Research

Research in the Bladder SPORE will be associated with either the developmental research program (for new pilot projects) or with one of five core, multidisciplinary projects:

Project 1: Forerunner Genes: A Novel Class of Early Detection Markers for Bladder Cancer

Project Leaders
Dr. Bogdan Czerniak, professor of pathology
Dr. Menashe Bar-Eli, professor of cancer biology
Dr. H. Barton Grossman, professor of urology
Dr. Keith Baggerly, biostatistician

Identification of early changes associated with the development of preneoplastic conditions would provide important clue on early events of carcinogenesis and could aid in the development of novel markers for early identification of occult neoplasia. We performed genome-wide search for losses of genetic material on multiple DNA samples corresponding to normal urothelium, in situ preneoplastic lesions and invasive carcinoma extracted from the entire mucosal surface of resected human bladders. The analysis of hits associated with growth advantage of preneoplastic lesions allowed us to identify six chromosomal regions mapping to 3q22.1, 5q22.2-5q23.3, 9q22.12, 10q26.1, 13q14 and 17p13 that may be critical for the development of bladder cancer and contain a distinct class of genes referred to as forerunner (FR) genes involved in the clonal expansion of precursor conditions. We concentrated our efforts on one of these regions, which contain a model tumor suppressor RB1. We used high-resolution whole-organ mapping with SNPs, which facilitated the identification of three positional candidate FR genes (ITM2B, P2RY5 and CHC1L) mapping contiguously to RB1. We also provide evidence that their inactivation is likely to be critical for the development of preneoplastic lesions. Using combined genetic and epigenetic mapping of the same region we have identified an additional FR gene contiguous to RB1 (GPR38). The expression, mutational and methylation analyses showed that at least two of the prototypic FR genes (ITM2B and GPR38) are frequently methylated in bladder cancer and their methylation can be detected in voided urine samples. This proposal describes our continuing studies focused on the role and function of the prototypic FR gene (ITM2B) contiguous to RB1. Our preliminary data implicate that this gene plays a key role in controlling proliferation of preneoplastic clone and is frequently methylated in bladder cancer. Therefore, in addition to the functional studies we propose to use the frequently methylated FR genes combined with other methylation markers for the detection of bladder cancer.

The specific aims for our study are as follows:

Clarify the role and function of the FR gene (ITM2B) in bladder cancer
Identify an effective methylation panel of genes, including FR genes for detecting bladder cancer in urine
Validate the methylation panel of biomarkers in a prospective cohort of patients at high risk of bladder cancer recurrence)

Project 2: Markers of Susceptibility as Predictors of Bladder Cancer Recurrence

Project Leaders
Dr. Xifeng Wu, professor of epidemiology
Dr. H. Barton Grossman, professor of urology
Dr. Christopher Amos, biostatistician

We plan to build upon the epidemiologic and genetic findings and the existing specimen and data repository from the initial SPORE project and our funded research to evaluate predictors of bladder cancer (BC) recurrence in 800 patients with superficial BC. We have previously identified several promising markers in pathways for tobacco carcinogen activation / detoxification and DNA damage / repair. Now we propose to extend our candidate gene approach to a pathway-based approach to systematically examine the joint influence of genetic polymorphisms in specific pathways on clinical outcome; to extend our panel from metabolism and DNA damage/repair pathways to inflammation, oxidative stress and the TRAIL-induced apoptosis pathways; to integrate clinical and epidemiologic data with the genetic data to build a quantitative risk assessment model for BC recurrence; to link our research to an ongoing BCG clinical trial on superficial BC; and finally to explore functional significance of the SNPs.

Our specific aims are as follows:

Specific Aim 1a

Construct a well-characterized cohort of 800 patients with superficial BC.

Specific Aim 1b

Assess frequencies of genetic predisposition markers in all 800 cases including SNPs in genes that are involved in inflammatory processes, oxidative stress and TRAIL induced apoptosis. Our hypothesis is that polymorphisms in these genes are associated with risk of BC recurrence, and may also have effects on BCG treatment response in patients with superficial BC.

