AnalysisandApplicationofMetastasis-relatedGenestoDisclosethe
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AnalysisandApplicationofMetastasis-relatedGenestoDisclosetheKeyProteinsandPathwaysInvolvedintheMetastasisofColorectalCarcinoma
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Guan-Zhen Yu, Ying Chen, Ya-Qiu Long, Dong Dong, Jie-Jun Wang *
Department of Oncology, Changzheng Hospital , Shanghai , China
*Corresponding author: Jie-jun Wang
【 Abstract 】 Background&Aims Metastasis-associated genomic alteration has been recognized as playing a critical role in the tomour progression toward metastasis. Here, we analyzed changes of gene profiles between primary tumor cells and metastatic tumor cells using cDNA and tissue microarrays. Methods Primary tumor and nodal metastatic cells were implanted into subcutaneousness of nude mice. Then growth and metastatic potential was observed. Samples from tumor in mice were extracted for RNA, which was then used for 13824 human genes array. In addition, protein expression of selected genes was determined by Tissue microarray and immunohistochemistry. Function of Grb2 was determined by In vitro wound assay and Western blot. Results Nearly 400 genes showed different expression between primary tumor cells and metastatic cells. Statistics showed that Grb2 was extremely higher in the metastases and Grb2- mediated Ras pathway was evaluated during metastasis. Subsequent examination of selected genes by Real-time RT-PCR and immunohistochemistry substantiated the reliability of our analysis. Blocking Grb2-SH2 domain signaling transduction could inhibit cell motility by inactivating erbB2 protein. Conclusions Overexpression of Grb2 may contribute to tumor growth, invasiveness and metastasis. This functional profile makes Grb2 a high priority target for anti-cancer-metastasis drug development.
Introduction
Colorectal carcinoma is one of the most common cancers in the world. Despite advances in the treatment and early diagnosis, this disease remains a heavy threat to life for a large number of people, due to the metastasis and companied complications. Significant effects have been shown on the primary tumor because of the ongoing new chemotherapeutic agents and improved drug effectiveness; however, almost all the patients suffered from fatal metastasis in the end, which awaits new targets and strategies. To achieve this goal, a comprehensive understanding of the mechanisms of colorectal cancer progression toward metastasis will be essential.
At the molecular level, activation of oncogenes and inactivation of tumor suppressor genes contribute to the development of colorectal carcinoma, which has been under intensive study for many years. However, in the progression toward metastasis, two distinct classes of genes may be much more important-metastasis activator (or metastasis-promoting) and metastasis suppressor genes [1] . Nevertheless, how these genetic alterations bring about the progress to metastasis of colorectal carcinomas remains unclear. According to previous studies, accumulations of mutant genes during the moving of metastatic cells tend to be accompanied by other genetic and epigenetic changes including loss of heteozygosity [2] , aberrant promoter methylation [3] , and/or gene amplifications, all of which can alter gene expression profiles. Therefore, genome-wide monitoring of gene expression is of great importance if we are to disclose the numerous and diverse events associated with metastasis.
Essentially, identification of metastasis genes involves comparison of cells that differ in metastatic potential. Several approaches, e.g., comparison of chromosome additions/deletions/ translations, differential display, subtractive hybridization or recently developed 'gene chip', have been developed to identify genes that are expressed differently between cell lines and/or tissues of different metastatic potential. Recently, proteomic technologies provide a new sight to analyze expression profiles in various tissues. However, all these methods have limitations. Except 'gene chip', they are labor intensive, time consuming, requiring large amounts of mRNA/protein, and perhaps undeveloped. cDNA microarray, a fully developed technique, provides high-throughput analysis of expression profiles by means of small-array slides spotted with cDNA although large amount of data sometimes can be obtained and a number of false positive data can be observed. Here, we used real-time RT-PCR and tissue microarray (TMA) to confirm parts of our results and go analysis to exclude false positives and select useful data.
