Molecular therapeutics is a recognized promising approach for melanoma, but relevant target genes remain elusive. We report that overload of the recently cloned H11/HspB8 induces apoptosis in 55% of examined melanoma cultures. Apoptosis was determined by activation of caspases-9 and -3 and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL), and was not seen in normal melanocytes. It was associated with H11/HspB8 complexation with transforming growth factor-b-activated kinase (TAK) 1 and activation of TAK1 and p38 mitogen activated protein 3 kinases. TAK1 was not bound, nor activated by the H11/HspB8 mutant W51C, which has dominant antiapoptotic activity. b-Catenin was phosphorylated by activated TAK1, inhibiting its nuclear accumulation and mictophthalmiaassociated transcription factor and cyclin dependent kinase 2 expression. The dominant-negative TAK1 mutant K63W inhibited b-catenin phosphorylation and caspase activation. The data indicate that H11/HspB8 overload causes melanoma growth arrest and apoptosis through TAK1 activation and suggest that H11/HspB8 is a promising molecular therapy target.

H11/HspB8 is a functionally distinct small heat shock protein. It causes growth arrest in melanocytes, associated with inhibition of cyclin E/cdk2 and trueβ~-catenin phosphorylation at the transcriptional activity site Ser552 and is silenced through DNA methylation in 27/35 (77%) melanoma tissues/early cultures. 5'-Aza-2-deoxycytidine (Aza-C) induces melanoma cell death correlated with the levels of H11/HspB8 DNA methylation (p<0.001). In lines with low/moderate H11/HspB8 methylation, PI3-K inhibition increases Aza-C-induced cell death. Aza-C Inhibits growth of melanoma xenografts related to the levels of H11/HspB8 methylation, and a non-methylated/non-TAK1 binding H11/HspB8 mutant confers Aza-C resistance. H11/HspB8 is a potential molecular marker for demethylation therapies.

Oncolytic virotherapy is a unique cancer therapeutic that encompasses tumour cell lysis through both virus replication and programmed cell death (PCD) pathways. Nonetheless, clinical efficacy is relatively modest, likely related to the immunosuppressive tumour milieu. Our studies use the herpes simplex virus type 2 (HSV-2)-based oncolytic virus D PK that has documented anti-tumour activity associated with virus replication, PCD and cancer stem cell lysis. They are designed to examine whether D PK-mediated oncolysis includes the ability to reverse the immunosuppressive tumour microenvironment by altering the balance of cytokines directly secreted by the melanoma cells and to define its mechanism. Here, we show that melanoma cells secreted the immunosuppressive cytokine IL-10, and that secretion was inhibited by D PK through virus replication and c-Jun N-terminal kinase/c-Jun activation. D PK-induced IL-10 inhibition upregulated surface expression of MHC class I chain-related protein A, the ligand for the activating NKG2D receptor expressed on NK-and cytotoxic T-cells. Concomitantly, D PK also upregulated the secretion of inflammatory cytokines TNF-a, granulocyte macrophage colony-stimulating factor and IL-1b through autophagy-mediated activation of Toll-like receptor 2 pathways and pyroptosis, and it inhibited the expression of the negative immune checkpoint regulator cytotoxic T-lymphocyte antigen 4. Pharmacologic inhibition of these processes significantly reduces the oncolytic activity of D PK.

The herpes simplex virus type 2 (HSV-2) protein ICP10PK has anti-apoptotic activity in virusinfected hippocampal cultures through activation of the Ras/Raf-1/MEK/ERK pathway (Smith et al. 2000;Perkins et al. 2002;Perkins et al. 2003). To exclude the possible contribution of other viral proteins to cell fate determination, we examined the survival of primary hippocampal cultures and neuronally differentiated PC12 cells transfected with ICP10PK from apoptosis caused by NGF withdrawal. NGF deprivation caused apoptosis in cultures mock-transfected or transfected with the kinase-negative ICP10 mutant p139™, but not in ICP10PK-transfected cultures. In one clone (PC47), ICP10PK inhibited caspase-3 activation through upregulation/stabilization of adenylate cyclase (AC), activation of PKA and MEK, and the convergence of the two pathways on ERK activation. The anti-apoptotic proteins Bag-1 and Bcl-2 were stabilized and the pro-apoptotic protein Bad was phosphorylated (inactivated). In another clone (PC70), ICP10PK inhibited apoptosis through MEKdependent upregulation of the anti-apoptotic protein XIAP (that inhibits the activity of processed caspase-3) and downregulation of the apoptogenic protein Smac/DIABLO. This may be cell-type specific, but the baculovirus p35 protein did not potentiate the neuroprotective activity of ICP10PK in PC12 cells, suggesting that ICP10PK inhibits both caspase activation and activity. The data indicate that ICP10PK inhibits apoptosis independent of other viral proteins and is a promising neuronal gene therapy platform.

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