Isoform-specific disruption of the TP73 gene reveals a critical role for TAp73γ in tumorigenesis via leptin.

TP73, a member of the p53 family, is expressed as TAp73 and ΔNp73 along with multiple C-terminal isoforms (α-η). ΔNp73 is primarily expressed in neuronal cells and necessary for neuronal development. Interestingly, while TAp73α is a tumor suppressor and predominantly expressed in normal cells, TAp73 is found to be frequently altered in human cancers, suggesting a role of TAp73 C-terminal isoforms in tumorigenesis. To test this, the TCGA SpliceSeq database was searched and showed that exon 11 (E11) exclusion occurs frequently in several human cancers. We also found that p73α to p73γ isoform switch resulting from E11 skipping occurs frequently in human prostate cancers and dog lymphomas. To determine whether p73α to p73γ isoform switch plays a role in tumorigenesis, CRISPR technology was used to generate multiple cancer cell lines and a mouse model in that Trp73 E11 is deleted. Surprisingly, we found that in E11-deificient cells, p73γ becomes the predominant isoform and exerts oncogenic activities by promoting cell proliferation and migration. In line with this, E11-deficient mice were more prone to obesity and B-cell lymphomas, indicating a unique role of p73γ in lipid metabolism and tumorigenesis. Additionally, we found that E11-deficient mice phenocopies Trp73-deficient mice with short lifespan, infertility, and chronic inflammation. Mechanistically, we showed that Leptin, a pleiotropic adipocytokine involved in energy metabolism and oncogenesis, was highly induced by p73γ,necessary for p73γ-mediated oncogenic activity, and associated with p73α to γ isoform switch in human prostate cancer and dog lymphoma. Finally, we showed that E11-knockout promoted, whereas knockdown of p73γ or Leptin suppressed, xenograft growth in mice. Our study indicates that the p73γ-Leptin pathway promotes tumorigenesis and alters lipid metabolism, which may be targeted for cancer management.

Genistein Combined Polysaccharide (GCP) Can Inhibit Intracrine Androgen Synthesis in Prostate Cancer Cells.

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 < 0.001) and cause decreased expression of intracrine androgen synthesis pathway enzymes (~2.5-fold decrease of 3ßHSD (p < 0.001), 17ßHSD (p < 0.001), CYP17A (p < 0.01), SRB1 (p < 0.0001), and StAR (p < 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 < 0.001). Genistein caused similar decreases in the expression of 17ßHSD and CYP17A (2.5-fold (p < 0.001) and 1.5-fold decrease (p < 0.01), respectively) compared to GCP, however it did not cause altered expression of the other intracrine androgen synthesis pathway enzymes; 3ßHSD, SRB1, and StAR. Our combined data indicate that GCP and/or genistein may have clinical utility and that further pre-clinical studies are warranted.

Combination of cyclin-dependent kinase and immune checkpoint inhibitors for the treatment of bladder cancer.

BACKGROUND: Perturbation of the CDK4/6 pathway is frequently observed in advanced bladder cancer. We investigated the potential of targeting this pathway alone or in combination with chemotherapy or immunotherapy as a therapeutic approach for the treatment of bladder cancer METHODS: The genetic alterations of the CDK4/6 pathway in bladder cancer were first analyzed with The Cancer Genome Atlas database and validated in our bladder cancer patient-derived tumor xenografts (PDXs). Bladder cancer cell lines and mice carrying PDXs with the CDK4/6 pathway perturbations were treated with a CDK4/6 inhibitor palbociclib to determine its anticancer activity and the underlying mechanisms. The combination index method was performed to assess palbociclib and gemcitabine drug-drug interactions. Syngeneic mouse bladder cancer model BBN963 was used to assess whether palbociclib could potentiate anti-PD1 immunotherapy. RESULTS: Of the 413 bladder cancer specimens, 79.2% harbored pertubations along the CDK4/6 pathway. Palbociclib induced G0/G1 cell cycle arrest but with minimal apoptosis in vitro. In mice carrying PDXs, palbociclib treatment reduced tumor growth and prolonged survival from 14 to 32 days compared to vehicle only controls (p = 0.0001). Palbociclib treatment was associated with a decrease in Rb phosphorylation in both cell lines and PDXs. Palbociclib and gemcitabine exhibited antagonistic cytotoxicity in vitro (CI > 3) and in vivo, but palbociclib significantly enhanced the treatment efficacy of anti-PD1 immunotherapy and induced CD8+ T lymphocyte infiltration in syngeneic mouse models. CONCLUSIONS: The CDK4/6 pathway is feasible as a potential target for the treatment of bladder cancer, especially in combination with immunotherapy. A CDK4/6 inhibitor should not be combined with gemcitabine.

