The authors have declared that no competing interests exist.
Conceived and designed the experiments: YWC. Performed the experiments: ZSL HCC YCY. Analyzed the data: YWC BCL. Contributed reagents/materials/analysis tools: ZSL HCC YCY. Wrote the paper: YWC BCL.
Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor that plays an important role in differentiation and pathogenesis. KLF4 has been suggested to act as an oncogene or tumor suppressor in different tumor types. However, the role of KLF4 in hepatocellular carcinoma (HCC) remains unclear. Here, we demonstrate that forced expression of Klf4 in murine HCC cell lines reduced anchorage-independent growth in soft agar as well as cell migration and invasion activities
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most frequent cause of cancer-related mortality worldwide, with 6,000,000 new cases diagnosed annually
KLF4, also known as gut-enriched krüppel-like factor/GKLF or epithelial/endothelial zinc finger/EZF, is a member of the krüppel-like factor (KLF) transcription factor family. Members of the family contain three domains of krüppel-like zinc fingers. KLF4 can both activate and repress genes that are involved in cell-cycle regulation and differentiation in epithelium and rises in response to DNA damage, serum starvation, and contact inhibition
Importantly, multiple lines of evidence showed that KLF4 can function as an oncogene or a tumor suppressor depending on the type of cancer involved
Recent studies identifying transcriptional targets of KLF4 revealed that it promotes the expression of epithelial-specific proteins and inhibits the epithelial to mesenchymal transition (EMT), indicating that it may function to sustain an epithelial phenotype
Using
Evaluation of murine HCC cell lines with different cell migration activity identified reduced levels of Klf4 mRNA and protein in HCC cells with high migration ability (
(A) Klf4 and β-actin protein levels were detected in murine HCC cell lines, MM189 with ectopic Klf4 expression (MM189 PB-Klf4) and its corresponding control (MM189 PB) by immunoblot assay. β-actin served as a loading control. (B) Representative anchorage-independent growth activity for MM189 cells with ectopic Klf4 expression (MM189 PB-Klf4) and its corresponding control (MM189 PB). The colonies were observed at lower magnification (40×) in the left panel. The relative activity was determined by normalizing the mean number of colonies in MM189 PB-Klf4 cells to that in MM189 PB cells. Bar, SE. *, p<0.05. (C) Representative data shows the relative migration activity of MM189 expressing Klf4 (MM189 PB-Klf4) and its vector control (MM189 PB). The migrated cells were observed at magnification (100×) in the left panel. The relative migration activity was defined by normalizing the mean of migrated cells/per field in MM189 PB-Klf4 cells to that in MM189 PB cells. Bar, SE. ***, p<0.001. (D) Representative data shows the relative invasion activity of MM189 expressing Klf4 (MM189 PB-Klf4) and its vector control (MM189 PB). The invaded cells were observed at magnification (100×) in the left panel. The relative invasion activity was defined by normalizing the mean of invaded cells/per field in MM189 PB-Klf4 cells to that in MM189 PB cells. Bar, SE. *, p<0.05.
To ascertain whether the effect of Klf4 on HCC anchorage-independent growth, migration and invasion correlated with
(A) Quantification of the weight of the tumor lesions in mice (n = 7) subcutaneously injected with MM189 PB-Klf4 or MM189 PB cells. Bar, SE. *, p<0.05. (B) The representative field for detection of Ki-67 expression by immunohistochemistry under the light microscope with 200× magnification in the left panel. The percentage of positive Ki-67 stain was defined as the intensity of positive nuclei divided by that of the total nuclei in the field. Bar, SE. ***, p<0.001. (C) Representative lung fields of nude mice after the delivery, via tail vein injection, of MM189 cells with ectopic Klf4 expression (MM189 PB-Klf4) or vector controls (MM189 PB). The boxed area in the upper panel was shown with macroscopic view. The upper panel was observed at lower magnification (40×) and the lower was for higher magnification (100×). (D) Quantification of weight of the lung lesions in mice (n = 6) injected with MM189 PB-Klf4 or MM189 PB cells. Bar, SE. *, p<0.05. (E) Quantification of total areas of the tumor lesions in lungs of mice (n = 6) injected with MM189 PB-Klf4 or MM189 PB cells. Bar, SE. *, p<0.05.
