EPAC-RAP1 Axis-Mediated Switch in the Response of Primary and Metastatic Melanoma to Cyclic AMP

Abstract

Cyclic AMP (cAMP) is an important second messenger that regulates a wide range of physiologic processes. In mammalian cutaneous melanocytes, cAMP-mediated signaling pathways activated by G-protein-coupled receptors (GPCR), like melanocortin 1 receptor (MC1R), play critical roles in melanocyte homeostasis including cell survival, proliferation, and pigment synthesis. Impaired cAMP signaling is associated with increased risk of cutaneous melanoma. Although mutations in MAPK pathway components are the most frequent oncogenic drivers of melanoma, the role of cAMP in melanoma is not well understood. Here, using the Braf(V600E)/Pten-null mouse model of melanoma, topical application of an adenylate cyclase agonist, forskolin (a cAMP inducer), accelerated melanoma tumor development in vivo and stimulated the proliferation of mouse and human primary melanoma cells, but not human metastatic melanoma cells in vitro The differential response of primary and metastatic melanoma cells was also evident upon pharmacologic inhibition of the cAMP effector protein kinase A. Pharmacologic inhibition and siRNA-mediated knockdown of other cAMP signaling pathway components showed that EPAC-RAP1 axis, an alternative cAMP signaling pathway, mediates the switch in response of primary and metastatic melanoma cells to cAMP. Evaluation of pERK levels revealed that this phenotypic switch was not correlated with changes in MAPK pathway activity. Although cAMP elevation did not alter the sensitivity of metastatic melanoma cells to BRAF(V600E) and MEK inhibitors, the EPAC-RAP1 axis appears to contribute to resistance to MAPK pathway inhibition. These data reveal a MAPK pathway-independent switch in response to cAMP signaling during melanoma progression.Implications: The prosurvival mechanism involving the cAMP-EPAC-RAP1 signaling pathway suggest the potential for new targeted therapies in melanoma. Mol Cancer Res; 15(12); 1792-802. ©2017 AACR.

Fig. 1.. cAMP signaling promotes melanoma tumor development and primary melanoma cell growth.

(A) Schematic of the experimental design for chemical activation of adenylate cyclases (ACs) in Braf^Ca/Pten^−/− mouse melanoma model. 4-HT: 4-hydroxitamoxifen (5mM); forskolin 10mM (FSK): AC activator; DMSO: vehicle control. (B) Tumor volume 6 weeks (40 – 44 days) after the 4-HT application followed by 14 days of topical application of either AC activator FSK or DMSO. Data show volume calculated using the formula for a prolate ellipsoid, (length × width²)/2. DMSO: n = 7 (4 females, 3 males; 25 tumors); FSK: n = 7 (4 females, 3 males; 26 tumors). (C) Histology and immunohistochemical analysis of mouse melanoma tumors. S100A4 was used as a melanoma marker and Ki67 as a marker of proliferative cells. Scale bar: 400μm (D) MTT assay showing the growth of melanoma cells isolated from the vehicle-treated mouse tumor. FSK in the medium was replenished every 48 h throughout the experiment. Values (mean± SD) from 5-6 replicates are shown. (E) Effect of treatment with FSK on human primary melanoma cell lines was assessed by MTT assay as described for (D). DMSO: Control; FSK: AC activator forskolin. Data (mean ± SD) from 5-6 replicates analyzed by unpaired Student’s t test are shown. P values: * indicates P ≤0.05; ** ≤0.01; *** ≤0.001 and **** ≤0.0001.

Fig. 2.. cAMP decreases survival of human metastatic melanoma cell lines.

(A) Effect of FSK on survival of melanoma cell lines was measured using MTT assay. Cells plated in 96 wells were treated with FSK for 72 h. Data (mean ± SD) from 5-6 replicates normalized to DMSO control are shown. (B) Western blot shows pERK levels after 8 h treatment with MEK inhibitor AZD6244 (A) or the BRAF(V600E) inhibitor PLX4032 (P) (0.1μM for MRA-4 and MRA-6 or 10μM for MRA-5) (left panel) and AC activation with 10μM forskolin (F) (right panel). (C) and (D) Survival measured by MTT assay after 72 h treatment with increasing concentrations of BRAF(V600E) inhibitor PLX4032 alone or in combination with the AC activator forskolin (FSK). Data (mean ± SD) from 5-6 replicates normalized to a DMSO control are shown.

