No abstract

Colorectal cancer is the third leading cause of cancer mortality in the United States. Oxidative stress and DNA damage have been associated with a variety of human pathophysiological conditions, including colon cancer. Complex DNA damage may manifest in double strand breaks (DSBs) and non-DSB, bi-stranded, oxidatively-induced clustered DNA lesions (OCDLs). To date, there is very limited knowledge regarding the possible accrual of OCDLs in human colon tumors or the OCDL repair efficiency of primary colonic malignant cells. Therefore, the impact of oxidatively-induced clustered DNA damage in the predisposition to or progression of colon cancer remains undefined. We hypothesized that colon adenocarcinoma tissue and cells would have increased levels of DNA damage compared with normal counterparts. Furthermore, we expected that malignant colon cells would exhibit a defective Base Excision Repair (BER) pathway. To test this hypothesis, normal and malignant colonic tissue from 10 consented female patients was collected and number of DNA lesions measured using neutral constant field gel electrophoresis. Quantitative measurement of OCDL difference in each group was achieved using QuantiScan analysis. DNA repair efficiency of colon tissue-derived primary normal and malignant cells was assessed by single-cell gel electrophoresis. Expression and localization of BER-associated proteins was determined through Western blot analysis and immunohistochemistry. We found that degree of complex DNA damage varied among individual subjects. Forty percent of patients had tumors with increased levels of APE1-induced OCDLs compared to normal counterparts. The vast majority (80%) of malignant tissue expressed higher levels of APE1, XRCC1 and PARP1, ranging from 3- to >10-fold, compared to normal peri-tumoral tissue. However, 20% of malignant tissue had reduced levels of the aforementioned proteins as compared to peri-tumoral matched normal tissue. Oxidatively-induced DNA damage was more efficiently repaired in primary cells derived from normal tissue compared to cells from paired malignant tissue (1 hour versus >6 hours) following treatment with 6 mM H2O2. Since the expression of most BER-associated proteins (e.g., XRCC1, OGG1, etc.) was similar in both malignant and normal cells, the DNA repair defect may be the result of a 2.5-fold lower expression of APE1 protein measured in these cancerous cells compared to normal primary cells. Furthermore, a differential cytoplasmic to nuclear translocation of APE1 was also observed in cancerous cells compared to normal cells during the DNA damage and repair process. In conclusion, these findings strongly indicate that there is variation in oxidatively-induced complex DNA damage – perhaps mediated through a defective BER pathway – among patients with colon cancer, thus underscoring the need for patient-specific approaches to diagnosis and treatment of this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3920. doi:10.1158/1538-7445.AM2011-3920

e14635 Background: The clinical relevance of cancer stem cells (CSC’s) in solid organ malignancies is unclear. ALDH1 is thought to be a universal marker of stemness and is expressed in normal and neoplastic stem cells. We sought to determine the effects of stemness, expressed as levels of ALDH1, on survival for similarly staged metastatic colon cancer patients. Methods: Patients with lymph node positive colon cancer and unresectable liver metastasis diagnosed from 2004-2009 were identified. Sections were prepared from paraffin blocks and stained with an ALDH1 antibody. To determine quantitative expression of ALDH1, morphometric analysis with a Nikon microscope, SPOT video camera, and ImagePro software was performed with expression defined as stained area/total area. Ten to twenty randomly selected fields from were assessed. Patients were determined to have high expression of ALDH1 if respective levels were above the mean. Data analysis was performed with JMP. Results: A total of 16 patients were identified, with a median age of 64 years. The majority of patients were female (63%) and black (69%). Few primary tumors were poorly differentiated (25%). Median survival was 21.5 months (range: 1-47). Mean expression of ALDH1 was 0.041+0.01 for normal crypts, 0.031+0.01 for lymph node metastases, and 0.062+0.02 for primary tumors. ALDH1 expression was high in 31.3%, 20%, and 31.3% of patients for normal colon crypts, lymph node metastases, and primary cancers, respectively. Increased expression ALDH1 in tumors was strongly correlated with mortality (p=0.0013) and male sex (p=0.0166), but not with age (p=0.71) or race (p=0.50). Increased expression of ALDH1 in primary tumors was associated with a decreased overall survival, 27 vs. 6 months (p=0.0015). Increased expression of ALDH1 in normal colon crypts and lymph node metastases was not associated with survival, 27 vs. 19 (p=0.18) and 21 vs. 22 months (p=0.35), respectively. Conclusions: Increased stemness in primary colon cancers, expressed as ALDH1 levels, is associated with poor survival for similarly staged metastatic patients. These data suggest that an increase in stem cell fraction predicts a more aggressive clinical course.