Discovery and characterization of small molecules that target the GTPase Ral.

The Ras-like GTPases RalA and RalB are important drivers of tumour growth and metastasis. 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 to a site on the GDP-bound form of Ral. The compounds RBC6, RBC8 and RBC10 inhibited the binding of Ral to its effector RALBP1, as well as inhibiting Ral-mediated cell spreading of murine embryonic fibroblasts and anchorage-independent growth of human cancer cell lines. The binding of the RBC8 derivative BQU57 to RalB was confirmed by isothermal titration calorimetry, surface plasmon resonance and (1)H-(15)N transverse relaxation-optimized spectroscopy (TROSY) NMR spectroscopy. RBC8 and BQU57 show selectivity for Ral relative to the GTPases Ras and RhoA and inhibit tumour xenograft growth to a similar extent to the depletion of Ral using RNA interference. Our results show the utility of structure-based discovery for the development of therapeutics for Ral-dependent cancers.

CD24 expression is important in male urothelial tumorigenesis and metastasis in mice and is androgen regulated.

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.

CD24 is an effector of HIF-1-driven primary tumor growth and metastasis.

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.

RREB1 transcription factor splice variants in urologic cancer.

RREB1 is an alternatively spliced transcription factor implicated in Ras signaling and cancer. Little is known about the expression of RREB1 isoforms in cell lines or human tumors, or about the clinical relevance of the latter. We have developed tools for IHC of RREB1 protein isoform-specific amplification of RREB1 mRNA and selective knockdown of RREB1 isoforms and use these to provide new information by characterizing RREB1 expression in bladder and prostate cancer cell lines and human tissue samples. Previously described splice variants RREB1α, RREB1ß, RREB1γ, and RREB1δ were identified, as well as the novel variant RREB1ε. Total and isoform-specific mRNA expression was lower in most but not all tumors, compared with normal tissues. RREB1 IHC performed on a bladder cancer TMA did not indicate a relationship between total RREB1 expression and overall survival after radical cystectomy for invasive bladder cancer. In contrast, in vitro proliferation studies using the UMUC-3 bladder cancer cell line after selective isoform-specific knockdown of expression indicate that RREB1α is not necessary for proliferation, but that RREB1ß may be required. These contributions should accelerate progress in the nascent RREB1 field by providing new reagents while also providing clues to the role of RREB1 isoforms in human cancer and raising the possibility of isoform-specific roles in human carcinogenesis and progression.

Src and caveolin-1 reciprocally regulate metastasis via a common downstream signaling pathway in bladder cancer.

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 nonmetastatic RT4 cells. Biochemical studies indicated that 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 whereas 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 seems warranted.

A gene expression ratio-based diagnostic test for bladder cancer.

PURPOSE: Bladder cancer is relatively common but early detection techniques such as cystoscopy and cytology are somewhat limited. We developed a broadly applicable, platform-independent and clinically relevant method based on simple ratios of gene expression to diagnose human cancers. In this study, we sought to determine whether this technique could be applied to the diagnosis of bladder cancer. EXPERIMENTAL DESIGN: We developed a model for the diagnosis of bladder cancer using expression profiling data from 80 normal and tumor bladder tissues to identify statistically significant discriminating genes with reciprocal average expression levels in each tissue type. The expression levels of select genes were used to calculate individual gene pair expression ratios in order to assign diagnosis. The optimal model was examined in two additional published microarray data sets and using quantitative RT-PCR in a cohort of 13 frozen benign bladder urothelium samples and 13 bladder cancer samples from our institution. RESULTS: A five-ratio test utilizing six genes proved to be 100% accurate (26 of 26 samples) for distinguishing benign from malignant bladder tissue samples (P < 10(-6)). CONCLUSIONS: : We have provided a proof of principle study for the use of gene expression ratios in the diagnosis of bladder cancer. This technique may ultimately prove to be a useful adjunct to cytopathology in screening urine specimens for bladder cancer.

Invasion and metastasis models for studying RhoGDI2 in bladder cancer.

Invasion and metastasis are the critical steps in cancer progression that lead to death from this disease. Intense investigation into the underlying mechanisms of metastasis has revealed a complex set of signaling pathways that regulate the process. Since the mid-1980s, it has been demonstrated that the Rho family of proteins plays a major role in these pathways. Proteins that regulate Rho, including guanine nucleotide exchange factors, GTPase-activating proteins, and Rho GDP dissociation inhibitors (RhoGDIs), have also been shown to contribute to cancer progression. Among this group of Rho-regulating proteins is RhoGDI2 (RhoGDIbeta/LyGDI/GDID4/RabGDIbeta). Our laboratory initially identified RhoGDI2 as a metastasis suppressor due to its differential expression between metastatically capable and poorly metastatic bladder cancer cell lines. Over the subsequent years, in vivo and in vitro systems have been used to model steps in the metastatic cascade and to test how the expression of RhoGDI2 affected those processes. This chapter describes several of the more significant methods used to investigate the role of RhoGDI2 in bladder cancer invasion and metastasis. These methods include an in vitro assay for invasion using bladder organ cultures, lung metastasis assays in immunocompromised murine hosts, polymerase chain reaction-based quantification of metastatic burden, and derivation of increasingly metastatic cell lines.

Differential diagnosis of solitary lung nodules with gene expression ratios.

OBJECTIVE: We have developed a new technique that uses the ratios of select gene expression levels to translate complex genomic data into simple clinically relevant tests for the diagnosis and prognosis of cancer. We determined whether select gene pair ratio combinations can be used to detect and diagnose lung cancer with high accuracy and sensitivity. METHODS: We used gene expression profiling data to train a ratio-based predictor model to discriminate among a set of samples (n = 145 total) composed of normal lung, small cell lung cancer, adenocarcinoma, squamous cell carcinoma, and pulmonary carcinoid (the training set). We then examined the optimal test in an independent set of samples (the test set, n = 122). Finally, we used one aspect of the test to determine whether the gene ratio technique was capable of detecting cancer in specimens from fine-needle aspirations performed ex vivo with normal lung (n = 14) and suspected tumor nodules (n = 15) acquired at our institution. RESULTS: We found that a ratio-based test with 23 genes could be used to classify training set samples with 90% accuracy. This same test was similarly accurate (88%) when applied to the test set of samples. We also found that this test was 87% and 100% accurate at detecting cancer in normal and tumorous fine-needle aspiration specimens, respectively. CONCLUSION: The gene expression ratio diagnostic technique is likely to aid in the differential diagnosis of solitary lung nodules in patients with suspected cancer and may also prove useful in developing lung cancer screening strategies that incorporate analysis of fine-needle aspiration specimens.

The metastasis-associated gene CD24 is regulated by Ral GTPase and is a mediator of cell proliferation and survival in human cancer.

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.

Identification of novel candidate oncogenes and tumor suppressors in malignant pleural mesothelioma using large-scale transcriptional profiling.

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.