Heterochromatin Protein1γ Is Required For Prostate Cancer Progression Through HP1γ/miR-451a/c-Myc Feedback Loop

Histone modification and chromatin remodeling play essential roles in genome reprogramming during tissue homeostasis and cancer development.Heterochromatin protein 1(HP1)was first identified in Drosophila as the heterochromatin-associated protein.HPI family members,consist of HPla,HP1β and HP1γ,are involved in a number of cellular processes,including senescence,DNA repair,cell cycle,cell differentiation and cancer development.HP1γ might be implicated in some cancer types,such as lung,breast,colon and esophageal cancers,with a small-scale screen of cancer tissues.A tissue microarray study demonstrated that both nuclear and cytoplasmic HP1γstaining intensities are elevated in prostate cancer,compared to benign tissues.Its expression is correlated with proliferative marker,Ki-67.However,it remains elusive how HP1γ regulates gene expression to facilitate prostate cancer development.Prostate cancer(PCa)is the second leading cause of cancer-related deaths in the United States,with approximately 26,120 men estimated to die in 2016.c-Myc signaling plays an important role in mediating castration resistance,epithelial-mesenchymal transition and docetaxel resistance in PCa cells.Thus,c-Myc could be a potential therapy target for prostate cancer.Although amplification of the c-Myc oncogene is present in a subset of advanced prostate tumors,the nuclear c-,Myc protein is up-regulated in cancer progression even in the absence of gene amplification.However,the mechanisms by which it is upregulated remain unresolved.In this thesis,we found that HP1γ is overexpressed in prostate cancer,not only in public datasets but also in our own cohort of PCa specimens.Of note,HPly expression is negatively correlated with the survival of patients.To explore the function of HP1γin PCa,we knocked down HPly in PC3 and DU145 cells with two shRNAs targeting different regions of HP1γ mRNA(shHPly-1 and shHPly-2).Comparing to the cells with control shRNA(shCTL),depletion of HPly in both PC3 and DU145 cell lines induced G2/M arrest and apoptosis,which leading markedly reduction of growth rate and the colony formation capability in soft agar.To check whether HPly is vital for prostate carcinogenesis in vivo,PC3 cells with HP1γ knockdown were s.c.inoculated into nude mice.We found that the cells with shHPlys xenografts grew much slower than that of shCTL,accompanied by reduction of proliferative index and induction of apoptotic rate.Thus,we demonstrated that HP1γ is required for prostate cancer proliferation and tumorigenesis.Next we tried to identify HPly’s downstream targets.Herein,we specifically focused on miRNAs which have been regarded as a new class of mediators in prostate cancer development.miRNA profiling analysis was conducted in HP1γ knockdown(shHP1γ-1)and control(shCTL)DU 145 cells.Among 32 cancer-related miRNAs we tested,miR-451a was up-regulated the most.We also showed that HP1γ binds to the promoter of miR-451a through ChlP assay.Loss of HP1γ reduced the methylated histone H3K9 on its promoter region,which accounts for the induction of miR-451a.So,our study firstly proved that HP1γ directly represses miR-451a expression through binding to methylated histone H3K9 on its promoter region.We further demonstrated that miR-451a,as a tumor suppressive miRNA,partially mediates the role of HP1γ in prostate cancer.To test how miR-451a can function as tumor suppressor in PCa cells,we performed bioinformative analysis and found c-Myc is a potential target of miR-451a.Therefore,by forced expression of miR-451a mimic,we found that the protein levels of c-Myc were downregulated.miR-451a mimics targeting the wild-type c-Myc-3’UTR in psiCheck2 reporter plasmid,and decreased luciferase activity.Such inhibition was abrogated in the mutant c-Myc-3’UTR plasmid.It suggests that c-Myc is a direct target of miR-451a in PCa cells.As we have demonstrated that miR-451a is negatively regulated by HP1γ,we assessed the mRNA and protein levels of c-Myc in PC3 and DU 145 cells expressing either shHP1γ-1 or shHP1γ-2.We found that loss of HP1γ inhibits the level c-Myc.Moreover,in the HP1γ-deficient PC3 cells inhibition of miR-451a reversed the reduction of c-Myc.It indicated that HP1γ induces c-Myc via directly repressing miR-451a expression.Interestingly,we found that c-Myc directly upregulates HP1γ expression through the E-box at HP1γ promoter region and HP1γ is partially responsible for c-Myc oncogenic activity in PCa.Given the fact that HP1γ is capable of inhibiting miR-451a,we also found that c-Myc represses miR-451a through HP1γ.It suggests a positive feedback among HP1γ,miR-451a and c-Myc exists in PCa.Furthermore,we confirmed the HP1γ/miR-451a/c-Myc regulatory axis in human prostate cancer specimens.Taken together,we showed that HP1γ is overexpressed in prostate cancer.HP1γ is required for PCa progression and partially responsible for c-Myc oncogenic activity in prostate cancer.Mechanistically,c-Myc directly upregulates HP1γ expression and HP1γ induces c-Myc via directly repressing miR-451a expression through binding to methylated histone H3K9 on its promoter region,which forms a positive feedback.The HP1γ/miR-451 a/c-Myc regulatory axis was confirmed in human prostate cancer specimens.This finding reveals alternative pathway that maintains c-Myc at high expression level in prostate cancer.