Pyruvate Kinase M2 Activates mTORC1 by Phosphorylating AKT1S1

In cancer cells, the mammalian target of rapamycin complex 1 (mTORC1) that requires hormonal and nutrient signals for its activation, is constitutively activated. We found that overexpression of pyruvate kinase M2 (PKM2) activates mTORC1 signaling through phosphorylating mTORC1 inhibitor AKT1 substrate 1 (AKT1S1). An unbiased quantitative phosphoproteomic survey identified 974 PKM2 substrates, including serine202 and serine203 (S202/203) of AKT1S1, in the proteome of renal cell carcinoma (RCC). Phosphorylation of S202/203 of AKT1S1 by PKM2 released AKT1S1 from raptor and facilitated its binding to 14-3-3, resulted in hormonal- and nutrient-signals independent activation of mTORC1 signaling and led accelerated oncogenic growth and autophagy inhibition in cancer cells. Decreasing S202/203 phosphorylation by TEPP-46 treatment reversed these effects. In RCCs and breast cancers, PKM2 overexpression was correlated with elevated S202/203 phosphorylation, activated mTORC1 and inhibited autophagy. Our results provided the first phosphorylome of PKM2 and revealed a constitutive mTORC1 activating mechanism in cancer cells.

Pyruvate kinase (E.C. 2.7.1.40) is a rate-limiting glycolysis enzyme that catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP, resulting in the formation of pyruvate and ATP 14 . Among the four pyruvate kinase isoforms expressed in mammals is the M1 isoform (PKM1), which is expressed in most adult tissues; the L and R isoforms, which are specifically expressed in liver and red blood cells 15,16 , respectively; and the M2 isoform (PKM2), which is expressed during embryonic development and in most adult cells, except in adult muscle, brain and liver cells 17 . The amino acid sequence of PKM2 is identical to PKM1, except for a 23 amino acid stretch (a.a. 378-434) at its C-terminus. The c-Myc-heterogeneous nuclear ribonucleoprotein-dependent alternative splicing of exon 9 and exon 10 of the transcript of the PKM gene result in PKM1 and PKM2, respectively 18 . Exon 9-containing PKM1 exists as a glycolytically active stable tetramer, and exon 10-containing PKM2 exists in a dynamic equilibrium between a glycolytically inactive dimer and a glycolytically active tetramer.
Proposed underlying tumorigenic mechanisms of PKM2 include facilitating anabolic metabolism by diverting glycolytic intermediary metabolites to anabolic pathways 17 . The introduction of PKM2, but not glycolytic active PKM1, into PKM2 knockdown cancer cells restored their ability to form tumor xenografts 17 , showing that the non-glycolytic functions of PKM2 are needed to sustain cancer growth. Moreover, switch PKM2 from dimer to tetramer by small molecule TEPP-46 inhibited oncogenic growth of xenograft tumors 19 , highlighting tumorigenic importance of dimeric PKM2. Upon stimulation by epidermal growth factor (EGF), interleukin-3 or apoptotic signals, dimeric PKM2 translocate into the nucleus and display various functions 20,21 . For example, nuclear PKM2 associates with chromatin 20,22 , binds to the C-terminus of Oct-4 and enhances Oct-4-mediated transcription 23 , binds to HIF1 to recruit the p300 transcriptional co-activator to enhance the hypoxic transcriptional response 24 and contributes to the transactivation of cyclin D and c-Myc 25,26 . Remarkably, the kinase activity of PKM2 is required for its nucleic actions, implying that PKM2 is either a protein kinase or that metabolites associated with PKM2 are required for these functions.
Dimeric PKM2 was initially found to phosphorylate histone H3 at T11 upon EGF receptor activation 27 , exhibit cysteine-dependent histone H1 phosphorylation activity 28 and activate the transcription of MEK5 by phosphorylating stat3 at Y705 29 . Later, a protein array assay showed that SAICAR-bound PKM2 phosphorylates an array of substrates and, in particular, activates Erk1/2 signaling to induce cell proliferation 30 . These findings suggested that PKM2 is a multi-specific protein kinase that regulates a number of substrates. Notably, the protein kinase activity of PKM2 was also challenged by some experiments 31 while results from yeast protein equivalent to PKM2 reassure protein kinase of PKM2 32 . A quantitative phosphoproteomic approach employs a fixed proteome as the substrates 33,34 made cell-wide screening of PKM2 substrates possible and may provide answers for whether PKM2 has protein kinase activity.

