Protein kinase A inhibitor proteins (PKIs) divert GPCR-Gαs-cAMP signaling toward EPAC and ERK activation and are involved in tumor growth.

The PKA-inhibitor (PKI) family members PKIα, PKIß, and PKIγ bind with high affinity to PKA and block its kinase activity, modulating the extent, and duration of PKA-mediated signaling events. While PKA is a well-known regulator of physiological and oncogenic events, the role of PKI proteins in these pathways has remained elusive. Here, by measuring activation of the MAPK pathway downstream of GPCR-Gαs-cAMP signaling, we show that the expression levels of PKI proteins can alter the balance of activation of two major cAMP targets: PKA and EPAC. Our results indicate that PKA maintains repressive control over MAPK signaling as well as a negative feedback on cAMP concentration. Overexpression of PKI and its subsequent repression of PKA dysregulates these signaling pathways, resulting in increased intracellular cAMP, and enhanced activation of EPAC and MAPK. We also find that amplifications of PKIA are common in prostate cancer and are associated with reduced progression free survival. Depletion of PKIA in prostate cancer cells leads to reduced migration, increased sensitivity to anoikis and reduced tumor growth. By altering PKA activity PKI can act as a molecular switch, driving GPCR-Gαs-cAMP signaling toward activation of EPAC-RAP1 and MAPK, ultimately modulating tumor growth.

Atypical chemokine receptors in tumor cell growth and metastasis.

Atypical chemokine receptors (ACKRs) are seven-transmembrane cell surface protein receptors expressed in immune cells, normal mesenchymal cells, and several tumor cells. As of this writing, six ACKRs have been characterized by diverse activities. They bind both cysteine-cysteine (CC) type and cysteine-X-cysteine (CXC)-type chemokines, either alone, or together with a ligand bound-functional G-protein coupled (typical) chemokine receptor. The major structural difference between ACKRs and typical chemokine receptors is the substituted DRYLAIV amino acid motif in the second intracellular loop of the ACKR. Due to this substitution, these receptors cannot bind Gαi-type G-proteins responsible for intracellular calcium mobilization and cellular chemotaxis. Although initially characterized as non-signaling transmembrane receptors (decoy receptors) that attenuate ligand-induced signaling by GPCRs, studies of all ACKRs have shown ligand-independent and ligand-dependent transmembrane signaling in both non-tumor and tumor cells. The precise function and mechanism of the differential expression of ACKRs in many tumors are not understood well. The use of antagonists of ACKRs ligands has shown limited antitumor potential; however, depleting ACKR expression resulted in a reduction in experimental tumor growth and metastasis. The ACKRs represent a unique class of transmembrane signaling proteins that regulate growth, survival, and metastatic processes in tumor cells, affecting multiple pathways of tumor growth. Therefore, closer investigations of ACKRs have a high potential for identifying therapeutics which affect the intracellular signaling, preferentially via the ligand-independent mechanism.

ß-Arrestins Regulate Stem Cell-Like Phenotype and Response to Chemotherapy in Bladder Cancer.

ß-Arrestins are classic attenuators of G-protein-coupled receptor signaling. However, they have multiple roles in cellular physiology, including carcinogenesis. This work shows for the first time that ß-arrestins have prognostic significance for predicting metastasis and response to chemotherapy in bladder cancer. ß-Arrestin-1 (ARRB1) and ß-arrestin-2 (ARRB2) mRNA levels were measured by quantitative RT-PCR in two clinical specimen cohorts (n = 63 and 43). The role of ARRBs in regulating a stem cell-like phenotype and response to chemotherapy treatments was investigated. The consequence of forced expression of ARRBs on tumor growth and response to Gemcitabine in vivo were investigated using bladder tumor xenografts in nude mice. ARRB1 levels were significantly elevated and ARRB2 levels downregulated in cancer tissues compared with normal tissues. In multivariate analysis only ARRB2 was an independent predictor of metastasis, disease-specific-mortality, and failure to Gemcitabine + Cisplatin (G+C) chemotherapy; ∼80% sensitivity and specificity to predict clinical outcome. ARRBs were found to regulate stem cell characteristics in bladder cancer cells. Depletion of ARRB2 resulted in increased cancer stem cell markers but ARRB2 overexpression reduced expression of stem cell markers (CD44, ALDH2, and BMI-1), and increased sensitivity toward Gemcitabine. Overexpression of ARRB2 resulted in reduced tumor growth and increased response to Gemcitabine in tumor xenografts. CRISPR-Cas9-mediated gene-knockout of ARRB1 resulted in the reversal of this aggressive phenotype. ARRBs regulate cancer stem cell-like properties in bladder cancer and are potential prognostic indicators for tumor progression and chemotherapy response.

Inhibition of androgen receptor promotes CXC-chemokine receptor 7-mediated prostate cancer cell survival.

