Cancer Theory Update: the Hexokinase-based Metabolic Oncogenic Process


A key component of many cancers, especially the most aggressive, is their ability to metabolize glucose at a highly elevated rate. The first step in the metabolism of glucose is its phosphorylation to form glucose 6-phosphate, which traps the molecule within the cell. This reaction is unable to proceed in vitro unless the protein hexokinase (HK) and magnesium cations are present; [HK] and [Mg2+] remain constant throughout the reaction.

Different forms of HK (I, II, III, and IV) are found in differing levels throughout the body. Each form displays unique functional characteristics.

Figure 1: Saturation Curves for HK I and IV


Most cancer cells reprogram cellular glucose metabolism to fulfill their anabolic demands. Among cancer cells, HCC cells probably display the most comprehensive reprogramming of glucose metabolism3. This is manifested by turning off the expression of certain enzymes required for the functionality of mature hepatocytes but not for HCC cells and by turning on the expression of enzymes that are required for the accelerated glucose metabolism in cancer cells. This metabolic distinction between HCC cells and normal hepatocytes has not yet been exploited to selectively target the cancer cells. A major distinction between HCC cells and normal hepatocytes is the enzymes that catalyze the first committed step in glucose metabolism. This step is catalyzed by GCK in normal hepatocytes, but GCK is suppressed in HCC cells; instead, HK2 expression is induced. HK2 expression is induced in many cancer cells. However, these cancer cells also express HK1, whereas human HCC cells generally do not express HK1. Because drugs that are delivered systemically tend to accumulate in the liver first, relatively low doses of HK2 inhibitors would selectively target HCC cells and not normal hepatocytes. Considering that systemic deletion of HK2 in mice does not elicit any severe physiological consequences, HK2 could be an excellent target for HCC therapy. We found that hepatic HK2 deletion inhibits hepatocarcinogenesis in mice and that its silencing in human HCC cells inhibits proliferation and tumorigenesis in vivo and increases sensitivity to cell death. Importantly, HK2 silencing synergized with metformin to inhibit tumor growth of human HCC cells. Since the metformin transporter is highly expressed in hepatocytes, the combination of HK2 inhibition and metformin might be an effective therapeutic avenue for HCC. Currently, the only FDA-approved therapeutic drug for HCC is sorafenib, but its efficacy in the treatment of HCC is relatively low. Our results that the combination of sorafenib and HK2 silencing markedly increased HCC cell death and synergistically inhibited tumor growth suggest that HK2 inhibition could significantly increase the efficacy of sorafenib. The studies described here showed that expression of GCK in HCC cells with HK2 KD did not restore ECAR and oncogenesis; this may explain why HCC cells induce the expression of HK2. Interestingly, a mitochondrial binding deficient mutant of HK2 did not restore ECAR and oncogenesis despite having similar in vitro kinase activity as WT hexokinase. Mitochondrial binding of hexokinases near VDAC may be required for access to ATP derived from OXPHO to efficiently phosphorylate glucose16. Our results provide experimental evidence for this hypothesis by showing that mitochondrial binding deficient HK2 cannot fully restore ECAR to the level of WT HK2. The MFA data showed that, despite markedly reduced glycolytic flux upon HK2 KD, glutamine flux did not significantly change. Interestingly, the flux to the oxidative and non-oxidative PPP did not change, although the mechanism is unclear. Overall, it appears that HCC cells are highly dependent on glutamine utilization for TCA flux. Notably, the importance of glutamine for tumor growth in vivo is controversial; glutamine utilization appears to be minimal in some in vivo tumors, except HCC tumors, which display high-glutamine utilization17, 18.

Although we did not find a significant change in the flux of glucose to the de novo serine biosynthesis pathway upon HK2 KD, the HK2 KD HCC cells increased the extracellular serine/glycine exchange by 2-fold. These results suggest that the HK2 KD cells require additional one-carbon units, which are generated during the conversion of serine to glycine. Consistently, we found that serine deprivation significantly reduced the proliferation of HK2 KD and not control cells. It is not clear why excess one-carbon units are required. It is tempting to speculate that it is required to generate NADH from the mitochondrial folate pathway to support the increase in OXPHO observed after HK2 KD. However, because the magnitude of the extra serine flux is small (~5 nmol/106 cells/h) relative to OXPHO flux (~150 nmol/106 cells/h), this might be unlikely.

