Oncogene (2015), 1–12 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc

ORIGINAL ARTICLE

Regulatory circuit of PKM2/NF-κB/miR-148a/152-modulated tumor angiogenesis and cancer progression Q Xu1,7, L-Z Liu2,7, Y Yin1,6, J He2, Q Li1, X Qian1, Y You4, Z Lu5, SC Peiper2, Y Shu3,8 and B-H Jiang1,2,8 Upregulation of the embryonic M2 isoform of pyruvate kinase (PKM2) emerges as a critical player in the cancer development and metabolism, yet the underlying mechanism of PKM2 overexpression remains to be elucidated. Here we demonstrate that IGF-1/IGFIR regulates PKM2 expression by enhancing HIF-1α-p65 complex binding to PKM2 promoter. PKM2 expression is regulated by miR-148a/152 suppression. PKM2 directly interacts with NF-κB p65 subunit to promote EGR1 expression for regulating miR-148a/ 152 feedback circuit in normal cells, but not in cancer cells because of the DNA hypermethylation of miR-148a and miR-152 gene promoters. The silencing of miR-148a/152 contributes to the overexpression of PKM2, NF-κB or/and IGF-IR in some cancer cells. We show that disruption of PKM2/NF-κB/miR-148a/152 feedback loop can regulate cancer cell growth and angiogenesis, and is also associated with triple-negative breast cancer (TNBC) phenotype, which may have clinical implication for providing novel biomarker(s) of TNBC and potential therapeutic target(s) in the future. Oncogene advance online publication, 23 February 2015; doi:10.1038/onc.2015.6

INTRODUCTION The metabolic phenotype of cancer cells differs from that of normal cells. Cancer cells have an elevated glucose uptake and lactate production, regardless of oxygen availability, relative to normal cells. This phenomenon is termed the Warburg effect, and it increases the availability of glucose-derived carbons needed for biosynthesis and cell growth.1,2 Pyruvate kinase is important for aerobic glycolysis, and its isoform PKM2 has a central role in the metabolism of cancer cells.3,4 Mounting evidence demonstrates that PKM2 is critical for maintaining the malignant phenotype, cell cycle progression, and tumor growth of cancer cells.5–8 PKM2 may act in its canonical role as pyruvate kinase, catalyzing the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP, or in a recently identified role as protein kinase.5,9 PKM2, which contains a nuclear-translocation sequence and an ATP binding domain, can phosphorylate histone H3 or STAT3 to after the epigenetic state of genes and activate transcription.9 PKM2 also interacts with other proteins, like β-catenin, and with transcriptions factors, including Oct-4 and HIF-1α, to exert its function.10,11 For example, the MUC1-C binds directly to PKM2 to stimulate PKM2 activity and regulate glycolysis.12 PKM2 also interacts with CD44, enhancing the glycolytic phenotype of cancer cells that are either deficient in p53 or exposed to hypoxia.13 The receptor tyrosine kinase/PI3K/ AKT/mTOR pathway is also involved in PKM2-regulated cell metabolism.7,14 Recent studies indicate that PKM2 expression can be regulated by miR-122 and miR-326.15,16

As with PKM2, insulin-like growth factor receptor (IGF-IR) overexpression is a hallmark of several kinds of cancer17 and deregulation of the IGF axis expression has been linked to a number of pathologies.18,19 However, how IGF-IR/PKM2 signaling is regulated in normal and tumor cells remains largely obscure. We demonstrate the novel regulatory circuit involving PKM2, NF-κB, EGR1 and miR-148a/152 respond to IGF-IR activation in breast cancer cells. This axis plays an important role in regulating cell proliferation, colony formation and angiogenesis. The levels of these proteins in this newly identified signal pathway are significantly correlated with triple-negative breast cancer (TNBC) phenotype. RESULTS miR-148a/152 inhibited the Warburg effect, and PKM2 is a direct target of miR-148a/152 We and others have reported that miR-148a and miR-152 affect cell growth, and apoptosis in different cell lines.20–23 To determine whether miR-148a and miR-152 regulate the Warburg effect, we performed an aerobic glycolysis analysis by using conditioned media collected from three cell lines (293T, MDA-MB-231 and H1299) for glucose consumption and lactate production. Both glucose consumption and lactate production levels were significantly decreased in these cells that stably overexpressed miR-148a or miR-152 compared with the control cells expressing a scrambled RNA (Figure 1a). To explore a possibility that PKM2 may

1 State Key lab of Reproductive Medicine, Department of Pathology, Collaborative Innovation Center for Cancer Personalized Medicine, Cancer Center, Nanjing Medical University, Nanjing, China; 2Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA; 3Department of Oncology, The First Affiliated Hospital of Nanjing Medical University, Collaborative Innovation Center for Cancer Personalized Medicine, Cancer Center, Nanjing Medical University, Nanjing, China; 4Department of Neurosurgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China; 5Brain Tumor Center and Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA and 6Department of Pathology, Anhui Medical University, Hefei, China. Correspondence: Professor Y Shu, Department of Oncology; The First Affiliated Hospital of Nanjing Medical University, Nanjing or Professor B-H Jiang, Department of Pathology, Nanjing Medical University, China; and Department of Pathology, Anatomy & Cell Biology, Thomas Jefferson University, 1020 Locust Street, Suite 334, Jefferson Alumni Hall, Philadelphia, PA 19107, USA. E-mail: [email protected] or [email protected] or [email protected] 7 These authors contributed equally to this work. 8 These authors contributed equally to this work. Received 20 January 2014; revised 17 December 2014; accepted 29 December 2014

