Human steroid sulfatase enhances aerobic glycolysis through induction of HIF1α and glycolytic enzymes

Human steroid sulfatase (STS) has been linked with poor prognosis in steroid-associated tumors and represents an important clinical target in cancers, yet the mechanism of STS-induced carcinogenesis remains unclear. To correlate STS with cancer metabolism, we determined the effects of STS on aerobic glycolysis. STS overexpression increased cellular levels of lactic acid, the final product of aerobic glycolysis. Moreover, STS suppressed the oxygen consumption rate (OCR), which represents mitochondrial respiration. Inhibition of STS by the specific inhibitor STX064 recovered STS-induced OCR repression and lactic acid over-production. DHEA, but not DHEA-S, suppressed the OCR level and enhanced lactic acid production. To understand the molecular mechanism of STS-induced cancer metabolism, we measured the expression of glycolytic enzymes hexokinase 2 (HK2) and pyruvate kinase M2 (PKM2), which was highly upregulated by STS and DHEA at both protein and mRNA levels. HIF1α is a key mediator of aerobic glycolysis, and STS enhanced HIF1α promoter activity, mRNA expression, and protein expression. Down-regulation of HIF1α by siRNA suppressed the HK2 and PKM2 expression induced by both STS and DHEA. HIF1α siRNA also recovered the OCR repression and lactic acid over-production induced by both STS and DHEA. To explore the mechanism in vivo, we produced transgenic mice overexpressing STS and found that STS expression was particularly enhanced in the lung. Consistent with our in vitro results, the expression of HIF1α, HK2, and PKM2 was also increased in mouse lung tissues. In conclusion, we suggest that STS may induce aerobic glycolysis through enhancing HIF1α expression.     

miR-625-5p/PKM2 negatively regulates melanoma glycolysis state

PKM2 plays an important role in cancer glycolysis, however, the link of PKM2 and microRNAs (miRNAs) in melanoma is still unclear. The study will investigate the role of miRNAs in regulating PKM2 mediated melanoma cell glycolysis. We found that high PKM2 expression in melanoma tissues and cell lines was positively associated with glycolysis. Further study indicated that miR-625-5p regulated PKM2 expression on mRNA and protein levels in melanoma cells. There was a negative relationship between miR-625-5p and PKM2 expression in the clinical melanoma samples. These findings provide an evidence that miR-625-5p/PKM2 plays a role in melanoma cell glucose metabolism.     

Pyruvate kinase type M2 is upregulated in colorectal cancer and promotes proliferation and migration of colon cancer cells

Pyruvate kinase type M2 (PKM2) has been reported to be involved in aerobic glycolysis and cell growth in various tumors. However, the expression pattern of PKM2 in colorectal cancer (CRC) and the correlation between PKM2 expression and CRC remains unclear. The aim of this study is to investigate PKM2 expression and its possible role in CRC. We found that expression of PKM2 was increased in CRC and the increased PKM2 expression was associated with later stage and lymph metastasis of the tumors. Knockdown of PKM2 suppressed the aerobic glycolysis and decreased lactate production of colon cancer RKO cells. Knockdown of PKM2 repressed proliferation and migration of the cells. Inhibition of PKM2 suppressed xenograft tumor growth of RKO cells in vivo. These results suggest that the expression of PKM2 plays a critical role in development of CRC, and it may provide a growth advantage for colon cancer cells. Thus, PKM2 might be a potential therapeutic target for CRC. ? 2012 IUBMB Life, 64(9): 775-782, 2012.     

