Our observations show that cAMP-dependent PKA activation and CREB phosphorylation are important events in RGS17 induced proliferation in lung tumor cells. This is evidenced by our results demonstrating RGS17 and PKA-dependent CREB phosphorylation, RGS17 regulation of CREB responsive gene expression, and RGS17-dependent forskolin stimulation of growth. There are many CREB-responsive gene products that may be effectors of PKA-CREB activation, including Cyclin D1, RIPK4/PKK (protein kinase C–associated NFκB activator; ref. 11
), and KCIP1 (antiapoptotic RGS17 interacting protein kinase C inhibitor), all of which we found to be affected by loss of RGS17. NFκB, which can be activated directly by PKA (12
), may also be involved in the proliferative and survival effects of RGS17 independently of CREB. PKA activity in RGS17-expressing cells is likely to have proliferative and survival effects through one or more of these effectors. Delineation of the roles of these effectors in RGS17-mediated tumor cell proliferation requires further investigation.
CREB has been described as a proto-oncogene having proliferative and survival effects on myeloid progenitors and has been implicated in their transformation in acute myeloid leukemia (13
). Furthermore, PKA and CREB have also been shown to play a role in the tumorigenesis of endocrine tissues, and various molecular aberrations in this pathway have been observed in endocrine tumors (14
). In a recent study, phosphorylated CREB was found to be an early marker for lung adenocarcinoma and the tobacco carcinogen 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone (NNK) was found to induce phosphorylation of CREB in a time-dependent manner (15
). Tumor-specific immunoreactivity to anti–phospho-CREB was detected in 43 of 47 human lung adenocarcinomas, suggesting that CREB-regulated gene expression is a critical event in lung carcinogenesis In another study, NNK was found to induce CREB phosphorylation in a PKA-dependent manner in human lung adenocarcinoma and small airway epithelial cells (16
). Interestingly, this activation of the PKA-CREB pathway was partially abrogated by inhibition of EGF receptor (EGFR) signaling, suggesting crosstalk between these signaling pathways. EGFR is often overexpressed or mutated in lung cancers and can be transactivated by several G-protein–coupled receptors (17
). Interestingly RGS17 expression has been found to be induced in rat astrocytes upon EGFR stimulation (18
). A very recent study showed that EGFR and PKA pathways are involved in oxidant induced survival of type II alveolar cells (19
). Given the importance of EGFR signaling to lung cancer and the role of PKA in EGFR signaling, enhancement of EGFR signaling by RGS17 through PKA in lung tumors is an attractive hypothesis.
Expression array analysis suggested that the expression of a variety of CNS and receptor-mediated signal transduction genes are coregulated with RGS17 expression. RGS17, being a RGS with expression and function in the brain, would be expected to be associated with genes of such function. Consistent with neurologic function, there is evidence that RGS17 may be involved in dopaminergic signaling. RGS17 and other RZ subfamily RGS proteins reduce D2R-mediated cAMP inhibition (4
), and RGS17 transcript is decreased in dopamine D1 receptor knockout mice (20
). Dopamine receptors are G-protein–coupled receptors that can up-regulate (D1R) or down-regulate (D2R) cAMP signaling (21
). As mentioned in the introduction, RGS17 can also abrogate μ-opioid induced cAMP inhibition (8
Interestingly, RGS17 knockdown resulted in a significant (P
< 0.05) increase in expression of only the FoxO4 and Hnt genes, both of which have tumor suppressive roles. FoxO4 has known tumor suppressive functions through the activation of p27KIP (22
) and suppresses HER-2–mediated tumorigenicity (22
). Hnt encodes Neurotrimin and is closely linked to its paralog opioid binding/cell-adhesion molecule–like, which is frequently lost in ovarian cancer and has tumor-suppressor function. Repression of these tumor suppressors by RGS17, or a combination of this with activation of CREB-responsive genes may lead to, or may be necessary for the proliferation of tumor cells.
The results presented here identify RGS17 as a cAMP-PKA-CREB–activating regulator of lung tumor cell proliferation. Our observation that expression of RGS17 is widely up-regulated in tumors provide further evidence that RGS17 plays a role in tumorigenesis. Interestingly, we also found RGS17 to be consistently up-regulated in prostate tumors, in which a role for G-protein and PKA signaling has been implicated (24
). The profound effect of loss of RGS17 and/or PKA inhibition on tumor cell growth establishes RGS17 and the cAMP-PKA-CREB pathway as potential targets of cancer therapeutics. Robust inhibition of both total and phospho-CREB in RGS17 knockdown cells accompanies a dramatic decrease in the growth rate of tumor cells and may likely be due to feedback autoregulation of CREB expression through CREs within the CREB promoter itself. Increased total CREB in RGS17 overexpressing cells also suggests a positive feedback mechanism of CREB transcriptional regulation, which has been well-documented (25
). Increased cAMP levels and proliferation are induced by overexpression of RGS17 in lung tumor cells. RGS17 overexpression is sufficient to promote proliferation, especially under low serum conditions, and partially rescue proliferation in cells under PKA inhibition. Because H89 inhibition of PKA cannot be expected to be 100% effective, we conclude that overexpresion of RGS17 overcomes inhibition through increased PKA activation rather than bypass through another pathway. Culture of lung tumor cells for 4 days on low serum reveal robust effects of RGS17 expression on proliferation, CREB phosphorylation and total CREB expression, indicating that sustained activation of CREB plays a critical role in RGS17 induced proliferation. These findings lead to a model for RGS17-induced proliferation where cAMP-PKA-CREB signaling is regulated by RGS17 through inhibition of adenylate cyclase inhibiting G(i)-coupled receptors permitting G(s)-coupled receptor signaling. Further investigation of the involvement of RGS17 and the cAMP-PKA-CREB pathway in receptor-mediated tumor phenotypes including those mediated by μ-opioid receptors and the recently genetically implicated nAChR family receptors on chromosome 15q will be vital to the better understanding of lung tumorigenesis. It will also be imperative to investigate the roles of specific CREB target genes in RGS17 induced tumor cell growth.