Published OnlineFirst August 10, 2010; DOI: 10.1158/1055-9965.EPI-10-0677 Cancer Research Article Epidemiology, Biomarkers & Prevention Expression of the Tumor Suppressor Krüppel-Like Factor 4 as a Prognostic Predictor for Colon Cancer Nilesh V. Patel1, Amr M. Ghaleb1, Mandayam O. Nandan1, and Vincent W. Yang1,2 Abstract Background: The zinc finger transcription factor Krüppel-like factor 4 (KLF4) regulates numerous physiologic processes, including proliferation, differentiation, and development. Studies also showed that KLF4 is involved in tumorigenesis and somatic cell reprogramming. Here, we aimed to assess whether KLF4 is a prognostic indicator for colon cancer. Methods: Levels of KLF4 were measured by immunohistochemical analysis of a tissue microarray contain- ing 367 independent colon cancer sections. Univariate data analysis was done in addition to construction of multivariate models with several clinicopathologic factors to evaluate KLF4 as an independent predictor of survival and cancer recurrence (disease-free survival). Results: Colon cancer tissues had significantly overall lower KLF4 levels compared with noncancer tissues (P < 0.0001). Using logistic regression, a trend was noted for decreased odds of KLF4 expression in higher stages of tumors. In univariate and multivariate analyses, KLF4 was a significant predictor of survival and recurrence. Conclusions: KLF4 expression is significantly downregulated in colon cancer, and loss of KLF4 is an independent predictor of survival and recurrence. Impact: These findings suggest that KLF4 may serve as a prognostic biomarker for colon cancer. Cancer Epidemiol Biomarkers Prev; 19(10); 2631–8. ©2010 AACR. Introduction markers independent of established clinicopathologic predictors of the disease. Despite substantial advances in the early diagnosis and Biomarker research in colorectal cancer is becoming treatment of colorectal cancer, it remains a disease with increasingly popular for a variety of clinical and research high morbidity and mortality. Approximately 150,000 applications. Precise biomarkers may be useful as a new cases of colon cancer were diagnosed last year in surrogate endpoint in preliminary studies, for the strati- the United States, making it the fourth most common fication of patients in clinical trials, and in the refinement cancer diagnosed in men and women (1). It is also the of disease prognosis. The debate surrounding the role of second most common cause of cancer-related death chemotherapeutics in American Joint Committee on despite a recent reduction in mortality due to increased Cancer (AJCC) stage II colon cancer provides an example screening and improvements in treatment for late-stage of such an application. Within the past decade, a number cancer (2). Studies have identified mutations in tumor of new systemic treatments for colon cancer, including suppressor genes and oncogenes that result in dysregula- oral fluropyrimidines, oxaliplatin, and irinotecan, have tion of numerous pathways leading to colorectal carcino- been shown to improve overall and disease-free survival genesis (3, 4). Nonetheless, future research in colorectal in stage III cancer patients (5). Unlike patients with third- cancer depends on the ability to conduct research relative stage cancer, current recommendations by the American to potential applications of available data, translate the Society of Clinical Oncology do not promote the routine mechanistic data into the clinical arena, and evaluate use of chemotherapeutics in stage II cancer (6). Although no clinical trial has proven overall benefit of adjuvant therapy in stage II, it is hypothesized that up to 20% of patients with a risk for recurrence similar to that of stage Authors' Affiliations: 1Division of Digestive Diseases, Departments of III disease would have benefited from chemotherapeutics Medicine and 2Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, Georgia (5). Currently, the American Society of Clinical Oncology suggests that patients with inadequately sampled nodes, Corresponding Author: Vincent W. Yang, Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, 615 T4 lesions, perforation, or poorly differentiated histology Michael Street, Atlanta, GA 30322. Phone: 404-727-5638; Fax: 404- should at least be considered for such treatment at the 727-5767. E-mail: [email protected] discretion of the treating physician and patient (6). How- doi: 10.1158/1055-9965.EPI-10-0677 ever, no studies have evaluated the benefit of such a rec- ©2010 American Association for Cancer Research. ommendation or whether the suggested criteria correctly www.aacrjournals.org 2631 Downloaded from cebp.aacrjournals.org on September 25, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst