Tetraspanins Affect Myeloma Cell Fate Via Akt Signaling and FoxO Activation.

The significance of cellular interaction with microenvironmental components to tumor progression is increasingly acknowledged. In myeloma (MM), the involvement of bone marrow is critically important to cell growth, survival and drug resistance. It is currently perceived that effective disease treatment must address these interactions. We have previously shown that re-expression of the eGFP fusion proteins CD81-N1 and CD82-N1 tetraspanins in MM cell lines (CAG, RPMI 8226) caused cell stress and massive death. Here we demonstrate that the anti-myeloma effect of CD81-N1 and CD82-N1 compared to mock involves: a down-regulation of Akt(Ser473) [20%–40%]; activation of FoxO transcription factors [evidenced in decreased phosphorylation (20%–40%) and elevated transcription of Gadd45A (70%–80%)]; and is accompanied by a decrease in phosphorylated mTOR(Ser2448) [30%] and mTOR/rictor activity [demonstrated in phosphorylation of Akt at Ser473 position] (p<0.05). Next, we investigated the involvement of PI3K, the prime activator of Akt. Our results show that addition of the PI3K inhibitor wortmannin, affected both the mock and tetraspanin transfected myeloma cells in the same manner. We deduce from these results that the CD81-N1/CD82-N1 induced death is independent of the PI3K signaling. This finding further supports the importance of mTOR/rictor rather than PI3K in the regulation of Akt activity under these circumstances. Based on publications that attribute JNK a regulatory role in FoxO4 activity, especially in stress related scenarios, we decided to examine the consequences of JNK inhibition on tetraspanin induced cell death. Indeed, a most significant rescue of 26% of the cells was demonstrated in the CD81-N1 transfected CAG. Moreover, we conjectured that the cell death was due to myeloma dependence on Akt activation. Hence, in a breast cancer cell line model characterized with variegated levels of Akt activation we demonstrated that the tetraspanin effect was also mediated via Akt and dependent on the ‘cell’s addiction’ to its activity. Significantly higher rates of cell death were observed in the Akt dependent MDA-MB-231 breast cancer cell line compared to Akt independent MCF-7 with both CD81-N1 and CD82-N1 overexpression (70% versus 50% and 50% versus 10%, respectively). Moreover, in CD82-N1 transfected MDA-MB-231 we observed a significant reduction in Akt(Ser473). We suggest that tetraspanins have modulator potential in MM and constitute a promising frontier to be explored in Akt dependent pathological conditions. Moreover, targeting the pathways (via the ‘tetraspanin switch’) rather than blocking a single factor may present a promising therapeutic strategy in a complex drug resistant disease such as myeloma.