Krüppel-like Factor 4 Promotes Esophageal Squamous Cell Carcinoma Differentiation by Up-regulating Keratin 13 Expression

Abstract

Squamous cell differentiation requires the coordinated activation and repression of genes specific to the differentiation process; disruption of this program accompanies malignant transformation of epithelium. The exploration of genes that control epidermal proliferation and terminal differentiation is vital to better understand esophageal carcinogenesis. KLF4 is a member of the KLF family of transcription factors and is involved in both cellular proliferation and differentiation. This study using immunohistochemistry analysis of KLF4 in clinical specimens of esophageal squamous cell carcinoma (ESCC) demonstrated that decreased KLF4 was substantially associated with poor differentiation. Moreover, we determined that both KLF4 and KRT13 levels were undoubtedly augmented upon sodium butyrate-induced ESCC differentiation and G1 phase arrest. Conversely, silencing of KLF4 and KRT13 abrogated the inhibition of G1-S transition induced by sodium butyrate. Molecular investigation demonstrated that KLF4 transcriptionally regulated KRT13 and the expression of the two molecules appreciably correlated in ESCC tissues and cell lines. Collectively, these results suggest that KLF4 transcriptionally regulates KRT13 and is invovled in ESCC cell differentiation.

Keywords: Kruppel-like factor 4 (KLF4); cell cycle; differentiation; keratin; transcription regulation.

FIGURE 1.

Reduced expression of KLF4 is associated with the dedifferentiation of ESCC. A, example case showed the expression of KLF4 in esophageal tumors by immunohistochemisry staining on the tissue microarray. Representative pictures of KLF4 in normal esophageal epithelium ➀, well differentiated ➁, moderately differentiated ➂, and poorly differentiated ➃ carcinoma tissues are shown (bar, 15 μm). Quantitative analysis of the KLF4 staining between ESCC tissues and the matched normal esophageal epithelia are shown on the right. B, KYSE150 cells were transiently transfected with pcDNA3.1 and pcDNA3.1-KLF4 vector. 48 h later, RT-PCR analysis was performed to examine mRNA expression of KLF4 and a selected group of terminal differentiation genes as indicated. GAPDH was used as internal control. The band intensities of mRNA expression level were quantified by densitometry and normalized against GAPDH. The data represent the means ± S.D. *, p < 0.05.

FIGURE 2.

Expression of KRT13 and KLF4 are correlated in esophageal squamous carcinoma cell lines and tissues. A, representative images of KRT13 staining are shown in normal esophageal epithelium ➀, well differentiated ➁, moderately differentiated ➂, and poorly differentiated ➃ carcinoma tissue sections (bar, 15 μm). Quantitative analysis of the KRT13 staining between ESCC tissues and the matched normal esophageal epithelia are shown on the right. B, statistically significant positive correlation between klf4 and KRT13 mRNA was observed by Pearson's method in esophageal squamous cell carcinoma cell lines and tissues in four independent published data sets (➀ GSE9982, ➁ GSE21293, ➂ GSE23400, and ➃ GSE33103). C, expression of KLF4 and KRT13 in a series of esophageal cancer cell lines was determined by RT-PCR (upper panel) and Western blot analysis (lower panel). D, KYSE150 cells were transiently transfected with the pcDNA3.1 and pcDNA3.1-KLF4 vector; cells were harvested, and Western blot was performed to measure the protein expression of KLF4 and KRT13. E, Western blot analysis showed the protein expression of KLF4 and KRT13 in KYSE30 and KYSE450 cells transfected with KLF4 siRNA pools (si-KLF4) or nontargeting control (nc). β-Actin serves as an internal control. The band intensities of KRT13 protein were quantified by densitometry and normalized against β-actin. The data represent the means ± S.D. *, p < 0.05.

FIGURE 3.

