Knockdown of pyruvate kinase M2 suppresses bladder cancer progression

Clinical specimens

Prior approval for the use of clinical samples for research purposes was obtained from the Second Xiangya Hospital of Central South University (Changsha, China). The institutional ethics and scientific committee approved this study. We collected pairs of fresh BCa tissues and normal adjacent tissues from 36 patients who underwent radical cystectomy and for whom related clinical parameters were available. Written consent was obtained from all patients.

Cell culture

T24, 5,637 and EJ cells were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and were maintained in RPMI 1640 (catalogue number: 11875119, Gibco, New York, USA) supplemented with 10% foetal bovine serum (catalogue number: 10099141C, Gibco). HCV29 and UM-UC-3 cells were provided by Shenzhen Second People’s Hospital (Shenzhen, China) and were cultured in Dulbecco’s modified Eagle medium (catalogue number: 11965092, Gibco) supplemented with 10% foetal bovine serum. Cells were maintained in a humidified 5% CO₂ environment at 37 °C.

RNA interference

The pLKO.1-puro vector was purchased from XIAMEN Anti-HeLa Biological Technology Trade Co. Ltd. (Xiamen, China) and was used to prepare plasmids encoding the shRNA of PKM2 (shPKM2) or negative control of shPKM2 (shCtrl). The corresponding primer sequences for shPKM2 were designed with DNAMAN (LynnonBiosoft, CA, USA) and synthesized according to the pLKO.1-puro vector specification and the human PKM2 gene (Table 1).

As previously described, lentiviruses that were concentrated were packaged using plasmids shPKM2 or shCtrl, and the TCID₅₀ of lentiviruses was detected [12]. According to the previous description, the obtained lentiviruses and T24 cells or UM-UC-3 cells overexpressing luciferase (Xiamen Immocell Biotechnology Co. Ltd., Xiamen, China) were used to generate stable PKM2 knockdown cell lines and their negative controls [13].

Measurement of glucose consumption

Cellular glucose metabolism rates were measured by following the conversion of 5-³H-glucose to ³H₂O as described previously [14]. In brief, when grown to approximately 80% confluence, the cells were incubated at 37 °C for 30 min in Krebs buffer (catalogue number: PH1832, Scientific Phygene, Fuzhou, China) without glucose after being washed twice with phosphate buffered solution (PBS, catalogue number: C0221A, Beyotime Biotechnology, Shanghai, China). Subsequently, the buffer was replaced with Krebs buffer containing 10 mM glucose spiked with 10 μCi of 5-³H-glucose. After 1 h, the supernatant samples were transferred to PCR tubes containing 0.2 N HCl (catalogue number: 10011018, Sinopharm Group, Beijing, China), and the amount of ³H₂O was determined by diffusion as described previously [14].

Measurement of lactate production

The cells grew to approximately 80% confluence in 12-well plates. The old supernatant was discarded. Then, the cells were incubated in fresh growth medium for 1 h after washing twice with PBS. The lactate produced by treated cells was measured using the PicoProbe™ Lactate Fluorometric Assay Kit (catalogue number: ab169557, abcam, Cambridgeshire, United Kingdom) according to the manufacturer’s instructions. The cell number was determined using a Multisizer 4e Coulter Counter (Beckman Coulter Inc., CA, USA).

Measurement of cell apoptosis

T24 cells with or without shPKM2 were collected and suspended in annexin V-fluorescein isothiocyanate and propidium iodide reagents (catalogue number: A211-01, Vazyme, Nanjing, China) at 27 °C for 30 min in the dark following the manufacturer’s instructions. The prepared cells were subjected to flow cytometry analysis (NovoCyte 1300, ACEA Biosciences Inc., CA, USA).

ROS detection

T24 cells with or without shPKM2 were loaded with a reactive oxygen species detection kit (catalogue number: S0033S, Beyotime Biotechnology) according to the manufacturer’s instructions. The cells were then subjected to flow cytometry analysis.

