Antonina V. Kurtova

Blocking PGE2-induced tumour repopulation abrogates bladder cancer chemoresistance

Cytotoxic chemotherapy is effective in debulking tumour masses initially; however, in some patients tumours become progressively unresponsive after multiple treatment cycles. Previous studies have demonstrated that cancer stem cells (CSCs) are selectively enriched after chemotherapy through enhanced survival. Here we reveal a new mechanism by which bladder CSCs actively contribute to therapeutic resistance via an unexpected proliferative response to repopulate residual tumours between chemotherapy cycles, using human bladder cancer xenografts. Further analyses demonstrate the recruitment of a quiescent label-retaining pool of CSCs into cell division in response to chemotherapy-induced damages, similar to mobilization of normal stem cells during wound repair. While chemotherapy effectively induces apoptosis, associated prostaglandin E2 (PGE2) release paradoxically promotes neighbouring CSC repopulation. This repopulation can be abrogated by a PGE2-neutralizing antibody and celecoxib drug-mediated blockade of PGE2 signalling. In vivo administration of the cyclooxygenase-2 (COX2) inhibitor celecoxib effectively abolishes a PGE2- and COX2-mediated wound response gene signature, and attenuates progressive manifestation of chemoresistance in xenograft tumours, including primary xenografts derived from a patient who was resistant to chemotherapy. Collectively, these findings uncover a new underlying mechanism that models the progressive development of clinical chemoresistance, and implicate an adjunctive therapy to enhance chemotherapeutic response of bladder urothelial carcinomas by abrogating early tumour repopulation.

A distinct role for Lgr5 + stem cells in primary and metastatic colon cancer

Cancer stem cells (CSCs) have been hypothesized to represent the driving force behind tumour progression and metastasis, making them attractive cancer targets. However, conclusive experimental evidence for their functional relevance is still lacking for most malignancies. Here we show that the leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) identifies intestinal CSCs in mouse tumours engineered to recapitulate the clinical progression of human colorectal cancer. We demonstrate that selective Lgr5+ cell ablation restricts primary tumour growth, but does not result in tumour regression. Instead, tumours are maintained by proliferative Lgr5− cells that continuously attempt to replenish the Lgr5+ CSC pool, leading to rapid re-initiation of tumour growth upon treatment cessation. Notably, CSCs are critical for the formation and maintenance of liver metastasis derived from colorectal cancers. Together, our data highlight distinct CSC dependencies for primary versus metastasic tumour growth, and suggest that targeting CSCs may represent a therapeutic opportunity for managing metastatic disease.

Three differentiation states risk-stratify bladder cancer into distinct subtypes

Current clinical judgment in bladder cancer (BC) relies primarily on pathological stage and grade. We investigated whether a molecular classification of tumor cell differentiation, based on a developmental biology approach, can provide additional prognostic information. Exploiting large preexisting gene-expression databases, we developed a biologically supervised computational model to predict markers that correspond with BC differentiation. To provide mechanistic insight, we assessed relative tumorigenicity and differentiation potential via xenotransplantation. We then correlated the prognostic utility of the identified markers to outcomes within gene expression and formalin-fixed paraffin-embedded (FFPE) tissue datasets. Our data indicate that BC can be subclassified into three subtypes, on the basis of their differentiation states: basal, intermediate, and differentiated, where only the most primitive tumor cell subpopulation within each subtype is capable of generating xenograft tumors and recapitulating downstream populations. We found that keratin 14 (KRT14) marks the most primitive differentiation state that precedes KRT5 and KRT20 expression. Furthermore, KRT14 expression is consistently associated with worse prognosis in both univariate and multivariate analyses. We identify here three distinct BC subtypes on the basis of their differentiation states, each harboring a unique tumor-initiating population.

Positive association of collagen type I with non-muscle invasive bladder cancer progression

PURPOSE Non-muscle invasive bladder cancers (NMIBC) are generally curable, while ~15% progresses into muscle-invasive cancer with poor prognosis. While efforts have been made to identify genetic alternations associated with progression, the extracellular matrix (ECM) microenvironment remains largely unexplored. Type I collagen is a major component of the bladder ECM, and can be altered during cancer progression. We set out to explore the association of type I collagen with NMIBC progression. EXPERIMENTAL DESIGN The associations of COL1A1 and COL1A2 mRNA levels with progression were evaluated in a multi-center cohort of 189 patients with NMIBCs. Type I collagen protein expression and structure were evaluated in an independent single-center cohort of 80 patients with NMIBCs. Immunohistochemical analysis was performed and state-of-the-art multi-photon microscopy was used to evaluate collagen structure via second harmonic generation imaging. Progression to muscle invasion was the primary outcome. Kaplan-Meier method, Cox regression, and Wilcoxon rank-sum were used for statistical analysis. RESULTS There is a significant association of high COL1A1 and COL1A2 mRNA expression in patients with poor progression-free survival (P=0.0037 and P=0.011, respectively) and overall survival (P=0.024 and P=0.012, respectively). Additionally, immunohistochemistry analysis of type I collagen protein deposition revealed a significant association with progression (P=0.0145); Second-harmonic generation imaging revealed a significant lower collagen fiber curvature ratio in patients with invasive progression (P = 0.0018). CONCLUSIONS Alterations in the ECM microenvironment, particularly type I collagen, likely contributes to bladder cancer progression. These findings will open avenues to future functional studies to investigate ECM-tumor interaction as a potential therapeutic intervention to treat NMIBCs.