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Figure 1. PKM2 is a target of miR-148a/152. (a) The cultured media from 293T, H1299 and MDA-MB-231 (MB-231) cells stably expressing a scrambled control miRNA (Scr), miR-148a or miR-152 were collected for the measurement of glucose consumption and lactate production levels. (b) The complementary pairing of miR-148a or miR-152 with PKM2 wild-type (WT) and mutant (Mut) 3′ UTR reporter constructs are shown (top). The reporter constructs containing the WT or Mut PKM2 3′ UTR regions were co-transfected with miR-Scr, miR-148a, miR-152 or with anti-miR-148a or anti-miR-152, anti-Scr inhibitor and β-gal plasmid into 293T cells. The relative luciferase/β-gal activities were analyzed in the cells 48 h after the transfection (bottom) (*Po0.05). (c) MB-231, T47D and A549 cells stably expressing miR-Scr, miR-148a or miR-152; or cells were transfected with anti-miR-148a, anti-miR-152, or anti-Scr inhibitors, and cultured for 72 h. Cells were lysed, and PKM2 protein levels were analyzed by immunoblotting. (d) 293T, A549, and T47D cells stably expressing Scr, miR-148a and miR-152 were infected with lentivirus carrying PKM2 or Scr. The cultured media were collected for the measurement of glucose and lactate (*Po 0.05 versus Scr; #Po0.05 versus miR-148a or miR-152).

be involved in miR-148a/152 inhibited the Warburg effect, we employed our recently developed miRNA algorithm ‘Targetsearch’ to obtain a list of possible mRNA targets of miR-148a/152.20 We identified a putative miR-148a/152-binding element (84–104 bp) in the 3′ untranslated region (UTR) of PKM2 mRNA. We constructed luciferase reporter plasmids containing the wild-type PKM2 3′ UTR or a 3′ UTR with mutation in the putative binding site. The luciferase activities from the wild-type construct were inhibited upon transfection of miR-148a/152 and were induced by transfection with miR-148a/152 inhibitors. Point mutations in putative binding site abrogated the effect of miR-148a/152, demonstrating that miR-148a and miR-152 specifically target the PKM2 3′ UTR by binding to the identified seed sequence (Figure 1b). We examined the PKM2 mRNA and protein levels in these cell lines. The protein levels of PKM2 were attenuated in cells that stably expressed miR-148a or miR-152, and were greatly increased in the miR-148a- or miR-152-knockdown cells by using miRNA inhibitors (Figure 1c). No significant differences were found in PKM2 mRNA levels (Supplementary Figure S1), suggesting that PKM2 expression is regulated at the translational level through miR-148a or miR-152. Notably, the miR-148a/152 inhibited the Warburg effect in different cells can be rescued though ectopic expression of PKM2 without the 3′UTR region in the cells that stably express miR-148a Oncogene (2015) 1 – 12

or miR-152 (Figure 1d). Taken together, our data demonstrate that miR-148a and miR-152 inhibit the Warburg effect through direct suppression of PKM2 expression. New mechanism of miR-148a and miR-152 expression regulated by EGR1 and DNA methylation in cancer cells To understand how miR-148a and miR-152 suppression may be regulated in cancer cells, we searched for potential transcription factors involved in miR-148a and miR-152 regulation within their 5-kbp promoter regions. We found that the miR-148a promoter contains three consensus binding sites for EGR1 at positions − 1503 to − 1493, − 1075 to − 1065 and − 593 to − 582 and that the miR-152 promoter contains at least two putative EGR1 binding sites at positions − 602 to − 553 and − 71 to − 60. EGR1 is known to bind to a GC-rich canonical sequence (GCGG[T]GGGCGG).24,25 The region from − 602 to − 553 in the miR-152 promoter contains three closely spaced EGR1 binding sites that were recognized as one EGR1 binding site cluster. To experimentally confirm that miR-148a and miR-152 are regulated by EGR1, we cloned four different fragments of miR-148a promoter and three different fragments of miR-152 promoter into pGL3 luciferase reporter plasmid. With these constructs, we observed 1.3–6.2-fold increases in luciferase activities compared with the pGL3-basic vector © 2015 Macmillan Publishers Limited

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Figure 2. EGR1 regulates transcription from miR-148a and miR-152 genes. (a) Schematic diagrams show the potential EGR1 binding sites of miR-148a and miR-152 promoters and the construction of miR-148a and miR-152 promoter luciferase reporters. Boxes indicate the distribution of the putative EGR1 binding sites (BS). Sequences of the consensus EGR1 binding sites are also shown. Each reporter plasmid was cotransfected with EGR1 expression plasmid and β-gal plasmid. The luciferase activities were normalized to the activity of β-gal, and values shown are normalized to the value of pGL3 basic construct (*P o0.05, **Po0.01). (b) 184A1 and MB-231 cells were treated with IGF-1 or infected with lentivirus carrying PKM2 or empty vector (control). Cross-linked chromatin was immunoprecipitated with antibodies specific for EGR1 and IgG. Purified DNA was analyzed by real-time PCR with five specific primer pairs shown in Supplementary Table S1 (*P o0.05 versus IgG; #Po0.05, ##P o0.01 versus Control). (c) Cells as indicated were transfected with plasmid encoding EGR1 or vector alone, or with siRNA targeting EGR1 or a scrambled control (si-Scr). miR-148a and miR-152 levels were assessed by real-time PCR (*P o0.05). (d) Results of bisulfite sequence of miR-148a and miR-152 promoter regions in 184A1 and MB-231 cells. (e) 5-Aza-dC-treated MB-231 cells were treated with IGF-1 or infected with lentivirus carrying PKM2 or empty vector (control). ChIP assays using anti-IgG or anti-EGR1 antibodies were performed and followed by analysis of real-time PCR (*P o0.05 versus IgG; ##Po 0.01 versus control). (f) MB-231 cells treated with or without 5-Aza-dC were transfected with siEGR1 or si-Scr. miR-148a and miR-152 levels were assessed by real-time PCR (**P o0.01 versus IgG; #P o0.05 versus 5-Aza-dC).

