Among the signaling pathways known to be constitutively active in melanoma, little is known about the oncogenic role of the PI3K/Akt pathway. The aim of the current study is to investigate the downstream signaling targets of this pathway in melanoma. Western blotting experiments demonstrated high levels of phospho-PDK1, phospho-Akt and phospho-GSK3β in all of the melanoma cell lines tested, confirming that this pathway is constitutively active. Inhibition of PI3K activity, using LY294002 (10 μM) or an adenovirus encoding for dominant negative (DN)-p85 PI3K, reduced growth of melanoma cells in 2D culture, and was associated with G1-phase cell cycle arrest, the increased expression of p27KIP-1 and the nuclear accumulation of FoxO. In contrast, inhibition of Akt –using either API-2 (10 and 30 μM), an adenovirus for DN-AKT, or mTOR –using rapamycin, did not inhibit cell growth, induce apoptosis or result in cell cycle arrest. This suggested that in these cell lines the downstream effects of blocking PI3K activity were largely independent of Akt and mTOR activity. Many groups have shown differences in cell signaling between 2D and 3D cell culture. With this in mind we investigated the effects of inhibiting the PI3K/Akt/mTOR pathways in a 3D collagen-implanted spheroid model. In these studies, LY294002 decreased the survival of spheroids from early stage melanoma cell lines, but not those from metastatic lines. Again, inhibiting Akt or mTOR had no effect on either the growth or survival of any of the melanoma cell lines - irrespective of tumor stage. When PI3K activity was inhibited in spheroids derived from melanoma metastases it was found that the cells remained viable but no longer invaded into the surrounding collagen. Another possible target of PI3K activity is the small GTPase Rac, which is also implicated in tumor cell invasion. Infection of collagen-implanted spheroids with DN-Rac completely blocked melanoma cell invasion into the surrounding collagen. It was further found that infection of melanoma cells with DN-Rac also blocked cell cycle entry and inhibited growth. In summary, it seems that the oncogenic effects of constitutive PI3K activity in melanoma are mediated more through the activity of Rac rather than Akt.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]