SMYD3 promotes hepatocellular carcinoma progression by methylating S1PR1 promoters

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. SET and MYND domain-containing protein 3 (SMYD3) has been shown to promote the progression of various types of human cancers, including liver cancer; however, the detailed molecular mechanism is still largely unknown. Here, we report that SMYD3 expression in HCC is an independent prognostic factor for survival and promotes the proliferation and migration of HCC cells. We observed that SMYD3 upregulated sphingosine-1-phosphate receptor 1 (S1PR1) promoter activity by methylating histone 3 (H3K4me3). S1PR1 was expressed at high levels in HCC samples, and high S1PR1 expression was associated with shorter survival. S1PR1 expression was also positively correlated with SMYD3 expression in HCC samples. We confirmed that SMYD3 promotes HCC cell growth and migration in vitro and in vivo by upregulating S1PR1 expression. Further investigations revealed that SMYD3 affects critical signaling pathways associated with the progression of HCC through S1PR1. These findings strongly suggest that SMYD3 has a crucial function in HCC progression that is partially mediated by histone methylation at the downstream gene S1PR1, which affects key signaling pathways associated with carcinogenesis and the progression of HCC.Subject terms: Liver cancer, Oncogenes     

Long noncoding RNA DLEU1 promotes proliferation and glycolysis of gastric cancer cells via APOC1 upregulation by recruiting SMYD2 to induce trimethylation of H3K4 modification

ObjectivesAPOC1 has been reported to promote tumor progression. Nevertheless, its impact on cell proliferation and glycolysis in gastric cancer (GC) remains to be probed. Hence, this study explored the related impacts and mechanisms.MethodsDLEU1, SMYD2, and APOC1 expression was detected in GC cells. Afterward, ectopic expression and knockdown experiments were conducted in GC cells, followed by measurement of cell proliferation, glucose uptake capability, lactic acid production, ATP content, extracellular acidification rate (ECAR), oxygen consumption rate (OCR), and GLUT1, HK2, and LDHA expression. In addition, interactions between DLEU1 and SMYD2 were analyzed with RIP and RNA pull down assays, and the binding of SMYD2 to APOC1 promoter and the methylation modification of SMYD2 in H3K4me3 were assessed with a ChIP assay. The ectopic tumor formation experiment in nude mice was conducted for in vivo validation.ResultsDLEU1, SMYD2, and APOC1 were highly expressed in GC cells. The downregulation of DLEU1 or APOC1 inhibited glucose uptake capability, lactic acid production, ECAR, the expression of GLUT1, HK2, and LDHA, ATP contents, and proliferation but augmented OCR in GC cells, which was also verified in animal experiments. Mechanistically, DLEU1 interacted with SMYD2 and recruited SMYD2 to APOC1 promoter to promote H3K4me3 modification, thus facilitating APOC1 expression. Furthermore, the effects of DLEU1 silencing on GC cell proliferation and glycolysis were negated by overexpressing SMYD2 or APOC1.ConclusionLncRNA DLEU1 recruited SMYD2 to upregulate APOC1 expression, thus boosting GC cell proliferation and glycolysis.     

Switching of Pyruvate Kinase Isoform L to M2 Promotes Metabolic Reprogramming in Hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is an aggressive tumor, with a high mortality rate due to late symptom presentation and frequent tumor recurrences and metastasis. It is also a rapidly growing tumor supported by different metabolic mechanisms; nevertheless, the biological and molecular mechanisms involved in the metabolic reprogramming in HCC are unclear. In this study, we found that pyruvate kinase M2 (PKM2) was frequently over-expressed in human HCCs and its over-expression was associated with aggressive clinicopathological features and poor prognosis of HCC patients. Furthermore, knockdown of PKM2 suppressed aerobic glycolysis and cell proliferation in HCC cell lines in vitro. Importantly, knockdown of PKM2 hampered HCC growth in both subcutaneous injection and orthotopic liver implantation models, and reduced lung metastasis in vivo. Of significance, PKM2 over-expression in human HCCs was associated with a down-regulation of a liver-specific microRNA, miR-122. We further showed that miR-122 interacted with the 3UTR of the PKM2 gene. Re-expression of miR-122 in HCC cell lines reduced PKM2 expression, decreased glucose uptake in vitro, and suppressed HCC tumor growth in vivo. Our clinical data and functional studies have revealed a novel biological mechanism involved in HCC metabolic reprogramming.     