(Figure 2a). Subsequently, we performed chromatin immunoprecipitation-qPCR (ChIP-qPCR) experiments in 184A1 and MDAMB-231 cells. In 184A1 cells, a significant signal was observed in the ChIP-qPCR assay with EGR1 antibody but not IgG antibody, and these signals were dramatically enhanced after IGF-1 treatment or PKM2 overexpression. Surprisingly, the there was little increase in EGR1 signals in response to IGF-1 or PKM2 stimulation in MDA-MB-231 cells (Figure 2b). The levels of miR-148a and miR-152 were enhanced by EGR1 overexpression, but suppressed by siRNA-mediated knockdown of EGR1 expression in 293T and 184A1 cells; on the contrary, only marginal changes in miR-148a/152 levels were observed in MDA-MB-231 cells (Figure 2c). We hypothesized that hypermethylation blocks EGR1 binding to the promoters of miR-148a and miR-152 since the CpG methylation regions are located within the region between transcription © 2015 Macmillan Publishers Limited

start site and EGR1 binding sites in the promoters of miR-148a and miR-152. Further analysis revealed hypermethylation of miR-148a and miR-152 promoters in MDA-MB-231 cells when compared with immortalized human mammary epithelial cells 184A1 (Figure 2d and Supplementary Figure S2a). Treatment of MDAMB-231 cells with 5-Aza-dC, a demethylation reagent, dramatically lowered methylation in the promoter regions of miR-148a and miR-152 in MDA-MB-231 cells (Supplementary Figure S2b) and restored EGR1 binding to the promoter of miR-148a/152 in response to IGF-1 treatment or PKM2 overexpression (Figure 2e). 5-Aza-dC also recovered the ability of EGR1 in regulation of miR-148a/152 expression in MDA-MB-231 cells (Figure 2f). These results are consistent with previous observation we and others have made that DNA methylation is responsible for the low expression levels of miR-148a/152 in breast cancer cells.20,26 Collectively, these results suggest that EGR1 regulates both Oncogene (2015) 1 – 12

PKM2/NF-κB/EGR1/miR-148a/152 in cancer development Q Xu et al

4 miR-148a and miR-152 expression by the binding of EGR1 with the miRNA gene promoters at several binding sites, creating a complex regulation feature through DNA methylation. PKM2 directly interacts with NF-κB p65 to regulate EGR1 expression To further examine whether PKM2 directly interacts with EGR1, we performed co-immunoprecipitation (Co-IP) using nuclear extracts prepared from three different cell types treated with or without IGF-1. We did not detect any interaction between PKM2 and EGR1 (Supplementary Figure S3a). EGR1 can be transcriptionally regulated by the p65 subunit of NF-κB through the proximal κB regulatory motif.27 In agreement with published data, we

observed that EGR1 expression was strongly induced in cells treated with TNF-α and were attenuated in p65 knockdown cells by treatment with a siRNA (Supplementary Figure S3b). Moreover, we observed that PKM2 overexpression significantly enhanced activity from a reporter construct driven by the EGR1 promoter, and increased endogenous EGR1 protein levels in untreated cells but not in cells treated with siRNA targeting p65 mRNA (Figure 3a and Supplementary Figure S3c). Compared to IgG background signals, the NF-κB p65 occupancy of EGR1 promoter was substantially increased with IGF-1 treatment or PKM2 overexpression (Figure 3b). Thus, it is possible that PKM2 regulates EGR1 expression through interaction with NF-κB p65 subunit. Co-IP analysis of nuclear extracts from the three different cell types showed that a weak band of identified as PKM2

Figure 3. PKM2 interacts with NF-κB p65 to regulate EGR1 expression. (a) MB-231, H1299 and 293T cells stably expressing PKM2 or Scr were transfected with si-p65 or si-Scr and grew for 72 h. Total proteins were collected and PKM2, p65, EGR1 and β-actin levels were determined by immunoblotting assay. (b) 184A1 cell were treated with IGF-1 or infected with lentivirus carrying PKM2 or empty vector (Scr Control). ChIP assays were performed by using anti-IgG or anti-p65 antibody, followed by analysis of real-time PCR (**Po0.01 versus IgG; ##Po 0.01 versus control). (c) Co-IP was performed using NE prepared from 293T, A549 or MB-231 cells treated with or without IGF-1. (d) The upper panel shows a schematic illustration of the exon 9 in PKM1 and exon 10 in PKM2. Co-IP was performed on 293T cells transfected with GAL4-E9 or GAL4-E10. (e) The upper panel shows a schematic illustration of p65 and its mutants. In the lower panel, 293T cells stably expressing PKM2 were transfected with Flag-tagged p65, or p65 RHD region, or p65TAD region. Forty-eight hours after transfection, cells were subjected to Co-IP analysis using the indicated antibodies for IP and blotting, with 10% of input proteins is indicated in the bottom panels. (f) Immunofluorescence assay was performed on 184A1 cells transfected with or without Flag-tagged RHD-p65 construct and treated with or without IGF-1. NE, nuclear extracts; NLS, nuclear localization signal; TAD, transactivation domain. Merge 1: Green+Red; Merge 2: Green+Red+Blue. Scale bar: 10 μm. Oncogene (2015) 1 – 12

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PKM2/NF-κB/EGR1/miR-148a/152 in cancer development Q Xu et al

