PKM2 promotes tumor angiogenesis by regulating HIF-1α through NF-κB activation
© Azoitei et al. 2016
Received: 29 July 2015
Accepted: 29 December 2015
Published: 6 January 2016
Initially identified as a molecule that regulates the final step of glycolysis, the M2 isoform of pyruvate kinase (PKM2) was recently reported to have a central role in the metabolic reprogramming of cancer cells as well as participating in cell cycle progression and gene transcription. Despite intensive efforts, the intricate molecular mechanisms through which PKM2 regulates tumor progression remain elusive.
The proliferation and apoptosis of various pancreatic cancer cells using lentiviral-mediated PKM2 abrogation were assessed in vitro via Western blot and flow cytometric assay while the in vivo experiments involved tumor xenograft on chicken chorionallantoic membranes and immunohistochemistry on human tissue specimens. In order to decipher the molecular mechanism of HIF-1α and p65/RelA regulation by PKM2 in cancer cells cultivated in hypoxic atmosphere or normoxia we involved various biochemical assays such as Western blotting, immunoprecipitation, reporter gene assay and ELISA.
Strong expression of PKM2 was observed in 68 % of human pancreatic adenocarcinoma specimens and almost all analyzed pancreatic cancer cell lines. Abrogation of PKM2 resulted in impaired proliferation and augmented apoptosis in vitro as well as impaired tumor growth and decreased blood vessel formation in vivo. Furthermore, deletion of PKM2 negatively impacted hypoxia-induced HIF-1α accumulation and promoter activity ultimately resulting in impaired secretion of VEGF.
Our study suggests that in hypoxic pancreatic tumors PKM2 interferes both with NF-κB/p65 and HIF-1α activation that ultimately triggers VEGF-A secretion and subsequent blood vessel formation.
The primary function of pyruvate kinase (PK) enzyme is to regulate the final rate-limiting step of glycolysis, which catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to adenosine disphosphate (ADP), yielding one molecule of pyruvate and one molecule of adenosine triphosphate (ATP) . There are four pyruvate kinase isoenzymes: PKR (expressed in red blood cells), PKL (the liver isoform), PKM1 (found in heart, brain and muscle) and PKM2 (upregulated in embryonic and tumor cells) . PKM1 and PKM2 isoforms are produced upon mutually exclusive alternative splicing of the PKM pre-mRNA, reflecting the inclusion of either exon 9 (PKM1) or exon 10 (PKM2), respectively . Due to its upregulation in various tumors, PKM2 has garnered attention and was demonstrated to play an essential role in tumor progression [4, 5]. It is worth noting that PKM2 expression levels vary among different types of cancer  suggesting that PKM2’s role in tumorigenesis depends on signaling context. Several studies indicate that PKM2 regulates apoptosis and proliferation [7–9]. While siRNA-mediated PKM2 ablation resulted in caspase-mediated apoptosis and tumor regression in vivo [10, 11], somatostatin-induced nuclear translocation of PKM2 was associated with the induction of cell death in a caspase-independent manner . A recent view on how elevated levels of PKM2 would benefit proliferating tumor cells is based on the recent findings that PKM2, but not PKM1, can translocate to the nucleus and act both as a protein kinase and as transcriptional coactivator for hypoxia-inducible factor alpha (HIF-1α) in HeLa cervical carcinoma cells . In this study, Luo and colleagues demonstrated that HIF-1α binds hypoxia response elements (HRE) within the first intron of human PKM2 that contains a HIF-1-binding site (5′-ACGTG-3′) followed by a 5′-CACA-3′ sequence. PKM2 physically interacts with HIF-1α in the nuclei of hypoxic human cancer cells and promotes transactivation of HIF-1α target genes by enhancing the recruitment of p300 to HRE sites . Similarly, phosphoinositide 3-kinase (PI3K) activation has been shown to increase PKM2 expression through HIF-1α-regulated transcription of the PKM gene [12, 13]. PKM2 has also been demonstrated to participate in transcriptional activation in response to epidermal growth factor (EGF)  and to interact, cooperate with, and be regulated by Oct-4 [9, 14]. Only very recently, PKM2 was reported to interact with NF-κB subunit p65/RelA and to promote tumor angiogenesis and cancer progression . In this study, the authors demonstrated that activation of IGF-1/IGF-1R induces HIF-1α/p65 complex formation, which thus binds to the PKM promoter region leading to PKM2 upregulation and PKM2-mediated breast cancer cell growth. Several studies indicated that control of HIF-1α gene by NF-κB provides an important, additional and parallel level of regulation over the HIF-1α pathway [16–19]. Moreover, in the absence of NF-κB, the HIF-1α gene is not transcribed and therefore no stabilization and activity is observed even after prolonged hypoxia [18, 19].
