Wild-type KRAS is a novel therapeutic target for melanoma contributing to primary and acquired resistance to BRAF inhibition.

Malignant melanoma reveals rapidly increasing incidence and mortality rates worldwide. By now, BRAF inhibition is the standard therapy for advanced melanoma in patients carrying BRAF mutations. However, only approximately 50% of melanoma patients harbor therapeutically attackable BRAF mutations, and overall survival after treatment with BRAF inhibitors is modest. KRAS (Kirsten Rat sarcoma) proteins are acting upstream of BRAF and have a major role in human cancer. Recent approaches awaken the hope to use KRAS inhibition (KRASi) as a clinical tool. In this study, we identified wild-type KRAS as a novel therapeutic target in melanoma. KRASi functions synergistically with BRAF inhibition to reduce melanoma proliferation and to induce apoptosis independently of BRAF mutational status. Moreover, acquired resistance to BRAF inhibitors in melanoma is dependent on dynamic regulation of KRAS expression with subsequent AKT and extracellular-signal regulated kinase activation and can be overcome by KRASi. This suggests KRASi as novel approach in melanoma-alone or in combination with other therapeutic regimes.

MicroRNA miR-125b controls melanoma progression by direct regulation of c-Jun protein expression.

A fundamental event in the development and progression of malignant melanoma is the deregulation of cancer-relevant transcription factors. We recently showed that c-Jun is a main regulator of tumor progression in melanoma and thus the most important member of the AP-1 transcription factor family for this disease. Interestingly, we revealed that c-Jun expression was regulated on the post-transcriptional level and therefore speculated that miRNAs could be involved in c-Jun regulation. We determined seed sequences for miR-125b and miR-527 in the coding region of c-Jun mRNA that hints at the direct involvement of miRNA-dependent regulation on the protein level. We found that the expression of miR-125b was significantly reduced in malignant melanoma cell lines and tissue samples compared with melanocytes, whereas miR-527 remained unchanged. In further functional experiments, treatment of melanoma cells with pre-miR-125b resulted in strong suppression of cellular proliferation and migration, supporting the role of miR-125b in melanoma. In addition, transfection of pre-miR-125b led to strong downregulation of c-Jun protein but not mRNA expression in melanoma cells. Luciferase assays using reporter plasmids containing the miR-125b seed sequence in the luciferase coding region confirmed the direct interaction with miR-125b. Furthermore, immunoprecipitation of Ago-2 revealed that c-Jun mRNA accumulated in the RNA-induced silencing complex after pre-miR-125b transfection in melanoma cells. In summary, we identified an important role for miR-125b in malignant melanoma. Moreover, we demonstrated post-transcriptional regulation of c-Jun by this miRNA and showed that c-Jun is a main mediator of the effects of miR-125b on melanoma cells.

GLI1-dependent transcriptional repression of CYLD in basal cell carcinoma.

CYLD is a deubiquitination enzyme that regulates different cellular processes, such as cell proliferation and cell survival. Mutation and loss of heterozygosity of the CYLD gene causes development of cylindromatosis, a benign tumour originating from the skin. Our study shows that CYLD expression is dramatically downregulated in basal cell carcinoma (BCC), the most common cancer in humans. Reduced CYLD expression in basal cell carcinoma was mediated by GLI1-dependent activation of the transcriptional repressor Snail. Inhibition of GLI1 restored the CYLD expression-mediated Snail signaling pathway, and caused a significant delay in the G1 to S phase transition, as well as proliferation. Our data suggest that GLI1-mediated suppression of CYLD has a significant role in basal cell carcinoma progression.

Post-transcriptional regulation controlled by E-cadherin is important for c-Jun activity in melanoma.

A central event in the development of malignant melanoma is the loss of the tumor-suppressor protein E-cadherin. Here, we report that this loss is linked to the activation of the proto-oncogene c-Jun, a key player in tumorigenesis. In vivo, malignant melanomas show strong expression of the c-Jun protein in contrast to melanocytes. Interestingly, c-Jun mRNA levels did not differ in the melanoma cell lines when compared to melanocytes, suggesting that c-Jun could be regulated at the post-transcriptional level. To uncover the link between E-cadherin and c-Jun, we re-expressed E-cadherin in melanoma cells and detected decreased protein expression and activity of c-Jun. Furthermore, c-Jun accumulation is dependent on active E-cadherin-mediated cell-cell adhesion and regulated via the cytoskeleton. Additionally, we determined that, with respect to c-Jun regulation, there are two melanoma subgroups. One subgroup regulates c-Jun expression via the newly discovered E-cadherin-dependent signaling pathway, whereas the other subgroup uses the MAPKinases to regulate its expression. In summary, our data provide novel insights into the tumor-suppressor function of E-cadherin, which contributes to the suppression of c-Jun protein translation and transcriptional activity independent of MAPKinases.

Isolation of high quality protein samples from punches of formalin fixed and paraffin embedded tissue blocks.

In general, it is believed that the extraction of proteins from formalin-fixed paraffin embedded samples is not feasible. However, recently a new technique was developed, presenting the extraction of non-degraded, full length proteins from formalin fixed tissues, usable for western blotting and protein arrays. In the study presented here, we applied this technique to punch biopsies of formalin fixed tissues embedded in paraffin to reduce heterogeneity of the tissue represented in sections, and to ensure analysing mainly defined cellular material. Successful extraction was achieved even from very small samples (0.7 mm(3)). Additionally, we were able to detect highly glycosylated proteins and protein modification, such as phosphorylation. Interestingly, with this technique it is feasible to extract high quality proteins from 14 year old samples. In summary, the new technique makes a great pool of material now usable for molecular analysis with high throughput tools.