Despite the lack of changes in the TCA cycle flux, we found an increase in OXPHO after HK2 KD, suggesting that OXPHO is increased independently of the TCA cycle. Although we don’t know the exact mechanism by which OXPHO is increased despite no change in the TCA flux, it could be partially attributed to an increase in coupling efficiency and the maximum use of respiration capacity in Huh7 HK2 KD cells. The increase in OXPHO was diminished if the cells were treated with metformin, which inhibits mitochondrial complex I. The combination of metformin and HK2 KD synergistically increased HCC cell death in vitro and tumor growth in vivo. The combination of metformin, which is approved for the treatment of diabetes, together with HK2 inhibition is an attractive approach for HCC therapy. Systemic administration of metformin primarily targets the liver;13 hepatocytes express relatively high levels of organic cation transporter (OCT1)19, the metformin transporter. HK2 is usually the only hexokinase expressed in HCC cells. Therefore, the combination of HK2 inhibition and metformin could selectively target HCC cells, especially since drugs that are systemically administered tend to accumulate in the liver first.

Our studies also uncovered a synergistic effect of HK2 KD and metformin on mTORC1 activity. Surprisingly, metformin alone in the range of 1–5 mM did not significantly affect AMPK and mTORC1 activities. However, the combination of HK2 KD and metformin inhibited mTORC1 activity, although it did not have a significant effect on AMPK activity. It is unclear why the combination of HK2 KD, which significantly decreased glycolysis, and metformin, which inhibits OXPHO, did not activate AMPK. One possibility is that the AMP/ATP and ADP/ATP ratios did not reach a threshold level sufficient to activate AMPK despite reduced glycolysis and OXPHO. Regardless, we observed that the combination of metformin and HK2 KD inhibited mTORC1 even when AMPK was depleted. Interestingly, it was previously reported that metformin inhibits mTORC1 by AMPK-independent mechanisms20, 21. We found that the combination of HK2 KD and metformin markedly increased the expression of REDD1, which inhibits mTORC1 by activating TSC2. Although we cannot completely exclude other mechanisms by which HK2 KD and metformin inhibit mTORC1, the attenuation of mTORC1 inhibition by REDD1 depletion suggest that this is a major mechanism. Notably, it was previously reported that REDD1 is induced by metformin in an AMPK-independent manner and in a p53-dependent manner20. However, we observed the induction of REDD1 even in Huh7 cells that carry a mutated p5322.

Finally, we found that HK2 KD in combination with sorafenib, the only FDA-approved drug for HCC, markedly increased HCC cell death and tumor growth in vivo. It remains to be determined whether small molecule inhibitors could be developed to inhibit specifically HK2. Both HK1 and HK2 are allosterically inhibited by their own catalytic product, G6P, but inorganic phosphate antagonizes the ability of G6P to inhibit HK1, and not HK25. Thus, it might be possible to develop G6P mimetics to inhibit HK2 activity. Using this approach, it was recently reported that some glucosamine derivatives that bind both the G6P and glucose binding sites in hexokinase could preferentially inhibit HK2 activity23. Some of these compounds are two order of magnitude more potent inhibitors of HK2 than HK1. We used one of these compounds (compound 34) on Huh7 HCC cells and found that it markedly inhibited ECAR in these cells with no apparent inhibition in control Skhep1 cells (Supplementary Fig. 8). More studies are required to determine the stability of the compound in different cell type and the feasibility of its use in vivo in whole animal. Taken together, our studies showed that HK2 is an ideal therapeutic target for HCC either by itself or in combination with metformin or sorafenib.


Cell culture and stable cell line generation

All cell lines used were confirmed free from mycoplasma contamination, as determined by PCR. The human HCC cell lines HepG2 and Huh7 and lentiviral packaging line 293FT were grown in Dulbecco’s Modified Essential Media (DMEM, Hyclone), 10% fetal bovine serum (FBS, Atlanta Biologicals) or with TET-approved FBS (Atlanta Biologicals) (for inducible KD lines), and 1× Pen/Strep (Fisher) at 37 °C in a 5% CO2 atmosphere. Glucose concentrations were kept at either (25 mM) or (5.5 mM). Doxycycline induction was at (900 ng/mL) for both Huh7 and HepG2 inducible HK2 KD cell lines. For HK2 KD, stable expression of HK2-targeting shRNA was produced from either pLKO.1-Puro or Tet-On (T.O.)-pLKO.1-Puro (for inducible expression) lentiviral vectors (Invitrogen). The sequence targeting human HK2 used was: 5′-CCAAAGACATCTCAGACATTG-3′. The pLenti-6-D-TOPO-Blast lentiviral vector (Invitrogen) was used for the overexpression of hexokinases: Rat WT HK2, rat mitochondrial non-binding HK2 mutant (MTD: ∆1–20), rat catalytic HK2 mutant (SA: S155A/S603A), and human GCK (liver variant). Transduced cells were antibiotic selected with puromycin for 6–8 days or with blasticidin for 7–10 days. Cells were then removed from antibiotic selection and grown for generation of frozen stocks at earliest passage; all experiments were performed with early passage cells.

