Human melanoma cells expressing V600E B-RAF are susceptible to IGF1R targeting by small interfering RNAs

Abstract

The type 1 insulin-like growth factor receptor (IGF1R) is overexpressed by malignant melanomas compared with benign naevi, and mediates proliferation, motility and protection from apoptosis. However, the utility of IGF1R targeting as anti-cancer therapy may be limited by activating mutations in downstream signaling intermediates. We previously showed that IGF1R knockdown blocked survival of prostate cancer cells in which Akt activation was deregulated by PTEN loss. The current study investigated effects of IGF1R targeting in cells harboring activating RAS-RAF mutations, found in 70-80% of human melanomas. We assembled a panel of eight human melanoma cell lines: two expressing wild-type (WT) B-RAF and N-RAS, two with activating N-RAS mutations and four harboring V600E B-RAF. We also generated isogenic cell populations overexpressing WT or V600E B-RAF. Cells expressing V600E B-RAF were relatively resistant to apoptosis. However, IGF1R gene silencing was capable of inducing significant inhibition of survival, enhancement of apoptosis, and Btwo-fold sensitization to cisplatin and temozolomide. These effects were independent of mutation status and were associated with reduced activation of Akt and also, unexpectedly, of ERKs. These results support development of IGF1R targeting as therapy for melanoma, regardless of the presence of activating mutations in the RAS-RAF pathway.

Insulin-Like Growth Factor System and Sporadic Malignant Melanoma

“…Nevertheless, recent studies showed that this effect is independent of B-RAF mutation status, suggesting that IGF-1R targeting may be an effective therapeutic approach for the treatment of melanoma and other tumors carrying activating mutations in the Ras/Raf-mitogen-activated protein kinase signaling pathway. 57 The same was true when antiapoptotic protein family and extracellular signal-regulated kinase signaling was analyzed. 57 Last, in the human melanoma cell line SK-MEL-2, IGF-1R expression was regulated by cyclooxygenase-2 expression, vitamin C, and IGF-2 via a feedback loop for amplification of IGF-1R expression by IGF-2.…”

“…57 The same was true when antiapoptotic protein family and extracellular signal-regulated kinase signaling was analyzed. 57 Last, in the human melanoma cell line SK-MEL-2, IGF-1R expression was regulated by cyclooxygenase-2 expression, vitamin C, and IGF-2 via a feedback loop for amplification of IGF-1R expression by IGF-2. In particular, vitamin C down-regulation of IGF-1R in the human melanoma cell line SK-MEL-2 determines the inhibition of cell proliferation.…”

Validation of the type 1 insulin-like growth factor receptor as a therapeutic target in renal cancer

“…Clonogenic assays were done as previously described (14,17). In brief, 48 h after siRNA transfection, cells were re-seeded into 10 cm dishes at 1,000 to 1,500 cells per dish, depending on the cell line.…”

“…Apoptosis Assay Forty-eight hours after siRNA transfection, CC-RCC cells were re-seeded at 40,000 cells per well into 96-well plates coated with poly-HEMA as previously described (17) to prevent adherence. Caspase 3/7 activation assays (Apo-1; Promega) were done according to the instructions of the manufacturer, by adding assay substrate, incubating at 37°C for up to 12 h, and analysis on a FluorScan Universal Microplate Spectrophotometer.…”

“…In other tumor types, chemosensitivity can be enhanced by blocking IGF signaling (24,25). Work from our laboratory has shown that IGF1R depletion enhances the chemosensitivity and radiosensitivity of prostate cancer and melanoma (14,17,26).…”