Interestingly, we observed that ectopic Klf4 expression in MM189 and BL322 cells altered the cell shape to a more epithelial morphology (
(A) Ectopic Klf4 expression shifts cell morphology from a mesenchymal- to an epithelial phenotype. Phase contrast microscopy with 200× magnification (upper panel). Note the cobblestone appearance of the Klf4-expressing cells. Cytoskelton F-actin proteins were stained with rodamine-phalloidin and viewed under fluorescence microscope with 630× magnification (lower panel, shown in grey mode). (B) Immunoblot analysis of epithelial and mesenchymal proteins in MM189 PB and MM189 PB-Klf4 cells. BL185 cells and 3T3L1 cells served as positive controls for the expression of E-cadherin and Vimentin, respectively. α-tubulin served as a loading control. (C) Quantitative RT-PCR demonstrated the relative mRNA levels for E-cadherin (Cdh1) and epithelial mesenchymal transition (EMT)-related transcription factors in MM189 PB-Klf4 and MM189 PB cells. All amplifications were normalized to an endogenous β-actin control. For each gene, the relative expression of mRNA in MM189 PB-Klf4 cells was normalized to that in MM189 PB cells. Bar, SE. **, p<0.01; ***, p<0.001. (D) Immunoblot analysis of Twist, Snail and Slug in MM189 PB and MM189 PB-Klf4 cells. α-tubulin served as a loading control.
The krüppel-like family of transcription factors regulate a diverse set of genes through direct binding to GC-rich promoter regulatory regions containing the CACCC consensus sequence
(A) Schematic representation of
To determine whether suppression of Slug expression is required for the phenotypes associated with ectopic Klf4 expression. MM189 PB-Klf4 cells were infected with a retroviral vector expressing mouse Slug, or empty vector, and ectopic Slug expression confirmed by immunoblot assay (
(A) Slug protein level was detected in HCC cell lines, MM189 with only ectopic Klf4 (MM189 PB-Klf4/PB) and MM189 with both Klf4 and Slug expression (MM189 PB-Klf4/PB-Slug) by immunoblot assay. α-tubulin served as a loading control. (B) Observations of morphological change by the simultaneous ectopic expression of Slug and Klf4 in MM189 cells from epithelial- to mesenchymal-like shape under phase contrast microscopy with 200× magnification (upper panel). Cytoskelton F-actin proteins were stained with rodamine-phalloidin and viewed under fluorescence microscope with 630× magnification (lower panel, shown in grey mode). (C) Immunoblot analysis of mesenchymal and epithelial proteins in MM189 PB-Klf4/PB and MM189 PB-Slug/PB-Klf4 cells. α-tubulin served as a loading control. (D) Representative data shows the relative migration activity of MM189 cells expressing Klf4/Slug (MM189 PB-Klf4/PB-Slug) and its vector control (MM189 PB-Klf4/PB). The migrated cells were observed at magnification (100×) in the upper panel. The relative migration activity was defined by normalizing the mean of migrated cells/per field in MM189 PB-Klf4/PB-Slug cells to that in MM189 PB-Klf4/PB cells. Bar, SE. ***, p<0.001. (E) Quantification of weight of the lung lesions in mice (n = 7) injected with MM189 PB-Klf4/PB or MM189 PB-Klf4/PB-Slug cells. Bar, SE. *, p<0.05. (F) Quantification of tumor area of the lung lesions in mice (n = 7) injected with MM189 PB-Klf4/PB or MM189 PB-Klf4/PB-Slug cells (p = 0.065). Bar, SE. (G) Quantification of the weight of the tumor lesions in mice (n = 12) subcutaneously injected with MM189 PB-Klf4/PB or MM189 PB-Klf4/PB-Slug cells. Bar, SE.
