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DNA damage, induced by either chemical carcinogens or environmental pollutants, plays an important role in the initiation of colorectal cancer. DNA repair processes, however, are involved in both protecting against cancer formation, and also contributing to cancer development, by ensuring genomic integrity and promoting the efficient DNA repair in tumor cells, respectively. Although DNA repair pathways have been well exploited in the treatment of breast and ovarian cancers, the role of DNA repair processes and their therapeutic efficacy in colorectal cancer is yet to be appreciably explored. To understand the role of DNA repair, especially homologous recombination (HR), in chemical carcinogen-induced colorectal cancer growth, we unraveled the role of RAD51AP1 (RAD51-associated protein 1), a protein involved in HR, in genotoxic carcinogen (azoxymethane, AOM)–induced colorectal cancer. Although AOM treatment alone significantly increased RAD51AP1 expression, the combination of AOM and dextran sulfate sodium (DSS) treatment dramatically increased by several folds. RAD51AP1 expression is found in mouse colonic crypt and proliferating cells. RAD51AP1 expression is significantly increased in majority of human colorectal cancer tissues, including BRAF/KRAS mutant colorectal cancer, and associated with reduced treatment response and poor prognosis. Rad51ap1-deficient mice were protected against AOM/DSS-induced colorectal cancer. These observations were recapitulated in a genetically engineered mouse model of colorectal cancer (ApcMin/+). Furthermore, chemotherapy-resistant colorectal cancer is associated with increased RAD51AP1 expression. This phenomenon is associated with reduced cell proliferation and colorectal cancer stem cell (CRCSC) self-renewal. Overall, our studies provide evidence that RAD51AP1 could be a novel diagnostic marker for colorectal cancer and a potential therapeutic target for colorectal cancer prevention and treatment.Implications:This study provides first in vivo evidence that RAD51AP1 plays a critical role in colorectal cancer growth and drug resistance by regulating CRCSC self-renewal.

β-Arrestins are classic attenuators of G-protein–coupled receptor signaling. However, they have multiple roles in cellular physiology, including carcinogenesis. This work shows for the first time that β-arrestins have prognostic significance for predicting metastasis and response to chemotherapy in bladder cancer. β-Arrestin-1 (ARRB1) and β-arrestin-2 (ARRB2) mRNA levels were measured by quantitative RT-PCR in two clinical specimen cohorts (n = 63 and 43). The role of ARRBs in regulating a stem cell-like phenotype and response to chemotherapy treatments was investigated. The consequence of forced expression of ARRBs on tumor growth and response to Gemcitabine in vivo were investigated using bladder tumor xenografts in nude mice. ARRB1 levels were significantly elevated and ARRB2 levels downregulated in cancer tissues compared with normal tissues. In multivariate analysis only ARRB2 was an independent predictor of metastasis, disease-specific-mortality, and failure to Gemcitabine + Cisplatin (G+C) chemotherapy; ∼80% sensitivity and specificity to predict clinical outcome. ARRBs were found to regulate stem cell characteristics in bladder cancer cells. Depletion of ARRB2 resulted in increased cancer stem cell markers but ARRB2 overexpression reduced expression of stem cell markers (CD44, ALDH2, and BMI-1), and increased sensitivity toward Gemcitabine. Overexpression of ARRB2 resulted in reduced tumor growth and increased response to Gemcitabine in tumor xenografts. CRISPR-Cas9–mediated gene-knockout of ARRB1 resulted in the reversal of this aggressive phenotype. ARRBs regulate cancer stem cell-like properties in bladder cancer and are potential prognostic indicators for tumor progression and chemotherapy response.

Supplementary Methods

Supplementary Table S1: Specimen and patient characteristics; Supplementary Table S2: Determination of the association between adjuvant treatment failure and demographic/clinical parameters or marker levels; Supplementary Table S3: qPCR primers and ARRB1-specific CRISPR guide RNA sequences; Supplementary Table S4: Source of various antibodies used for immunofluorescence and Western blotting; Supplementary Figure S1: Kaplan Meier plot for stratification of patients into higher risk and lower risk for Disease-Specific Mortality (DSM) based on the ARRB2 arrestin level; Supplementary Figure S2: Expression of cytokeratins and Bmi-1 following modulation of ARRB2 levels in 5637 cells; Supplementary Figure S3: Expression of SOX2 following modulation of ARRB2 levels in bladder cancer cell lines; Supplementary Figure S4: Response of ARRB2 overexpressing transfectants to Cisplatin; Supplementary Figure S5: Expression ABC transporters in ARRB2-modulated bladder cancer cells; Supplementary Figure S6: Effect of ARRB2 overexpression on Cancer Stem Cell markers in vivo; Supplementary Figure S7: ARRB1 depletion affects clonogenic growth, spheroid formation and expression of CSC markers

