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Polo-like kinase 1 (PLK1) is an essential cell-cycle regulator that is frequently overexpressed in various human cancers. To determine whether Plk1 overexpression drives tumorigenesis, we established transgenic mouse lines that ubiquitously express increased levels of Plk1. High Plk1 levels were a driving force for different types of spontaneous tumors. Increased Plk1 levels resulted in multiple defects in mitosis and cytokinesis, supernumerary centrosomes, and compromised cell-cycle checkpoints, allowing accumulation of chromosomal instability (CIN), which resulted in aneuploidy and tumor formation. Clinically, higher expression of PLK1 positively associated with an increase in genome-wide copy-number alterations in multiple human cancers. This study provides in vivo evidence that aberrant expression of PLK1 triggers CIN and tumorigenesis and highlights potential therapeutic opportunities for CIN-positive cancers. Significance: These findings establish roles for PLK1 as a potent proto-oncogene and a CIN gene and provide insights for the development of effective treatment regimens across PLK1-overexpressing and CIN-positive cancers.

Metastasis is lethal in most bladder cancer patients. Expression of CD24, a glycosyl phosphatidylinositol (GPI)-linked sialoglycoprotein and cancer stem cell marker, is associated with metastatic progression in multiple cancer types, yet the role of CD24 in this process remains unclear. While developing a murine model of human metastatic bladder cancer, we observed that tumor cell CD24 expression correlated with a propensity to metastasize to the lung. Our immunohistochemical evaluation of 60 paired primary and metastatic human bladder cancer samples revealed increased intensity (P < 0.001) and frequency (P < 0.001) of CD24 expression in metastases. To directly evaluate the role of CD24 in metastatic colonization, we manipulated CD24 expression in human bladder cancer cell lines using short hairpin RNA depletion, cDNA overexpression, and fluorescence-activated cell sorting selection. Although suppression of CD24 reduced acute tumor cell retention in the lungs of mice inoculated intravenously with cancer cells, this differential retention was no longer apparent after 24 hours, prompting us to evaluate the role of CD24 in lung colonization. Here, CD24 was found necessary for subsequent development of lung metastases. We next treated clinically detectable lung metastases in mice with anti-CD24 antibody and observed reduced tumor growth and prolonged survival. These findings suggest that CD24 is a lynchpin of metastatic progression and a promising therapeutic target for antimetastatic therapy. Cancer Res; 71(11); 3802–11. ©2011 AACR.

Substantial effort has been devoted to in vitro testing of candidate chemotherapeutic agents. In particular, the United States National Cancer Institute Developmental Therapeutics Program (NCI-DTP) Human Tumor Cell Line Screen has screened hundreds of thousands of compounds and extracts, for which data on more than 40,000 compounds tested on a panel of 60 cancer cell lines (NCI-60) are publically available. In tandem, gene expression profiling has brought about a sea change in our understanding of cancer biology, allowing discovery of biomarkers or signatures able to characterize, classify, and prognosticate clinical behavior of human tumors. Recent studies have used tumor profiling matched to clinical trial outcome data to derive gene expression models predicting therapeutic outcomes, though such efforts are costly, time-consuming, tumor type-specific, and not amenable to rare diseases. Furthermore, addition of new or established drugs to multidrug combinations in which such models are already available requires the entire model to be re-derived. Can the aforementioned in vitro testing platform, coupled to the universal language of genomics, be used to develop, a priori, gene expression models predictive of clinical outcomes? Recent advances, including the CO-eXpression ExtrapolatioN (COXEN) algorithm, suggest that development of these models may be possible and raise important implications for future trial design and drug discovery. Cancer Res; 70(5); 1753–8

Ral GTPases are important mediators of transformation, tumorigenesis, and cancer progression. We recently identified the metastasis-associated protein CD24, a glycosyl phosphatidyl inositol–linked surface protein, as a downstream target of Ral signaling by profiling the expression of RalA/B–depleted bladder carcinoma cells. Because CD24 is highly expressed in bladder and many other tumor types, we sought to determine if this protein plays an essential role in maintaining the malignant phenotype. Here, we show that loss of CD24 function in cell lines derived from common tumor types is associated with decreased rates of cell proliferation, clonogenicity in soft agar, changes in the actin cytoskeleton, and induction of apoptosis. Given these phenotypes, we evaluated a human bladder cancer tissue microarray by immunohistochemistry for CD24 to determine if CD24 is a prognostic cancer biomarker. Multivariate analysis showed that increased CD24 expression correlated with shorter patient disease-free survival (P = 0.07). In conclusion, we show that CD24 is a novel and functionally relevant Ral-regulated target and a potentially important prognostic marker. We suggest that these insights may lead to future therapeutic approaches that seek to eliminate CD24 function in cancer cells. (Cancer Res 2006; 66(4): 1917-22)