The multifaceted regulation and functions of PKM2 in tumor progression

Tumor cells undergo metabolic rewiring from oxidative phosphorylation towards aerobic glycolysis to maintain the increased anabolic requirements for cell proliferation. It is widely accepted that specific expression of the M2 type pyruvate kinase (PKM2) in tumor cells contributes to this aerobic glycolysis phenotype. To date, researchers have uncovered myriad forms of functional regulation for PKM2, which confers a growth advantage on the tumor cells to enable them to adapt to various microenvironmental signals. Here the richness of our understanding on the modulations and functions of PKM2 in tumor progression is reviewed, and some new insights into the paradoxical expression and functional differences of PKM2 in distinct cancer types are offered.     

HMGCR inhibition stabilizes the glycolytic enzyme PKM2 to support the growth of renal cell carcinoma

Renal cell carcinoma (RCC) is responsible for most cases of the kidney cancer. Previous research showed that low serum levels of cholesterol level positively correlate with poorer RCC-specific survival outcomes. However, the underlying mechanisms and functional significance of the role of cholesterol in the development of RCC remain obscure. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) plays a pivotal role in RCC development as it is the key rate-limiting enzyme of the cholesterol biosynthetic pathway. In this study, we demonstrated that the inhibition of HMGCR could accelerate the development of RCC tumors by lactate accumulation and angiogenesis in animal models. We identified that the inhibition of HMGCR led to an increase in glycolysis via the regulated HSP90 expression levels, thus maintaining the levels of a glycolysis rate-limiting enzyme, pyruvate kinase M2 (PKM2). Based on these findings, we reversed the HMGCR inhibition-induced tumor growth acceleration in RCC xenograft mice by suppressing glycolysis. Furthermore, the coadministration of Shikonin, a potent PKM2 inhibitor, reverted the tumor development induced by the HMGCR signaling pathway.

Why do low levels of serum cholesterol positively correlate with poor renal cell carcinoma survival outcomes? This study shows that inhibition of the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase stabilizes pyruvate kinase M2 by up-regulating HSP90 expression, enhancing glycolysis and tumor growth in renal cell carcinoma.     

TSP50 promotes the Warburg effect and hepatocyte proliferation via regulating PKM2 acetylation

Metabolic reprogramming is a hallmark of malignancy. Testes-specific protease 50 (TSP50), a newly identified oncogene, has been shown to play an important role in tumorigenesis. However, its role in tumor cell metabolism remains unclear. To investigate this issue, LC–MS/MS was employed to identify TSP50-binding proteins and pyruvate kinase M2 isoform (PKM2), a known key enzyme of aerobic glycolysis, was identified as a novel binding partner of TSP50. Further studies suggested that TSP50 promoted aerobic glycolysis in HCC cells by maintaining low pyruvate kinase activity of the PKM2. Mechanistically, TSP50 promoted the Warburg effect by increasing PKM2 K433 acetylation level and PKM2 acetylation site (K433R) mutation remarkably abrogated the TSP50-induced aerobic glycolysis, cell proliferation in vitro and tumor formation in vivo. Our findings indicate that TSP50-mediated low PKM2 pyruvate kinase activity is an important determinant for Warburg effect in HCC cells and provide a mechanistic link between TSP50 and tumor metabolism.Subject terms: Cancer metabolism, Oncogenes, Tumour biomarkers

Gao et al. shows that testes-specific protease 50 (TSP50) binds to PKM2 and promotes the Warburg effect by increasing PKM2 K433 acetylation level and PKM2 acetylation site (K433R) mutation remarkably abrogated the TSP50-induced aerobic glycolysis, cell proliferation in vitro and tumor formation in vivo. Our study reveals a link between an oncogene and a key enzyme in HCC glycolysis, which provides new ideas for human HCCs treatment with TSP50 as the target.     