Supplementary Figures and Legends 1-3

Purpose:Poor prognosis of patients with muscle-invasive bladder cancer that often metastasizes drives the need for discovery of molecular determinants of bladder cancer progression. Chondroitin sulfate proteoglycans, including CD44, regulate cancer progression; however, the identity of a chondroitinase (Chase) that cleaves chondroitin sulfate from proteoglycans is unknown. HYAL-4 is an understudied gene suspected to encode a Chase, with no known biological function. We evaluated HYAL-4 expression and its role in bladder cancer.Experimental Design:In clinical specimens, HYAL-4 wild-type (Wt) and V1 expression was evaluated by RT-qPCR, IHC, and/or immunoblotting; a novel assay measured Chase activity. Wt and V1 were stably expressed or silenced in normal urothelial and three bladder cancer cell lines. Transfectants were analyzed for stem cell phenotype, invasive signature and tumorigenesis, and metastasis in four xenograft models, including orthotopic bladder.Results:HYAL-4 expression, specifically a novel splice variant (V1), was elevated in bladder tumors; Wt expression was barely detectable. V1 encoded a truncated 349 amino acid protein that was secreted. In bladder cancer tissues, V1 levels associated with metastasis and cancer-specific survival with high efficacy and encoded Chase activity. V1 cleaved chondroitin-6-sulfate from CD44, increasing CD44 secretion. V1 induced stem cell phenotype, motility/invasion, and an invasive signature. CD44 knockdown abrogated these phenotypes. V1-expressing urothelial cells developed angiogenic, muscle-invasive tumors. V1-expressing bladder cancer cells formed tumors at low density and formed metastatic bladder tumors when implanted orthotopically.Conclusions:Our study discovered the first naturally-occurring eukaryotic/human Chase and connected it to disease pathology, specifically cancer. V1-Chase is a driver of malignant bladder cancer and potential predictor of outcome in patients with bladder cancer.

Hasanali et al Supplementary Materials

Four Supplementary Figures and Seven Supplementary Tables

Purpose:Gemcitabine-based chemotherapy regimens are first-line for several advanced cancers. Because of better tolerability, gemcitabine + cisplatin is a preferred neoadjuvant, adjuvant, and/or palliative chemotherapy regimen for advanced bladder cancer. Nevertheless, predicting treatment failure and overcoming resistance remain unmet clinical needs. We discovered that splice variant (V1) of HYAL-4 is a first-in-class eukaryotic chondroitinase (Chase), and CD44 is its major substrate. V1 is upregulated in bladder cancer and drives a malignant phenotype. In this study, we investigated whether V1 drives chemotherapy resistance.Experimental Design:V1 expression was measured in muscle-invasive bladder cancer (MIBC) specimens by qRT-PCR and IHC. HYAL-4 wild-type (Wt) and V1 were stably expressed or silenced in normal urothelial and three bladder cancer cell lines. Transfectants were analyzed for chemoresistance and associated mechanism in preclinical models.Results:V1 levels in MIBC specimens of patients who developed metastasis, predicted response to gemcitabine + cisplatin adjuvant/salvage treatment and disease-specific mortality. V1-expressing bladder cells were resistant to gemcitabine but not to cisplatin. V1 expression neither affected gemcitabine influx nor the drug-efflux transporters. Instead, V1 increased gemcitabine metabolism and subsequent efflux of difluorodeoxyuridine, by upregulating cytidine deaminase (CDA) expression through increased CD44–JAK2/STAT3 signaling. CDA inhibitor tetrahydrouridine resensitized V1-expressing cells to gemcitabine. While gemcitabine (25–50 mg/kg) inhibited bladder cancer xenograft growth, V1-expressing tumors were resistant. Low-dose combination of gemcitabine and tetrahydrouridine abrogated the growth of V1 tumors with minimal toxicity.Conclusions:V1/Chase drives gemcitabine resistance and potentially predicts gemcitabine + cisplatin failure. CDA inhibition resensitizes V1-expressing tumors to gemcitabine. Because several chemotherapy regimens include gemcitabine, our study could have broad significance.

Background The clinical manifestation of benign prostatic hyperplasia (BPH) is causally linked to the inflammatory microenvironment and proliferation of epithelial and stromal cells in the prostate transitional zone. The CXC-chemokine interleukin-8 (IL-8) contributes to inflammation. We evaluated the expression of inflammatory cytokines in clinical specimens, primary cultures, and prostatic lineage cell lines. We investigated whether IL-8 via its receptor system (IL-8 axis) promotes BPH. Methods The messenger RNA and protein expression of chemokines, including components of the IL-8 axis, were measured in normal prostate (NP; n = 7) and BPH (n = 21), urine (n = 24) specimens, primary cultures, prostatic lineage epithelial cell lines (NHPrE1, BHPrE1, BPH-1), and normal prostate cells (RWPE-1). The functional role of the IL-8 axis in prostate epithelial cell growth was evaluated by CRISPR/Cas9 gene editing. The effect of a combination with two natural compounds, oleanolic acid (OA) and ursolic acid (UA), was evaluated on the expression of the IL-8 axis and epithelial cell growth. Results Among the 19 inflammatory chemokines and chemokine receptors we analyzed, levels of IL-8 and its receptors (CXCR1, CXCR2), as well as, of CXCR7, a receptor for CXCL12, were 5- to 25-fold elevated in BPH tissues when compared to NP tissues (P ≤ .001). Urinary IL-8 levels were threefold to sixfold elevated in BPH patients, but not in asymptomatic males and females with lower urinary tract symptoms (P ≤ .004). The expression of the IL-8 axis components was confined to the prostate luminal epithelial cells in both normal and BPH tissues. However, these components were elevated in BPH-1 and primary explant cultures as compared to RWPE-1, NHPrE1, and BHPrE1 cells. Knockout of CXCR7 reduced IL-8, and CXCR1 expression by 4- to 10-fold and caused greater than or equal to 50% growth inhibition in BPH-1 cells. Low-dose OA + UA combination synergistically inhibited the growth of BPH-1 and BPH primary cultures. In the combination, the drug reduction indices for UA and OA were 16.4 and 7852, respectively, demonstrating that the combination was effective in inhibiting BPH-1 growth at significantly reduced doses of UA or OA alone. Conclusion The IL-8 axis is a promotor of BPH pathogenesis. Low-dose OA + UA combination inhibits BPH cell growth by inducing autophagy and reducing IL-8 axis expression in BPH-epithelial cells.

