Splicing Mechanisms And Roles Of A Newly Identified Cancer-related PIAS3 Isoform

Transcription regulation is the link between gene and protein expression.As an important post-transcription regulation,alternative splicing is a key molecular mechanism for increasing the functional diversity of the eukaryotic proteomes,which has become the hot-pot field in the eukaryotic gene expression regulation.Alternative splicing is closely related to the occurrence of tumor development.Cancer cells may transform the normal gene to create oncogenic isoforms;or change the tumor-suppressor to a function deficient isoform,so as to promote their proliferation,evade apoptotic signals and immune surveillance.Cancer-specific splice isoforms,which may bestow survival advantages to cancer cells,often result in rapid death of the human subject.Dysregulated alternative splicing has been another key feature of cancer.The significance and modulation of alternative splicing has become the most ascendant field of life science research.PIAS3 was identified as an inhibitor of STAT3 and can prevent the recruitment of STAT3 to its downstream target gene promoter to inhibit STAT3 transcription activity.And as a transcriptional regulation factor,PIAS3 regulates a wide variety of tumor associated signals,such as MITF,NF?B and Smad signals.In vitro studies have shown that PIAS3 functions as a tumor suppressor.Overexpression of PIAS3 can inhibit proliferation signal of various types of cancer,or restore the drug sensitivity of lung cancer or ovarian cancer and endometrial cancer to induce apoptosis.However,the research results on PIAS3 expression are very different.People found that PIAS3 expression is lower than the normal tissue in many kinds of cancer,but other studies have also shown the up-regulation of PIAS3 expression in different tumor tissues.The contradiction between the expressions of PIAS3 in different tumors,even in the same kind of tumor suggested the more complicated regulatory mechanism in PIAS3 expression,which needs to be further researched.Until now,two isoforms of PIAS3?(64KD)and PIAS3?(68KD)were identified in mice.However,only one type of 68 KD human PIAS3 has been identified in various human tissues.In this thesis research,we found an innovative existence of a cancer related PIAS3 short isoform by alternative splicing.We discussed the regulatory mechanism of the selective splicing,and revealed the bio-significance of this process in cancer.First,we identified a truncated PIAS3 isoform with 87-121 amino acids deletion in human cancer.When Western Blot was performed to survey PIAS3 expression in human normal prostate tissue and several cancer cell lines,we found one band sized 68 KD of PIAS3 protein expressed in BPH tissue,and a smaller size than 68 KD of PIAS3 protein also detected in all of the five tumor cell lines interestingly.The shorter PIAS3 isoform was amplified and sequenced,showing with the deletion of a 105 bp sequence(PIAS3?mRNA),encoding 35aa(87-121aa)in-frame extension of the EXON2 of PIAS3.In order to distinguish the two human PIAS3 isoforms,we named the longer 68 KD protein in the normal prostate tissue as human PIAS3 L,and shorter 64 KD protein in cancers as PIAS3 S.Then we compared two human PIAS3 isoforms(PIAS3L and PIAS3S)and two mouse PIAS3 isoforms(PIAS3? and PIAS3?)and found they were highly identical isoforms,indicating that human PIAS3 S identified in tumors to be a product of an mRNA splicing mechanism from PIAS3 L of normal tissue,with a selective splicing mechanism similar as mice maybe involved.Further we studied and identified the specific mechanism for PIAS3 splicing.We analyzed the 105 bp sequence to distinguish between the Exon2 regions of PIAS3 L and PIAS3 S,in which GT-AG boundary rule of intron was observed and a branchpoint A site was located in the middle.Notably,the branchpoint A site was overlapped by potential binding sites of both STAT3 and AR.We found the consistency of AR and PIAS3 L expression;and the consistency of STAT3 and PIAS3 S expression in various of cells and tissues.The overexpression and gene silence experiments confirmed that AR protein was capable of up-regulating the mRNA and protein level of PIAS3 L,whereas STAT3 protein was capable of