PKM2 Substrates Survey by Quantitative Phosphoproteomic Approach.
To identify substrates of PKM2, we adopted a quantitative phosphoproteomic approach 34,35 . Briefly, proteome of RCC were cross-linked onto Sephoraose-4B resin to prevent endogenous kinases from accessing to their substrates. Alkaline phosphatase was used to remove existing phosphorylation in the proteome. Following removal of alkaline phosphatase by washing, the phosphorylation free proteome was re-phosphorylated by PKM2 with PEP as the phospho-donor. PKM2-untreated and -treated tryptic peptides library were each labeled with hydrogen and deuterium formaldehyde and sodium cyanoborohydride (NaBH 3 CN), respectively. Phospho-peptides were enriched by Ti4 + -IMAC microspheres and the relative abundance between PKM2-untreated and -treated phosphor-peptides were compared by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) (Fig. 1A).
Consistent with that wild-type PKM2, not its inactive PKM2 K270M mutant 36 increased the levels of the phosphoserine (P-Ser), phosphothreonine (P-Thr) and phosphotyrosine (P-Tyr) of RCC proteome in a PEP dependent manner (Fig. 1B), a total of 974 residues in 405 proteins, including 13 of the PKM2 substrates previously identified by the protein array assay 30 , were identified as potential PKM2 substrates (Table S1). A twofold increase in the phosphorylation levels of treated over untreated peptides was employed as cutoff for high-confidence (> 99%) in vitro substrates identification, as this ratio was well beyond the range of inherent variability.
Bioinformatic Characterization of PKM2 Substrates. Among the positively identified substrates of PKM2, 876 are serines, 81 are threonines and 17 are tyrosines, confirmed that PKM2 is a multi-specific protein kinase. WebLogo analysis 37 showed that PKM2 prefers to phosphorylate serine or threonine residues spanned by acidic amino acids ( Fig. 2A). Motif-x analysis 38 revealed no strong consensus sequences in the substrates of PKM2 ( Figure S1A). Moreover, as estimated by NetSurfP 1.1 39 , 95.95% of the phosphorylation sites of PKM2 are solvent exposed (Fig. 2B), and the vast majority (96.36%) of the resides phosphorylated by PKM2 are predicted 40 to be within the disordered regions of proteins ( Figure S1B). These results were consistent with the fact that PKM2 predominantly phosphorylates sites in coils (87.87%) rather than in more ordered regions, such as α -helices (11.65%) and β -sheets (0.48%) (Fig. 2C).
Gene ontology annotations 42 revealed that the substrates of PKM2 are involved in diverse biological functions, ranging from RNA processing to mitosis (Fig. 2E). The mechanisms of actions of PKM2 substrates include protein binding, DNA binding, RNA binding, helicases, histone demethylases and transcription factors ( Figure S1C). Remarkably, KEGG GO enrichment analysis revealed that PKM2 substrates are enriched in prostate, endometrial thyroid, colorectal, chronic myeloid leukemia and non-small cell lung cancer pathways, as well as in cancer Scientific RepoRts | 6:21524 | DOI: 10.1038/srep21524 associated pathways, such as DNA repair and cell cycle pathways (Fig. 2F), in line with the facts that PKM2 is preferentially expressed in most cancers. Collectively, these results suggest that PKM2 phosphorylates an array of substrates and regulates a plethora of cancer associated cellular functions.
Tyrosine substrates of PKM2 were not subject to bioinformatics analysis because the number of identifications was insufficient for statistical analysis, due to that insufficient extraction of membrane fraction, where most phospho-tyrosine proteins are located. In line with this expectation, we did see increase of phosphor-tyrosine in the membrane fraction by PKM2 overexpression ( Figure S1D).