Abstract 5470: Blocking wound-induced tumor repopulation between chemotherapy cycles as a novel approach to abrogate chemoresistance

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Acquired chemoresistance remains a major clinical issue in the management of advanced solid cancers. Initial response to cytotoxic chemotherapy is common, but certain patients progressively become unresponsive after multiple chemotherapy cycles. While causes of drug resistance are multiple and complex, here we approach this problem from a new angle: we studied whether repopulation of residual surviving cancer cells between chemotherapy cycles contributes to progressive chemoresistance. Currently the identity of repopulating cancer cells following chemotherapy is unknown, and the underlying molecular mechanisms that initiate tumor repopulation remain poorly understood. In the present study we use bladder cancer as a model and report that quiescent cancer stem cells (CSCs) are unexpectedly recruited to proliferate and repopulate residual tumors in response to chemotherapy-induced damage. This phenomenon is similar to how normal resident tissue stem cells mobilize to wound sites for tissue repair. We further investigate whether blockade of this wound-induced CSC repopulation can provide an innovative approach to abrogate chemoresistance. Previously we showed that cytokeratin 14 (CK14) marks the most primitive bladder cancer cells and abundance of CK14+ cancer cells in patients correlates with poor survival. Here, we followed the standard clinical chemotherapy regimen with gap periods to allow recovery of normal tissues between treatment cycles. While one cycle of gemcitabine and cisplatin effectively reduced tumor growth in vivo, a generalized expansion of CK14+ CSCs occurred in residual tumors during these gap periods between cycles. Further analysis revealed the induction of a “wound-response” gene signature in residual tumors and active recruitment of quiescent CSCs into proliferation in response to chemotherapy-induced damage. We demonstrated that prostaglandin E2 (PGE2) released by neighboring dying cancer cells could induce CSC expansion in a paracrine manner. This undesirable CSC expansion could be abrogated by a PGE2 neutralizing antibody and Celecoxib, an FDA approved COX2 inhibitor that blocks PGE2 signaling. In vivo administration of Celecoxib blocked the induction of “wound-response” gene signature and significantly attenuated progressive development of chemoresistance in xenograft tumors, including primary xenografts derived from a patient who failed chemotherapy.These results revealed a new mechanism by which CSCs contribute to therapeutic resistance via repopulating residual tumors between chemotherapy cycles. Repopulation was initiated by dying cells that induced wound response and recruitment of CSCs to repair chemotherapy-induced damages. Therapeutic intervention with Celecoxib effectively blocked this process and improved chemotherapeutic response in bladder tumors, supporting further validation in other solid cancers. Citation Format: Antonina V. Kurtova, Jing Xiao, Qianxing Mo, Senthil Pazhanisamy, Ross Krasnow, Seth P. Lerner, Fengju Chen, Terrence Roh, Erica Lay, Philip L. Ho, Keith S. Chan. Blocking wound-induced tumor repopulation between chemotherapy cycles as a novel approach to abrogate chemoresistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5470. doi:10.1158/1538-7445.AM2015-5470

Abstract 4801: Stromal-mediated collagen I signal in promoting bladder cancer progression

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Bladder cancer is the fifth most common malignancy, which is mostly incurable as invasive disease. It is clinically important to study the mechanisms underlying bladder cancer progression. While the tumor microenvironment is widely established to play an active role in epithelial cancers, its contribution to bladder cancer remains unexplored. The presence of cancer-associated fibroblasts (CAFs), characterized by the co-expression of vimentin, alpha-smooth muscle actin, and tenascin C, has been associated with invasive bladder cancer. Nevertheless, functional contributions of CAFs to bladder cancer progression have not been studied. Here,we report the successful isolation and molecular characterization of bladder CAFs. We further investigate their functional roles on bladder cancer progression, with an emphasis on stromal secreted collagen I in the paracrine activation of discoidin domain receptor (DDR1) signaling in neighboring bladder cancer cells. Using bioinformatics analysis we found that invasive bladder cancer patients with elevated expression of CAF genes have a poorer survival than those with lower CAF gene expression. Subsequently, we isolated and characterized CAFs from patient-derived tissues. Co-transplantation of CAFs and bladder cancer cells as xenograft tumors revealed high collagen I (COL1) deposition in these tumors formed, while molecular analyses uncovered CAFs as the primary source of COL1. Further experiments verified that COL1 as a single extracellular matrix component could phenocopy the tumor phenotype resembling those co-transplanted with CAFs and cancer cells. Further, pre-stimulation with COL1 could also enhance metastatic colonization of bladder cancer cells to lung. Molecular analysis of these COL1 stimulated cancer cells revealed up-regulation of the collagen receptor DDR1, but not integrins. Immunohistochemical analysis confirmed the presence of DDR1+ cancer cells adjacent to CAFs in the primary tumor site, with enhanced and exclusive expression of DDR1 in paired lung metastasis. Oncomine analysis showed that invasive bladder cancer expressed higher mRNA levels of COL1 and DDRs than non-invasive cancer, indicating that collagen I-DDR1 interaction may be a generalized phenomenon during invasive bladder cancer progression. To delineate the molecular mechanism downstream to collagen I-DDR1 we studied the interaction of DDR1 and STAT3, a factor we previously reported to drive invasive bladder cancer progression. Stimulation of bladder cancer cells with COL1 revealed a time kinetic increase in total and activated DDR1 protein associated with STAT3 phosphorylation. Further analysis of lung metastasis confirmed the co-localization of DDR1 and nuclear active STAT3. Collectively, these findings uncovered a role of CAFs in bladder cancer progression via stromal mediated collagen I signaling and warrant further analysis of therapeutic options to target signaling components downstream to collagen I. Citation Format: Antonina V. Kurtova, Jing Xiao, Erica J. Lay, Qianxing Mo, Seth P. Lerner, David R. Rowley, Keith S. Chan. Stromal-mediated collagen I signal in promoting bladder cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4801. doi:10.1158/1538-7445.AM2014-4801