BACKGROUND Differential expression of chemokines/chemokine receptors in colorectal cancer (CRC) may enable molecular characterization of patients' tumors for predicting clinical outcome. OBJECTIVE To evaluate the prognostic ability of these molecules in a CRC cohort and the CRC TCGA-dataset. METHODS Chemokine (CXCL-12α, CXCL-12β, IL-17A, CXCL-8, GM-CSF) and chemokine receptor (CXCR-4, CXCR-7) transcripts were analyzed by RT-qPCR in 76 CRC specimens (normal: 27, tumor: 49; clinical cohort). RNA-Seq data was analyzed from the TCGA-dataset (n= 375). Transcript levels were correlated with outcome; analyses: univariate, multivariable, Kaplan-Meier. RESULTS In the clinical cohort, chemokine/chemokine receptor levels were elevated 3-10-fold in CRC specimens (P⩽ 0.004) and were higher in patients who developed metastasis (P= 0.03 - < 0.0001). CXCR-4, CXCR-7, CXCL-12α, CXCL-8, IL-17 and GM-CSF levels predicted metastasis (P⩽ 0.0421) and/or overall survival (OS; P⩽ 0.0373). The CXCR-4+CXCR-7+CXCL-12 marker (CXCR-4/7+CXCL-12 (α/b) signature) stratified patients into risk for metastasis (P= 0.0014; OR, 2.72) and OS (P= 0.0442; OR, 2.7); sensitivity: 86.67%, specificity: 97.06%. In the TCGA-dataset, the CXCR-4/7+CXCL-12 signature predicted metastasis (P= 0.011; OR, 2.72) and OS (P= 0.0006; OR: 4.04). In both datasets, the signature was an independent predictor of clinical outcome. CONCLUSIONS Results of 451 specimens from both cohorts reveal that the CXCR-4/7+CXCL-12 signature potentially predicts outcome in CRC patients and may allow earlier intervention.

INTRODUCTION AND OBJECTIVE:Due to better tolerability, Gemcitabine+Cisplatin is a preferred chemotherapy regimen for advanced bladder cancer (BC). Predicting treatment failure and overcoming resist...

RAD51-associated protein 1 (RAD51AP1) plays an integral role in homologous recombination by activating RAD51 recombinase. Homologous recombination is essential for preserving genome integrity and RAD51AP1 is critical for D-loop formation, a key step in homologous recombination. Although RAD51AP1 is involved in maintaining genomic stability, recent studies have shown that RAD51AP1 expression is significantly upregulated in human cancers. However, the functional role of RAD51AP1 in tumor growth and the underlying molecular mechanism(s) by which RAD51AP1 regulates tumorigenesis have not been fully understood. Here, we use Rad51ap1-knockout mice in genetically engineered mouse models of breast cancer to unravel the role of RAD51AP1 in tumor growth and metastasis. RAD51AP1 gene transcript was increased in both luminal estrogen receptor–positive breast cancer and basal triple-negative breast cancer, which is associated with poor prognosis. Conversely, knockdown of RAD51AP1 (RADP51AP1 KD) in breast cancer cell lines reduced tumor growth. Rad51ap1-deficient mice were protected from oncogene-driven spontaneous mouse mammary tumor growth and associated lung metastasis. In vivo, limiting dilution studies provided evidence that Rad51ap1 plays a critical role in breast cancer stem cell (BCSC) self-renewal. RAD51AP1 KD improved chemotherapy and radiotherapy response by inhibiting BCSC self-renewal and associated pluripotency. Overall, our study provides genetic and biochemical evidences that RAD51AP1 is critical for tumor growth and metastasis by increasing BCSC self-renewal and may serve as a novel target for chemotherapy- and radiotherapy-resistant breast cancer. Significance: This study provides in vivo evidence that RAD51AP1 plays a critical role in breast cancer growth and metastasis by regulating breast cancer stem cell self-renewal.

