During Human Melanoma Progression AP‐1 Binding Pairs are Altered with Loss of c‐Jun In Vitro

Abstract

We demonstrated previously that c‐Jun, JunB and c‐Fos RNA were dysregulated in metastatic melanoma cells compared with normal human melanocytes. The purpose of this study was to evaluate the distribution in composition of AP‐1 dimers in human melanoma pathogenesis. We investigated AP‐1 dimer pairing in radial growth phase‐like (RGP) (w3211) and vertical growth phase‐like (VGP) (w1205) human melanoma cells and metastatic cell lines (cloned from patients, c83‐2c, c81‐46A, A375, respectively) compared with melanocytes using electrophoretic mobility shift assay (EMSA), Western blot and transfection analyses. There are progressive variations in AP‐1 composition in different melanoma cell lines compared with normal melanocytes, in which c‐Jun, JunD and FosB were involved in AP‐1 complexes. In w3211, c‐Jun, JunD and Fra‐1 were involved in AP‐1 binding, while in w1205, overall AP‐1 binding activity was decreased significantly and supershift binding was detected only with JunD antibodies. In metastatic c81‐46A and A375 cells, only JunD was involved in AP‐1 binding activity, but in a third (c83‐2c) c‐Jun, JunD and Fra‐1 were present. Western blot evaluation detected c‐Jun in melanocytes and w3211, but this component was decreased significantly or was not detectable in w1205, c81‐46A and A375 cells. In contrast, JunD protein was elevated in c81‐46A and c83‐2c cells compared with melanocytes and RGP and VGP cell lines. Normal melanocytes and c83‐2c cells (which have c‐Jun involved in AP‐1 binding), transfected with c‐Jun antisense and treated with cisplatin, showed higher viability compared with untransfected cells, while in c81‐46A cells (in which only JunD is detectable) no change in cell viability was observed following treatment with cisplatin and c‐jun antisense transfection. A dominant‐negative c‐Jun mutant (TAM67) significantly increased the soft agar colony formation of w3211 and c83‐2c cells. These results suggest that components of AP‐1, especially c‐Jun, may offer a new target for the prevention or treatment of human melanoma progression.

Alterations in Activating Protein 1 Composition Correlate with Phenotypic Differentiation Changes Induced by Resveratrol in Human Melanoma

“…Results from this current investigation, together with our previous observations that AP-1 composition has been altered during progression (Yamanishi et al, 1991;Yang et al, 2004), indicate that the abnormalities of AP-1 dimer composition in human melanoma cells may offer a unique preventive and therapeutic target for intervention of human melanoma.…”

“…To investigate whether elevated Fra-2 could interfere with MHC class I and Fas/CD95 expression, we also transiently overexpressed Fra-2 in human melanoma A375 cells, which only has JunD detectable in the AP-1 complex (Yang et al, 2004). The expression level of MHC class I and Fas/CD95 were detected by cytofluorometric analysis.…”

“…These results suggested that aberrant redox status in metastatic melanomas might be associated with a quantitative or qualitative difference in AP-1 signaling. Our previous in vitro investigations showed that during human melanoma progression AP-1 DNA binding pairs were distinctly altered and were associated with elevated drug resistance and cell malignancy (Yang et al, 2004). To investigate the mechanisms of resveratrol action, we extended our studies to its effects on AP-1 signaling.…”

Specific c-Jun target genes in malignant melanoma

“…The enrichment of DNA fragments displayed as peaks in the promoter/enhancer regions of specific target genes (ENCODE) were localized to the promoter/enhancer regions of FosB, NFATC2, WEE1, PVR, MAP1LC3B and LGALS3 [17][18][19][20][21][22] ( Fig. 2, Table 2).…”

Targeting Nitric Oxide Signaling with nNOS Inhibitors As a Novel Strategy for the Therapy and Prevention of Human Melanoma

“…3A). Immunoblotting analysis showed that with nNOS depletion, the expression levels of JunD, MMP-1, APE/Ref-1, and Bcl-2 were significantly reduced, which are well-known genes that contribute to melanoma proliferation and invasion (58,59,61).…”