Bruce M. Boman - US grants

DESCRIPTION (provided by applicant): Stem cells (sc) are almost certainly the cells of origin of intestinal cancer. But, sc mechanisms are obscure because there are no specific markers to identify SO. Our long-term objective is to use animal models to develop & test research tools for identifying adult intestinal sc in vivo. Here we will test one tool, GFP-Tcf4 transgenic mice. We designed this mouse line to display green fluorescence protein positive (GFP+) cells when Tcf4 is activated. Tcf4 is important because 1) intestinal tumors have activated Tcf4 due to cancer-causing Apc mutations & 2) available evidence suggests that Tcf4 activation in normal crypts is unique to sc. Thus, a marker for Tcf4 activation may be a marker for sc. We crossed GFP-Tcf4 mice with ApcMin mice because the latter are at high risk for intestinal cancer & to assess effects of Apc mutations on Tcf4 activation. AIM 1: To identify (ID), isolate & characterize GFP+ enterocytes from GFP-Tcf4 mice. GFP+ enterocytes in these mice appear localized to the crypt base (where SC reside). To quantify this finding, we will a) use fluorescence microscopy/advanced image analysis (of intestinal sections) measuring in vivo GFP signal intensity along the crypt axis, & b) flow cytometry sorting (of isolated fixed, GFP+ enterocytes derived from purified whole crypts). Hypothesis 1(H1): GFP+ enterocytes are only in the crypt base, are a small % of crypt cells, stain for nuclear (-catenin ((c). AIM 2. To see, using microscopy & cytometry as above, if the GFP reporter responds to changes in Tcf4 activation. H2: The # of crypt cells that are GFP+ & show nuclear (c localization are increased in mutant crypts (normal-appearing mucosa [1 Apc mutation], adenomatous mucosa [2 mutations]). AIM 3. To isolate viable GFP+ & GFP- enterocytes. Here we will isolate viable (not fixed) enterocytes & sort them by flow cytometry. H3: Prevention of clumping in single cell suspensions of enterocytes will permit flow cytometric sorting of viable cells; GFP+ but not GFP- cells will show nuclear (c staining & test positive for Hoechst dye efflux, a functional SC property. Significance. If predictions hold, Aim 1 results will suggest that GFP expression ID's intestinal SC (or sc-like cells); Aim 2 results, that the reporter responds to changes in Tcf4 activation; Aim 3 results, that viable GFP+ intestinal cells are isolatable and characterizable hence can be tested to see if they have other sc properties. One could then evaluate the role of SC in initiation & promotion of intestinal neoplasms.

[unreadable] DESCRIPTION (provided by applicant): Our long-term objective is to identify and characterize populations of intestinal stem cells (SC). Developing the ability to identity SC in colonic crypts will make possible investigation into their putative role in the origin of colon cancer. Our immediate objective is to develop markers for colonic stem cell enriched (SCE) preparations. Array-based gene expression profiling will be used to characterize colonic SCE preparations made based on anatomical and functional properties of SC (location at the bottom of the crypt & clonogenicity). We will investigate SCE preparations from purified normal crypts (Aim 1) & from crypts that contain mutant APC (from familial adenomatous polyposis [FAP] patients; Aim 2) to compare their gene expression profiles. We will test two hypothesis: H1: Genes selectively expressed in crypt base cells (compared to the whole crypt) are also selectively expressed in clonogenic cells (compared to the whole crypt). H2: Genes selectively expressed in crypt base cells and/or clonogenic cells have increased expression in FAP crypts compared to normal crypts. Tissue samples from surgical colectomy specimens will be collected, colonic crypts purified, and microarray analysis done to characterize SCE preparations. AIM 1: To determine gene expression profiles for SCE preparations from normal human crypts. Task 1.a: evaluate microarray profiles for SCE preparations from the bottom of purified crypts. Task 1.b evaluate microarray profiles for SCE preparations from crypt clonogenic cells from colonies in soft agarose. Task 1.c determine which genes are common to profiles in both I.a and 1.b and build a targeted array. AIM 2: To determine gene expression patterns and levels for SCE marker genes in abnormal crypts, (FAP crypts). Task 2: compare gene expression levels and patterns in purified whole crypts from FAP vs. normal individuals using the targeted array (1 .c). We predict: i) there is a unique gene expression pattern for each SCE preparation (base & clonogenic cells), and ii) these patterns are similar. We also predict that genetic markers for SCE preparations have increased levels of expression in FAP crypts compared to normal crypts. Our results will provide gene expression profiles that might serve as specific markers for colonic SC populations or at least for SCE preparations. With markers for SCE preparations, future research could lead, through various experimental approaches, to specific markers for colonic SC. With specific markers for SC, we could directly test the idea that colon cancer initiation occurs because APC mutation leads to SC overproduction. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): 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.

DESCRIPTION (provided by applicant) Our AP4 center will focus on hereditary colorectal cancer (HCC) because: I) HCC, including Hereditary Non-polyposis Colon Cancer (HNPCC) and Familial Adenomatous Polyposis (FAP) are subsets (approximately 3%, approximately 1%) of colorectal cancer (CRC) and individuals carrying an HCC trait (a germline MMR or APC mutation) are at extremely high risk for CRC. 2) Our Director, Bruce Boman, MD, PhD, has great familiarity with basic and clinical HCC issues and is President of an HCC-dedicated organization of academic investigators - the Collaborative Group of the Americas for Inherited Colorectal Cancer (CGA-ICC). 3) Our team has an established track record of working with biotechnology and industry in cancer research. 4) Genetically defined subsets of CRC have tumors with different etiotogic mechanisms, patterns of mutations, biological behaviors and responses to treatments. The strategy of defining genetic subsets of CRC to develop more effective anti-cancer drugs is applicable to HNPCC and FAP carriers and patients because these are genetically well-defined cohorts. 5) Our team is poised to address pressing needs in the HCC field with promising research projects: P1- A need to identify all trait carriers before they develop CRC. Our solution is a serum-based immunoassay that quantifies wild type MMR or APC proteins in lymphocytes; P2/P3- A need for chemopreventives for trait carriers. We will develop, for FAP, mimetics of 15-LOX-1 & inhibitors of Beta-catenin/TCF-4/Survivin signaling. P4 - A need to detect HCC early, before advanced disease occurs. We will develop serum markers for early HCC detection. P5/P6 - A need for treatments for trait carriers with advanced disease. We will develop prototype new agents based on (a) the anti-proliferative effects of uroguanylin and (b) a GC-C Protein-based vaccine that elicits an immune response that attacks micrometastases but not the colon. The Center will also develop resources that promote collaborative, multidisciplinary HCC research projects among Center partners (e.g., database of HCC patients and researchers; tissue bank). Anticipated Outcomes of the planning year: i) identification of academic and industry partners and initial projects and establishment of collaborative, multidisciplinary working relationships among these partners; ii) planning and holding a successful Partners meeting (which will include discussions of the AP4 program, our Center research focus and administration, research projects, partner commitment and Center funding, intellectual property rights, strategic planning, 5 y application); iii) development of a detailed Strategic Plan; iv) financial and other commitments of partners; v) submission of an outstanding and fundable 5 y AP4 grant application.