Previous studies are usually based on comparison of two cells of different metastatic potential, and/or between the primary tumor and the secondary metastases from same/different patients [4] . However, cell lines have a different gene expression profile from primary tumor cells after long times of passages [5] . Tumor tissues include too much non-malignant cells, such as normal epithelium and stroma cells, which have effect on tumor cell growth and migration [6] . To overcome these limitations and to detect exact differences between primary tumor cells and metastatic cells, we used two methods. Firstly, we implanted primary tumor cells and metastatic tumor cells into nude mice, where tumor cells grow faster than other stuff, so pupative pure tumor cells could be obtained at a limited time. Secondly, laser microdissecton (LMD) was taken to collect malignant cells. Then, after performing RNA extracting and amplification, we were able to compare gene expression profiles of colorectal primary cancer cells with those of metastatic cancer cells using cDNA microarray slides containing 13824 human cDNA fragments. Finally, we selected common up-/down-regulated genes in both results.
In this study, we reported detection of 376 genes, the expression of which was changed during progression toward metastasis of colorectal cancer. Among these genes, 190 genes showed up- regulated expression and 186 ones showed down-regulated expression in metastatic cells compared with primary tumor cells. TMA, a newly developed technique, is another high-throughput method for analyzing gene expression profiles by means of small array slide spotted with hundreds of tissue samples. TMA is especially useful in confirming and investigating results obtained from cDNA microarray. In our work, five gene products were screened using immunohistochemistry and TMA (containing normal mucosa, primary tumor tissues, and nodal metastases). After that, we used go analysis and Fisher's exact test to find valuable information and then used pathway-analysis to investigate changes of some pathways. Finally, we found that Grb2 was more common and extremely higher in metastatic tissues than primary tumor tissues. Genes involved Grb2-mediated Ras pathway was also widely changed. So, we used Grb2-SH2 domain-binding antagonist to counterpart its role of metastasis potential in colorectal cancer cell lines, which turned to be effective. All these results not only disclose the complex nature of changes in genetic activity during colorectal cancer metastasis but also provide valuable information that should prove useful for identifying novel therapeutic targets.......（后略) ......
Guan-Zhen Yu, Ying Chen, Ya-Qiu Long, Dong Dong, Jie-Jun Wang *
Department of Oncology, Changzheng Hospital , Shanghai , China
*Corresponding author: Jie-jun Wang
【 Abstract 】 Background&Aims Metastasis-associated genomic alteration has been recognized as playing a critical role in the tomour progression toward metastasis. Here, we analyzed changes of gene profiles between primary tumor cells and metastatic tumor cells using cDNA and tissue microarrays. Methods Primary tumor and nodal metastatic cells were implanted into subcutaneousness of nude mice. Then growth and metastatic potential was observed. Samples from tumor in mice were extracted for RNA, which was then used for 13824 human genes array. In addition, protein expression of selected genes was determined by Tissue microarray and immunohistochemistry. Function of Grb2 was determined by In vitro wound assay and Western blot. Results Nearly 400 genes showed different expression between primary tumor cells and metastatic cells. Statistics showed that Grb2 was extremely higher in the metastases and Grb2- mediated Ras pathway was evaluated during metastasis. Subsequent examination of selected genes by Real-time RT-PCR and immunohistochemistry substantiated the reliability of our analysis. Blocking Grb2-SH2 domain signaling transduction could inhibit cell motility by inactivating erbB2 protein. Conclusions Overexpression of Grb2 may contribute to tumor growth, invasiveness and metastasis. This functional profile makes Grb2 a high priority target for anti-cancer-metastasis drug development.
Introduction
Colorectal carcinoma is one of the most common cancers in the world. Despite advances in the treatment and early diagnosis, this disease remains a heavy threat to life for a large number of people, due to the metastasis and companied complications. Significant effects have been shown on the primary tumor because of the ongoing new chemotherapeutic agents and improved drug effectiveness; however, almost all the patients suffered from fatal metastasis in the end, which awaits new targets and strategies. To achieve this goal, a comprehensive understanding of the mechanisms of colorectal cancer progression toward metastasis will be essential.
