Charles Giardina - US grants

laboratory mouse; nonsteroidal antiinflammatory agent; tumor necrosis factor alpha

[unreadable] DESCRIPTION (provided by applicant): The employment of chemical weapons by Iraq during the Iran-Iraq War (1980-1988) resulted in over 100,000 military and civilian casualties among Iranians. Both organophosphates (nerve agent) and alkylating compounds (mustards) were used, with mustards being the most-frequently utilized. Mustard gas is a known mutagen and chronic, occupational exposure is carcinogenic in humans. However, it is not dear whether an acute exposure to mustard agent (e.g., in a battlefield or terrorist scenario) is sufficient to initiate lung carcinogenesis. We propose performing a molecular analysis of lung cancers obtained from individuals exposed to mustard gas during the Iran-Iraq War. We will focus on the p53 tumor suppressor gene since the frequency, type and position of p53 mutations are dependent on the nature of the primary carcinogen. Paraffin-embedded lung tumors from mustard-exposed and nonexposed Iranian military veterans and civilians are available for analysis (approximately 50 to 100 per group). A limited set of tissue from cancer-free mustard gas victims is also available from an on-going Iranian study to analyze the p53 mutation load at early stages of carcinogenesis. The following studies are proposed: 1. A sequence analysis of the p53 tumor suppressor gene will be performed on DNA isolated from lung tumor samples from mustard gas-exposed and non-exposed Iranian cohorts. A characteristic mutational fingerprint in tumors from mustard gas victims would implicate mustard gas as the primary carcinogen. 2. p53 status in non-cancer tissue from mustard gas exposed and non-exposed individuals will be determined. This analysis will include an examination of p53 stabilization, down-stream gene activation (p21 and Mdm2 expression) and mutation. The tissue analyzed will be derived from a current Iranian study of mustard gas victims referred to the clinic for hemoptysis evaluation. Successful completion of these studies will help establish mustard gas as the relevant carcinogen in Iranian mustard gas victims, a conclusion that would have associated ethical and legal implications. These studies would also set the groundwork for a more detailed genetic examination of these carcinogen-induced tumors, and could facilitate the development of genetic tests to determine cancer risk in mustard gas victims. Finally, these studies will provide unique insight into the ability of a powerful mutagen to initiate carcinogenesis in humans after a single, high intensity exposure. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The recent identification and development of small molecules that activate p53 open new possibilities for colon cancer treatment and prevention where the mutational inactivation of p53 is typically a late event in the adenoma-carcinoma sequence. Our preliminary data indicate that p53 in AOM-induced mouse colon tumors is primed for activation by the recently developed Mdm2 inhibitor Nultin-3, making these tumors hypersentive to this compound. We propose that stimulating p53 activity at selected stages of carcinogenesis will serve to suppress colon cancer development. To test this hypothesis, we will determine if the Mdm2 inhibitor Nutlin-3 can induce stasis or regression of colon tumors in the mouse AOM model (Aim 1). We will also determine whether Nutlin-3 can selectively activate p53 and apoptosis in human colon adenomas and mouse aberrant crypt foci (using an ex vivo culture format; Aim 2). Finally, we will determine whether the expression levels of the epigenetically regulated ARF gene sensitizes colon cancer and adenoma cells to Nutlin-3 or dietary agents that affect the p53 pathway (Aim 3). The long term goals of these exploratory studies are to: 1) establish p53/Mdm2 as a target for colon cancer prevention agents, 2) determine the stage of carcinogenesis when Mdm2 inhibitors are most effective and 3) determine the contribution of ARF to sensitizing transformed cells to agents that activate p53. PUBLIC HEALTH RELEVANCE:Colon polypectomy is an important tool for colon cancer prevention. However, limitations to this approach include the incomplete removal of sessile polyps and the missed detection of small adenomas. Our goal is to develop safe and effective pharmacological or dietary agents, to be used in conjunction with present treatment practices, to suppress the risk of colon cancer development in high risk patients. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We propose to develop a novel and innovative system for studying very early stages of colon cancer development. This in vitro culture system involves promoting embryonic stem cell (ESC) differentiation into intestinal organoids through a definitive endoderm intermediate, and then initiating cell transformation through oncogene expression or tumor suppressor gene knockdown. This experimental system, which we have been developing with mouse and human ESCs, is anticipated to be robust and highly flexible and enable cancer biologists to address fundamental questions relating to very early events in colon carcinogenesis. Advantages of this system include an in vitro format compatible with the rapid analysis of nutrients, small molecules, siRNAs or other biologics. Furthermore, intestinal organoids can be generated from human ESCs, allowing the detailed study of intestinal cells from individuals with distinct colon cancer risks. In the proposed studies we will assess the utiliy of intestinal organoids for studying the effects of Braf oncogene activation. Our interest in Braf mutated cells stems from the fact that BRAF-mutated cancers frequently develop in the human proximal colon where they can be difficult to detect and remove, making effective chemoprevention of these cancers an important goal. Intestinal organoids will be prepared from mouse ESCs in which Braf oncogene become activated during intestinal lineage commitment (using Villin- Cre/LSL-BrafV600E mice) and are regulated by their native promoters. We will then evaluate how oncogene expression influences the organoids in relationship to endogenous intestinal tissues in vivo. This work will focus on oncogene effects on cellular turnover dynamics, intestinal stem cell expansion and cellular differentiation. Since Braf activation is associated wih epigenetic alterations, we will also determine the effect of oncogene activation on the expression of epigenetic modifying factors and the silencing of growth regulatory genes. We will also determine the ability of cell growth modulating chemopreventive agents to correct molecular and proliferation defects in oncogene-expressing intestinal organoids. Finally, we will develop high throughput assay systems to screen natural compound and pharmacological agent libraries for novel chemopreventive agents that selectively inhibit the growth of Braf-expressing intestinal cells. We anticipate indentifying novel chemopreventive agents (or agent combinations) for further development in subsequent proposals. Finally, establishing the organoid system for cancer research could facilitate the study many factors and variables that impact cancer promotion and prevention. PUBLIC HEALTH RELEVANCE: The KRAS and BRAF oncogenes can be activated early in colon carcinogenesis and stimulate a complex combination of pro-growth and anti-growth signaling pathways that ultimately determine whether the cells can progress to colon cancer or not. In this proposal we will derive intestinal cells from embryonic stem cells to establish a simple system to study the complex events that follow KRAS and BRAF oncogene activation. Our focus will be to determine the role of epigenetics in promoting the growth effects of the oncogenes with an eye towards understanding how the pro-growth signaling might be suppressed for colon cancer prevention.

