The Roles Of Hzitmp7 Involved In Prostate Cancer Progression And Its Function In STAT3 Signaling Regulation

Cancer has been one of the most complicated diseases in the world and gain attentions as a global public health problem.Prostate cancer is the most common malignant disease in older men with a potential threat to male reproductive system.Androgen/androgen receptor(AR)signaling is widely considered to be the core of the development of prostate cancer.The abnormal activation of AR signal is crucial in thedevelopment of prostate cancer,especially in themalignant transformation processof hormone-independent state.It mainly manifested in two aspects.On one hand,AR mutations causes AR pathway activation with a ultra-sensitive phenomenon to a quite low level of androgen,and promotes malignant proliferation of prostate cells.On the other hand,due to the abnormal expression of AR regulatory co-factors,or due synergistic effects of AR co-factors together with AR,AR signal is enlarged,or AR downstream target genes is activated bypass AR,result in prompting malignant proliferation of prostate cancer cells.Therefore,the identification of AR co-regulated factors is the core issue in the malignant transformation and the development of prostate cancer.Hzimp7(human zinc finger-containing,Miz1,PIAS-like protein on chromosome 7)is a novel co-transcription activation factor of AR.Hzimp7 with highly homologous MIZ PIAS family structure domain,which is defined as the PIAS similar protein(ZIMZ2).So far the function studies focused on this novel protein is still very limited.The expression its significanceof hzimp7 in tumors have not yet to be explained.The associated signalpathway regulated by hzimp7 in tumors has not been reported.However,the few the existing studies indicated some features of hzimp7.Hzimp7 is demonstrated high expressed in reproductive system,which is the most abundant in the testicular tissue,and also exist in the prostate,heart,brain,pancreas and ovary,while is less or none in other organizations.In addition,as the regulation protein of AR,hzimp7 could enlarge AR signal.As well,a new members of PIAS family,hzimp7 coordinate with PIAS3 to regulate ARtranscriptional activity for AR downstream PSA gene activation.All these results indicate the significance of hzimp7 as co-transcription activation factors of AR.Nevertheless,the expression of hzimp7 in prostate cancer and its correlation with tumor are still not clear.Moreover,the data that hzimp7 genes directly associated with prostatic cell proliferation ability has not been reported.Especially,the roles of hzimp7 involved in the core proliferation signal pathways of cancer need to be clarified.Therefore,this project conducted a systematic study on the above questions.In histological aspect,high flux tissue chips of prostate and breast cancer were immunohistological stained by hzimp7,to reveal the relevance between the expression of hzimp7 and the malignant degree of tumor.In details,Kaplan-meiersurvival analysis with prostate cancer was taken in 53 clinicalcasesto clarify the association of hzimp7 with the development of prostate cancer.In cellaspects,the relationship between hzimp7 genes with the cellproliferation ability of prostate cancer,and the mechanism that hzimp7 involved in STAT3 signal pathway,which is key proliferation signal pathway in tumor,were studied to expound the physiological significance of hzimp7 in the process of cell proliferation.First of all,we detected expression level of hzimp7 in prostate and breast cancers using tissue microarray,and studied the correlation between hzimp7 with tumor malignant degree in these representativehormone-dependent tumors.According to the immunostainingdata from prostate cancer tissue chip of 3 cases of normal prostate tissue and 31 cases of prostate cancer tissues,we found that hzimp7 only expressed a small amount in the cytoplasm in normal prostate tissue,incontrast,the expression level of hzimp7 is significantly increased in both cytoplasm and nucleus in prostate cancer tissues compared with normal prostate tissue.In addition,accompanied with malignant degree increased,the expression of hzimp7 was increased.This finding was confirmed by the immunostaining result of 4 cases of normal breast tissue and 29 cases of breast cancer.Consistent with prostate cancer,hzimp7 expression enhanced in breast cancer,and accompanied by deterioration of breast cancer,hzimp7 expression increased.All of