BACKGROUND: The systemic effect of intracavernosal liposomal bupivacaine (Exparel) injection during inflatable penile prosthesis (IPP) implantation on systemic hemodynamics has not been clarified. AIM: To evaluate whether intraoperative intracavernosal Exparel injection affects systemic hemodynamics. METHODS: We studied 56 consecutive patients who underwent IPP implantation under general anesthesia using the transverse scrotal approach. Exparel [10 ml of 1.3% (13.3 mg/ml)] was instilled bilaterally intracavernosally via a 25-gauge needle around 30 min after starting the surgery. All patients graded their postoperative pain using a standard 10-point scale. OUTCOMES: Perioperative vital signs at defined time intervals and postoperative pain were monitored. The presence and degree of postoperative pain using a standard 10-point scale. RESULTS: Medians and interquartile ranges (IQR) of the patients’ age [64 years (57.3–69.3)], operation time [85 min (78–96)], and estimated blood loss were [75 ml (29–100)] were recorded. Although the median preoperative systolic noninvasively measured blood pressure of 131 mm/Hg (IQR 122–139) fell 25% to 98 mm/Hg (IQR 90–100) (P < .001) after anesthesia started, there was no significant decrease in blood pressure between before and after Exparel injection. The perioperative pulse rate and pulse oximetry oxygen saturation were stable. Neither arrhythmia nor convulsion occurred. Patients reported postoperative pain as 0 (IQR 0–2.25) on a scale of 0–10. CLINICAL IMPLICATIONS: Intraoperative intracavernosal Exparel injection is safe and effective in patients undergoing IPP implantation surgery. STRENGTH AND LIMITATIONS: This is the first report to describe systemic hemodynamics of intracavernosal injection of Exparel. Limitations are lack of blood concentration of Exparel at various time points and long-term follow-up for pain assessment. CONCLUSION: Although limited by a lack of the blood concentration of Exparel and short follow-up for pain assessment, we concluded that Intraoperative intracavernosal Exparel injection does not affect systemic hemodynamics.

The 5-year survival rate of patients with metastatic renal cell carcinoma (mRCC) is

Background: Molecular markers of clinical outcome may aid in designing targeted treatments for bladder cancer. However, only a few bladder cancer biomarkers have been examined as therapeutic targets. Methods: Data from The Cancer Genome Atlas (TCGA) and bladder specimens were evaluated to determine the biomarker potential of the hyaluronic acid (HA) family of molecules – HA synthases, HA receptors and hyaluronidase. The therapeutic efficacy of 4-methylumbelliferone (4MU), a HA synthesis inhibitor, was evaluated in vitro and in xenograft models. Results: In clinical specimens and TCGA data sets, HA synthases and hyaluronidase-1 levels significantly predicted metastasis and poor survival. 4-Methylumbelliferone inhibited proliferation and motility/invasion and induced apoptosis in bladder cancer cells. Oral administration of 4MU both prevented and inhibited tumour growth, without dose-related toxicity. Effects of 4MU were mediated through the inhibition of CD44/RHAMM and phosphatidylinositol 3-kinase/AKT axis, and of epithelial–mesenchymal transition determinants. These were attenuated by HA, suggesting that 4MU targets oncogenic HA signalling. In tumour specimens and the TCGA data set, HA family expression correlated positively with β-catenin, Twist and Snail expression, but negatively with E-cadherin expression. Conclusions: This study demonstrates that the HA family can be exploited for developing a biomarker-driven, targeted treatment for bladder cancer, and 4MU, a non-toxic oral HA synthesis inhibitor, is one such candidate.

Purpose: Poor prognosis of patients with muscle-invasive bladder cancer that often metastasizes drives the need for discovery of molecular determinants of bladder cancer progression. Chondroitin sulfate proteoglycans, including CD44, regulate cancer progression; however, the identity of a chondroitinase (Chase) that cleaves chondroitin sulfate from proteoglycans is unknown. HYAL-4 is an understudied gene suspected to encode a Chase, with no known biological function. We evaluated HYAL-4 expression and its role in bladder cancer. Experimental Design: In clinical specimens, HYAL-4 wild-type (Wt) and V1 expression was evaluated by RT-qPCR, IHC, and/or immunoblotting; a novel assay measured Chase activity. Wt and V1 were stably expressed or silenced in normal urothelial and three bladder cancer cell lines. Transfectants were analyzed for stem cell phenotype, invasive signature and tumorigenesis, and metastasis in four xenograft models, including orthotopic bladder. Results: HYAL-4 expression, specifically a novel splice variant (V1), was elevated in bladder tumors; Wt expression was barely detectable. V1 encoded a truncated 349 amino acid protein that was secreted. In bladder cancer tissues, V1 levels associated with metastasis and cancer-specific survival with high efficacy and encoded Chase activity. V1 cleaved chondroitin-6-sulfate from CD44, increasing CD44 secretion. V1 induced stem cell phenotype, motility/invasion, and an invasive signature. CD44 knockdown abrogated these phenotypes. V1-expressing urothelial cells developed angiogenic, muscle-invasive tumors. V1-expressing bladder cancer cells formed tumors at low density and formed metastatic bladder tumors when implanted orthotopically. Conclusions: Our study discovered the first naturally-occurring eukaryotic/human Chase and connected it to disease pathology, specifically cancer. V1-Chase is a driver of malignant bladder cancer and potential predictor of outcome in patients with bladder cancer.

