SIAH3 is frequently epigenetically silenced in cancer and regulates mitochondrial metabolism.

Of the seven in absentia homologue (SIAH) family, three members have been identified in the human genome. In contrast to the E3 ubiquitin ligase encoding SIAH1 and SIAH2, little is known on the regulation and function of SIAH3 in tumorigenesis. In this study, we reveal that SIAH3 is frequently epigenetically silenced in different cancer entities, including cutaneous melanoma, lung adenocarcinoma and head and neck cancer. Low SIAH3 levels correlate with an impaired survival of cancer patients. Additionally, induced expression of SIAH3 reduces cell proliferation and induces cell death. Functionally, SIAH3 negatively affects cellular metabolism by shifting cells form aerobic oxidative phosphorylation to glycolysis. SIAH3 is localized in the mitochondrion and interacts with proteins involved in mitochondrial ribosome biogenesis and translation. We also report that SIAH3 interacts with ubiquitin ligases, including SIAH1 or SIAH2, and is degraded by them. These results suggest that SIAH3 acts as an epigenetically controlled tumor suppressor by regulating cellular metabolism through the inhibition of oxidative phosphorylation.

Biomarker RIPK3 Is Silenced by Hypermethylation in Melanoma and Epigenetic Editing Reestablishes Its Tumor Suppressor Function.

For several decades, cancers have demonstrably been one of the most frequent causes of death worldwide. In addition to genetic causes, cancer can also be caused by epigenetic gene modifications. Frequently, tumor suppressor genes are epigenetically inactivated due to hypermethylation of their CpG islands, actively contributing to tumorigenesis. Since CpG islands are usually localized near promoters, hypermethylation of the promoter can have a major impact on gene expression. In this study, the potential tumor suppressor gene Receptor Interacting Serine/Threonine Protein Kinase 3 (RIPK3) was examined for an epigenetic regulation and its gene inactivation in melanomas. A hypermethylation of the RIPK3 CpG island was detected by bisulfite pyrosequencing and was accompanied by a correlated loss of its expression. In addition, an increasing RIPK3 methylation rate was observed with increasing tumor stage of melanomas. For further epigenetic characterization of RIPK3, epigenetic modulation was performed using a modified CRISPR/dCas9 (CRISPRa activation) system targeting its DNA hypermethylation. We observed a reduced fitness of melanoma cells by (re-)expression and demethylation of the RIPK3 gene using the epigenetic editing-based method. The tumor suppressive function of RIPK3 was evident by phenotypic determination using fluorescence microscopy, flow cytometry and wound healing assay. Our data highlight the function of RIPK3 as an epigenetically regulated tumor suppressor in melanoma, allowing it to be classified as a biomarker.