To confirm the relationship between KLF4 expression levels and HCC pathogenesis, we confirmed whether down-regulation of KLF4 was found in human HCC. We analyzed KLF4 expression profiles using existing cDNA microarray data sets deposited in Oncomine
(A) Decreased KLF4 mRNA levels in HCC tissues (N = 225) in comparison with normal liver tissues (N = 220)
KLF4 was identified as a tumor suppressor with loss of expression in a series of cancers
Several pieces of data presented in this manuscript strongly support the hypothesis that KLF4 acts as a tumor suppressor in HCC. Ectopic Klf4 expression decreased anchorage-independent growth of HCC cells in culture, as well as their tumorigenic growth in vivo. This reduced tumor growth was associated with decreased staining for the proliferation marker Ki-67. More recently, KLF4 has been shown to inhibit the migration and invasion activities in several cancer models, suggesting its potential role as a metastasis suppressor
Based on data mining using Oncomine and validation by qRT-PCR using a small collection of HCC samples, we have demonstrated that KLF4 mRNA is down-regulated in most of HCC tissues compared with normal liver tissues
While we did not observe a consistent effect of Klf4/KLF4 expression on cell cycle progression in HCC cell lines (
The reduction of Slug mRNA observed with ectopic Klf4 expression led us to examine the ability of Klf4 to transcriptionally regulate Slug gene expression. Using both ChIP and luciferase reporter assays, we found that endogenous Klf4-containing transcription complex binds to and represses the Slug promoter. Our current results suggest the possibility that Klf4 indirectly repress the promoter through interactions with other transcriptional repressor. This location (−300 bp) was different from that by software prediction, indicating the possibility that the down-regulated Slug by Klf4 was mediated by interaction with other transcription repressors but not by directly binding to the Slug promoter. Moreover, our data did not rule out the possibility that unpredicted Klf4 binding site was located within −300 bp. Additional studies will be needed to provide these mechanistic details.
In KLF family, down-regulation of KLF6, an early event of hepatocarcinogenesis, was also demonstrated to contribute to pathogenesis of HCC
In summary, our data demonstrate that KLF4 acts as a tumor suppressor in HCC, at least in part by repressing SLUG expression. Whereas further studies are required to characterize the reciprocal regulation between KLF4 and SLUG as well as the mechanisms leading to down-regulation of KLF4 in HCC, our findings provide new insights into a potential role and mechanism by which KLF4 inhibits tumorigenesis and metastasis of HCC.
All animal studies were performed in strict accordance with the recommendations in the guidelines for the Care and Use of Laboratory Animals of National Health Research Institutes, Taiwan. The protocol was approved by the Institutional Animal care and Use Committee of National Health Research Institutes (Protocol No: NHRI-IACUC-100047-A and NHRI-IACUC-100136-A). Animals were housed with abundant food and water. All efforts were made to minimize suffering.
HCC tumor specimens were obtained from Taiwan Liver Cancer Network (TLCN). Informed consent was obtained from each patient before surgery. The study protocol (Protocol No: EC1001207) was viewed and approved by the Institutional Review Board of National Health Research Institutes and the user committee of TLCN.
Total RNA from 10 pairs of HCC tumor specimens and their tumor-adjacent tissues were obtained from TLCN. Clinical parameters and pathological features were provided by TLCN.
The MM189, BL322 and BL185 murine HCC cell lines have been previously described
All complementary DNA (cDNA) expression constructs were generated in pBABE-puro or pBABE-neo expression vectors (Addgene). cDNA encoding wild type mouse Klf4, human KLF4 and mouse Slug was generated by reverse transcription PCR (RT-PCR) amplification of RNA isolated from mouse or human HCC cell lines using the Superscript III first strand synthesis system (Invitrogen) according to the manufacturer’s protocol. The primers for amplified cDNA are listed in
Immunoblot assay was performed as previously described
Soft agar assays were performed as previously described
Migration and invasion assays were performed in transwell assay as described in our previous studies
Subcutaneous tumor growth and lung colonization assays were conducted as described previously
The sections were dewaxed in xylene and rehydrated in alcohol series of a decreasing concentration. The sections were heated by microwave for 20 min in citrate buffer (pH 6.0) for antigen retrieval and incubated with 3% hydrogen peroxide for blocking the endogenous peroxidase activity. After incubation in blocking serum for 20 min, the samples were reacted with anti-Ki-67 (NCL-Ki67p, Novacastra laboratories) or E-cadherin (BD Bioscience) antibodies for 30 min according to the instructions (Vector Lab). Chromogenic detection was performed with Vector ABC Kit (Vector Lab). Sections were counterstained with hematoxylin and viewed under light-field microscope. Using the imaging software (Image J, NIH), the percentage of positive Ki-67 stain was defined as the total intensity of positive nuclei of tumor cells divided by that of the total nuclei in the field (original magnification X200).