Studies indicate that molecular subtypes in muscle invasive bladder cancer predict the clinical outcome. We evaluated whether subtyping by a simplified method and established classifications could predict the clinical outcome.We subtyped institutional cohort 1 of 52 patients, including 39 with muscle invasive bladder cancer, an Oncomine™ data set of 151 with muscle invasive bladder cancer and TCGA (The Cancer Genome Atlas) data set of 402 with muscle invasive bladder cancer. Subtyping was done using simplified panels (MCG-1 and MCG-Ext) which included only transcripts common in published studies and were analyzed for predicting metastasis, and cancer specific, overall and recurrence-free survival. TCGA data set was further analyzed using the Lund taxonomy, the Bladder Cancer Molecular Taxonomy Group Consensus and TCGA 2017 mRNA subtype classifications.Muscle invasive bladder cancer specimens from cohort 1 and the Oncomine data set showed intratumor heterogeneity for transcript and protein expression. MCG-1 subtypes did not predict the outcome on univariate or Kaplan-Meier analysis. On multivariate analysis N stage (p ≤0.007), T stage (p ≤0.04), M stage (p=0.007) and/or patient age (p=0.01) predicted metastasis, cancer specific and overall survival, and/or the cisplatin based adjuvant chemotherapy response. In TCGA data set publications showed that subtypes risk stratified patients for overall survival. Consistently the MCG-1 and MCG-Ext subtypes were associated with overall but not recurrence-free survival on univariate and Kaplan-Meier analyses. TCGA data set included 21 low grade specimens of the total of 402 and subtypes associated with tumor grade (p=0.005). However, less than 1% of muscle invasive bladder cancer cases are low grade. In only high grade specimens the MCG-1 and MCG-Ext subtypes could not predict overall survival. On univariate analysis subtypes according to the Bladder Cancer Molecular Taxonomy Group Consensus, TCGA 2017 and the Lund taxonomy were associated with tumor grade (p0.0001) and overall survival (p=0.01 to0.0001). Regardless of classification, subtypes had about 50% to 60% sensitivity and specificity to predict overall and recurrence-free survival. On multivariate analyses N stage and lymphovascular invasion consistently predicted recurrence-free and overall survival (p=0.039 and 0.003, respectively).Molecular subtypes reflect bladder tumor heterogeneity and are associated with tumor grade. In multiple cohorts and subtyping classifications the clinical parameters outperformed subtypes for predicting the outcome.

INTRODUCTION AND OBJECTIVES:Despite many available treatments, >90% of metastatic renal cell carcinoma (mRCC) patients die within 5 years. Due to its modest efficacy, Sorafenib (SF) is a second lin...

INTRODUCTION AND OBJECTIVES:Muscle invasive bladder cancer (MIBC) causes the majority of morbidity and mortality in BCa patients. Prognosis of MIBCa patients may be improved by identification of no...

β-Arrestins are classic attenuators of G-protein–coupled receptor signaling. However, they have multiple roles in cellular physiology, including carcinogenesis. This work shows for the first time that β-arrestins have prognostic significance for predicting metastasis and response to chemotherapy in bladder cancer. β-Arrestin-1 (ARRB1) and β-arrestin-2 (ARRB2) mRNA levels were measured by quantitative RT-PCR in two clinical specimen cohorts (n = 63 and 43). The role of ARRBs in regulating a stem cell-like phenotype and response to chemotherapy treatments was investigated. The consequence of forced expression of ARRBs on tumor growth and response to Gemcitabine in vivo were investigated using bladder tumor xenografts in nude mice. ARRB1 levels were significantly elevated and ARRB2 levels downregulated in cancer tissues compared with normal tissues. In multivariate analysis only ARRB2 was an independent predictor of metastasis, disease-specific-mortality, and failure to Gemcitabine + Cisplatin (G+C) chemotherapy; ∼80% sensitivity and specificity to predict clinical outcome. ARRBs were found to regulate stem cell characteristics in bladder cancer cells. Depletion of ARRB2 resulted in increased cancer stem cell markers but ARRB2 overexpression reduced expression of stem cell markers (CD44, ALDH2, and BMI-1), and increased sensitivity toward Gemcitabine. Overexpression of ARRB2 resulted in reduced tumor growth and increased response to Gemcitabine in tumor xenografts. CRISPR-Cas9–mediated gene-knockout of ARRB1 resulted in the reversal of this aggressive phenotype. ARRBs regulate cancer stem cell-like properties in bladder cancer and are potential prognostic indicators for tumor progression and chemotherapy response.

INTRODUCTION: Despite many available treatments, >90% of metastatic renal cell carcinoma (mRCC) patients die within 5 years. Due to its modest efficacy, Sorafenib (SF) is a second line treatment for mRCC. Molecular basis for SF failure is unknown. Hymecromone (HC) is a non−toxic orally bioavailable agent used in Europe for improving liver health. We examined the molecular basis of SF failure in clinical specimens and provided mechanistic evidence for why SF+HC could be a potent therapeutic combination against mRCC. METHODS: UGT1A9 (A9) levels and SF glucuronidation were measured in kidney specimens (45 normal; 86 tumor) by qPCR and a glucuronidation assay using tumor microsomes. Response to SF+HC combination in vector-only (EV), A9-overexpressing (A9) and A9-knockdown (A9-KO) 786-0 and Caki-1 transfectants, primary RCC spheroids, and endothelial cells was examined by proliferation, apoptosis, motility and invasion assays. Target analysis was performed by immunoblot. Treatment efficacy was evaluated in Caki−1 s.c. and orthotopic xenograft models, with bioluminescence imaging. RESULTS: A9 has been shown to glucuronidate and inactivate SF. A9 levels were 8-10-fold elevated in mRCC patients’ tumors compared to non-mRCC patients and normal kidney tissues. A9 levels were an independent predictor of metastasis (P=0.022; efficacy: 73%; sensitivity: 93.8%). Tumor microsomes from mRCC patients’ glucuronidated SF 5-fold higher than from non-mRCC patients. In RCC cells, HC treatment downregulated A9 levels, A9 promoter activity and inhibited SF-glucuronidation. SF (5 µM) and HC (20-40 µg/ml) combination inhibited RCC cell and primary tumor spheroid proliferation (>90%), motility/invasion (>80%), and induced apoptosis (5-fold). While A9 transfectants were resistant to SF+HC treatment, A9-KO cells were sensitive to SF alone. Endothelial cells co-cultured with A9 transfectants were resistant to SF+HC treatment. SF+HC upregulated apoptosis effectors, but downregulated CD44, RHAMM, and phospho-Met levels in EV but not A9 transfectants. HC (100 or 200 mg/kg) and SF (30 mg/kg) oral combination abrogated Caki−1 tumor growth (>80%) without serum/tissue toxicity. HC+SF treatment decreased A9 levels, Ki67 index and microvessel density. A9-overexpressing tumors were resistant to treatment. CONCLUSION: This is the first study to demonstrate that A9 overexpression in mRCC is likely a mechanism of SF failure and A9 downregulation can re-sensitize cells. HC+SF is an orally bioavailable and non-toxic combination that may be an effective therapeutic strategy for mRCC. Citation Format: Andre R. Jordan, Jiaojiao Wang, Luis E. Lopez, Daley S. Morera, Vinata B. Lokeshwar. Novel molecularly targeted combination for renal cell carcinoma [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 2080.

