Hypoxia-Induced YAP Activation Promotes Chemoresistance In HCC Cells

Objectives:As a critical stage during the progression of hepatocellular carcinoma (HCC), the hypoxic microenvironment (Hypoxia) could induce reduced susceptibility to chemotherapy drugs, escape from apoptosis and invasion and metastasis in HCC cells. However, the molecular mechanisms and signaling pathways that involved in hypoxia-mediated malignant process of HCC remain elusive. Thus it is crucial to explore the mechanisms underlying hypoxia promoted HCC malignancy and identify novel targets for intervention of hypoxic HCC. Mounting evidences revealed that the carcinogenic factor Yes-associated protein (YAP) plays a key role in HCC development. Our preliminary study found that YAP could be activated by hypoxic microenvironment. Therefore, this study aims to investigate how hypoxia induce YAP, and further explore the mechanisms underlying the activation of YAP mediated by hypoxia, and explore the small-molecule inhibitors capable of interrupting hypoxia-induced activation of YAP, and ultimately, sensitizing hypoxic HCC cells to chemotherapeutic drugs. These findings suggest a novel approach for overcoming hypoxia resistance, whereby targeting YAP activation can resulted in increase apoptotic cell death and proliferation inhibition, thus provide experimental evidences and theoretical basis for novel strategies to counteract hypoxia related HCC malignancy.Methods:Human HCC cells HepG2, SMMC-7721, Bel-7402 and HepG2 xenografted nude mice model were employed to validate that tumor hypoxia could induce YAP nuclear translocalization and increase YAP-mediated transcriptional activity in HCC cells, and investigate YAP protein stability maintained by hypoxia. (1) Confluent-plated (25?30 × 104 cells per 1 mL culture medium, after 36 h, cells grow to confluence) HCC cells (HepG2, SMMC-7721 and Bel-7402) were cultured under normoxia (20% O2) or hypoxia (1% O2) for 24 hours, the subcellular localization of YAP was observed by immunofluorescence in HCC cells. (2) After cultured with above-mentioned conditions, human HCC cell HepG2 were collected for cell fractionation experiment and Western Blot to detect nuclear YAP in HCC cell. (3) With small interfering RNA (siRNA) silencing YAP and Real-time PCR, the mRNA level of CTGF and AREG (the target genes of YAP) under normoxia and hypoxia were determined. (4) Using HepG2 xenografted nude mice, hypoxyprobe PIMO staining, Cryostat sections technique and immunofluorescence experiments, how intratumoral hypoxia influence YAP protein expression and subcellular localization was observed. (5) Cultured time-dependent YAP protein expression under normoxia and hypoxia were analyzed by Western Blot. (6) The phosphorylation level of YAP (Ser127) under normoxia and hypoxia was also detected by Western Blot. (7) Blocking protein synthesis with the inhibitor cycloheximide (CHX), the protein degradation rate of YAP under normoxia and hypoxia were determined with Western Blot.Human HCC HepG2, SMMC-7721, Bel-7402 cell lines were used to investigate the effect of HIF-1? on hypoxia-induced YAP activation, further explore the regulatory mechanisms and explore small molecular inhibitors to suppress hypoxia-induced YAP activation. (1) After treated with HIF-1 accumulation agent cobalt chloride (CoCl2), the subcellular localization of YAP in the HCC cells (HepG2, SMMC-7721 and Bel-7402) was observed by immunofluorescence experiment. (2) Total and phosphorylated YAP protein levels were detected by Western Blot when HIF-1 was accumulated by CoCl2. (3) Using HepG2 transfected with HIF-la plasmid to overexpress HIF-la, total YAP protein was detected by Western Blot. (4) With siRNA silencing HIF-1? under hypoxia, YAP subcellular localization was determined by immunofluorescence staining for endogenous YAP and total YAP protein was detected by Western Blot. (5) Blocking YAP protein ubiquitin-dependent degradation with the proteasome inhibitor MG132, the ubiquitination of YAP under normoxia, hypoxia and CoCl2 treatment were determined by experiments combined with co-immunoprecipitation and Western Blot. (6) Stimulated with normoxia, hypoxia and CoCl2 treatment, the total protein and phosphorylation level of MST kinase and LATS1 kinase (the upstream kinases of YAP) were determined by Western Blot. (7) The mRNA and protein levels of HMGCR, upstream signal of YAP were determined by Real-time PCR and Western Blot respectively. (8) Using siRNA silencing HMGCR and mevalonic acid (MVA) under hypoxia, YAP subcellular localization was determined by immunofluorescence and total YAP protein was detected by Western Blot. (9) Treated with HMGCR inhibitor pravastatin and atorvastatin with/without MVA under hypoxia, YAP subcellular localization was determined by immunofluorescence and total YAP protein was detected by Western Blot. (10) Using HepG2 treated with atorvastatin under hypoxia, cell fractionation experiment and Western Blot were used to detect nuclear YAP in HCC cell. (11) Treated with pravastatin and atorvastatin with/without MVA under hypoxia, the mRNA levels of CTGF and AREG were detected by Real-time PCR. (12) Treated with pravastatin and atorvastatin under hypoxia, the total protein and phosphorylation level of LATS1 kinase were determined by Western Blot.The interruption of hypoxia-activated YAP with either siRNA or statins could increased the susceptibility of HCC cells to chemotherapeutic drugs under hypoxia through apoptotic cell death. (1) Using siRNA silencing YAP under hypoxia, meanwhile treated with different concentrations of sorafenib or SN38 (active metabolite of irinotecan), the proliferation inhibition rate of HepG2, SMMC-7721 and Bel-7402 was measured by SRB staining and IC50 values were