Different from their normal counterparts, tumor cells avidly convert glucose to lactate to produce energy even when abundant oxygen exists. This distinct metabolic phenotype termed as aerobic glycolysis serves as a metabolic advantage as it provides dividing cells both energy and glycolytic intermediates (phosphor-metabolites) that are required as precursors for the synthesis of nucleic acids, amino acids, and lipids (1–3). Furthermore, acidification of the extracellular microenvironment due to increased lactate production may facilitate tumor cell invasion and metastasis (3, 4). In addition, recent studies indicate that aerobic glycolysis avoids production of reactive oxygen species (ROS), which may disturb the redox state and induce cell apoptosis in cancers (5, 6). Therefore, it has even been proposed that aerobic glycolysis is an early and essentially irreversible step that leads to tumorigenesis (7).
The exact molecular mechanisms underlying cancers' reliance on glycolysis remain unclear. It is likely to be a combination of direct oncogenic stimulation and epigenetic responses to the hypoxic tumor environment. There has been a long-held belief that the switch of tumor glucose metabolism from oxidative phosphorylation to aerobic glycolysis is due to the defects of mitochondrial oxidative phosphorylation (7). But the mechanisms seem to be intricate. Despite intensive studies, how tumor cells facilitate lactate production and block mitochondrial oxidative phosphorylation remains largely unknown. Recent results show that upregulation of glycolytic enzymes plays a crucial role in cancer. Among these enzymes, pyruvate kinase type M2 (PKM2) comprises one of the most unregulated genes in cancers (8–10). It is shown that expression of PKM2 in cancer cells is essential for the establishment of aerobic glycolysis and provides a selective growth advantage for tumor cells in vivo (10). Pyruvate kinase (PK) catalyzes the formation of pyruvate from phosphoenolpyruvate (PEP), the rate-limiting final step in glycolytic cascade. There are four PK isoforms: L, R, M1, and M2. They are expressed, respectively, in specific tissues. The M2 isoform, a splice variant of M1 is expressed during embryonic development (11) and tumor formation. It has been reported that tumor cells exclusively express PKM2 (10, 12). In contrast to PKM1 that exists only as a highly active tetramer, PKM2 can shift between a tetrameric form that has a high affinity for its substrate PEP and a dimeric form with a low PEP affinity (12). A high level of the dimeric form of PKM2 coincides with increased amounts of glycolytic phosphor-metabolites needed by biosynthetic processes. A high level of the PKM2 tetramer facilitates production of ATP and lactate. Studies have shown that most PKM2 exists mainly as a dimeric form in tumor cells. Some viral oncoproteins like HPV-16E7 and pp60v-Src kinase (12, 13) as well as cellular phosphotyrosine signaling (14, 15) can induce the dimerization of PKM2, but the Ras oncogene induces the tetramerization of PKM2 (16, 17). Recently, Kosugi et al. (18) found that PKM2 activity is also regulated by direct binding of oncoprotein MUC1-C. Another important metabolic regulator of PKM2 is fructose 1,6-bisphosphate. High level of this glycolytic intermediate reflects sufficient amounts of phosphor-metabolites and induces reassociation of the dimeric form of PKM2 to the highly active tetrameric form, thus shifting tumor metabolism from synthesis of cell building blocks to energy regeneration (12). So it is believed that PKM2 plays a key role in the channeling of glucose carbons either into catabolic or into anabolic pathways [14, 15]. But not all of the tumors express PKM2 only; there might be different expressing patterns and roles of PKM2 in different tumors (19, 20).