5 immunoprecipitated with anti-p65 was detected in absence of IGF-1 stimulation (Figure 3c). In contrast, a stronger band was detected upon IGF-1 treatment. Similar results were obtained by using an anti-PKM2 antibody, whereas PKM1, produced from an alternatively spliced product of the PKM2 gene, failed to interact with p65 (Supplementary Figure S3d). As PKM1 and PKM2 differ only by the presence of a single exon, we hypothesize that this region of the encoded protein is responsible for PKM2 interaction with p65. We transfected 293T cells with the GAL4-E9 plasmid that contains the PKM1-specific exon 9 or with the GAL4-E10 plasmid that contains the PKM2-specific exon 10 and subjected extracts from these cells to a Co-IP assay with anti-p65 antibody. p65 strongly bound to the GAL4-E10 fusion protein but not to GAL4E9, indicating that the E10 domain of PKM2 is critical for interaction with p65 (Figure 3d). To determine which domain of p65 is necessary for the interaction with PKM2, constructs encoding Flag fused to fulllength p65, Flag fused to the Rel homology domain (RHD) spanning amino acids 1 to 304 (RHD-p65), or to C-terminal transactivation domain (TAD) spanning amino acids 292 to 551 (TAD-p65) were transfected into 293T cells. Full-length of p65 and the RHD-p65 fragment were co-immunoprecipitated by anti-PKM2 antibodies. Similar results were obtained by using anti-Flag antibodies (Figure 3e). These results suggest that PKM2 specifically interacts with p65 through the RHD region. To determine the subcellular localization of the PKM2/p65 complex, cells treated with or without IGF-1 were immunostained using PKM2 (green) and p65 (red) antibodies. PKM2 was mainly sequestered in the cytoplasm, whereas p65 was located in both cytoplasm and nucleus. IGF-1 treatment resulted in accumulation of PKM2 and p65 or RHD-p65 in nuclei of multiple cells (Figure 3f and Supplementary Figure S3e). IGF-IR and PKM2 regulate miR-148a/152 expression to form a negative feedback loop and IGF-1 regulates PKM2 expression through enhancing HIF-1α-p65 complex binding to PKM2 promoter We found that IGF-IR overexpression or IGF-1 treatment increased nuclear levels of PKM2, p65 and EGR1 expression; and while IGF-IR knockdown decreased these proteins expression levels (Figures 4a and b). Furthermore, either PKM2 or p65 knockdown abrogated IGF-IR-induced PKM2-p65 complex formation and EGR1 upregulation (Supplementary Figure S4a and b). These results strongly suggest that IGF-IR/PKM2 regulate miR-148a/152 expression in a negative feedback loop involving EGR1 expression. To test this possibility, we first examined miR-148a/152 expression in untreated cells or cells treated with IGF-1. IGF-1 stimulation or IGF-IR overexpression significantly increased the expression levels of miR-148a and miR-152 in 293T and 184A1 cells, but not in MDAMB-231 cells; whereas knockdown of PKM2 or p65 abrogated the IGF-1/IGF-IR-induced miR-148a/152 accumulation, indicating that the PKM2/p65 complex plays a pivotal role in IGF-1-mediated regulation of miR-148a/152 expression (Figure 4c). We further observed that EGR1 knockdown resulted in the miR-148a/152 downregulation independent of alterations of IGF-IR or PKM2 expression (Figure 4d). Consistent with our recent findings that IGF-IR is a direct target of miR-148a/152,20 we confirmed that miR-148a/152 targets IGF-IR in multiple types of cells. Similarly overexpression of miR-148a/152 suppressed p65 and EGR1 proteinslevels in these cells, while inhibition of miR-148a/152 induced these protein expression (Figure 4e). Given that HIF-1α is a coactivator with p65 in the regulation of PKM2,11 and IGF-1 treatment increased PKM2 expression (Figure 4g), we aimed to determine whether IGF-1-promoted PKM2 expression through enhancing HIF-1α-p65 complex binding to PKM2 promoter. Similar to EGF treatment, IGF-1 treatment also enhanced the interaction between HIF-1α and p65 (Figure 4f). Knockdown of either HIF-1α © 2015 Macmillan Publishers Limited

or p65 abrogated IGF-1-induced HIF-1α-p65 complex binding to PKM2 promoter as well as IGF-1-induced PKM2 expression (Figures 4g and h). Collectively, these results suggested that HIF-1α and p65 interaction is required for HIF-1α-p65 complex binding to the promoter of PKM2 and upregulation of PKM2 upon IGF-1 stimulation. More importantly, we demonstrated that there is a novel feedback loop through EGR1-induced overexpression of miR-148a/152 to suppress IGF-IR/PKM2 signals in normal cells. In breast cancer cells, DNA hypermethylation abrogated EGR1 occupancy in the promoters of miR-148a and miR-152 to disrupt this feedback regulatory circuit, which may contribute to much higher levels of IGF-IR and PKM2 in some cancer cells for playing an important role in cancer development and angiogenesis. PKM2 promotes breast cancer cell proliferation, colony formation, and angiogenesis To test the functional relevance of PKM2 in breast cancer cells, we performed cell proliferation and tumor angiogenesis analyses of two breast cancer cells. The results demonstrated that ectopic expression of PKM2 strongly promoted breast cancer cell proliferation, colony formation, tube formation and angiogenesis; while inhibition of endogeneous PKM2 significantly decreased these functions (Figures 5a and d). These results indicated that PKM2 is important for breast cancer cell proliferation and tumor angiogenesis. To test whether overexpression of HIF-1α or p65 is sufficient to rescues PKM2 knockdown-inhibited breast cell growth. PKM2 knockdown cells were infected with adenovirus carrying HIF-1α or GFP, or transfected with pCMV-p65 or pCMV-Scr plasmid. Western-blotting confirmed that a high expression levels of HIF-1α or p65 were achieved in the cells compared with the control cells (data not shown). Cell proliferation results showed that forced expression of HIF-1α or p65 partially restored PKM2 knockdown-inhibited breast cell growth (Supplementary Figure 5). Next, we tested the biological function role of PKM2/p65 in IGF-1R-induced breast cancer cell proliferation and tumor angiogenesis. We transfected breast cancer cell lines stably expressing IGF-IR with siRNA targeting PKM2 or p65. Our results showed that knockdown of PKM2 or p65 expression partially abrogated IGF-IR-promoted breast cancer cell proliferation, colony formation, tube formation and angiogenesis, suggesting PKM2/ p65 plays crucial roles in IGF-IR-induced breast cancer cell functions. (Figures 5e and h). Clinical relevance of PKM2 and NF-κB overexpression and miR-148a/152 suppression in triple-negative breast cancer cells We evaluated PKM2 expression levels in 20 pairs of breast cancer tissue specimens and their matched adjacent normal tissues. PKM2 protein levels were much higher in cancer tissues than those in normal tissues (Supplementary Figure S6a). Next, we examine PKM2, NF-κB and miR-148a/152 expression levels in 47 pairs of invasive ductal carcinoma specimens and their matched adjacent normal tissues, which include 18 TNBC subtypes and 29 non-TNBC subtypes of breast cancer. We demonstrated that the expression levels of miR-148a and miR-152 were significantly lower in TNBC samples than those in non-TNBC samples (Figures 6a and b). TNBC showed high DNA methylation levels in miR-148a and miR-152 promoter regions, which were inversely correlated the levels of miR-148a and miR-152 expression, suggesting that DNA hypermethylation is responsible for the silencing of miR148a/152 expression in these breast cancer tissues (Figures 6c and d and Supplementary Figure S6b), which is consistent with our previous suggestion in cancer metastasis.20 TNBC tissues also showed higher levels of PKM2 and nuclear NF-κB expression than non-TNBC samples (72 versus 43%; 56 versus 27%). Correlation studies in 47 breast cancer specimens demonstrated that miR-148a and miR-152 expression levels inversely correlated with Oncogene (2015) 1 – 12

