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It has been demonstrated that immunotherapy using gene-modified dendritic cells (DC) expressing tumor-associated antigens is an effective way to elicit antigen-specific anti-tumor response. Our previous studies using gene-modified DC expressing the human melanoma immunodominant antigen MART-1 to immunize against mouse melanoma tumors, results in a robust antigen-restricted immune response against its murine counterpart. However, a significant proportion of tumor cells remain resistant to this immunotherapeutic modality and they can be manifested as slow and late-developing or recurrent immune-resistant tumors. Tumor cells exhibit immune escape properties that promote their survival such as modulation of antigen expression/presentation, secretion of immunosuppressive factors and resistance to apoptosis. Moreover, the activation of Nuclear Factor κB (NF-κB) has been associated with increase expression of anti-apoptosis genes in tumor cells. Activation of NF-κB in tumor cells can be induced by the engagement of the TNF receptor superfamily members and their associated adaptor molecules. Therefore, we hypothesize that the tumor immune-resistance can be partially acquired by changes in expression of apoptosis-related genes that can be mediated by activation of anti apoptosis-related signaling pathways associated to the TNFR superfamily members. In this study we examined the differential apoptosis-related gene expression profile between immune-sensitive and immune-resistant B16 mouse melanoma cells by focus cDNA microarray, observing the up-regulation of key anti apoptotic genes such as Mcl-1, Survivin, Traf6 and Tank in the immune-resistant melanoma cells (B16R). We confirmed this differential gene expression by semi-quantitative RT-PCR. Noteworthy, the most significant change in gene expression observed in the immune-resistant population was two-fold increase of Traf6 transcripts. In order to determine the specific role of Traf6 up-regulation in tumor immune-resistance, we transiently knocked down the expression of Traf6 in B16R cells using Traf6-specific small inhibitory RNA (siRNA). Further, we examined the in vitro sensitivity of Traf6/siRNA treated and untreated B16R cells to TNF-α-mediated apoptosis, observing a significant increase (30 % ± 5 SEM) in sensitivity to the cytotoxic effect of TNF-α (1000 U/mL) when compared to the untreated control group. These results suggest a possible molecular mechanism of acquired immune-resistance of melanoma tumor cells that can be defeated by selectively targeting anti apoptosis-related genes such as Traf6 resulting in a more effective DC-based immunotherapy against melanoma.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]