Specific Aim 1c

Build up risk assessment model for BC recurrence. We will integrate clinical and epidemiologic data with the genetic data from the studies described above as well as from the initial grants to build a quantitative risk assessment model for BC recurrence. Genetic variations on metabolic enzyme genes, microenvironment genes and DNA repair genes will be available from our other funding sources to be incorporated into the model

Specific Aim 2

Assess determinants of BCG treatment response in 200 patients with superficial BC enrolled in a clinical trial. We will measure protein levels of IL-2, IL-8 and TRAIL in voided urine specimens. We will correlate these protein levels as well as SNPs in pathways relevant to the the action of BCG (especially genes involved in inflammation, oxidative stress and TRAIL apoptosis pathways with recurrence rates. Our hypothesis is that specific genotypes in genes related to the mechanism of BCG action may negatively affect the treatment response of patients with BC to this therapy. As a secondary aim, we will use experimental approaches and/or computational approaches to verify or discover the functional impact of promising polymorphisms. Our hypothesis is that SNPs associated with bladder cancer recurrence alter the function of their genes. The ability to rapidly screen individuals for BC recurrence risk using minimally invasive procedures (blood and urine samples) has immense clinical value. These markers will be useful for identifying patient subgroups at high risk for recurrence who should undergo more intensive screening. Markers of treatment response could be used to design individualized treatment plans.

Project 3: Death Receptors in Bladder Cancer Therapy

Project Leaders
Dr. David McConkey, professor of cancer biology
Dr. Randall Millikan, associate professor in genitourinary medical oncology
Dr. Ashish Kamat, assistant professor of urology
Dr. Yu Shen, biostatistician

Interferons and other immunomodulators (i.e., BCG) are used as front-line therapy in patients with bladder cancer, but their mechanisms of action remain unclear. Interferons have potent anti-angiogenic effects that appear to contribute to their anti-tumor activities. In addition, in studies performed by our laboratories during the first cycle of SPORE funding, we demonstrated that interferon-a (IFNa) induced apoptosis in a subset (6/20) of human bladder cancer cell lines and that IFNa-induced expression of tumor necrosis factor apoptosis-inducing ligand (TRAIL) was involved. In parallel studies we demonstrated that some bladder cancer cells are IFN-resistant because they are resistant to TRAIL. Importantly, we showed that resistance to IFN or TRAIL could be reversed by incubating them with the proteasome inhibitor, bortezomib (PS-341, Velcade) or the histone deaceylase inhibitor, SAHA, and our preliminary data strongly suggest that they do so by promoting the p53-independent accumulation of the cyclin-dependent kinase inhibitor, p21Waf-1/Cip-1. The overall goal of the studies proposed in this competing renewal is to use this information to design an effective, death receptor-based therapeutic strategy for the treatment of urothelial cancer.

To this end, we propose the following specific aims:

Define the role of p21 in bortezomib- or SAHA-mediated sensitization of urothelial carcinoma cells to IFN or TRAIL
Evaluate the efficacy and toxicity of TRAIL- or IFN-based combination therapies in orthotopic bladder tumors in vivo
Develop methods to measure pharmacodynamic markers of biological response to bortezomib-based therapy in patients

These experiments will complement parallel studies being performed in Projects 4 and 5, where alternative strategies to enhance TRAIL sensitivity and/or bypass the TRAIL pathway altogether are being explored. The most important distinction between this project and previous work with biological agents is that it appears that cytostatic agents promote TRAIL-induced apoptosis in tumor cells, whereas they can undermine the effects of many (if not most) conventional chemotherapeutic agents. All of the compounds being studied are either already FDA-approved for the treatment of cancer or are in the process of being evaluated in Phase I-III clinical trials. Therefore, an important feature of this project is that we will open a clinical trial of our most promising combination by the 4th year of SPORE funding

Project 4: Biology and Therapeutic Targeting of the Epidermal Growth Factor Receptor in Bladder Cancer

Project Leaders
Dr. Menashe Bar-Eli, professor of cancer biology
Dr. David McConkey, professor of cancer biology
Dr. Colin P. N. Dinney, professor of urology
Dr. Arlene Siefker-Radtke, professor of genitourinary medical oncology
Dr. Yu Shen, biostatistician

