Integrated Molecular and Clinical Analysis of AKT Activation in Metastatic Melanoma

Abstract

PURPOSE: Activation of the phosphoinositide 3-kinase (PI3K)-AKT pathway has been implicated in melanoma based primarily on the prevalence of mutations in PTEN and NRAS. To improve our understanding of the regulation and clinical significance of the PI3K-AKT pathway in melanoma, we quantitatively measured the levels of phosphorylated AKT, its substrate GSK3alpha/beta, and its negative regulator PTEN in clinical metastases. Results were compared with mutational status, clinical outcomes, and sites of metastasis. EXPERIMENTAL DESIGN: DNA and protein were isolated from dissected frozen melanoma metastases (n = 96). Activating mutations of BRAF, NRAS, AKT, PIK3CA, and KIT were detected by mass spectroscopy genotyping. Phosphorylated AKT (Ser473 and Thr308), P-GSK3alpha/beta, and PTEN protein expression were measured by reverse-phase protein array. A panel of human melanoma cells lines (n = 58) was analyzed for comparison. RESULTS: BRAF-mutant tumors had higher levels of P-AKT-Ser473 (P = 0.01), P-AKT-Thr308 (P = 0.002), and P-GSK3alpha/beta (P = 0.08) than NRAS-mutant tumors. Analysis of individual tumors showed that almost all tumors with elevated P-AKT had low PTEN levels; NRAS-mutant tumors had normal PTEN and lower P-AKT. Similar results were observed in melanoma cell lines. Stage III melanoma patients did not differ in overall survival based on activation status of the PI3K-AKT pathway. Brain metastases had significantly higher P-AKT and lower PTEN than lung or liver metastases. CONCLUSIONS: Quantitative interrogation of the PI3K-AKT pathway in melanoma reveals unexpected significant differences in AKT activation by NRAS mutation and PTEN loss, and hyperactivation of AKT in brain metastases. These findings have implications for the rational development of targeted therapy for this disease. (Clin Cancer Res 2009;15(24):7538-46).

Figure 1

RPPA analysis of AKT activation in melanoma clinical specimens and correlation with activating mutations. A, Average expression levels of P-AKT-Ser473 (blue), P-AKT-Thr308 (red), P-GSK3α/β (yellow), and PTEN (green) in BRAF-mutant or NRAS-mutant tumors or those that are wild-type (WT) for both genes. *, P<.05 for BRAF versus NRAS; **, P<.01 for BRAF versus NRAS; +, P<.05 for WT versus NRAS. B, Expression of P-AKT-Thr308 in individual BRAF- or NRAS-mutant tumors or those that are wild-type for both genes(○). Each circle represents one tumor and is colored to scale by the level of PTEN expression in the tumor (green = low, red = high). Tumors with AKT1(), AKT3(), and KIT(△) mutations are indicated. C, Expression of P-AKT-Ser473. D, Expression of P-GSK3α/β.

Figure 2

Comparison of RPPA results with IHC analysis of P-AKT. IHC staining for P-AKT-Ser473 on formalin-fixed, paraffin-embedded tissues from melanoma lymph node metastases. Black circle outlines the tumor deposit in each lymph node. A, Tumor with high P-AKT-Ser473 by RPPA. B, Tumor with low P-AKT-Ser473 by RPPA.

Figure 3

AKT activation in human melanoma cell lines. A, RPPA analysis of levels of P-AKT-Ser473 in cells following serum starvation for 24 h. Cells are grouped on the basis of mutations in BRAF and NRAS. Cell lines with AKT3() and KIT(△) mutations are indicated. B, Levels of P-AKT-Thr308. C, Western blotting of human melanoma cells grown in serum-replete medium. Mutations in BRAF (“B”), NRAS (“N”), and KIT (“K”) are indicated, as are secondary alterations in the AKT pathway (“A”, AKT3-E17K; “P”, loss of PTEN expression). The same cell lysate (WM46) was run in the first lane of the first gel and the last lane of the second gel to ensure comparable immunoblot results. Loading was confirmed by ERK2 staining.

Figure 4

AKT activation and clinical outcomes. A, Unsupervised hierarchical clustering of 56 regional(lymph node, soft tissue) metastases based on the expression levels of P-AKT-Ser473, P-AKT-Thr308, and PTEN. For comparative analyses, tumors in the upper cluster are categorized as P-AKT High/PTEN Low; tumors in the lower cluster as P-AKT Low/PTEN High. B, Kaplan-Meier overall survival (OS) analysis from the date of surgery for patients with P-AKT High/PTEN Low(red line) or P-AKT Low/PTEN High(blue line). C, OS from the date of melanoma diagnosis.

Figure 5

AKT activation by metastatic site. A, Average expression levels of P-AKT-Ser473 (blue), P-AKT-Thr308 (red), P-GSK3α/β (yellow), and PTEN (green) in melanoma metastases resected from the brain, liver, and lung, as determined by RPPA. *, P<.05 brain versus lung; **, P<.05 brain versus liver and lung; ***, P<.01 brain versus lung and liver. B, Expression of P-AKT-Thr308 (by RPPA) in individual tumors. Each symbol represents one tumor and is colored to scale by the level of PTEN expression in the tumor (green = low, red = high). The shapes of data points indicate mutations in BRAF(○), NRAS(△), or neither(i.e., wild-type for both:□). The tumors with mutations in AKT1 or KIT are labeled. C, Expression of P-AKT-Ser473.