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The ubiquitin-proteasome system (UPS) is responsible for up to 90% of intracellular protein degradation. Alterations in UPS are extensively involved in the development and advancement of malignant pathologies. Thus, the components of the UPS can become potential targets for cancer therapeutics. KPC1 is an E3 ubiquitin ligase component of the UPS that regulates key pathways and processes in cancer. KPC1 sustains the ubiquitination of cytoplasmic p27, determining its elimination and transition between cell-cycle phases. KPC1 also regulates NF-κB signaling by inducing ubiquitination of p105 to allow subsequent proteasomal processing to the functional form p50. It has been shown that the KPC1-p50 duo is reduced or absent in multiple malignancies and that therapeutic reinforcement of the functional axis can exhibit significant tumor suppressor activity. Here, we highlight the potential role of KPC1 as a tumor suppressor by fully describing its crucial role in p27 signaling and the canonical NF-κB pathway.

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Purpose: Abnormal activation of the NF-κB pathway induces a more aggressive phenotype of cutaneous melanoma. Understanding the mechanisms involved in melanoma NF-κB activation may identify novel targets for this pathway. KPC1, an E3 ubiquitin ligase, is a regulator of the NF-κB pathway. The objective of this study was to investigate the mechanisms regulating KPC1 expression and its clinical impact in melanoma.Experimental Design: The clinical impact of KPC1 expression and its epigenetic regulation were assessed in large cohorts of clinically well-annotated melanoma tissues (tissue microarrays; n = 137, JWCI cohort; n = 40) and The Cancer Genome Atlas database (TCGA cohort, n = 370). Using melanoma cell lines, we investigated the functional interactions between KPC1 and NF-κB, and the epigenetic regulations of KPC1, including DNA methylation and miRNA expression.Results: We verified that KPC1 suppresses melanoma proliferation by processing NF-κB1 p105 into p50, thereby modulating NF-κB target gene expression. Concordantly, KPC1 expression was downregulated in American Joint Committee on Cancer stage IV melanoma compared with early stages (stage I/II P = 0.013, stage III P = 0.004), and low KPC1 expression was significantly associated with poor overall survival in stage IV melanoma (n = 137; HR 1.810; P = 0.006). Furthermore, our data showed that high miR-155-5p expression, which is controlled by DNA methylation at its promoter region (TCGA; Pearson's r −0.455; P < 0.001), is significantly associated with KPC1 downregulation (JWCI; P = 0.028, TCGA; P = 0.003).Conclusions: This study revealed novel epigenetic regulation of KPC1 associated with NF-κB pathway activation, promoting metastatic melanoma progression. These findings suggest the potential utility of KPC1 and its epigenetic regulation as theranostic targets. Clin Cancer Res; 23(16); 4831–42. ©2017 AACR.

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Purpose: Abnormal activation of the NF-κB pathway promotes a more aggressive phenotype of cutaneous malignant melanoma. Understanding the mechanisms regulating the NF-κB pathway in melanoma is of critical importance. KPC1 (RNF123) is an E3 ubiquitin ligase that leads to proteasomal processing of precursor NF-κB1 p105 into mature p50, one of the most important steps in the NF-κB pathway regulation. We demonstrated novel epigenetic mechanisms affecting KPC1 expression that lead to an abnormal activation of the NF-κB pathway, which was significant during tumor progression in melanoma patients. Experimental Design: Initially using melanoma cell lines, we investigated the functional interactions between KPC1 and NF-κB, and the epigenetic regulations of KPC1, including microRNA targeted interaction and DNA methylation. The clinical impact of KPC1 expression and these epigenetic regulations were further assessed in large cohorts of clinically well-annotated melanoma tissues (tissue micro-arrays; n=137, JWCI cohort; n=40) and melanoma TCGA database cohort (n=370). High-throughput RNA sequencing, reverse-phase protein array and human methylation 450k platform were utilized for comprehensive analyses. Results: Initially using metastatic melanoma cell lines, we verified that KPC1 promotes processing of NF-κB1 p105 into p50, thereby modulates NF-κB-target gene expression and suppresses melanoma cell proliferation. Concordantly in melanoma tissue, KPC1 expression was down-regulated in AJCC stage IV melanoma compared to early stages (JWCI cohort stage I/II p=0.013, stage III p=0.004), whereby low KPC1 expression was significantly associated with poor overall survival in stage IV melanoma (tissue micro-arrays, n=137, Hazard Ratio 1.810, p=0.006). Furthermore, epigenetic mechanisms regulating KPC1, particularly miR-155-5p and DNA methylation level at its promoter region, was shown to be significant in melanoma lines. This regulatory mechanism was validated in clinical melanoma tissues. High miR-155-5p expression, which is negatively regulated by its promoter DNA methylation level (melanoma TCGA database cohort; Pearson's r -0.455, p Conclusions: We identified miR-155-5p, which is epigenetically controlled by its promoter methylation, has a regulatory role on KPC1 expression. These interactions promote to down-regulation of KPC1 and abnormal NF-κB pathway activation, leading to highly proliferative melanoma cells and poor clinical outcomes. These findings suggest utility of KPC1 expression level for stratification of stage IV melanoma patients, and the importance of the miR-155-5p-KPC1-NF-κB-axis in controlling melanoma proliferation. Citation Format: Yuuki Iida, Aaron Ciechanover, Diego M. Marzese, Keisuke Hata, Matias Bustos, Shigeshi Ono, Jinhua Wang, Matthew P. Salomon, Kevin Tran, Yelena Kravtsova-Ivantsiv, Gordon B. Mills, Michael A. Davies, Dave S.B. Hoon. Epigenetic regulation of KPC1 ubiquitin ligase has a regulatory role on the NF-κB pathway in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3541.