Introduction:In the first part of this thesis,the author have made an in-depth and comprehensive systematic review of S-phase kinase-associated protein 2（Skp2）through extensive literature search in relation to the ubiquitin proteasome system（UPS）with more emphasis given to its substrates,regulatory mechanisms,and its role in cancer development and its potential as a target of anticancer drugs.Accordingly,Skp2 is a well-characterized oncoprotein that belongs to the FBXL subclass of F-box proteins.It has a molecular weight of 45 kDa and is localized in nucleus and cytoplasm.Skp2 is broadly expressed in several tissue types.As a key substrate recognizing component of the SCF（S-phase kinase-associated protein 1（Skp1）-Cullin1-F-box）E3 ubiquitin ligase,Skp2 regulates the stability and activity of proteins mainly by specifically targeting them for UPS-mediated degradation.As such it influences numerous cellular functions such as cell proliferation,cell cycle progression,transcription and apoptosis.Skp2 is overexpressed in multiple human cancers including non-small cell lung cancer,prostate cancer,breast carcinomas,cervical cancer,osteosarcoma and gastric cancer.Deregulated Skp2 function promotes neoplastic transformation.Targeting Skp2 at different levels may represent a novel and a promising approach for the treatment of different human cancers with Skp2 overexpression.However,despite its immense and well-established role in UPS-mediated protein turnover,much is unknown about the function of Skp2 independent of the ubiquitination pathway.Previously,Skp2 has been reported to regulate RhoA gene transcription and p300 function independent of its E3 ligase activity raising the possibility of other previously unexplored roles of Skp2 in gene expression independent of its E3 ligase activity.Due to this,in the second part of this project,we have investigated the role of Skp2 in the regulation of lysine-specific demethylase（LSD1）expression and function independent of its E3 ligase activity in gastric cancer.Research work purpose:The main aim of this project was to explore the relationship between Skp2 and LSD1 oncoproteins,and to identify the mechanism of LSD1 regulation by Skp2 through gene silencing（knockdown and knockout）and overexpression experiments in gastric cancer.Moreover,the study aimed at exploring the effect of Skp2 on LSD1 demethylase substrates and the expression or the activity of key cellular proteins including human epidermal growth factor receptor 2,androgen receptor and Akt kinase with respect to gastric cancer oncogenesis.Methods:In this project,MGC-803,MKN-45 and SGC-7901 gastric cancer cell lines stably expressing lentiviral-mediated shRNA oligonucleotides against Skp2 mRNA were constructed for knockdown experiments.Moreover,CRISPR/Cas9-mediated Skp2 knockout MGC-803 and MKN45 cells were also generated.LSD1 knockout MGC-803 cells generated by CRISPR/Cas9mediated were also used.MGC-803 cells that overexpress Skp2 was generated by transfecting Skp2 plasmid using pIRES2-EGFP bicistronic lentiviral expression vector.Western blotting was used to detect the expression of Skp2,LSD1,human epidermal growth factor receptor 2（HER2）,ubiquitin specific peptidase 7（USP7）,androgen receptor（AR）,p27,and protein kinase B（Akt）and epidermal growth factor receptor（EGFR）activation from single cell-derived Skp2 depleted and Skp2 overexpressing gastric cancer cell lines.Histone protein methylation status（H3K4me1 and H3K4me2）was also determined from nuclear protein fractions extracted from these cells.Moreover,western blotting was also used to detect Skp2,pAkt,AR and HER2 protein expression from total protein samples obtained from single-cell derived LSD1 knocked out MGC-803 cells.Furthermore,quantitative polymerase chain reaction（qPCR）reaction was performed to analyze expression of LSD1 and p300 genes in Skp2 depleted MGC-803,MKN-45 and SGC-7901 cell lines,and Skp2 overexpressing MGC-803 cell as well as to assess the efficiency of Skp2 knockout in MGC-803 and MKN-45 cells.Cycloheximide（CHX）assay