Identification Of Novel LSD1 Non-histone Substrates In Regulating Myoblast Cells Autophagy And Osteosarcoma Cells Proliferation

PART Ⅰ: LSD1 NEGATIVELY REGULATES AUTOPHAGY IN MYOBLAST CELLS BY DRIVING PTENDEGRADATIONLysine-specific demethylase 1(LSD1)is a well characterized transcriptional regulator functioning on the chromatin to remove mono-and di-methyl groups from lysine 4 or lysine 9 of histone 3(H3K4 or H3K9).Apart from histone substrate,LSD1 also has non-transcriptional activities via targeting non-histone substrates that participate in diverse biological processes.During the process of growth and differentiation of myoblast cells,it’s unclear whether LSD1 could regulate autophagy of myoblast cells by targeting its non-histone substrates.In our study,we found that LSD1 negatively regulates autophagy in myoblast C2C12 cells by pharmacological inhibition and gene knockdown of LSD1.By determining the signal cascades and performing rescue assay,we found that LSD1 induces autophagy by regulating AKT/m TORC1 signaling pathway in myoblast cells.Further studies showed that inhibition of LSD1 can result in the increase of PTEN protein level in a transcription-independent mechanism.LSD1 interacts with PTEN and promotes ubiquitination of PTEN,consequently,the stability of PTEN decreased.Together,our findings identify a novel biological function of LSD1 in autophagy,mediated by regulating the stability of PTEN and the activity of AKT/m TORC1.Moreover,LSD1 plays an important role in myoblast differentiation.Our work will gain a better understanding of the non-histone substrates and functions of LSD1.PART Ⅱ: COMBINED LSD1 AND CDK4/6 INHIBITION IS SYNERGISTIC AGAINST PROLIFERATION OFOSTEOSARCOMA CELLSOsteosarcoma is the most common primary bone tumors,often occurs to teenagers and the children.Presently,new targeted therapeutic strategies are still required to improve the survival rate for osteosarcoma.Both the epigenetic studies and cell cycle checkpoint related studies will provide better drug targets for the treatment of osteosarcoma.The role and regulation mechanism of LSD1 in osteosarcoma,and the biological function of LSD1 inhibitor for preclinical application need to be explored.In our study,under the treatment of LSD1 inhibitor,we found the G1 phase arrest of osteosarcoma cells by flow cytometry assay and the decrease of protein level of CDK4.In vivo ubiquitination assays demonstrated that LSD1 regulate the ubiquitination and degradation of CDK4 dependent on its enzymatic activity.To explore the regulational site of CDK4,truncations and mutations of CDK4 were constructed.Further studies demonstrated that LSD1 interacts with the peptides of CDK4 ranging from the 1st amino acid to the 96 th amino acids,and the ubiquitination site of CDK4 may located in K211,K225,K282 and K297,and SCF-SKP2 does not participate in this process.The clony formation and cell viability assay determined both LSD1 inhibitor and CDK4 inhibitor can inhibit the proliferation of osteosarcoma cells,and the two inhibitors have a significantly combination effect.We anticipate that through this study about CDK4,we will gain a better understanding of precisely how LSD1 is functioning to regulate cell cycle and of how CDK4 protein is regulated by its post-translational modifications and that this will provide new targets and approaches to osteosarcoma treatment.