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vimentin. Similarly, in tumor tissues from AOM/DSS-treated mice, KLF4 levels were negatively correlated with
mesenchymal markers including SNAI2, β-catenin, and vimentin and positively correlated with the epithelial marker
E-cadherin.

Conclusion: These findings suggest that the loss of KLF4 expression is a potentially significant indicator of EMT in
CRC.

Keywords: Krüppel-like factor 4, colorectal cancer, epithelial-mesenchymal transition

INTRODUCTION
Colorectal cancer (CRC) is the third leading cause of the cancer-related deaths often with metastasis to
[1,2]
the liver, lung, and bone . Epithelial-mesenchymal transition (EMT) is a transdifferentiation process
that allows a polarized epithelial cell to assume a mesenchymal cell phenotype, characterized by
enhanced migratory and invasiveness capacity, elevated resistance to apoptosis, and increased synthesis
[3]
of extracellular matrix (ECM) components . During the EMT process, epithelial cells lose apical-basal
polarity that is accompanied by reorganization of cytoskeleton and reprogramming of the signaling
pathways that allow for an increase in motility and the development of an invasive phenotype. This
multistep complex process is characterized by modifications in the expression of a host of transcription
factors and specific cell-surface proteins, as well as reorganization and expression of cytoskeletal
[3]
proteins, and production of enzymes that degrade the ECM . A change in some of these factors, such
as upregulation of TWIST, SNAI1, SNAI2, ZEB1, vimentin, and N-cadherin, and downregulation of
[4,5]
E-cadherin and tight junction proteins such as ZO-1 is indicative of progression of EMT . In CRC, EMT
has been strongly associated with the invasive and metastatic phenotype, thereby generating the life-
threatening manifestations of metastatic disease cancer. The activation of the EMT program has been
suggested to be the critical mechanism for the acquisition of malignant phenotypes by epithelial cancer
[3]
cells .

Krüppel-like factor 4 (KLF4) belongs to the family of zinc-finger transcriptions factors that play critical
roles during development, proliferation, differentiation, and homeostasis, as well as development and
progression of many diseases including inflammation and carcinogenesis [6-8] . In the digestive tract,
KLF4 is predominantly expressed in differentiated cells of the villus and surface epithelium of the small
intestine and colon, respectively [9-13] . Importantly, evidence indicates that KLF4 functions as a tumor
suppressor that inhibits progression of CRC . It has been shown that loss of KLF4 expression is associated
[12]
with the early stage of CRC development and that KLF4 is a prognostic indicator for CRC survival and
recurrence [14,15] . Recently, we demonstrated that KLF4 also plays a protective role against tumor formation
during inflammation-induced colorectal tumorigenesis [16-18] . The biochemical mechanisms triggering the
acquisition of the invasive phenotype and the subsequent systemic spread of the cancer cell have been
areas of intensive research. Here, we demonstrate that KLF4’s role in colorectal tumorigenesis extends to its
ability to regulate EMT.

METHODS
Samples from patients
Surgical specimens of resected colorectal cancer specimen obtained from Stony Brook University and
State University of New York Downstate were used in this study. In total, 12 specimens were processed
for immunofluorescence. All samples were of Caucasian origin, with 2 female and 10 male. One sample
was qualified as stage I, one as stage 2, two as stage 3, and eight as stage IV [Table 1]. The protocol for the
sample collection was originally approved by the Institutional Review Board by the State University of