Yehia Daaka

A role for the G12 family of heterotrimeric G-proteins in prostate cancer invasion

Many studies have suggested a role for the members of the G12 family of heterotrimeric G proteins (Gα12 and Gα13) in oncogenesis and tumor cell growth. However, few studies have examined G12 signaling in actual human cancers. In this study, we examined the role of G12 signaling in prostate cancer. We found that expression of the G12 proteins is significantly elevated in prostate cancer. Interestingly, expression of the activated forms of Gα12 or Gα13 in the PC3 and DU145 prostate cancer cell lines did not promote cancer cell growth. Instead, expression of the activated forms of Gα12 or Gα13 in these cell lines induced cell invasion through the activation of the RhoA family of G proteins. Furthermore, inhibition of G12 signaling by expression of the RGS domain of the p115-Rho-specific guanine nucleotide exchange factor (p115-RGS) in the PC3 and DU145 cell lines did not reduce cancer cell growth. However, inhibition of G12 signaling with p115-RGS in these cell lines blocked thrombin- and thromboxane A2-stimulated cell invasion. These observations identify the G12 family proteins as important regulators of prostate cancer invasion and suggest that these proteins may be targeted to limit invasion- and metastasis-induced prostate cancer patient mortality.

Prostaglandin E2 Promotes Lung Cancer Cell Migration Via EP4-βArrestin1-c-Src Signalsome

Many human cancers express elevated levels of cyclooxygenase-2 (COX-2), an enzyme responsible for the biosynthesis of prostaglandins. Available clinical data establish the protective effect of COX-2 inhibition on human cancer progression. However, despite these encouraging outcomes, the appearance of unwanted side effects remains a major hurdle for the general application of COX-2 inhibitors as effective cancer drugs. Hence, a better understanding of the molecular signals downstream of COX-2 is needed for the elucidation of drug targets that may improve cancer therapy. Here, we show that the COX-2 product prostaglandin E2 (PGE2) acts on cognate receptor EP4 to promote the migration of A549 lung cancer cells. Treatment with PGE2 enhances tyrosine kinase c-Src activation, and blockade of c-Src activity represses the PGE2-mediated lung cancer cell migration. PGE2 affects target cells by activating four receptors named EP1 to EP4. Use of EP subtype-selective ligand agonists suggested that EP4 mediates prostaglandin-induced A549 lung cancer cell migration, and this conclusion was confirmed using a short hairpin RNA approach to specifically knock down EP4 expression. Proximal EP4 effectors include heterotrimeric Gs and βArrestin proteins. Knockdown of βArrestin1 expression with shRNA significantly impaired the PGE2-induced c-Src activation and cell migration. Together, these results support the idea that increased expression of the COX-2 product PGE2 in the lung tumor microenvironment may initiate a mitogenic signaling cascade composed of EP4, βArrestin1, and c-Src which mediates cancer cell migration. Selective targeting of EP4 with a ligand antagonist may provide an efficient approach to better manage patients with advanced lung cancer. Mol Cancer Res; 8(4); 569–77. ©2010 AACR.

Identification of βArrestin2 as a corepressor of androgen receptor signaling in prostate cancer

Androgen receptor (AR) signaling regulates the development and homeostasis of male reproductive organs, including the prostate. Deregulation of AR and AR coregulators, expression, or activity is involved in the initiation of prostate cancer and contributes to the transition of the disease to hormone-refractory stage. The ubiquitous βArrestin proteins are now recognized as bona fide adapters and signal transducers with target effectors found in both the cytosol and nucleus. Here, we provide evidence that βArrestin2 forms a complex with AR and acts as an AR corepressor in androgen-dependent prostate cancer cells. Accordingly, the forced overexpression of βArrestin2 diminishes, and knockdown of βArrestin2 expression with RNAi increases the androgen-induced prostate-specific antigen (PSA) gene expression. βArrestin2 serves as an adapter, bringing into close proximity the Mdm2 E3 ligase and AR, thereby promoting AR ubiquitylation and degradation. Human prostate tissues evidence an inverse relationship between the expression of βArrestin2 and AR activity: glands that express high levels of βArrestin2 exhibit low expression of PSA, and those glands that express low levels of βArrestin2 evidence elevated PSA levels. We conclude that βArrestin2 acts as a corepressor of AR by serving as a scaffold for Mdm2 leading to the AR ubiquitylation and degradation.

