Expression of ral GTPases, their effectors, and activators in human bladder cancer.

PURPOSE: The Ral family of small G proteins has been implicated in tumorigenesis, invasion, and metastasis in in vitro and animal model systems; however, a systematic evaluation of the state of activation, mutation, or expression of these GTPases has not been reported in any tumor type. EXPERIMENTAL DESIGN: We determined the activation state of the RalA and RalB paralogs in 10 bladder cancer cell lines with varying Ras mutation status. We sequenced RalA and RalB cDNAs from 20 bladder cancer cell lines and functionally evaluated the mutations found. We determined the expression of Ral, Ral activators, and Ral effectors on the level of mRNA or protein in human bladder cancer cell lines and tissues. RESULTS: We uncovered one E97Q substitution mutation of RalA in 1 of 20 cell lines tested and higher Ral activation in cells harboring mutant HRAS. We found overexpression of mRNAs for RalA and Aurora-A, a mitotic kinase that activates RalA, in bladder cancer (both P < 0.001), and in association with tumors of higher stage and grade. RalBP1, a canonical Ral effector, mRNA and protein was overexpressed in bladder cancer (P < 0.001), whereas Filamin A was underexpressed (P = 0.004). We determined that RalA mRNA levels correlated significantly with protein levels (P < 0.001) and found protein overexpression of both GTPases in homogenized invasive cancers. Available data sets suggest that RalA mRNA is also overexpressed in seminoma, glioblastoma, and carcinomas of the liver, pancreas, and prostate. CONCLUSION: These findings of activation and differential expression of RalA and RalB anchor prior work in model systems to human disease and suggest therapeutic strategies targeting both GTPases in this pathway may be beneficial.

Depletion of major vault protein increases doxorubicin sensitivity and nuclear accumulation and disrupts its sequestration in lysosomes.

The major vault protein (MVP) is the major constituent of the vault particle, the largest known ribonuclear protein complex. To date, vaults have no clear function, although their low expression levels in de novo chemosensitive and curable tumors, such as testicular cancer, make them attractive candidates as contributors to intrinsic drug resistance. Here, we show that MVP knockdown in human bladder cancer cells via small interfering RNA results in sensitization toward doxorubicin in two distinct exposure protocols. The drug was detected in the nucleus immediately following addition and was subsequently sequestered to lysosomes, predominantly located adjacent to the nucleus. MVP knockdown leads to increased sensitivity toward doxorubicin and an enhanced nuclear accumulation of the drug as well as a loss of its perinuclear sequestration. Not only doxorubicin subcellular distribution was perturbed by MVP knockdown but lysosomal markers, such as pH-sensitive LysoSensor, pinocytosed dextran conjugates after 24-h chase period, and the lysosomal specific antigen Lamp-1, also showed a markedly different staining compared with controls. Lysosomes appeared dispersed through the cytoplasm without a clear organization adjacent to the nucleus. Microtubules, however, appeared unperturbed in cells with reduced MVP expression. Based on these data, we hypothesize that MVP and, by extension, vault complexes are important for lysosomal function and may influence cellular drug resistance by virtue of this role.

A novel model to identify interaction partners of the PTEN tumor suppressor gene in human bladder cancer.

Phosphatase and tensin homolog (PTEN), deleted on chromosome 10, is a potent tumor suppressor. PTEN expression is reduced in advanced bladder cancer and reduction correlates with disease stage. To gain insights into the function of PTEN in human bladder cancer by identifying its binding partners, we developed a novel IPTG inducible PTEN expression system and evaluated this system in the PTEN null UMUC-3 human bladder cancer xenograft model. In this model, induction of PTEN in vivo resulted in reduced tumor growth. We used mass spectrometry to identify PTEN interaction partners in these cells, which identified known interaction partners major vault protein (MVP) and paxillin as well as a novel interaction partner, TRK fused gene (TFG). In conclusion, using a biologically relevant model system to dissect PTEN tumor suppressor function in human bladder cancer, we identified three molecules important for many cellular functions in complex with PTEN.

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.

RalA and RalB: antagonistic relatives in cancer cell migration.

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 approximately 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.

The role of Ras superfamily proteins in bladder cancer progression.

PURPOSE: The prognosis of patients with bladder cancer is strongly dependent on whether the lesion is superficial or invasive at initial presentation. In addition, a significant fraction of patients presenting with superficial disease have invasive tumor during followup. Understanding how superficial bladder cancer progresses to invasive forms of the disease is of paramount importance for early diagnosis and successful treatment. Molecular mechanisms underlying bladder cancer progression are being elucidated. We reviewed the roles that members of the Ras superfamily of monomeric G proteins, an important class of cellular regulator, have in bladder cancer and its progression. MATERIALS AND METHODS: We performed MEDLINE searches focusing on members of the Ras superfamily of monomeric G proteins and their involvement in transitional cell carcinoma, which is the most common form of bladder cancer. General involvement in cancer of key superfamily members, focusing on mechanisms and downstream pathways, was also reviewed through MEDLINE and manual bibliographic searches. RESULTS: With more than 100 members in humans the Ras superfamily is a diverse group of monomeric G proteins. These proteins regulate many cellular processes, such as cell cycle progression, actin cytoskeletal dynamics and membrane traffic. Members of the Ras and Rho family are also known to be involved in human cancer through mutation, over expression and dysregulation. In this review we focus on bladder cancer. In particular we focus on how H-Ras, RalA/B and RhoGDI2, a regulator of Rho family members, participate in bladder cancer progression and how their participation may be related to other molecules associated with bladder cancer progression, such as epidermal growth factor receptor, p53 and PTEN (phosphatase and tensin homologue deleted on chromosome 10). CONCLUSIONS: The findings discussed offer the hopeful possibility that signaling pathways mediated by Ras superfamily members may offer new opportunities for diagnostic and therapeutic interventions in bladder cancer.

Ras superfamily monomeric G proteins in carcinoma cell motility.

With over 100 members in humans, the Ras superfamily is a diverse group of monomeric G proteins participating in many cellular processes. Members of the Ras, Rho, and Arf families have been shown to regulate cell motility in fibroblasts and epithelial cells. Ras and Rho family members are also widely involved in human tumorigenesis, either through activating mutations or by overexpression. In this review, tools for studying carcinoma cell migration are discussed and evidence for regulation of carcinoma cell motility by specific Ras superfamily members is summarized. Novel emerging mechanisms of migration in carcinoma cells involving RhoC and Ral are also discussed.

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.