Nuclear translocation of PKM2 modulates astrocyte proliferation via p27 and β-catenin pathway after spinal cord injury

Aberrant functionality of the cell cycle has been implicated in the pathology of traumatic SCI. Although it has been reported that the expressions of various cell cycle related proteins were altered significantly following SCI, detailed information on the subject remains largely unclear. The embryonic pyruvate kinase M2 (PKM2) is an important metabolic kinase in aerobic glycolysis or the warburg effect, however, its functions in central nervous system (CNS) injury remains elusive. Here we demonstrate that PKM2 was not only significantly upregulated by western blot and immunohistochemistry but certain traumatic stimuli also induced translocation of PKM2 into the nucleus in astrocytes following spinal cord injury (SCI). Furthermore, the expression levels and localization of p-β-catenin, p27, cyclin D1 and PCNA were correlated with PKM2 after SCI. In vitro, we also found that PKM2 co-immunoprecipitation with p-β-catenin and p27 respectively. Knockdown of PKM2 apparently decreased the level of PCNA, cyclinD1, p27 in primary astrocyte cells. Taken together, our findings indicate that nuclear translocation of PKM2 promotes astrocytes proliferation after SCI through modulating cell cycle signaling. These discoveries firstly uncovered the role of PKM2 in spinal cord injury and provided a potential therapeutic target for CNS injury and repair.     

Specific Pyruvate Kinase M2 Inhibitor,Compound 3K,Induces Autophagic Cell Death through Disruption of the Glycolysis Pathway in Ovarian Cancer Cells

Ovarian cancer is a common cause of death among gynecological cancers. Although ovarian cancer initially responds to chemotherapy, frequent recurrence in patients remains a therapeutic challenge. Pyruvate kinase M2 (PKM2) plays a pivotal role in regulating cancer cell survival. However, its therapeutic role remains unclear. Here, we investigated the anticancer effects of compound 3K, a specific PKM2 inhibitor, on the regulation of autophagic and apoptotic pathways in SK-OV-3 (PKM2-overexpressing human ovarian adenocarcinoma cell line). The anticancer effect of compound 3K was examined using MTT and colony formation assays in SK-OV-3 cells. PKM2 expression was positively correlated with the severity of the tumor, and expression of pro-apoptotic proteins increased in SK-OV-3 cells following compound 3K treatment. Compound 3K induced AMPK activation, which was accompanied by mTOR inhibition. Additionally, this compound inhibited glycolysis, resulting in reduced proliferation of SK-OV-3 cells. Compound 3K treatment suppressed tumor progression in an in vivo xenograft model. Our findings suggest that the inhibition of PKM2 by compound 3K affected the Warburg effect and induced autophagic cell death. Therefore, use of specific PKM2 inhibitors to block the glycolytic pathway and target cancer cell metabolism represents a promising therapeutic approach for treating PKM2-overexpressing ovarian cancer.     

PKM2 coordinates glycolysis with mitochondrial fusion and oxidative phosphorylation

A change in the metabolic flux of glucose from mitochondrial oxidative phosphorylation (OXPHOS) to aerobic glycolysis is regarded as one hallmark of cancer. However, the mechanisms underlying the metabolic switch between aerobic glycolysis and OXPHOS are unclear. Here we show that the M2 isoform of pyruvate kinase (PKM2), one of the rate-limiting enzymes in glycolysis, interacts with mitofusin 2 (MFN2), a key regulator of mitochondrial fusion, to promote mitochondrial fusion and OXPHOS, and attenuate glycolysis. mTOR increases the PKM2:MFN2 interaction by phosphorylating MFN2 and thereby modulates the effect of PKM2: MFN2 on glycolysis, mitochondrial fusion and OXPHOS. Thus, an mTOR-MFN2-PKM2 signaling axis couples glycolysis and OXPHOS to modulate cancer cell growth.     

