Vinata Lokeshwar - US grants

DESCRIPTION: (adapted from the investigator's abstract) Two critical factors that affect the prognosis of bladder cancer patients are high-grade invasive tumors and the high rate of tumor recurrence. Hyaluronidase is an enzyme that degrades hyaluronic acid (HA), a glycosaminoglycan, into small angiogenic fragments. Two hyaluronidases have been identified as potential "molecular markers" of high-grade bladder tumors (BT). The long-term objectives of this proposal are to elucidate functions of these hyaluronidases in BT progression and to develop a simple, non-invasive and accurate test for early detection and post-therapy surveillance of bladder cancer. To test the hypothesis that BT-derived hyaluronidases are structurally distinct from other known hyaluronidases and are expressed in a tumor, tissue, or cell specific manner, BT-derived hyaluronidases will be isolated, purified, cDNA cloned and sequenced (Aim 1). These enzymes will be characterized for substrate specificity and sensitivity to inhibitors and their expression will be examined at protein and mRNA levels in normal tissues, tumor tissues, and tumor cells (Aim 2). To test whether elevated BT-derived hyaluronidase levels confer metastatic phenotype to otherwise indolent BT cells, low-grade BT cells will be transfected with cDNAs of these enzymes and following orthotopic implantation in nude mice metastasis will be examined. In addition, the role of angiogenic HA fragments on functions of BT cells and BT-derived endothelial cells which regulate BT progression will be examined (Aim 3). Urinary HA levels are elevated in BT patients and urinary hyaluronidase levels are elevated in patients with high-grade BT. To test whether a HA-hyaluronidase test can detect bladder tumor recurrence and indicate its malignant potential, urinary HA-hyaluronidase levels in 150 BT patients will be measured at scheduled surveillance visits and results will be compared with cystoscopic or urine cytology findings (Aim 4). The proposed study could result in identifying and functionally characterizing a new marker of high-grade BT which could improve diagnosis and treatment of bladder cancer.

DESCRIPTION (provided by applicant): Despite superb efforts of patient advocacy groups, our insight into the etiology, diagnosis and treatment for interstitial cystitis (IC) is still inadequate. Since the luminal glycosaminoglycan (GAG) layer protects the bladder urothelium from noxious substances in urine, alterations in urothelial GAGs may be associated with IC pathophysiology. Urinary levels of total (i.e., non-sulfated and sulfated) GAGs and hyaluronic acid (HA) are elevated in IC patients with severe disease, as judged by the validated O'Leary-Sant questionnaire and clinical index. These patients' urine also contain one or more unique GAG species and a high molecular mass HA species. Urinary total GAG levels and GAG profile appear to be accurate markers for monitoring disease severity, regardless of the type of treatment the patients are undergoing. IC urothelial cultures secrete higher levels of matrix metalloproteinases (MMPs)-2 and -9, when compared with normal urothelial cells, suggesting an association between MMPs and this disease. This proposal is designed to investigate the involvement of IC specific GAGs, high molecular mass HA and MMPs in the pathophysiology of IC and how GAG-like substances may bring about symptom relief. Furthermore, to evaluate in a multi-center trial the usefulness of total urinary GAG and HA levels, GAG and HA profiles and urinary MMP levels in the follow-up of IC patients. To identify IC-specific GAGs and their cellular source, these GAGs will be purified from IC patients' urine and primary urothelial culture conditioned media (CM), by sequential liquid chromatographies, digestion with GAG-degrading enzymes and HPLC (Aim 1). The cellular basis of qualitative and quantitative alterations in GAGs, will be evaluated by performing a pair-wise comparison of GAG levels and GAG profile in urine, tissue extracts and urothelial CM from IC patients with varying degrees of disease severity. The involvement of IC-specific GAGs in IC pathophysiology and of GAG-like substances in causing symptom relief, will be evaluated by cDNA microarray analysis of changes in gene expression in normal and IC urothelial cells treated with IC-specific GAGs and pentosan polysulfate, respectively (Aim 2). To understand their association with IC, HA levels; HA profile and MMP levels will be analyzed in urine, tissue extracts and urothelial CM from IC patients with varying degrees of disease severity. Changes in normal urothelial gene expression following treatment with high molecular mass HA will be evaluated by cDNA microarray analysis, and compared with gene expression in IC urothelial cells, to reveal the involvement of HA in IC pathophysiology (Aim 3). In a multi-center trial, the usefulness of total GAG levels, GAG and HA profile and MMP levels in IC patient follow-up and their use for monitoring treatment response will be evaluated (Aim 4). The proposed study will reveal the function and diagnostic potential of urothelial GAGs (including HA) and MMPs in 1C pathophysiology. Furthermore, it might yield a test or a combination of tests that can be used in the follow-up of IC patients and for monitoring 9 treatment responses.