In this study, we investigated the role of PKM2 in angiogenesis of hypoxic pancreatic tumors. We found that PKM2 is expressed in human pancreatic adenocarcinoma and controls VEGF-A secretion by regulating both HIF-1α and NF-κB. Our study favors a signaling mechanism which places the HIF system as a downstream effector of NF-κB biological functions and indicate PKM2 as a kinase that acts upstream of these two transcription factors in hypoxic pancreatic tumors.
Cell lines and reagents
Human pancreatic cancer cell lines used in the study are: Capan1, adenocarcinoma cells derived from pancreatic metastatic site, #ATCC HTB-79; Panc1, a pancreatic epitheloid carcinoma cell line, #ATCC CRL-1469; BxPC3, pancreas adenocarcinoma cells, #ATCC CRL-1687 and Mia Paca-2 carcinoma cells, #ATCC CRL-1420. PaTu2 and PancTu1 pancreatic adenocarcinoma cells were kindly provided by Prof. Simone Fulda, Institute for Experimental Cancer Research in Pediatrics, Frankfurt, Germany. BxPC3 and Capan1 were used for in vivo investigations due to their ability to form tumors. Due to higher transient transfection efficacy, PaTu2 and Capan1 were involved in reporter assays and ELISA. Cell lines of early passages were cultured in DMEM (Invitrogen, Germany) supplemented with 10 % fetal calf serum (FCS: Biochrom / Millipore, Germany), 1 % penicillin/streptomycin. BAY 87-2243 was purchased from Seleckchem (#S7309), TEPP-46 was from Millipore (#5.05 487.0001).
Short hairpins, plasmids, lentiviral transduction and transfection
PKM2-specific shRNAs originate from the “MISSION shRNA Library” designed and developed by the TRC at the Broad Institute of MIT and Harvard. Two PKM2 hairpins (NM_182471.1-1706s1c1- #2 and NM_182471.1-1493s1c1- #4) that showed high efficacy knock-down were selected. The TRC lentiviral human p65/RelA shRNAs were purchased from Thermo Scientific, GE Dharmacon (#RHS4533-EG5970). The most efficient two p65-specific shRNAs (TRCN0000014684-F12 and TRCN0000014687-G3) were used for experiments. The pcDNA3-YFP-p65 expression plasmid was a kind gift from Dr. Franz Oswald, University Hospital of Ulm; pcDNA3-HIF-1α was obtained from Addgene (HIF-1α, #18949). High-titer virus-containing supernatants of HEK293FT cells after transient co-transfection of lentiviral vectors with pMD2.G and psPAX2 packaging vectors were used for lentiviral mediated transduction of cancer cells.
Cancer cells plated in 12-well dishes were transfected with 330 ng/ml of the respective promoter plasmid as indicated in the figure legends using Lipofectamine 2000 (Invitrogen #15338-100) or Fugene HD (Promega #E231A) according to the manufacturer instructions. Luciferase activity was determined using the Dual Luciferase Assay Kit (Promega, Mannheim, Germany). Firefly luciferase units were normalized with Renilla luciferase after co-transfection with 17 ng/ml pRL-TK plasmid (Promega, Germany).
BxPC3 and Capan1 pancreatic cancer cells (1 × 106) transduced with shPKM2 or non-coding shRNA were xenografted within a 5 mm silicon ring on the surface of the chorionallantoic membrane (CAM) of chicken eggs 8 days after fertilization. Four days after implantation (day 12 after fertilization), tumors were retrieved, fixed in formalin and further subjected to immunohistochemistry.
Immunohistochemistry of CAM and mouse tumors
Formalin-fixed tumors were embedded in paraffin using standard procedures. The 5 μm sections were processed and stained with antibodies directed against Ki67 (1:100; Dako, clone MIB-1), desmin (1:80; Dako, clone D33) and von Willebrand Factor VIII (1:100; Biocare Medical, #CP039B).
Patient samples and ethics statement
All patient samples had been submitted to the Institutes of Pathology of the Bundeswehrkrankenhaus Ulm or the University Hospital Münster for routine diagnostic purposes between 2003 and 2015. Patient and clinic-pathological data including tumor localization and site of metastasis were obtained from the respective pathology report archives. All samples have been anonymized thoroughly for the use in the study. This study has been approved by the local ethics committee of the University of Münster, Germany (No. 2015-102-f-S).