Western blotting analysis

Cells were lysed in ice-cold RIPA buffer (catalogue number: P0013C, Beyotime Biotechnology), and 20 µg of protein was separated by electrophoresis on 8% denaturing SDS‒PAGE gels. After the separated proteins were transferred to membranes, the membranes were incubated with 5% bovine serum albumin (catalogue number: ST2254-20g, Beyotime Biotechnology) in PBS at room temperature for 2 h, followed by incubation with primary antibodies overnight at 4 °C. The next day, the membranes were incubated with the appropriate secondary antibodies at room temperature for 1 h. The chemiluminescent substrate kit (catalog number: WP20005, Invitrogen, CA, USA) was added to the membrane and the protein bands were visualized using X-ray film. The signal intensity was quantified using ImageJ software (version 8, National Institutes of Health, Maryland, USA). The study employed primary antibodies against PKM2 (catalogue number: 4053, 1:1000, Cell Signaling Technology, Boston, USA), vimentin (catalogue number: 46173, 1:1000, Cell Signaling Technology), E-cadherin (catalogue number: 3195, 1:1000, Cell Signaling Technology), fibronectin (catalogue number: 26836, 1:1000, Cell Signaling Technology), or β-actin (catalogue number: 4970, 1:1000, Cell Signaling Technology), and secondary horseradish peroxidase (HRP)-linked antibodies against mouse IgG (catalogue number: 7076, 1:2000, Cell Signaling Technology) or rabbit IgG (catalogue number: 7074, 1:2000, Cell Signaling Technology).

Cell proliferation assay

For the cell proliferation assay, cells were seeded in 96-well plates at an initial density of 3 × 10³ cells/well. The cells were stained with cell counting kit-8 (CCK-8, catalogue number: C0037, Beyotime Biotechnology) for 1 h at 37 °C at each time point. Then, the absorbance of each well was measured at 450 nm using a SpectraMax^® Absorbance Plate Reader (supplier number: PLUS 384, Molecular Devices, San Francisco, USA). All experiments were performed in triplicate.

Transwell assay

Migration and invasion assays were performed using transwell plates (Corning, Shanghai, China) with 8-μm-pore membranes precoated without or with Matrigel (catalogue number: E1270, Sigma-Aldrich, Shanghai, China), respectively. In total, 1 × 10⁵ T24 cells with or without shPKM2 were plated in the upper chambers of the transwells. After a 24-h incubation, the cells that crossed the membrane were stained with 0.5% crystal violet, and six random fields per membrane were counted. The migrated or invaded cells were counted and photographed under a light microscope (Motic, Xiamen, China).

Wound healing assay

T24 cells infected with virus containing shCtrl or shPKM2 were seeded into 6-well plates (Corning, Shanghai, China) and grown to 100% confluence. A straight-line wound was created by scratching the monolayer with a 200 μL pipette tip (Corning, Shanghai, China). The cells were continuously cultured in serum-free medium for 24 h and observed under a microscope (Motic, Xiamen, China).

Immunohistochemical (IHC) staining

Deparaffinized and rehydrated tissue sections were pretreated with 3% peroxidase in methanol (catalogue number: 10014108, Sinopharm Group) for 10 min at room temperature. Antigen retrieval was performed by boiling the sections in citrate buffer (catalogue number: P4809, Sigma-Aldrich) (pH 6.0) for 30 min. After preincubation with 10% normal goat serum (catalogue number: C0265, Beyotime Biotechnology) in PBS for 1 h at room temperature, the sections were incubated with PKM2 antibody (1:200, catalogue number: 4053, Cell Signaling Technology) overnight at 4 °C and then with HRP-polymer anti-rabbit IgG (catalogue number: KIT-5010, MXB Biotechnologies, Fuzhou, China) for another hour. The PKM2 signal was visualized by DAB (catalogue number: P0202, Beyotime Biotechnology) staining.

Animal studies

All animal experiments were approved by the Ethical Committee on Animal Experiments of the Animal Care Committee of Xiamen University (Xiamen, China).

Twenty-eight 6-week-old male nude mice provided by Shanghai Laboratory Animal Centre (Shanghai, China) were randomly divided into four groups with 7 mice in each group. For tumour growth assays, 5 × 10⁶ T24 cells expressing shCtrl or shPKM2 were subcutaneously injected into their lower backs and allowed to grow for 7 weeks (n=7 per group). Starting one week after injection, the tumour volume was measured weekly using a Vernier calliper. After 7 weeks of tumour growth, the nude mice were euthanized with 100% CO₂ gas at a flow rate of 30–70% of the chamber volume per minute. The tumours were harvested, and the weight and volume of each tumour were measured with an electronic balance and Vernier calliper, respectively.