PKM2/NF-κB/EGR1/miR-148a/152 in cancer development Q Xu et al

6 PKM2 and NF-κB expression levels (Figures 6a, c and d). Taken together, these results demonstrate for the first time that the PKM2/NF-κB upregulation and miR-148a/152 suppression are strongly associated with TNBC phenotype. DISCUSSION PKM2 is a critical enzyme for glycolytic metabolism and the Warburg effect that is preferentially expressed in embryonic tissue

and cancer cells. During tumorigenesis, PKM2 is overexpressed, leading to the switch from regular cell metabolism to aerobic glycolysis, which is this alternate pathway for cell biosynthetic pathways.8,28 Constitutive activation of IGF-IR is also a common event in human cancer, and we have found that similar to EGF, IGF-1/IGF-IR induces HIF-1α-p65 complex formation, which binds to PKM2 promoter region, leading to PKM2 upregulation. Furthermore, IGF-1/IGF-IR activation stimulates cytoplasmic PKM2 translocated into nucleus. Nuclear PKM2 can function as a

Figure 4. IGF-IR activation promotes PKM2 nuclear-translocation and miR-148a/152 expression. (a–b) Immunoblotting analysis of IGF-IR, p-IGFIR, and β-actin levels in cytoplasmic extracts (Cyto); and PKM2, p65, EGR1 and H2A levels in nuclear extracts (NE) in (a) cells stably expressing IGF-IR, or IGF-1-treated 184A1 and 293T cells; and in (b) 293T, 184A1 and MB-231 cells transfected with si-Scr or siRNA targeting IGF-IR. (c) Realtime PCR analysis of miR-148a and miR-152 levels in 184A, 293T and MB231 cells transfected with siRNAs against PKM2 (siPKM2), p65 (sip65) or scrambled control (si-Scr); followed by treatment with or without IGF-1 or infected with lentivirus carrying IGF-IR or control vector as indicated. (d) Real-time PCR analysis of miR-148a and miR-152 levels in 293T and 184A1 cells infected with lentivirus carrying IGF-IR, PKM2; or transfected with siEGR1 or si-Scr (*Po0.05 versus control; #Po0.05 versus IGF-IR or PKM2). (e) Immunoblotting of IGF-IR, EGR1 and p65 levels in 293T, H1299 and MB-231 cells stably expressing Scr, miR-148a or miR-152; or transfected with anti-miR-148a, anti-miR-152 inhibitor or anti-Scr. (f) Co-IP was performed using nuclear extracts (NE) prepared from 184A1 or MB-231 cells treated with or without IGF-1. (g) MB-231 cells were transfected with si-Scr, siHIF-1α or sip65 for 24 h, then was treated with IGF-1 for 12 h or infected with lentivirus carrying IGF-1R or empty vector (Scr Control) for 24 h. The expression levels of PKM2 and β-actin were tested by immunoblotting analysis. (h) MB-231 cells were transfected with si-Scr, si-HIF-1α or si-p65 for 24 h, then treated with IGF-1 for 12 h. ChIP assays were performed by using anti-IgG, anti-p65 or anti-HIF-1α antibodies, followed by analysis of real-time PCR (*Po 0.05; **Po0.01). Oncogene (2015) 1 – 12

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Figure 4.

(Continued)

protein kinase alone or through interactions with other factors, such as β-catenin or HIF-1α, to regulate gene transcription.10 The molecular mechanisms that control the constitutive activation of IGF-IR/PKM2 signals remain puzzling. The evidence that the glucose-dependent PKM2 acetylation leads to decrease of PKM2 levels provides one possible way for cells to suppress the over-activation of IGF-IR/PKM2 signals.6 Recent studies demonstrate that PKM2 is a target of miR-326 and miR-122 in glioma and hepatocellular carcinoma cells.15,16 We found that the levels of miR-326 and miR-122 were not changed by IGF-IR or PKM2 alterations. Interestingly, miR-148a/152 directly target PKM2 and IGF-IR, and miR-148a/152 are positively regulated by IGF-IR and PKM2, suggesting a novel feedback loop that regulates PKM2 activity. We demonstrated that PKM2, but not PKM1, is a novel binding partner of p65. PKM1 and PKM2 are alternatively spliced products of PKM2 gene and differ by only 23 amino acids within a single alternatively spliced exon. Inclusion of exon 10 results in the M2 isoform that has an allosteric pocket structure, which allows PKM2 to bind to fructose 1,2-bisphosphate and interact with other proteins.29 PKM2 binds to the RHD domain of p65, enhancing p65 occupation in EGR1 promoter region to stimulate EGR1 expression. We also found that the transcription factor EGR1 regulated the expression of miR-148a and miR-152 genes. These results reveal a novel mechanism involving in the complex regulation of PKM2/p65/EGR1 axis and miR-148a/152 expression. IGF-IR/PKM2 activation stimulates EGR1 expression to activate miR-148a/152 transcription, which in turn suppresses IGF-IR/PKM2 signals through downregulation of IGF-IR and PKM2 protein levels. DNA hypermethylation abrogated EGR1 occupancy at the promoters of miR-148a and miR-152 leading to inhibition of miR-148a/152 expression in breast cancer cells. However, in normal cells, the promoters of miR-148a and miR-152 are regulated by EGR1 for decreasing IGF-IR/PKM2 activation. Disruption of this feedback regulatory circuit by DNA methylation may contribute to cancer development. The results of our current study not only support the idea that PKM2 has a crucial role in breast cancer cell proliferation and angiogenesis, but also elucidate the important roles of HIF-1α and p65 in PKM2-mediated cell growth. Furthermore, we highlighted the roles of PKM2/p65 complex in IGF-IR signal pathway for promoting breast cancer cell proliferation and angiogenesis. PKM2 has garnered attention in recent years as it is upregulated in many tumors and plays an essential role in tumor progression.8,9,30 However, two studies on PKM2 expression in © 2015 Macmillan Publishers Limited

