Phosphatidylinositol-3-kinase as a therapeutic target in melanoma

Abstract

Purpose: Phosphatidylinositol-3 kinases (PI3K) are critical for malignant cellular processes including growth, proliferation, and survival, and are targets of drugs in clinical development. We assessed expression of PI3K in melanomas and nevi, and studied associations between PI3K pathway members and in vitro response to a PI3K inhibitor, LY294002.

Experimental design: Using Automated Quantitative Analysis, we quantified expression of p85 and p110alpha subunits in 540 nevi and 523 melanomas. We determined the IC(50) for LY294002 for 11 melanoma cell lines and, using reverse phase protein arrays, assessed the association between levels of PI3K pathway members and sensitivity to LY294002.

Results: p85 and p110alpha tend to be coexpressed (P < 0.0001); expression was higher in melanomas than nevi (P < 0.0001) for both subunits, and higher in metastatic than primary melanomas for p85 (P < 0.0001). Although phospho-Akt (pAkt) levels decreased in all cell lines treated with LY294002, sensitivity was variable. We found no association by t tests between baseline p85, p110alpha, and pAkt levels and sensitivity to LY294002, whereas pS6 Ser(235) and Ser(240) were lower in the more resistant cell lines (P = 0.01 and P = 0.004, respectively).

Conclusions: Expression of p85 and p110alpha subunits is up-regulated in melanoma, indicating that PI3K is a good drug target. Pretreatment pS6 levels correlated with sensitivity to the PI3K inhibitor, LY294002, whereas PI3K and pAkt did not, suggesting that full activation of the PI3K pathway is needed for sensitivity to PI3K inhibition. pS6 should be evaluated as a predictor of response in melanoma patients treated with PI3K inhibitors, as these drugs enter clinical trials.

Fig. 1

Schema of the PI3K pathway. Growth factor receptors, integrins, activated Ras, or PTEN loss stimulate the PI3K/Akt/mTOR pathway. The products of PI3K activate Akt, which in turn controls key cellular processes by phosphorylating mediators of apoptosis, transcription, cell cycle progression, and translation. Arrows, activating phosphorylation; perpendicular lines, inhibitory phosphorylation.

Fig. 2

A–D, AQUA of in situ protein levels (A, for p85; B, for p110α): AQUA uses S100 to create a tumor or nevus mask (two panels on left, at ×10 and ×40). S100 staining was both nuclear and cytoplasmic (middle). 4′, 6-Diamidino-2-phenylindole defines the nuclear compartment (second from right) within the tumor mask. p85 (A) and p110α (B) expression is measured within the cytoplasmic compartments, within the tumor mask (right), and each clinical case is assigned a score based on pixel intensity per unit area within the tumor mask. ANOVA was used to compare p85 (C) and p110α (D) expression in benign nevi, primary, metastatic, and all malignant (primary and metastatic combined) specimens.

Fig. 3

Western blots showing levels of pAkt before and after exposure to 50 μmol/LLY294002 for1h, normalized to β-actin loading. Levels of pAkt decreased in all cell lines, regardless of sensitivity to LY294002.

Fig. 4

Unsupervised hierarchical clustering of cell lines (rows) and baseline levels of biomarkers (columns). The cell lines in red are more resistant to LY294002, and those in black are more sensitive. Red, high expression; green, low expression; brightest green, lowest expression. Expression levels represent the average levels of repetitive measurements for each marker, normalized to the loading for the cell line. Levels of pS6 (red) tend to be higher in the more sensitive cell lines than the resistant cell lines.