Naito, T. et al. examined the tumour induction abilities of the HT1376, 5,637, T24, and UM-UC-3 cell lines, and found that the tumour formation rate of UM-UC-3 cells injected into the mouse bladder was up to 90% [15]. Therefore, UM-UC-3 cells were used for the orthotopic BCa model. In brief, nude mice were anaesthetized with inhaled 1.5% isoflurane (Abbott Laboratories, Shanghai, China) using a nose cone delivery device, and 5 × 10⁵ UM-UC-3 cells expressing luciferase and shCtrl or shPKM2 were injected into the bladders (n=7 per group). Tumour growth at day 28 was monitored using the live animal IVIS Lumina II system (Caliper Life Sciences, Massachusetts, USA).

Analysis of data from high-throughput sequencing

T24 cells expressing shPKM2 or shCtrl were sent to Beijing Novogene Technology Co., Ltd (Beijing, China). For high-throughput sequencing. Principal component analysis (PCA) was used to evaluate the obtained data for intergroup differences and intragroup sample duplication. Gene expression differences between groups were compared using DESeq2 software (1.20.0), with padj <0.05 and |log2FC|>0 indicating significant differences. Differentially expressed genes from obtained datasets were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) and Disease Ontology (DO) enrichment analysis was performed using clusterProfiler software (3.4.4), with a p value <0.05 as the threshold for significant enrichment. Gene set enrichment analysis (GSEA) of the obtained data was performed KEGG and DO datasets with the following website: http://www.broadinstitute.org/gsea/index.jsp.

Statistics

All statistical analyses were performed with SPSS 19.0. Differences in mean values between two groups were analysed by Student’s t-test. Single-factor analysis of variance (ANOVA) was used to analyse the significance of differences between multiple groups, followed by Bonferroni’s post hoc correction. p<0.05 was considered to indicate statistical significance.

PKM2 is highly expressed in BCa tissues

PKM2 expression levels in BCa have rarely been documented. To this end, we collected 36 pairs of matched BCa tissues and normal tissues and detected the protein and mRNA levels of PKM2. The results showed that both the protein levels (Figure 1A and B) and mRNA levels (Figure 1C and D) were significantly higher in BCa tissues (n=36) than in normal tissues (n=36), suggesting that PKM2 may be involved in BCa progression. Importantly, high PKM2 protein and mRNA expression levels were tightly associated with tumour grade, tumour stage and lymph node metastasis (Figure 1E and G & Table 3). In accordance with the clinical findings, PKM2 expression levels were also higher in BCa cell lines than in the nonmalignant epithelial cell line HCV29 (Figure 1H). Together, these data support the notion that PKM2 is overexpressed in BCa in clinical and experimental settings.

Figure 1:

PKM2 is upregulated in bladder cancer (BCa) samples and cell lines. (A) Representative images of IHC staining showing that PKM2 is upregulated in BCa tissues compared to normal bladder tissues. (B) Top, PKM2 protein levels in normal bladder tissues (N) and BCa tissues (B) measured by Western blotting. β-Actin served as a loading control. Bottom, statistical analysis of PKM2 expression in normal bladder tissues and BCa tissues. The data are presented as the mean ± standard error of the mean (SEM). (C) qRT‒PCR analysis of PKM2 expression in human BCa tissues and matched normal bladder tissues from 36 patients with BCa. Data are presented as the log2-fold change (relative PKM2 mRNA expression in a tumour sample/relative mRNA expression in the matched normal bladder tissue sample) to show the relative expression in every paired sample. The difference in relative expression between all the normal bladder samples and tumour samples is shown. (D) PKM2 mRNA levels were significantly increased in BCa samples (BCa tissues: n=36, matched normal bladder tissues: n=36). The expression of each gene was normalized to that of β-actin. The data are presented as the means ± SEMs. (E, F, G) Correlation between PKM2 mRNA expression and BCa clinical stage (E; Ta-T1: n=14; T2-T4: n=22), pathological grade (F; grade I: n=14; grade III: n=22) and metastasis status (G; M0: n=20; M1: n=16). The data are presented as the means ± SEMs. (H) PKM2 expression levels were increased in BCa cell lines compared to normal epithelial cells (HCV29). The data are presented as the means ± standard deviations (SD). ^**p<0.01; ^***p<0.001.