these data suggest the importance of hzimp7 in the process of tumor progression.Then,we explored the relationship between the expression level of hzimp7 and the prognosis of prostate cancer.We complete survival information and divided 53 prostate cancer patients into groups based on the expression level of hzimp7,and analyzed 10 years of survival rate with Kaplan-Meiersurvival analysis method.We found that the 10 years survival rate was 65% in low expression hzimp7 group,and the survival rate is only 24.2% in high expression hzimp7 group.These two groups have significant statistical difference(P < 0.01).The results suggest that hzimp7 is a risk factor of the prognosis of prostate cancer,as for that the higher its expression level is,the worse prognosis is.Next,we studied the association between hzimp7 gene and proliferation of tumor cells.It was found that hzimp7 can promote the proliferation of prostate cancer cells,and blocking hzimp7 gene by shRNA inhibits cell proliferation.The regulation of hzimp7 in cell proliferation is shown in a dose-dependent manner.These results suggest that the hzimp7 can enhance the proliferation of prostate cancer cells.During the cell proliferation progression induced by hzimp7,prostate cancer cells lose the ability to proliferate when we blocked STAT3 gene by siRNA.These results suggest that STAT3 gene is essential in hzimp7-induced cell proliferation.STAT3 signal is one of the core signals in a wide variety of tumor associated with proliferation including prostate cancer.Members of PIAS family could directly inhibit STAT3 signal,hzimp7 as a novel member of PIAS family,its roles in STAT3 signalregulation is not clear.We further systemic revealed the regulatory mechanism of hzimp7 in STAT3 signal with luciferase reportor experiments.Results show that hzimp7 could activate the transcriptional downstream of STAT3 signal,with a dose-dependent manner.And we found that hzimp7 can promote STAT3 phosphorylation,and then activate the expression of Cyclin D1 and Bcl-2,in Western blot experiments,which are STAT3 downstream genes and closely related to proliferation.It is proved by immunoprecipitation experiment that STAT3 and hzimp7 can interact each other and form compoundsin prostate cancer LNCaPcells.Because of hzimp10,homologous protein of hzimp7,could not activate STAT3 signal,we constructed GST-hzimp7 fusion proteinsbased on the two largest different domains between hzimp7 and hzimp10,and combinated it to STAT3 binding with GST-pulldown experiment.We found that the GST-hzimp7(740-850-aa)protein is a key functional domains for the direct recognization of hzimp7 and STAT3.Finally,we studied the function of hzimp7 in the cellular positioning of STAT3.Physiological function of STAT3 mainly realized by the STAT3Y705 phosphorylation.We use three different eukaryotic expression plasmid of STAT3,STAT3-WT(wild type),STAT3-DN(inactivation)and STAT3-CA(activated).The 705-amino acids tyrosine(Y)of STAT3-DN was mutated to phenylalanine(F)by point mutation technology.So STAT3-DN which loosed the phosphorylation sites can not be activated.And the same site of STAT3-CA was mutated to cysteine(C)which allow STAT3-CA to form dimers immediately.So that even without tyrosine phosphorylation,STAT3-CA still can achieve the effect of STAT3 activation.Luciferase assay show that both STAT3-WTand STAT3-CA can activate transcription of STAT3 downstream signal,and hzimp7 can enhance this progress.STAT3-DN had lost the ability of regulating STAT3 signal,also the function of hzimp7 was blocking.We studied the role of hzimp7 during STAT3 transferring to nuclear.We found that in prostate cancer cell line of LNCaP,wild-type STAT3 was originally located in the cytoplasm,the overexpression of hzimp7 can transfer STAT3 into nuclear.And for STAT3-CA,which is located in the nucleus by itself,can stay in the cytoplasm when hzimp7 gene was blocking.These results underscore the essential roles of hzimp7 in activating STAT3 signal pathway.In conclusion,we proved that a novel transcription factor hzimp7 was closely related to the tumor progression and prostate cancer prognosis in histology.At the cytology level,we found that hzimp7 promoted the proliferation of prostate cancer cells and regulated the main proliferation signal of STAT3 pathway.At the same time,this study provide evidence that hzimp7 is expected to be a new marker in the diagnosis of cancers,and evidences for hzimp7 as a new members of PIAS family.