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MAPK and PI3K pathways

BRAF in melanoma
We previously demonstrated that virtually all melanomas have high constitutive activity in the MAPK pathway. The reasons for this increased signaling activity are manyfold and include activating mutations in BRAF in over 65% of melanomas, activating N-Ras mutations in 15% of melanomas, autocrine stimulation of growth factor receptors, such as c-met and FGFR1, activation through ?v?3 integrin binding, and Notch1 signaling. Constitutive activity in the Ras/Raf/MEK/ERK MAPK pathway contributes to the oncogenic phenotype of melanoma through its effects on cell proliferation, invasion and survival. In experimental systems, the role of BRAF in melanoma looks convincing. In vitro studies have shown that V600E BRAF is an oncogene in immortalized mouse melanocytes and that selective downregulation of V600E BRAF using RNAi causes cell death and reversal of the melanoma phenotype Increased BRAF activity also suppresses the activity of the melanocyte-specific transcription factor micropthalmia (MITF), diverting the melanoma cells from a differentiation pathway into a highly proliferative state. Activation of the MAPK pathway through the growth factors SCF/c-kit ligand and bFGF, in combination with activation of the G-protein coupled receptor EDNRB, drives melanocytes in human skin grafted to SCID mice to uncontrolled proliferation resembling malignant transformation. Thus, the BRAF/MEK pathway is an ideal candidate for targeted melanoma therapy.

PI3 kinase pathway
Activity in the PI3K/Akt pathway arises either through paracrine growth factor loops, for example through IGF-1 stimulation, PI3K gene mutations or loss of PTEN expression. Melanomas generally lack PI3K and Akt mutations, but PTEN is lost in 30% of cell lines and approximately 10% of clinical melanoma specimens. Transduction of melanocytes with the p85 gene of PI3K leads, in combination with other oncogenes, to malignant transformation. Akt is the central player in the PI3K/Akt-mediated oncogenic behavior of melanoma and it phosphorylates a number of important downstream targets in the regulation of apoptosis. Of the three Akt family members, Akt1-3, about 50% of melanoma cells have constitutive activity in Akt3. Inhibition of Akt activity in melanoma, using either PI3K inhibitors or selective RNAi to Akt3, both reduce growth and induce some degree of apoptosis.

Figure 1 - Intracellular signaling for the MAP kinase and PI3 kinase pathways. Growth factors activate signaling cascades for the MAP kinase pathway resulting in activation of RAS, RAF, MEK, and ERK. They also activate the PI3 kinase cascade resulting in PKB/AKT activation, which in turn blocks endogenous GSK3ß.

3D spheroid model development and responses to BRAF/MEK and PI3K inhibition
There is evidence that the tumor microenvironment modulates the response to therapy. We drew upon our long experience in developing organotypic models to investigate whether modulating the microenvironment and growing the melanoma cells in 3D culture would impact their response to MEK/PI3K inhibitors. For assays, melanoma cells were first grown under non-adherent conditions on top of agar until they formed 3D spheroids. After 48 to 72 hours, the spheroids were harvested, mixed with collagen, plated out, overlayed with media and left to grow. Cells emerged from the spheroids to invade into the surrounding collagen in a stage-specific manner. Spheroids grown from cells derived from earlier stage lesions (RGP and VGP; WM35 and WM793) were less invasive into the collagen than those from metastases (1205Lu) (Figure 4A), and all remained viable (Figure 4B). Spheroids were treated with either U0126 or LY294002 and left to grow for 72 hours. Under these conditions it was noted that the most aggressive melanoma cells were completely resistant to U0126, no longer underwent apoptosis and continued to invade into the collagen (Figure 5). The apparent resistance to the MEK inhibitors of metastatic melanoma cells is indeed supported by early clinical studies on MAPK inhibitors which demonstrate little benefit to melanoma patients.

Similar experiments were performed with the 3D spheroid model using the PI3K inhibitor LY294002. Again it was noted that only the melanoma cell lines from the early-stage RGP and VGP lesions had reduced cell viability (data not shown). As with U0126, the cell lines derived from metastatic lines were resistant to the effects of LY294002 in 3D culture. The same results were also seen following treatment of melanoma cells with inhibitors of Src (SU6656) and Rho kinase (ROCK). Implantation of the spheroids into collagen was critical to the drug resistance, as spheroids left on-top of the agar were not drug resistant.

Figure 2 - Inhibition of melanoma cells in spheroids implanted in collagen using a MEK inhibitor. Melanoma cells were grown under non-adherent conditions for 72 hours until spheroids had formed. Spheroids were then harvested and implanted into a collagen gels before being treated with UO126 (1 and 10?M). After 72 hours cells were treated with a cell viability kit, where living cells stain green and dead cells red. In all cases, data shown are representative of three experiments. Magnification (WM35, WM793) X 10. Magnification (1205Lu, C8161) X4. U0126 inhibited only the growth of early-stage melanoma cell lines (WM35 and WM793) but not of metastatic melanoma cells (1205Lu and C8161). Note: Three of the lines (WM35, WM793, 1205Lu) were V600E BRAF mutated and one, (C8161) was wild-type for both N-Ras and BRAF.

Related References:

1 - Smalley, K.S.M., Lioni, M., Palma, M.D., Xiao, M., Desai, B., Eghazi, S., Hansson, J., W, H., King, A.J., Flaherty, K.T., Herlyn, M., Nathanson, K.L.: Cyclin D1 mediates BRAF inhibitor resistance in a sub-set of BRAF-V600E mutated melanomas. Molec. Can. Ther: 7: 2876-2883, 2008. PMID18790768

2 - Smalley, K.S.M., Xiao, M., Villanueva, J., Nguyen, T.K., Flaherty, K.T., Letrero, R., Nathanson, K.L., Herlyn, M.: CRAF inhibition induces Bcl-2 dependent apoptosis in melanomas with non V600E BRAF mutations. Oncogene 28: 85-94, 2009. PMID18794803

3 - Xie, P., Williams, D.S., Atilla Gokumen, G.E., Milk, L., Xiao, M., Smalley, K.S., Herlyn, M., Meggers, E., Marmorstein, R.: Structure-based design of an organoruthenium phosphotidyl-inositol-3-kinase inhibitor revieals a switch governing lipid kinase potency and selectivity. ACS Chem. Biol. 3: 305-316, 2008. PMID18484710 (NIHMS 53988)

4 - Lee, J., Li, L., Brafford, P., Sproesser, K., Haass, N., Smalley, K., van den Eijnden, M., Halloran, M., Tsai, J., Bollag, G., Herlyn, M: PLX4032, a potent inhibitor of the B-Raf V600E oncogene, selectively inhibitrs V600E-positive melanomas. Pigment Cell Melanoma Res. 23, 820-827, 2010. PMID20973932

5 - Vultur, A., Villanueva, J., Herlyn, M. BRAF inhibitor unveils its potential against advanced melanoma. Cancer Cell 18: 301-302, 2010. PMID20951940