Inactivation of the p53 tumor suppressor protein is considered essential for the development of most or all human cancers. Over 50% of cancers harbor TP53 mutations that destroy p53 function. In most cases, missense mutations in one TP53 allele are followed by loss-of-heterozygosity (LOH), so tumors express only a mutant, inactive form of p53. TP53 mutations and LOH have been linked, in many cases, with poor therapy response, increased tumor aggressiveness, and decreased long-term survival. Despite this, remarkably little is known about how TP53 point mutations are acquired, how LOH occurs, or the cells involved. Approximately 15% of human colorectal cancers (CRCs) are microsatellite unstable (MSI) due to somatic or inherited defects in DNA mismatch repair (MMR). Interestingly, TP53 mutations are less frequent in MSI (MMR deficient) CRCs than cancers where MMR is intact. This has suggested MMR defects do not give rise to TP53 mutations in MSI tumorigenesis, though experiments to directly address this question are lacking. Wt p53 is expressed at low levels due to MDM2, an E3-ligase that binds p53 and promotes its degradation. Small molecule MDM2 antagonists are being developed as therapeutic agents. Nutlin-3a (Nutlin) is an MDM2 antagonist that stabilizes/activates p53 by blocking MDM2-p53 binding. Nutlin causes robust growth arrest/apoptosis in p53 wt cells and human tumor xenografts. Importantly, only p53-null or p53-mutant cells can grow and form colonies in Nutlin. In this grant proposal, we have leveraged the powerful growth inhibitory activity of Nutlin to select p53-mutated cells and examine how TP53 mutations arise and how LOH occurs. Preliminary data suggest MMR-deficient CRC cells acquire TP53 mutations at greater rate than repair-proficient counterparts. Further, these initiating TP53 mutations appear more prevalent in cells that express certain cancer stem cell (CSC) markers (CD44), but not CSC marker-negative cells. Finally, TP53 LOH in MSI cells occurs through a copy-neutral mechanism. Based on these findings we hypothesize 1) MMR deficiency gives rise to initiating point mutations in p53, 2) TP53 LOH in MSI cells occurs through a copy-neutral mechanism, and 3) CD44(+) are either more prone to acquire TP53 mutations, or p53 mutations convert CD44low cells into CD44hi and thus may increase cancer "stemness".

Public Health Relevance

Inactivating mutations in p53 contribute to tumor development and are observed in over 50% of all human cancers. In most cases, mutations in one TP53 allele are followed by loss-of-hetrozygosity (LOH), such that the cancer expresses only a mutant, inactive form of the protein. TP53 mutations and LOH have been linked with poor therapy response, increased tumor aggressiveness, and decreased long-term survival. Surprisingly, however, little is known about how TP53 point mutations are acquired, how LOH occurs, or the cells involved. We have developed an innovative strategy to select cells that have acquired TP53 mutation or undergone LOH. Using this strategy, we will determine the extent to which specific DNA repair defects contribute to TP53 mutation acquisition and the mechanism of LOH in human colon cancer cells. We will also test the relationship between p53 mutation/LOH and the extent to which stem-cell properties are expressed in colon cancer. These studies could ultimately reveal novel factors or pathways that could be targeted for cancer treatment/prevention.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21CA178733-01
Application #
8571884
Study Section
Special Emphasis Panel (ZCA1-SRLB-J (M1))
Program Officer
Johnson, Ronald L
Project Start
2013-07-22
Project End
2015-06-30
Budget Start
2013-07-22
Budget End
2014-06-30
Support Year
1
Fiscal Year
2013
Total Cost
$215,197
Indirect Cost
$63,807
Name
Rush University Medical Center
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
068610245
City
Chicago
State
IL
Country
United States
Zip Code
60612