A NEW TUMOR SUPPRESSOR GENE CANDIDATE REGULATED BY THE NON-CODING RNA PCA3 IN HUMAN PROSTATE CANCER

Prostate cancer is the second leading cause of cancer-related death and the most common non-skin cancer in men in the USA. Considerable advancements in the practice of medicine have allowed a significant improvement in the diagnosis and treatment of this disease and, in recent years, both incidence and mortality rates have been slightly declining. However, it is still estimated that 1 man in 6 will be diagnosed with prostate cancer during his lifetime, and 1 man in 35 will die of the disease. In order to identify novel strategies and effective therapeutic approaches in the fight against prostate cancer, it is imperative to improve our understanding of its complex biology since many aspects of prostate cancer initiation and progression still remain elusive. The study of tumor biomarkers, due to their specific altered expression in tumor versus normal tissue, is a valid tool for elucidating key aspects of cancer biology, and may provide important insights into the molecular mechanisms underlining the tumorigenesis process of prostate cancer. PCA3, is considered the most specific prostate cancer biomarker, however its biological role, until now, remained unknown. PCA3 is a long non-coding RNA (ncRNA) expressed from chromosome 9q21 and its study led us to the discovery of a novel human gene, PC-TSGC, transcribed from the opposite strand and in an antisense orientation to PCA3. With the work presented in this thesis, we demonstrate that PCA3 exerts a negative regulatory role over PC-TSGC, and we propose PC-TSGC to be a new tumor suppressor gene that contrasts the transformation of prostate cells by inhibiting Rho-GTPases signaling pathways. Our findings provide a biological role for PCA3 in prostate cancer and suggest a new mechanism of tumor suppressor gene inactivation mediated by non-coding RNA. Also, the characterization of PCA3 and PC-TSGC led us to propose a new molecular pathway involving both genes in the transformation process of the prostate, thus providing a new piece of the jigsaw puzzle representing the complex biology of prostate cancer.

CELLULAR AND DEVELOPMENTAL FUNCTIONS OF THE XENOPUS ARVCF-CATENIN:KAZRIN COMPLEX

Xenopus ARVCF (xARVCF), a member of p120-catenin subfamily, binds cadherin cytoplasmic domains to enhance cadherin metabolic stability, or when dissociated, modulates Rho-family GTPases. We previously found that xARVCF binds directly to Xenopus KazrinA (xKazrinA), a widely expressed, conserved protein that bears little homology to established protein families. xKazrinA is also known to influence keratinocyte proliferation-differentiation and cytoskeletal activity. In my study, I first evaluated the expression pattern of endogenous Kazrin RNA and protein in Xenopus embryogenesis as well as in adult tissues. We then collaboratively predicted the helical structure of Kazrin’s coiled-coil domain, and I obtained evidence of Kazrin’s dimerization/oligomerization. In considering the intracellular localization of the xARVCF-catenin:xKazrin complex, I did not resolve xKazrinA in a larger ternary complex with cadherin, nor did I detect its co-precipitation with core desmosomal components. Instead, screening revealed that xKazrinA binds spectrin. This suggested a potential means by which xKazrinA localizes to cell-cell junctions, and indeed, biochemical assays confirmed a ternary xARVCF:xKazrinA:xβ2-spectrin complex. Functionally, I demonstrated that xKazrin stabilizes cadherins by negatively modulating the RhoA small-GTPase. I further revealed that xKazrinA binds to p190B RhoGAP (an inhibitor of RhoA), and enhances p190B’s association with xARVCF. Supporting their functional interaction in vivo, Xenopus embryos depleted of xKazrin exhibited ectodermal shedding, a phenotype that could be rescued with exogenous xARVCF. Cell shedding appeared to be caused by RhoA activation, which consequently altered actin organization and cadherin function. Indeed, I was capable of rescuing Kazrin depletion with ectopic expression of p190B RhoGAP. In addition, I obtained evidence that xARVCF and xKazrin participate in craniofacial development, with effects observed upon the neural crest. Finally, I found that xKazrinA associates further with delta-catenin and p0071-catenin, but not with p120-catenin, suggesting that Kazrin interacts selectively with additional members of the p120-catenin sub-family. Taken together, my study supports Kazrin’s essential role in development, and reveals KazrinA’s biochemical and functional association with ARVCF-catenin, spectrin and p190B RhoGAP.