August 10, 2010; DOI: 10.1158/1055-9965.EPI-10-0677 Patel et al. predict recurrence risk. Biomarkers may prove especially in duplicate because 2-fold redundancy permits accurate useful in further stratifying stage II patients into those analysis of protein expression (34). who have a high risk for recurrence and those who do The paraffin-fixed colon tissue microarray was con- not, the former group being more likely to benefit from structed between 1989 and 1996 by the National Cancer chemotherapeutics. Several biomarkers such as S-phase Institute's Cancer Diagnosis Program of the National fractions and vascular endothelial growth factor expres- Cancer Institute (35). The microarray was assembled sion have been evaluated in this regard (7, 8). using 367 cores of colon cancer (49 stage I, 122 stage II, Krüppel-like factors (KLF) are a family of evolutionarily 144 stage III, and 52 stage IV), 37 cores of adenomatous conserved mammalian zinc finger transcription factors polyps, 34 cores of normal colon tissues matched to tu- named for their homology with Krüppel, a Drosophila mor sections, and an additional 40 normal colon sections melanogaster protein (9). KLFs are involved in a diverse from individuals with diverticulosis. Of the colon cancer array of fundamental biological processes, including cases, 5-year follow up data were complete on 96 stage proliferation, differentiation, development, and apoptosis II tumors (26 recurred) and 125 stage III tumors (10-12). Among the KLF family, KLF4 (also called gut- (65 recurred). Of all stage II and III tumors, 45 were cen- enriched KLF or GKLF) was one of the first identified sored before the 5-year follow up was complete. Nonco- (13, 14). In addition to regulating many important phys- lon tissue cores were embedded on the microarrays for iologic processes, KLF4 has been shown to play a role in internal control of staining. In addition, cores from colon pathologic conditions such as cancer and inflammation cancer cell lines were embedded as additional internal (15-20). More recently, KLF4 was shown to play a crucial controls. These cell lines were characterized and authen- role in the reprogramming of somatic cells into induced ticated by the National Cancer Institute under American pluripotent stem cells (21-25). Type Culture Collection guidelines (35). Of all patients, Expression of KLF4 is enriched in epithelial tissues, 418 were Caucasian, 12 were African American, and 11 including the gut (13, 14). In the intestine, KLF4 is highly identified with another race. Two hundred and seven expressed in the postmitotic, terminally differentiated subjects were male, and 233 were female (gender of one in- epithelial cells at the luminal surface (26, 27). In vitro, dividual was unknown). Mean age was 68.62 (SD, 12.48). KLF4 inhibits proliferation by blocking cell cycle prog- Additional covariate data were collected on tumor depth, ression at the G1/S boundary (28). In addition, KLF4 nodal status, metastasis, histology, location, and degree of mediates the cell cycle checkpoint functions of the tumor dysplasia as assessed by an independent pathologist. suppressor p53 following DNA damage (29-31). More- Selection of patients across various stages was done to over, loss of expression of KLF4 due to several reasons, ensure enough power to detect differences in recurrence including loss of heterozygosity, promoter hypermethy- within stage II and stage III cancer independently. The lation, and loss-of-function mutations, has been docu- cases were chosen to detect a difference in the prevalence mented in a small set of colorectal cancer specimens rate of 0.35 within stage II or III tumors that recurred and (32). This tumor suppressive effect of KLF4 is supported those that did not within the 5-year follow-up period. To by in vivo evidence in which mice heterozygous for detect differences in a binary outcome marker across var- Klf4 manifest increased tumor burden when bred to the ious stages of disease, enough stage I and IV tumors were ApcMin mice that are genetically predisposed to intestinal also included, so >80% power was available to detect a adenoma formation (17). From a mechanistic perspective, 0.30 difference in prevalence rate of KLF4 (see Guidance KLF4 inhibits Wnt signaling, a key pathway in colorectal for Statistical Analysis of Biomarker Data Generated carcinogenesis, by inhibiting the activity of β-catenin, a from the NCI Colon Tissue Microarray; ref. 36). mediator of Wnt signaling (33). Because of its putative tumor suppressor function, Immunohistochemistry KLF4 may serve as a prognostic indicator for colon can- The microarrays were treated with xylene for deparaf- cer. Here, we measured KLF4 expression levels in