Fig. 3.. PKA activity is required for the survival of human primary but not metastatic melanoma cells.

(A) Effect of treatment with 5μM H89 (PKA inhibitor) on the growth of primary (upper panels) or metastatic (lower panels) melanoma cells. H89 in the medium was replenished every 48 h and surviving cells were estimated using MTT assay. Data (mean ± SD) from 5-6 replicates analyzed by unpaired Student’s t test are shown. P values: * indicates P ≤0.05; ** ≤0.01; *** ≤0.001 and **** ≤0.0001. (B) and (C) Western blot analysis show the effect of 8 h treatment with 5μM H89 on pCREB and pERK of primary (panel B) and metastatic (panel C) melanoma cells.

Fig. 4.. Effects of elevated [cAMP] on metastatic melanoma cells are not dependent on CREB

(A) Metastatic melanoma cells were transduced with CREB shRNA or scrambled shRNA (control) lentivirus and transduced cells were selected using puromycin. Western blot shows CREB knockdown, pCREB and pERK levels 6 days post-transduction. (B) Effect of cAMP elevation by treatment with forskolin (F) on the clonogenicity of control and CREB-knockdown melanoma cells. Forskolin or DMSO (D) was replenished every 72 h for three weeks. Data (mean ± SD) from 3 replicates analyzed by unpaired Student’s t test are shown. P values: * indicates P ≤0.05; ** ≤0.01; *** ≤0.001 and **** ≤0.0001.

Fig. 5.. EPAC has opposite roles in primary and metastatic melanoma cells.

(A) Western blot shows RAP1-GTP levels after GST-RalGDS-RBD fusion protein pulldown from melanoma cells treated with DMSO (D) or 5μM EPAC inhibitor ESI-09 (E) for 8 h. (B) Effect of EPAC inhibitor ESI-09 or DMSO (replenished every 48 h) on the growth of primary melanoma (top panel: ESI-9 at 5μM) and metastatic (bottom panel; ESI-09 at 10μM) melanoma cells. Data (mean ± SD) from 5-6 replicates analyzed by unpaired Student’s t test are shown. P values: * indicates P ≤0.05; ** ≤0.01; *** ≤0.001 and **** ≤0.0001. (C) Effect of EPAC inhibition by ESI-09 on sensitivity of melanoma cells to BRAF(V600E) inhibitor PLX4032. Cells plated in 96-well plates were treated with DMSO or 5 μM ESI-09 and increasing concentrations of PLX4032 for 72 h and cell survival was assessed using MTT assay. Data from 5-6 replicates normalized to a DMSO control are shown. (D) Western blot shows the effect of 8 h treatment with 5μM ESI-09 on pERK in metastatic melanoma cells. Western blots shown in Figure 5A and 5B were performed simultaneously using same lysates from same experiment; therefore, same β-Tubulin is used.

Fig. 6.. Switch in the role of RAP1 signaling in primary vs. metastatic melanoma cells.

Western blot analysis of pERK in (A) primary melanoma cells transfected with RAP1A/B siRNA (4 pooled siRNAs) or scrambled control siRNA and harvested at 48 h and (B) metastatic melanoma cells transfected with RAP1A or RAP1B siRNAs (pool of two for each) or scrambled siRNA and harvested at 72 h. MTT assay (C) shows the effect of RAP1A/B knockdown on the growth of primary melanoma cells after 48 h of transfection and (D) the effect of RAP1A or RAP1B knockdown on metastatic melanoma cells after 72 h of transfection. Data (mean ± SD) from 5-6 replicates analyzed by unpaired Student’s t test are shown. P values: * indicates P ≤0.05; ** ≤0.01; *** ≤0.001 and **** ≤0.0001. (E) A schematic model for the switch in cAMP signaling response in primary and metastatic melanoma cells mediated by EPAC-RAP1 signaling. Although a critical role for cAMP-PKA-CREB signaling axis has been well established, the role of cAMP-EPAC-RAP1 axis in melanocytes remains to be investigated.