Colorectal cancer (CRC) is a particularly frequent and significant health problem worldwide. About 1,200,000 new cases are diagnosed of CRC all over the world every year. In the United States, CRC is the third most commonly diagnosed cancer and the third highest cause of cancer-related deaths (21). The incidence of CRC has also been increasing rapidly in the Asia in the last few decades (22). Although the survival of CRC patients has dramatically improved with the progress in various therapeutic treatment applied nowadays, approximately 45% of patients will die mainly from metastatic disease in 5 years after diagnosis (23). So, intensive study in the biology of CRC and especially the development of new effective and targeted therapies are needed. PKM2 plays an important role in cancer and might serve as an attractive target for cancer therapy. However, the expression of PKM2 and association of PKM2 expression level and CRC are not clear. In this work, we set out to determine the expression pattern of PKM2 in CRC and its role in growth of colon cancer cells. Our results indicated that expression of PKM2 was increased in CRC, which was associated with later stage and lymph metastasis of the tumors. Inhibition of PKM2 suppressed the proliferation of colon cancer cells in vitro and tumor growth in vivo. Our results suggest that PKM2 plays a critical role in CRC, and it is a potential target for CRC therapy.
MATERIALS AND METHODS
Tissues of CRC and the adjacent normal epithelia were obtained from surgical specimens immediately after resection from patients undergoing primary surgical treatment in the Department of Surgery, Affiliated Zhongshan Hospital of Fudan University. The samples were flash-frozen in liquid nitrogen and stored at −80 °C. All the CRC tissues were routinely stained with H&E and confirmed to have at least 80% malignant cells before experiment. Written informed consents approving tissue donation for research purposes was obtained from patients before tissue collection.
Cell Culture and Reagents
Human colon cancer RKO, SW480, HT29 cells were maintained in Dulbecco's modified Eagle medium (DMEM, Gibco), human colon cancer HCT116 cells in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco). Then cells were incubated at 37 °C with 5% CO2. PKM1 antibody was from Abgent (San Diego, CA). PKM2 antibody was from Cell Signaling (Beverly, MA). Beta-actin antibody was from Sigma (St. Louis, MO).
Immunoblotting and Histochemical Staining
Immunoblotting was done as described (24). Briefly, cells or grinded tissues were lysed on ice for 30 min in radio immunoprecipitation assay (RIPA) buffer (100 mM Tris, 150 mM NaCl, 1% Triton, 1% deoxycholic acid, 0.1% SDS, 1 mM ethylene diamine tetraacetic acid (EDTA), and 2 mM NaF) supplemented with 1 mM sodium vanadate, 1 mM leupeptin, 1 mM aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 1 mM pepstatin A. Then supernatant was collected after centrifugation at 12,000g for 15 min, and protein concentrations were determined using protein assay reagent from Bio-Rad (Hercules, CA). Equal amounts of proteins were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitro-cellulose membrane. PKM1 and PKM2 proteins were detected by immunoblotting using specific antibodies, respectively. Histochemical staining of the tissues was performed as described (25). Briefly, tissues were fixed in 4% buffered formalin for 24 h and paraffin-embedded by conventional method. Then 3-μm-thick sections were heat-immobilized, deparaffinized, and rehydrated in a series of increasing ethanol concentrations. Then sections were incubated in 10 mM citrate buffer (pH 6.0) in microwave for 10 min, followed by incubation with 1.5% block serum for 60 min. Sections were then incubated with antibodies against PKM1 or PKM2 at 4 °C overnight and then with the fluorescein isothiocyanate (FITC)-conjugated secondary antibodies for 60 min at room temperature in the dark.
Real-time PCR was done as described (24). Briefly, tissues were grinded with liquid nitrogen, then powdered tissues were transferred to tubes with 1 ml Trizol (Invitrogen), and then total RNA was abstracted following the manufacturer's protocol. RNA was quantified by measuring A260 and A280 absorbance in a Nanodrop spectrophotometer (Thermo scientific). Real-time PCR was performed on Applied Biosystems Step Two Real-Time PCR System (Applied Biosystems, Foster City, CA) using the comparative threshold cycle (Ct) quantization method. Real-time Master Mix (Toyobo, Osaka, Japan) was used to detect and quantify the expression levels of target genes. Actin was used as internal control. The primers used are as follows: PKM2: 5′-CCATTACCAGCGACCCCACAG-3′ (F), 5′-GGGCACGTGGGCGGTATCT-3′ (R); ACTIN: 5′-GATCATT GCTCCTCCTGAGC-3′ (F), 5′-ACTCCTGCTTGCTGATCCA C-3′ (R).