Methionine Adenosyltransferase 1a (MAT1A) Enhances Cell Survival During Chemotherapy Treatment and is Associated with Drug Resistance in Bladder Cancer PDX Mice.

Bladder cancer is among the top ten most common cancers, with about ~380,000 new cases and ~150,000 deaths per year worldwide. Tumor relapse following chemotherapy treatment has long been a significant challenge towards completely curing cancer. We have utilized a patient-derived bladder cancer xenograft (PDX) platform to characterize molecular mechanisms that contribute to relapse following drug treatment in advanced bladder cancer. Transcriptomic profiling of bladder cancer xenograft tumors by RNA-sequencing analysis, before and after relapse, following a 21-day cisplatin/gemcitabine drug treatment regimen identified methionine adenosyltransferase 1a (MAT1A) as one of the significantly upregulated genes following drug treatment. Survey of patient tumor sections confirmed elevated levels of MAT1A in individuals who received chemotherapy. Overexpression of MAT1A in 5637 bladder cancer cells increased tolerance to gemcitabine and stalled cell proliferation rates, suggesting MAT1A upregulation as a potential mechanism by which bladder cancer cells persist in a quiescent state to evade chemotherapy.

Correlation of Platinum Cytotoxicity to Drug-DNA Adduct Levels in a Breast Cancer Cell Line Panel.

Platinum drugs, including carboplatin and oxaliplatin, are commonly used chemotherapy drugs that kill cancer cells by forming toxic drug-DNA adducts. These drugs have a proven, but modest, efficacy against several aggressive subtypes of breast cancer but also cause several side effects that can lead to the cessation of treatment. There is a clinical need to identify patients who will respond to platinum drugs in order to better inform clinical decision making. Diagnostic microdosing involves dosing patients or patient samples with subtherapeutic doses of radiolabeled platinum followed by measurement of platinum-DNA adducts in blood or tumor tissue and may be used to predict patient response. We exposed a panel of six breast cancer cell lines to 14C-labeled carboplatin or oxaliplatin at therapeutic and microdose (1% therapeutic dose) concentrations for a range of exposure lengths and isolated DNA from the cells. The DNA was converted to graphite, and measurement of radiocarbon due to platinum-DNA adduction was quantified via accelerator mass spectrometry (AMS). We observed a linear correlation in adduct levels between the microdose and therapeutic dose, and the level of platinum-DNA adducts corresponded to cell line drug sensitivity for both carboplatin and oxaliplatin. These results showed a clear separation in adduct levels between the sensitive and resistant groups of cell lines that could not be fully explained or predicted by changes in DNA repair rates or mutations in DNA repair genes. Further, we were able to quantitate oxaliplatin-DNA adducts in the blood and tumor tissue of a metastatic breast cancer patient. Together, these data support the use of diagnostic microdosing for predicting patient sensitivity to platinum. Future studies will be aimed at replicating this data in a clinical feasibility trial.

COX-2/sEH Dual Inhibitor PTUPB Potentiates the Antitumor Efficacy of Cisplatin.

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.

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.

Microchamber Cultures of Bladder Cancer: A Platform for Characterizing Drug Responsiveness and Resistance in PDX and Primary Cancer Cells.

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.

A diagnostic microdosing approach to investigate platinum sensitivity in non-small cell lung cancer.

The platinum-based drugs cisplatin, carboplatin and oxaliplatin are often used for chemotherapy, but drug resistance is common. The prediction of resistance to these drugs via genomics is a challenging problem since hundreds of genes are involved. A possible alternative is to use mass spectrometry to determine the propensity for cells to form drug-DNA adducts-the pharmacodynamic drug-target complex for this class of drugs. The feasibility of predictive diagnostic microdosing was assessed in non-small cell lung cancer (NSCLC) cell culture and a pilot clinical trial. Accelerator mass spectrometry (AMS) was used to quantify [14 C]carboplatin-DNA monoadduct levels in the cell lines induced by microdoses and therapeutic doses of carboplatin, followed by correlation with carboplatin IC50 values for each cell line. The adduct levels in cell culture experiments were linearly proportional to dose (R2 = 0.95, p < 0.0001) and correlated with IC50 across all cell lines for microdose and therapeutically relevant carboplatin concentrations (p = 0.02 and p = 0.01, respectively). A pilot microdosing clinical trial was conducted to define protocols and gather preliminary data. Plasma pharmacokinetics (PK) and [14 C]carboplatin-DNA adducts in white blood cells and tumor tissues from six NSCLC patients were quantified via AMS. The blood plasma half-life of [14 C]carboplatin administered as a microdose was consistent with the known PK of therapeutic dosing. The optimal [14 C]carboplatin formulation for the microdose was 107 dpm/kg of body weight and 1% of the therapeutic dose for the total mass of carboplatin. No microdose-associated toxicity was observed in the patients. Additional accruals are required to significantly correlate adduct levels with response.

