Functional analysis of the potential tumour suppressor SIAH3

Of the seven in absentia homolog (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 about the regulation and function of SIAH3 in cancer development. Therefore, this work focuses on the widely overlooked family member SIAH3 and its role in tumorigenesis. This study is based on cell culture experiments using human cell lines, followed by RNA and protein analyses as well as the investigation of cellular metabolism. Since SIAH3 is missing the RING domain, the part that mediates the ubiquitin transfer to substrates, SIAH3 does not function as an enzymatically active ligase. This work describes 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. In addition to its regulation on gene level, SIAH3 is also controlled on protein level. SIAH3 interacts with SIAH1 and SIAH2, which in turn mediate proteasomal degradation of SIAH3. Functionally, cell culture experiments showed that induced expression of SIAH3 reduces cell proliferation and induces cell death. Concerning the underlying mechanisms, the results of my work suggest different possibilities. First, SIAH3 is involved in Hippo signalling, interacting with LATS2 and activating tumour suppressive target genes like GADD45A and ANKRD1. Second, SIAH3 negatively affects cellular metabolism by shifting cells from aerobic oxidative phosphorylation to glycolysis. Microscopy demonstrated that SIAH3 localises to mitochondria, with the N-terminal S3UNS domain being responsible for translocation. Proteome analyses revealed reduced levels of proteins involved in the respiratory chain complexes I, III, IV as well as the ATP synthase upon SIAH3 induction, explaining the decline in oxidative phosphorylation. The interactome showed that SIAH3 binds proteins involved in mitochondrial ribosome biogenesis and translation. Furthermore, SIAH3 interacts with several ubiquitin ligases, recruiting at least SIAH1 to the mitochondrion. I also showed that SIAH3 interacts with mitochondrial importers like CHCHD4 und TIMM50, which opens a third opportunity how SIAH3 could affect mitochondrial metabolism. These findings expand our understanding of SIAH3’s function beyond its known role in PINK1-Parkin mediated mitophagy, suggesting that SIAH3 acts as an epigenetically controlled tumour suppressor by regulating cellular metabolism through the inhibition of oxidative phosphorylation. And finally, this work demonstrates that the silenced SIAH3 gene can be reactivated by epigenetic editing, providing a basis for restoring SIAH3’s function and potentially adding a small but important piece to the puzzle of cancer therapy.

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Functional analysis of the potential tumour suppressor SIAH3

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