Our broad long-term objective is to characterize GI stem cell (SC) based mechanisms underlying colorectal cancer (CRC) development because this could lead to identification of vulnerable molecular processes in tumor cells and thus to discovery of more effective anti-cancer drugs and/or chemopreventive agents. Since evidence indicates that colonic SC are the cells of origin of CRC, developing the ability to identify colonic SC (Aim 1) should make possible isolation of SC populations and, consequently, studies into the role of SC mechanisms in CRC development. Because a mutation in the APC gene initiates most CRC cases, and because recent evidence indicates that APC-based mechanisms regulate SC population size, then correlating changes in APC-based functions with changes in colonocyte properties along the normal crypt axis (Aim 2) should shed light on how APC-based mechanisms are involved in the regulation of SC and how SC mechanisms are involved in CRC. Given that markers for colonic SC are currently lacking, a necessary condition for SC identification is knowing that they reside at the bottom of the crypt, and thus use of fresh tissues rather than cultured cells is required in our proposed study.
In Aim 1 we will use an innovative strategy to identify potential markers for colonic SC by applying proteomic analysis (2D gels/mass spectrometry) to the investigation of freshly isolated human colonic crypt subsections (bottom third of crypts where SC reside). Because evidence indicates that when SC contain mutant APC (as in FAP) the crypt SC population expands and there are substantially more SC, our first hypothesis is that (a) proteomic analysis will show that expression of some proteins (spot intensity) in histologically normal-appearing FAP crypts is significantly higher than in control crypts, and (b) antibodies generated to at least some such proteins will show staining at the normal crypt bottom and increases in the number of positively-stained crypt bottom cells in FAP. Identifying SC markers will enable investigators to grow SC and study SC changes during tumorigenesis.
In Aim 2 we will use normal human crypt specimens to distinguish varying patterns, along the crypt axis, of APC phosphorylation, subcellular localization and interactions with binding proteins. Our second hypothesis is that known differences in APC concentration and subcellular localization along the crypt axis correlate with specific patterns in APC phosphorylation & binding to other proteins. These correlations will suggest mechanisms by which APC regulates SC population size and by which APC mutation leads to SC overpopulation and CRC.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21DK063014-01
Application #
6560305
Study Section
General Medicine A Subcommittee 2 (GMA)
Program Officer
May, Michael K
Project Start
2003-01-21
Project End
2004-12-31
Budget Start
2003-01-21
Budget End
2003-12-31
Support Year
1
Fiscal Year
2003
Total Cost
$148,450
Indirect Cost
Name
Thomas Jefferson University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
053284659
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
Patel, Bhavinkumar B; Li, Xin-Ming; Dixon, Maketa P et al. (2011) APC +/- alters colonic fibroblast proteome in FAP. Oncotarget 2:197-208
Yeung, Anthony T; Patel, Bhavinkumar B; Li, Xin-Ming et al. (2008) One-hit effects in cancer: altered proteome of morphologically normal colon crypts in familial adenomatous polyposis. Cancer Res 68:7579-86
Patel, Bhavinkumar B; Li, Xin-Ming; Dixon, Maketa P et al. (2007) Searchable high-resolution 2D gel proteome of the human colon crypt. J Proteome Res 6:2232-8
Li, Xin-Ming; Patel, Bhavinkumar B; Blagoi, Elena L et al. (2004) Analyzing alkaline proteins in human colon crypt proteome. J Proteome Res 3:821-33
Boman, Bruce M; Walters, Rhonda; Fields, Jeremy Z et al. (2004) Colonic crypt changes during adenoma development in familial adenomatous polyposis: immunohistochemical evidence for expansion of the crypt base cell population. Am J Pathol 165:1489-98