PKM2 Phosphorylates S202 and S203 of AKT1S1. The phosphorylation levels of serine 202 (S202) and serine 203 (S203) of mTORC1 inhibitor AKT1S1 were increased by 177.0 fold and 137.7 fold, respectively, by PKM2 treatment (Fig. 3A, Table S1), suggested that functions of AKT1S1 and activity of mTORC1 may be regulated by PKM2. This hypothesis was further confirmed. When they are co-expressed in HEK293T cells, PKM2 was co-purified with AKT1S1 ( Fig. 3B) and AKT1S1 was also co-purified with PKM2 (Fig. 3C), showed that PKM2 and AKT1S1 interact with each other. Moreover, incubation of purified AKT1S1 with recombinant PKM2 resulted in an elevation of the P-Ser levels of AKT1S1 in a PEP dependent manner (Fig. 3D), confirmed that PKM2 phosphorylates AKT1S1 directly. However, supplementation TEPP-46 to the culture media to promote Proteins in RCC lysate were treated as indicated in the blue brackets, the resulted peptides were labeled, mixed and enriched for phosphopeptides, followed by LC-MS/MS analysis to identify PKM2 substrates. (B) Proteins in a lysate of RCC were de-phosphorylated with alkaline phosphatase and precipitated with acetone. The redissolved proteins were treated with recombinant PKM2 or PKM2 K270M (K270M), employing PEP as the phosphor-donor. The levels of P-Ser, P-Thr and P-Tyr in proteins were determined after treatments.
Phosphorylation of S202/S203 of AKT1S1 Dissociates AKT1S1 from Raptor. Phosphorylation on sites of AKT1S1, such as on threonine 246, releases AKT1S1 from raptor and activates mTORC1 45 . We thus  tested whether the phosphorylation on S202/S203 of AKT1S1 by PKM2 also affects the AKT1S1-raptor interaction. Purified AKT1S1 treated by recombinant PKM2 and PEP had increased P-S202/203 levels but significantly lower ability to pull down raptor (Fig. 4A), imply that phosphorylation on S202/S203 of AKT1S1 decrease interaction between AKT1S1 and raptor. Supporting this notion, overexpression of PKM2, but not kinase inactive PKM2 K270M , decreased the interaction between AKT1S1 and raptor in HEK293T cells (Fig. 4B). Moreover, PKM2 overexpression failed to alter the interactions between raptor and AKT1S1 S202A,S203A (2SA) or AKT1S1 S202E,S203E (2SE) (Fig. 4C,D). These results collectively suggested that PKM2 regulates AKT1S1-raptor interaction by phosphorylating S202/203 of AKT1S1. Furthermore, overexpression of PKM2 in HEK293T cells greatly decreased the interaction between the S202 to alanine AKT1S1 (S202A) mutant and raptor, but only slightly decreased the interaction between the S203 to alanine AKT1S1 (S203A) mutant and raptor (Fig. 4E), suggest that PKM2 mediates AKT1S1-raptor interaction mainly through phosphorylating S203 of AKT1S1.
Activation of mTOR signaling usually requires nutritional and hormonal signals 7 . However, PKM2 overexpression alone activated mTOR signaling under serum starvation (Fig. 5H), amino acids starvation (Fig. 5I) or both (Fig. 5J). These results showed that PKM2 overexpression is sufficient to activate mTOR signaling, regardless the existence of growth factors and amino acids signals.
Numerical values below the gels indicate quantification of the bands relative to untreated AKT1S1 (hereinafter). (E) Flag-tagged AKT1S1 was co-expressed with PKM2. P-ser levels of AKT1S1 from cells cultured with and without TEPP-46 (100 nM) supplementation were determined. (F) Flag-tagged AKT1S1 was co-expressed with PKM2 or PKM2 Y105F (Y105F), P-ser levels of AKT1S1 purified from different cells were determined. (G) Flag-tagged AKT1S1 was co-expressed with either HA-tagged PKM2 or HA-tagged PKM2 K270 mutant (K270M) in HeLa cells. The P-Ser levels of Flag bead-purified AKT1S1 from each culture were determined and quantified. (I) Purified AKT1S1 was treated with either purified PKM2 or purified K270M. The P-S202, P-S203 and P-S202/203 levels of each treated AKT1S1 were determined by site-specific antibodies and quantified. The relative intensities of phosphorylation signals were normalized to those of untreated AKT1S1. (J) Flag-tagged PKM2 or Flag-tagged K270M was overexpressed in HEK293T cells. The endogenous P-S202/203 levels of AKT1S1 of each culture were determined and the relative intensities of P-S202/203 signals were normalized to that of HEK293T cells. (K) The endogenous P-S202/203 levels of HEK293T cells before and after PKM2 knockdown by independent shRNAs were compared. The PKM2 knockdown efficiency was confirmed by western blot.
