Growth suppression by dual BRAF(V600E) and NRAS(Q61) oncogene expression is mediated by SPRY4 in melanoma

Abstract

The underlying forces that shape mutational patterns within any type of cancer have been poorly characterized. One of the best preserved exclusionary relationships is that between BRAF(V600E) and NRAS(Q61) in melanomas. To explore possible mechanisms which could explain this phenomenon, we overexpressed NRAS(Q61) in a set of BRAF(V600E) melanoma lines and vice versa. Controlled expression of a second activating oncogene led to growth arrest ("synthetic suppression") in a subset of cells, which was accompanied by cell cycle arrest and senescence in several melanoma cell lines along with apoptosis. Through differential gene expression analysis, we identified SPRY4 as the potential mediator of this synthetic response to dual oncogene suppression. Ectopic introduction of SPRY4 recapitulated the growth arrest phenotype of dual BRAF(V600E)/NRAS(Q61) expression while SPRY4 depletion led to a partial rescue from oncogenic antagonism. This study thus defined SPRY4 as a potential mediator of synthetic suppression, which is likely to contribute to the observed exclusivity between BRAF(V600E) and NRAS(Q61R) mutations in melanoma. Further leverage of the SPRY4 pathway may also hold therapeutic promise for NRAS(Q61) melanomas.

Fig. 1

Differential cell growth response upon rival oncogene expression. The rival oncogene was induced with doxycycline (50–100 ng/ml) and subjected for 6 days cell viability assays using cell-titer-glow reagent. a A panel of four isogeneic stable NRAS(Q61)/Tet-On BRAF(V600E) and five BRAF(V600E)/Tet-On-NRAS(Q61R) mutant cell lines and an immortalized primary human melanocyte line (Pmel) were assayed for cell viability at fifth day following rival oncogene induction with doxycycline. Cell lines showing antagonism such as b two NRAS(Q61R)/Tet-On-BRAF(V600E) and c two BRAF(V600E)/Tet-On-NRAS(Q61R). Cell lines showing cooperativity/neutral such as d one BRAF(V600E)/Tet-On-NRAS(Q61R) and one NRAS(Q61R)/Tet-On-BRAF(V600E). The protein expression was confirmed by western blotting. For each cell line, cell viability was performed independently more than three times in triplicates. Student’s t test, doxycycline vs. no-doxycycline, ^◇p ≤ 0.05

Fig. 2

Ectopic induction rival oncogene inhibit melanoma cell growth and enhance associated phenotypes. On fifth day following rival oncogene induction with doxycycline (50–100 ng/ml), three antagonistic NRAS* + iBRAF* lines (SK-MEL-119^NRAS* and WM1361^NRAS*, GMEL^BRAF*) and two neutral BRAF* + iNRAS* (SK-MEL-63^NRAS* and A375^BRAF*) lines were assayed for their phenotypic dependence. a Senescence was detected by senescence-associated expression of β-galactosidase (SA-β-gal) staining, (b) different phases of cell cycle were detected with PI staining and c apoptosis was detected by Annexin-V staining and d colony formation was detected by 0.1% of crystal violet in 24 well plates. Student’s t test, doxycycline vs. no-doxycycline, ^◇p ≤ 0.05. Bar, 40 µm. The data presented are representative of three independent experiments

Fig. 3

Rival oncogene upregulates SPRY4 expression in growth suppressive melanoma cells. a The overall workflow and heat map results of microarray analysis of mRNA isolated from three melanoma cell lines as indicated. log2-fold differences as obtained by [log2(+Dox/oncogene) − log2(no Dox/oncogene)] − [log2(+Dox/vector) − log2(no Dox/vector)]. b DAVID enrichment scores (ES) for set of all genes that were significantly increased or decreased by at least twofold among antagonistic; SK-MEL-119^NRAS* + iBRAF*, GMEL^BRAF* + iNRAS* and neutral; A375^BRAF* + iNRAS* cell lines. c DAVID enrichment of growth suppressive overlapping significantly up- and downregulated (=2 folds) genes in SK-MEL-119^NRAS* + iBRAF*, GMEL^BRAF* + iNRAS* cell lines but not A375. d SPRY4 transcripts were among the most upregulated ones in both SK-MEL-119^NRAS* and Gmel^BRAF*, but not A375^BRAF* cell lines

Fig. 4

SPRY4 as a mediator of oncogene antagonism. a qPCR validating SPRY4 upregulation upon rival oncogene expression in all the four cell lines showing antagonism (SK- MEL-119^NRAS*, WM1361^NRAS*, MGH-CH-1^BRAF* and GMEL^BRAF* but b not the neutral SK-MEL-63^NRAS* and A375^BRAF* cell lines. After normalization to GUSB1 RNA, data were presented as mean ± SD of triplicates experiment. c Parallel increases in the protein level was also confirmed in two suppressive cell lines but not in two neutral cell lines after induction with/without 50–100 ng/ml of doxycycline for 2–3 days by western blotting. Student’s t test, doxycycline vs. no-doxycycline, ^◇p ≤ 0.05. The data presented are representative of three independent experiments

Fig. 5

SPRY4 overexpression inhibits in vitro and in vivo cell growth. a Following third day of SPRY4 overexpression, two antagonistic lines, SK-MEL-119^NRAS*, GMEL^BRAF* and two neutral lines, SK-MEL-63^NRAS* and A375^BRAF*, were subjected for 6 days cell viability assays. Also, SK-MEL-119^NRAS* cells were subcutaneously injected in NSG mice at 0.2 million per flank per animal into 8 mice per group. b Tumor growth curve and tumor burden tolerated by mice was measured every seventh day for 7 weeks and plotted (mean ± SD, N = 8). c A representative image of H&E and IHC in 5 µm sections of xenograft tumors. Protein levels of SPRY4, positive staining of SA-ß-galactosidase in cytoplasm, and Ki-67 mainly detected in the nuclei. TUNEL-positive cells significantly increased in SPRY4 overexpressed tumors. Student’s t test, vector vs. SPRY4, ◇p ≤ 0.05. Bar, 150 µm. Measurement of percentage area and pixel value statistics of three defined selections covered by the stained cells using analyze tool in ImageJ software

Fig. 6

Rival oncogene-induced growth suppression is rescued by SPRY4 silencing in vitro. a Western blot analysis and quantitative densitometry of the protein expression in SK- MEL-119^NRAS* + iBRAF*cells that ectopically express rival oncogenes and siRNA SPRY4 constructs. Total cell lysate extracts at 48 h were probed with antibodies for BRAF(V600E), BRAF(WT), NRAS(Q61R), NRAS(WT), SPRY4 and internal loading control GAPDH. b Silencing of SPRY4 abrogates the growth inhibition effect of rival oncogene expression in SK-MEL-119^NRAS* cells as compared to siRNA nontarget control (siNTC) vector. c Differential regulation of SPRY4 and p21 proteins upon second oncogene induction. d SPRY4 overexpression and (e) SPRY4 silencing effect on p21 protein expression in the presence of rival oncogene, among suppressive (Red) and nonsuppressive (Green) cell lines was confirmed by western blotting. Student’s t test, doxycycline vs. no-doxycycline, ^◇p ≤ 0.05. The data presented are representative of three independent experiments