The Lipolysis-stimulated Lipoprotein Receptor (LSR) Regulates Hippo-YAP Signaling To Inhibit Cell Growth In Hepatocellular Carcinoma

I.Objective Hepatocellular carcinoma(HCC)is the sixth most prevalent cancer and the third most frequent cause of cancer-related death.Because of the early metastasis,stronger invasion,poor treatment efficacy,5-year overall survival rate for all stages of patients with HCC is just 18%,moreover,for patients with distant stage the 5-year survival rate is only 3% in the USA.Several therapeutic methods like surgical resection and transplantation are still not efficient and have kinds of drawbacks.Thus,discovering approaches to avoid recurrence and metastasis of HCC and to provide novel target therapeutic strategies is of outmost importance in HCC.The Hippo pathway is a key regulator of organ size and tissue homeostasis and is dysregulated in many human cancers including human liver cancer.YAP is the major effector of Hippo pathway and high expression in nuleus of liver cancer cell.The Hippo pathway is crucial in organ size control,cell contact inhibition and homeostasis.Deregulation of Hippo pathway leads to kinds of cancer.However,the mechanisms are still not clear.The lipolysis-stimulated lipoprotein receptor(LSR)is a type I single-pass transmembrane protein that is mainly expressed in the liver.LSR is a newly discovered molecular component of tricellular contacts localized in most epithelial tissues and has a barrier function.It also regulates negatively plasma triglyceride levels measures at the postprandial stage.Recently the mechanism of LSR in cancer have draw the attenion to researchers,but the results are controversail.On the one hand,several studies have reported that loss of LSR enhances tumor progression in bladder cancer and endometrial cancer.It has been shown that loss of LSR can promote cell invasion and migration via upregulation of TEAD1/AREG which are the downstream protein of Hippo pathway in human endometrial cancer and another study showed that knockdown of LSR would upregulate YAP target genes in bladder cancer.Besides as 14-3-3s taking part in protein trafficking by altering YAP localization,LSR can bind to 14-3-3s directly.All in all,YAP serves as an independent prognostic marker of HCC,and LSR and YAP may collaborate to play roles in the development of HCC.On the other hand,LSR functions in the promotion of aggressive breast cancer and poor patient outcome.These suggest that LSR may be involved in multiple types of tumor,unfortunately the detailed relationship of LSR and liver cancer has not been investigated.However all the studies simply discribed correlation between LSR and YAP and the detailed mechanism of how LSR relulate YAP and take part in Hippo pathway are remain uncertain.In the present study,we investigated the behavior and roles of LSR in liver cancer cells in vivo and in vitro.Furthermore,we focused on whether LSR influence the liver cancer malignancy via Hippo pathway and how it takes place.The data demonstrate a novel mechanism of actions by LSR,suggests that LSR may represent a novel promising target to fight liver cancer,and also provide theoretical foundation for researching new targeted drugs to liver cancer.II.Methods 1.Tissue specimens.All the human liver cancer tissue specimens and adjacent normal control tissues were collected from the First Affiliated Hospital of dalian medical university,a total of 3 cases.All experiments were carried out in accordance with the approved guidelines and with the declaration of Helsinki.2.Western blot and IHC to detect the expression level of endogenous LSR in liver cancer tissue and paied normal liver tissue.3.In order to calculated the hazard ratio(HR)in Fig.1C,the data were obtained from Gene Expression Omnibus(GEO,accession number: GSE 10143 and GSE 27150)and The Cancer Genome Atlas(TCGA)Program with the help of PROGgene V2(http://genomics.jefferson.edu/proggene/),a total of 454 cases.The synthesis of data were performed as previously described using Review Manager(Computer program.Version 5.3.Copenhagen: The