoting FlnA nuclear localization and enhancing androgen responsiveness in CaP.

BACKGROUND Docetaxel is the first line treatment for castration resistant prostate cancer (CRPC). However, docetaxel resistance rapidly develops. Identifying the critical mechanisms giving rise to docetaxel resistance is the major challenge in advanced prostate cancer.

To develop bladder cancer-specific ligands using a combinatorial chemistry approach.We performed a high-throughput one-bead one-compound combinatorial chemistry approach to identify ligands that bound to bladder transitional cell carcinoma cells. The whole-cell binding assay allowed successful identification of a few peptides that bound selectively to bladder cancer cells. Single cell suspensions derived from clinical bladder cancer specimens and cell lines were used to determine the binding specificity. Studies with mouse xenografts were performed to determine the in vivo binding and targeting efficiency, specificity, and biodistribution of one of the ligands.One cyclic peptide named PLZ4 (amino acid sequence: cQDGRMGFc) was identified that could selectively bind to bladder cancer cell lines and all of the 5 primary bladder cancer cells from human patients, but not to normal urothelial cells, cell mixtures from normal bladder specimens, fibroblasts, and blood cells. Comparison of PLZ4 binding to cell lines of different cancer origins showed that it was bladder cancer-specific (P0.05). PLZ4 could bind to tumor cells treated with urine at pH 6.0, but not to noncancerous cells collected from the urine of 4 patients actively being treated with intravesical Bacillus Calmette-Guerin therapy. In vivo and ex vivo imaging studies showed that PLZ4 linked to Cy5.5 fluorescent dye administered via tail vein injection was specifically taken up in mouse xenografts developed from excised fresh human bladder cancer specimens. Several ligands contain the same DGR motif, but only PLZ4 was bladder cancer-specific. We performed alanine walk and rainbow bead coding experiments, and found that the C-terminal GF residues were also important for cell binding and modulated the binding specificity.PLZ4 has the potential to be used for targeted therapy and imaging detection during diagnosis and follow-up/surveillance of noninvasive and advanced bladder cancer.

The self-renewal capacity ascribed to embryonic stem cells (ESC) is reminiscent of cancer cell proliferation, raising speculation that a common network of genes may regulate these traits. A search for general regulators of these traits yielded a set of microRNAs for which expression is highly enriched in human ESCs and liver cancer cells (HCC) but attenuated in differentiated quiescent hepatocytes. Here, we show that these microRNAs promote hESC self-renewal, as well as HCC proliferation, and when overexpressed in normally quiescent hepatocytes, induce proliferation and activate cancer signaling pathways. Proliferation in hepatocytes is mediated through translational repression of Pten, Tgfbr2, Klf11, and Cdkn1a, which collectively dysregulates the PI3K/AKT/mTOR and TGFβ tumor suppressor signaling pathways. Furthermore, aberrant expression of these miRNAs is observed in human liver tumor tissues and induces epithelial–mesenchymal transition in hepatocytes. These findings suggest that microRNAs that are essential in normal development as promoters of ESC self-renewal are frequently upregulated in human liver tumors and harbor neoplastic transformation potential when they escape silencing in quiescent human hepatocytes. Mol Cancer Res; 10(7); 979–91. ©2012 AACR.

