RGS17, an overexpressed gene in human lung and prostate cancer, induces tumor cell proliferation through the cyclic AMP-PKA-CREB pathway

Abstract

We have identified RGS17 as a commonly induced gene in lung and prostate tumors. Through microarray and gene expression analysis, we show that expression of RGS17 is up-regulated in 80% of lung tumors, and also up-regulated in prostate tumors. Through knockdown and overexpression of RGS17 in tumor cells, we show that RGS17 confers a proliferative phenotype and is required for the maintenance of the proliferative potential of tumor cells. We show through exon microarray, transcript analysis, and functional assays that RGS17 promotes cyclic AMP (cAMP)-responsive element binding protein (CREB)-responsive gene expression, increases cAMP levels, and enhances forskolin-mediated cAMP production. Furthermore, inhibition of cAMP-dependent kinase prevents tumor cell proliferation, and proliferation is partially rescued by RGS17 overexpression. In the present study, we show a role for RGS17 in the maintenance of tumor cell proliferation through induction of cAMP signaling and CREB phosphorylation. The prevalence of the induction of RGS17 in tumor tissues of various types further implicates its importance in the maintenance of tumor growth.

Figure 1

Overexpression of RGS17 in human cancers. A, in four published microarray data sets for lung cancer versus normal control tissue, and one for prostate cancer versus normal control tissue, RGS17 was highly expressed in tumor samples compared with controls. AD, adenocarcinoma; LCNC, large cell neuroendocrine carcinoma; LCC, large cell carcinoma; SCC, squamous cell carcinoma; nN, number of normal samples; nT, number of tumor samples. P values were calculated by two tailed Student’s t test. B, qRT-CPR detecting RGS17 transcript accumulation in lung tumors of various pathologies and prostate tumors compared with patient-matched normal control tissue. RGS17 expression is induced in lung and prostate tumors. IB, immunoblot.

Figure 2

RGS17 is required for maintenance of proliferative rate of tumor cell lines. A, RGS17 transcript was measured by qRT-PCR. Proliferation was measured by MTT assay for several days after seeding at 500 cells per well. H1299 cells exhibit slowed growth and decreased tumor size in nude mice when RGS17 transcript is knocked down. Control cells were injected into the right flank of each mouse, whereas shRGS knockdown cells were injected into the left flank of each mouse. B, Hct116 cells show slowed growth and decreased tumor size in nude mice when RGS17 transcript is knocked down. C, DU145 cells show slowed growth when RGS17 transcript is knocked down. D, Western blot showing 3HA-tagged RGS17 overexpression in H1299 cells. RGS17 overexpression in H1299 cells increases growth rate as measured by MTT assay.

Figure 3

Validation of array data and RGS17-dependent cAMP-responsive gene expression. A, RT-PCR validation of array data on cAMP-responsive gene regulation upon loss of RGS17. qRT-PCR on H1299 with RGS17 knockdown compared with H1299 with vector control, including cAM-regulated FoxP2, Cyclin D1, and KCIP-1, as well as tumor suppressor FoxO4 which was determined by array to be up-regulated upon RGS17 knockdown. B, qRT-PCR on tumor versus matched normal controls. KCIP-1 expression correlates with RGS17 expression in colon and prostate tumors. C, fold induction of cAMP responsive genes by forskolin. Forskolin induction of gene expression is decreased with loss of RGS17 expression.

Figure 4

RGS17 induces cAMP formation, CREB phosphorylation, and growth stimulation by forskolin. A, cAMP activity in arbitrary units (AU) and the fold induction of cAMP formation by forskolin is decreased with loss of RGS17. Western blotting shows that CREB phosphorylation is lost with RGS17 knockdown. p/t CREB, ratio of phospho-CREB signal to total CREB signal. B, cells under 1% serum were treated with DMSO or 10 μmol/L forskolin and monitored for proliferation using MTT assay. RGS17 is required for forskolin stimulation of growth in H1299 cells. C, 3HA-RGS17 overexpression in H1299 cells increases cAMP levels and enhances forskolin induction of cAMP formation. Western blotting shows that 3HA-RGS expression modestly increases phospho-CREB levels in relation to total CREB.

Figure 5

RGS17 overexpression protects against PKA inhibitor mediated inhibition of CREB phosphorylation and growth arrest. A, H1299 cells with vector or shRGS-2 were plated at low density (500 cells per well of 12-well dish) and allowed to grow under 1% serum with or without H89. H89 arrests cell growth in both vector and shRGS-2 cells. B, Western blotting shows that H89 causes decreased phosphorylation of CREB in both vector and shRGS-2 cells under 1% serum for 12 h. When cells are treated with 1% serum long term (4 d), total CREB and phospho-CREB are reduced in cells with RGS17 knockdown. C, overexpression of 3HA-RGS17 (RGS) protects H1299 cells from growth arrest induced by H89. D, Western blotting shows that CREB phosphorylation is inhibited by H89 and that 3HA-RGS17 overexpression partially protects CREB phosphorylation.