Gab2-mediated signaling promotes melanoma metastasis

Abstract

Metastatic melanoma is a disease with a poor prognosis that currently lacks effective treatments. Critical biological features of metastasis include acquisition of migratory competence, growth factor independence, and invasive potential. In an attempt to identify genes that contribute to melanoma pathogenesis, a genome-wide search using bacterial artificial chromosome array comparative genomic hybridization and single nucleotide polymorphism arrays in a series of 64 metastatic melanoma samples and 20 melanoma cell lines identified increased copy numbers of Gab2 located on 11q14.1. Gab2 is an adaptor protein that potentiates the activation of the Ras-Erk and PI3K-Akt pathways and has recently been implicated in human cancer; however, its role in melanoma has not been explored. In this study, we found that Gab2 was either amplified (approximately 11%) and/or overexpressed (approximately 50%) in melanoma. Gab2 protein expression correlated with clinical melanoma progression, and higher levels of expression were seen in metastatic melanomas compared with primary melanoma and melanocytic nevi. We found that overexpression of Gab2 potentiates, whereas silencing of Gab2 reduces, migration and invasion of melanoma cells. Gab2 mediated the hyperactivation of Akt signaling in the absence of growth factors, whereas inhibition of the PI3K-Akt pathway decreased Gab2-mediated tumor cell migration and invasive potential. Gab2 overexpression resulted in enhanced tumor growth and metastatic potential in vivo. These studies demonstrate a previously undefined role for Gab2 in melanoma tumor progression and metastasis.

Figure 1

Identification of increased copy numbers of 11q13.5-14.1 in melanoma. A: Hierarchical clustering of DNA copy number data from melanoma tumor samples and cell lines show subclusters delineating two independent amplicons on 11q13.2 and 11q13.5-14.1, harboring cyclin D1 and Gab2, respectively. All of the 64 tumor samples consisted of metastatic melanomas. Of the 20 melanoma cell lines, 5 were derived from primary (WM39, WM1552, WM793, WM278, and WM35) and 15 from metastatic melanomas. B: 11q14.1 DNA copy number data showing focal amplifications with Gab2 at the center of the amplicon. C: A representative case showing Gab2 locus amplification by fluorescent in situ hybridization . D: SNP signal intensity colorgram verifying the 11q13.5-14.1 amplicon in melanoma samples. E: Gene expression analysis of Gab2 by qRT-PCR in tumors with increased copy numbers of 11q14.1 shows consistent up-regulation of Gab2 (means ± SD, n = 3, P < 0.05).

Figure 2

Gab2 is overexpressed in metastatic melanoma. Total cell lysates were isolated and Gab2 levels were measured by Western blot analysis. β-Actin was used as a loading control. Quantitation of Gab2 expression was determined by densitometry and was normalized with β-actin. The BRAF and NRAS mutation status of the cell lines is indicated. All melanoma cell lines with a BRAF mutation harbor the BRAF^V600E mutation. The WM1361 cell line has the NRAS^Q61R mutation. Gab2 is expressed at very low levels, if any, in a normal human melanocyte cell line and primary melanoma cells, whereas it is expressed at high levels in metastatic cell lines.

Figure 3

Gab2 expression correlates with clinical tumor progression. A: Quantitative analysis of Gab2 protein expression (AQUA-based analysis) in a series of nevi, primary melanomas, and metastatic melanomas is shown. Although the variance is large, there are significantly higher expression levels of nonnuclear Gab2 in metastatic lesions compared with both primary melanomas and nevi. The score for each case is the average of two observations from two unique tissue spots of a single block tissue microarray. Data shown in this figure is from 18 nevi and 15 primary tumors and 15 metastatic tumors. B: Representative examples of primary and metastatic melanoma immunostaining of Gab2 are shown.

Figure 4

Gab2 promotes tumor cell migration and invasion in vitro. A and B: Western blot analysis, migration and Matrigel invasion assays of control (scrambled) or Gab2 knockdown with siRNA of metastatic melanoma cell lines (Mewo, A2058, and Ht144 cells) are shown. Gab2 knockdown resulted in decreased migration (means ± SD, n = 3). Gab2 silencing resulted in a significant reduction of the invasive ability of the cells (means ± SD, n = 3, *P < 0.05). C and D: Western blot analysis of forced Gab2 expression in primary melanoma cell lines (WM793, WM278, WM1552, and WM3862) as well as migration and Matrigel invasion assay results are shown. Forced expression of Gab2 in primary melanoma cells resulted in a significant increase of melanoma cell migration and invasion (means ± SD, n = 3, *P < 0.05). E: Representative examples of vector and Gab2-expressing primary melanoma cells (WM793) invading through the Matrigel are shown.

Figure 5

Gab2 enhances tumor growth and metastatic capability of melanoma cells in vivo. A: In vivo tumor growth of the WM3862 primary melanoma cell line expressing either vector or Gab2 was determined. Mice were injected with 1 × 10⁶ cells expressing either Gab2 or vector alone subcutaneously. Tumors were measured with a caliper in two dimensions and mice were sacrificed when tumor surface area reached 1.5 cm². Kaplan-Meier curves show that mice injected with Gab2-overexpressing cells reached tumor burden threshold faster than the vector group (*P < 0.05). All mice injected with Gab2-overexpressing cells reached the tumor burden threshold between days 16 to 21, whereas mice injected with vector alone reached this threshold between days 26 to 46. B: Representative tumor sections stained with H&E and Gab2 (by immunohistochemistry) are shown. Note that Gab2 overexpression alters melanoma cell morphology from epitheloid to spindle in subcutaneous tumors. C: In vivo metastasis of WM3862 cell line expressing either vector or Gab2 was evaluated. Mice were injected with vector or Gab2-overexpressing WM3862 cells via the tail veins. Seven of eleven (63%) mice in the Gab2-overexpressing group developed lung metastasis whereas none in the vector group had lung tumors (*P < 0.05). D: Representative examples of lung sections stained with H&E and Gab2 are shown. Note that Gab2-expressing tumor cells form nodules within the lung and invade into the lung parenchyma.

Figure 6

Gab2 promotes tumor cell migration and invasion via Akt activation. A: Gab2 silencing in the Mewo metastatic melanoma cell line using siRNA technology shows decreased Akt phosphorylation. B: Western blot analysis showing vector or Gab2 forced expression into primary melanoma cell lines WM793, WM278, WM1552, and WM3862 after serum starvation for 24 hours. Gab2 overexpression results in Akt and PDK1 phosphorylation in melanoma cells. C: Treatment of Gab2-overexpressing primary melanoma cells with the PI3K inhibitor, LY294002 (25 μmol/L), for 24 hours results in a significant decrease in tumor cell migration and invasion potential (means ± SD, n = 3, *P < 0.05).