DESCRIPTION (provided by applicant): Due to tumor heterogeneity in progression and frequent recurrence, the management of bladder cancer (BCa) patients is both challenging and costly. Hyaluronic acid (HA) is a glycosaminoglycan and HYAL1 is a tumor cell-derived hyaluronidase (HAase) that degrades HA into angiogenic fragments. HYAL1, HA and HA-synthase (HAS) are molecular determinants of BCa growth, invasion and angiogenesis, and are also accurate markers of BCa diagnosis. The objectives of this proposal are to examine the mechanism by which HA-HYAL1 system promotes BCa growth and progression, how HYAL1 gene expression is regulated in BCa, and do HA-HYAL1 family of markers have any prognostic potential for BCa patients. Sulfated HA compounds inhibit HYAL1 activity and inhibit BCa growth by causing cell cycle arrest and apoptosis. 4-methylumbelliferone (4-MU) inhibits HA synthesis and inhibits BCa cell growth by inducing apoptosis. 4-MU and sHA are non-toxic and orally bioavailable. To examine how HYAL1 and HA regulate BCa growth and progression, we will evaluate the effects of sHA compounds and 4-MU in vitro and in an orthotopic BCa model that metastasizes to lung. The intracellular mechanisms by which sHA and 4-MU induce cell cycle arrest and/or apoptosis and affect HA receptor-mediated signaling in BCa and normal cells will also be examined (Aim 1). Currently, it is unknown how HYAL1 expression is regulated in normal and tumor tissues. Cloning of HYAL1 promoter showed that DNA methylation and certain transcription factors are involved in HYAL1 gene regulation in BCa cells. To examine the mechanism of HYAL1 regulation, a promoter methylation and HYAL1 expression will be evaluated in a cohort of normal and BCa tissues. Interaction between a transcription factor and HYAL1 promoter will be evaluated in BCa cells and tissues by EMSA and ChiP assay and siRNA transfection. Regulation of methylation and HYAL1 promoter activity by HA-HYAL1 signaling will also be evaluated (Aim 2). In BCa, the expression of HA, HYAL1 and HA-syntheses (HAS1, 2, 3) is elevated. The efficacy of HA-HYAL1 family of markers to predict disease progression, tumor recurrence and treatment response will be evaluated in a multi-center retro-prospective study that involves tissue microarrays (Aim 3). This study should reveal how HA-HYAL1 promotes BCa growth and progression and whether it can be controlled by HA and HAase inhibitors. It will also reveal how HYAL1 expression is regulated in BCa tissues and may suggest ways to control it. The study should establish whether HA- HYAL1 family members can predict prognosis for BCa patients. PUBLIC HEALTH RELEVANCE: The study should reveal how HA-HYAL1 promotes BCa growth and progression and whether it can be controlled by HA and HAase inhibitors. It will also reveal how HYAL1 expression is regulated in BCa tissues and may suggest ways to control it. The study should establish whether HA-HYAL1 family members have a place in the clinical management of BCa patients for predicting prognosis.