TMPRSS2-ERG fusions unexpectedly identified in men initially diagnosed with nonprostatic malignancies.

Background: TMPRSS2-ERG gene fusions are frequently found in prostate cancer and are pathognomomic for prostatic origin. In a series of cancer cases assayed with comprehensive genomic profiling (CGP) in the course of clinical care, we reviewed the frequency of TMPRSS2-ERG fusions in patient tumors of various histologic subtypes. Methods: Frequency of TMPRSS2-ERG fusions was determined in comprehensive genomic profiles from 64,263 cancer cases submitted to Foundation Medicine to assess genomic alterations suggesting benefit from targeted therapy. Genomic results from an index case of prostate cancer that underwent evolution from adenocarcinoma to pure squamous cell carcinoma are presented. Results: TMPRSS2-ERG fusions were identified for 0.86% (250/29030) of male patients and not found for female patients (0/35233). TMPRSS2-ERG fusions were detected in six tumors that were classified as squamous carcinoma, five of which were of unknown primary site. The index case is a patient with a large left retrovesical mass diagnosed as squamous carcinoma by morphologic examination and a history of Gleason 9 prostate cancer with prior prostatectomy and salvage radiation therapy. TMPRSS2-ERG was detected by genomic profiling in the squamous cell tumor, the primary adenocarcinoma of the prostate, and in a metachronous prostatic adenocarcinoma metastasis. Based on these results, the patient received androgen deprivation therapy. A phylogenetic tree demonstrating clonal and histopathologic evolution of prostate cancer in the index patient was constructed. Conclusions: In this large CGP dataset, TMPRSS2-ERG fusion was seen in ~30% of prostate cancers regardless of histologic type; the fusion was on occasion detected in advanced cancers not initially carrying a diagnosis of prostate carcinoma. CGP of advanced cancers in men may reveal prostatic origin by detection of the pathognomomic TMPRSS2-ERG fusion gene.

Microdose-Induced Drug-DNA Adducts as Biomarkers of Chemotherapy Resistance in Humans and Mice.

We report progress on predicting tumor response to platinum-based chemotherapy with a novel mass spectrometry approach. Fourteen bladder cancer patients were administered one diagnostic microdose each of [14C]carboplatin (1% of the therapeutic dose). Carboplatin-DNA adducts were quantified by accelerator mass spectrometry in blood and tumor samples collected within 24 hours, and compared with subsequent chemotherapy response. Patients with the highest adduct levels were responders, but not all responders had high adduct levels. Four patient-derived bladder cancer xenograft mouse models were used to test the possibility that another drug in the regimen could cause a response. The mice were dosed with [14C]carboplatin or [14C]gemcitabine and the resulting drug-DNA adduct levels were compared with tumor response to chemotherapy. At least one of the drugs had to induce high drug-DNA adduct levels or create a synergistic increase in overall adducts to prompt a corresponding therapeutic response, demonstrating proof-of-principle for drug-DNA adducts as predictive biomarkers. Mol Cancer Ther; 16(2); 376-87. ©2016 AACR.

Diagnostic Microdosing Approach to Study Gemcitabine Resistance.

Gemcitabine metabolites cause the termination of DNA replication and induction of apoptosis. We determined whether subtherapeutic "microdoses" of gemcitabine are incorporated into DNA at levels that correlate to drug cytotoxicity. A pair of nearly isogenic bladder cancer cell lines differing in resistance to several chemotherapy drugs were treated with various concentrations of 14C-labeled gemcitabine for 4-24 h. Drug incorporation into DNA was determined by accelerator mass spectrometry. A mechanistic analysis determined that RRM2, a DNA synthesis protein and a known resistance factor, substantially mediated gemcitabine toxicity. These results support gemcitabine levels in DNA as a potential biomarker of drug cytotoxicity.

Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer.