Cell Proliferation Assay
To determine cell proliferation, cells were plated in six-well plates at a density of 5 × 104 cells per well, and the total cell numbers were obtained every 24 h by a hemocytometer for a 1–5 day period. Time zero was taken 16 h after seeding. Cell population doublings (PDLs) were calculated from the initial (I) and final cell number (E) according to the following equation: PDLs per 5 days = (log(E) − log(I))/log 2 (26).
Cell Migration Assay
For migration, cells were resuspended (5 × 104) with FBS-free DMEM medium and seeded onto Transwell Permeable Support inserts with 8 μm microporous membrane (Costar, Corning, MA) in a 24-well plate. The lower chamber of the transwell was filled with 600 μl of DMEM containing 20% FBS. After incubation at 37 °C for 30 h, the cells on the upper surface of the membrane were wiped out with a cotton swab. The cells adherent to the outer surface of the membrane were fixed and stained with the three-step stain set (Richard-Allan Scientific, Kalamazoo, MI), photographed and counted under a microscope. Four fields were randomly counted per filter in each group, and the experiment was performed three separate times.
Measurement of Glucose Metabolism
The glucose metabolism rates were measured as described previously . Briefly, cells were washed twice by phosphate buffered saline (PBS) and then incubated in Krebs buffer without glucose for 30 min. The Krebs buffer was then replaced with Krebs buffer containing 10 mM glucose spiked with 10 mCi of 5-3H-glucose. In 1 h, 3H2O generated was separated chromatographically and determined using a liquid scintillation counter.
Measurement of Lactate Production
Cells were plated in a 6-cm dish and grown to approximate 80% confluence. The old medium was discarded, and the cells were washed twice and switched to fresh medium. Then the cells were incubated for one more hour, and the medium was collected. The lactate in the medium was measured with fluorescence-based assay kits (BioVision). Production of lactate was calculated from the difference between the concentrations in the medium before and after culturing. Cell number was counted using a Coulter particle analyzer (10).
Measurement of Oxygen Consumption
Cellular oxygen consumption rates were measured using a water-jacketed (37 °C) anaerobic chamber fitted with a polarographic oxygen electrode as described previously (8). The electrode was calibrated with 150 mM NaCl equilibrated to room air at 37 °C (corresponding to 199 nmol O2 per ml).
The lentivirus for PKM2 RNA interference (shPKM2) and control was prepared by GenePharm (Shanghai, China). The targeting sequence for PKM2 and control sequence were as described previously (10). The cells were infected with the lentivirus in the presence of 5 μg/ml polybrene. After selection in 0.2 μg/ml puromycin for 1 week, stable cell lines were obtained.
Four-week-old male nude mice (BALB/cA-nu/nu) were obtained from Shanghai Experimental Animal Center and maintained in pathogen-free conditions. The mice were separated into two groups and were injected subcutaneously with shPKM2 or control cells (2 × 106) at each flank. Tumor formation was assessed every 2 days, and tumors were dissected and weighed 40 days after injection. All animals were maintained and used in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute of Nutritional Sciences.
Statistical analysis was performed by SPSS12.0 for windows. SD was calculated from three independent experiments except where indicated. P < 0.05 indicates a significant difference.
Expression of PKM2 Was Increased in Human CRC
Sixty pairs of human CRC samples (primary tumor tissues and paired normal colorectal tissues) were tested. We determined the mRNA levels of PKM2 in these tissues and found that 43 of 60 (71.7%) of colorectal tumors had increased PKM2 mRNA (Fig. 1A). Univariate analysis indicated that the PKM2 mRNA levels were significantly different between tumors and paired normal colorectal tissues (P < 0.01). Statistic analysis indicated that the increased expression of PKM2 is associated with more lymph node metastasis and later rimary tumor, regional lymph nodes, distant metastasis staging (Table 1). Immunohistochemical staining of the tissues gave similar results (Fig. 1B). We did PKM1 staining of the tissues and the results indicated that the normal colorectal mucosa showed strong PKM1 staining, while the malignant cells showed a selective expression of PKM2 (Fig. 1B). Western blot results also indicated an increased expression of PKM2 in CRC tissues (Fig. 1C).
