Effect of the DASH diet on the sodium-chloride cotransporter and aquaporin-2 in urinary extracellular vesicles.

The dietary approach to stop hypertension (DASH) diet combines the antihypertensive effect of a low sodium and high potassium diet. In particular, the potassium component of the diet acts as a switch in the distal convoluted tubule to reduce sodium reabsorption, similar to a diuretic but without the side effects. Previous trials to understand the mechanism of the DASH diet were based on animal models and did not characterize changes in human ion channel protein abundance. More recently, protein cargo of urinary extracellular vesicles (uEVs) has been shown to mirror tissue content and physiological changes within the kidney. We designed an inpatient open label nutritional study transitioning hypertensive volunteers from an American style diet to DASH diet to examine physiological changes in adults with stage 1 hypertension otherwise untreated (Sacks FM, Svetkey LP, Vollmer WM, Appel LJ, Bray GA, Harsha D, Obarzanek E, Conlin PR, Miller ER 3rd, Simons-Morton DG, Karanja N, Lin PH; DASH-Sodium Collaborative Research Group. N Engl J Med 344: 3-10, 2001). Urine samples from this study were used for proteomic characterization of a large range of pure uEVs (small to large) to reveal kidney epithelium changes in response to the DASH diet. These samples were collected from nine volunteers at three time points, and mass spectrometry identified 1,800 proteins from all 27 samples. We demonstrated an increase in total SLC12A3 [sodium-chloride cotransporter (NCC)] abundance and a decrease in aquaporin-2 (AQP2) in uEVs with this mass spectrometry analysis, immunoblotting revealed a significant increase in the proportion of activated (phosphorylated) NCC to total NCC and a decrease in AQP2 from day 5 to day 11. This data demonstrates that the human kidney's response to nutritional interventions may be captured noninvasively by uEV protein abundance changes. Future studies need to confirm these findings in a larger cohort and focus on which factor drove the changes in NCC and AQP2, to which degree NCC and AQP2 contributed to the antihypertensive effect and address if some uEVs function also as a waste pathway for functionally inactive proteins rather than mirroring protein changes.NEW & NOTEWORTHY Numerous studies link DASH diet to lower blood pressure, but its mechanism is unclear. Urinary extracellular vesicles (uEVs) offer noninvasive insights, potentially replacing tissue sampling. Transitioning to DASH diet alters kidney transporters in our stage 1 hypertension cohort: AQP2 decreases, NCC increases in uEVs. This aligns with increased urine volume, reduced sodium reabsorption, and blood pressure decline. Our data highlight uEV protein changes as diet markers, suggesting some uEVs may function as waste pathways. We analyzed larger EVs alongside small EVs, and NCC in immunoblots across its molecular weight range.

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.

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.

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.

RhoGDI2: a new metastasis suppressor gene: discovery and clinical translation.

The greatest risk for morbidity and mortality caused by bladder cancer is due to metastasis. For this reason, we have developed a paradigm for discovering the molecular mechanisms underlying bladder cancer progression to an invasive and metastatic phenotype. Results of microarray gene expression analysis of a cell culture model were parsed by identifying overlapping genes that correlate with increasing stage and grade of human tumors. One gene identified by this method, RhoGDI2, was tested in various in vitro and in vivo model systems and confirmed to be a metastasis suppressor gene. Using a similar strategy of gene identification by concordance of microarray gene expression results from cells expressing RhoGDI2 and human bladder cancers, two molecular effectors of RhoGDI2 signaling were identified. These targets, endothelin-1 and Neuromedin U are excellent potential targets for therapeutic intervention in the metastatic cascade.

The role of Ral A in epidermal growth factor receptor-regulated cell motility.

