| Background: The latest global cancer report shows that liver cancer is predicted to be the sixth most common cancer and the fourth leading cause of cancer-related deaths,with about 841,000 new cases and 782,000 deaths in each year.The incidence and mortality of liver cancer in men is two to three times higher than in women,making liver cancer ranks fifth in terms of global cases and second in terms of deaths for males.Hepatocellular carcinoma(HCC)accounts for 75% ～ 85% of primary liver cancer and is one of the most common malignant tumors in China.At present,there are many treatments for hepatocellular carcinoma,among which liver transplantation and hepatectomy are the most effective radical treatment.Despite the improvement of resection techniques in recent years,the five-year survival rate of HCC patients after liver resection can only reach about 30%.Tumor recurrence and metastasis after liver resection are the main causes of treatment failure and death of HCC patients.Therefore,control of recurrence and metastasis is critical to further improve the survival of HCC patients.Cerebral cavernous malformation(CCM)are vascular lesions characterized by abnormally dilated capillaries in the brain,which can result in cerebral haemorrhages,focal neurological defects and seizures.Mutations of three protein-encoding genes(KRIT1/CCM1,CCM2/MGC4607 and PDCD10/CCM3)are linked with CCM progression.The three CCM proteins can interact with a collection of signaling,cytoskeletal and adaptor proteins and play a vital role in a range of basic cellular processes including cell apoptosis,proliferation,migration and polarity.Although these three genes have been extensively studied in CCM disease,their role in tumors is still not very clear.Some studies have shown KRIT1 and CCM2 are downregulated in melanoma and neuroblastoma respectively and act as tumor suppressor to inhibit tumor proliferation and progression.However,PDCD10 can lead to more severe form of CCM disease and have more complicated biological functions.Many researches have revealed that PDCD10 participates in cell apoptosis,proliferation,migration and embryonic angiogenesis,which are all critical factors associated with tumor progression.Purpose: The expression of CCM1,CCM2 and PDCD10 in HCC is preliminarily analyzed.At the same time,the role and molecular mechanism of PDCD10 in the invasion and metastasis of HCC are further explored.Besides,potential intervention measures are also investigated.Method: In this study,the expression of CCM1,CCM2 and PDCD10 in HCC tumor tissues and normal liver tissues will be analyzed in multiple databases(such as TCGA and GEO),and the relationship between the expression of these three genes and the prognosis of HCC patients in TCGA will also be analyzed.By analyzing data from public databases,we found that PDCD10 is the only one that is significantly upregulated in HCC tissues compared with normal liver tissues in all databases,and HCC patients with high PDCD10 expression had a significantly worse prognosis than those with low PDCD10 expression.Therefore,we focused on the role of PDCD10 in the follow-up experiments.We randomly selected 30 pairs HCC tissues and corresponding adjacent non-tumorous liver tissues from our own specimen biobank and verified the expression of PDCD10 by q RTPCR,western blotting and immunohistochemical staining.To further explore the clinical significance of PDCD10 expression in HCC,we constructed a training cohort and a validation cohort.According to scores of immunohistochemical staining,patients in the two cohorts were divided into PDCD10 high expression group and PDCD10 low expression group,which were further used to compare the relationship between PDCD10 expression and clinicopathological features and prognosis of HCC patients.The univariate and multivariate analysis were used to determine whether PDCD10 is an independent risk factor for prognosis of HCC patients.To explore the effects of PDCD10 on the biological behavior of HCC cells,we selected appropriate HCC cell lines to knock down and overexpress PDCD10 and observed the effects of PDCD10 intervention on the proliferation,migration,invasion,growth and metastasis of HCC cells in vitro and in vivo.We will further investigate whether PDCD10 knockdown or overexpression affects EMT(epithelial-mesenchymal transformation,EMT)of HCC cells.In terms of mechanism,we first analyzed multiple databases to find the signaling pathways regulated by PDCD10 and verified the results by a series of experiments.Finally,this study will also determine how PDCD10 regulates the corresponding signaling pathway and search potential intervention measures,which will lay a theoretical foundation for the treatment of HCC.Result: PDCD10 m RNA and protein expression were significantly upregulated in HCC tissues compared with adjacent non-tumorous liver tissues.Interestingly,HCC with microvascular invasion(MVI)had significantly higher PDCD10 expression than those without MVI.Nodular HCC(NHCC,a type of HCC with the worst prognosis)had significantly higher PDCD10 expression than small HCC(SHCC)and solitary large HCC(SLHCC,a specific type of large HCC with relatively good prognosis),while there was no significant difference between SLHCC and SHCC.Meanwhile,the m RNA and protein expression of PDCD10 in HCC cell lines with highly