Expression of Dickkopf genes is strongly reduced in malignant melanoma.

The Dickkopf (DKK) genes were originally identified as factors inducing head formation in Xenopus. The genes code for inhibitors that are involved in Wnt signaling. We speculate that loss of DKK expression plays a role in development or progression of malignant melanoma. Thus, we evaluated melanoma cell lines and tissue samples of malignant melanoma for loss of DKK, especially DKK-3 transcription. We found that DKK-1, -2 and -3 were downregulated or lost in all cell lines and in most of the tumor samples analysed. Reduced DKK-3 expression occurred as early as in primary tumors detected by both immunohistochemical and reverse transcription-polymerase chain reaction RT-PCR analysis. Functional assays with stable DKK-3 transfected cell lines revealed that DKK-3 expression increased cell-cell adhesion and decreased cell migration. Further, downregulation of fibronectin, snail-1 and re-expression of E-cadherin was found in the DKK-3 expressing cell clones supporting a role of DKK-3 in tumor progression. Our studies thus indicate that loss of DKK-3 expression may contribute to melanoma progression.

Expression of Hugl-1 is strongly reduced in malignant melanoma.

The human gene Hugl-1 (Llgl/Lgl1) has significant homology to the Drosophila tumor suppressor gene lethal(2)giant larvae (lgl). The lgl gene codes for a cortical cytoskeleton protein, Lgl, that is involved in maintaining cell polarity and epithelial integrity. We speculate that Hugl-1 might play a role in epithelial-mesenchymal transition (EMT) and that loss of Hugl-1 expression plays a role in the development or progression of malignant melanoma. Thus, we evaluated melanoma cell lines and tissue samples of malignant melanoma for loss of Hugl-1 transcription. We found that Hugl-1 was downregulated or lost in all cell lines and in most of the tumor samples analysed, and that these losses were associated with advanced stage of the disease. Reduced Hugl-1 expression occurred as early as in primary tumors detected by both immunohistochemical and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Functional assays with stable Hugl-1-transfected cell lines revealed that Hugl-1 expression increased cell adhesion and decreased cell migration. Further, downregulation of MMP2 and MMP14 (MT1-MMP) and re-expression of E-cadherin was found in the Hugl-1-expressing cell clones supporting a role of Hugl-1 in EMT. Our studies thus indicate that loss of Hugl-1 expression contributes to melanoma progression.

Influence of the cytoplasmic domain of E-cadherin on endogenous N-cadherin expression in malignant melanoma.

E-cadherin is known to be an important molecule in epithelial-mesenchymal transition (EMT). Malignant transformation of melanocytes frequently attends with loss of E-cadherin expression and induction of expression of mesenchymal molecules like N-cadherin. The switch of the cadherin class is an interesting phenomenon of melanoma cells and in EMT in general. Therefore, we analysed the capacity of E-cadherin to regulate expression of N-cadherin in melanocytic cells. Our experiments revealed that melanoma cells downregulate endogenous N-cadherin expression after transient transfection of full-length E-cadherin, but also of the cytoplasmic domain of E-cadherin. Therefore, we concluded that the extracellular domain of E-cadherin and cell-cell contacts are not necessary for negative regulation of N-cadherin. Melanoma cells re-expressing full-length or cytoplasmatic E-cadherin have reduced NFkappaB activity in comparison to mock-transfected cells. Downregulation of NFkappaB activity, either directly or by re-expression of E-cadherin, led to a suppression of N-cadherin promoter activity and N-cadherin expression. Consequently, an NFkappaB-binding site in the N-cadherin promoter was characterized. In summary, our results suggest that N-cadherin is directly regulated by E-cadherin. Loss of E-cadherin induces NFkappaB activity and N-cadherin expression in tumorigenic EMT.

Loss of E-cadherin leads to upregulation of NFkappaB activity in malignant melanoma.

Malignant transformation of melanocytes frequently coincides with loss of E-cadherin expression. Here, we show that loss of E-cadherin leads to induction of nuclear factor kappa B (NFkappaB) activity in melanoma cell lines. Melanoma cells show constitutively active NFkappaB, whereas no activity is found in primary melanocytes. After re-expression of E-cadherin in melanoma cells, strong downregulation of NFkappaB activity was found. Consistently, NFkappaB activity was induced in primary human melanocytes after inhibition of E-cadherin activity by functionally blocking anti-E-cadherin antibodies. Interestingly, re-expression of E-cadherin-blocked p38 MAPK activity and the p38 MAPK inhibitors SB203580 and SB202190 almost completely prevented NFkappaB activation in melanoma cells. Furthermore, cytoplasmatic beta-catenin induced p38 and NFkappaB activation in malignant melanoma. To our knowledge, this is the first report suggesting a correlation between E-cadherin and NFkappaB activity in melanocytes and melanoma cells. In summary, we conclude that loss of E-cadherin and cytoplasmatic beta-catenin induces p38-mediated NFkappaB activation, potentially revealing an important mechanism of tumorigenesis in malignant melanomas.