Attenuation of PKM2 suppresses BCa cell proliferation and invasion

A rapid proliferation rate and strong invasive ability are two hallmarks of cancer cells [16, 17]. To determine whether PKM2 affects these two processes, we first constructed three shRNAs against PKM2, all of which effectively decreased PKM2 levels (Figure 2A and B). We chose the best shRNA, shPKM2-1, for the subsequent experiments. The CCK-8 assay results revealed that PKM2 knockdown decreased the proliferation rate of T24 cells (Figure 2C). Furthermore, shPKM2 strongly decreased the migratory ability of T24 cells, as indicated by wound healing (Figure 2D) and Matrigel-free Transwell assays (Figure 2E). Accordingly, the invasive ability of T24 cells, as monitored by the Matrigel-based transwell assay, was also decreased by shPKM2 (Figure 2F). Mechanistically, silencing PKM2 markedly affected epithelial-mesenchymal transition (EMT)-related genes such as E-cadherin, vimentin, and fibronectin (Figure 2G). PKM2 silencing decreased the expression of Twist1, Snai1, Zeb1, LDHA, GLUT1, and c-Myc, while no significant change in β-catenin, Slug or Zeb2 expression was observed (Figure 2H ).

Figure 2:

Attenuation of PKM2 suppresses BCa cell proliferation and invasion. (A, B) The knockdown efficiency of shPKM2 was detected by Western blotting (A) and qRT‒PCR (B) and showed that PKM2 was successfully silenced by shRNA in T24 cells. β-actin was used as a loading control. The data are presented as the means ± SDs. (C) T24 cell proliferation was dramatically suppressed by shRNA targeting PKM2. The data are presented as the means ± SEMs. (D) Knockdown of PKM2 in T24 cells inhibited cell migration, as indicated by the wound-healing assay. (E, F) Transwell assays revealed that shRNA-mediated PKM2 knockdown abrogated the migration (E) and invasion (F) of T24 cells. The data are presented as the means ± SEMs. (G) Silencing PKM2 in T24 cells decreased the expression levels of EMT-associated genes. β-actin was used as a loading control. (H) Silencing PKM2 in T24 cells decreased Twist1, Zeb1, LDHA, Snai1, GLUT1, and c-Myc expression but did not significantly affect β-catenin, Slug or Zeb2 expression. The data are presented as the means ± SEMs. These experiments were performed three times independently (n=3). ^*p<0.05; ^**p<0.01; ^***p<0.001; ns: not significant.

All these findings indicate that PKM2 acts as an oncogenic factor to promote BCa progression, likely by regulating EMT.

PKM2 deficiency abrogates aerobic glycolysis and sensitizes BCa cells to cisplatin treatment

Because PKM2 is a key enzyme required for glycolysis, we sought to explore glycolytic activity after manipulating the levels of PKM2. As shown in Figure 3A, attenuating PKM2 expression in T24 cells considerably reduced glucose consumption and lactate production, indicating the important role of PKM2 in mediating aerobic glycolysis. Consistently, increased ROS levels were observed in PKM2-deficient T24 cells (Figure 3B). Importantly, shPKM2 sensitized T24 cells to cisplatin treatment, as reflected by the increase in apoptosis rate and the decrease in viability rate after exposure to cisplatin (Figure 3C).

Figure 3:

PKM2 deficiency abrogates aerobic glycolysis and sensitizes BCa cells to cisplatin treatment. (A) PKM2 deficiency suppressed glucose uptake and lactate production. Equal numbers of shCtrl and shPKM2 T24 cells were separately seeded into 24-well plates. Forty-eight hours later, the levels of glucose and lactate in the culture medium were measured by a spectrophotometer. The data are presented as the means ± SDs. (B) ROS levels, which were measured by flow cytometry, in T24 cells were increased when PKM2 was knocked down by shRNA. The data are presented as the means ± SDs. (C) PKM2 knockdown resulted in increased apoptosis of T24 cells and sensitization of T24 cells to cisplatin treatment. T24 cells with or without PKM2 expression were treated with 20 µM cisplatin for 24 h and then collected for cell viability and apoptosis detection by flow cytometry. The data are presented as the means ± SDs. These experiments were performed three times independently (n=3). ^*p<0.05; ^**p<0.01; ^***p<0.001.