The Ral family of small G proteins has been implicated in tumorigenesis, invasion, and metastasis. However, little emphasis has been placed on clarifying the individual roles of the two Ral proteins, RalA and RalB, in these processes in view of their high sequence homology. Here we analyze the separate contributions of RalA and RalB in regulating cell migration, a necessary component of the invasive phenotype, in two human cancer cell lines; UMUC-3, a bladder carcinoma line, and the prostate carcinoma line, DU145. Although inhibiting RalA protein expression by ∼80% with two different small interfering RNA duplexes had no effect on migration, inhibiting RalB expression to the same extent with two different duplexes resulted in a marked reduction in migration. Inhibiting RalB expression did trigger a significant loss of actin cytoskeleton fibers in UMUC-3 that was not seen with inhibition of RalA expression. Interestingly, simultaneous inhibition of RalA and RalB expression had no effect on migration. However, dual inhibition of RalA and RalB expression in UMUC-3 did result in an almost total loss of actin fibers as well as a reduction in proliferation, particularly in reduced serum conditions. These results suggest that RalA and RalB have different roles in cell migration and that they may in fact act as antagonists with regard to this phenotype. As further verification of this hypothesis, we found that expression of constitutively active RalA inhibited migration, whereas expression of constitutively active RalB stimulated migration, consistent with this model. In summary, we present the first demonstration that despite their significant sequence homology, RalA and RalB have nonoverlapping and opposing functions in cancer cell migration but overlapping functions in cell growth.

Hypoxia drives malignant progression in part by promoting accumulation of the oncogenic transcription factor hypoxia inducible factor–1α (HIF-1α) in tumor cells. Tumor aggressiveness also relates to elevation of the cancer stem cell–associated membrane protein CD24, which has been causally implicated in tumor formation and metastasis in experimental models. Here, we link these two elements by showing that hypoxia induces CD24 expression through a functional hypoxia responsive element in the CD24 promoter. HIF-1α overexpression induced CD24 mRNA and protein under normoxic conditions, with this effect traced to a recruitment of endogenous HIF-1α to the CD24 promoter. Short hairpin RNA–mediated attenuation of HIF-1α or CD24 expression reduced cancer cell survival in vitro and in vivo at the levels of primary and metastatic tumor growth. CD24 overexpression in HIF-1α–depleted cancer cells rescued this decrease, whereas HIF-1α overexpression in CD24-depleted cells did not. Analysis of clinical tumor specimens revealed a correlation between HIF-1α and CD24 levels and an association of their coexpression to decreased patient survival. Our results establish a mechanistic linkage between 2 critically important molecules in cancer, identifying CD24 as a critical HIF-1α transcriptional target and biologic effector, strengthening the rationale to target CD24 for cancer therapy. Cancer Res; 72(21); 5600–12. ©2012 AACR.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through base pairing with other mRNA transcripts. MiRNA expression signatures have characterized many phenotypes in bladder cancer, including grade, stage, and metastasis. However, simultaneous mRNA changes accompanying the miRNA profile, along with the miRNA:mRNA interaction, have not been systematically studied in these phenotypes. To gain insights into the miRNA regulatory network in bladder cancer metastasis, we profiled miRNA and mRNA expression in two pairs of metastatic and non-metastatic cell lines. Interactions between differentially expressed miRNAs and mRNAs were predicted using 6 software programs. The candidate miRNA:mRNA pairs were further selected by their clinical relevance, including their expression in grade, stage, and tumor in 2 cohorts totaling 256 human tumors. We identified more miRNA:mRNA pairs with up-regulated miRNAs and down-regulated genes in metastatic cells than the pairs with down-regulated miRNAs and up-regulated target genes. The novel overexpression of one of the miRNAs, hsa-miR-146a, was confirmed by quantitative RT-PCR in metastatic cell lines. It has been shown that decreased level of hsa-miR-146a is associated with progression of prostate cancer and polymorphism in hsa-miR-146a gene is associated with prostate cancer risk. We are carrying out in vitro and in vivo experiments on the top 5 scoring pairs ranked by the number of prediction software and clinical relevance. Our results suggest the significance of miRNA regulatory network in cancer metastasis and provide a list of potential miRNA:mRNA pairs for further investigation. Citation Format: Yuanbin Ru, Katerina J. Kechris, Steven C. Smith, George A. Calin, Dan Theodorescu. Significance of microRNA:target interaction in bladder cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2899. doi:10.1158/1538-7445.AM2013-2899

