Keith Syson Chan - US grants

DESCRIPTION (provided by applicant): Bladder cancer is the second most common urological malignancy in the United States and is usually not curable at its advanced stage. However, basic research funding geared specifically to diseases in this organ site is relatively meager. It has long been observed that human tumors are heterogenous, as defined by histology or biological properties (i.e. anchorage independent growth property, proliferation and differentiation status). One model hypothesizes the existence of a hierarchy in tumors, which may explain the heterogeneity of tumor cell population within a tumor. This model proposes that rare tumor-initiating cells with stem cell-like properties (cancer stem cells) exist, which can self-renew and differentiate into phenotypically diverse tumor cells that reconstitute the heterogeneity of original tumor. In the current proposal, I describe for the first time the successful isolation and initial characterization of a rare CD44 positive, bladder cancer stem cells (CSCs) from patient tumor. I hypothesize that CSCs exist in all patient bladder tumors and therefore can be prospectively isolated based on their unique biological properties. The primary experimental approaches will be utilizing a combination of cell surface markers (Aim 1A, B&C) and constructs that report on self-renewal signaling pathways (Aim2A). Localization of bladder CSCs and the correlation of their expression level to disease progression will be determined by immunohistochemical analysis in tissue sections and high throughput tissue microarrays (Aim1 D). Further, in order to define a molecular signature" for bladder CSCs, two separate approaches will be taken (Aim3): (I) a small scale molecular screen to look at specific "sternness genes" at mRNA level by quantitative RT-PCR (Aim3Ai), and utilization of cutting edge microfluidics western technologies to look at these candidates at protein level (Aim3Aii);(II) a genome wide screen using HEEBO microarrays (Aim 3B). Preclinical targeting of self- pathways unique to bladder CSCs (Aim2B) in comparison to the MARK pathway (Aim2C) will reveal drug targets in vivo. I hypothesize that these CSCs are in fact the "roots", and therefore ideal targets for future anti-cancer therapies. My long term goal is to enrich for a highly purified population of bladder CSCs, further characterize their biological properties (e.g. ability to invade, and migrate to distal organs), and to develop specific strategies to target such CSCs in attempt to eradicate advanced stage bladder cancers.

DESCRIPTION (provided by applicant): Bladder cancer, the fifth most common malignancy in the U.S. with approximately 70,000 new cases diagnosed each year, is essentially incurable after it has begun to progress. Much of the difficulty lies in the scarcity of mechanistic insightsin the functional involvement of the fibroblastic microenvironment, and its interactions with the epithelial cancer compartment in modulating early bladder cancer invasion. The applicant and his collaborators have made considerable strides in closing this gap. They were among the first to isolate and characterize human bladder cancer stem cells. Recently, they showed that an expansion of phenotypic cancer stem cells correlates with a poorer clinical outcome and bladder cancer invasion. Their proposal aims to extend these findings by elucidating the functional contribution of cancer-associated fibroblasts (CAFs) in the modulation of bladder cancer stem cells and tumor progression. This application has its foundation via demonstrating that bladder cancer patients with an elevated expression of CAF genes also have muscle-invasive cancer and a poorer survival. Moreover, activated CAFs localize immediately adjacent to phenotypic cancer stem cells, suggesting a possible functional interaction between these two cell types. Additional findings indicate an important role of collagen I (COL1) excreted by CAFs, which can bind to discoidin domain receptor tyrosine kinase 1 (DDR1) on bladder cancer cells and modulate their tumorigenic properties. This progress has led to a novel working hypothesis - that COL1-DDR1 signaling between activated CAFs and bladder cancer cells is one of the mechanisms exerted by CAFs to regulate cancer stem cells and tumor progression. Three specific research aims will be pursued. Aim 1 seeks to establish the functional involvement of CAFs in the regulation of cancer stem cells and tumor progression, with an emphasis on using primary patient specimens. To further define the clinical significance of CAFs, we will utilize tw independent patient cohorts to assess the expression of CAFs/COL1 in relation to bladder cancer differentiation and selected clinical prognostic information. Aim 2 will employ both gain-of-function and loss-of-function strategies to define the functional contributions of DDR1 on bladder cancer cells to mediate the biological properties promoted by CAFs. These studies will be aided by the availability of newly isolated bladder CAFs and the applicant's expertise in isolating cancer stem cells for subsequent analysis. Finally, Aim 3 will access Stat3 and additional downstream mechanisms related to DDR1, using established proteomics profiling approach. The applicant intends to uncover druggable targets within this pathway for early bladder cancer intervention, as the long-term goal of this proposal.

