Malignant pleural mesothelioma (MPM) is a highly lethal, poorly understood neoplasm that is typically associated with asbestos exposure. We performed transcriptional profiling using high-density oligonucleotide microarrays containing approximately 22,000 genes to elucidate potential molecular and pathobiological pathways in MPM using discarded human MPM tumor specimens (n = 40), normal lung specimens (n = 4), normal pleura specimens (n = 5), and MPM and SV40-immortalized mesothelial cell lines (n = 5). In global expression analysis using unsupervised clustering techniques, we found two potential subclasses of mesothelioma that correlated loosely with tumor histology. We also identified sets of genes with expression levels that distinguish between multiple tumor subclasses, normal and tumor tissues, and tumors with different morphologies. Microarray gene expression data were confirmed using quantitative reverse transcriptase-polymerase chain reaction and protein analysis for three novel candidate oncogenes (NME2, CRI1, and PDGFC) and one candidate tumor suppressor (GSN). Finally, we used bioinformatics tools (ie, software) to create and explore complex physiological pathways. Combined, all of these data may advance our understanding of mesothelioma tumorigenesis, pathobiology, or both.

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 Ras-like GTPases RalA and B are important drivers of tumor growth and metastasis1. Chemicals that block Ral function would be valuable as research tools and for cancer therapeutics. Here, we used protein structure analysis and virtual screening to identify drug-like molecules that bind a site on the GDP-form of Ral. Compounds RBC6, RBC8 and RBC10 inhibited Ral binding to its effector RalBP1, Ral-mediated cell spreading in murine fibroblasts and anchorage-independent growth of human cancer cell lines. Binding of RBC8 derivative BQU57 to RalB was confirmed by isothermal titration calorimetry, surface plasma resonance and 15N-HSQC NMR. RBC8 and BQU57 show selectivity for Ral relative to Ras or Rho and inhibit xenograft tumor growth similar to depletion of Ral by siRNA. Our results show the utility of structure-based discovery for development of therapeutics for Ral-dependent cancers.

Hypoxia drives malignant progression in part by promoting accumulation of the oncogenic transcription factor 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 (HRE) 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. shRNA 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 while 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 co-expression to decreased patient survival. Our results establish a mechanistic linkage between two critically important molecules in cancer, identifying CD24 as a critical HIF-1α transcriptional target and biological effector, strengthening the rationale to target CD24 for cancer therapy.

Overexpression of CD24, a glycosyl phosphatidylinositol-linked sialoglycoprotein, is associated with poor outcome in urothelial carcinoma and contributes to experimental tumor growth and metastasis. However, the requirement for CD24 ( Cd24a in mice) in tumorigenesis and spontaneous metastasis from the orthotopic site remains uncharacterized. Using N -butyl- N -(4-hydroxybutyl) nitrosamine induction of invasive and metastatic bladder cancer, we show that Cd24a -deficient male mice developed fewer bladder tumors than C57BL/6 control male mice. Evaluating only mice with evidence of primary tumors, we observed that Cd24a- deficient male mice also had fewer metastases than wild-type counterparts. In parallel observations, stratification of patients based on CD24 immunohistochemical expression in their tumors revealed that high levels of CD24 are associated with poor prognosis in males. In female patients and mice the above observations were not present. Given the significant role of CD24 in males, we sought to assess the relationship between androgen and CD24 regulation. We discovered that androgen receptor knockdown in UM-UC-3 and TCCSUP human urothelial carcinoma cell lines resulted in suppression of CD24 expression and cell proliferation. Androgen treatment also led to increased CD24 promoter activity, dependent on the presence of androgen receptor. In vivo, androgen deprivation resulted in reduced growth and CD24 expression of UM-UC-3 xenografts, and the latter was rescued by exogenous CD24 overexpression. These findings demonstrate an important role for CD24 in urothelial tumorigenesis and metastasis in male mice and indicate that CD24 is androgen regulated, providing the foundation for urothelial bladder cancer therapy with antiandrogens.