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through base pairing with other mRNA transcripts. MiRNA expression signatures have characterized many phenotypes in bladder cancer, including grade, stage, and metastasis. However, simultaneous mRNA changes accompanying the miRNA profile, along with the miRNA:mRNA interaction, were not systematically studied in these phenotypes. To gain insights into the miRNA regulatory network in bladder cancer metastasis, we profiled miRNA and mRNA expressions in two pairs of metastatic and non-metastatic cell lines. Interactions between differentially expressed miRNAs and mRNAs were predicted using six software. We identified 194 miRNA:mRNA pairs between 18 up-regulated miRNAs and 66 down-regulated genes in metastatic cells; while the down-regulation of miRNAs and the increased expression of target genes were rare. The overexpression of one of the miRNAs, hsa-miR-146a, is confirmed by quantitative RT-PCR in metastatic cell lines. The 66 potential target genes are enriched in cytoskeleton organization and actin binding, underlining the importance of cell motility in cancer metastasis. Analysis of microarray data from two patient cohorts indicated lower expression of the 66 miRNA target genes in tumors than in normal bladder tissues. Our results suggest the significance of miRNA regulatory network in cancer metastasis and provide a list of potential miRNA:mRNA pairs for further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3391. doi:1538-7445.AM2012-3391

A perennial issue in biomedical research regards validation of the relevance of experimentally tractable model systems to disease characteristics of interest. In the study of many types of cancer, this often operationally means selection of a cell line or a number of cell line models for perturbation within in vitro or in vivo models of disease progression or treatment. However, many classic cell lines are of uncertain provenance, may have been isolated from tumors staged and graded decades ago in a fashion of limited interpretability with regards to current care, or have exhibited drifting phenotypes in intervening years. One example is that of the classic bladder cancer cell line, T24, isolated from a sample of “verified grade III papillomatosis of the bladder” in 1973, which has given rise to isogenically verified metastatic variants like T24T (1). We recently have reported an algorithm, called COXEN (COeXpression ExtrapolatioN), which uses concordant gene expression signatures of complex clinicopathologic phenotypes to infer interpretable and predictable relationships between disparate cell line and tumor populations, despite significantly different global transcriptional differences. While prior reports have used COXEN to predict chemotherapeutic response in clinical trials a priori based on signatures from cell lines (2,3), herein we report an application of the reverse analysis, using signatures of clinicopathologic characteristics in tumors including invasion, nodal metastasis, and poor survival to select from a panel of forty bladder cancer cell lines models reflecting with highest fidelity the transcriptional patterns underlying the parameter of interest. We find that this approach validates some cell line model systems of bladder cancer and proposes new uses for others. This approach, widely adaptable to other tumor histologies, may provide important guidance for selection of appropriate models to test therapies targeted at clinically important phenotypes. (1) Harding MA et al. Cancer Res 2002. (2) Lee JK et al. PNAS 2007. (3) Williams et al. Cancer Res 2009. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 50. doi:10.1158/1538-7445.AM2011-50

Ral GTPases, which include RalA and RalB, mediate essential phenotypes in model cancer systems; however, demonstration of the clinical relevance of these molecules has been lacking. To address this concern, we develop two types of gene expression signatures as surrogates for Ral GTPase status in tumors and characterize their nosologic and prognostic performance across several epithelial malignancies. Given Ral's well characterized role in regulating a variety of transcriptional pathways including AP1, NFkB, STAT3B, forkhead family members, and RREB1, the first type of signature is related to transcription downstream of RalA and RalB and was derived empirically from expression profiling of cells depleted of these molecules. This signature was then applied to human tumor gene expression data by using a nearest neighbor algorithm for classification. Consistent with prior findings of a role for these molecules in model surrogates of cancer progression(1,2), presence of the Ral transcriptional signature characterized the muscle invasive status of human bladder cancer (P Secondly, we have assayed constitutively-active RalA and RalB mutants, finding that either induces accelerated experimental metastasis of UMUC3 bladder cancer cells in mice (logrank P=0.01), similar to results for other tumor types (4). This made us question whether signatures of metastasis from tumor profiling could be associated with differential Ral activation. We assayed the activation of RalA and RalB across 40 cell lines, then mapped individual tumor expression of metastatic genes to these cell lines in silico. Using this methodology to compare tumors to cell lines of known Ral activation, we found association of higher Ral activation with nodal metastasis in bladder cancer and with visceral metastasis in breast cancer, both P (1) Oxford, Cancer Res, 2005 (2) Lim, Curr Biol, 2006 (3) Sowalsky, Oncogene, 2009 (4) Yin, Mol Cell Biol, 2007 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5063.

