Study On C-Myc Deubiquitination

Ubiquitination is a tightly controlled cascade through the action of three classes of enzymes that either targets substrate proteins for degradation by 26S proteasome or changes the sublocalization and activity of protein to regulate their function without affecting their stability. This proteolytic system can lead to numerous cellular outcomes, including protein degradation, cell growth and differertiation, cell immune response, cell apoptosis, sorting and trafficking of transmembrane proetins. Yet, as the case of phosphorylation and acetylation, such a covalent conjugation is reversible and disassembled by deubiquitinating enzymes, whose functions are as important but less well understood. Virtually every cellular process that requires temporally or spatially regulated protein-protein interactions is regulated by the balance of ubiquitylation and deubiquitylation. The past years have witnessed that some of the most vital cellular responses and functions are controlled by deubiquitinases, such as cell signaling, cell cycle progression, DNA damage repair. Deubiquitinases regulate protein ubuiquitination level either by binding directly the substrates or indirectly binding the E3 ligases of substrates. Aberrant ubiquitination regulated by E3 ligases and deubiquitinases underlies many disease state, including cancer. USP37 belongs to cysteine proteases subfamily, which is reported to regulat a variety of substrates. USP37 removes degradative polyubiquitin from cell cycle regulator CyclinA,leading to cell cycle progression from G1 to S. USP37 can also modulate PLZF/RARA stability and cell transformation. Besides, as a downstream target gene of REST, USP37 binds with p27, promoted its deubiquitination and stabilization and blocked neuronal cell proliferation. Based on these study, We can draw a conclusion that USP37 regulates specific substrates in a cell-type and context-dependent manner. Interestingly, USP37 expression is high and associated with poor prognosis in non-small cell lung cancers. Yet, the exact role of USP37 in lung tumorigenesis is still unclear.Transcriptional factor c-Myc is expressed widely in a variety of tissues and species, and is estimated to be involved in 15% of all human genes, affecting a number of coordinates physiological processes necessary for cell cycle progression, cell metabolism, cell differentiation and senescence, protein systhesis, mitochondria function. Deregulation of c-Myc leads to as many as 20% cancers. c-Myc has an N-terminal transactivation domain and performs its role in transcription with the bHLH (basic helix-loop-helix)-containing protein Max to bind specific DNA sequences of its target genes. The cellular expression level of c-Myc is tightly controlled at multiple levels, including forming heterodimers with Max, transcription, mRNA stability and a variety of post-translational modification such as phosphorylation, acetylation, ubuiquitination. It is reported that c-Myc is a protein with quite a short half-life. Thus, ubiquitination may play an important role in the regulation of its turnover. Many differernt E3 ligases can target c-Myc for K48 poly-ubiquitination and regulate the stability of c-Myc, such as Fbw7, SKP2, HectH9. Phosphorylations at serine 62 (Ser62) and threonine 58 (Thr58) are essential for the interaction of c-Myc with its E3 ligase Fbw7. Correspondingly, the reported deubiquitinase of c-Myc is USP28, which can indirectly regulate the stability of c-Myc in a Fbw7-dependent manner. Mutants of c-Myc lacking the ability of binding Fbw7 cannot be stabilized by USP28. However, the tumor suppressor Fbw7 is frequently mutated or deleted in a variety of cancers. Therefore, it is essential to search for deubiquitinases that directly target c-Myc for deubiquitination. Moreover, data from other group showed that USP28 cannot affect the growth of lung cancer cells. Thus, it is critical to test whether there exists any DUB for c-Myc in lung tumorigenesis.Here, in our study we found a novel deubiquitinating enzyme (DUB) that directly binds and stabilizes c-Myc via via molecular biology and cellular biology methods. USP37 binds, deubiquitinates c-Myc and then affects the expression of c-Myc downstream target genes, leading to their transcriptional activation and involvement in lung cancer cell proliferation and Warburg effect. Furthermore, we analyzed a series of human lung cancer specimens by immunohistochemical (IHC). Our data showed that USP37 mRNA level is inversely correlated with USP37 gene methylation. Also, there exists a dramatic positive correlation between the expression of c-Myc and that of USP37. Our study suggests that USP37 is a potential therapeutic target for the treatment of lung cancer.The major parts of our research are as follows:1. Identification of c-Myc’s new deubiquitinasesA series of USP plasmids were overexpressed in H1299 lung cancer cells. The endogenous protein levels of c-Myc were d using western blotting, quantified and normalized to actin. The normalized data were used to compare with the control group transfected with empty vector. As previous reported,USP28 significantly increased c-Myc protein levels.To our surprise, coexpression of USP37 dramatically increased the protein level of c-Myc as well.We cotransfected the c-Myc and USP37 in 293T cells and found that USP37 can specifically stabilize c-Myc but not Klf4,which is dependent on its deubiquitinase activity as DUB dead mutant USP37DD (USP37 C350S), in which the catalytic cysteine 350 has been replaced by Ser can not stabilize c-Myc. The result was confirmed by a luciferase-based protein degradation system (LPDS) assay. Additionally, the half life of c-Myc in H1299 cells transfected with or without USP37 was analyzed by a cycloheximide (CHX) chase assay. The half-life of endogenous c-Myc USP37 was significantly prolonged from approximately 13.6 to 37.4 min by USP37, indicating that USP37 regulates the stability of c-Myc on post-translation level.Collectively,our results showed that USP37 specifically stablizes c-Myc in a deubiquitinase activity-dependent level.2. Study