In bladder cancer, increased Caveolin-1 (Cav-1) expression and decreased Src expression and kinase activity correlate with tumor aggressiveness. Here, we investigate the clinical and functional significance if any, of this reciprocal expression in bladder cancer metastasis. We evaluated the ability of tumor Cav-1 and Src RNA and protein expression to predict outcome following cystectomy in 257 patients enrolled in two independent clinical studies. In both, high Cav-1 and low Src levels were associated with metastasis development. We overexpressed or depleted Cav-1 and Src protein levels in UMUC-3 and, RT4 human bladder cancer cells and evaluated the effect of this on actin stress fibers, migration using transwells and lung metastasis following tail vein inoculation. Cav-1 depletion or expression of active Src in metastatic UMUC-3 cells decreases actin stress fibers, cell migration and metastasis, while, Cav-1 overexpression or Src depletion increased the migration of non-metastatic RT4 cells. Biochemical studies indicated Cav-1 mediates these effects via its phosphorylated form (pY14), whereas Src effects are mediated through phosphorylation of p190RhoGAP and these pathways converge to reduce activity of RhoA, RhoC and Rho effector ROCK1. Treatment with a ROCK inhibitor reduced UMUC-3 lung metastasis in vivo, phenocopying the effect of Cav-1 depletion or expression of active Src. Src suppresses while Cav-1 promotes metastasis of bladder cancer through a pharmacologically tractable common downstream signaling pathway. Clinical evaluation of personalized therapy to suppress metastasis development based on Cav-1 and Src profiles appears warranted.

Little is known about which genes mediate metastasis in bladder cancer, which accounts for much of the mortality of this disease. We used human bladder cancer cell lines to develop models of two clinically common metastatic sites, lung and liver, and evaluated their gene expression with respect to human tumor tissues. Parental cells were injected into either the murine spleen to generate liver metastases or tail vein to generate lung metastases with sequential progeny derived by re-injection and comparisons made of their organ-specific nature by crossed-site injections. Both genomic and transcriptomic analyses of organselected cell lines found salient differences and shared core metastatic profiles, which were then screened against gene expression data from human tumors. The expression levels of laminin V gamma 2 (LAMC2) contained in the core metastatic signature were increased as a function of human tumor stage, and its genomic location was in an area of gain as measured by comparative genomic hybridization. Using immunohistochemistry in a human bladder cancer tissue microarray, LAMC2 expression levels were associated with tumor grade, but inversely with nodal status. In contrast, in node-negative patients, LAMC2 expression was associated with visceral metastatic recurrence. In summary, LAMC2 is a novel biomarker of bladder cancer metastasis that reflects the propensity of cells to metastasize via either lymphatic or hematogenous routes. Metastasis represents a critical event in the natural history of disease for a cancer patient and is associated with significant reduction in patient survival.1 However, although a significant body of research has shed light on this process, less is known about the nature of organspecific metastasis.2,3 In vivo metastasis assays using cancer cell lines passaged through mouse models have been used to discover the role of specific genes relevant to metastatic behavior. 4 For example, in breast cancer, recent reports have addressed the question of the molecular regulation of organ site-specific metastasis. Using the spontaneously metastatic MDA-MB-231 human breast cancer cell line, one group reported the in vivo selection of sublines of differing metastatic potential to bone, lung, and adrenal gland, 5,6 and then evaluated in both animal models and metastatic tumor tissues from patients the gene expression profiles associated with site-specific metastasis. 7Bladder cancer is the fifth most commonly diagnosed cancer in the United States, 8 and the molecular lesions characterizing bladder carcinogenesis and progression are beginning to be better elucidated. 9 However, little is known about the pathways that regulate general metastatic propensity or organ site-specific tropism to lung or liver, which are two common sites of dissemination in this and other tumor types. Our prior work has focused on the discovery and investigation of genes that regulate lung metastasis, such as the metastasis suppressor RhoGDI2, by comparing metastatic T24T cells to their isogenic nonmetastatic relativ...

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