We previously demonstrated that H2 relaxin (RLN2) facilitates castrate-resistant (CR) growth of prostate cancer (CaP) cells through PI3K/Akt/β-catenin-mediated activation of the androgen receptor (AR) pathway. As inhibition of this pathway caused only ~50% reduction in CR growth, the goal of the current study was to identify additional RLN2-activated pathways that contribute to CR growth. Next-generation sequencing-based transcriptome and gene ontology analyses comparing LNCaP stably transfected with RLN2 versus LNCaP-vector identified differential expression of genes associated with cell proliferation (12.7% of differentially expressed genes), including genes associated with the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) and nuclear factor-kappaB (NF-κB) pathways. Subsequent molecular analyses confirmed that the cAMP/PKA and NF-κB pathways play a role in facilitating H2 relaxin-mediated CR growth of CaP cells. Inhibition of PKA-attenuated RLN2-mediated AR activity inhibited proliferation and caused a small but significant increase in apoptosis. Combined inhibition of the PKA and NF-κB signaling pathways via inhibition of PKA and Akt induced significant apoptosis and dramatically reduced clonogenic potential, outperforming docetaxel, the standard of care treatment for CR CaP. Immunohistochemical analysis of tissue microarrays in combination with multispectral quantitative imaging comparing RLN2 levels in patients with benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia, and CaP determined that RLN2 is significantly upregulated in CaP vs BPH (p = 0.002). The combined data indicate RLN2 overexpression is frequent in CaP patients and provides a growth advantage to CaP cells. A near-complete inhibition of RLN2-induced CR growth can be achieved by simultaneous blockade of both pathways.

Context MicroRNAs (miRNA) are noncoding RNAs that post-transcriptionally regulate gene expression. Their altered expression and function have been observed in most urologic cancers. MiRNAs represent potential disease biomarkers and novel therapeutic targets. Objective To review and evaluate the evidence implicating miRNAs in the pathogenesis of prostate cancer (PCa), bladder cancer (BCa), and renal cancer. Evidence acquisition A systematic review was performed using PubMed and Embase to search for reports using strings for microRNA, non-coding RNA, cancer, prostate, bladder, and renal cancer. Identified manuscripts were retrieved and references searched. Selected studies were required to concentrate on the role of miRNA in these urologic cancers. Evidence synthesis We reviewed articles that focus on this topic. More than 40 miRNAs have been implicated in urologic cancer and many target common carcinogenic pathways. In particular, apoptosis avoidance, cell proliferation, epithelial-to-mesenchymal transition, angiogenic signalling, and the generation of androgen independence are targeted or facilitated by more than one miRNA. Little work has been done to evaluate the translational applications for this knowledge to date. Novel therapeutic strategies have been developed and are under investigation to selectively modulate miRNAs; such work would potentially enable personalised tumour therapy. Conclusions MiRNAs appear to be important modulators of urologic cancer. Their expression is frequently altered in these tumours, and many are functionally implicated in their pathogenesis. They require evaluation to determine the translational role and therapeutic potential for this knowledge.

BACKGROUND: Men on active surveillance for clinical stage I nonseminomatous germ cell tumor (NSGCT) undergo frequent computed tomography imaging to avoid delayed detection of disease. Irradiation from frequent imaging and chemotherapy upon progression may place patients at increased risk of a second malignancy. In this study, the authors sought to identify such an increased risk among men who chose initial surveillance for NSGCT. METHODS: The authors utilized data from the Surveillance, Epidemiology and End Results Program and stratified the cohort based on whether they underwent retroperitoneal lymph node dissection (RPLND). A propensity-score model was used to adjust for covariates, and a competing-risks regression analysis was performed to estimate cumulative incidence rates of second malignancy. Incidence risk ratios were predicted by using the cumulative incidence rates per 10,000 patients. RESULTS: There was no statistically significant increase in the incidence of a secondary malignancy for the entire cohort of testicular cancer survivors. However, when the analysis was restricted to patients with clinical stage I NSGCT, nonsurgical management only in those aged >45 years was an independent predictor of developing a second malignancy. For every 10,000 patients with stage I NSGCT who chose to forego RPLND, an absolute excess incidence of 22, 52, and 73 secondary malignancies would be diagnosed at 5 years, 10 years, and 15 years, respectively. CONCLUSIONS: The current results indicated that patients aged >45 years who forego RPLND for T1 or T2 clinical stage I NSGCT are more likely to develop a second malignancy than those who do undergo RPLND. Nonsurgical management of NSGCT may be associated with more long-term health risks than primary RPLND. Cancer 2011;. © 2011 American Cancer Society.