The shPKM2 group exhibited slower tumour growth than the control group

It is worth evaluating the contribution of PKM2 to BCa progression in a xenograft mouse model. Thus, we subcutaneously implanted shCtrl- or shPKM2-expressing T24 cells (5 × 10⁶) into nude mice and monitored tumour growth weekly. Tumours in mice in the shPKM2 group had a slower growth rate than that in control group (Figure 4A and B, and Table 4). To further confirm the in vivo function of PKM2, we utilized an orthotopic mouse model. Here, we first generated luciferase-expressing UM-UC-3 cells with or without shPKM2 transfection and then inoculated these cells (5 × 10⁵) into the bladders of nude mice. Tumour progression was evaluated via an in vivo imaging system (IVIS). After 6 weeks, a dramatic reduction in the luciferase signal was observed in the shPKM2 group compared to the control group (Figure 4C), suggesting that PKM2 knockdown suppresses BCa growth. Collectively, these two mouse models support the hypothesis that PKM2 plays an oncogenic role in BCa progression.

Figure 4:

PKM2 is indispensable for in vivo BCa growth. (A) T24 cells (5 × 10⁶) were mixed with Matrigel (1:1) and subcutaneously implanted into nude mice. Tumour progression in the shCtrl (n=7) and shPKM2 (n=7) groups was assessed weekly by determine tumour volume with callipers. The data are presented as the means ± SEMs. (B) At 7 weeks after nude mice were subcutaneously implanted with T24 cells, the tumours were excised, photographed and weighed, and the results are shown in the figure. The upper picture comprises images of the tumours whose short and long diameters are shown in Table 4, and the lower picture is the quantitation of tumour weight. shCtrl group: n=7; shPKM2 group: n=5. Noticeably, no tumours were observed in the two nude mice implanted with shPKM2-expressing T24 cells. The data are presented as the means ± SEMs. (C) Luciferase-expressing UM-UC-3 cells (5 × 10⁵) were orthotopically injected into the bladder. The size of tumours generated by shCtrl (n=7) and shPKM2 (n=7) cells was monitored by an IVIS imaging system (left). Quantitation of photon flux as assessed by bioluminescence measurements (right). The data are presented as the means ± SEMs. ^*p<0.05; ^**p<0.01; ^***p<0.001.

PKM2 is associated with various diseases and biological processes

We performed high-throughput sequencing on three groups of T24 cells with stable PKM2 knockdown and control cells, and assessed the differences between groups and gene expression distribution (Figure 5A and B). In total, 630 genes were upregulated and 1,103 genes were downregulated in PKM2 knockdown cells compared with control cells (Figure 5C and D). The genes co-expressed with PKM2 are enriched in many biological processes and various diseases, such as thermogenesis and respiratory system cancer (Figure 6). Gene enrichment analysis showed that PKM2 may be associated with bladder cancer, bladder disease and DNA replication (Figure 7A and B). Knockdown of PKM2 increased the mRNA levels of the related genes CXCL8, E2F1, GSTP1, HBEGF, HRAS, IL-6, MCM2, MCM5, MCM7, MUC1, MUC5AC, NGF, PCNA, POLD1, RASSF1, RNASEH2C, THBS1 and TP53 (Figure 7C).

Figure 5:

Changes in gene expression in PKM2-silenced cells were explored by analysing data obtained by high-throughput sequencing. (A) Boxplot of gene expression distribution in sequencing samples. (B) Principal component analysis (PCA) plot of sequenced samples. (C) Volcano plot of differentially expressed genes. (D) Heatmap of differentially expressed gene clustering.

Figure 6:

Enrichment analysis of data obtained by high-throughput sequencing with significance values. (A) Scatter plot of differentially enriched KEGG gene sets. (B) Scatter plot of differentially enriched DO gene sets.

Figure 7:

PKM2 may be associated with bladder cancer, bladder disease and DNA replication. (A) GSEA of sequenced samples in the KEGG dataset and DO dataset. (B) Rank metric score of genes associated with bladder cancer, DNA replication and bladder disease. (C) The mRNA levels of functional genes were verified by qRT‒PCR.