PGE2 promotes angiogenesis through EP4 and PKA Cγ pathway

Inflammation is increasingly recognized as a critical mediator of angiogenesis, and unregulated angiogenic response is involved in human diseases, including cancer. Proinflammatory prostaglandin E2 (PGE2) is secreted by many cell types and plays important roles in the process of angiogenesis via activation of cognate EP1-4 receptors. Here, we provide evidence that PGE2 promotes the in vitro tube formation of human microvascular endothelial cells, ex vivo vessel outgrowth of aortic rings, and actual in vivo angiogenesis. Use of EP subtype-selective agonists and antagonists suggested EP4 mediates the prostaglandin-induced tube formation, and this conclusion was substantiated with small interfering RNA to specifically knockdown the EP4 expression. EP4 couples to Gαs, leading to activation of protein kinase A (PKA). Inhibition of PKA activity or knockdown of PKA catalytic subunit γ with RNAi attenuates the PGE2-induced tube formation. Further, knocking down the expression of Rap1A, HSPB6, or endothelial NO synthase, which serve as PKA-activatable substrates, inhibits the tube formation, whereas knockdown of RhoA or glycogen synthase kinase 3β that are inactivated after phosphorylation by PKA increases the tube formation. These results support the existence of EP4-to-PKA angiogenic signal and provide rationale for use of selective EP4 signal inhibitors as a probable strategy to control pathologic angiogenesis.

Targeting Gβγ Signaling to Inhibit Prostate Tumor Formation and Growth

Prostate cancer starts as androgen-dependent malignancy and responds initially to androgen ablative therapy. Beneficial effects of androgen ablation, however, are often temporary and the cancer reappears as androgen-independent tumor, suggesting the existence of additional factors responsible for progression of the disease. Attention has focused on receptor tyrosine kinases as the growth mediators of androgen-independent prostate cancer; overexpression of epidermal growth factor receptors or their ligand heparin-bound epidermal growth factor, for example, promotes transition to androgen independence. Emerging data demonstrate involvement of another class of cell membrane-anchored receptors, the heterotrimeric guanine-binding (G) protein-coupled receptors (GPCRs) in prostate cancer. In vitro, stimulation of many endogenous GPCRs induces mitogenic signaling and growth of prostate cancer cells. The GPCRs transduce mitogenic signals via activated G proteins in the form of Gα-GTP and Gβγ subunits. Here, we show that expression of a Gβγ inhibitor peptide derived from carboxy terminus of G protein-coupled receptor kinase 2 obliterates serum-regulated prostate cancer cell growth in vitro and prevents prostate tumor formation in vivo. We also demonstrate that inhibition of Gβγ signaling retards growth of existing prostate tumors by inducing cell death. These data establish a central role for heterotrimeric G proteins in prostate cancer and suggest targeted inhibition of Gβγ signaling may serve as specific molecular therapy tool to limit pathologic growth of advanced prostate cancer.

Prostaglandin E2 Regulates Renal Cell Carcinoma Invasion through the EP4 Receptor-Rap GTPase Signal Transduction Pathway

Prognosis for patients with early stage kidney cancer has improved, but the treatment options for patients with locally advanced disease and metastasis remain few. Understanding the molecular mechanisms that regulate invasion and metastasis is critical for developing successful therapies to treat these patients. Proinflammatory prostaglandin E2 plays an important role in cancer initiation and progression via activation of cognate EP receptors that belong to the superfamily of G protein-coupled receptors. Here we report that prostaglandin E2 promotes renal cancer cell invasion through a signal transduction pathway that encompasses EP4 and small GTPase Rap. Inactivation of Rap signaling with Rap1GAP, like inhibition of EP4 signaling with ligand antagonist or knockdown with shRNA, reduces the kidney cancer cell invasion. Human kidney cells evidence increased EP4 and decreased Rap1GAP expression levels in the malignant compared with benign samples. These results support the idea that targeted inhibition of EP4 signaling and restoration of Rap1GAP expression constitute a new strategy to control kidney cancer progression.

Aberrant PGE2 metabolism in bladder tumor microenvironment promotes immunosuppressive phenotype of tumor-infiltrating myeloid cells

Bladder cancer is associated with enhanced inflammation and characterized by deregulated prostanoid metabolism. Here we examined prostaglandin E₂ (PGE₂) metabolism and myeloid cell subsets that infiltrate tumor tissue using two xenograft models of human bladder cancer. Human bladder tumor xenografts implanted into athymic nude mice become highly infiltrated with host CD11b myeloid cells of bone marrow origin. Fast growing SW780 bladder tumor xenografts were infiltrated with heterogeneous CD11b myeloid cell subsets including tumor-associated macrophages and myeloid-derived suppressor cells. In contrast, majority of myeloid cells in tumor tissue from slow growing bladder cancer Urothel 11 displayed more immature, homogenous phenotype and comprised mostly MHC II class-negative myeloid-derived suppressor cells. We demonstrate that human bladder tumors secrete substantial amounts of PGE₂. Normal bone marrow myeloid cell progenitors cultured in the presence of a bladder tumor-conditioned medium, which is enriched for PGE₂, failed to differentiate into mature APCs and acquired phenotype of the myeloid-derived suppressor cells or inflammatory macrophages with up-regulated chemokine receptor CXCR4. Collectively our data demonstrate that enhanced cancer-related inflammation and deregulated PGE₂ metabolism in tumor microenvironment promote immunosuppressive pro-tumoral phenotype of myeloid cells in bladder cancer. These data also suggest that not only local tumor microenvironment but other factors such as stage of cancer disease and pace of tumor growth could markedly influence the phenotype, differentiation and immune function of myeloid cells in tumor tissue.