was performed to assess the effect of Skp2 on posttranslational LSD1 stability.Cell growth and proliferation assay,clonogenic assay and cell cycle progression analysis were performed to evaluate the effect of Skp2 on gastric cancer cell phenotypes.Finally,the effect of Skp2 on in vivo tumorigenesis was assessed in SCID mice.Results:1-Skp2 regulates transcriptional and post-transcriptional LSD1 expression:Western blot and quantitative polymerase chain reaction（qPCR）analyses revealed that Skp2 depletion using lentiviral mediated shRNA or CRISPR/Cas9 downregulated the expression of both LSD1 mRNA and LSD1 protein in MGC-803,MKN-45 and SGC-7901 gastric cancer cell lines.The mRNA level of LSD1 was decreased by 2.45-fold,1.73-fold and 2.17-fold upon Skp2 knockdown in MGC-803,MKN-45 and SGC-7901 Skp2 knockdown cells compared to the control groups,respectively.Similarly,the LSD1 mRNA expression was reduced by 1.63-fold and 1.61-fold in Skp2 knockout MGC-803 and MKN-45 cells compared to the control groups,respectively.On the other hand,induction of Skp2 overexpression in MGC-803 cells markedly upregulated LSD1 expression both at the mRNA and protein levels.In Skp2 overexpressing MGC-803 cells,the level of LSD1 mRNA expression was increased by 2.86-fold compared to the control groups.2-Skp2 regulates p300 mRNA expression:Furthermore,Skp2 depletion significantly suppressed p300 gene expression in MGC-803 and MKN-45 cells.Analysis of qPCR results showed that Skp2 knockdown decreased the p300 mRNA expression by 2.45-fold and 2.61-fold compared to the control groups in MGC-803 and MKN-45 cells,respectively.Skp2 knockout also produced a 1.8-fold decrease in the expression of p300 mRNA in MGC-803 cells compared to the corresponding control groups.By contrast,Skp2 overexpression in MGC-803 cells upregulated p300 mRNA expression by more than 1.95-fold.3-Skp2 regulates USP7 expression:Western blot analysis further revealed that Skp2 depletion decreased the level of ubiquitin specific peptidase 7（USP7）deubiquitinase in MGC-803 and MKN-45.In contrast,Skp2 overexpression increased USP7 protein expression level in MGC-803 cells.Moreover,CHX chase assay revealed that Skp2 increased LSD1 protein stability.4-Skp2 modulates LSD1-mediated histone demethylation:Assessment of the effect of Skp2 on LSD1 demethylase activity by western blotting revealed that the mono-and dimethylation of histone H3 K4（H3K4me2 and H3K4me1）was elevated upon Skp2 depletion in MGC-803,MKN-45 and SGC-7901 cells.On the other hand,Skp2 overexpression reduced the mon-methylation of histone H3 K4（H3K4me1）in MGC-803 cells.5-Skp2 and LSD1 regulate HER2 and AR expression:HER2 expression was decreased upon Skp2 depletion in MGC-80,MKN-45 and SGC-7901 cell lines.Moreover,LSD1 knockout also decreased HER2 expression in MGC-803 cells.By contrast,Skp2 overexpression caused increased HER2 expression in MGC-803 cells.Similarly,Skp2 depletion led to a decline in the expression of androgen receptor protein in MGC-803 and MKN-45 cells.LSD1 knockout also reduced AR level in MGC-803 cells.In contrast,Skp2 overexpression increased AR protein level in MGC-803 cells.6-Skp2 and LSD1 promote Akt activation,and Skp2 also regulates EGFR activation:Evaluation of Akt activation by phosphorylation revealed that Skp2 depletion decreased S473 Akt phosphorylation in MGC-803,MKN-45 and SGC-7901 cells.Similarly,LSD1 knockout decreased S473 phosphorylation of Akt in MGC-803 cells.On the other hand,Skp2 overexpression led to elevated S473 Akt phosphorylation in MGC-803 cells.Furthermore,Skp2 depletion decreased Y1068 EGFR activation in MGC-803 and MKN-45 cells.7-Skp2 regulates p27 degradation,cell cycle progression and cell growth and proliferation:The level of p27 was elevated upon Skp2 depletion in MGC-803,MKN-45 and SGC-7901 cells.On the other hand,Skp2 overexpression decreased p27 level in MGC-803 cells.Skp2 depletion also reduced the proportion of cells entering the S-phase by arresting them at the G1 phase in SGC-7901 cells.Cell growth and