DEVELOPMENTAL AND CELLULAR FUNCTIONS OF DELTA-CATENIN

Catenins have diverse and powerful roles in embryogenesis, homeostasis or disease progression, as best exemplified by the well-known beta-catenin. The less studied delta-catenin likewise contains a central Armadillo-domain. In common with other p120 sub-class members, it acts in a variety of intracellular compartments and modulates cadherin stability, small GTPase activities and gene transcription. In mammals, delta-catenin exhibits neural specific expression, with its knock-out in mice correspondingly producing cognitive defects and synaptic dysfunctions. My work instead employed the amphibian, Xenopus laevis, to explore delta-catenin’s physiological functions in a distinct vertebrate system. Initial isolation and characterization indicated delta-catenin’s expression in Xenopus. Unlike the pattern observed for mammals, delta-catenin was detected in most adult Xenopus tissues, although enriched in embryonic structures of neural fate as visualized using RNA in-situ hybridization. To determine delta-catenin’s requirement in amphibian development, I employed anti-sense morpholinos to knock-down gene products, finding that delta-catenin depletion results in developmental defects in gastrulation, neural crest migration and kidney tubulogenesis, phenotypes that were specific based upon rescue experiments. In biochemical and cellular assays, delta-catenin knock-down reduced cadherin levels and cell adhesion, and impaired activation of RhoA and Rac1, small GTPases that regulate actin dynamics and morphogenetic movements. Indeed, exogenous C-cadherin, or dominant-negative RhoA or dominant-active Rac1, significantly rescued delta-catenin depletion. Thus, my results indicate delta-catenin’s essential roles in Xenopus development, with contributing functional links to cadherins and Rho family small G proteins. In examining delta-catenin’s nuclear roles, I identified delta-catenin as an interacting partner and substrate of the caspase-3 protease, which plays critical roles in apoptotic as well as non-apoptotic processes. Delta-catenin’s interaction with and sensitivity to caspase-3 was confirmed using assays involving its cleavage in vitro, as well as within Xenopus apoptotic extracts or mammalian cell lines. The cleavage site, a highly conserved caspase consensus motif (DELD) within Armadillo-repeat 6 of delta-catenin, was identified through peptide sequencing. Cleavage thus generates an amino- (1-816) and carboxyl-terminal (817-1314) fragment each containing about half of the central Armadillo-domain. I found that cleavage of delta-catenin both abolishes its association with cadherins, and impairs its ability to modulate small GTPases. Interestingly, the carboxyl-terminal fragment (817-1314) possesses a conserved putative nuclear localization signal that I found is needed to facilitate delta-catenin’s nuclear targeting. To probe for novel nuclear roles of delta-catenin, I performed yeast two-hybrid screening of a mouse brain cDNA library, resolving and then validating its interaction with an uncharacterized KRAB family zinc finger protein I named ZIFCAT. My results indicate that ZIFCAT is nuclear, and suggest that it may associate with DNA as a transcriptional repressor. I further determined that other p120 sub-class catenins are similarly cleaved by caspase-3, and likewise bind ZIFCAT. These findings potentially reveal a simple yet novel signaling pathway based upon caspase-3 cleavage of p120 sub-family members, facilitating the coordinate modulation of cadherins, small GTPases and nuclear functions. Together, my work suggested delta-catenin’s essential roles in Xenopus development, and has revealed its novel contributions to cell junctions (via cadherins), cytoskeleton (via small G proteins), and nucleus (via ZIFCAT). Future questions include the larger role and gene targets of delta-catenin in nucleus, and identification of upstream signaling events controlling delta-catenin’s activities in development or disease progression.