Rap1GAP regulates renal cell carcinoma invasion

Although patients with localized and regional kidney tumors have a high survival rate, incidence of mortality significantly increases for patients with metastatic disease. It is imperative to decipher the molecular mechanisms of kidney tumor migration and invasion in order to develop effective therapies for patients with advanced cancer. Rap1, a small GTPase protein, has been implicated in cancer cell growth and invasion. Here, we profile migratory and invasive properties of commonly used renal cell carcinoma (RCC) cell lines and correlate that with expression and function of the Rap inactivator Rap1GAP. We report that levels of Rap1GAP inversely correlate with invasion but not migration. We also report that forced over-expression of Rap1GAP decreases invasion of RCC cells but does not impact their rate of proliferation. Low expression levels of Rap1GAP in RCC cells are due, at least in part, to promoter hypermethylation. Rescued expression of Rap1GAP with a demethylating drug, decitabine (5-azadC), decreases the RCC SN12C cell invasion of collagen, fibronectin, and Matrigel matrices. RCC cell lines express distinct levels of cell adhesion proteins and the forced over-expression of Rap1GAP attenuated levels of both cadherins and integrins that are known to regulate the cancer cells invasion. These results demonstrate that targeted restoration of Rap1GAP expression may serve as a potential therapeutic approach to reduce metastasis of kidney cancers.

Arf GTPase-activating Protein AGAP2 Regulates Focal Adhesion Kinase Activity and Focal Adhesion Remodeling

Focal adhesions are specialized sites of cell attachment to the extracellular matrix where integrin receptors link extracellular matrix to the actin cytoskeleton, and they are constantly remodeled during cell migration. Focal adhesion kinase (FAK) is an important regulator of focal adhesion remodeling. AGAP2 is an Arf GTPase-activating protein that regulates endosomal trafficking and is overexpressed in different human cancers. Here we examined the regulation of the FAK activity and the focal adhesion remodeling by AGAP2. Our results show that FAK binds the pleckstrin homology domain of AGAP2, and the binding is independent of FAK activation following epidermal growth factor receptor stimulation. Overexpression of AGAP2 augments the activity of FAK, and concordantly, the knockdown of AGAP2 expression with RNA interference attenuates the FAK activity stimulated by epidermal growth factor or platelet-derived growth factor receptors. AGAP2 is localized to the focal adhesions, and its overexpression results in dissolution of the focal adhesions, whereas knockdown of its expression stabilizes them. The AGAP2-induced dissolution of the focal adhesions is independent of its GTPase-activating protein activity but may involve its N-terminal G protein-like domain. Our results indicate that AGAP2 regulates the FAK activity and the focal adhesion disassembly during cell migration.

G-protein coupled receptor kinase 5 regulates prostate tumor growth.

PURPOSE The limited success of cancer therapeutics is largely attributable to the ability of cancer to become resistant to conventional cytotoxic chemotherapy. Thus, further identification of signaling molecules and pathways that influence tumorigenesis is needed to increase the overall therapeutic options. GRKs, originally recognized for their conserved role in GPCR signal control, have now emerged as regulators of additional biological molecules and functions. MATERIALS AND METHODS We used Western blot analysis to determine GRK expression in prostate cancer and RNA interference to establish the role of GRK5 in prostate cancer growth and progression through the cell cycle. RESULTS GRK5 was expressed highly in the aggressive prostate cancer PC3 cell line and its silencing by RNA interference attenuated in vitro cell proliferation. PC3 cells that stably expressed lentiviral small hairpin RNA and targeted GRK5 evidence reduced xenograft tumor growth in mice. This was reversed by rescuing expression with wild-type but not with kinase inactive K215R GRK5, implying the need of GRK5 kinase activity for tumor growth. To investigate possible cellular mechanism(s) for GRK5 in cell growth regulation we tested whether kinase activity would impact cell cycle progression. Like forced over expression of kinase-inactive K215R GRK5, GRK5 knockdown led to G2/M arrest in the cell cycle. Also, evidence revealed that the loss of GRK5 activity resulted in decreased cyclin D1 expression, Rb protein phosphorylation and E2F target gene expression involved in cell cycle control. CONCLUSIONS Results provide direct evidence that GRK5 has an immediate role in the regulation of prostate tumor growth.