Immunohistochemistry and image acquisition of human samples
Immunohistochemistry for PKM2 and CD31 was performed on 4 μm FFPE tissue slides that displayed the invasive margin of the tumors on a Ventana BenchMark Autostainer (Ventana, Tucson, USA) using PKM2 antibody (Abcam, #ab55602, 1:1000) and mouse monoclonal antibody against CD31 (clone JC70, Ventana, Tucson, USA, prediluted) following the manufacturer’s protocol. Light microscopy and image acquisition was performed using a Leica DM6000B light microscope (Leica, Wetzlar, Germany) and the Diskus Mikroskopische Diskussion image acquisition software (Carl Hilgers, Königswinter, Germany). PKM2 staining intensity was graded as negative/weak, moderate, or strong.
Western blot and immunoprecipitation
Whole cell extracts were prepared using IP lysis buffer containing 10 mM Tris-HCl, 5 mM EDTA, 50 mM NaCl, 50 mM NaF and 1 % Triton X100 supplemented with Complete Protease inhibitor Cocktail and PhosStop tablets (Roche). Lysates were subjected to SDS-PAGE and proteins transferred to PVDF membranes (Millipore, Massachusetts, USA). For coIP, protein extracts (0.5 -1.5 mg) were incubated with 2 μg antibody and Protein G-Sepharose (GE Healthcare). Membranes were blocked with 5 % non-fat dry milk in phosphate buffered saline (PBS) containing 0.2 % Tween 20 and incubated over night at 4 °C with specific antibodies. For subsequent washes 0.2 % Tween 20 in PBS was used. The following antibodies were used: PKM2 (Abcam, #ab55602); cleaved PARP (Cell Signaling, #9542S); HIF-1α (BD Transduction Laboratories, #610959); p65/RelA C-20 (Santa Cruz, #sc-372), GFP (Roche, # 1052 1400) and β-actin (Sigma, #A1978). Bands were quantified by densitometric analysis using ImageJ software http://imagej.net.
Annexin-V/propidium iodide (PI) staining was performed via flow cytometry according to the manufacturer’s guidelines as previously described . Flow cytometric analysis was performed using FACSCalibur (Becton Dickinson) and the data was analyzed by FlowJo software.
The hypoxic environment was generated using the Anaerocult A Mini Kit (Merck, Darmstadt, Germany #1.01611.0001). Cells were incubated under low oxygen atmosphere for 8–24 h at 37 °C.
Statistical analysis was performed with GraphPad Prism 5.0 software (GraphPad Software, Inc, La Jolla, CA, USA). Statistical significance was assessed by an unpaired student t test. p < 0.05 or less was considered significant. The quantitative analysis of immunohistology staining was performed with Image J software (National Institute of Health, Bethesda, MD, USA) using the Colour Deconvolution plugin.
PKM2 is highly expressed in human pancreatic tumor specimens
Effect of PKM2 depletion on tumor cell proliferation and tumor cell viability
PKM2 depletion in pancreatic cancer cells results in impaired tumor growth and angiogenesis in vivo
Hypoxia induces the nuclear translocation of PKM2 and enhances its interaction with HIF-1α
PKM2 knock-down is associated with impaired hypoxia-induced HIF-1α accumulation and VEGF secretion
The nuclear translocation and interaction with HIF-1α posed the question whether PKM2 was able to regulate the expression of this oxygen sensor protein under hypoxic conditions. In our experimental setup using pancreatic tumor cells, abrogation of PKM2 negatively impacted hypoxia-induced accumulation of HIF-1α (Fig. 4d, Additional file 4: Figure S4A-B). Furthermore, impaired HIF-1α accumulation was associated with significant reduction of transcriptional activation of the HIF-responsive element (HRE), a HIF-1α-docking site present in promoters that contain the RCGTG sequence (Fig. 4e). Since upregulation of VEGF-A, an essential driver of blood vessel formation, occurs mainly via stabilization of HIF-1α, we sought to investigate whether PKM2 contributes to VEGF secretion in hypoxic pancreatic cancer cells. ELISA experiments demonstrate that impaired HIF-1α accumulation following depletion of PKM2 was corroborated with impaired low oxygen-driven VEGF promoter activity (Fig. 4f) and decreased levels of hypoxia-induced secreted VEGF-A (Fig. 4g). Same effects were obtained with either TEPP-46, a selective activator molecule reported to force PKM2 into the tetrameric form triggering thus its nuclear exclusion  or BAY 87-2243, an HIF-1α-specific inhibitor. Incubation with TEPP-46 or BAY 87-2243 resulted in impaired transcriptional activity of both HIF-1α and VEGF-A in hypoxic pancreatic cancer cells (Additional file 5: Figure S5A–D).