invasive capacity(such as Hep3 B,HCCLM3 and MHCC-97H)were significantly higher than that in immortalized normal liver cell line L02.Immunohistochemical staining also confirmed that HCC tissues with MVI had the highest expression of PDCD10,followed by HCC tissues without MVI,and adjacent non-tumorous liver tissues had the lowest expression of PDCD10.These results demonstrated that PDCD10 expression is upregulated in HCC and may be associated with invasion and metastasis of HCC.To explore the clinical significance of PDCD10,we followed the relevant requirements of REMARK guidelines and randomly selected 160 HCC patients who undergo liver resection in Xiangya Hospital of Central South University and meet the inclusion criteria to establish a training cohort.At the same time,140 HCC patients who meet the inclusion criteria in the Affiliated Cancer Hospital of Xiangya School of Medicine,Central South University were randomly selected as a validation cohort.Statistical analysis combing with clinical information showed that PDCD10 expression was associated with adverse clinicopathological characteristics of HCC patients in both cohorts.Furthermore,Kaplan-Meier analysis using postoperative follow-up data found that overall survival and diseasefree survival of HCC patients with high PDCD10 expression were significantly worse than those with low PDCD10 expression in both the training and validation cohort,which was consistent with the results in public database.Finally,univariate and multivariate analysis by Cox proportional risk regression model showed that high PDCD10 expression was an independent prognostic risk factor for overall and disease-free survival of HCC patients in both cohorts.We can conclude that PDCD10 could be a new biomarker to determine prognosis of HCC patients.Studies have shown that PDCD10 could regulate proliferation,apoptosis and participate in cell migration.To explore the role of PDCD10 on biological behavior of HCC cells,we selected Hep3 B with relatively high PDCD10 expression and Hep G2 with relatively low PDCD10 expression for subsequent experiments.After knocking down of PDCD10 in Hep3B by lentivirus,the proliferative,migratory and invasive capacity of Hep3 B were significantly reduced,while PDCD10 overexpression in Hep G2 cells could significantly promote its proliferation,migration and invasion.Then,we constructed subcutaneous tumor model and orthotopic tumor model in nude mice and monitored the growth of orthotopic tumors using in vivo imaging system.The results showed that PDCD10 knockdown inhibited not only tumor growth,but also intrahepatic and pulmonary metastasis,while PDCD10 overexpression could significantly promote tumor growth and metastasis.Intriguingly,PDCD10 overexpression led to a fusion-like mesenchymal phenotype,and the expression of epithelial marker E-cadherin is significantly decreased,while the expression of mesenchymal marker Vimentin is significantly increased,which indicates the occurrence of EMT in HCC cells.Knocking down of PDCD10 could lead to an opposite result.Therefore,PDCD10 promotes HCC growth,metastasis and EMT in vitro and in vivo.Analysis of STRING protein interaction database and TCGA database found that PDCD10 is closely related to Hippo signaling pathway.Subsequently,a series of experiments confirmed that PDCD10 overexpression lead to dephosphorylation of YAP,a key protein of Hippo signaling pathway.Dephosphorylated YAP translocated to nuclei and enhanced the transcriptional activity of TEAD transcription factors by playing a role of transcriptional regulation,thus promoting the expression of downstream effector genes related to tumor proliferation,migration,invasion and EMT.Low PDCD10 expression resulted in high phosphorylation status of YAP and thus proteasome degradation.Further analysis showed that PDCD10 was closely related to protein phosphatase2A(PP2A)complex.PDCD10 directly bound the core catalytic subunit PP2 Ac,improved its enzyme activity and promoted the binding of PP2 Ac to YAP protein,resulting in the dephosphorylation and activation of YAP.In HCC cells with high PDCD10 expression,PP2 Ac knockdown could inhibit their increased migratory and invasive ability and reverse the EMT process.However,PP2 Ac overexpression in HCC cells with low PDCD10 expression could lead to similar phenotypes of high PDCD10 expression.Interestingly,we found that LB100,an inhibitor of PP2 Ac,could significantly reduce the activity of PP2 Ac in HCC cells with high PDCD10 expression,and inhibit growth and pulmonary metastasis of HCC cells with high PDCD10 expression in vivo,but had no significant effect on HCC cells with low PDCD10 expression.These results revealed the molecular mechanism by which PDCD10 promotes HCC progression,and LB100 may be a potential therapeutic agent for the treatment of HCC with high PDCD10 expression.Conclusion: We found that PDCD10 is significantly upregulated in HCC.PDCD10 binds PP2 Ac to improve its enzyme activity and promote the dephosphorylation of YAP,and then activate the expression of downstream effector genes,ultimately leading to the progression of HCC.PDCD10 is a potential therapeutic target and important biomarker for predicting prognosis of HCC patients,which is of great importance to clinical application. |
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