Alteration Of Invasiveness And Metastatic Potential Of Hepatocellular Carcinoma After Radiation, Its Molecular Basis And Intervention-an Experimental Study

Hepatocellular carcinoma(HCC) is the third most common cause of death from cancer in the world and the second cancer killer in China.Surgical resection remains the most effective treatment for HCC.Unfortunately,less than 20%of patients with HCC are candidates for resection at the time of diagnosis because of multinodular pattern,vascular invasion and liver cirrhosis.Radiotherapy has been used to treat HCC for half century.Recently,with the advances of biology and technology,such as 3-dimensional conformal radiotherapy(3D-CRT),radiotherapy has become more important for the treatment of HCC.However,it is reported that even after radiotherapy with curative dosage,residual cancer can be detected in more than 60%of patients.We did observe that even tumor was "eradicated" after radiotherapy,but several months later some of the tumors relapsed or metastasized quickly with poor prognosis.Questions raise based on this observation:does radiotherapy promote the invasiveness and metastatic potential of residual cancer? What is the molecular background in this process? What can be done for intervention to improve the outcome of radiotherapy? Our present study was designed to clarify these issues by treating nude mice bearing human HCC orthotopic xenografts with fractionated radiotherapy.After treatment the invasiveness and metastatic potential of residual HCC was evaluated and possible molecular background was investigated.We found that radiation indeed promotes the invasion and metastasis of residual cancer through upregulation of TMPRSS4,which can induce epithelial-mesenchymal transition(EMT),a major determinant of metastasis. Administration of TMPRSS4 inhibitor AEBSF or combined use of IFN-αand aspirin can inhibit the invasion and metastasis caused by radiation,and may be a novel approach to improve outcome of radiotherapy for HCC.1.Radiation increased the invasiveness of MHCC97L cells in vitro through upregulation of MMP-2 and VEGF Alteration in the invasiveness of MHCC97L cells after radiation was examined using the Matrigel invasion assay.The invasiveness of MHCC97L cells were significantly increased after radiation at a time and dose-dependent manner,and reached the maximum at 48h after 8 Gy radiation.Forty eight hours after radiation at different dose(0,4,8Gy),the number of cells that passed through the transwell inserts was 7.2±1.9,13.2±2.7(P＜0.05) and 31.2±7.2(P＜0.005) respectively,while there was no significant difference 96 hours after radiation.This increase in invasiveness was associated with upregulation of expression or activity of MMP-2 and VEGF.The levels of protein for MMP-2 and VEGF were determined by Western blot analysis and showed a significant increase after exposure to 8 Gy for 48 hours.In order to see if the increase in MMP-2 and VEGF secretion correlates with changes in the expression of the corresponding genes,we tested their mRNA levels by quantitative RT-PCR.There was a dose-dependent increase in MMP-2 and VEGF mRNA expression and the maximal level was observed at dose of 8 Gy;MMP-2 activity was examined by zymographic analysis and was increased in a time and dose dependent manner,attaining a maximum at 48 hours after 8 Gy radiation.2.Aspirin inhibited the activity/expression of MMP-2 and VEGF and thereby suppressed radiation augmented invasiveness of MHCC97L cells.The invasiveness of MHCC97L cell was significantly enhanced after radiation at doses of 4 Gy and 8 Gy.However,when aspirin(2mM) was added before radiation, the invasion rate dropped near to the level of unirradiated cells.Furthermore,aspirin significantly inhibited the radiation induced increase in VEGF and MMP-2 secretion, as well as the MMP-2 activity.Taken together,these findings suggest that the concomitant use of aspirin during radiation could inhibit the radiation enhanced invasion through suppressing the expression or activity of VEGF and MMP-2.3.The invasion and metastasis of residual HCC after radiotherapy in vivoIn order to further investigate the invasion and metastasis potential of residual HCC in vivo,metastatic animal model of human HCC was established by orthotopic implantation of histologically intact MHCC97L HCC tissue into the liver of nude (nu/nu) mice.Four weeks after implanation,the tumors were irradiated at a dose of 2Gy/d,from Monday to Friday for two weeks,and the total radiation dose was 20Gy. The mice were sacrificed 2 or 30 days afer radiation and the residual cancer were replanted into the liver of other nude mice.These mice were sacrificed 6 weeks after replantation.The replanted tumor volume of 2 days- postradiotherapy group and its control group are 1.61±0.51 and 2.25±0.52cm~3(p＜0.05),and the incidence of lung metastasis was 12.5%and 66.7%(P＜0.05);while tumor volume of 30 dayspostradiotherapy group and its control group are 2.60±0.61 and 2.15±0.71cm~3(p＞0.05),and the incidence of lung metastasis was 100%and 63.6%(P＜0.05), respectively.Significant tumor inhibition was only found between control and the 2 days-postradiotherapy group,but not between control and 30 days-postradiotherapy group.The incidence of lung metastasis and inhepatic dissemination were markedly increased in the 30 days-postradiotherapy group.These results suggested that 30 days after radiation the invasiveness and metastatic potential of residual HCC were significantly enhanced.4.Changes in the invasion and metastasis related gene expression profile of residual HCC in nude mice after radiotherapy.In order to investigate the mechanism involved in this process,invasion and metastasis related cDNA microarray was used to analyse changes of gene expression profile after radiotherapy.The results showed that KISS1 and TIMP2,two factors