PKM2 contributes to VEGF secretion in pancreatic cancer cells via activation of NF-κB transcription factors
PKM2 regulates HIF-1α -induced VEGF secretion in a NF-κB/p65-dependent manner
The primary function of pyruvate kinase M2 isoform (PKM2) is to catalyze the phosphorylation from phosphoenolpyruvate (PEP) to pyruvate as the last step of glycolysis to generate ATP. Incidentally, tumor cells need a permanent supply of glycolytic intermediates [25, 26] and an impaired activity of PKM2 (through dimer formation) was observed to favor rapid cell proliferation. Several studies from recent years demonstrated that PKM2 may play a far broader role in promoting cancer progression than has previously been appreciated. However, the intricate molecular mechanisms through which PKM2 exerts its tumorigenic role remain elusive.
In this study, we have identified PKM2 as a crucial molecule for progression of pancreatic cancer in which the tumor microenvironment has been reported to be highly hypoxic . Accordingly, we found moderate to strong PKM2 expression in all examined human pancreatic adenocarcinoma samples. Depletion of PKM2 was associated with impaired proliferation and augmented tumor cell death in vitro, while the in vivo tumor xenograft experiments revealed a close association between impaired tumor growth and decreased blood vessel formation. These findings, together with earlier studies showing that PKM2 promotes angiogenesis by binding to TEM8 - tumor endothelial factor 8 , suggest that PKM2 might contribute to tumor growth by regulating crucial molecules involved in tumor vascularization. Such a molecule is the well characterized VEGF. The fact that VEGF is directly regulated by HIF-1α, an oxygen sensor protein reported to interact with PKM2  urged us to evaluate the role of PKM2 with respect to HIF-1α stabilization and VEGF secretion in hypoxic pancreatic tumors. Firstly, we showed that PKM2 translocates to the nucleus in pancreatic cancer cells cultivated in hypoxic conditions. Secondly, the interaction between PKM2 and HIF-1α is augmented in hypoxic cancer cells. Lastly, abrogation of PKM2 prevented hypoxia-mediated HIF-1α accumulation and HIF-1α promoter activity, which negatively impacted VEGF secretion by pancreatic cancer cells deprived of oxygen. Together, these findings suggest that PKM2 is required for HIF-1α mediated VEGF transcriptional activity and secretion. Hypoxia not only results in the accumulation of HIF-1α, but also activates the NF-κB transcription factors . Because PKM2 was reported to directly interact with the NF-κB p65 subunit to promote EGR1 expression , we explored the possibility whether PKM2 interferes with NF-κB activation by hypoxia. Indeed, PKM2 expression arrest was mirrored by impaired hypoxia-driven promoter activity of NF-κB and its target gene VEGF. Ectopic expression of p65 restored VEGF transcription after PKM2 ablation inferring that the kinase regulates VEGF via NF-κB p65 subunit. This finding is also supported by the fact that PKM2 interacts with p65 in pancreatic cancer cells. It has been previously shown that activation of NF-κB and transcript abundance of p65 are diminished in HIF-1α-deficient mice  and that the NF-κB subunits translocated to the nucleus interact with the NF-κB binding sites in the HIF-1α promoter, thus increasing HIF-1α promoter activity and expression [18, 24]. The fact that HIF-1α itself contributes to the activation of the NF-κB pathway adds a further level of complexity to the cross-talk between these molecules. Our data demonstrate that PKM2 is able to regulate the NF-κB as well as the HIF-1α signaling pathways and suggest that the kinase acts upstream of these two transcription factors in pancreatic tumors. Interestingly, ectopic expression of p65 restored HIF-1α transcriptional activity in hypoxic cells featuring PKM2 knock-down. In the same cells however, overexpression of HIF-1α showed only marginal effect on NF-κB transcription. While ectopic expression of p65/RelA resulted in augmented HIF-1α transcription already in normoxic condition, the overexpression of PKM2 had no impact on HIF-1 promoter activity in cells with abrogated p65. These data place the NF-κB p65 subunit below PKM2 and upstream of HIF-1α in the hypoxia signaling axis.
Since in human pancreatic ductal adenocarcinoma (PDAC) HIF-1α expression highly correlates with tumor size and poor prognosis  it will be a demanding task to dissect the different signaling states and the potential of feed-back signaling loops which may be of great importance for therapeutic targeting of pancreatic tumors using PKM2 inhibitors currently under intense development.
We thank Susanne Bobrovich for excellent technical assistance and Omar Elekad for participation to the study during his internship in the laboratory. We are thankful to Dr. Franz Oswald at Ulm University for providing the pcDNA3-YFP-p65 construct. The work and positions of NA and KD were supported by the Deutsche Forschungsgemeinschaft, grant AZ:96-1 to NA.
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