Scientific RepoRts | 6:21524 | DOI: 10.1038/srep21524 PKM2 Inhibits Autophagy by Activating mTORC1. The activation of mTORC1 is known to prevent autophagy 48,49 , a catabolic pathway induced by harmful stimuli, including nutrient deprivation. Changes of markers of autophagosome formation, including a decrease of p62 and an increase in the conversion of LC3B-I to LC3B-II 50,51 , were prevented by PKM2 overexpression in HEK293T, HeLa, HCT116 p53+/+ and HCT116 p53−/− cells cultured under serum starvation (Fig. 6A, S5A-C). These results were echoed by an immunofluorescence assay that revealed PKM2 overexpression in HeLa cells reversed serum starvation-induced LC3B overexpression (Fig. 6B), and showed that PKM2 overexpression prevents serum starvation-induced autophagy. Moreover, the autophagy-relieving effect of PKM2 was abolished by shutting down mTORC1 signaling with rapamycin (Fig. 6C), and chloroquine-induced mTORC1-independent autophagy was not responsive to PKM2 overexpression (Fig. 6D). These results suggested that PKM2 inhibits autophagy by mediating mTORC1 activity. Furthermore, consistent with that TEPP-46 abrogates PKM2's protein kinase activity (see Fig. 3E), the autophagy preventive effects of PKM2 were abolished by TEPP-46 (Fig. 6E), confirmed that PKM2 prevents autophagy through its protein kinase activity. Lastly, the overexpression of AKT1S1, but not 2SA, induced autophagy in Purified raptor was incubated with AKT1S1, AKT1S1 + PEP or AKT1S1 + PEP + PKM2. After incubation, AKT1S1 was purified by Flag beads. The amount of raptor copurified with AKT1S1 and the PS202/203 level of AKT1S1 were determined and normalized to that of AKT1S1 incubated with raptor without other components. (B) Raptor and AKT1S1 were co-expressed with either PKM2 or K270M in HEK293T cells. The amount of raptor co-precipitated with AKT1S1 from different cells was determined. (C) Raptor was co-expressed with either AKT1S1 or 2SA in HEK293T cells. The amount of raptor co-immunoprecipitated with AKT1S1 or 2SA in the presence and absence of PKM2 was compared. (D) Raptor was co-expressed with either AKT1S1 or 2SE in HEK293T cells. The amount of raptor co-immunoprecipitated with AKT1S1 or 2SE in the presence and absence of PKM2 was determined and raptor signals were normalized to that co-expressed with AKT1S1 alone. (E) Raptor was co-expressed with either S202A or S203A in HEK293T cells. The amount of raptor co-immunoprecipitated with S202A or S203A in the presence and absence of PKM2 was determined and compared. (F) 14-3-3 and AKT1S1 were co-expressed with either PKM2 or K270M in HEK293T cells. The amount of 14-3-3 co-immunoprecipitated with AKT1S1 was compared under different coexpression conditions. HeLa cells, and overexpression of PKM2 only rescued autophagy induced by AKT1S1 overexpression (Fig. 6F), showed that PKM2 inhibits autophagy through phosphorylating S202/203 of AKT1S1.

Correlation of PKM2 Expression with AKT1S1 S202/203 Phosphorylation, mTOR Activation and Autophagy Inhibition in Cancers.
To confirm that PKM2 expression activates mTOR signaling and inhibits autophagy, we analyzed markers of each molecular events in renal cell carcinoma (RCC) and breast cancer samples. Immunohistochemistry (IHC) analysis was performed to 10 samples of each tumor that contained both normal and cancer tissues to show differential expression of each marker in normal and cancer tissues. In both RCC (Fig. 7A, S6A) and breast cancer (Fig. 7B, S6B), PKM2 was overexpressed in cancer tissues, alongside with elevated phosphorylation of S202/203, phosphorylation of mTOR and phosphorylation of 4EBP that signaled activation of mTOR signaling, and elevated p62 that signaled inhibited autophagy. These results provided evidence that PKM2 activates mTOR signaling and inhibits autophagy in vivo.