Up-regulation of KRT13 upon KLF4 is mediated through the GKRE in KRT13 promoter. A, GKRE resides at −411 to −399 bp upstream of the KRT13 ATG codon and is highly conserved in different species. B, luciferase reporter assay was performed using different vectors containing predicted KLF4-binding sites upstream of the KRT13. GKRE is indicated by arrowhead. Bars represent the means ± S.D. C, luciferase reporter assay was performed using increased amounts of pcDNA3.1-KLF4 as indicated. Bars represent the means ± S.D. D, EMSA were performed using 3 fmol of labeled oligonucleotides and 5 μg of nuclear extracts (NE) obtained from HEK293T cells transfected with pcDNA3.1-KLF4. Competitor oligonucleotides were added at 50- and 200-fold molar excess of the labeled probe. In experiments involving antibody, 2 μg of anti-KLF4 polyclonal antibody was added to the reaction. The positions of free probe (F) and shifted bands (S) are indicated. M1, M2, and M3 are the mutant oligonucleotides used as competitor DNA. E, KYSE150 cells were transfected with pcDNA3.1-KLF4, and 48 h later, ChIP assays were performed with antibodies against KLF4 or tubulin, IgG control, respectively. The presence of the KRT13 5′-flanking region containing GKRE or not was assayed by quantitative RT-PCR. Bars represent the means ± S.D. F, luciferase reporter assay was performed using different vectors containing wild-type (WT) or mutant-type (MT) KLF4-binding sites upstream of the KRT13. Putative KLF4-responsive sequences (AAGG) are underlined. Mutant of the KLF4-binding site is represented by shading. Bars represent the means ± S.D. *, p < 0.05.

FIGURE 4.

KLF4 and KRT13 expression are regulated during SB and calcium-induced esophageal cancer cell differentiation. A, KYSE150 cells were incubated with 4 mm SB for the indicated time, and the expression of KLF4 and KRT13 was determined by Western blot. B, KYSE150 cells were incubated with increasing concentrations (0–5 mm) of SB for 48 h. The expression of KLF4 and KRT13 was determined by Western blot. C, KYSE150 and KYSE450 cells were incubated with 2.4 mm CaCl₂ for the indicated times, and the expression of KLF4 and KRT13 was determined by Western blot.

FIGURE 5.

KRT13 promotes SB-induced cell growth inhibition. A, empty vector or KRT13-transfected KYSE150 cells were seeded at 1 × 10⁵ cells per well in conventional medium treated or untreated (control) with 4 mm SB on 6-well plates; cells were stained with propidium iodide and analyzed by flow cytometry at 48 h (lower panel), and Western blotting was performed to measure the protein expression of KRT13 (upper panel). B, cell viability of KRT13-transfected KYSE150 cells compared with empty vector-transfected KYSE150 cells treated or untreated (con) with 4 mm SB at selected time points. Bars represent the means ± S.D. C, KYSE450 cells were transfected with KRT13 siRNA (siKRT13) or nontargeting control (nc) and treated or untreated (control) with 4 mm SB for 48 h. Cell cycle distribution was assessed by flow cytometry. Bars represent the means ± S.D. (left panel). Western blot analysis shows the knockdown of KRT13 in KYSE450 cells (right panel). β-Actin served as an internal control. *, p < 0.05.

FIGURE 6.

KLF4 and KRT13 are required for SB-induced cell cycle arrest. A, KYSE450 cells transfected with KLF4 siRNA pools (si-KLF4) or nontargeting control (nc) treated or untreated with 4 mm SB for 48 h. Western blotting was performed to determine the expression of KLF4. β-Actin served as an internal control. B, KYSE450 cells transfected with KLF4 siRNA pools (siKLF4) or nontargeting control (nc) were seeded at 1.5 × 10⁵ cells per well in conventional medium and treated or untreated (control) with 4 mm SB on 6-well plates; cells were stained with propidium iodide and analyzed by flow cytometry at 48 h. Bars represent the means ± S.D. C, KYSE450 cells were cotransfected FLAG-KRT13 or vector with KLF4 siRNA (siKLF4) or nontargeting control (nc) and treated with 4 mm SB for 48 h. Western blot analysis showed the expression of KLF4 and KRT13 in KYSE450 cells. β-Actin served as an internal control. D, cell viability at selected time points of each indicated treatment was analyzed by MTS cell proliferation array. Bars represent the means ± S.D. *, p < 0.05.