At the molecular level, activation of oncogenes and inactivation of tumor suppressor genes contribute to the development of colorectal carcinoma, which has been under intensive study for many years. However, in the progression toward metastasis, two distinct classes of genes may be much more important-metastasis activator (or metastasis-promoting) and metastasis suppressor genes [1] . Nevertheless, how these genetic alterations bring about the progress to metastasis of colorectal carcinomas remains unclear. According to previous studies, accumulations of mutant genes during the moving of metastatic cells tend to be accompanied by other genetic and epigenetic changes including loss of heteozygosity [2] , aberrant promoter methylation [3] , and/or gene amplifications, all of which can alter gene expression profiles. Therefore, genome-wide monitoring of gene expression is of great importance if we are to disclose the numerous and diverse events associated with metastasis.
Essentially, identification of metastasis genes involves comparison of cells that differ in metastatic potential. Several approaches, e.g., comparison of chromosome additions/deletions/ translations, differential display, subtractive hybridization or recently developed 'gene chip', have been developed to identify genes that are expressed differently between cell lines and/or tissues of different metastatic potential. Recently, proteomic technologies provide a new sight to analyze expression profiles in various tissues. However, all these methods have limitations. Except 'gene chip', they are labor intensive, time consuming, requiring large amounts of mRNA/protein, and perhaps undeveloped. cDNA microarray, a fully developed technique, provides high-throughput analysis of expression profiles by means of small-array slides spotted with cDNA although large amount of data sometimes can be obtained and a number of false positive data can be observed. Here, we used real-time RT-PCR and tissue microarray (TMA) to confirm parts of our results and go analysis to exclude false positives and select useful data.
Previous studies are usually based on comparison of two cells of different metastatic potential, and/or between the primary tumor and the secondary metastases from same/different patients [4] . However, cell lines have a different gene expression profile from primary tumor cells after long times of passages [5] . Tumor tissues include too much non-malignant cells, such as normal epithelium and stroma cells, which have effect on tumor cell growth and migration [6] . To overcome these limitations and to detect exact differences between primary tumor cells and metastatic cells, we used two methods. Firstly, we implanted primary tumor cells and metastatic tumor cells into nude mice, where tumor cells grow faster than other stuff, so pupative pure tumor cells could be obtained at a limited time. Secondly, laser microdissecton (LMD) was taken to collect malignant cells. Then, after performing RNA extracting and amplification, we were able to compare gene expression profiles of colorectal primary cancer cells with those of metastatic cancer cells using cDNA microarray slides containing 13824 human cDNA fragments. Finally, we selected common up-/down-regulated genes in both results.
In this study, we reported detection of 376 genes, the expression of which was changed during progression toward metastasis of colorectal cancer. Among these genes, 190 genes showed up- regulated expression and 186 ones showed down-regulated expression in metastatic cells compared with primary tumor cells. TMA, a newly developed technique, is another high-throughput method for analyzing gene expression profiles by means of small array slide spotted with hundreds of tissue samples. TMA is especially useful in confirming and investigating results obtained from cDNA microarray. In our work, five gene products were screened using immunohistochemistry and TMA (containing normal mucosa, primary tumor tissues, and nodal metastases). After that, we used go analysis and Fisher's exact test to find valuable information and then used pathway-analysis to investigate changes of some pathways. Finally, we found that Grb2 was more common and extremely higher in metastatic tissues than primary tumor tissues. Genes involved Grb2-mediated Ras pathway was also widely changed. So, we used Grb2-SH2 domain-binding antagonist to counterpart its role of metastasis potential in colorectal cancer cell lines, which turned to be effective. All these results not only disclose the complex nature of changes in genetic activity during colorectal cancer metastasis but also provide valuable information that should prove useful for identifying novel therapeutic targets.......（后略) ......
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