Discussion
In the current study, we report as far as we acknowledged the first phosphorylome of PKM2, shed lights on the plethora functions of PKM2 52 . Our proteomic approach was validated by that substrates we identified overlapped some of the substrates previously identified by protein microarray assays 30 (Table S1). However, by no mean our identifications were complete since some of the documented PKM2 substrates, such as T11 of H3 27 and Y705 of stat3 29 , were not identified by the current survey. Nevertheless, close to 70% of the PKM2 substrates are located in the nucleus (Fig. 2D) and PKM2 substrates are enriched in various cancer pathways (Fig. 2F), consistent with the fact that the protein kinase activity of PKM2 plays critical roles in cancer biology. Interestingly, although no consensus sequence was identified for PKM2, the phosphorylation site preference of PKM2 is similar to that of Casein Kinase II (CK2), a conserved protein serine/threonine kinase that preferentially phosphorylates serine and threonine sites spanned by acidic amino acids 33,35 , suggested that PKM2 may, like CK2, may play a plethora of roles in proliferation, apoptosis, differentiation, transformation and carbohydrate metabolism regulation 53,54 .
The finding that PKM2 phosphorylates S202/203 of AKT1S1 revealed a linkage between PKM2 overexpression and mTOR activation in cancer cells. Previous phosphorylomic studies had repeatedly found that Ser202 and Ser203 are phosphorylated by unidentified upstream kinases [55][56][57][58] . The biologic significance of S202/203 phosphorylation was therefore uncharacterized. Our study showed that S202/203 of AKT1S1 are phosphorylated by PKM2 and their phosphorylation activates mTORC1 by relieving AKT1S1 from raptor. Importantly, mTOR signaling activation by PKM2 is independent of nutritional signals (Fig. 5H-J). This relieved cancer cells from restrains of nutrients and hormonal signals that are subject to fluctuate, ensures constant growth/proliferation advantages for them over normal cells. Moreover, activation of mTORC1 by PKM2 highlights the how PKM2 overexpression promotes cancerous metabolism. PKM2 promotes accumulation of glycolytic intermediates for biosynthesis 17 as well as activates the mTOR signaling, which utilizes intermediates for macromolecules biosynthesis 4 . PKM2 thus simulates those of mutations of tumor suppressor p53 or oncogene c-Myc, which not only promote aerobic glycolysis to accumulate glycolytic intermediates 59,60 but also activate anabolic processes 61,62 . This, together with that mTOR activation was reportedly to increase PKM2 expression 63 , suggests that there exist a positive regulatory loop between mTORC1 and PKM2. When either PKM2 is overexpressed or mTOR signaling is activated, PKM2 expression and mTOR signaling are concomitantly activated through this loop. Therefore, when either PKM2 or mTORC1 are activated, both anabolic intermediates accumulation and utilization and autophagy inhibition will be resulted (Fig. 7C). All these effects facilitate the onset of cancers.
In RCC and breast cancer samples, strong correlations had been found among PKM2 overexpression, S202/203 phosphorylation, mTOR activation and autophagy inhibition (Fig. 7), consistent with that PKM2 overexpression and mTOR activation are both exist in RCC and breast cancers 5,64,65 and support that PKM2 activates mTOR signaling in vivo. Given that PKM2 expression and its protein kinase activity is involved in cell cycle regulation 66 , inhibiting protein kinase activity of PKM2 may represent a mean to curb anabolic metabolism and cancer progression.

Materials and Methods
Cell culture, materials and antibodies. HEK293T and HeLa cells were cultured in DMEM containing 10% NCS. HCT116 +/+ and HCT116 −/− were cultured in McCoy′ 5A containing 10% FBS. Cells were transfected with plasmids using polyethylenimine (PEI, linear, 25KDa). AKT1S1 S202A, S202E, S203A and S203E were to those of untreated cells. (D) The levels of P-T37/46-4EBP were determined by immunofluorescence in HeLa cells and PKM2 overexpressing HeLa cells with and without rapamycin supplementation in the culture media, respectively. Bar scales are 25 μ m. (E) EGF effects on the levels of endogenous P-T389-S6K and P-T37/46-4EBP of HEK293T cells and PKM2 knockdown HEK293T cells were determined and quantified relative to that of non-treated cells. (F) AKT1S1 was knocked down by shRNA in HEK293T cells (bottom). The levels of P-T389-S6K and P-T37/46-4EBP in response to PKM2 overexpression were determined in AKT1S1 knockdown cells after shRNA resistant AKT1S1, 2SA and 2SE were each re-introduced into cells. P-T389-S6K and P-T37/46-4EBP signals were normalized relative to untreated cells. (G) Growth curves of HEK293T cells, the PKM2 overexpressing HEK293T cells, the AKT1S1 knockdown HEK293T cells and PKM2 overexpressing AKT1S1 knockdown HEK293T cells were determined. Shown are the average values (n = 3) with SD. Knockdown efficiency of AKT1S1 is demonstrated in (F). (H-J) The levels of endogenous P-T389-S6K and P-T37/46-4EBP of HEK293T cells were detected under serum starvation (SS, H), amino acids starvation (AAs Starv., I) and both (J) were detected. Amino acids starvation was achieved by culturing cells in basal DMEM with all other ingredients except amino acids.