Table 1. Relationship between PKM2 expression and clinical features of CRC
Increased PKM2 (n = 43), n(%)
Nonincreased PKM2 (n = 17), n(%)
χ2 test was performed to analyze the correlations of PKM2 mRNA levels with clinical and pathologic parameters. Tumor stage was based on TNM. T: tumor, N: lymph node, M: metastasis.
Expression of PKM2 is Essential for Aerobic Glycolysis in RKO Cells
Next we examined the expression patterns of PK in several CRC cell lines including RKO, HCT116, HT29, and SW480. All cells examined exclusively expressed PKM2 (Fig. 2A). As PKM2 is overexpressed in CRC, we then set out to assess its significance in glucose metabolism of tumor cells. We found that knockdown of PKM2 led to a significant decrease of glucose metabolism rate in RKO cells (Fig. 2B). The lactate production rate was also decreased (Fig. 2C). These results suggest that inhibition of PKM2 suppressed the aerobic glycolysis of RKO cells. In addition, we found that the consumption of oxygen was not affected significantly (Fig. 2D).
Expression of PKM2 Promotes Cell Proliferation and Migration in RKO Cell Lines
Then we determined the effect of PKM2 on proliferation of RKO cells. The results showed that knockdown of PKM2 inhibited proliferation of RKO cells (Fig. 3A). The cell PDL was significantly decreased if PKM2 was inhibited (Fig. 3B). As increased PKM2 is associated with later tumor stage in CRC (Table 1), we next determined whether or not PKM2 played a role in migration of RKO cells. The results showed that knockdown of PKM2 suppressed migration of the cells (Figs. 3C and 3D).
PKM2 Promotes Tumor Growth In Vivo
Finally, we determined the effects of knockdown of PKM2 on growth of RKO xenografts in vivo. Nude mice were injected subcutaneously with RKO cells at each flank (2 × 106/each site). The tumor growth was monitored over a 40-day period. As shown in Figure 4, knockdown of PKM2 inhibited the in vivo tumor growth of RKO cells (Figs. 4A and 4B). The shPKM2 cells developed less tumors than the control cells did (Fig. 4C). Mice injected with the shPKM2 cells showed delayed tumor development (Fig. 4D).
Aerobic glycolysis has widely been accepted to be an essential component of the malignant phenotype and a hallmark of invasive cancers. However, how cancer cells establish this special metabolism is unclear. Overexpression of glycolytic genes is found to play critical roles in the establishment of tumor aerobic glycolysis (8). PKM2 is one of the most highly expressed genes and plays a crucial role in malignant transformation and tumor glucose metabolism. The tumor cells exclusively express PKM2 (27) and upregulation of PKM2 has been found in a few types of tumors (8–10), including CRC although with lower sample numbers (28). Here, we demonstrated that PKM2 is upregulated in colon cancer tissues compared with normal mucosa (Fig. 1). Statistical analysis indicated that the increased expression of PKM2 is correlated with later tumor stage and more lymph node metastasis of CRC (Table 1). Our results suggest that the renascence of PKM2 may facilitate development of CRC. It should be noted that the expression of PKM2 was also found in healthy colon although at a lower level (Fig. 1), indicating that PKM2 is not specific to cancer cells.
