Metastatic Potential Of Liver Cancer Originates From Primary Tumor

Primary liver cancer(PLC) is one of the most common and fatal carcinomas in the world.It takes the third place of cancer-related death globally and the second in China (more than half of the cases are in China).Though some of the patients have survived for a long time through early diagnosis and comprehensive therapy,however,metastasis and recurrence has become the major obstacle for further improvement of prognosis.The 5-year recurrent rate was as high as 60%after curative resection.At this moment, prediction and intervention of metastatic recurrence are still not available.Therefore it is urgently needed to investgate the molecular basis underlying tumor metastasis.Although some histopathological methods such as TMN stages,Edmondson's grades make important guidelines for the clinical therapy of liver cancer,it's hard to predict the metastatic potential of the tumors.High-throughput molecular biological techniques,such as microarray technology,provide the possibility to screen molecular alterations associated with tumor carcinogenesis and progression in a genome-wide scale. It has been widely used in molecular classification,metastatic prediction and response to treatment in multiple tumor types.In our former study using cDNA microarray containing 9180 genes,we analyzed the expression profiles of 40 HCC samples without or with metastases and that of their intrahepatic metastases in a genome-wide scale.Unexpectedly,we found that the gene expression profiles of metastatic lesions was very similar to that of their primary tumors (p＞0.05).However,the gene expression profiles of primary HCC with metastasis was significantly different from that of primary HCC without metastasis(153 genes with significance,p＜0.001),regardless of tumor size,encapsulation and age of patient.On these grounds,we proposed for the first time the new hypothesis that genes favoring HCC metastasis progression are initiated relatively early in the primary tumors(Ye QH, et al.Nat Med 2003),which challenging the traditional metastatic theories of metastasis being a nonrandom and highly selective process and arising in late stages(Fidler,et al. Nat Rev Cancer 2003).In order to further validate the hypothesis that genes favoring HCC metastasis progression are initiated in rimary tumors,we analyze the gene expression profiles of pure tumor cells from PLC without and with extrahepatic metastases and that of their metastases using laser capture microdissection(LCM) and gene chips in this study.At the same time,we will try to uncover some genes which may play important roles in PLC metastatic progression and may become the molecular targets for early prediction and intervention of tumor metastasis.PART ONE Exploring the Metastatic Origination of Primary Liver Cancer with LCM, Two-cycle RNA Amplification and Expression ProfilesPurpose:To explore the metastatic origination of primary liver cancer by comparing the gene expression profiles of paired PLC tumors(primary and extrahepatic metastases),metastasis-free small liver cancer,paired gastrointestinal tumor with liver metastases and normal liver tissues.Method:A total of eleven paired PLC tumors with their extrahepatic metastases (including 5 lymph node metastases,3 adrenal metastases and 3 lung metastases), ten metachronous lung metastases from PLC,ten small liver cancers without metastasis,nine paired gastrointestinal tumors and their liver metastases and five normal liver tissues were enrolled in this sutdy.LCM was performed to enrich pure tumor cells and hepatocytes.Total RNA was extracted using Picopure RNA kit from Arcturus.RNA qulity and quantity were checked by Agilent 2100 Bioanalyzer.Affymetrix two-cycle amplification method was used to get more than 10μg cRNA for Affymetrix Hu133 2.0 Plus Chip examination.The expression level of candidate metastatic genes was validated by qRT-PCR.Results:BRBArray and Mas 5 softwares were employed to analyze the gene expression profiles associated with PLC metastasis.No difference was found between the gene expression profiles of PLC and their matched extrahepatic metastasis. However,significant difference was found between the gene expression profiles of PLC with and without metastasis.There were 379 genes with significance between NM group(Non-metastasis) and LM group(Lung metastasis),1085 genes between NM and LN group(Lymph node Metastasis) and 1603 genes between NM and GIM group(Gastrointestinal Metastasis).12 genes were overlapped among these three significant gene groups,.PGK1 was found to be highly elevated in LM group comparing with NM.qRT-PCR examination validated the high expression level in LM group(p＜0.0002),indicating that it may relate to the lung metastasis process of PLC.Conclusion:The gene expression profiles of primary liver cancers are similar to that of their paired extrahepatic meatstases but significantly different from that of primary tumors without metastasis.This further validate the hypothesis we speculated in our former study that genes favoring HCC metastasis progression are initiated in rimary tumors.And PGK1 may be a potential molecule both as predicting tumor marker and potential therapeutic target for lung metastasis of primary liver cancer.PART TWO Exploring the Metastatic Origination of Primary Liver Cancer with LCM and SNP Array techniquesPurpose:To further explore the metastatic origination of primary liver cancer by comparing the SNP expression profiles of paired PLC tumors(primary and extrahepatic metastases) using LCM and SNP array techniques.Methods:A total of eleven paired PLC tumors with their extrahepatic metastases (including 5 lymph node metastases,3 adrenal metastases and 3 lung metastases) and nine paired gastrointestinal tumors and their liver metastases were enrolled in this sutdy.LCM was performed to enrich pure tumor cells.DNA was extracted for SNP expression profiles examination usingAffymetrix SNP 6.0 arrays.Reslults:Genotyping Console 2.0 and Nexus 3.0 softwares were employed to analyze the SNP expression profiles.The DNA copy number variants were similarly between primary tumors and the paired metastases. Conclusion:The hypothesis of genes favoring HCC metastasis progression are initiated in rimary tumors was further validated by LCM and SNP arrays.