proliferation was significantly suppressed by Skp2 depletion in MGC-803 and SGC-7901 cells.In contrast,Skp2 overexpression accelerated the growth and proliferation of MGC-803 cells.Moreover,clonogenic growth assay revealed that Skp2 knockdown or deletion significantly suppressed the clonogenic property of MGC-803 and SGC-7901 cells.8-Skp2 promotes in vivo tumorigenesis:Evaluation of the effect of Skp2 on in vivo tumor growth revealed that Skp2 knockdown profoundly suppressed gastric cancer tumorigenesis in mouse models in vivo.Both tumor weight and tumor volume were decreased by almost 7-fold in ShSkp2 groups compared with the shNC groups.Conclusions and the creative achievements:Skp2 has been reported to play a huge role in UPSmediated protein turnover in different human cancers.In this project,we discovered a novel role of Skp2 in gene regulation independent of its E3 ligase activity.We found that Skp2 is a new regulator of LSD1 expression primarily at the gene level possibly through upregulating p300 transcriptional co-activator expression and recruiting it to the transcription factor Myc complex which binds to the promoter region of LSD1 gene in gastric cancer.Moreover,our findings suggest that Skp2 might be involved in posttranslational regulation of LSD1 stability through upregulating the expression of deubiquitinase notably the USP7 enzyme.Furthermore,we also demonstrated that the role of Skp2 goes beyond mere regulation of LSD1 expression and its stability.Skp2 modulates LSD1 functions including its demethylase activity on mono-and di-methylated H3 core histones（H3K4me1 and H3K4me2）.Moreover,Skp2 also regulated AR and HER2 expressions possibly through LSD1.Skp2 and LSD1 might also act in concert to regulate activation of growth and survival pathways such as the PI3K/Akt pathways in gastric cancer.Skp2 has also been found to increase EGFR activation in gastric cancer cells.The functional outcome of all these effects of Skp2 is regulation of cancer cell growth and proliferation,cell cycle progression,and in vivo tumorigenesis.The discovery of Skp2 in modulating LSD1 gene expression indicates that the role of Skp2 is not limited to regulation of posttranslational protein stability and activity in cancer.Moreover,Skp2-mediated regulation of LSD1-dependent cellular functions would further highlight the role these oncoproteins play in modulating carcinogenesis.Furthermore,the discovery of Skp2mediated regulation of LSD1 is a new mechanism of regulation for LSD1 in cancer.To our knowledge,this is the first report demonstrating that Skp2 is involved in regulating LSD1 mRNA and protein expression,and possibly its posttranslational stability independent of its E3 ubiquitin ligase activity in gastric cancer.The discovery of interaction between Skp2 and LSD1 proteins which have well-established and key oncogenic functions in different human cancers would have a paramount significance to understand the molecular mechanism of carcinogenesis.Furthermore,it would pave a new avenue to further explore novel cross-talk mechanisms between Skp2 and oncogenic cancer signaling pathways that regulate gene expression epigenetically such as chromatin-modifying enzymes,and would also serve as an important venture in the effort to discover key therapeutic targets.Overall,owing to the critical roles both Skp2 and LSD1 plays in cancer development,understanding their interaction at the molecular level would have a paramount importance in cancer therapy especially for drug molecules that target Skp2 and LSD1 pathways,and for development of dual-acting new inhibitors.The findings of this project would shed light on the unforeseen aspects of Skp2 functions and on the mechanism of LSD1 regulation in gastric cancer.Moreover,this study would also provide insight for further studies aimed at exploring novel Skp2 targets that do not require its E3 ligase activity,and mechanistic and functional studies focused on LSD1.The emerging Skp2 inhibitors could also exploit this line of axis as their site of action.