INTRODUCTION AND OBJECTIVE: The majority of morbidity and mortality in bladder cancer (BCa) patients is due to muscle invasive bladder cancer (MIBC). The prognosis of patients with MIBC could be improved by identification of novel biomarkers and therapeutic agents. Chondroitin sulfate proteoglycans are known to promote tumor growth and metastasis. However, a Chondroitinase (Chase) that degrades chondroitin sulfate has not been identified. HYAL4, a member of the glycosaminoglycan (GAG) degrading enzyme-family, potentially has Chase activity; however, HYAL4 (HY4) has not be studied in any biological system, normal or disease. In this study, we evaluated the expression of all six members of the GAG-degrading enzyme family in BCa specimens and evaluated HY4 functions in preclinical models of BCa. METHODS: Cohort 1: 79 bladder tissues (normal (NBL) = 31; tumor (TBL) = 52); cohort 2: 40 cystectomy specimens from MIBC patients who received adjuvant Gemcitabine plus cisplatin (G+C) treatment for metastatic disease with chemotherapy. Gene expression was measured by q-PCR in both cohorts. HY4 was either overexpressed (HY4) or knocked-down in immortalized normal urothelial (Urotsa) and 3 BCa cell lines. Transfectants were assayed for Chase activity, anchorage independent growth, motility, invasion, molecular signaling. Tumor growth and metastasis in intravesical examined in HT1376 bladder orthotopic model. RESULTS: In cohort 1 HYAL1, HYAL4 mRNA levels were significantly (6-13-fold) elevated in TBL tissues when compared to NBL tissues (P 80% accuracy. HY4-expressing transfectants secreted HY4 and Chase activity in their conditioned media. Overexpression of HY4 in normal urothelial and BCa cells significantly increased, while its knockdown abrogated, anchorage-independent growth, invasion, chemotactic motility (> 3-fold). HY4 expression induced Gemcitabine resistance (IC50: vector: 3.1 nM; HY4: 126 nM). HY4 expression upregulated a stem cell signature. Urothelial cells expressing HY4 formed tumors in NOD/SCID mice. HY4-expressing BCa cells metastasized to lung and spleen in an orthotopic BCa model. Citation Format: Daley S. Morera, Sarrah S. Lahorewala, Soum D. Lokeshwar, Andre R. Jordan, Vinata B. Lokeshwar. HYAL4: The first study on a potential molecular driver and biomarker of invasive bladder 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 1259.

INTRODUCTION AND OBJECTIVE: Molecular subtypes have been suggested to predict outcome in patients with muscle invasive bladder cancer (MIBC). The major subtypes reported have been “luminal” and “basal”; however, there is little consensus regarding subtype-signatures and the clinical parameters used to evaluate outcome. The objective of this study was to examine using multiple datasets, if molecular subtypes are distinct entities within MIBC and whether they predict clinical outcome. METHODS: We analyzed transcriptome data from all 402 MIBC samples in The Cancer Genome Atlas (TCGA) and 151 high-grade MIBC samples from three datasets in Oncomine™. Transcript levels were also measured in 52 bladder tumor specimens with clinical follow-up (cohort-1). Samples were subtype-scored using gene panels (GP-11, GP-30, BASQ) consisting only of those genes common among published studies. Correlation of subtypes to recurrence/progression-free survival (R/PFS), metastasis, cancer-specific survival (CSS) and overall-survival (OS) was examined by logistic regression, Cox proportional Hazards model and Kaplan-Meier analyses. RESULTS: Based on GP-11 subtype-scoring, >75% of the MIBC TCGA-dataset samples were mixed, i.e. neither pure-luminal nor pure-basal. When tumors were categorized as pure-luminal, pure-basal, or mixed, the subtypes could not predict OS or R/PFS. Consistent with published studies, when tumors were categorized as luminal or basal the subtypes predicted OS (P=0.051); sensitivity: 53.9%; specificity: 62%. Subtypes also correlated with tumor-grade (P=0.0005); most low-grade MIBC cases (16/21) in the TCGA-dataset were luminal. However, MIBC is rarely low-grade and subtypes could not predict OS (P=0.131) when only high-grade cases were included. Subtypes were not significant prognosticators in multivariate analyses. GP-30 and BASQ panels validated these results. In the Oncomine-dataset and cohort-1, subtypes could not predict metastasis, CSS, or OS. CONCULUSIONS: Our study of multiple datasets reveals that molecular subtypes may reflect tumor-heterogeneity but are likely not distinct entities within MIBC. Furthermore, molecular subtypes have limited prognostic capability for MIBC patients. Although there is a need to individualize patient care, further examination into the molecular subtypes of MIBC is needed before their incorporation into clinical practice. Citation Format: Sarrah S. Lahorewala, Daley S. Morera, Jiaojiao Wang, Vinata B. Lokeshwar. Results from multiple datasets including the TCGA reveal limited clinical significance of molecular subtypes in bladder 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 462.

