ORIGINAL ARTICLE High expression of PKA regulatory subunit 1A protein is related to proliferation of human melanoma cells G Mantovani 1 , S Bondioni 1 , AG Lania 1 , M Rodolfo 2 , E Peverelli 1 , N Polentarutti 3 , T Veliz Rodriguez 3 , S Ferrero 4 , S Bosari 4 , P Beck-Peccoz 1 and A Spada 1 1 Endocrine Unit, Department of Medical Sciences, University of Milan, Fondazione Ospedale Maggiore IRCCS, Milan, Italy; 2 Unit of Melanoma Genetics, Istituto Nazionale per lo Studio e la Cura dei Tumori IRCCS, Milan, Italy; 3 Istituto Clinico Humanitas IRCCS, Department of Immunology and Inflammation, Rozzano, Milan, Italy and 4 Pathology Unit, Department of Medicine, Surgery and Dentistry, AO San Paolo and Fondazione Ospedale Maggiore IRCCS, Milan, Italy The cAMP–protein kinase A (PKA) pathway is the major signal transduction pathway involved in melanocyte- stimulating hormone receptor-mediated signaling and melanin production, whereas its role in the control of melanocyte proliferation is still controversial. In this study, we evaluated the effects of selective activation of the different PKA regulatory subunits type 1A (R1A) and type 2B (R2B) on melanocyte proliferation. Immunohis- tochemistry demonstrated that normal melanocytes lacked R1A protein whereas this subunit was highly expres- sed in all human melanomas studied (N ¼ 20) and in six human melanoma cell lines. Pharmacological activation of the R2 subunits by the cAMP analogue 8-Cl-cAMP inhibited proliferation and increased caspase-3 activity by 68.77 ± 10.5 and 72 ± 9% respectively, in all cell lines with the exception of the only p53-mutated one. Similar effects were obtained by activating R2 subunits with other analogues and by silencing R1A expression. The antiproliferative and proapoptotic effects of 8-Cl-cAMP were comparable to those observed with commonly used antitumoral drugs. Moreover, 8-Cl-cAMP potentiated the effects of these drugs on both cell proliferation and caspase-3 activity. In conclusion, this study first reports that human melanomas are characterized by a high R1/R2 ratio and that pharmacological and genetic manipu- lations able to revert this unbalanced expression cause significant antiproliferative and proapoptotic effects in melanoma cells. Oncogene (2008) 27, 1834–1843; doi:10.1038/sj.onc.1210831; published online 1 October 2007 Keywords: melanomas; PKA; cAMP; R1/R2 ratio; proliferation Introduction Cyclic AMP is implicated in the regulation of a variety of cell functions that are, at least in part, related to protein phosphorylation through the activation of protein kinase A (PKA). In addition to the control of differentiated functions, cAMP inhibits or stimulates cell proliferation depending on the cell type. Recently, mutations of genes involved in cAMP signaling and resulting in the constitutive activation of cAMP forma- tion have been identified as cause of endocrine neoplasia (Landis et al., 1989; Weinstein et al., 1991; Kirschner et al., 2000; Lania et al., 2001). In particular, germ-line mutations of the gene encoding the PKA type 1A regulatory subunit (R1A) have been demonstrated to be involved in the pathogenesis of Carney complex (Kirschner et al., 2000), while activating mutations of the Gs-a gene lead to the McCune–Albright syndrome (Weinstein et al., 1991). Dramatic changes in the proportion of the two PKA regulatory subunits, R1 and R2, occur during ontogenic development, differentiation processes and neoplastic transformation, indicating distinct roles for these isoen- zymes in growth control (Cho-Chung, 1990; Tortora et al., 1990). In particular, several studies support the view that R1 is related to cell proliferation whereas R2 is primarily involved in tissue differentiation (Tortora et al., 1990; Cho-Chung et al., 1995). Accordingly, in a variety of human cancer cell lines, transformation coincides with a sharp increase in R1, while R2 over- expression reverts the malignant into a non-transformed phenotype (Handschin and Eppenberger, 1979; Rohlff et al., 1993; Nesterova et al., 1996; Cho et al., 2000; Cho-Chung and Nesterova, 2005). Changes in PKA regulatory subunit expression in tumors are not exclusively due to mutational events, as demonstrated in tumoral pituitary cells that are characterized by low or absent expression of the R1A subunit protein due to proteasome-dependent protein degradation, this unba- lanced expression resulting in cell proliferation (Lania et al., 2004). Interestingly, the two human diseases known to be determined by mutations in the cAMP-dependent path- way, that is Carney complex and McCune–Albright Received 16 March 2007; revised 29 August 2007; accepted 3 September 2007; published online 1 October 2007 Correspondence: Dr A Spada, Endocrine Unit, Department of Medical Science, University of Milan, Fondazione, Policlinico, Ospedale Maggiore IRCCS, Via F Sforza, 35, Milan 20122, Italy. E-mail: anna.spada@unimi.it Oncogene (2008) 27, 1834–1843 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc