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. 2014 Oct 28;111(43):15526-31.
doi: 10.1073/pnas.1407717111. Epub 2014 Oct 13.

Role of pyruvate kinase M2 in transcriptional regulation leading to epithelial-mesenchymal transition

Atsushi Hamabe  1 , Masamitsu Konno  2 , Nobuhiro Tanuma  3 , Hiroshi Shima  3 , Kenta Tsunekuni  4 , Koichi Kawamoto  1 , Naohiro Nishida  2 , Jun Koseki  5 , Koshi Mimori  6 , Noriko Gotoh  7 , Hirofumi Yamamoto  8 , Yuichiro Doki  9 , Masaki Mori  10 , Hideshi Ishii  11
Affiliations

Role of pyruvate kinase M2 in transcriptional regulation leading to epithelial-mesenchymal transition

Atsushi Hamabe et al. Proc Natl Acad Sci U S A. .

Abstract

Pyruvate kinase M2 (PKM2) is an alternatively spliced variant of the pyruvate kinase gene that is preferentially expressed during embryonic development and in cancer cells. PKM2 alters the final rate-limiting step of glycolysis, resulting in the cancer-specific Warburg effect (also referred to as aerobic glycolysis). Although previous reports suggest that PKM2 functions in nonmetabolic transcriptional regulation, its significance in cancer biology remains elusive. Here we report that stimulation of epithelial-mesenchymal transition (EMT) results in the nuclear translocation of PKM2 in colon cancer cells, which is pivotal in promoting EMT. Immunoprecipitation and LC-electrospray ionized TOF MS analyses revealed that EMT stimulation causes direct interaction of PKM2 in the nucleus with TGF-β-induced factor homeobox 2 (TGIF2), a transcriptional cofactor repressor of TGF-β signaling. The binding of PKM2 with TGIF2 recruits histone deacetylase 3 to the E-cadherin promoter sequence, with subsequent deacetylation of histone H3 and suppression of E-cadherin transcription. This previously unidentified finding of the molecular interaction of PKM2 in the nucleus sheds light on the significance of PKM2 expression in cancer cells.

Keywords: colorectal cancer; epithelial–mesenchymal transition; invasion; pyruvate kinase M2; transforming growth factor-β–induced factor homeobox 2.

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Conflict of interest statement

Conflict of interest statement: This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology; a Grant-in-Aid from the Third Comprehensive 10-year Strategy for Cancer Control, Ministry of Health, Labor, and Welfare; a grant from the Kobayashi Cancer Research Foundation; a grant from the Princess Takamatsu Cancer Research Fund, Japan; a grant from the National Institute of Biomedical Innovation; and a grant from the Osaka University Drug Discovery Funds. A.H. is a research fellow of the Japan Society for the Promotion of Science. Partial support was received from Taiho Pharmaceutical Co., Ltd. (to J.K., M.M., and H.I.), Chugai Co., Ltd., Yakult Honsha Co., Ltd., Merck Co., Ltd., Takeda Science Foundation, and Takeda Medical Research Foundation (to M.K., N.N., M.M., and H.I.) through institutional endowments.

Figures

Fig. 1.
Fig. 1.
PKM2 translocates into the nucleus during EMT. (A) Schematic representation of the procedure for EMT induction. The cells incubated for 48 h after seeding are defined as pre-EMT, and the cells cultured with 2.5 ng/mL TGF-β1 and 10 ng/mL EGF are defined as post-EMT. (B) Photomicrographs of the morphological change in SW480 cells. The cells were stained using the Diff-Quik Kit (Sysmex Corp.). The number of hours indicates the period since EMT induction was initiated. (Scale bar, 100 μm.) (C) Relative transcript (mRNA) levels of CDH1, VIM, ZEB1, and SNAI2 after induction of EMT for 0, 48, and 96 h. The values at 0 h (pre-EMT) have been normalized to 1, and the data are expressed as fold. (D) Relative mRNA levels of PKM1, PKM2, and pyruvate kinase (total PK) after induction of EMT for 0, 48, and 96 h. (E) Western blot assays of E-cadherin, vimentin, and PKM2 expression in pre-EMT and post-EMT cells. Post-EMT cells were harvested at 72 h. (F) Western blot assays of PKM1, PKM2, α-tubulin, and histone H3 in nuclear and cytoplasmic lysates prepared from SW480 cells. With normalization to cytoplasmic tubulin or nuclear histone H3 blots, the relative intensities of PKM2 blots are shown in comparison with those in the pre-EMT condition. (G) SW480 cells were treated with dimethyl suberimidate for 30–60 min, immediately followed by whole cell lysis. The monomer and dimer states of PKM2 were analyzed by Western blot assay. Columns represent the average of at least three independent experiments; error bars represent the SD of the mean from triplicate results. *P < 0.05.
Fig. 2.
Fig. 2.
PKM2 is required for EMT induction. (A) Phase-contrast photomicrographs of SW480 cells transfected with siControl or siPKM2 after EMT induction for 48 h. (B) Relative transcript (mRNA) levels of CDH1 and VIM after EMT induction in cells transfected with siControl or siPKM2 for 48 h. (C) Western blot assays of E-cadherin, vimentin, PKM2, and β-actin expression in pre-EMT and post-EMT cells. Post-EMT cell samples were harvested at 72 h. With normalization to β-actin as a control, the relative intensities of E-cadherin and vimentin are shown in comparison with those in the control pre-EMT condition. Note that siPKM2 knockdown works efficiently in post-EMT cells. (D) Invasive behavior of SW480 cells treated with siControl or siPKM2. (E) Schematic procedure for establishing PKM1 OE or PKM2 OE SW480 cells. (F) Western blot assays of PKM1, PKM2, and β-actin expression in WT SW480 cells, cells stably expressing shRNA constructs targeting pyruvate kinase (shPK), and shPK cells overexpressing either PKM1 or PKM2 constructs. (G) Relative mRNA levels of CDH1, VIM, and ZEB1 after EMT induction in PKM1 OE or PKM2 OE SW480 cells for 72 h. (H) Western blot assays of E-cadherin, vimentin, and β-actin expression in PKM1 OE and PKM2 OE cells. Post-EMT cell samples were harvested at 72 h. Column values = average of at least three independent experiments; error bars represent SD from the mean of triplicate experiments. *P < 0.05.
Fig. 3.
Fig. 3.
Interaction between nuclear PKM2 and TGIF2 mediates EMT induction. (A) Polyacrylamide gel electrophoresis of proteins immunoprecipitated with anti-PKM2 antibody in the nucleic lysate of cells cultured under normal conditions, with EGF alone, or with TGF-β1 and EGF. The band detected in samples of cells stimulated with TGF-β1 and EGF was excised and analyzed by MS. (B) Western blot assays of immunoprecipitated samples of nucleic lysates with anti-PKM2 or anti-TGIF2 antibody. Samples were harvested after the cells were treated as indicated for 72 h. (C) Western blot assays of TGIF2 and β-actin expression in cells transfected with siControl or siTGIF2. (D) Relative transcript (mRNA) levels of CDH1, VIM, and ZEB1 after induction of EMT in cells transfected with siControl or siTGIF2 for 72 h. (E) Western blot analysis of TGIF2, E-cadherin, PKM2, and β-actin expression in pre-EMT and post-EMT cells transfected with siControl or siTGIF2. Post-EMT samples were harvested at 72 h, when siRNA inhibition was profound. (F) Relative mRNA levels of CDH1 and VIM after EMT induction in PKM1 OE and PKM2 OE cells. Post-EMT samples were harvested at 72 h. (G) Western blot analysis of E-cadherin and β-actin after EMT induction in PKM1 OE and PKM2 OE cells transfected with siTGIF2. Post-EMT samples were harvested at 72 h. Column values = average of at least three independent experiments; error bars represent SD from the mean of triplicate experiments. *P < 0.05.
Fig. 4.
Fig. 4.
TGIF2 binds to the CDH promoter and recruits HDAC3 during EMT. (A) Schematic diagram showing the positions of two sets of primers designed to cover the promoter region of the CDH1 gene. (B) ChIP assays were performed with IgG and anti-TGIF2 antibody, followed by qPCR (mean ± SD, n = 3). ChIP samples were harvested from the nucleic lysate of SW480 cells treated as indicated for 72 h. (C) Western blot assays of immunoprecipitated samples of nucleic lysate with anti-TGIF2 antibody. Each sample was harvested after the cells were treated as indicated for 72 h. (D) ChIP assays were performed with IgG and anti-acetylated H3K9 antibody, followed by qPCR (mean ± SD, n = 3). ChIP samples were harvested from the nucleic lysate of SW480 cells treated as indicated for 72 h. Column values = average of at least three independent experiments; error bars represent SD from the mean of triplicate experiments. *P < 0.05.
Fig. 5.
Fig. 5.
The immunohistochemistry. (A) Staining at the invasive front, showing an inverse correlation between PKM2, E-cadherin, and TGIF2 expression. (Scale bar, 100 μm.) (B) The representative cases are shown for staining for PKM2, TGIF2, and E-cadherin. Invasive fronts of tumors were stained by anti-PKM2, anti-E-cadherin, and anti-TGIF2 antibodies, and the intensities were assigned to positive and negative groups. With regard to TGIF2 staining, under the microscopic observation, cases with more than 50% of cells stained in nucleus were designated as positive, whereas the others were negative. (C) The 10 positive and 10 negative cases for cellular PKM2 were examined for nuclear TGIF2 and membranous E-cadherin. (D) Theoretical model illustrating the functional roles of PKM2 and TGIF2 in regulating CDH1 transcription during EMT.
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