Tumor cell motility is one of the rate-limiting steps of invasion, which defines progression toward a more malignant phenotype. Elevated expression of epidermal growth factor (EGF) receptor in many cancers is associated with progression of superficial to invasive forms of the disease and is sometimes found in tumors that also have activating Ras mutations, suggesting that both events contribute to tumor invasion. Here we show that EGF stimulates motility in human tumor cell lines, which harbor activating Ha-RasV12 via a novel signal transduction pathway mediated by the small GTP-binding proteins RalA and RhoA but independent of Rac1 and Cdc42. On EGF stimulation, RalA localizes to the cell membrane. In addition, activation of RalA and expression of Rho were increased by EGF stimulation in both the nonmetastatic and metastatic variants of the same cell line. However, elevated levels of constitutively activated RalA were only found in the metastatic variant. This is the first demonstration of an essential role for Ral in EGF-mediated cell motility and its potential contribution to tumor metastasis in human cancer.

Profiling the evolution of human metastatic bladder cancer.

Pulmonary metastases frequently develop in patients with aggressive bladder cancer, yet investigation of this process at the molecular level suffers from the poor availability of human metastatic tumor tissue and the absence of suitable animal models. To address this, we developed progressively more metastatic human bladder cancer cell lines and an in vivo bladder-cancer lung-metastasis model, and we successfully used these to identify genes of which the expression levels change according to the degree of pulmonary metastatic potential. By initially intravenously injecting the poorly metastatic T24T human urothelial cancer cells into nude mice, and then serially reintroducing and reisolating the human tumor cells from the resultant mouse lung tumors, three derivative human lines with increasingly metastatic phenotypes, designated FL1, FL2, and FL3, were sequentially isolated. To identify the genes associated with the most lung-metastatic phenotype, the RNA complement from the parental and derivative cells was evaluated with oligonucleotide microarrays. In doing so, we found 121 genes to be progressively up-regulated during the transition from T24T to FL3, whereas 43 genes were progressively down-regulated. As expected, many of the genes identified in these groups could, according to the ascribed functions of their protein product, theoretically participate in tissue invasion and metastasis. In addition, the magnitude of gene expression changes observed during the metastatic transition correlated with the in vivo propensity for earlier lung colonization and decreased host survival. To additionally define which genes found in the experimental system were of relevance to human bladder cancer lung metastasis, we evaluated gene expression profiles of 23 primary human bladder tumors of various stages and grades, and then we compared these gene expression profiles to the altered profiles in our model cell lines. Here we found that the expression of epiregulin, urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP)14, and tissue inhibitor of metalloproteinase (TIMP-2) were consistently and progressively up-regulated when viewed as a function of tumor stage in tissues of patients versus the metastatic potential seen in the mouse lung model. The strong correlation of these four markers between the experimental and clinical situations helps validate this system as a useful tool for the study of lung metastasis and defines targets of therapy that may reduce the incidence of this process in patients.

Functional genomic comparison of lineage-related human bladder cancer cell lines with differing tumorigenic and metastatic potentials by spectral karyotyping, comparative genomic hybridization, and a novel method of positional expression profiling.

We have recently characterized T24T, an invasive and metastatic variant of the T24 human bladder cell line, resulting in a model for bladder cancer progression. To gain additional insight into the repertoire of genetic changes that may be responsible for the invasive and metastatic phenotype, we used spectral karyotyping (SKY) in combination with comparative genomic hybridization (CGH) in these cells. To assess the functional significance of the genetic differences found between the two cell lines, we have developed a positional expression profiling (PEP) method for comparing gene expression data obtained from oligonucleotide microarrays based upon chromosomal position. Using SKY and CGH, we were able to define the genetic changes in the cell lines, and in addition, resolve the identity of all marker chromosomes from our initial karyotyping and G-band analysis. PEP analysis revealed important similarities and differences when compared with the cytogenetic data, allowing insights of how genomic structural changes affect gene expression on a regional scale. The shape of the expression profiles for chromosomes 8, 12, and X correlated well with the numerical imbalances revealed by CGH and SKY, whereas regions like 10q, gained in T24T compared with T24, was not associated with changes in gene expression. Furthermore, we have shown that 12p, a region of agreement between CGH and PEP harbors RhoGDI2, a candidate gene, the expression of which inversely correlates with bladder tumor progression, demonstrating the usefulness of this multimodal approach in identifying promising genetic changes that may be responsible for the invasive phenotype.