calculated. (2) Using siRNA silencing YAP under hypoxia, meanwhile treated with different concentrations of doxorubicin, cisplatin or 4-HPR, the proliferation inhibition rate of HepG2 was measured by SRB staining and IC50 values were calculated. (3) Using siRNA silencing YAP under hypoxia, meanwhile treated with sorafenib or SN38, the apoptosis rate of HepG2 cells was determined by FACS following PI staining. (4) Using siRNA silencing YAP under hypoxia, meanwhile treated with sorafenib or SN38, cell apoptotic marker PARP cleavage was analyzed by Western Blot. (5) Treated with combination with atorvastatin/pravastatin and sorafenib or SN38 under hypoxia respectively, the proliferation inhibition rate of HepG2 was measured by SRB staining and IC50 values were calculated.Results:1) Tumor hypoxia could induce nuclear translocation and activation of YAP, and maintain YAP protein stability through reducing the degradation of YAP.Immunofluorescence experiments showed that compared to normoxia control, hypoxia could significantly induce nuclear translocation of endogenous YAP in HCC cells. Cell fractionation analyses showed that YAP predominantly localized in the nucleus when HCC cell HepG2 was cultured under hypoxia. Importantly, Immunofluorescence staining with anti-PIMO and anti-YAP antibodies in tissue sections of HepG2 xenograft showed that YAP overexpressed and located in nucleus under intratumoral hypoxia in vivo. The relative mRNA levels of YAP target genes CTGF and AREG were significantly up-regulated by hypoxia, as indicated by the results achieved from Real-time PCR analyses. With the siRNA silencing of YAP under hypoxia, the enhanced mRNA levels of CTGF and AREG would be attenuated. Further study showed that the protein levels of YAP decreased gradually in a time-dependent manner under normoxia, while YAP remained relatively stable under hypoxia, even with the co-incubation of a protein synthesis inhibitor, cycloheximide (CHX). In addition, the phosphorylation of YAP at serine 127 which lead to the proteasomal degradation of the total protein decreased under hypoxia.2) Hypoxia-induced YAP nuclear translocalization and stability maintaining in HCC cells was not mediated by HIF-1?; instead, the HMGCR/mevalonate pathway was involved, and the HMGCR inhibitor statins could prevent the nuclear translocation of YAP and subsequent transactivation of YAP target genes.Although HIF-la was accumulated by CoCl2 treatment under normoxia, little impact on the subcellular localization of YAP was observed using immunofluorescence analyses, which was different from that under hypoxia. Further studies using Western Blot analyses revealed that HIF-la accumulated by CoCl2 under normoxia failed to accumulate comparable total protein level of YAP as that increased under hypoxia, despite the reduction of phosphorylated YAP. Similar observations were also achieved from those HepG2 cells with the exogenous HIF-la overexpression under normoxia. In consistent with these results, siRNA of HIF-la failed to attenuate hypoxia-caused reduction of YAP phosphorylation, increment of YAP protein and nuclear translocation of YAP. Using MG-132 to prevent the degradation of ubiquitinated YAP, co-immunoprecipitation assay combined with Western Blot showed that compared with normoxia control, YAP ubiquitination was decreased under hypoxia, but enhanced with exposure to CoCl2 treatment. Further studies showed that phosphorylation of LATS1 kinase drastically reduced under hypoxia while remained high level under normoxia and CoCl2 treatment. The Real-time PCR results showed that the mRNA level of HMGCR (an upstream regulator of YAP) was significantly up-regulated by hypoxia. When HMGCR was suppressed by siRNA or its inhibitors atorvastatin and pravastatin under hypoxia, the hypoxia-mediated YAP activation and stabilization were repressed, and the prohibition could be rescued by mevalonic acid (MVA).3) YAP silence or Statins could sensitize the HCC cells to anti-cancer agents by inducing apoptosis under hypoxia.Compared to HCC cell lines under normoxia, the hypoxic cells were more resistant to anti-cancer agents including sorafenib, SN38, doxorubicin, cisplatin and 4-HPR, as indicated by decreased inhibition ratio under hypoxia, determined by SRB assays. The hypoxic resistance to chemotherapeutic agents could be attenuated by YAP silence or Statins. FACS analyses following PI staining showed that compared with normoxia, treatment with sorafenib or SN38, the apoptosis ratio of HepG2 cells decreased under hypoxia, while silencing YAP with siRNA under hypoxia reinforce the apoptotic cell death. Western Blot results showed that PARP was cleaved significantly by sorafenib or SN38 under normoxia, whereas left almost intact under hypoxia. The resistance to PARP cleavage under hypoxia in sorafenib or SN38-treated cells could be abolished by siRNA of YAP.Conclusions:The present study reveals that hypoxia induced nuclear translocation and activation of YAP in HCC cells. Mechanistically, HMGCR/mevalonate-LATS1 kinase axis-mediated reduced phosphorylation of YAP at serine 127 and attenuated ubiquitin-proteasome degradation resulted in the nuclear translocation and increased total protein level of YAP in hypoxic HCC cells. As inhibitors of HMGCR, statins suppressed hypoxia-activated YAP and reduced total YAP protein under hypoxia. In addition, inteference of YAP with either siRNA or HMGCR inhibitor statins increased the susceptibility of HCC cells to chemotherapeutic agents under hypoxia by apoptosis induction. Our findings suggests that YAP could be regarded as a promising therapeutic target for hypoxic HCC, thus provides new ideas and theoretical basis for the development of novel therapeutic strategies for HCC treatment.