Nordic Cochrane Centre,The Cochrane Collaboration,2014).4.To downregulate the expression of LSR in Hep3 B and Huh7 cells,these cells were infected by sh RNA lentiviral particles(Santa Cruz;code: sc-97082V)in the presence of polybrene and puromycin.Subsequently,two LSR knocked down clones,KD-LSR-I and KD-LSR-II,were used for this study.5.In order to evaluate the effect of LSR in vivo and in vitro,we conducted clone formation,xenograft nude mice assay and 3D sphere assay using the stablely knocked down cells.6.Western blot to evaluate the Hippo pathway protein after LSR stablely knocked down.In order to obtain the V5-LSR-WT and V5-LSR-Y623 A stably overexpressing cells,we conducted the LSR-Y623 A mutation using the site-directed mutagenesis,and used the p CDH lentivirus the infect SNU449 cells.Western blot to evaluate the Hippo pathway protein using the stablely overexpressed cell lines.7.Immunofluorescence to detect the endogenous YAP localization after LSR knocked out.8.The cell fractionation assay was performed to detect the localization of endogenous YAP after LSR knocked out.9.Co-immunoprecipitation to detect the interaction of LSR and YAP,and the influence factor of it.The laser confocal used to observe the co-localization of LSR and YAP.10.In order to obtain the V5-LSR-Y623 A stably overexpressing cells,we conducted the LSR-Y623 A mutation using the site-directed mutagenesis,and used the p CDH lentivirus the infect SNU449 cells.Lentiviral constructs and stable cell lines.The p CDH packaging plasmids ps PAX2 and p MD2 G were co-transfected into HEK293T cells for virus production.Cells were selected 1.5μg/ml puromycin in culture medium.Western blot to evaluate the Hippo pathway protein using the stablely overexpressed cell lines.Co-immunoprecipitation and the laser confocal were used to observe the interaction and co-localization of LSR and YAP.III.Results Part 1: 1.Distribution of LSR in liver cancer and its high expression of LSR reduced risk of death.To identity the relationship between LSR and liver cancer,we initially examined the expression of LSR in liver cancer tissues with Western blot and LSR was highly expressed in normal liver tissues.After that immunohischemical staining for LSR was performed using paraffin embedded sections of liver cancer tissues and paired normal tussues.LSR showed stronger staining in normal tissues than in liver cancer tissues.In addition,to evaluate if the abnormal expression of LSR in HCC tissues was associated with the survival of patient,we obtained the data from GSE 10143,GSE 27150 and TCGA.Importantly,the forest plots of these data suggested that,compared to low expression of LSR,high expression of LSR reduced risk of death by 11%(high expression of LSR vs.low expression of LSR: HR = 0.89,95% CI,0.81 and 0.97,P = 0.008,).This suggests that LSR might inhibit the development and progression of HCC.Taken together,the above results indicate a possible inverse association between liver LSR expression and development of liver cancer.2.Loss of LSR promotes cell proliferation.LSR is a component of tricelluler tight junctions(t TJs)which regulates the organ size and contact inhibition,suggesting that LSR may also play a role in cell proliferation.To prove our hypothesis,we screened the endogenous LSR expressin in liver cancer cell lines and then we constructed stable LSR-knockdown Hep3 B and Huh7 cell lines(Hep3B-KD-LSR and Huh7-KD-LSR)and respective negative control cell lines(Control)by lentivirus-mediated sh RNA interference.We found that LSR knockdown inhibited liver cancer cell colony formation(P<0.01).In an immunocompromised nude mouse xenograft model,injection of liver cancer cell.Consistent with the in vitro results,LSR knockdown significantly induced xenografted tumor growth in these mice with control Hep3 B or Huh7 cell injection(P<0.01).LSR knockdown significantly induced the 3D sphere formation in the low attachment plate(P,0.01).Part 2 1.LSR regulates YAP activity and Hippo-YAP signaling plays a role in LSR tumor suppressor function.It is known that loss of LSR can induce m