that inbibit invasion and metastasis,were down-regulated in residual cancer 2 days after radiotherapy,while TMPRSS4,which can promote invasion and metastasis,was significantly up-regulated in residual cancer 30 days after radiotherapy.Furthermore, BAI1 was down-regulated in residual cancer 30 days after radiotherapy.5.Validation of cDNA microarray results by quantitative RT-PCR and westen blotIn order to verify the above-mentioned changes in KISS1,TIMP2,TMPRSS4 and BAIl from the cDNA microarray analyses,we tested the effect of radiation on their mRNA levels by quantitative RT-PCR.Compared with control group,the KISS 1(P＜0.01) and TIMP2(P＜0.05) mRNA expression in residual cancer 2 days postradiotherapy were significantly increased.The TMPRSS4 mRNA expression of residual cancer 30 days postradiotherapy was much higher than that of control group and residual cancer 2 days after radiotherapy(P＜0.01),but there was no significant difference in BAI mRNA expression(P＞0.05) between these groups.We further analysed the protein level of TMPRSS4 in these 3 groups and western blot result showed that TMPRSS4 protein was also significantly enhanced in residual cancer 30 days postradiotherapy.These findings indicate that TMPRSS4 is a key oncogene that related to radiation induced invasiveness and metastasis.6.Western blot analysis of EMT related molecules and COX-2According to literature,up-regulation of TMPRSS4 can inhibit the expression of E-cadherin and facilitate EMT,we further detected epithelial marker of E-cadherin and mesenchymal marker of Vimentain and N-cadherin.SIP1/ZEB2,an E-cadherin transcriptional repressor,was also detected by western blot.The results showed that expression of SIP1,Vimentain and N-cadherin was significantly increased while expression of E-cadherin was inhibited.Administration of TMPRSS4 inhibitor AEBSF inhibited the expression of TMPRSS4 and SIP1 and up-regulated the expression of E-cadherin,together with reduced invasion and metastasis in vivo after radiation.It is concluded that radiation promoted the invasiveness and metastasis through TMPRSS4 induced EMT.We also analysed some molecules that were not included in the cDNA microarray by western blot,and found that COX-2 expression was significantly increased in the residual cancer 30 days postradiotherapy.Since there is an inverse relationship between COX-2 and E-cadherin expression,enhanced expression of COX-2 also contributes to EMT.7.Combined administration of IFN-αand aspirin or pseudomonas aeruginosa vaccine(PA) inhibits the growth and metastasis of HCC in vivoWe previously reported that High-dose(1.5×10~7 U/kg·d) and long-term therapy with IFN-αinhibits tumor growth and recurrence in nude mice bearing human HCC xenografts.However,in clinical practice some patients can not stand high-dose therapy with IFN-αbecause of the side effects.Since COX-2 expression is increased after radiation,we choose combined administration of low-dose IFN-α(7.5×10~6 U/kg·d) and aspirin(15mg/kg·d) to inhibit the growth and metastasis of HCC in vivo, which was as effective as high-dose IFN-α(1.5×10~7 U/kg·d).Furthermore, administration of PA intraperitoneally also significantly inhibit the growth and metastasis of HCC through induction of apoptosis.Thereby we choose combined administration of low-dose IFN-αand aspirin or PA to inhibit the radiation induced invasion and metastasis. 8.Combined administration of low-dose IFN-αand aspirin or PA inhibits the dissemination and metastasis of residual cancerThirty days afer radiation,the residual cancer were resected and replanted into the liver of other nude mice.The mice were divided into control group,low-dose IFN-αplus aspirin treatment group and PA treatment group.Six weeks after that,the mice were sacrificed.Low-dose IFN-αplus aspirin or PA treatment significantly decreased the dissemination in liver and completely inhibited the lung metastasis.These findings may provide novel approach to enhance the efficacy of radiotherapy for HCC.Conclusions1.The invasion and metastasis of HCC in vivo was inhibited in early phase and then enhanced in the late phase after radiation.2.Radiation enhanced the invasion and metastasis of residual cancer through upregulation of TMPRSS4,which inhibited the expression of E-cadherin and induced EMT.3.Combined administration of low-dose IFN-αand aspirin or TMPRSS4 inhibitor AEBSF inhibited the dissemination and metastasis of residual cancer through suppression of COX-2 and TMPRSS4 and upregulation of E-cadherin.PA can inhibit the dissemination and metastasis through inducing the apoptosis of HCC.4.Combined administration of low-dose IFN-αand Aspirin could be a potential therapeutic approach to inhibit radiation induced invasion and metastasis and improve the efficacy of radiotherapy for HCC.The potential application of this work1.Clarifying the molecular basis of radiation induced invasion and metastasis of residual HCC helps to instruct the combined therapy postradiotherapy.2.Combined administration of low-dose IFN-αand Aspirin significantly inhibited the dissemination and metastasis of residual HCC postradiotherapy,thereby provides a new clue to improve the outcome of radiotherapy.3.Combined administration of low-dose IFN-αand Aspirin significantly inhibited the growth and metastasis of HCC in vivo,thus provides a new approach for treatment of HCC.Originalities of this work 1.For the first time to clarify the alteration and molecular basis of invasion and metastasis potential of residual HCC after radiation in vivo,and point out that upregulation of TMPRSS4 and induced EMT might play a major role.2.For the first time to point out that combined administration of low-dose IFN-αand aspirin significantly inhibited the growth and metastasis of HCC in vivo, and also suppressed the dissemination and metastasis of residual cancer postradiotherapy.