Abstract
Hyperactivation of transforming growth factor-β (TGF-β) signaling pathway is a common feature of hepatocellular carcinoma (HCC) progression. However, the driver factors leading to enhanced TGF-β activity are not well characterized. Here, we explore the mechanisms that loss of Krüppel-like factor 4 (KLF4) exacerbates oncogenic TGF-β signaling in human HCC. The expression of KLF4 and TGF-β signaling components in primary HCC and their clinicopathologic relevance and significance was evaluated by using tissue microarray and immunohistochemistry. Cellular and molecular impacts of altered KLF4 expression and TGF-β signaling were determined using immunofluorescence, western blot, reverse-transcriptase PCR, chromatin immunoprecipitation and promoter reporter assays. Loss of KLF4 expression in primary HCC closely correlated with decreased Smad7 expression, increased p-Smad2/3 expression and independently predicts reduced overall and relapse-free survival after surgery. TGF-β signaling components were expressed in most HCC cells, and activation of TGF-β signaling promoted cell migration and invasion. Enforced KLF4 expression blocked TGF-β signal transduction and inhibited cell migration and invasion via activation of Smad7 transcription, whereas deletion of its C-terminal zinc-finger domain diminished this effect. KLF4 protein physically interacts with the Smad7 promoter. Promoter deletion and point mutation analyses revealed that a region between nucleotides –15 bp and –9 bp of the Smad7 promoter was required for the induction of Smad7 promoter activity by KLF4. Our data indicate that KLF4 suppresses oncogenic TGF-β signaling by activation of Smad7 transcription, and that loss of KLF4 expression in primary HCC may contribute to activation of oncogenic TGF-β signaling and subsequent tumor progression.
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Abbreviations
- ChIP:
-
chromatin immunoprecipitation
- FFPE:
-
formalin-fixed paraffin-embedded
- KLF4:
-
Krüppel-like factor 4
- EMT:
-
epithelial-to-mesenchymal transition
- ZDF:
-
zinc-finger domain
- PBS:
-
phosphate-buffered saline
- PBS-T:
-
PBS containing 0.1% Triton X-100
- PCR:
-
polymerase chain reaction
- qPCR:
-
quantitative PCR
- RT:
-
reverse transcription
- siRNA:
-
small interfering RNA
- TGF:
-
transforming growth factor
- TMA:
-
tissue microarray
- UTR:
-
untranslated region.
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Acknowledgements
We thank Don Norwood for editorial comments. The work is supported in part by grants R01-CA129956 and R01-CA148954 from the National Institutes of Health (K Xie), grant #81101623 from the National Natural Science Foundation of China and grant #81101623 and #81672846 from Science and Technology Commission of Shanghai Municipality (H Sun).
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Sun, H., Peng, Z., Tang, H. et al. Loss of KLF4 and consequential downregulation of Smad7 exacerbate oncogenic TGF-β signaling in and promote progression of hepatocellular carcinoma. Oncogene 36, 2957–2968 (2017). https://doi.org/10.1038/onc.2016.447
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DOI: https://doi.org/10.1038/onc.2016.447
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