Project Summary/Abstract A critical step in colon cancer development occurs when a cell within a field of Apc- heterozygote mutant cells undergoes Apc loss of heterozygosity (LOH). Understanding the mechanism and cellular consequences of an Apc LOH event could provide important information on how this event might be suppressed to reduce cancer risk. Critical questions that remain unanswered include: Do the events leading to Apc LOH include a failure of cytokinesis? Are all cells that undergo an LOH event at equivalent risk of progression? What genetic and environmental factors can impact Apc LOH? We propose to develop an intestinal organoid system as a malleable experimental platform to study Apc LOH. We will utilize intestinal organoids generated from ESCs since these ?mini-intestines? can be genetically manipulated and grown in the quantities necessary to detect the relatively rare Apc LOH event. We propose to develop this organoid system to study Apc LOH in real-time by linking the ApcMin allele to GFP and the Apc-wild type allele to an RFP reporter and the diphtheria toxin receptor (DTR). We will use these organoids to enumerate and isolate cells that have undergone Apc LOH. We will initially determine how p16 and p53 affect Apc LOH frequency, but envision these organoids being used to assess the impact of a broad range of mutations and polymorphisms. We will study the nature of cells emerging from an Apc LOH event, focusing on their proliferative capacity and their relationship to normal ISCs and cancer stem cells. Finally, we will assess the role of tetraploid cells as precursors for Apc LOH and determine if a novel mitosis-targeting agent that selectively induces apoptosis of tetraploid cells can reduce the frequency of Apc LOH. These exploratory studies are anticipated to establish an experimental system for studying a critical event in colon cancer development; the emergence of Apc-mutant cells in a pre-malignant tissue field. In the long term we envision establishing an integrated experimental pipeline to understand how environmental and genetic factors impact Apc LOH, how cells that have undergone Apc LOH progress to cancer stem cells, and ultimately, how an individual's risk of colon cancer risk can be reduced.