Pyruvate kinase M2 facilitates colon cancer cell migration via the modulation of STAT3 signalling

Understanding the mechanisms of colorectal cancer (CRC) metastatic progression is essential to reducing its morbidity and mortality. Pyruvate kinase (PK) catalyses the final step of glycolysis and has been identified as a critical regulator of glucose consumption. However, the mechanisms and roles of PKM1 and PKM2 in the regulation of CRC cell migration and cell adhesion remain elusive. Here, we report that PKM2 rather than PKM1 drives CRC cell migration and cell adhesion, whereas PKM attenuation reverses these phenomena. Furthermore, the overexpression of PKM2 significantly increases the expression of N-cadherin, MMP-2, MMP-9, STAT3, Snail-2, pFAK and active β1-integrin, while E-cadherin expression is suppressed. More importantly, the results indicated that PKM2 overexpression facilitates STAT3 nuclear translocation, and it is required for PKM2 function in the regulation of migration and adhesion associated signalling. In addition, the dimeric form of PKM2, which lacks the pyruvate kinase activities but possesses protein kinase activity, is critical for CRC cell migration and cell adhesion. Overall, this study suggests that PKM2 overexpression promotes CRC cell migration and cell adhesion by regulating STAT3-associated signalling and that PKM2 may serve as a therapeutic target for CRC metastasis.     

MUC1-C oncoprotein regulates glycolysis and pyruvate kinase M2 activity in cancer cells

Aerobic glycolysis in cancer cells is regulated by multiple effectors that include Akt and pyruvate kinase M2 (PKM2). Mucin 1 (MUC1) is a heterodimeric glycoprotein that is aberrantly overexpressed by human breast and other carcinomas. Here we show that transformation of rat fibroblasts by the oncogenic MUC1-C subunit is associated with Akt-mediated increases in glucose uptake and lactate production, consistent with the stimulation of glycolysis. The results also demonstrate that the MUC1-C cytoplasmic domain binds directly to PKM2 at the B- and C-domains. Interaction between the MUC1-C cytoplasmic domain Cys-3 and the PKM2 C-domain Cys-474 was found to stimulate PKM2 activity. Conversely, epidermal growth factor receptor (EGFR)-mediated phosphorylation of the MUC1-C cytoplasmic domain on Tyr-46 conferred binding to PKM2 Lys-433 and inhibited PKM2 activity. In human breast cancer cells, silencing MUC1-C was associated with decreases in glucose uptake and lactate production, confirming involvement of MUC1-C in the regulation of glycolysis. In addition, EGFR-mediated phosphorylation of MUC1-C in breast cancer cells was associated with decreases in PKM2 activity. These findings indicate that the MUC1-C subunit regulates glycolysis and that this response is conferred in part by PKM2. Thus, the overexpression of MUC1-C oncoprotein in diverse human carcinomas could be of importance to the Warburg effect of aerobic glycolysis.     

Beta‐elemene inhibits breast cancer metastasis through blocking pyruvate kinase M2 dimerization and nuclear translocation

Pyruvate kinase M2 (PKM2), playing a central role in regulating aerobic glycolysis, was considered as a promising target for cancer therapy. However, its role in cancer metastasis is rarely known. Here, we found a tight relationship between PKM2 and breast cancer metastasis, demonstrated by the findings that beta‐elemene (β‐elemene), an approved drug for complementary cancer therapy, exerted distinct anti‐metastatic activity dependent on PKM2. The results indicated that β‐elemene inhibited breast cancer cell migration, invasion in vitro as well as metastases in vivo. β‐Elemene further inhibited the process of aerobic glycolysis and decreased the utilization of glucose and the production of pyruvate and lactate through suppressing pyruvate kinase activity by modulating the transformation of dimeric and tetrameric forms of PKM2. Further analysis revealed that β‐elemene suppressed aerobic glycolysis by blocking PKM2 nuclear translocation and the expression of EGFR, GLUT1 and LDHA by influencing the expression of importin α5. Furthermore, the effect of β‐elemene on migration, invasion, PKM2 transformation, and nuclear translocation could be reversed in part by fructose‐1,6‐bisphosphate (FBP) and L‐cysteine. Taken together, tetrameric transformation and nuclear translocation of PKM2 are essential for cancer metastasis, and β‐elemene inhibited breast cancer metastasis via blocking aerobic glycolysis mediated by dimeric PKM2 transformation and nuclear translocation, being a promising anti‐metastatic agent from natural compounds.