(A) The cumulative incidence of melanoma versus latency in the FVB mice following doxycycline-induced the deletion of Ikkb and/or expression of HRas^V12 was analyzed according to the method described by Gray (80). Melanoma incidence among doxycyline-treated Ikkb^wt mice reached 40% during 120 days compared with only 5% melanoma in Ikkb^Δ/Δ mice treated with doxycycline. (B) Western blot analysis of tumors from Ikkb^wt and Ikkb^Δ/Δ mice treated with doxycycline to induce expression of mutant Ras and Cre recombinase. Tumor lysates from 3 tumors of either Ikkb^wt or Ikkb^Δ/Δ mice were subjected to Western blot analysis and staining with antibody to murine Ikkβ and Ikkγ. (C) Spontaneous amelanotic melanoma (arrow) arising in the ear. (D) Spontaneous amelanotic melanomas (arrows) in tail and back. (E) Spontaneous amelanotic melanoma skin tumors (arrows) arising in the back skin. (F) Spontaneous amelanotic melanoma skin tumors (arrows) in the anus.

(A) E19 melanocytes cultured with or without doxycycline were subjected to apoptosis analysis by Annexin V. (B) Expression of the indicated proteins in melanocytes cultured with or without doxycycline-induced Ikkb deletion and/or HRas^V12 expression were analyzed by immunoblotting, using specific antibodies. Immunoblot of β-actin was used as a loading control. Protein expression was quantitated and normalized to that of the same genetic background cells, without doxycycline-induced HRas^V12 expression. Mean ± SD represents 3 independent experiments statistically analyzed between Ikkb^wt and Ikkb^Δ/Δ cells with HRas^V12 expression. (C) Western blot of p53 expression in Ikkb^Δ/Δ E19 melanocytes stably expressing lentiviral shRNA p53 or shRNA control. The expression of p53 protein in cells with p53 knockdown (9.6%) is shown in comparison with that (100%) of shRNA control–expressing cells. (D) E19 melanocytes with shRNA-mediated p53 knockdown and/or doxycycline-induced Ikkb knockout were subjected to Annexin V apoptosis analysis. shRNA-mediated p53 knockdown significantly reduced apoptosis in cells null for Ikkb (P < 0.01). (E) Ikkb^Δ/Δ and Ikkb^wt E19 melanocytes were treated with or without doxycycline, and p53 mRNA levels were examined using RT-PCR. There was no significant change in p53 mRNA level upon deletion of Ikkb. (F) Phosphorylation of p53 (S15) and the expression of p53 protein in Ikkb^Δ/Δ and Ikkb^wt E19 melanocyte lysates were immunoblotted with phospho-p53 (S15 antibody), and the band density is reported as the ratio between cells treated with versus without doxycycline. (G) The phosphorylation status of Mdm2 (S166) was determined by immunoblot with the β-actin–loading control. There was significant reduction of Mdm2 phosphorylation following the deletion of Ikkb (P < 0.05). *P < 0.05, **P < 0.01.

(A) Cytokine profiles were determined in the serum of melanoma-bearing or tumor-free mice by cytokine array (n = 4). 6Ckine, chemokine (C-C motif) ligand 21; CTACK, cutaneous T cell–attracting chemokine; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; sTNFR, soluble tumor necrosis factor receptor; TARC, thymus activation regulated chemokine; TIMP, tissue inhibitors of metalloproteinase. (B) IL-6 levels in the tissues of Ikkb^wt mice with or without melanoma were determined. Supernatant (Sup) IL-6 secreted by the cultured E19 melanocytes and melanoma cells derived from melanoma in Ikkb^wt mice was measured by ELISA. (C) FVB mice were xenografted with melanoma cells from Ikkb^wt mice. These mice were treated with doxycycline and/or BMS-345541. Tumor volume was measured, and tumor lysate was prepared for IL-6 ELISA assay. Each value represents the mean from 5 mice. BMS, BMS-345541. (D) The E19 melanocytes from Ikkb^wt or Ikkb^Δ/Δ mice were cultured with or without doxycycline, and the supernatant IL-6 was determined by ELISA assay. (E) The cell lysates were prepared from the cultured cells above and subjected to immunoblotting for phospho-Stat3. The total Stat3 was probed as a loading control. The blots were scanned and quantified. Each value was from 3 independent experiments.