[unreadable] DESCRIPTION (provided by applicant): The overall objective of this proposal is to target the tumor-associated hyaluronic acid-hyaluronidase (HA-HAase) system to develop novel therapeutics for prostate cancer (CaP). HA is a glycosaminoglycan that promotes tumor growth and metastasis and HAase is an endoglycosidase that degrades HA into angiogenic fragments. HYAL1 is a tumor-derived HAase, secreted by CaP cells. Combined HA-HYAL1 expression in CaP tissues is an independent prognostic indicator for predicting CaP progression. Blocking HYAL1 expression in CaP cells, by antisense cDNA transfection, decreases tumor growth, invasion and vascularization in xenografts. Sulfated HA (sHA) polymer and small sHA oligosaccharides are potent inhibitors of HYAL1 activity which inhibit CaP cell growth by causing cell-cycle arrest and inhibit the growth of CaP xenografts. These agents decrease activation of focal adhesion kinase and down regulate androgen receptor levels and activity. 4-methyl-umbelliferone (4-MU), used as a dietary supplement for the treatment of liver aliments, inhibits HA synthesis. By blocking pericellular HA formation, 4-MU induces apoptosis in CaP cells by caspase-8 activation and inhibits the growth of CaP xenografts. Both sHA and 4-MU are orally bioavailable, active against both androgen dependent and independent CaP cells and show no histologic or systemic toxicity upon long-term treatment. The main hypothesis to be tested in the proposed project is that, sHA and 4-MU, either alone or in combination, will abrogate CaP growth and its progression. The efficacy of sHA compounds to inhibit cell growth and invasion will be evaluated in vitro. Inhibition of tumor growth, metastasis and angiogenesis will be examined in 3 CaP xenograft models (i.e., orthotopic tumor implant, induced metastasis and intra-osseous growth models). Effect of sHA on HA-HYAL1 mediated intracellular signaling will also be investigated (Aim 1). Effect of 4-MU in controlling CaP cell growth, invasion and motility will be investigated in vitro. Inhibition of tumor growth and metastasis by 4-MU, either alone or in combination with sHA, will be examined in the three CaP xenograft models, as described above. Mechanism of 4-MU induced apoptosis and abrogation of HA- mediated intracellular signaling will also be investigated (Aim 2). Efficacy of sHA and 4-MU, either alone or in combination, as stage-specific treatments for CaP growth and metastasis will be investigated in two transgenic models of CaP (Aim 3). Relevance: This is the first study that proposes to evaluate inhibitors of the HA-HYAL1 system as therapeutic agents for the treatment of CaP. If proven efficacious, sHA compounds and 4-MU will be a novel class of glycosaminoglycan-based target-specific anti-tumor drugs. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application seeks funds to provide 25 travel awards of $1000 each to trainee investigators (graduate students, residents, clinical and postdoctoral fellows and faculty below the rank of Assistant Professor) to participate in the 8th World Basic Urological Research Congress. This is a joint meeting between the Society for Basic Urologic Research (SBUR) and the European Society for Urological Research (ESUR) and is also the 19th Annual Fall SBUR Meeting. The World Congress will be held between November 5th and 8th, 2009, in New Orleans, Louisiana. SBUR is a society of scientists specializing in research on benign and malignant urologic diseases. The Society has held its independent Fall Annual Symposia for 18 consecutive years with great success. Timely subject of the year is addressed at each meeting with special emphasis on a cross-fertilization of urology and another relevant field of research. The 2009 meeting will focus on the theme "Molecular Targets for Diagnostics and Therapeutics in Urology". The meeting's objectives are to accelerate the translation of new diagnostic and prognostic markers and treatments to the patient bedside, identify and exploit novel molecular and cellular targets, and learn about new experimental strategies, concepts and cutting-edge technologies. Key advancements in benign and malignant diseases of the bladder, prostate, and kidneys will be emphasized through a mini-symposium on dialogues in clinical and translational research. The trainees will have opportunities to network among peers and senior members of both Societies, to present their data at poster sessions and, in selected cases, as podium presentations. Invited speakers at the meeting are leaders in their respective fields, offering extensive knowledge and experience. The atmosphere of the meetings is friendly and informal, providing encouragement and unique opportunities for trainees to participate and establish collaborations. In order for such interactions to occur, these trainees need support for their travel expenses. If funded, this grant will contribute significantly to the educational objectives of this meeting. The objective of this 8th World Urological Research Congress (19th Fall Society for Basic Urologic Research Meeting) is to strengthen the ongoing dialogue between basic and clinical scientists in our study of urologic disease and genitourinary tract health. Our goals are to accelerate the translation of new markers and treatments to the patient bedside, by identifying and exploiting novel molecular and cellular targets, and by learning about new experimental strategies, concepts and cutting-edge technologies. Additional features of this meeting include, a job fair and a satellite "Grants - How to" interactive forum between meeting attendees and experts from granting agencies.