breast cancer report contradictory results;31,32 our data support the report that PKM2 is highly expressed in breast cancer samples. TNBC is defined by the lack of expression of estrogen receptor, progesterone receptor and HER2, and TNBC is characterized by high proliferation rates, poor differentiation, early relapse and rapid progression and mortality.33 TNBC patients currently have few therapeutic options, highlighting the importance of understanding the molecular mechanisms associated with TNBC. Here, we demonstrated that activity of the PKM2/NF-κB/miR-148a/152 signaling pathway was significantly correlated with TNBC occurrence. Hypermethylation of miR-148a and miR-152 gene promoters was observed in TNBC samples compared to nonTNBC samples. The silencing of miR-148a/152 at least partly contributes to expression of high levels of PKM2 and NF-κB in TNBC. miR-148a downregulation is associated with colorectal cancer and pancreatic cancer prognosis.34,35 PKM2 overexpression is correlated with glioma as well as gastric tumor malignancy and prognosis.30,36 Thus novel regulation of PKM2 by miR-148a/152 has clinical implications in several different types of cancers. In summary, our study for the first time demonstrated that PKM2 interacts with p65 to activate EGR1 and miR-148a and miR-152 expression to overcome IGF-IR/PKM2 signals that are important for maintaining normal cell function. Our results contribute to better understanding of the mechanisms through which IGF-IR/PKM2 and PKM2/NF-κB/miR-148a/152 pathways regulate tumor angiogenesis and cancer progression and may guide development of future therapeutic interventions. MATERIALS AND METHODS Cell lines and reagents HEK293T, 184A1, T47D, MDA-MB-231, H1299 and A549 cells were obtained from American Type Culture Collection and were cultured according to the manufacturer’s instructions. IGF-1 and 5-aza-2′-deoxycytidine were purchased from Sigma (St. Louis, MO, USA). Antibodies against IGF-IR, p-IGF-IR, EGR1, Flag and PKM2 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against p65 and GAL4 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against PKM1 and HIF-1α were from Signalway Biotechnology (College Park, MD, USA) and BD Pharmingen (San Jose, CA, USA), respectively. All siRNA oligonucleotides were purchased from Thermo Scientific (Carlsbad, CA, USA). Oncogene (2015) 1 – 12

PKM2/NF-κB/EGR1/miR-148a/152 in cancer development Q Xu et al

8 Tissue samples A collection of 47 pairs of invasive ductal carcinoma specimens and adjacent normal tissues were obtained from the tissue banks of Anhui Medical University and Nanjing Medical University. All tumor samples were collected immediately after the surgical removal and snap-frozen in liquid nitrogen. None of the patients received preoperative chemotherapy. Cases were classified and selected based on diagnosis using the Anatomic Pathology system, and no information regulated by HIPPA was included in the study. Before preparing RNA, DNA, protein samples, sections were stained for histological evaluation and tumor cell evaluation. Only those cases with 470% tumor cell population in the sections were used in this study as cancer tissue samples. The experimental procedures were approved by the

Institutional Review Board of the Nanjing Medical University and Anhui Medical University.

Immunoblotting and RT-qPCR analysis Cells were lysed in RIPA lysis and extraction buffer with protease inhibitors (Thermo scientific) and the nuclear proteins were extracted using nuclear extraction Kit (Cayman Chemical, Ann Arbor, MI, USA). The supernatants were run on 8 or 10% gradients SDS-PAGE gels. Proteins were transferred to a nitrocellulose membrane and blocked with 5% non-fat milk before overnight incubation with primary antibodies as indicated in Figures. Protein bands were detected by incubation with horseradish peroxi-

Figure 5. PKM2 promotes breast cancer cell proliferation and angiogenesis. (a–d) MB-231 and T47d cells were infected with lentivirus carrying PKM2 or control vector, or transfected with siPKM2 or si-Scr. Cells were used to analyze for (a) cell proliferation assay, (b) colony formation assay, (c) HUVEC tube formation assay using conditioned medium and (d) angiogenesis responses using chicken chorioallantoic membrane (CAM) assay. The representative plugs from each group were shown, and relative angiogenesis responses were mean +s.d. from 10 CAMs (right panel). (* or # indicated Po0.05, ** or ## indicated P o0.01. * or ** indicates significant difference when compared with that of the control). (e–f) MB-231 and T47D cells stably expressing IGF-IR were transfected with siRNA targeting PKM2, p65 or a scrambled control (Scr). Cells were used to analyze for (e) cell proliferation assay, (f) colony formation assay, MB-231 cells stably expressing IGF-IR were transfected with siRNA targeting PKM2, p65 or Scr and (g) HUVEC tube formation assay by using conditioned medium (mean ± s.e.m. reported; n = 3), and (h) angiogenesis assay on the CAM (means ± s.e. shown; n = 6; *P o0.05, **Po 0.01 versus vector; #Po0.05, ##P o0.01 versus IGF-1). Oncogene (2015) 1 – 12

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9

Figure 5.