RNAs of AREG and TEAD1,cell migration,invasion and proliferation via Hippo/YAP pathway.To investigate how LSR influence the Hippo/YAP pathway,we tested the expression of key proreins of Hippo/YAP pathway in stable knockdown cell lines via Western blot.Western blot demonstrated that YAP and LATS2 were not changed whereas p-YAP was reduced dramatically and the YAP target gene Cyr61 and CTGF was induced.Consistent with these results,knockdown LSR also revealed increased nuclear and decreased cytoplasmic YAP staining.To further confirm this shift,Western blot was performed on both cytoplasmic and nuclear cell extracts from LSR-knockdown cell and control cells.Cytoplasmic expression of YAP decreased while nuclear expression of YAP increased.p-YAP expression was the opposite.Lamin A+C and tubulin confirmed differential extractionn of the nuclei and the cytoplasm respectively.To gain further mechanistic insight into LSR-mediated regulation of YAP,we constructed V5-LSR plasmid,then co-transfect the indicated plasmids with or without XMU-MP-1,and immunoprecipitated myc,blotted with indicated antibodies.As shown in Fig.2F,we found LSR interacted with YAP and the interaction could be reduced by the enzymatic activity of MST2 and LATS2,the effect also could be reverted by the inhibitor of MST2 and LATS2. 2.LSR interacts with YAP.Domain organization and key modifications of LSR.The above results imply that LSR perhaps regulates Hippo pathway in a manner dependent on YAP.Interestingly,we found one PPXY motif and one RPRARS motif.The PPXY motif is conserved in LSR across species.To confirm this conclusion,we generated LSR tyrosine-623 A single mutant plasmid.Dramatically,we found YAP exhibited no bingding to LSR tyrosine-623 A.By immunofluorescence staining,we indeed observed colocalization of ectopically expressed wile-type LSR with YAP but LSR tyrosine 623 A did not.These data demonstrate that PPXY is the key component by which LSR interacts with YAP.To substantiate the physiological function of LSR,we next conduct overexpression of LSR lentivirus and LSR-tyrosine-623 A lentivirus,then we generated cells stably overexpression LSR-WT and LSR tyrosine 623 A cell lines respectly using a relatively low expression cell line(SNU449),as shown in(Fig.4D),overexpression of LSR-WT could induce p-YAP and reduce Cyr61,CTGF,but did not affect YAP expression,while overexpression of LSR tyrosine-623 A reverted that.Collectively,these data support that LSR induce YAP phosphorylation(serine 127)and the PPXY motif plays a key role.3.14-3-3 contributes to LSR tumor suppressor function.To examin the role of 14-3-3s,we took the LSR serine 493 A mutant plasmid which did not have an interaction with 14-3-3 into coimmunoprecipitation,found the interaction between LSR and YAP was weaken than the wild type.Next,we examined the effect of overexpression of LSR serine 493 A.As expected,the overexpression of LSR serine 493 A in SNU449 can also induce p-YAP expression,reduce Cyr61 and CTGF expression,but have little influece on YAP.The results that were further confirmed by immunofluorescence staining in SNU449 cells.In control cells,YAP was mainly located in the nucleus.When it come to overexpression of LSR,YAP was mainly located in the cytoplasm.When we overexpression of LSR tyrosing 623 A,YAP was still mainly located in the nucleus.Consistently,overexpression of LSR serine 493 A,YAP was partially in cytoplasm.Altogether,these data suggested that 14-3-3 is involed in LSR regulating Hippo pathway via YAP as a part of Hippo pathway.IV.Conclusions 1、 LSR was more abundant in the normal liver than in tumor tissues and high expression of LSR reduced the risk of death by 11%.2、 loss of LSR promotes liver cancer cells proliferation and sphere formation.3、 LSR acts as tumor suppressor or function through YAP or YAP play a role in LSR-mediated tumor suppression.4、 Interaction with YAP is central for LSR-mediated liver cancer cells function.5、 There is a impact of MST1/LATS1 on LSR-mediated liver cancer cells function.6、 14-3-3s play an important role in LSR/YAP interaction.