Identification and characterization of Xenopus Kazrin, a nucleocytoplasmic shuttling protein that interacts with ARVCF catenin

In common with other members of the p120-catenin subclass of catenins, ARVCF-catenin appears to have multiple cellular and developmental functions. In Xenopus, our lab recently demonstrated that xARVCF- and Xp120-catenins are each essential for early vertebrate embryogenesis, being functionally linked to Rho-family GTPases (RhoA, Rac) and cadherin metabolic stability. For the project described here, the yeast two-hybrid system was employed to screen a Xenopus laevis neurula library for proteins that interact with xARVCF, resulting in the identification of the Xenopus homolog of Kazrin (xKazrin). Kazrin is a variably-spliced protein of unknown function that has been shown to interact with periplakin and envoplakin, components of desmosomal junctions. Kazrin's primary sequence is highly conserved across vertebrate species and is composed of an amino-terminal nuclear export sequence (NES), a carboxy-terminal nuclear localization sequence (NLS) and a central predicted coiled-coil domain. In vitro and in vivo authenticity tests demonstrated that xARVCF-catenin interacts directly with xKazrin via xARVCF's Armadillo and carboxy-terminal regions and xKazrin's coiled-coil domain. The interaction of xARVCF-catenin with xKazrin is specific and does not extend to the related Xp120-catenin. xKazrin co-localized with E-cadherin at sites of cell-cell contact and could be co-immunoprecipitated with components of the cadherin complex. xKazrin was also present in the cytoplasm and nucleus. Suggestive of a nuclear role, mutation of xKazrin's predicted NLS resulted in nuclear exclusion, while deletion of the predicted NES resulted in loss of sensitivity to nuclear export inhibitors. Within Xenopus embryos, xKazrin was expressed across all developmental stages and appeared at varying levels in adult tissues. Morpholino depletion of xKazrin from Xenopus embryos resulted in axial elongation abnormalities and loss of tissue integrity after neurulation. Over-expression of xKazrin had no effect, while over-expression of a NLS mutant resulted in a mild phenotype similar to that seen in xKazrin depleted embryos. Interestingly, the axial phenotype resulting from reduced xKazrin levels was largely rescuable by xARVCF over-expression. In conjunction with xARVCF-catenin, xKazrin has properties consistent with its function at cell-cell contact sites and in the nucleus. ^

p21 activated kinase 5 links multiple GTPase signaling pathways at mitochondria

The p21-activated kinase 5 (PAK5) is a serine/threonine protein kinase associated with the group 2 subfamily of PAKs. Although our understanding about PAK5 is very limited, it is receiving increasing interest due to its tissue specific expression pattern and important signaling properties. PAK5 is highly expressed in brain. Its overexpression induces neurite outgrowth in neuroblastoma cells and promotes survival in fibroblasts. ^ The serine/threonine protein kinase Raf-1 is an essential mediator of Ras-dependent signaling that controls the ERK/MAPK pathway. In contrast to PAK5, Raf-1 has been the subject of intensive investigation. However due to the complexity of its activation mechanism, the biological inputs controlling Raf-1 activation are not fully understood. ^ PAKs 1-3 are the known kinases responsible for phosphorylation of Raf-1 on serine 338, which is a crucial phosphorylation site for Raf-1 activation. However, dominant negative versions of these kinases do not block EGF-induced Raf-1 activation, indicating that other kinases may regulate the phosphorylation of Raf-1 on serine 338. ^ This thesis work was initiated to test whether the group 2 PAKs 4, 5 and 6 are responsible for EGF-induced Raf-1 activation. We found that PAK5, and to a lesser extent PAK4, can activate Raf-1 in cells. Our studies thereafter focused on PAK5. With the progress of our study we found that PAK5 does not significantly stimulate serine 338 phosphorylation of Triton X-100 soluble Raf-1. PAK5, however, constitutively and specifically associates with Raf-1 and targets it to a Triton X-100 insoluble, mitochondrial compartment, where PAK5 phosphorylates serine 338 of Raf-1. We further demonstrated that endogenous PAK5 and Raf-1 colocalize in Hela cells at the mitochondrial outer membrane. In addition, we found that the mitochondria-targeting of PAK5 is determined by its C-terminal kinase domain plus the upstream proximal region, and facilitated by the N-terminal p21 binding domain. We also demonstrated that Rho GTPases Cdc42 and RhoD associate with and regulate the subcellular localization of PAK5. Taken together, this work suggests that the mitochondria-targeting of PAK5 may link Ras and Rho GTPase-mediated signaling pathways, and sheds light on aspects of PAK5 signaling that may be important for regulating neuronal homeostasis. ^