Cells on collagen–coated slips were fixed and followed the protocols as previously described
Total RNA was isolated from cell lines as previous described
293T cells were transfected in 6-well plates, using Polyjet transfection reagent (SignaGen lab) according to the manufacturer’s protocol, with
Chromatin immunoprecipitation (ChIP) assay was performed using the mouse Klf4 Chromatin Immunoprecipitation Kit (R&D). Cells were grown to near confluency on 10-cm dishes and cross-linked, lysed, followed by sonication in lysis buffer. After centrifugation, some supernatant was processed as the input after DNA purification. The reminder of the samples were immunoprecipitated overnight with specific antibodies to Klf4 or control IgG, then streptavidin conjugated agarose beads (Sigma) added and incubated for 1 hour. After washing and reversal of cross-links, followed by phenol-chloroform extraction and isopropanol precipitation, DNA was suspended in 50 µl of sterile H2O. PCR was performed using 5 µl of immunoprecipitated DNA as template and the following gene specific primers corresponding to –225/−436 of the mouse
Data were expressed as mean±standard error of the mean (SE). The unpaired 2-tailed
Reduced Klf4 expression in cells with high migration activity. (A) Relative migration activity of two murine HCC cell lines. The relative migration activity was defined by normalizing the mean of migrated cells/per field in MM189-B cells to that in MM189-A cells. Bar, SE. ***, p<0.001. (B) Quantitative RT-PCR demonstrating the relative mRNA levels of Klf4 in two murine HCC cell lines. All amplifications were normalized to an endogenous β-actin control. The relative expression of Klf4 mRNA in MM189-B cells was normalized to that in MM189-A cells. Bar, SE. ***, p<0.001. (C) Protein levels of Klf4 and β-actin were detected in two murine HCC cell lines by immunoblot assay. β-actin served as a loading control.
(TIF)
Ectopic Klf4 expression inhibited colony formation, migration and invasion in BL322 cells. (A) Klf4 and β-actin protein levels were detected in murine HCC cell line BL322 with ectopic Klf4 expression (BL322 PB-Klf4) and its corresponding control (BL322 PB) by immunoblot assay. β-actin served as a loading control. (B) Representative anchorage-independent growth activity for BL322 cells with ectopic Klf4 expression (BL322 PB-Klf4) and its corresponding control (BL322 PB). The colonies were observed at lower magnification (40×) in the left panel. The relative activity was determined by normalizing the mean number of colonies in BL322 PB-Klf4 cells to that in BL322 PB cells. Bar, SE. *, p<0.05. (C) Representative data shows the relative migration activity of BL322 cells expressing Klf4 (BL322 PB-Klf4) and its vector control (BL322 PB). The relative migration activity was defined by normalizing the mean of migrated cells/per field in BL322 PB-Klf4 cells to that in BL322 PB cells. Bar, SE. *, p<0.05. (D) Representative data shows the relative invasion activity of BL322 cells expressing Klf4 (BL322 PB-Klf4) and its vector control (BL322 PB). The relative invasion activity was defined by normalizing the mean of invaded cells/per exp in BL322 PB-Klf4 cells to that in BL322 PB cells. Bar, SE. *, p<0.05.
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Knockdown of Klf4/KLF4 enhanced cell migration in HCC cell lines. (A) Klf4 and α-tubulin protein levels were detected in MM189 with Klf4 shRNA expression (MM189 Klf4 sh) and vector controls (MM189 PLKO-GFP) by immunoblot assay. α-tubulin served as a loading control. (B) Relative migration activity of MM189 with Klf4 knockdown (MM189 Klf4 sh) and its vector control (MM189 PLKO-GFP). The relative migration activity was defined by normalizing the mean of migrated cells/per field in MM189 Klf4 sh cells to that in MM189 PLKO-GFP cells. Bar, SE. ***, p<0.001. (C) KLF4 and β-actin protein levels were detected in PLC5 cells with KLF4 shRNA expression (PLC5 KLF4 sh) and vector controls (PLC5 PLKO-GFP) by immunoblot assay. β-actin served as a loading control. (D) Relative migration activity of PLC5 with KLF4 knockdown (PLC5 KLF4 sh) and its vector control (PLC5 PLKO-GFP). The relative migration activity was defined by normalizing the mean of migrated cells/per field in PLC5 KLF4 sh cells to that in PLC5 PLKO-GFP cells. Bar, SE. ***, p<0.001.