Chloroquine diphosphate salt (c6628-25 g) was from Sigma-aldrich, TEPP46 was from Cayman. Antibodies were either home-made or commercially purchased.
Quantitative phosphorylomic analysis. Tryptic peptides of PKM2 treated and untreated proteome of HEK293T cells were heavy and light labeled, respectively, as described in text. The Ti 4+ -IMAC microspheres were used to enrich the phosphopeptides following the reported protocol. For mass spectrometric analysis, a quaternary surveyor MS pump (Thermo, San Jose, CA) coupled with a LTQ Orbitrap XL mass spectrometer (Thermo, CA) was used. Data analysis and detailed parameters are described in the supplementary information. Phosphorylation levels increased more than twofold by PKM2 are selected as candidate substrates of PKM2.
Western blot, dot blot and immunofluorescence analysis. Standard procedures were followed for western blot, dot blot and Immunofluorescence analysis. Western blot signals, dot blot signals were obtained by detecting chemiluminescence on Typhoon FLA 9500 (GE Healthcare). Scoring of Immunofluorescence of LC3B was analyzed using ANOVA with Turkey's post-test (One-way ANOVA for comparisons between groups, Two-way ANOVA for comparisons of magnitude of changes between different groups from different cell lines). Representative IHC (left) and statistic (right, n = 10) results are shown. For RCC samples, normal and tumor tissues are marked by N and T, respectively. For breast cancer, tumor and normal tissues are marked by red and yellow arrows, respectively. Pathologic results were confirmed by experienced pathologists. Bar scales were 100 μ m, heights of breast cancer samples were compressed to 1/2. (C) Schematic diagram of the PKM2-mTORC1 regulatory loop. Overexpression of PKM2 leads accumulation of anabolic intermediates and activation of mTOR signaling that promotes utilization of anabolic intermediates and inhibits autophagy. Meanwhile, mTOR activates PKM2 to form a positive loop to enhance the anabolic processes. Signals that activate either PKM2 or mTORC1 can result in both anabolic functions.
Scientific RepoRts | 6:21524 | DOI: 10.1038/srep21524 Bioinformatics analysis. All analysis was performed using published software. The gene ontology annotations were downloaded from QuickGO database, while the enrichment analyses were performed with a hypergeometric distribution. The enrichment analyses of KEGG pathways were carried out using DAVID. All the heatmaps were visualized with the ggplot2 program (http://had.co.nz/ggplot2/) in the R package (http:// www.r-project.org/). The amino acid preferences were visualized with WebLogo, while the comparisons of amino acid preferences were visualized with Two Sample Logo.
Human Samples and Mutation Screening. Human samples are acquired from Ruijing Hospital, Shanghai Jiaotong University. Informed consents from the patients were obtained. The procedures related to human subjects were carried out in accordance with the approved guidelines approved by Ethic Committees of Shanghai Jiaotong University.
Immunohistochemistry. Tissue sections were prepared form the formalin-fixed paraffin embedded specimens. Antigen retrieval of renal cell carcinoma or breast cancer specimens was performed by incubating the slides in Tris-EDTA buffer (pH 8.4) at 99 °C for 60 minutes. The endogenous peroxidase activity was inactivated in solution of methanol with 3% H2O2. The slides were incubated with primary antibody for 60 minutes and secondary antibody for 8 minutes, followed by DAB Chromagen stainning for 8 minutes. All procedures were performed using stainer (BenchMark XT, Ventana) and the slides were scanned by scanner (Ventana iScan Coreo). The quantification of IHC results were performed by an experienced pathologist. The intensity was calculated according to positive areas and positive degree. Sections were staining with PKM2 (1:100), P-S202/203-AKT1S1 (1:30), P-S2448-mTOR (1:100), P-T37/46-4EBP1 (1:500), p62 (1:200) and LC3B (1:100) antibody using an Ultraview Detection Kit.
Statistical Analysis. Significant differences between groups were determined using Student's T test. The significance level for statistical testing was set at p < 0.05.