Krüppel-like factor 8 induces epithelial-to-mesenchymal transition and promotes invasion of pancreatic cancer cells through transcriptional activation of four and a half LIM-only protein 2

Abstract

Pancreatic cancer (PC) is one of the most aggressive types of cancer with an extremely poor prognosis. Invasive growth and early metastasis is one of the greatest challenges to overcome for the treatment of PC. Numerous previous studies have indicated that the transcription factor Krüppel-like factor 8 (KLF8) and nuclear cofactor four and a half LIM-only protein 2 (FHL2) serve important roles in tumorigenesis and tumor progression; however, their roles in PC remain elusive. The present study revealed that KLF8 and FHL2 expression is aberrantly co-overexpressed in PC tissue samples and associated with tumor metastasis. Furthermore, a positive correlation between the expression levels of KLF8 and FHL2 was observed. Subsequently, the present study identified KLF8 as a critical inducer of epithelial-to-mesenchymal transition (EMT) and invasion. Of note, the present study demonstrated that KLF8 overexpression induced a strong increase in FHL2 expression, and subsequent promoter reporter assays determined that KLF8 directly bound and activated the FHL2 gene promoter. Furthermore, FHL2 knockdown in KLF8-overexpressing cells partially reversed the EMT and invasive phenotypes. The present study identified KLF8-induced FHL2 activation as a novel and critical signaling mechanism underlying human PC invasion.

Keywords: Krüppel-like factor 8; epithelial-to-mesanchymal transition; four and a half LIM-only protein 2; invasion; pancreatic cancer.

Figure 1.

Expression levels of KLF8 and FHL2 in PC tissue samples and cell lines. Patients with PC with lymph node metastases exhibited higher (A) KLF8 and (B) FHL2 mRNA expression levels compared with those exhibited by patients without lymph node metastases (*P<0.001). (C) WB analysis of KLF8 and FHL2 protein expression levels in pancreatic cell lines: Miapaca-2, Capan-1, Bxpc-3, Panc-1 and SW1990. GAPDH was used as the internal control. (D) The patients with high expression levels of KLF8 also demonstrated high FHL2 expression levels. (E) Protein expression levels of KLF8 and FHL2 in tumor samples of patients with PC with or without lymph node metastasis by WB analysis. GAPDH expression levels were used as internal controls. KLF8, Krüppel-like factor 8; FHL2, four and a half LIM-only protein 2; PC, pancreatic cancer; WB, western blot.

Figure 2.

KLF8 upregulates FHL2 expression to induce EMT and promote cell invasion. (A) KLF8, FHL2, E-cadherin and Slug expression levels in stable KLF8 transfectants in Panc-1 cells were detected by WB. GAPDH was used as the internal control. (B) Quantification of an invasion assay performed with or without KLF8 transfection. (C) Expression of epithelial-to-mesenchymal transition-associated biomarkers, including E-cadherin, Slug and FHL2, were detected by WB 72 h following transfection with KLF8-scr or KLF8-FHL2 siRNA. (D) Quantification of an invasion assay performed with KLF8-scr or KLF8-FHL2 siRNA. Invasive potential of Panc-1 stable transfectants following transfection with (E) vector, (F) KLF8, (G) KLF8-scr siRNA and (H) KLF8-FHL siNRA. Morphology of Panc-1 cells transfected with (I) vector, (J) KLF8, (K) KLF8-scr siRNA and (L) KLF8-FHL2-siRNA cells, visualized by phase-contrast microscopy. Magnification, ×100. *P<0.05 vs. other group. KLF8, Krüppel-like factor 8; FHL2, four and a half LIM-only protein 2; EMT, epithelial-to-mesenchymal transition; scr, scrambled; siRNA, short interfering RNA; WB, western blotting.

Figure 3.

FHL2 is a direct transcriptional activation target of KLF8. (A) The luciferase reporter constructs contained the FHL2 promoter with potential KLF8 binding sites upstream of a luciferase gene. (B) Transcriptional activities of reporters in the transient transfected cells were detected using a dual-luciferase assay, and the results are expressed as fold-change of relative luciferase units. *P<0.001; ^#P>0.05. (C) Primer sequences used for site-directed mutations. Site-directed mutagenesis of GT-box in pLuc55 was performed to generate mutations, and the mutated nucleotides were marked with square frames. WT, wild-type; MT, mutant; KLF8, Krüppel-like factor 8; FHL2, four and a half LIM-only protein 2; EMT, epithelial-to-mesenchymal transition; luc, luciferase.