In order to assess the significance of PKM2 in CRC, an effective PKM2 short hairpin RNA (shRNA) was used, and series of experiments were performed in RKO cells. As PK catalyzes a rate-limiting step in glycolytic cascade, we first evaluated the impact of PKM2 on tumor glucose metabolism. Results showed that knockdown of PKM2 markedly decreased the rate of glucose metabolism in RKO cells (Fig. 2B). The lactate production was also decreased (Fig. 2C). However, the oxygen consumption was little affected (Fig. 2D). These results imply that, in PKM2 expressing cells, glucose is mainly converted to lactate with little oxygen consumed. So, coinciding with the findings in other cancers (10), CRC cells also prefer to metabolize glucose to lactate even enough oxygen exists. Expression of PKM2 plays a crucial role in the metabolic remodeling in RKO cells.
We found that the expression of PKM2 is important for proliferation of RKO cells (Fig. 3). As we know, glucose metabolism provides tumor cells both ATP and phosphor-metabolites required for cell proliferation. So, as an essential glycolytic enzyme, it is neither surprising nor unexpected that PKM2 plays a crucial role in maintaining proliferation of the cells. As described above, PKM2 is also expressed in some other types of cells. So, inhibition of expression of PKM2 may also affect the proliferation of other cells. The superiority of PKM2 compared with PKM1 might be that it can balance energy supply and glycolytic phospho-metabolite pools by switching between two quaternary conformations (12–18). As is mentioned above, PKM2 can exist as an active tetrameric and an inactive dimeric form. So the transition of PKM2 according to cells' demand between the two conformations channels glucose carbons either into catabolic or into anabolic pathways and optimizes tumor cell proliferation, growth, and survival (29). Another contradiction to be mentioned in tumor cells is that in contrast with normal proliferating cells, tumor cells have to survive in environments with insufficient oxygen and nutrient supplies (29, 30). So the expression of PKM2 induces a switch to anaerobic metabolism, which allows maintenance of metabolic activities and ATP supply in the absence of oxygen. Recent studies (29) have indicated that an inactive dimer of PKM2 can rescue tumor cells from glucose starvation-induced apoptotic cell death and can optimize tumor cell metabolic activity, proliferation, growth, and survival by economizing the less glucose available to synthetic processes.
We found that overexpression of PKM2 was correlated with later tumor stage in CRC patients, suggesting that PKM2 acts to promote tumor progression and metastasis. As migration has been found to be important and necessary for tumor metastasis formation (31–33), we determined the effects of PKM2 on migration of RKO cells, and the results showed that knockdown of PKM2 blocked the cell migration significantly. In fact several researches have indicated that aerobic glycolysis is associated with increased incidence of metastasis in multiple tumors (34–37).
A few studies indicated that the increased lactate production by aerobic glycolysis might be an important factor that facilitated tumor cell metastasis (38–41). Although the molecular mechanisms are not clearly understood, lactate has been found to increase migration of various cancer cell lines (37, 41). Recent data indicated that transforming growth factor beta (TGF-β) signaling pathway might be a mediator of the lactate-associated effects on migration of cancer cells (42). Moreover, lactate stimulates some cytokines, such as vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), resulting lactate-induced angiogenesis and increased tumor metastasis (43, 44). Another reason for PKM2-enhanced tumor metastasis is that, by favoring aerobic glycolysis, tumor cells generate bicarbonic and lactic acids, which render the extracellular environment more acidic, and potentially favoring tumor invasion by pH-dependent activation of cathepsins and metalloproteinases (45).
Our studies demonstrate that PKM2 is upregulated in CRC, and it is essential for the aerobic glycolysis in colon cancer cells. Our results showed that PKM2 regulates cell proliferation and migration and plays an important role in tumor growth of colon cancer cells. Thus, PKM2 may be function dependent or independent of its enzymatic activity in tumorigenesis and metastasis. Furthermore, recent studies suggested that silencing of PKM2 can increase chemotherapy sensitivity to docetaxel in lung cancer cells (46). Therefore, evaluating PKM2 status in CRC may aid targeted therapy selection for this disease.
The work was supported by Natural Science Foundation of China (30970586) and Chinese Academy of Sciences (KSCX2-EW-R-08).