PROJECT SUMMARY Metastatic progression of the primary tumor accounts for the majority of cancer deaths. While the initial steps of the metastatic cascade are rather well defined, identification of targets to block this process remains a major clinical challenge. Previous studies have elegantly investigated the mechanistic contribution of tumor cell intrinsic properties that promote metastasis in bladder urothelial carcinomas. However, the functional significance of the tumor microenvironment and its contribution to this complicated process is not well characterized, and therefore, warrants investigation. The long-term goal of this renewal application is to continue explore how collagens?a major extracellular matrix component of the microenvironment?act as a ligand to mediate crosstalk with their receptor on tumor cells to facilitate the metastatic cascade. We will investigate the downstream regulatory mechanisms of collagen receptor signaling in both the primary tumor and metastatic sites, and to exploit these regulatory processes as a revolutionizing approach to target metastases. Such innovative approaches to perturb collagen-cancer crosstalk?not only at the primary tumor but also at the metastatic niche?will move the field forward by providing a new conception in metastatic disease management, and likely extend beyond bladder carcinomas to other cancer types.

PROJECT SUMMARYTO APPLICATION This application is in direct response to the RFA-CA-19-049 ?Revision Applications for Mechanisms of Cancer Drug Resistance?, to investigate a non-classical mechanism driving the chemoresistance of locally advanced bladder carcinomas. Advanced bladder cancer claims approximately 18,000 deaths annually in the United States; yet, funding and research devoted to this cancer-type are significantly under-proportioned. A major clinical setback for bladder cancer treatment lies in the poor patient response towards chemotherapy with treatments providing only a dismal 5% improvement in overall survival. Thus, the goal of this application aims to understand the less-studied biology, i.e., cancer cell-extrinsic mechanisms, causing chemotherapy drug resistance. The success of chemotherapy is long thought to depend on its direct cytotoxic effects upon tumor cells and this has attracted much research interest in the past decades. However, there is growing evidence, as shown by our own research and others, that successful chemotherapy is also dependent on: 1) feedforward signals between dying cancer cells and the stromal fibroblast microenvironment, and 2) the active role(s) of collagens secreted from cancer associated fibroblasts to serve as a protective niche for chemoresistant cancer cells. Our proposal will leverage existing patient-derived cancer associated fibroblasts and patient-derived primary tumor cultures/xenografts developed from the parent grant NCI CA175397, which allow us to venture into a new research direction: i.e., how cell death associated release of extracellular factors impacts the fibrotic environment, consequently serves as a protective niche to fuel chemoresistant cancer cells? This proposal is innovative to elucidate the intricate feedforward loops between dying cancer cells, cancer-associated fibroblasts, and residual cancer stem cells during the emergence of chemoresistance. The success of this study will be innovative and impactful to challenge the current paradigm that chemotherapy drug resistance is primarily driven by cancer cell-intrinsic properties. Indeed, success of our proposal will establish an original concept: Dying cancer cells provide feedforward signals to cultivate a collagen-rich fibrotic environment, consequently initiating a ?vicious cycle? of epithelial-stromal feedforward signaling loop to fuel chemoresistance. Also, our proposal will provide new insights into the rational design of future intervention strategies to ?break? this vicious cycle, and thus, improve chemotherapy response. Such a concept likely extends beyond bladder carcinomas into other cancer types.

PROJECT SUMMARY This application is in response to PAR-19-183: Biology of Bladder Cancer. Muscle invasive bladder cancer (MIBC) claims approximately 18,000 deaths annually in the United States. Funding and research devoted to this cancer-type are significantly under-proportioned. An unmet clinical need for MIBC treatment lies in the poor patient response towards chemotherapy, with treatments providing only a dismal 5% improvement in overall survival. The long-term goal of this application is to address this urgent need for adjuvant therapies to improve chemotherapeutic response. The success of chemotherapy is historically thought to solely depend on its direct cytotoxic effects on tumor cells. However, there is growing evidence, as shown by our own research and others, that chemotherapeutic efficacy is also dependent on 1) successful prevention of cancer stem cells in repopulating residual tumors and 2) an effective anti-tumoral immune response. These two phenomena are often investigated separately but their possible synergy has been overlooked. Our research project is conceptually innovative to examine a common upstream pathway that regulates both tumor repopulation and immune response. We hypothesize that the inhibition of this common pathway will provide an effective therapeutic target for clinical translation. Our specific aims include: Aim 1) Decipher this pathway by investigating the non-canonical downstream mechanism leading to the extracellular release of pleiotropic factors. This is significant, since these extracellular factors can modulate both tumor repopulation and immune response. Aim 2) Evaluate how these extracellular factors and their cognate receptors drive the repopulation of quiescent cancer stem cells. Aim 3) Investigate how inhibition of this upstream pathway can collectively abrogate tumor repopulation and immunosuppression, and thus, enhance chemotherapeutic response. Success of this proposal will pose drug targets capable of augmenting patient response to chemotherapy. Moreover, these findings will provide insights to how these drugs can reestablish an immunostimulatory tumor microenvironment in MIBCs. In summary, the studies outlined in this proposal are significant to address an unmet need, i.e., to improve a dismal response of MIBC patients to standard chemotherapy. The conceptual advance from this study will likely extend beyond MIBC to benefit patients from other epithelial malignancies.