Role Of Akt Signaling In The Malignant Progression Of Melanoma

The Akt(or protein kinase B,PKB)pathway is one of the most important cell signaling pathway which involved in cell survival,growth,proliferation,metabolism and migration of the cells.Overexpression of phosphor-Akt has been detected in a variety of tumor cells,where Akt activation appears to promoted excessive proliferation,inhibit apoptosis,and enhance metastasis.So,proteins involved in Akt activation and signaling are potential targets for therapeutic intervention.In fact,drugs directed against some of these proteins are now in clinical trials for treating cancers.Although Akt signaling activity was shown to gradually increase throughout the tumorigenesis of melanoma(from normal melanocytes to melanoma as well as malignant melanoma),the regulation of Akt-mediated signaling pathways remain unclear.In this study,we focused on investigation of the regulatory mechanisms of Akt signaling during malignant progression of melanoma and to evaluate the efficacy of a compound for the treatment of melanoma by inhibition of Akt signaling pathway.In Chapter 1,the regulatory mechanisms of Akt kinase were studied in melanoma cells with PTEN activity.Homogenous B16F1 and B16BL6 melanoma cell lines with maximum differences in malignancy were identified by cell proliferation,adhesion,migration,and invasion assays.The high-malignancy B16BL6 cells exhibited a remarkable increase in Akt activity compared to low-malignancy B16F1 cells.The phosphatase and tensin homolog(PTEN)protein,a tumor suppressor,retained its function in B16F1 and B16BL6 cells as no deletion or mutation was detected by RNA interference(RNAi)of PTEN,application of a PTEN inhibitor,or by direct gene sequencing.The Na+/H+ exchange regulatory factor 1(NHERF1),another upstream regulator of Akt,exhibited altered subcellular localization in these cells.In low-malignancy B16F1 cells,expression of NHERF1 was detected in the nucleus,and the nuclear NHERF1 inhibited both the expression of phosphor-Akt and in vivo tumorigenesis.In malignant B16BL6 cells,however,NHERF1 localized to the cytoplasm and promoted the expression of phosphor-Akt,in vitro cell proliferation,and in vivo tumorigenesis.Furthermore,RNAi of NHERF1 indicated that the subcellular localization of PTEN was similar to NHERF1.In B16BL6 cells,cytoplasmic NHERF1 prevented the cytoplasm to nucleus translocation of PTEN,leading to the up-regulation of nuclear phosphor-Akt.To further analyze the mechanism of NHERF1-mediated regulation of Akt phosphorylation,sequence alignment and co-immunoprecipitation(Co-IP)assays were performed.The results demonstrated that the effects of cytoplasmic NHERF1 might be related to a direct interaction of the PDZ binding domain of PTEN and the PDZ domain of NHERF1.In Chapter 2,we identified functional differences among the three isoforms of Akt in melanoma cells.Overexpression of different Akt isoforms was induced in melanoma cells.Immunofluorescence and Western blotting assay revealed that Aktl and Akt2 localized to the cytoplasm while Akt3 localized to the nucleus.In addition,Western blotting and Co-IP indicated significant phosphorylation only of overexpressed nuclear Akt3.Expression of total phosphor-Akt decreased only after RNAi interference of endogenous Akt3,demonstrating that total Akt phosphorylation was mainly phosphor-Akt3 rather than phosphor-Aktl or phosphor-Akt2.Moreover,cell proliferation and colony formation rate decreased markedly after RNAi of Akt3,while no significant difference was detected after RNAi of Akt2 and only slightly decreased proliferation was observed following RNAi of Aktl.Thus,hyper-phosphorylation of nuclear Akt3 was crucial for tumorigenesis and the malignant progression of melanoma.A new organically synthesized compound,SBF-1,with high anti-tumor activity,was then tested on melanoma cells(Chapter 3).Western blot analysis indicated that hyper-phosphorylation of Akt was downregulated by SBF-1 and that treated melanoma cells were arrested in G1 phase.Further studies were conducted to investigate the mechanism of this phenomenon,and the results indicated that SBF-1 had no effect on the expression and phosphorylation of Akt-upstream proteins except PDK1 kinase.A docking test performed to analyze the interaction of SBF-1 and its potential receptor indicated that a stable complex was formed between SBF-1 and the substrate binding domain of PDK1.In addition,SBF-1 inhibited PDK1 activity,and reduced the expression of phosphor-Akt protein,as indicated by an in vitro kinase assay and protein overexpression analysis.A tumor model was established in mice by injection of melanoma cells via the flank to detect the tumor suppressing activity of SBF-1.Compared to cisplatin treatment,SBF-1 downregulated the Akt signaling pathway,inhibited tumor cell growth,and prolonged survival,all with fewer toxic side effects than observed with cisplatin.In addition,a spontaneous metastatic tumor model was established by injection of melanoma cells through the foot pad.The results showed that SBF-1 inhibited the proliferation of primary tumor and remarkably prevented tumor cells from invading the draining lymph nodes,while an in vitro adhesion assay demonstrated that the adhersion of B16BL6 cells on fibronectin was also inhibited.These effects of SBF-1 may probably through down-regulation of integrin ?4 caused by inhibition of Akt phosphorylation.In conclusion,the present study first reported overexpressoin of cytoplasmic NHERF1 during malignant progression of melanoma cells and a concomitant reduction in nuclear localization of the tumor suppressor PTEN.Reduced nuclear PTEN was associated with enhanced phosphor-activation of nuclear Akt and the development of melanoma cells.In addition,analysis of the subcellular localization of Akt isoforms revealed that Akt 1 and Akt2 were mainly cytoplasmic,while Akt3 localized to the nucleus and was crucial for melanoma tumorigenesis and malignant progression.Finally,we examined the anti-melanoma efficacy of a novel compound,SBF-1,and demonstrated that mice treated with SBF-1 exhibited both reduced tumor growth and metastasis,in addition to prolonged survival,by inhibiting Akt activity.Thus,SBF-1 is a potential drug for the clinical treatment of melanoma.