* DESCRIPTION (provided by applicant): Summary: More than 1/3rd of renal cell carcinoma (RCC) patients either have or develop metastatic-RCC (mRCC) despite nephrectomy and adjuvant treatments. Survival of mRCC patients at 5-years is < 10%. The major goal of this project is to develop a dietary supplement-based prevention and treatment strategy against mRCC. Nexavar? (SF) is an FDA approved oral angiogenesis inhibitor which improves overall survival by 12- 18%, causes disease stabilization for about 8-weeks and has a high failure rate. Although the cause of SF failure is unknown, its glucuronidation by UDP-Glucuronyltransferase-1A9 (A9) is a plausible one, because glucuronidation inactivates SF. Preliminary results presented in this application show for the first time that when compared to the SF-responders, A9 levels and SF-glucuronidation are significantly higher in tumors from those patients who fail SF treatment. Furthermore, a non-toxic dietary supplement Hymecromone (HC), consumed extensively in Asia and Europe, inhibits SF glucuronidation by downregulating A9 expression. In RCC and endothelial cells, the combination of HC and SF inhibited viability, motility, invasion and capillary formation. By inhibiting novel molecular targets in RCC and stromal cells, including A9, the combination abrogated signaling pathways that drive RCC cell survival, metastasis and angiogenesis. At concentrations less than ten-fold of those used for consumption, HC when combined with SF completely eliminated tumor growth in a SF-resistant RCC model, without toxicity. Tissue and plasma levels of SF and HC were well above the doses needed for the activity of HC+SF. The central hypothesis is that by inhibiting novel targets, HC+SF combination abrogates RCC and endothelial cell functions leading to the prevention and elimination of RCC growth, angiogenesis and metastasis. To test this hypothesis, the molecular basis of HC+SF activity will be examined in RCC and stromal cell co-cultures. Next the bioavailability and toxicity of HC+SF will be evaluated, along with the analysis of the molecular targets of HC and SF, as biomarkers, to predict RCC metastasis and response to SF. Finally, the efficacy of the HC+SF combination, to prevent tumor growth and metastasis, will be examined in spontaneously metastatic-orthotopic RCC models. Impact: This study should lead to an effective strategy for the prevention and control of mRCC that combines HC, a non-toxic dietary supplement, with SF. Evaluation of activity, bioavailability and toxicity in pre-clinical models and prediction of response may advance this dietary combination for clinical application.

