The phosphatidylinositol 3-kinase/AKT pathway is activated frequently in human cancer, and it has been implicated in tumor cell proliferation, survival, and chemoresistance. In this study, we addressed the role of AKT in cellular responses to the therapeutic methylating agent temozolomide (TMZ), and we investigated the possible link between TMZ-induced modulation of AKT function and activation of ataxia-telangiectasia and Rad3-related (ATR)-and ataxia telangiectasia mutated (ATM)-dependent signaling pathways. We found that clinically relevant concentrations of TMZ caused activation of endogenous AKT in lymphoblastoid cells, and in colon and breast cancer cells, and that this molecular event required a functional mismatch repair system. Transfection of a dominant-negative kinasedead form of AKT1 into breast cancer cells abrogated TMZinduced activation of endogenous AKT, and it markedly enhanced cell sensitivity to the drug. Likewise, exposure of the MMR-proficient cell lines to the AKT inhibitor D-3-deoxy-2-O-methyl-myo inositol 1-[(R)-2-methoxy-3-(octadecyloxy)-propyl hydrogen phosphate] (SH-5) impaired AKT phosphorylation in response to TMZ, and it significantly increased cell chemosensitivity. Furthermore, small interfering RNA (siRNA)-mediated reduction of AKT1 expression in colon cancer cells potentiated the growth inhibitory effects of TMZ. Inhibition of ATM expression in colon cancer cells by siRNA did not impair TMZ-induced activation of AKT, whereas siRNA-mediated inhibition of ATR prevented AKT activation in response to the drug and increased cell chemosensitivity. These results strongly support the hypothesis that clinical benefit could be obtained by combining TMZ with inhibitors of the AKT pathway. Moreover, they provide the first evidence of a novel function of ATR as an upstream activator of AKT in response to DNA damage induced by O 6 -guanine-methylating agents.The serine/threonine protein kinase AKT is a critical regulator of major cellular processes, including gene expression, glycogen metabolism, migration, proliferation, and survival (for review, see Bellacosa et al., 2005). Three isoforms of AKT, encoded by three different genes, are present in mammalian cells. Each isoform comprises an NH 2 -terminal pleckstrin homology domain, a catalytic protein kinase domain, and a short COOH-terminal region. All AKT isoforms contain two main regulatory phosphorylation sites, one within the kinase domain (Thr308/AKT1, Thr309/AKT2, Thr305/ AKT3), and one in the COOH-terminal region (Ser473/AKT1, Ser474/AKT2, Ser472/AKT3).This study was supported by the Italian Ministry of Health. Article, publication date, and citation information can be found at

The abnormal expression of several microRNAs has a causal role in tumorigenesis with either antineoplastic or oncogenic functions. Here we demonstrated that miR-126 and miR-126* play a tumor suppressor role in human melanoma through the direct or indirect repression of several key oncogenic molecules. The expression levels of miR-126&126* were elevated in normal melanocytes and primary melanoma cell lines, whereas they markedly declined in metastatic cells. Indeed, the restored expression of miR-126&126* in two advanced melanoma cell lines was accompanied by a significant reduction of proliferation, invasion and chemotaxis in vitro as well as of growth and dissemination in vivo. In accordance, the reverse functional effects were obtained by knocking down miR-126&126* by transfecting antisense LNA oligonucleotides in melanoma cells. Looking for the effectors of these antineoplastic functions, we identified ADAM9 and MMP7, two metalloproteases playing a pivotal role in melanoma progression, as direct targets of miR-126&126*. In addition, as ADAM9 and MMP7 share a role in the proteolytic cleavage of the HB-EGF precursor, we looked for the effectiveness of this regulatory pathway in melanoma, confirming the decrease of HB-EGF activation as a consequence of miR-126&126*-dependent downmodulation of ADAM9 and MMP7. Finally, gene profile analyses showed that miR-126&126* reexpression was sufficient to inactivate other key signaling pathways involved in the oncogenic transformation, as PI3K/AKT and MAPK, and to restore melanogenesis, as indicated by KIT/MITF/TYR induction. In view of this miR-126&126* wide-ranging action, we believe that the replacement of these microRNAs might be considered a promising therapeutic approach.