The epidermal growth factor receptor (EGFR) is overexpressed during bladder cancer progression, and clinically relevant EGFR antagonists inhibit the growth of tumor xenografts in vivo. However, EGFR inhibitors not displayed consistent activity in clinical trials performed in other disease sites, which has dampened clinical enthusiasm for aggressively developing them further. Studies performed within the context of non-small cell lung cancer demonstrated that clinical responses were linked to activating mutations within the EGFR tyrosine kinase domain, suggesting that a better understanding of the biological effects of EGFR inhibitors on tumor cells will help to identify tumors that will respond (biologically or clinically) to therapy. In preliminary studies conducted during the first cycle of SPORE funding, we found that EGFR inhibitors (gefitinib or cetuximab) blocked cell cycle progression in 8/20 human bladder cancer cell lines, and we have identified molecular mechanisms that appear to mediate these effects. Although none of the cell lines nor any of 75 primary tumors contained activating EGFR kinase domain mutations, preliminary data suggests that over 50% of primary tumors express a truncated form of the EGFR (EGFRvIII) that mediates ligand-independent signaling and promotes tumor growth in other model systems. Therefore, the overall goal of this project is to define the biological properties associated with EGFR-dependent growth in bladder cancer cells and to develop pharmacodynamic markers that can be used to monitor them in tissue biopsies harvested during therapy.

We propose the following specific aims:

Define the molecular mechanisms underlying the antiproliferative effects of EGFR inhibitors in urothelial carcinoma cells
Determine the functional significance of EGFR-vIII expression in urothelial carcinoma
Define the role of the host response in the angiogenesis inhibition observed in response to EGFR-directed therapy
Evaluate the activity of combined therapy with EGFR inhibitors and TRAIL

An important component of this project is a clinical trial designed to test the effects of cetuximab on pharmacodynamic markers in primary tumors. Thus, we are in a unique position to directly test the validity of the hypotheses we have generated in our preclinical studies and to exploit them in the design of more effective, EGR-based therapeutic strategies

Project 5: Improving Gene Therapy for Superficial Bladder Cancer

Project Leaders
Dr. William Benedict, professor of genitourinary medical oncology
Dr. Colin P. N. Dinney, professor of urology
Dr. Yu Shen, biostatistician

In the first cycle of SPORE funding, we developed a novel, reproducible model for human superficial bladder cancer in athymic mice. With this model we demonstrated gene transfer and efficacy of intravesical interferon-a gene therapy when the adenoviral vector was administered with Syn3 (Ad-IFN-a/Syn3). We showed that Ad-IFN-a/Syn3 induced caspase-3 activation and apoptosis in some human bladder cancer cells that were insensitive to recombinant IFNa protein and that cell death occurred via direct and indirect (bystander) effects. Moreover, normal urothelial cells appear to be resistant to Ad-IFN-a/Syn3. Although the exact mechanisms underlying the effects of Ad-IFN-a/Syn3 are unknown, the preclinical data were compelling enough to launch a Phase I study, which is scheduled to open in the fourth quarter of 2005. In this renewal we propose to define the unique mechanisms by which Ad-IFN-a mediates its antitumor activity. We also propose to develop pharmacodynamic urine-based markers that will allow us to track the biological activity of Ad-IFN-a/Syn3, focusing on the possibility that increased levels of soluble annexin 1 correlate with transduction efficiency, tumor cell apoptosis or both. Finally, by using an elegant whole organ mapping technique developed by our colleagues in Project 1, we propose to accurately map the expression of interferon-a, in a proof-of-principle experiment that will follow the Phase I trial.

To this end, we propose three specific aims:

To define the role of annexin-1 in apoptosis induced by Ad-IFNs
To identify the molecular mechanisms involved in the Ad-IFN-induced bystander effect
To evaluate transgene expression of IFN-a and the activity of Ad-IFN/Syn3 following intravesical instillation into the bladder of patients with urothelial carcinoma

These studies have the potential to establish a new paradigm for the therapy of superficial bladder cancer, which might qualitatively change the natural history of this disease.

Developmental Research Program

Directors
Dr. David McConkey, professor of cancer biology
Dr. H. Barton Grossman, professor of urology
Dr. Anita Sabichi, associate professor of clinical cancer prevention

The aim of the SPORE Developmental Research Program is to provide initial funding for promising pilot studies in bladder cancer. The research is designed to generate clinically testable hypotheses. It is also geared toward reducing the incidence and mortality of bladder cancer or improving the quality of life for bladder cancer patients.