Regulation of Net1A subcellular localization by the small GTPase Rac1

Activation of Rho family small G proteins is thought to be a critical event in breast cancer development and metastatic progression. Rho protein activation is stimulated by a family of enzymes known as guanine nucleotide exchange factors (Rho GEFs). The neuroepithelioma transforming gene 1 (Net1) is a Rho GEF specific for the RhoA subfamily that is overexpressed in primary breast tumors and breast cancer cell lines. Net1 isoform expression is also required for migration and invasion of breast cancer cells in vitro. These data indicate that Net1 may be a critical regulator of metastatic progression in breast cancer. Net1 activity is negatively regulated by sequestration in the nucleus, and relocalization of Net1 outside the nucleus is required to stimulate RhoA activation, actin cytoskeletal reorganization, and oncogenic transformation. However, regulatory mechanisms controlling the extranuclear localization of Net1 have not been identified. In this study, we have addressed the regulation of Net1A isoform localization by Rac1. Specifically, co-expression of constitutively active Rac1 with Net1A stimulates the relocalization of Net1A from the nucleus to the plasma membrane in breast cancer cells, and results in Net1A activation. Importantly, Net1A localization is also driven by endogenous Rac1 activity. Net1A relocalizes outside the nucleus in cells spreading on collagen, and when endogenous Rac1 expression was silenced by siRNA, Net1A remained nuclear in spreading cells. These data indicate that Rac1 controls the localization of the Net1A isoform and suggests a physiological role for Net1A in breast cancer cell adhesion and motility.

Increased geranylgeranylated K-Ras contributes to antineoplastic effects of farnesyltransferase inhibitors.

The Ras family of small GTPases (N-, H-, and K-Ras) is a group of important signaling mediators. Ras is frequently activated in some cancers, while others maintain low level activity to achieve optimal cell growth. In cells with endogenously low levels of active Ras, increasing Ras signaling through the ERK and p38 MAPK pathways can cause growth arrest or cell death. Ras requires prenylation – the addition of a 15-carbon (farnesyl) or 20-carbon (geranylgeranyl) group – to keep the protein anchored into membranes for effective signaling. N- and K-Ras can be alternatively geranylgeranylated (GG’d) if farnesylation is inhibited but are preferentially farnesylated. Small molecule inhibitors of farnesyltransferase (FTIs) have been developed as a means to alter Ras signaling. Our initial studies with FTIs in malignant and non-malignant cells revealed FTI-induced cell cycle arrest, reduced proliferation, and increased Ras signaling. These findings led us to the hypothesis that FTI induced increased GG’d Ras. We further hypothesized that the specific effects of FTI on cell cycle and growth result from increased signal strength of GG’d Ras. Our results did show that increase in GG’d K-Ras in particular results in reduced cell viability and cell cycle arrest. Genetically engineered constructs capable of only one type of prenylation confirmed that GG’d K-Ras recapitulated the effect of FTI in 293T cells. In tumor cell lines ERK and p38 MAPK pathways were both strongly activated in response to FTI, indicating the increased activity of GG’d K-Ras results in antiproliferative signals specifically through these pathways. These results collectively indicate FTI increases active GG’d K-Ras which activates ERK and p38 MAPKs to reduced cell viability and induce cell cycle arrest in malignant cells. This is the first report that identifies increased activity of GG’d K-Ras contributes to antineoplastic effects from FTI by increasing the activity of downstream MAPKs. Our observations suggest increased GG’d K-Ras activity, rather than inhibition of farnesylated Ras, is a major source of the cytostatic and cytotoxic effects of FTI. Our data may allow for determination of which patients would benefit from FTI by excluding tumors or diseases which have strong K-Ras signaling.