up-regulating the mRNA and protein level of PIAS3 S.RIP experiments provide the evidence that STAT3 and AR functioned as novel RBPs binding to PIAS3? pre-mRNA of 105 bp.Especially the co-immunoprecipitation experiments demonstrated that the STAT3 recruited U2AF35 to promote PIAS3 pre-mRNA splicing by spliceosome,and AR was the antagonist in this process.The above results provided a novel splicing mechanism to generate cancer related PIAS3 S isoform,by which STAT3 binds to PIAS3? pre-mRNA and recruits U2AF35 to 3' site of potential intron to promote pre-spliceosome formation for PIAS3 S mRNA splicing.However,AR disrupted the recruitment of U2AF35 by STAT3.These results provide a novel splicing mechanism hypothesis that has not been reported.Based on the above study,we further revealed the bio-significance of the aberrant splicing of PIAS3.STAT3 and AR signals are persistently activated in many types of cancer cells,especially in prostate cancer.First we compared the functions between different PIAS3 isoforms in STAT3/AR signaling regulation.PIAS3 L,but not PIAS3 S,significantly inhibited STAT3/AR-mediated transcriptional activity,since the PIAS3? protein itself played a STAT3/AR signaling inhibitory function.Then we identified the binding effect of each PIAS3 isoform to STAT3/AR.We detected a specific binding between PIAS3 L and STAT3/AR,but not between PIAS3 S and STAT3/AR,and revealed that PIAS3? region was the central region for PIAS3 L and STAT3/AR binding.In addition,we compared the different roles of two PIAS3 isoforms in STAT3/AR cellullar localization,demonstrated the involvement of the PIAS3? region in STAT3/AR cellular localization,and addressed the existence of PIAS3? region as the crucial factor to maintain STAT3/AR in the cytoplasm and be not able to entering the nucleus.These results suggested that PIAS3? region,the different region between two PIAS3 isoforms,functioned as a central region in STAT3/AR signal inhibition.Next,we further investigated the function of each PIAS3 isoform on tumor cancer proliferation and tumorigenesis.PIAS3 L remarkably inhibited cancer cell lines growth,clone formation,significantly down-regulated proliferative gene,BCL-2,PCNA,Ki67,CyclinD1 and survivin.Further,in nude-mice xenograft models,we found that PIAS3 S did not affect tumorigenesis significantly compared with the control group.However,PIAS3 L and PIAS3? significantly inhibited the tumorigenesis in vivo,with tumor incidence only about 1/6 of that in the control,and with a much smaller size,indicating the remarkable inhibition of tumorigenesis by the PIAS3? region.Finally,to confirm all above in vitro and in vivo data,we collected 63 cases of prostate cancer samples of different clinical stages,and used PIAS3? region(87-121aa)specific antibodies to detect the expression of PIAS3 L isoform in these tissues.We found that the reduction of PIAS3 L expression was closely associated with the increase in Gleason score in prostate cancer tissues,whereas PIAS3 S was not reduced,by detection with the PIAS3 FL antibody.The results strongly indicated that the expression of PIAS3 L decreased with the increase tendency in malignancy of clinical prostate cancer.We compared the survival rates in the 63 cases of prostate cancer.The survival rate was high in the PIAS3 L High expression group,whereas it was low in the PIAS3 L Low expression group,with significant difference(p=0.019),which addressed the crucial role of PIAS3 L expression level as an indicating factor for prostate cancer survival.Further,we revealed the relationship between PIAS3 L expression and STAT3 or AR localization in human tissue samples.It implied that reduced PIAS3 L was associated with the increased nuclear translocation and signal activation of STAT3 and AR.These results showed that PIAS3? region played the core roles for PIAS3 gene in its inhibition of tumor signal to ensure the well survival.We provided new insights into alternative splicing mechanism of PIAS3 related to cancer,modulated by STAT3;and addressed the bio-significance of the spliced region for its tumor suppressor roles in STAT3/AR signals inhibition,tumorigenesis inhibition,and clinical cancer survival,providing a new target for tumor prediction and therapy.