(Continued)

dase-conjugated antibodies, and visualized with an enhanced chemiluminesence reagent. Total RNAs were isolated using Trizol (Invitrogen, Waltham, MA, USA), and RT-qPCR assays were performed to test the expression levels of miR-148a and miR-152 according to the manufacturer’s instructions (Applied Biosystems, Carlsbad, CA, USA) using an ABI Prism 7900HT instrument.

Immunohistochemistry assay Immunohistochemistry analysis of PKM2 and nuclear NF-κB expression levels in adjacent normal breast tissues and breast cancer tissues were as described previously.20 Tumor samples were fixed with Bouin’s solution for 24 h, washed with 70% ethanol, and processed by the paraffin-embedded method. The tissues sections (5-μm thick) were deparafinized in xylenes and hydrated in a gradual series of ethanol and then heat immobilized or pepsin immobilized according to the manufacturer’s instructions. The slides were probed with anti-PKM2 or anti-p65 antibodies overnight at room temperature. After incubation with secondary antibody, the signal was developed using the DAB Histochemistry Kit (Invitrogen). The intensity of staining was graded according © 2015 Macmillan Publishers Limited

to the following criteria: 0 (no staining); 1 (weak staining = light yellow), 2 (moderate staining = yellow brown) and 3 (strong staining = brown).

In situ hybridization Sections (5-μm) of paraffin-embedded specimens were deparaffinized in xylenes, then rehydrated through an ethanol dilution series (from 100– 70%). Slides were submerged in diethyl pyrocarbonate-treated water and subjected to proteinase K digestion and 0.2% glycine treatment, refixed in 4% paraformaldehyde, and treated with acetylation solution; slides were rinsed thrice with PBS between treatments. Slides were prehybridized in hybridization solution (50% formamide, 5x SSC, 500 μg/ml yeast tRNA, and 1x Denhardt’s solution) at 50 °C for 30 min. Then, 10 pmol probe (LNAmodified and DIG labeled oligonucleotide; Exiqon, Vedbaek, Denmark) complementary to miR-148a or to miR-152 was added to the 150 μl of hybridization solution and hybridized for 2 h at a temperature of 20–25 °C below the calculated Tm of the LNA probe following washes in SSC with increasing stringency at the same temperature as of the hybridization. After incubation with anti-DIG-HRP Fab fragments conjugated to horseradish peroxidase, the hybridized probes were detected by incubating with Oncogene (2015) 1 – 12

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10

Figure 6. Clinical relevance of PKM2, NF-κB and miR-148a/152 expression in breast cancer. (a) Immunohistochemical (IHC) staining of PKM2 and nuclear NF-κB and ISH staining of miR-148a/152 in representative normal, non-TNBC and TNBC specimens (×200 and × 1000 magnification). Scale bar: 100 μm. (b) miR-148a and miR-152 expression in non-TNBC and TNBC samples was determined using real-time PCR. (c) Percentages of specimens showing low (score: 0-1) or high (score: 2–3) levels of PKM2, nuclear NF-κB and miR-148a/152 methylation in non-TNBC and TNBC samples. (d) Spearman correlation analyses between miR-148a/152 and PKM2, nuclear NF-κB and DNA methylation status in miR-148a/152 promoter regions in breast cancer specimens. (e) The diagram summarizes our findings: IGF-IR activation induces translocation of PKM2 into nucleus, where PKM2 interacts with p65 to form a functional complex. The PKM2/p65 complex induces NF-κB-driven EGR1 expression and subsequently, EGR1 directly binds to the promoters of miR-148a and miR-152 and activates their transcription. In addition, IGF-1/IGF-1R activation enhances HIF-1α-p65 complex formation and subsequently binds to PKM2 promoter, leading to PKM2 upregulation.

3′3-diaminobenzidine solution, and nuclei were counterstained with Carazzi’s Haematoxylin.

Plasmids and luciferase assay For the miRNA-target analysis, two fragments of PKM2-WT or PKM2-Mut were cloned downstream of the luciferase gene in pMIR-reporter plasmid. MDA-MB-231 cells were transfected using Lipofectamine (Invitrogen) with 200 ng of luciferase reporter plasmid, 100 ng of β-galactosidase (β-gal) plasmid, and 100 nmol of pre-miR-148a, pre-miR-152 or negative control precursor. Luciferase activities were measured 48 h after transfection using β-gal for normalization. For the promoter analysis, four different fragments of miR-148a promoter, three different fragments of miR-152 promoter, or one fragment of three tandem copies of the NF-κB sequence upstream of EGR1 promoter were cloned upstream of the luciferase gene in the pGL3-Luc reporter vector. HEK293T cells overexpressing EGR1 were transfected with 200 ng of luciferase reporter plasmid and 100 ng of β-gal plasmid. Experiments were performed in triplicate in three independent experiments. All primers used for plasmid construction are listed in Supplementary Table S1. Oncogene (2015) 1 – 12

DNA methylation analysis The methylation status of the promoters of miR-148a and miR-152 were analyzed by methylation-specific PCR and bisulfate-sequencing PCR as previously described.20 Genomic DNAs were modified with sodium bisulfite using the EpiTect Kit (Qiagen, Valencia, CA, USA) and analyzed by methylation-specific PCR (MSP). The methylation proportion of miR-148a and miR-152 in breast cancer specimens was assessed and scored as follows: 0 (only U band), 1 (U band is stronger than M band), 2 (M band is stronger than U band), and 3 (only M band).