The atypical C-X-C chemokine receptor 7 (CXCR7) has been implicated in supporting aggressive cancer phenotypes in several cancers including prostate cancer. However, the mechanisms driving overexpression of this receptor in cancer are poorly understood. This study investigates the role of androgen receptor (AR) in regulating CXCR7. Androgen deprivation or AR inhibition significantly increased CXCR7 expression in androgen-responsive prostate cancer cell lines, which was accompanied by enhanced epidermal growth factor receptor (EGFR)-mediated mitogenic signaling, promoting tumor cell survival through an androgen-independent signaling program. Using multiple approaches we demonstrate that AR directly binds to the CXCR7 promoter, suppressing transcription. Clustered regularly interspaced short palindromic repeats (CRISPR) directed Cas9 nuclease-mediated gene editing of CXCR7 revealed that prostate cancer cells depend on CXCR7 for proliferation, survival and clonogenic potential. Loss of CXCR7 expression by CRISPR-Cas9 gene editing resulted in a halt of cell proliferation, severely impaired EGFR signaling and the onset of cellular senescence. Characterization of a mutated CXCR7-expressing LNCaP cell clone showed altered intracellular signaling and reduced spheroid formation potential. Our results demonstrate that CXCR7 is a potential target for adjuvant therapy in combination with androgen deprivation therapy (ADT) to prevent androgen-independent tumor cell survival.

Bioactive natural products for chemoprevention and treatment of castration-resistant prostate cancer.

Prostate cancer (PCa), a hormonally-driven cancer, ranks first in incidence and second in cancer related mortality in men in most Western industrialized countries. Androgen and androgen receptor (AR) are the dominant modulators of PCa growth. Over the last two decades multiple advancements in screening, treatment, surveillance and palliative care of PCa have significantly increased quality of life and survival following diagnosis. However, over 20% of patients initially diagnosed with PCa still develop an aggressive and treatment-refractory disease. Prevention or treatment for hormone-refractory PCa using bioactive compounds from marine sponges, mushrooms, and edible plants either as single agents or as adjuvants to existing therapy, has not been clinically successful. Major advancements have been made in the identification, testing and modification of the existing molecular structures of natural products. Additionally, conjugation of these compounds to novel matrices has enhanced their bio-availability; a big step towards bringing natural products to clinical trials. Natural products derived from edible plants (nutraceuticals), and common folk-medicines might offer advantages over synthetic compounds due to their broader range of targets, as compared to mostly single target synthetic anticancer compounds; e.g. kinase inhibitors. The use of synthetic inhibitors or antibodies that target a single aberrant molecule in cancer cells might be in part responsible for emergence of treatment refractory cancers. Nutraceuticals that target AR signaling (epigallocatechin gallate [EGCG], curcumin, and 5α-reductase inhibitors), AR synthesis (ericifolin, capsaicin and others) or AR degradation (betulinic acid, di-indolyl diamine, sulphoraphane, silibinin and others) are prime candidates for use as adjuvant or mono-therapies. Nutraceuticals target multiple pathophysiological mechanisms involved during cancer development and progression and thus have potential to simultaneously inhibit both prostate cancer growth and metastatic progression (e.g., inhibition of angiogenesis, epithelial-mesenchymal transition (EMT) and proliferation). Given their multi-targeting properties along with relatively lower systemic toxicity, these compounds offer significant therapeutic advantages for prevention and treatment of PCa. This review emphasizes the potential application of some of the well-researched natural compounds that target AR for prevention and therapy of PCa.

ß-Arrestin-2 Counters CXCR7-Mediated EGFR Transactivation and Proliferation.

UNLABELLED: The atypical 7-transmembrane chemokine receptor, CXCR7, transactivates the EGFR leading to increased tumor growth in several tumor types. However, the molecular mechanism of CXCR7 ligand-independent EGFR transactivation is unknown. We used cDNA knock-in, RNAi and analysis of mitogenic signaling components in both normal prostate epithelial cells and prostate cancer cells to decipher the proliferation-inducing mechanism of the CXCR7-EGFR interaction. The data demonstrate that CXCR7-induced EGFR transactivation is independent of both the release of cryptic EGFR ligands (e.g., AREG/amphiregulin) and G-protein-coupled receptor signaling. An alternate signaling mechanism involving ß-arrestin-2 (ARRB2/ß-AR2) was examined by manipulating the levels of ß-AR2 and analyzing changes in LNCaP cell growth and phosphorylation of EGFR, ERK1/2, Src, and Akt. Depletion of ß-AR2 in LNCaP cells increased proliferation/colony formation and significantly increased activation of Src, phosphorylation of EGFR at Tyr-1110, and phosphorylation/activation of ERK1/2 compared with that with control shRNA. Moreover, ß-AR2 depletion downregulated the proliferation suppressor p21. Stimulation of ß-AR2-expressing cells with EGF resulted in rapid nuclear translocation of phosphorylated/activated EGFR. Downregulation of ß-AR2 enhanced this nuclear translocation. These results demonstrate that ß-AR2 is a negative regulator of CXCR7/Src/EGFR-mediated mitogenic signaling. IMPLICATIONS: This study reveals that ß-AR2 functions as a tumor suppressor, underscoring its clinical importance in regulating CXCR7/EGFR-mediated tumor cell proliferation. Mol Cancer Res; 14(5); 493-503. ©2016 AACR.