INTRODUCTION AND OBJECTIVES: Muscle invasive bladder cancer (MIBCa) causes the majority of morbidity and mortality in BCa patients. Prognosis of MIBCa patients may be improved by ide ntification of novel prognostic biomarkers and therapeutic targets. Chondroitin sulfate proteoglycans are known to promote tumor growth and metastasis. However, a Chondroitinase (Chase) that degrades chondroitin sulfate has not been identified. HYAL4, a member of the glycosaminoglycan (GAG) degrading enzyme-family, potentially has Chase activity; however, HYAL4 (HY4) has not be studied in any biological system, normal or disease. The objective of this study was to evaluate the expression of all six members of the GAG-degrading enzyme family in bladder cancer (BCa) specimens and to investigate HY4 functions in preclinical models of BCa. METHODS: Q-PCR was performed to measure mRNA levels of GAG-degrading enzyme family genes in 59 bladder tissues (normal (NBL) = 25; tumor (TBL) = 38). HY4 expression was also measured in 40 cystectomy specimens from MIBCa patients who later were treated for metastatic disease with Gemcitabine plus cisplatin (G+C) chemotherapy. HY4 was either stably expressed or knocked-down in immortalized urothelial and BCa cell lines. Transfectants were assayed for Chase activity, anchorage independent growth, motility, invasion, molecular signaling. Tumor growth and metastasis was monitored by bioluminescence imaging. RESULTS: Among the 6 genes, HYAL1 and HY4 mRNA levels were significantly (6-13-fold) elevated in TBL tissues when compared to NBL tissues (P 80% accuracy. HY4-expressing transfectants secreted HY4 and Chase activity in their conditioned media. Overexpression of HYAL-4 in normal urothelial and BCa cells significantly increased, while its knockdown abrogated, anchorage-independent growth, invasion, and chemotactic motility (> 3-fold). HY4 expression induced Gemcitabine resistance (IC50: vector: 3.1 nM; HY4: 126 nM). HY4 expression upregulated a stem cell signature. Urothelial cells expressing HY4 formed tumors in NOD/SCID mice. HY4-expressing BCa cells metastasized to lung and spleen in an orthotopic BCa model. CONCLUSION: This first study on HY4 shows that it is a novel molecular determinant of MIBCa and Gemcitabine resistance and a potential marker for clinical outcome. Citation Format: Sarrah S. Lahorewala, Daley S. Morera, Andre R. Jordan, Vinata B. Lokeshwar. HYAL4: A molecular driver and potential marker of invasive 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 2655.

Introduction: Using RNA-seq (HiSeq) transcriptome data in The Cancer Genome Atlas (TCGA) bladder cancer (BCa) dataset two molecular subtypes, basal and luminal, were identified in muscle invasive BCa (MIBCa). Recurring reported markers of the basal subtype are KRT5, KRT6A, KRT14 high and KRT20, GATA3, UPK3A, FOXA1 low. The luminal subtype is the opposite pattern of expression. Double-Negative (DN) subtype was defined as no expression of the 7 markers. Luminal, basal and DN subtypes were reported as predictors of better, poor and worst prognosis, respectively, based on overall survival (OS). EGFR is defined as a squamous differentiation marker associated with poor prognosis. The objective was to assess the clinical significance of these subtypes in TCGA data with validation in datasets from Oncomine. Methods: TCGA dataset containing 407 MIBCa patients was accessed through Xena Browser. The dataset included patient demographics, clinical parameters, OS, recurrence-free survival (RFS) and the transcript levels (log2(nor._count+1) of basal and luminal markers. All available follow-up data was included (27.9±28.49; max: 166 months). 25 BCa datasets (n=360 MIBCa patients) containing these same variables were accessed through Oncomine. High and low levels were stratified by median. Association of individual basal and luminal markers and the subtypes with clinical and outcome variables was analyzed by univariate and multivariate analyses. Kaplan-Meier analysis was performed to stratify patients into risk groups for OS and RFS. Results: In TCGA data, neither basal nor luminal markers levels significantly correlated with metastasis or lymphovascular invasion (LVI); P = 0.2 to 0.9. Only KRT5 significantly but inversely correlated with lymph node (LN) positivity; P=0.011. EGFR levels did not correlate with metastasis, LVI, or LN positivity; P > 0.05. In TCGA dataset 77 (18.9%), 50 (12.3%) and 14 (3.4%) expressed basal, luminal and DN subtypes, respectively; 266 (66.4%) patients did not conform to any group. In univariate or multivariate analyses, the subtypes also did not correlate with metastasis, LVI or LN status. Only luminal subtype associated with better OS; P=0.003. However no subtype significantly correlated with RFS. In KM analysis no subtype stratified patients regarding RFS (P>0.2). Oncomine BCa datasets validated these results. Conclusion: TCGA and Oncomine datasets show that the majority of MIBCa tissues express a mixed pattern of basal and luminal markers. Furthermore, basal, luminal or DN subtypes do not associate with clinical parameters or prognosis of MIBCa patients. Citation Format: Daley S. Morera, Daniel Belew, Andre R. Jordan, Vinata B. Lokeshwar. Molecular subtypes in muscle invasive bladder cancer: Evaluation of clinical significance [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 4614.

