Regulation Of C-Myc By Proteasome Activator REGγ

Deregulated c-Myc expression always occurrs in virous malignant tumors. Under normal growth conditions, ectopic expression of the oncogenic transcription factor c-Myc contributes to infinite cell prolifearation as well as tumorigenesis. In constract, it has been previously represented that elevated c-Myc are more prone to induce apoptosis under certain condition and cell context, such as cytokines deprivation or growth factors withdrawal. This process is mediated through a variety of distinct mechanisms, presumably due to trans-induction of specific pro-apoptotic genes or through repression of pro-survival genes. Here we provide solid evidence to support assumption that REGy can inhibit c-Myc function by dual regulation mechanisms, containing both transcriptional level and post-translational level. In fact, overexpressed REGy in vitro appears to dramatically induce c-Myc protein proteolysis, however downregulation of c-Myc could be restored by MG132. This findng highlights REGy alter c-Myc stability. Consistantly, half life of c-Myc generally changed when treated with protein synthesis inhibitor cycloheximide (CHX) upon depletion of REG. To determine whether two proteins bound together and delineate which domain is required for efficiently binding, coimmunoprecipitation assays were carried out. It turned out that a C-terminal fragment (amino acids 251-439) symboling the helix-loop-helix/leucine zipper (HLH/LZ) domain, which was used to form heterodimerization with Max, was necessary; while an N-terminal fragment (amino acids 85-96) region of REGy was critical. These observations demonstrated that both exogenous and endogenous c-Myc and REGy associated with each other in a super stable complex. In order to confirm the effect of REGy on the enrichment of endogenous c-Myc, we performed cervical cancer cell line Hela, defined with tumor suppressor p53 mutation background, to investigate REGy function on cell cycle progression. In Hela cell line with aberrant constitute expression of c-Myc, proliferation could be antagonized by higher REGy protein, which was able to bind with c-Myc physically and dramatically blocked stability of c-Myc, thereby inhibited cell ability of cellular hyperproliferation and transformation. Furthermore, absence of REGy also leaded to upregulation transcriptional activity of c-Myc responsive genes. It is revealed that nuclear-localized c-Myc was sharply increased in mouse embryo fibroblasts (MEF) lacking REGy. Unexpectedly, REGy deficiency also results in significant increase of c-Myc mRNA level, which could be arrested by blocking Wnt/β-catenin signaling pathway. c-Myc is one of Wnt target genes regulated by transcript factor complex, which composed by TCF/LEF and stabilized β-catenin. Highly conserved canonical Wnt signaling is esstential for overproliferation of cancer cells and can be actived by secreted wnt protein binds with cell surface receptor. Our study indicated that antiapoptotic Wnt signaling played important role in c-Myc mediated oncogenic transformation and REGy might serve to protect hyperactivation of Wnt signaling. Most strikingly, reduced expression of endougenous REGy lost ability preventing c-Myc accelerated cell survial, as measured by anchorage-independent growth in soft agar and Edu incorporation assay. Therefore, besides many E3 ubiquitin ligases mediating c-Myc degradation, REGy did fufill another shortcut way accomplishing the maintainance of checkpoint for controlling proper abudance of c-Myc protein without ATP energy consumption.In drosophila melanogaster, the biological significance of REGy has not been clarified. To gain further understanding of the specific relationship between c-Myc and REGy regarding physiological aspect, we sought out to utilize well established Gal4-UAS system.It is known that REGy and c-Myc both have one orthologue, named dREG and diminutive/dmyc, respectively. In the course of wing development, we characterized impaired function of dREG resulted in dmyc mRNA accumulation, subsequently followed by substantial cell death and developmental defect. Morover, loss of function of dREG seemed to activate wingless signaling pathway in wing dics, argued by increased downstream gene Nkd(naked cuticle)-lacZ expression, which was a readout of Wnt activity. Most importantly, severe apoptosis phenotype caused by depletion of REG can be rescued by dmyc knockdown. Additionally, it should be noted that dmyc facilitate programmed cell death-apoptosis in wing discs was p53-independent. In summary, our research in drosophila made it convenient to assess conserved biological role of myc, which tightly linked to REG.Collectively, we identified c-Myc as a novel substrate of proteasome activator REGy both in vertebrates and invertebrates. Our proof elucidated an insight that c-Myc can be degraded in ATP and ubiquitin independent manner, which was uncovered first time. These findings probably reflected both coordinated influence that REGy inhibited c-Myc-induced apoptosis in terms of normal epithelial cells while attenuated Myc-induced overgrowth in cancer cells. This kind of promising innovation of Myc protein adjustion may afford implication and be a rationale for shedding light on basic molecular mechanism of REGy misregulation at several stages of tumor development, by which we can explore more therapeutic medicine drugs to cure cancer and other diseases in human beings.