Characterization Of UHRF1 Stability And Its Role In Metabolic Reprogramming In Cancer

As a key epigenetic regulator,UHRF1 plays an important role in the maintenance of DNA methylation.As a multiple functional domain protein,UHRF1 has also been shown to play important roles in early embryonic development,cell cycle,apoptosis and DNA damage response.Furthermore,UHRF1 is frequently highly expressed in various human cancers and its overexpression often correlates with the aggressiveness of human cancers.UHRF1 is therefore considered as a potential biomarker and therapeutic target for cancer diagnosis and prognosis.So far,there is no reported inhibitor that targets directly at UHRF1.In this thesis I first aimed to identify small molecules which specific target UHRF1 proteins for degradation.By using a high throughput screening strategy,we have identified the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin(17-AAG)as a compound that can effectively induce UHRF1 degradation in various cancer cells.We present evidence that UHRF1 interacts with Hsp90 chaperone complex and is a novel Hsp90 client protein.Both Hsp90 inhibitors,17-AAG and 17-DMAG,can induce UHRF1 ubiquitination and subsequently a ubiquitin-dependent proteasome degradation in various cancer cells,and this process is dependent on Hsp70.Interestingly,mechanistic study indicates that two previously identified ubiquitin E3 ligases for Hsp90 client proteins,CHIP and CUL5,are not required for Hsp90 inhibitor-induced UHRF1 degradation.In addition,Hsp90-inhibitor-induced UHRF1 degradation is dependent neither on UHRF1's intrinsic E3 ligase activity nor the E3 ligase complex SCF?-TrCP that has been implicated in regulation of UHRF1 stability.Consistent with previous publications,we observed that UHRF1 overexpression in NIH3T3 fibroblast cells enhanced cell proliferation,and knockdown of UHRF1 in different cancer cells led to reduced proliferation and increased apoptosis.Furthermore,knockdown of UHRF1 reduced the inhibitory effect of Hsp90 inhibitors on cancer cell proliferation,suggesting that Hsp90 inhibitors may suppress cancer cell proliferation in part through its induced UHRF1 degradation.In summary,our results identify UHRF1 as a novel Hsp90 client protein and reveal a role of Hsp90 chaperone in maintaining UHRF1 protein stability.In this thesis I also aimed to investigate the potential role of UHRF1 in regulating cellular metabolism.UHRF1 is known to stimulate cell proliferation,although it is unclear if UHRF1 does so through its effect on DNA methylation.We hypothesized that UHRF1 may directly regulate cell proliferation through modulating the activity of one or more metabolic regulators.The AMP-activated protein kinase(AMPK)is a central regulator of cellular metabolism and metabolic homeostasis and does so by sensing cellular energy status in mammalian cells.In various cancer cells,AMPK suppresses tumor growth through negatively regulating aerobic glycolysis(the Warburg effect).Various energy stresses result in reduced intracelluar ATP and increased AMP/ADP.Binding of AMP and/or ADP can directly induce allosteric activation of AMPK or causing a conformational change that promotes AMPK phosphorylation by upstream kinases LKB1 or Ca2+ dependent CaMKK?.We present evidence that UHRF1 can interact with AMPK and may regulate cancer aerobic glycolysis through inhibiting AMPK activation.We found that UHRF1 knockdown in various cancer cell lines resulted in elevated AMPK phosphorylation and phosphorylation of its substrate ACC1,whereas UHRF1 overexpression led to inhibition of AMPK activity.This inhibitory effect on AMPK is most likely independent on UHRF1's role in DNA maintenance methylation,as an UHRF1 mutant deficient of E3 ligase activity that is inactive in DNA maintenance methylation is fully active in inhibiting AMPK activation.Mechanistically,UHRF1 specifically interacts with AMPKal/a2 and inhibits the interaction between upstream kinase LKB1 and AMPKa2 but not AMPKal.Thus,we present evidence that UHRF1 may regulate cancer cell metabolism by modulating AMPK activation and this effect on cellular metabolism appears to be independent of its role in DNA maintenance methylation.Finally,given AMPK's important roles in cell proliferation,our study adds a new layer of evidence for UHRF1 as a potential target for cancer therapy.