(A) Ikkb^f/f mice crossed with TetOn-Cre mice resulted in the Ikkb^f/f-TetO-Cre strain of mice. These mice were further interbred to obtain the additional genetic background of TetOn-Hras^v12, Ink4a/Arf^–/–, and melanocyte-specific Tyr-rtTA. These mice were named Ikkb^Δ/Δ mice, and their counterparts lacking TetOn-Cre were used as control Ikkb^wt mice. On refers to tetracycline/doxycycline-inducible expression of Cre and/or Hras^v12 gene in the TetOn system. Off refers to absence of Cre and/or Hras^v12-inducible gene expression when tetracycline/doxycycline is absent. (B) E19 melanocytes isolated from Ikkb^wt and Ikkb^Δ/Δ mice were treated with doxycycline (Dox) and stained with specific antibodies to Ikkβ (green) or S-100 (red), and nuclei were counterstained with Hoechst (blue). Original magnification, ×40. (C) Expression of the indicated proteins and their phosphorylation status were analyzed by immunoblotting, after the above cells were treated or not treated with doxycycline. Blot band densities from cultures treated with doxycycline were quantitated and normalized to the blot band density from cells of the same genetic background without doxycycline-induced HRas^V12 expression. The fold change (fold) represents the value of 3 independent experiments and was statistically analyzed. **P < 0.01. (D) Cellular Ikk kinase activity (KA) was examined in vitro by monitoring phosphorylation of the GST-IκB (aa 1–54) substrate by the immunoprecipitated Ikk complex. ³²P-labeled–Iκbα (³²P-Iκbα) indicates phosphorylated GST-IκB (aa 1–54). Fold indicates the ratio of the immunoblotted protein band density from the doxycycline-treated culture to the without doxycycline treatment control.

(A) Melanoma lesions arising on the tail were photographed. (B and C) Sections of melanoma (B) and nonmelanoma skin (C) were stained with H&E. (D–I) Immunohistochemistry of factor VIII in melanoma tissue (D), S-100 in melanoma section (E) and nonmelanoma skin (F), and Mart1 in melanoma section (G and H) and nonmelanoma skin (I). Note the thickening of the epidermis over the cutaneous melanoma lesion at the arrows. Original magnification, ×20 (numerical aperture, 0.5) (B and C); ×40 (numerical aperture, 1.0) (D–I).

(A) Melanocytes derived from either E19 Ikkb^wt or E19 Ikkb^Δ/Δ mice were synchronized by double-thymidine block and cell cycle was analyzed after 4 days of doxycycline induction for expression of HRas^V12 and/or deletion of Ikkb. The percentage of cells in each phase of the cell cycle (G₀/G₁, S, G₂/M) is indicated by red, white, or yellow, respectively. (B and C) Lysates from early passage E19 melanocytes, prepared by the indicated doxycycline treatment, were analyzed for expression of cell cycle checkpoint proteins by immunoblotting with antibodies as indicated. β-actin was used as a loading control. The protein expression by immunoblotting was quantified using the ImageJ program. Each value was from 3 independent experiments. The difference in the specific protein expression between Ikkb^wt cells and Ikkb^Δ/Δ cells, upon the doxycycline-induced HRas^V12 expression, was analyzed statistically. (*P < 0.05, **P < 0.01).

Mutation-activated HRas^V12 in Ink4a/Arf^–/– melanocytes triggers the Ikkβ/NF-κB signal transduction pathway, leading to inhibition of the apoptosis machinery (p53, p21, Bcl2, caspase 3 cleavage), a transcriptional burst of IL6 accompanied by activation of Stat3, cell cycle progression (with induction of Rb phosphorylation, aurora kinase, Cdk4, survivin), cell proliferation, and immortalization when p16/p19 are deleted. Deletion of Ikkb in these cells blocks cell cycle progression (reduced Rb phosphorylation, aurora kinase, Cdk4, and survivin expression), reduces proliferation, and enhances p53-mediated apoptosis. When p53 is knocked down in cells null for Ikkb, the induction of apoptosis is reversed, indicating that p53 induction (indicated by bold font) is required for the effects of loss of Ikkb on inhibition melanoma tumor growth. Therefore, deletion of Ikkb emerges as an effective target for treating melanoma tumors that express wild-type p53.