RhoGDI2 is an invasion and metastasis suppressor gene in human cancer.

To discover novel metastasis suppressor genes that are clinically relevant in common human cancers, we used isogenic human bladder cancer cell lines and used DNA microarray technology to identify genes whose expression diminishes as a function of invasive and metastatic competence. We then evaluated the expression profile of such genes in 105 pathologically characterized tumors from seven common organ sites, and we identified one gene, RhoGDI2, whose expression was diminished as a function of primary tumor stage and grade. When RhoGDI2 was transferred back into cells with metastatic ability that lacked its expression, it suppressed experimental lung metastasis but did not affect in vitro growth, colony formation, or in vivo tumorigenicity. In addition, RhoGDI2 reconstitution in these cells blocked invasion in an organotypic assay and led to a reduction of in vitro motility. These results indicate that RhoGDI2 is a metastasis suppressor gene, a marker of aggressive human cancer, and a promising target for therapy.

PTEN can inhibit in vitro organotypic and in vivo orthotopic invasion of human bladder cancer cells even in the absence of its lipid phosphatase activity.

Recent studies have found a higher frequency of the PTEN tumor-suppressor gene alterations in invasive bladder carcinoma than in superficial disease, suggesting that PTEN is important in this process. A role of PTEN in bladder cancer invasion is further suggested by the fact that PTEN is a regulator of cell motility, a necessary component of tumor invasion. However, it is unknown whether PTEN is mechanistically involved in 'in vivo' tumor invasion or merely an epiphenomenon and, if the former is true, whether this process is dependent on its protein or lipid phosphatase activities. To address these issues, we stably transfected several commonly used human bladder cancer cell lines with known invasive phenotypes with either wild-type PTEN constructs or those deficient in the lipid phosphatase (G129E) or both protein and lipid phosphatase (G129R) activities. Here we show that chemotaxis was inhibited by both the wild-type and G129E mutant of PTEN but not by G129R-transfected cells. Using a novel organotypic in vitro invasion assay, we evaluated the impact of wild-type and mutant PTEN transgene expression on the invasive ability of T24T, a human bladder cancer cell line with a functionally impaired PTEN. Results indicate that the G129E mutant blocks invasion as efficiently as wild-type PTEN transfection. In contrast to the wild-type gene, this mutant has no effect on cell clonogenicity in agar. To further establish the role of PTEN in tumor invasion, we evaluated vector- and PTEN-transfected T24T cells in an orthotopic in vivo assay that faithfully reproduces human disease. Microscopic examination of murine bladders at the completion of this experiment parallels the results obtained with the organotypic assay. Our results are the first demonstration: (1) that the inhibitory effects of PTEN on cell motility translate into suppression of in vivo invasion; (2) that PTEN can inhibit tumor invasion even in the absence of its lipid phosphatase activity; (3) how organotypic in vitro approaches can be used as surrogates of in vivo invasion allowing rapid dissection of molecular processes leading to this phenotype while reducing the number of animals used in research.

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.

RhoGDI signaling provides targets for cancer therapy.

Rho GDP-Dissociation Inhibitors (RhoGDIs) are important regulators of the Rho family of small GTPases. The expression of RhoGDIs is altered in a variety of cancers and they have been shown to mediate several processes during tumorigenesis and cancer progression. Using examples of RhoGDI-mediated signaling and expression patterns in endothelial cells as well as pancreatic, breast, and bladder cancer, the multitude of potential cancer therapeutic targets presented by a better understanding of their function is illustrated. Several novel therapeutic strategies are proposed for intervening in RhoGDI signaling, and potential complications arising from their implementation are discussed.