The promyelocytic leukemia zinc finger (PLZF) protein has been described as a transcriptional repressor of homeobox (HOX)-containing genes during embryogenesis. As we previously demonstrated a functional link between overexpression of HOXB7 and melanoma progression, we investigated the lack of PLZF as the possible cause of HOXB7 constitutive activation in these neoplastic cells. Accordingly, we found PLZF expression in melanocytes, but not in melanoma cells, a pattern inversely related to that of HOXB7. PLZF retroviral gene transduction was then performed in a panel of melanoma cell lines, and tumorigenicity was compared with that of empty vector-transduced control cell lines. Evaluation of in vitro migration, invasion and adhesion indicated that PLZF gene transduction induced a less malignant phenotype, as confirmed through in vivo studies performed in athymic nude mice. This reduced tumorigenicity was not coupled with HOXB7 repression. In order to find more about the molecular targets of PLZF, the gene expression profiles of PLZF-and empty vector-transduced A375 melanoma cells were analysed by Atlas Cancer macroarray. Among several genes modulated by PLZF enforced expression, of particular interest were integrin avb3, osteonectin/SPARC and matrix metalloprotease-9 that were downmodulated, and the tyrosinase-related protein-1 that was upregulated in all the analysed samples. This profile confirms the reduced tumorigenic phenotype with reversion to a more differentiated, melanocyte like, pattern, thus suggesting a suppressor role for PLZF in solid tumors. Moreover, these results indicate that PLZF and HOXB7 are functionally independent and that their coupled deregulation may account for most of the alterations described in melanomas.

BRAF inhibitors (BRAFi) have proven clinical benefits in patients with BRAF-mutant melanoma. However, acquired resistance eventually arises. The effects of BRAFi on melanoma cell proliferation and survival have been extensively studied, and several mechanisms involved in acquired resistance to the growth suppressive activity of these drugs have been identified. Much less is known about the impact of BRAFi, and in particular of dabrafenib, on the invasive potential of melanoma cells. In the present study, the BRAF-mutant human melanoma cell line A375 and its dabrafenib-resistant subline A375R were analyzed for invasive capacity, expression of vascular endothelial growth factor receptor (VEGFR)-2, and secretion of VEGF-A and matrix metalloproteinase (MMP)-9, under basal conditions or in response to dabrafenib. The consequences of inhibiting the PI3K/AKT/mTOR pathway on A375R cell responses to dabrafenib were also evaluated. We found that A375R cells were more invasive and secreted higher levels of VEGF-A and MMP-9 as compared with A375 cells. Dabrafenib reduced invasiveness, VEGFR-2 expression and VEGF-A secretion in A375 cells, whereas it increased invasiveness, VEGF-A and MMP-9 release in A375R cells. In these latter cells, the stimulating effects of dabrafenib on the invasive capacity were markedly impaired by the anti-VEGF‑A antibody bevacizumab, or by AKT1 silencing. A375R cells were not cross-resistant to the PI3K/mTOR inhibitor GSK2126458A. Moreover, this inhibitor given in combination with dabrafenib efficiently counteracted the stimulating effects of the BRAFi on invasiveness and VEGF-A and MMP-9 secretion. Our data demonstrate that melanoma cells with acquired resistance to dabrafenib possess a more invasive phenotype which is further stimulated by exposure to the drug. Substantial evidence indicates that continuing BRAFi therapy beyond progression produces a clinical benefit. Our results suggest that after the development of resistance, a regimen combining BRAFi with bevacizumab or with inhibitors of the PI3K/AKT/mTOR pathway might be more effective than BRAFi monotherapy.