In most patients, bladder cancer metastasis is incurable with current chemotherapy. To try to address this challenge, we have recently employed the novel COXEN algorithm (1) for in silico drug discovery. This approach utilized publicly available gene expression and in vitro sensitivity data for >40k compounds in NCI-60 cell line screen, from which concordant gene expression prediction models from COXEN enabled our discovery of an imidoazacridinone drug, NSC-637993, with high activity in vitro against forty bladder cancer cell lines (2). Examination of additional “hits” provided by COXEN revealed another compound in this class, C1311 (NSC-645809, Symadex) which is undergoing clinical trials in other tumor types. Our objective was to develop the foundation for use of C1311 in bladder cancer, alone and in rational combination with other clinically relevant agents. Herein, we demonstrate in a panel of 40 human bladder cancer cells that in vitro cytotoxicity profile for C1311 closely correlates that of NSC-637993 and compares favorably to that of standard of chemotherapeutics. Using genome-wide patterns of synthetic lethality of C1311 with ORF knockouts in budding yeast, we determined that combining C1311 with taxanes could provide mechanistically rational combinations for patients that have failed first line therapy with platinum based chemotherapy. To evaluate the biological relevance of these yeast findings in bladder cancer, we evaluated C1311 singly and in doublet combination with paclitaxel in the hollow fiber assay in mice, observing striking efficacy of the agents in vivo. Last, applying COXEN gene expression prediction from 40 bladder cancer cell lines to human patient tumors with known cisplatin-based chemotherapy response data (N=30) (3), we provide evidence that signatures of C1311 sensitivity exist even within patients exhibiting no change or progressive disease on standard of care agents. Taken together, these findings provide an exemplar for COXEN-based drug discovery and development, while demonstrating the utility of yeast genetics in guidance of rational combinations in vivo. Most importantly, the high activity of these agents in vitro and in vivo calls for clinical trials of these agents, while COXEN provides genomic biomarkers that may be used for patient selection. (1) Crunkhorn S. Nat Rev Drug Discov 2007;6:782-3. (2) Lee JK et al. PNAS 2007;104:13086-91. (3) Als et al. Clin Cancer Res. 2007 Aug 1;13(15 Pt 1):4407-14. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 747.

The use of systemic neoadjuvant cisplatin-based chemotherapy prior to radical cystectomy for clinically muscle invasive but clinically node negative urothelial carcinoma (UC) has been shown in a recent meta-analysis of randomized trials to confer a 5-7% survival benefit (1). This relatively modest effect, therapy toxicity, and treatment delay in non-responsive patients has diminished enthusiasm and use of this approach. In contrast, defining patients that would benefit most would provide a compelling reason for use of such therapy. Despite negative clinical staging, up to 30% of patients have lymph node metastasis at cystectomy, the most important predictor for subsequent disease relapse. Here we develop a gene expression-based predictor of node positive disease at cystectomy in clinically node negative patients that could be applied before definitive resection using tissue from the patient's endoscopic diagnosis. Importantly, we develop a predictor that can be applied on formalin fixed paraffin embedding (FFPE) tumor samples, for facile deployment of this clinical tool. We first performed an analysis of correlation of gene expression between fresh frozen (FF) and FFPE across 32 paired tissues samples preserved both ways, using bootstrapping to select only array probes that maintain robust correlation irrespective of tissue preservation method. Using the “robust” gene set, we identified a subset of genes that were differentially expressed between node positive and node negative disease found at cystectomy in a cohort of 90 FFPE tumor specimens we profiled by oligonucleotide microarrays. A model based on these genes was then optimized for prediction of node positive disease on an independent set of 91 FF specimens profiled on the same microarray platform (2), using a weighted kNN algorithm. A final, 108-feature (probeset) predictor was then tested on a third cohort of 185 FFPE samples from a prospective randomized clinical trial (AUO-AB 05/95) (3). On the independent dataset, the predictor exhibited favorable discriminant characteristics (AUC=0.70, 95%CI 0.62 to 0.78, P References 1. Cochrane Database Syst. Rev. 2005 2. Sanchez-Carbayo. JCO 2005. 3. Lehmann. JCO 2005. 4. Paik. NEJM 2004. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-65.