on the mechanism of c-Myc stability regulation mediated by USP371) USP37 regulates c-Myc protein stabilityEndogenous USP37 was down-regulated in the H1299 lung cancer cell line with two different siRNAs, and c-Myc expression was analyzed. We found endogenous c-Myc protein evidently decreased after USP37 was knocked-down. Yet, there was no evident change of c-Myc mRNA level. The effect of USP37 on c-Myc stability was blocked by addition of proteasome inhibitor MG132. Moreover, the half-life of c-Myc was reduced from 16.7 to 8.3 min by knocking-down USP37.Downregulating USP37 also significantly reduces steady-state levels of c-Myc induced by fresh serum refeeding, which substantially induced the expression of the c-Myc protein. Together, these data indicate that endogenous USP37 may regulate c-Myc protein stability in lung cancer cells.2) USP37 is a direct binding partener of c-MycWe cotransfected USP37 and c-Myc into 293T cells and Our data showed that HA-c-Myc could strongly interact with USP37-FLAG. An interaction between endogenous c-Myc and USP37 was also detected in H1299 cells.In addition, our immunofluorescence result showed that USP37 and c-Myc were colocalized in the nucleus. In vitro translated c-Myc protein can be pulled-down by bacterially purified His-USP37, but not His-USP15, indicating that USP37 can directly bind c-Myc. Moreover, we proved c-Myc deletion mutant lacking BoxIII domain lost the ability to interact with USP37 and could not be stabilized by USP37.3) USP37 deubiquitinated c-MycGiven that USP37 can stabilize c-Myc in a deubiquitinase activity dependent manner, the effect of USP37 on c-Myc ubiquitination was tested. Expression plasmids encoding His-ubiquitin and HA-c-Myc with or without wild-type USP37 or USP37DD were cotransfected into HEK293T cells. The ubiquitinated proteins were purified using Ni-NTA resin under denaturing condition. Our data showed that USP37 can markly deubiquitinate c-Myc but USP37 DD cannot. The same result was obtained by an immunoprecipitation-based ubiquitination assay. Next, the ubiquitination level of c-Myc was significantly increased while deleting endogenous USP37 by siRNA in H1299 lung cancer cells. An in vitro deubiquitination assay was performed to test whether USP37 can directly deubiquitinate c-Myc. Purified USP37 markedly reduced the ubiquitination level of immunoprecipitated c-Myc. To sum up, our results indicate that USP37 is a direct DUB with activity toward c-Myc that may protect c-Myc from polyubiquitination-mediated degradation.3. USP37 regulates lung cancer cell proliferation and Warburg effect via targeting c-Myc stablization1) USP37 promotes c-Myc-mediated Lung cancer cell growthc-Myc is a well-known oncoprotein related with many kinds of human cancers by ptomoting cancer cell proliferation.As metioned above,USP37 stablizes c-Myc by binding with and deubiquitinating c-Myc.We transfected two different siUSP37 into H1299 lung cancer cells and detect cell growth by MTT assay. We found that depletion of USP37 significantly blocked the growth of lung cancer cells. We knocked-down both USP37 and c-Myc to test whether USP37 regulate lung cancer cell proliferation by targeting c-Myc. Our data showed that USP37 lost the ability to promote cell growth without c-Myc, indicating the effect of USP37 on cell growth is dependent on c-Myc. To further confirm the result proved above, we overexpressed USP37 or USP37DD in H1299 lung cancer cells, measured the effect of USP37 on cell growth by clone formation assay and found that USP37 can increase lung cancer cell growth on a deubiquitinase activity-dependent manner. Furthermore, downregulation of USP37 can block lung cancer cell growth via targeting c-Myc. Together, our data indicate that c-Myc is likely a major target of USP37 to regulate lung cancer cell growth.3) USP37 promotes c-Myc-mediated Warburg effect in Lung cancer cellsIt is reported that warburg effect promotes cancer cell growth and tumorigenesis where cancer cells take up more glucose to produce lactate by glycolysis, even under the condition lacking oxygen. We measured the effect of USP37 on Warburg effect by testing the glucose uptake and lactate production and found that depletion of USP37 took up less glucose and produced more lactate. Besides, we test the expression of c-Myc downstream LDHA and Glutl that are reported to invove in Warburg effect. Our data showed that loss of USP37 significantly downregulates both LDHA and Glutl expession,which is dependent on the expression of c-Myc. Collectively, our data indicate that as the effect of USP37 on c-Myc, USP37 is important to lung cancer cell proliferation and Warburg effect, and leads to lung cancer.4. Study on the correlation of the expression of USP37 with c-Myc in lung cancerThe abundance of c-Myc is high in a variety of cancers. As USP37 can stabilize c-Myc, we found that USP37 levels were dramatically increased in around 64% human lung cancer tissues by immunohistochemical (IHC) analyses. Next, we examined the expression of USP37 data from TCGA database and found that USP37 mRNA levels are significantly upregulated in both lung adenocarcinoma and squamous cell carcinoma, not any other cancer types. Furthermore, we found that the levels of USP37 are inversely correlated with USP37 gene methylation. Next, our staining results showed that USP37 levels are statistically positively correlated with c-Myc in lung cancer by IHC. Together, our data indicate that the regulation of c-Myc by USP37 may occur in lung cancer.All the data surport the conclusion that we identified a direct DUB USP37 as a novel regulatior of oncoprotein c-Myc. We found that overexpression of USP37 directly deubiquitinates c-Myc and stabilizes c-Myc while down-regulating USP37 reduces c-Myc protein level. Thus, USP37 promotes lung cancer cell proliferation and the Warburg effect via targeting c-Myc. Also, our data showed that the expression of USP37 and c-Myc is positively correlated in serial sections of human lung cancer specimens by immunohistochemical (IHC) analyses. Collectively, USP37 is a novel DUB of c-Myc stability and function.