Virus production Lentiviral plasmids expressing miR-148a, miR-152 and control were obtained from Open Biosystems (Pittsburgh, PA, USA) and plasmids expressing IGF-IR, EGR1 and PKM2 were obtained from GeneCopoeia (Rockville, MD, USA). The viruses were generated by transfection of HEK293T cells with 10 μg transducing vector and packaging vectors (6.7 μg sPAX2 and 3.3 μg CMVVSV-G). Virus particles in the medium were harvested after 24 h and harvested every 12 or 24 h thereafter. The collected viral supernatants were filtered and transduced into targeted cells. © 2015 Macmillan Publishers Limited

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11 ChIP-qPCR assay

Metabolism assay

ChIP-qPCR assays were performed using ChIP Assay Kit (Qiagen). Cells were crosslinked by addition of 1% formaldehyde at room temperature for 10 min and quenched in 0.125 M glycine. The cells were rinsed with PBS buffer, detached with trypsin and centrifuged. DNA was immunoprecipitated from the sonicated cell lysates by using antibodies against EGR1 or IgG overnight at 4 °C and collected by incubation with protein A beads for 2 h. The complexes were eluted in elution buffer, treated with proteinase K. DNA was purified by QIAqucick PCR purification Kit (Qiagen) and was used as a template for quantitative-PCR analysis. Fold enrichment was calculated based on Ct as 2 − Δ(ΔCt), where ΔCt = CtIP—CtInput and Δ (ΔCt) = ΔCtantibody—ΔCtIgG. Primer sequences used in this study are listed in Supplementary Table S1.

HEK293T, MDA-MB-231 and H1299 cells that stably overexpressed miR-148a or miR-152 or those cells with PKM2 overexpression were cultured for 48 h. The conditioned mediums were collected and the glucose and lactate levels were measured by using glucose assay kit and lactate assay kit (BioVision, Milpitas, CA, USA), respectively. The glucose consumption and lactate production were normalized to cell number.

Coimmunoprecipitation assay HEK293 cells were transfected with pFlag-p65 or pFlag-p65 (1-304) or pFlag-p65 (292-551) expression vectors for 48 h and treated with 50 nmol of IGF-1 for 6 h. Whole cell extracts were harvested, then incubated with 1 mg of anti-FLAG, anti-PKM2 or control IgG antibodies (Santa Cruz) overnight at 4 °C in immunoprecipitation (IP) buffer. Protein G-agarose beads (10 ml) were added and incubated further for 1 h. The beads were washed five times with 1 ml of IP buffer. Antibody-bound complexes were eluted by boiling in loading sample buffer. Immunoprecipitates were denatured and proteins were analyzed by immunoblotting. To test PKM2 and EGR1 protein–protein interaction assay, the HEK293 cells infected with lentivirus expressing EGR1 and treated with 50 nmol of IGF-1 for 1 h. The whole cell extracts were incubated with 1 mg of anti-PKM2, anti-EGR1 or control IgG and preformed the experiment as above.

Statistical analysis The results were analyzed using the SPSS 13 statistical software (Chicago, IL, USA). Quantitative variables were analyzed using Student's t-test between two groups or one-way analysis among multiple groups. The correlations were analyzed using Spearman’s rank test. The differences were considered significant at Po 0.05.

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This work was supported in part by National Natural Science Foundation of China (81320108019, 81270736, 81071642), by Jiangsu Province Clinical Science and Technology Project (BL2012008), the Priority Academic Program Development of Jiangsu Higher Education Institutions (JX10231801), Jiangsu Province’s Key Discipline of Medicine (XK201117), and by National Institutes of Health grants R01ES020868 and R21CA175975.

Immunofluorescence assay

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Cells were transfected with or without expression vector for Flag-p65 (RHD). After incubation for 48 h, cells were treated with or without 50 nmol of IGF-1 for 1 h. Cells were fixed with 4% formaldehyde, permeabilized with PBS containing 0.5% Triton X-100 at 4 °C, and blocked with 2% bovine serum albumin (BSA) and 0.24% goat serum (Sigma) in PBST (PBS containing 0.1% Triton X-100). Cells were immunostained with antibodies against PKM2, p65 or Flag in PBST containing 2% BSA overnight at 4 °C. The antigen-primary antibody complex was detected using fluorescence isothiocyanate (FITC)-labeled goat anti-rabbit secondary antibodies (Santa Cruz) and tetramethyl rhodamine isothiocyanate (TRITC)-labeled goat antimouse secondary antibody (Santa Cruz). Microscopic observation was performed under a fluorescence microscope (Nikon).

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Cell proliferation and soft agar colony formation assay For cell proliferation assay, cells were trypsinized, counted and plated into triplicate wells of 12-well plates at 1 × 104 cells per well. Total cells were harvested and counted using hemocytometer after 5 days of culture. For colony formation assay, 1 ml of 0.5% solidified SeaPlaque agarose (BMA, ME, USA) was added to each well of 6-well plates, then 5 × 103 cells were mixed with 2 ml of 0.5% SeaPlaque and added onto the top of the well. After 12 days of culture, colonies were fixed with 100% methanol for 15 min and stained with 0.1% crystal violet. Colonies with diameter more than 1.5 mm were counted. The experiments were performed with three replicates, and repeated for three times.

Tube formation assay Matrigel (50 μl; BD Biosciences, San Jose, CA, USA) was placed into each well of a 96-well plate and polymerized for 1 h at 37 °C. HUVEC cells in 50 μl conditional medium were added to each well and incubated for 6–12 h. Images were captured with a digital camera. The capillary tubes were quantified by determination of length. Each condition was assessed in triplicate.

Chicken chorioallantoic membrane assay The white fertilized chicken eggs were incubated at 37 °C under conditions of constant humidity. An artificial air sac was created over a region containing small blood vessels in the CAM. The cells were implanted onto the CAM. Angiogenesis responses were analyzed 4 days after the implantation, and the numbers of total blood vessels were counted by two observers in a double-blind manner. © 2015 Macmillan Publishers Limited

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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

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