Development and Characterization of Novel Ras Inhibitors

Ras genes are mutated in 15% of human cancers. Ras GTPases operate as molecular switches regulating cellular processes including proliferation, differentiation, and apoptosis. The three main isoforms of Ras – H-Ras, K-Ras, and N-Ras – inhabit distinct nanodomains of the plasma membrane and intracellular compartments including the Golgi. However, the role of single endogenous Ras isoforms on these compartments remains unclear as most studies have utilized ectopically expressed and mutant forms of Ras proteins. In an effort to develop novel tools that will allow us to abrogate individual endogenous Ras isoforms, we targeted the catalytic domain of p120RasGAP to the plasma membrane with the hypervariable region (HVR) of H-Ras (GAP-CTH) or K-Ras (GAP-CTK) and to the Golgi using the HVR of H-Ras with insertion of a point mutation (GAP-CTH181S). We performed GST-RBD pull-downs on cells expressing each GAP construct and stimulated with epidermal growth factor (EGF). We found that GAP-CTH and GAP-CTK specifically inhibited H-Ras or K-Ras, respectively. However, we did not detect any effect of GAP-CTH181S on Ras activation. Additionally, we used confocal microscopy to verify the ability of GAP constructs to abrogate Ras activation in distinct sub-cellular compartments. We found that GAP-CTH inhibits H-Ras activation on the plasma membrane, while GAP-CTK inhibits K-Ras activation on the plasma membrane. On the contrary, GAP-CTH181S inhibited H-Ras activation on the Golgi. We also analyzed the effects of these GAP constructs on the activation of ERK and Akt in response to EGF stimulation. We found that EGF stimulation of the MAPK pathway was inhibited by GAP-CTK but none of the other GAP constructs, while Akt activation was not inhibited by any GAP construct. Finally, we assayed cellular proliferation and differentiation. We found that GAP-CTK and GAP-CTH were equipotent inhibitors of cellular growth, whereas GAP-CTH181S was less potent. We also found that GAP-CTK and GAP-CTH inhibited differentiation with similar potency, while GAP-CTH181S was more potent. This approach may be adapted to investigate any Ras-dependent signaling pathway. Therefore, it has the potential to become a powerful tool for studying Ras isoform-specific signaling outputs.

αvβ8 Integrin Interacts with RhoGDI1 to Regulate Rac1 and Cdc42 Activation and Drive Glioblastoma Cell Invasion

As an interface between the circulatory and central nervous systems, the neurovascular unit is vital to the development and survival of tumors. The malignant brain cancer glioblastoma multiforme (GBM) displays invasive growth behaviors that are major impediments to surgical resection and targeted therapies. Adhesion and signaling pathways that drive GBM cell invasion remain largely uncharacterized. Here we have utilized human GBM cell lines, primary patient samples, and pre-clinical mouse models to demonstrate that integrin αvβ8 is a major driver of GBM cell invasion. β8 integrin is overexpressed in many human GBM cells, with higher integrin expression correlating with increased invasion and diminished patient survival. Silencing β8 integrin in human GBM cells leads to impaired tumor cell invasion due to hyperactivation of the Rho GTPases Rac1 and Cdc42. β8 integrin associates with Rho GDP Dissociation Inhibitor 1 (RhoGDI1), an intracellular signaling effector that sequesters Rho GTPases in their inactive GDP-bound states. Silencing RhoGDI1 expression or uncoupling αvβ8 integrin-RhoGDI1 protein interactions blocks GBM cell invasion due to Rho GTPase hyperactivation. These data reveal for the first time that αvβ8 integrin, via interactions with RhoGDI1, suppresses activation of Rho proteins to promote GBM cell invasiveness. Hence, targeting the αvβ8 integrin-RhoGDI1 signaling axis may be an effective strategy for blocking GBM cell invasion.