INTRODUCTION AND OBJECTIVE: Hyaluronic acid (HA) family of molecules, HA-synthases (HAS-1,2,3), HA-receptors (CD44, RHAMM) and hyaluronidase (HYAL-1) are markers for bladder cancer (BCa) diagnosis and predicting prognosis. HA-family promotes tumor growth and metastasis by inducing epithelial mesenchymal transition (EMT). 4-Methylumbelliferone (4-MU) is an orally bioavailable dietary supplement that inhibits HA synthesis. We evaluated the expression of HA family and EMT markers in bladder tissues as well as the antitumor effects of 4-MU as a potential targeted therapeutic agent in preclinical models of BCa. METHODS: Quantitative PCR was used to measure mRNA expression of HA-family and EMT genes (β-catenin, Twist, and Snail) in 72 bladder tissue specimens (28 normal; 44 tumor); follow-up: 20.3±2.5 months; median 17 months. The effect of 4-MU (0-0.6 mM) on cell proliferation, apoptosis, intracellular signaling, and the expression of HA receptors, and EMT genes were examined in BCa cell lines by Q-PCR, immunoblotting, proximal ligation and PI-3K activity assays. Mechanism of action was analyzed by HA addition and mAkt overexpression. Effect of oral administration of 4-MU (100, 200-mg/kg) on tumor growth was analyzed in subcutaneous xenografts. RESULTS: HAS-1, -2 -3, HYAL-1 and Snail levels were 10-20-fold elevated in BCa tissues as compared to normal bladder (P3-fold) via the death receptor pathway. 4-MU downregulated HA-signaling, specifically transcript and/or protein levels of CD44, RHAMM, p-Akt, β-catenin, pβ-catenin(S552). Snail and twist were downregulated by 2-5-fold, but pβcatenin((T41/S45), pGSK-3α/β and E-cadherin levels were increased. 4-MU also inhibited CD44/PI-3K complex formation and PI-3K activity. HA addition or myristoylated Akt expression attenuated 4-MU effects. In xenograft studies, 4-MU oral treatment abrogated tumor growth of established tumors (vehicle, day 50: 766±221 mm3; 4-MU: 128±61, day 50) by abrogating HA-synthesis. No weight loss or serum or organ toxicity was observed in treated mice. CONCULUSION: This study demonstrates that HA-family signaling is upregulated in BCa and can be specifically targeted for treatment by a non-toxic dietary supplement. Support: 7R01 CA072821-16; 7R01CA176691-03 Citation Format: Daley S. Morera, Andre Jordan, Vinata B. Lokeshwar. Biomarker-driven targeted oral treatment strategy for bladder cancer [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 1146. doi:10.1158/1538-7445.AM2017-1146

Introduction: Five-year survival of metastatic renal cell carcinoma (mRCC) patients is < 10% and African American (AA) males have the highest incidence. Identification of the molecular determinants of mRCC and racial disparity in RCC is critical for biomarker development and targeted therapy. SDCT2 is expressed in kidney epithelial cells and is a succinate and citrate transporter, but its role has not been examined in any benign diseases or cancer. We examined SDCT2 expression in normal and RCC tissues and correlated it with clinical outcome and racial disparity. We also evaluated the biological functions and molecular signaling regulated by SDCT2 in RCC cells. Methods: Differential gene expression in the matched normal and RCC tissues (n=6/category) was evaluated by microarray analysis; results were validated by quantitative-PCR and immunoblotting in normal and RCC tissues from 53 patients (White=21; Hispanic=19; AA=13). VHL+ and VHL- RCC cells were stably transfected with a Flag-tagged SDCT2 construct. Transfectants were characterized for cell proliferation, cell cycle, motility, succinate/citrate transport and reactive oxygen species (ROS) measurement assays under normoxia and hypoxia (1% O2); cell death and senescence pathway markers were also evaluated. SDCT2 induction was evaluated following 5-azacytidine plus Trichostatin A treatment Results: SDCT2 was 63- and 100-fold downregulated in low- and high-stage RCC tissues, respectively. Q-PCR validation showed that SDCT2 levels were 40-fold downregulated in tumor tissues when compared to normal kidney (P Conclusion: This is the first study on a functional biomarker in RCC, SDCT2, that is a possible novel tumor suppressor gene. SDCT2 loss promotes RCC growth, survival and inhibits cellular senescence and its downregulation correlates with metastasis and racial disparity. Support: Grant NCI/NIH 5R01CA72821; 5R01CA176691 Citation Format: Andre R. Jordan, Martin Hennig, Daley S. Morera, Soum D. Lokeshwar, Asif Talukder, Lokeshwar Vinata. Prognostic significance and tumor suppressive functions of SDCT2 in renal cell carcinoma [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 1549. doi:10.1158/1538-7445.AM2017-1549