We report herein the development, functional and molecular characterization of an isogenic, paired bladder cancer cell culture model system for studying platinum drug resistance. The 5637 human bladder cancer cell line was cultured over ten months with stepwise increases in oxaliplatin concentration to generate a drug resistant 5637R sub cell line. The MTT assay was used to measure the cytotoxicity of several bladder cancer drugs. Liquid scintillation counting allowed quantification of cellular drug uptake and efflux of radiolabeled oxaliplatin and carboplatin. The impact of intracellular drug inactivation was assessed by chemical modulation of glutathione levels. Oxaliplatin- and carboplatin-DNA adduct formation and repair was measured using accelerator mass spectrometry. Resistance factors including apoptosis, growth factor signaling and others were assessed with RNAseq of both cell lines and included confirmation of selected transcripts by RT-PCR. Oxaliplatin, carboplatin, cisplatin and gemcitabine were significantly less cytotoxic to 5637R cells compared to the 5637 cells. In contrast, doxorubicin, methotrexate and vinblastine had no cell line dependent difference in cytotoxicity. Upon exposure to therapeutically relevant doses of oxaliplatin, 5637R cells had lower drug-DNA adduct levels than 5637 cells. This difference was partially accounted for by pre-DNA damage mechanisms such as drug uptake and intracellular inactivation by glutathione, as well as faster oxaliplatin-DNA adduct repair. In contrast, both cell lines had no significant differences in carboplatin cell uptake, efflux and drug-DNA adduct formation and repair, suggesting distinct resistance mechanisms for these two closely related drugs. The functional studies were augmented by RNAseq analysis, which demonstrated a significant change in expression of 83 transcripts, including 50 known genes and 22 novel transcripts. Most of the transcripts were not previously associated with bladder cancer chemoresistance. This model system and the associated phenotypic and genotypic data has the potential to identify some novel details of resistance mechanisms of clinical importance to bladder cancer.

Development and Characterization of Bladder Cancer Patient-Derived Xenografts for Molecularly Guided Targeted Therapy.

BACKGROUND: The overarching goal of this project is to establish a patient-derived bladder cancer xenograft (PDX) platform, annotated with deep sequencing and patient clinical information, to accelerate the development of new treatment options for bladder cancer patients. Herein, we describe the creation, initial characterization and use of the platform for this purpose. METHODS AND FINDINGS: Twenty-two PDXs with annotated clinical information were established from uncultured unselected clinical bladder cancer specimens in immunodeficient NSG mice. The morphological fidelity was maintained in PDXs. Whole exome sequencing revealed that PDXs and parental patient cancers shared 92-97% of genetic aberrations, including multiple druggable targets. For drug repurposing, an EGFR/HER2 dual inhibitor lapatinib was effective in PDX BL0440 (progression-free survival or PFS of 25.4 days versus 18.4 days in the control, p = 0.007), but not in PDX BL0269 (12 days versus 13 days in the control, p = 0.16) although both expressed HER2. To screen for the most effective MTT, we evaluated three drugs (lapatinib, ponatinib, and BEZ235) matched with aberrations in PDX BL0269; but only a PIK3CA inhibitor BEZ235 was effective (p<0.0001). To study the mechanisms of secondary resistance, a fibroblast growth factor receptor 3 inhibitor BGJ398 prolonged PFS of PDX BL0293 from 9.5 days of the control to 18.5 days (p<0.0001), and serial biopsies revealed that the MAPK/ERK and PIK3CA-AKT pathways were activated upon resistance. Inhibition of these pathways significantly prolonged PFS from 12 day of the control to 22 days (p = 0.001). To screen for effective chemotherapeutic drugs, four of the first six PDXs were sensitive to the cisplatin/gemcitabine combination, and chemoresistance to one drug could be overcome by the other drug. CONCLUSION: The PDX models described here show good correlation with the patient at the genomic level and known patient response to treatment. This supports further evaluation of the PDXs for their ability to accurately predict a patient's response to new targeted and combination strategies for bladder cancer.

Bridging tumor genomics to patient outcomes through an integrated patient-derived xenograft platform.

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.

The role of microRNA in castration-resistant prostate cancer.

INTRODUCTION: Castration-resistant prostate cancer (CRPC) has a historically low median survival rate, but recent advances and discoveries in microRNAs (miRNAs) have opened the potential for new prognostication modalities to enhance therapeutic success. As new chemotherapies and immunotherapies are developed, there is an increasing need for precision and stratification of CRPC to allow for optimization and personalization of therapy. METHODS: A systematic literature review was conducted via electronic database resulting in the selection of 42 articles based on title, abstract, study format, and content by a consensus of all participating authors. Most selected articles were published between 2002 and 2013. In this review, we discuss the robustness of miRNAs as a biomarker platform, miRNAs associated with prostate cancer, and recent discoveries of miRNA associations with CRPC. RESULTS: The associations discovered have been of interest owing to the ability to differentiate between CRPC and localized prostate cancer. With the evaluation of multiple miRNAs, it is possible to provide a profile regarding tumor characteristics. Furthermore, actions of miRNAs on CRPC tumor cells have the ability to suppress metastatic phenotypes. CONCLUSION: miRNAs may have a growing role in CRPC prognostication and may potentially transform into a therapeutic potential.