c-Myc is targeted to the proteasome for degradation in a SUMOylation-dependent manner, regulated by PIAS1, SENP7 and RNF4

Abstract

y These authors equally contributed to this work.Keywords: c-Myc, PIAS1, proteasome, SUMO, RNF4, SENP7 c-Myc is the most frequently overexpressed oncogene in tumors, including breast cancer, colon cancer and lung cancer. Post-translational modifications comprising phosphorylation, acetylation and ubiquitylation regulate the activity of c-Myc. Recently, it was shown that c-Myc-driven tumors are strongly dependent on the SUMO pathway. Currently, the relevant SUMO target proteins in this pathway are unknown. Here we show that c-Myc is a target protein for SUMOylation, and that SUMOylated c-Myc is subsequently ubiquitylated and degraded by the proteasome. SUMO chains appeared to be dispensable for this process, polymerization-deficient SUMO mutants supported proteolysis of SUMOylated c-Myc. These results indicate that multiple SUMO monomers conjugated to c-Myc could be sufficient to direct SUMOylated c-Myc to the ubiquitin-proteasome pathway. Knocking down the SUMO-targeted ubiquitin ligase RNF4 enhanced the levels of SUMOylated c-Myc, indicating that RNF4 could recognize a multi-SUMOylated protein as a substrate in addition to poly-SUMOylated proteins. Knocking down the SUMO E3 ligase PIAS1 resulted in reduced c-Myc SUMOylation and increased c-Myc transcriptional activity, indicating that PIAS1 mediates c-Myc SUMOylation. Increased SUMOylation of c-Myc was noted upon knockdown of the SUMO protease SENP7, indicating that it also could regulate a multi-SUMOylated protein in addition to poly-SUMOylated proteins. C-Myc lacks KxE-type SUMOylation consensus motifs. We used mass spectrometry to identify 10 SUMO acceptor lysines: K52, K148, K157, K317, K323, K326, K389, K392, K398 and K430. Intriguingly, mutating all 10 SUMO acceptor lysines did not reduce c-Myc SUMOylation, suggesting that SUMO acceptor lysines in c-Myc act promiscuously. Our results provide novel insight into the complexity of c-Myc post-translational regulation.

RNF4-Dependent Oncogene Activation by Protein Stabilization

“…However, RNF4-dependent protein stabilization as well as its transcriptional and biological activities were not affected by targeting SAE2 and inhibition of SUMOylation. SUMOylation was also reported to target c-Myc for degradation, and to maintain a c-Myc signature in MDA-MB-231 cells overexpressing MycER (González-Prieto et al, 2015; Kessler et al, 2012). Thus, differences in c-Myc levels and the biological context might account for these distinct cellular responses.…”

SUMOylation-Mediated Regulation of Cell Cycle Progression and Cancer

“…However, it is currently unclear, how SUMOylation exactly promotes c-Myc-driven tumorigenesis. Knocking down SUMO ligase PIAS1 reduced the SUMOylation levels of c-Myc and led to increased expression of a cMyc driven reporter gene [73], however inhibiting SAE2 repressed genes normally induced by c-Myc [69]. Despite these mechanistic questions, the finding that c-Myc driven tumors are dependent on a functioning SUMOylation system open up exciting new therapeutic opportunities to block this key oncogene [74].…”

“…E) However, the presence of multiple SUMO moieties can promote ubiquitylation via SUMO targeted ubiquitin ligases (Stubl) as described as a potential mechanism for c-Myc. SUMOylation via PIAS1 promotes ubiquitylation via the Stubl RNF4 and subsequent proteasomal degradation, thereby reducing transcriptional activity [73]. liver Overexpression of SUMO1 [111] colon Overexpression of SUMO1 [112] lip Overexpression of SUMO1 [113] gastric Upregulation of SUMO1 pseudogene3 [114] SUMO activating enzyme SAE1/2 gastric Overexpression of SAE2 [115] lung Overexpression of SAE2 [116] breast Low expression of SAE1 and SAE2 correlates with better survival [69] SUMO conjugating enzyme UBC9 lung Overexpression of UBC9 [117] primary colon and primary prostate Overexpression of UBC9 [64] metastatic breast, prostate and lung Downregulation of UBC9 [64] breast High levels of UBC9 correlate with higher risk for cancer and poor response to chemotherapy [118,119] multiple myeloma Overexpression of UBC9 [120] ovaries Overexpression of UBC9 [63] brain Overexpression of UBC9 [121] SUMO ligases PIAS1, PIAS2, PIAS3, PIASγ…”

Ubiquitin-dependent and independent roles of SUMO in proteostasis

“…These modifications led to the rapid degradation of c-Myc in a proteasome-dependent manner. The authors showed that mutagenesis of all SUMOylation sites which were identified by mass spectrometry did not lead to a reduced SUMOylation of c-Myc, suggesting that the attachment of SUMO to a lysine residue might be promiscuous within the protein (36). These results are compatible with the idea that SUMO and ubiquitin modifications might be arbitrary in proteins that are targeted for degradation and the exact location of the modification plays a subordinate role here.…”