(TIF)
Induction of morphological change by ectopic Klf4 expression in BL322 cells. (A) Observations of morphological change by ectopic expression of Klf4 from a mesenchymal- to an epithelial phenotype under phase contrast microscopy with 200× magnification (left panel). Cytoskelton F-actin proteins were stained with rodamine-phalloidin and viewed under fluorescence microscope with 630× magnification (right panel, shown in grey mode). (B) Quantitative RT-PCR demonstrating the relative mRNA levels for E-cadherin (Cdh1) and epithelial mesenchymal transition (EMT)-associated transcription factors in Klf4 expressing cells (BL322 PB-Klf4) and vector controls (BL322 PB). All amplifications were normalized to an endogenous β-actin control. The relative expression of mRNA in BL322 PB-Klf4 cells was normalized to that in BL322 PB cells. Bar, SE. *, p<0.05; **, p<0.01. (C) Immunoblot analysis of Twist, Snail and Slug in BL322 PB and BL322 PB-Klf4 cells. α-tubulin served as a loading control.
(TIF)
Ectopic expression or knockdown of Klf4/KLF4 changed the E-cadherin and Slug/SLUG mRNA expression in HCC cell lines. (A) KLF4 and α-tubulin protein levels were detected in human HCC cell line HuH-7 with ectopic KLF4 expression (HuH-7 PB-KLF4) and its corresponding control (HuH-7 PB) by immunoblot assay. α-tubulin served as a loading control (upper panel). Quantitative RT-PCR demonstrating the relative mRNA levels of CDH1, SNAIL and SLUG in HuH-7 cells with ectopic KLF4 expression (HuH-7 PB-KLF4) and vector controls (HuH-7 PB). All amplifications were normalized to an endogenous β-actin control. The relative expression of mRNA in HuH-7 PB-KLF4 cells was normalized to that in HuH-7 PB cells. Bar, SE. *, p<0.05. (B) KLF4 and α-tubulin protein levels were detected in human HCC cell line SK-HEP1 with ectopic KLF4 expression (SK-HEP1 PB-KLF4) and its corresponding control (SK-HEP1 PB) by immunoblot assay. α-tubulin served as a loading control (upper panel). Quantitative RT-PCR demonstrating the relative mRNA levels of CDH1, SNAIL and SLUG in SK-HEP1 cells with ectopic KLF4 expression (SK-HEP1 PB-KLF4) and vector controls (SK-HEP1 PB). All amplifications were normalized to an endogenous β-actin control. The relative expression of mRNA in SK-HEP1 PB-KLF4 cells was normalized to that in SK-HEP1 PB cells. Bar, SE. **, p<0.01; ***, p<0.001. (C) Quantitative RT-PCR demonstrating the relative mRNA levels of Cdh1, Snail and Slug in MM189 cells with Klf4 knockdown (MM189 Klf4 sh) and vector controls (MM189 PLKO-GFP). All amplifications were normalized to an endogenous β-actin control. The relative expression of mRNA in MM189 Klf4 sh cells was normalized to that in MM189 PLKO-GFP cells. Bar, SE. *, p<0.05; ***, p<0.001. (D) Quantitative RT-PCR demonstrating the relative mRNA levels of CDH1, SNAIL and SLUG in PLC5 cells with KLF4 knockdown (PLC5 KLF4 sh) and its vector controls (PLC5 PLKO-GFP). All amplifications were normalized to an endogenous β-actin control. The relative expression of mRNA in PLC5 KLF4 sh cells was normalized to that in PLC5 PLKO-GFP cells. Bar, SE. **, p<0.01.
(TIF)
Effect of ectopic Klf4/KLF4 expression on cell cycle analysis by flow cytometry. (A) Ectopic Klf4 expression did not change the cell cycle in MM189 cells using propidium iodide staining. (B) Ectopic KLF4 expression did not change the cell cycle in HuH-7 cells using propidium iodide staining. (C) Ectopic KLF4 expression led to G1 arrest in SK-HEP1 cells using propidium iodide staining.
(TIF)
A trace expression of E-cadherin was induced by ectopic Klf4 expression in MM189 PB-Klf4 tumors. The representative field for detection of E-cadherin expression in positive and negative control tissue sections (upper panel), sections from MM189 PB and MM189 PB-Klf4 tumors (middle panel) by immunohistochemistry under the light microscope with 400× magnification. The lower panel was demonstrated that the representative field for detection of Klf4 expression in sections from MM189 PB and MM189 PB-Klf4 tumors by immunohistochemistry under the light microscope with 400× magnification.
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Materials and Methods.
(DOC)
The authors thank Zhi-Xiang Li and Ju-Ching Yu for assistance in construction of plasmids. We thank Dr. Cheng-Wen Wu for proving the