18.CANCER PROTEOMICS PROJECT
Suppression of Intestinal Polyposis in Mdr1-deficient ApcMin/+ Mice
Aberrant transactivation of a certain set of target genes by the b-catenin and TCF/LEF (T-cell factor/lymphoid enhancer factor) complex has been considered crucial for the initiation of intestinal tumorigenesis. The human MDR1(ABCB1) gene contains multiple b-catenin-TCF4-binding elements in its promoter and is one of the immediate targets of the complex. The biological involvement of MDR1 in intestinal tumorigenesis was explored based on the following evidence. 1) Aberrant induction of the Mdr1a (Abcb1a) gene product, p-glycoprotein, associated with nuclear accumulation of the b-catenin protein, was observed even in nascent microscopic adenomas of Min mice. 2) Mdr1-deficient Min (ApcMin/+ Mdr1a/b-/- ) mice developed significantly fewer intestinal polyps than did ApcMin/+ Mdr1a/b+/+ mice. And 3) Inhibitors of p-glycoprotein, verapamil and cyclosporin A, had a suppressive effect on the in-vitro polypoid growth of IEC-6 intestinal epithelial cells expressing stabilized (
N89) b-catenin protein. Inhibitors of p-glycoprotein may be included in a novel class of chemopreventive agents against colorectal carcinogenesis.
Transcriptomic and Proteomic Analyses of Early Colorectal Carcinogenesis
The transcriptomic and proteomic studies were performed to identify the population of genes and proteins whose expression was regulated by induction of b-catenin protein stabilized by deletion of the N-terminal GSK3b (glycogen synthase kinase-3b) phosphorylation sites under strict control of the tetracycline-regulatory system in the rat small intestinal epithelial cell line IEC-6 using the 2D-DIGE (two-dimensional differential image gel electrophoresis) and GeneChipTM oligonucleotide microarray systems. A panel of the proteins whose expression was affected by the induction included the proteins which have not been implemented in colorectal carcinogenesis. Among the proteins identified by mass spectrometry, manganese superoxide dismutase (MnSOD), which was upregulated in IEC-6 cells expressing stabilized b-catenin protein, was selected for further study. An immunohistochemical study revealed that MnSOD was overexpressed in adenoma and adenocarcinoma cells of familial adenomatous polyposis (FAP) patients in parallel with the accumulation of b-catenin, suggesting the possible contribution of MnSOD to colorectal carcinogenesis.
Construction of a Database for Two-dimensional Gel Electrophoresis
2D-DIGE, in which proteins are labeled with multiple fluorescent dyes and separated according to their isoelectric point and molecular weight, was employed and modified as a cancer proteomic study tool. To evaluate the potentials of the 2D-DIGE system for cancer studies and to perform rapid spot identification, a 2D-DIGE database, which consisted of 2D patterns and annotation of protein spots, was constructed. Among 2,000 protein spots observed in colon cancer cells, DLD-1, 343 spots were identified using mass spectrometry and a database search. They were grouped based on their molecular function according to the Panther Categories in the Celera Discovery System. The majority of identified proteins was a family of nucleic acid binding protein, oxidoreductases, cytoskeletal proteins and chaperons, and the remaining proteins were distributed functionally over a broad range. Although one receptor and six proteins for signal transduction were observed, proteins with a lower amount, such as transcription factors and cell cycle regulators, were not observed. These results indicate that, while 2D-DIGE has a limited potential to assess the mechanisms of cancer biology, it will be useful for biomarker development because the broad range of proteins are observed with the information of expression level. The comparison of protein spot distribution of DLD-1 cells and biopsied tissues of human colon cancer were also performed. Most of the identified spots in the 2D database of DLD-1 cells were also observed in 2D images of human colon cancer tissues. These results suggest that the database for 2D-DIGE using DLD-1 cells is useful for rapid spot identification of clinical materials.
Application of Laser Microdissection for Proteomic Studies of Cancer
To link the pathological observation and the proteomic information of cancer tissues, newly developed fluorescent dyes were applied to the linkage of two-dimensional gel electrophoresis (2D-PAGE) and laser microdissection. In this application, the population of specific cells including tumor cells and their normal counterparts were isolated under microscopic observation, and the proteins extracted from the isolated cells were labeled with sensitive fluorescent dye and separated by 2D-PAGE. This application shortens the microdissection time considerably compared with that for previous 2D-PAGE using colorimetric staining, and enables quantitative study with a broad dynamic range of fluorescent signals. The proteomic study of adenoma tissues in Min mice revealed that 37 protein spots showed considerable changes in amount compared with normal small intestinal epithelial tissues. The changed proteins identified by mass spectrometric analysis included prohibitin, 14-3-3zeta, tropomyosin 3 and Hsp84, whose aberrant expression has not been reported previously in adenoma and colorectal cancer. The cancer tissues of lung, pancreas, colon and liver were also subjected to the proteomic study using this application and their 2D maps were constructed to find the alterations associated with carcinogenesis.