DESCRIPTION (provided by applicant): Castration resistant prostate cancer (CRPC) is incurable as the current anti-CRPC treatments do not extend life beyond a few months. Cytochrome P450 3A4 (3A4) is an enzyme that metabolizes and inactivates a number of xenobiotics, including anti-CRPC drugs Abiraterone and Docetaxel. Although, such metabolism usually occurs in the liver, preliminary data show elevated expression of 3A4 (mRNA and protein) and activity in prostate cancer (PCa) cells, when compared to normal prostate cells. Further, a dietary supplement, 4-methylumbelliferone (MU) downregulates 3A4 expression in PCa cells; consequently MU downregulates 3A4 activity and enhances cytotoxic effects of anti-CRPC drugs. Interestingly, a 3A4-variant (3A41B) generated by DNA polymorphism correlates with PCa metastasis and has high promoter activity. This exploratory project is designed to address an understudied area in PCa biology: is elevated expression of 3A4 in PCa cells a mechanism for their reduced sensitivity to anti-CRPC agents? Further, does downregulation of 3A4 expression in PCa cells, by a non-toxic dietary supplement, enhance the sensitivity of PCa cells to anti- CRPC agents? The role of 3A4 in PCa cells will be examined by determining whether modulation of 3A4 levels (over expression and silencing) and MU treatment alters the sensitivity of PCa cells to anti-CRPC agents. Furthermore, does 3A4 expressed in PCa cells inactivate anti-CRPC drugs and whether MU prevents this inactivation? Studies will also investigate the mechanism by which MU downregulates 3A4 levels; specifically, whether MU increases the degradation of 3A4 mRNA or transcriptionally silences both 3A4 wild type and variant 3A41B promoters. Efficacy of anti-CRPC agents, either alone or in combination with MU will be tested to inhibit the growth and progression of castration-resistant prostate tumors in xenograft models. PCa cells used in the xenograft will be those in which 3A4 expression is either up- or down-regulated. Impact: This exploratory project should reveal the role of 3A4 expressed in PCa cells in modulating the response of PCa cells to anti-CRPC agents. Furthermore, does combination with a non-toxic dietary supplement that specifically targets 3A4 expression enhances the sensitivity of PCa cells to anti-CRPC agents. If successful, the project has potential for clinical translation, in which based on the 3A4 levels in tumors a 3A4 inhibitor could be added to improve response to anti-CRPC treatments.

ABSTRACT: The current clinical practice of invasive diagnostic workup, frequent recurrence following bladder tumor resection, and high propensity of muscle invasive bladder cancer (MIBCa) for metastasis, contribute to the significant morbidity and mortality associated with bladder cancer (BCa); one of the costliest cancer to manage clinically. Painless hematuria is the common presenting symptom among BCa patients. However, only 15-25% of patients with visible hematuria and about 10% of the patients with microscopic hematuria actually have BCa. Patients presenting with hematuria undergo urological workup that involves cystoscopy and CT- urography. Cystoscopy is invasive and uncomfortable and both procedures associate with considerable risk for morbidity and are costly. Frequent BCa recurrence requires surveillance by cystoscopy every 3 to 6 months. Non-invasive biomarkers, including urine cytology, can reduce the invasive and costly workup among patients with hematuria or for those under surveillance for monitoring BCa recurrence. However, existing biomarkers are not used in clinic due to their sub-optimal efficacy and/or high false-positive rate. Further, biomarkers are not used for predicting clinical outcome in terms of metastasis or treatment response after a bladder removal surgery. In pilot studies, two new non-invasive tests based on a novel member of a glycosaminoglycan family, showed high accuracy to detect BCa, for non-invasively evaluating its grade and for early detection of BCa recurrence. Biomarker levels in BCa tissues correlated with clinical outcome. This project is designed to evaluate a hypothesis that this two urine tests can accurately diagnose BCa and non-invasively evaluate its grade among patients needing urological workup for hematuria, or to monitor BCa recurrence. Furthermore the tissue-based biomarkers accurately predict clinical outcome. The efficacy of both urine tests will be evaluated for BCa diagnosis and for evaluation of its grade among patients undergoing urological workup for hematuria (Aim 1). Efficacy of these urine tests will be compared to cystoscopy findings in patients under surveillance for monitoring BCa recurrence. The tests? findings will also be evaluated for predicting future recurrence (Aim 2). The biomarkers? expression in BCa tissues will be examined for predicting the development of MIBCa, metastasis and overall clinical outcome (Aim3). Impact: The study may result in two validated urine tests for non-invasively and accurately detecting BCa with grade evaluation as an added benefit. This could substantially reduce the number of patients undergoing urological workup for hematuria or surveillance cystoscopies for monitoring BCa recurrence. Tissue biomarkers, if found to be accurate prognosticators, could reduce unnecessary invasive bladder re-resections for MIBCa, and aid in early intervention and individualized treatment for patients with advanced BCa. Overall, if efficacious, these biomarkers could reduce BCa-related morbidity, and costs, while improving clinical outcome by early predictions.