Kruppel-like factor 4 induces p27Kip1 expression in and suppresses the growth and metastasis of human pancreatic cancer cells

Abstract

The zinc finger transcription factor Krüppel-like factor 4 (KLF4) has been implicated in both tumor suppression and progression. However, its function in pancreatic cancer has not been well characterized. Here, we show that pancreatic cancer cell lines expressed various levels of KLF4 RNA and protein. Ectopic expression of KLF4 by FG and BxPC-3 pancreatic cancer cells resulted in cell cycle arrest and marked inhibition of cell growth in vitro and attenuation of tumor growth and metastasis in an orthotopic mouse model. Overexpression of KLF4 also led to significant induction of p27(Kip1) expression, at both the RNA and protein levels, in a dose- and time-dependent manner, indicating that KLF4 transcriptionally regulates the expression of p27(Kip1). Chromatin immunoprecipitation assays consistently showed that KLF4 protein physically interacts with the p27(Kip1) promoter. Promoter deletion and point mutation analyses indicated that a region between nucleotides -435 and -60 of the p27(Kip1) promoter and intact of the three KLF4-binding sites within that region were required for the full induction of p27(Kip1) promoter activity by KLF4. Our findings suggest that KLF4 transactivates p27(Kip1) expression and inhibits the growth and metastasis of human pancreatic cancer.

Figure 1

KLF4 suppresses human pancreatic cancer cell growth in vitro and in vivo. A, KLF4 expression in human pancreatic cancer cell lines at the mRNA level (by Northern blot analysis; A1) and at the protein level (by Western blot analysis; A2). B, FG and BxPC-3 cells were transduced with Ad-KLF4 or Ad-EGFP at MOIs of 20 (FG cells; B1) or 15 (BxPC-3 cells; B2) and assessed for proliferation by cell counting at 1 to 3 d after transduction. C, growth kinetics of KLF4-transduced FG cells (C1) and BxPC-3 cells (C2) injected into the subcutis of nude mice. D, BxPC-3 cells with reduced KLF4 protein expression (by Western blot analysis; D1) grew larger tumors in nude mice (D2, representative tumor sizes; D3, averages of tumor weight). Bars, SE. *, P < 0.05 versus Ad-EGFP or nontarget control (Non-T).

Figure 2

Altered KLF4 expressions affect cell cycle progression of pancreatic cancer cells. FG cells (A) or BxPC-3 cells (B) were transduced with Ad-KLF4 at the indicated MOI; Ad-EGFP was used as a control or to adjust the total MOI to be equal in each treatment condition. Cells were harvested at 40 h after transduction, fixed in 70% ethanol, and then stained with propidium iodide for FACS analysis. C, parental BxPC-3 cells and BxPC-3 cells stably carrying nontarget (Non-T) or specific target (T-16) shRNAs were cultured in DMEM containing 1% colony-stimulating factor for 24 h; cells were harvested and processed for FACS analysis. Bars, SE. *, P < 0.05 versus Ad-EGFP control.

Figure 3

KLF4 up-regulates p27^Kip1 expression in human pancreatic cancer cells. A, FG cells were transduced with adenoviral KLF4 or EGFP (control) at a MOI of 20 and expression of KLF4 and cell cycle–related proteins was assessed at the indicated times by Western blotting. B, p27^Kip1 expression was determined in FG and BxPC-3 cells at 48 h after transduction with Ad-KLF4 at indicated MOI as noted for Fig. 2. C, BxPC-3 cells were treated with control (scrambled) siRNA or the indicated volumes of KLF4 siRNA (with control siRNA used to adjust the total volume to 10.0 μL/well); p27^Kip1 expression was determined 48 h later by Western blotting. D, transduction of FG cells with an EGFP-KLF4 fusion protein followed by immunofluorescence showed colocalization of KLF4 and p27^Kip1 in the nuclei. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 4

KLF4 transcriptionally activates p27^Kip1 promoter. A, FG cells were transduced with adenoviral KLF4 or EGFP as described for Fig. 2, and KLF4 and p27^Kip1 expression was measured 36 h later by Northern blotting. B, a full-length p27^Kip1 promoter reporter (p27PF) was transfected into FG and BxPC-3 cells with pcDNA3.1-KLF4 or pcDNA3.1 (control) as shown and p27^Kip1 promoter activity was determined 48 h later by dual luciferase assay. Results are expressed as the “fold” changes of the RR of pcDNA3.1-KLF4 vector versus pcDNA3.1 control vector. C, a series of 5′ deletion mutants based on the 3,568-bp p27^Kip1 promoter were transfected into HEK 293 cells by using a KLF4 expression vector or control vector, and promoter activities were measured with a dual luciferase assay. Results are expressed as fold changes of the RR of KLF4 expression vector versus pcDNA3.1 control vector. Bars, SE. *, P < 0.05 versus the value of pcDNA3.1 control vector.

Figure 5

Binding of KLF4 to the p27^Kip1 promoter in vivo and in vitro. A, schematic structure of p27^Kip1 promoter. The locations and sequences of putative KLF4-binding sites and forward (P1-F) and reverse (P1-R) PCR primers flanking the p27^Kip1 promoter were shown. B1, binding of endogenous KLF4 to p27^Kip1 promoter. Chromatin fragments were prepared from BxPC-3 cells and ChIP assay was performed using a control IgG or anti-KLF4 antibody. Chromatin fragments without IgG or antibody were used as input controls. The PCR was performed using two sets of primers (P1, for p27^Kip1 promoter; P2, for p27^Kip1 3′-UTR as a control) as described in Materials and Methods. B2, binding of exogenous KLF4 to p27^Kip1 promoter. Chromatin fragments were prepared from FG cells (PBS), FG cells transduced with Ad-EGFP (Ad-EGFP), or Ad-KLF4 harboring FLAG-tagged KLF4 gene (Ad-KLF4) and ChIP assay was performed using a specific anti-FLAG antibody. The PCR was performed using two sets of primers (P1, for lanes 2–5 and 10–12; P2, for lanes 6–9 and 13–16). C, EMSA. Nuclear protein was extracted from BxPC-3 cells transduced with Ad-KLF4. Radioisotope-labeled oligo #3 corresponding to #3 KLF4-binding site on p27 promoter was used as a probe. The probe was incubated with nuclear protein in the presence of control IgG (lane 1), anti-Sp1 (lane 2), anti-KLF4 (lane 3), cold wild-type oligo #3 (1:50 ratio; lane 4), or mutant oligo #3 (1:50 ratio; lane 5). Shifted bands were marked as “Sp1,” “Sp3,” or “KLF4”; supershifted bands “SS” were indicated by arrows; and free probes were marked as “FP.” D, p27 promoter activity. The p27 promoter reporter constructs with site-directed mutagenesis of three putative KLF4-binding sites were transfected into BxPC-3 cells in triplicate with KLF4 expression vector or control pcDNA3 vector and incubated for 48 h. The relative p27 promoter activities were determined and the activities in KLF4 groups were expressed as fold changes of that in their respective controls. This was a representative experiment of three with similar results. *, statistical significance (P < 0.01) compared with respective control groups.