Transient transfection with REDD1 and AMPKα1/2 siRNA (Sigma) was employed to KD REDD1 and AMPK protein in Huh7 cells. Experiments were conducted by plating 8 × 105 cells per 6-cm plate for next day transfection using Lipofectamine RNAiMax (Invitrogen). A final concentration of (50 µM) siRNA was used in culture media without antibiotics overnight. Cells were split the next day and treated with (2.5 mM) metformin the following day for 24 h before collecting.

Proliferation assays

Hexokinase 2 also known as HK2 is an enzyme which in humans is encoded by the HK2 gene on chromosome 2.[5][6] Hexokinases phosphorylate glucose to produce glucose-6-phosphate (G6P), the first step in most glucose metabolism pathways. This gene encodes hexokinase 2, the predominant form found in skeletal muscle. It localizes to the outer membrane of mitochondria. Expression of this gene is insulin-responsive, and studies in rat suggest that it is involved in the increased rate of glycolysis seen in rapidly growing cancer cells. [provided by RefSeq, Apr 2009][6]

Further readingStructure[edit]

HK2 is one of four highly homologous hexokinase isoforms in mammalian cells.[7][8][9][10][11]


The HK2 gene spans approximately 50 kb and consists of 18 exons. There is also an HK2 pseudogene integrated into a long interspersed nuclear repetitive DNA element located on the X chromosome. Though its DNA sequence is similar to the cDNA product of the actual HK2 mRNA transcript, it lacks an open reading frame for gene expression.[10]


This gene encodes a 100-kDa, 917-residue enzyme with highly similar N- and C-terminal domains that each form half of the protein.[10][10][12] This high similarity, along with the existence of a 50-kDa hexokinase (HK4), suggests that the 100-kDa hexokinases originated from a 50-kDa precursor via gene duplication and tandem ligation.[10][11] Both N- and C-terminal domains possess catalytic ability and can be inhibited by G6P, though the C-terminal domain demonstrates lower affinity for ATP and is only inhibited at higher concentrations of G6P.[10][10] Despite there being two binding sites for glucose, it is proposed that glucose binding at one site induces a conformational change which prevents a second glucose from binding the other site.[13] Meanwhile, the first 12 amino acids of the highly hydrophobic N-terminal serve to bind the enzyme to the mitochondria, while the first 18 amino acids contribute to the enzyme’s stability.[9][11]


As an isoform of hexokinase and a member of the sugar kinase family, HK2 catalyzes the rate-limiting and first obligatory step of glucose metabolism, which is the ATP-dependent phosphorylation of glucose to G6P.[11] Physiological levels of G6P can regulate this process by inhibiting HK2 as negative feedback, though inorganic phosphate (Pi) can relieve G6P inhibition.[8][10][10][11] Pi can also directly regulate HK2, and the double regulation may better suit its anabolic functions.[8] By phosphorylating glucose, HK2 effectively prevents glucose from leaving the cell and, thus, commits glucose to energy metabolism.[10][10][12] Moreover, its localization and attachment to the OMM promotes the coupling of glycolysis to mitochondrial oxidative phosphorylation, which greatly enhances ATP production to meet the cell’s energy demands.[14][15] Specifically, HK2 binds VDAC to trigger opening of the channel and release mitochondrial ATP to further fuel the glycolytic process.[8][15]

Another critical function for OMM-bound HK2 is mediation of cell survival.[8][9] Activation of Akt kinase maintains HK2-VDAC coupling, which subsequently prevents cytochrome c release and apoptosis, though the exact mechanism remains to be confirmed.[8] One model proposes that HK2 competes with the pro-apoptotic proteins BAX to bind VDAC, and in the absence of HK2, BAX induces cytochrome c release.[8][15] In fact, there is evidence that HK2 restricts BAX and BAK oligomerization and binding to the OMM. In a similar mechanism, the pro-apoptotic creatine kinase binds and opens VDAC in the absence of HK2.[8] An alternative model proposes the opposite, that HK2 regulates binding of the anti-apoptotic protein Bcl-Xl to VDAC.[15]

In particular, HK2 is ubiquitously expressed in tissues, though it is majorly found in muscle and adipose tissue.[8][10][15] In cardiac and skeletal muscle, HK2 can be found bound to both the mitochondrial and sarcoplasmic membrane.[16] HK2 gene expression is regulated by a phosphatidylinositol 3-kinaselp70 S6 protein kinase-dependent pathway and can be induced by factors such as insulin, hypoxia, cold temperatures, and exercise.[10][17] Its inducible expression indicates its adaptive role in metabolic responses to changes in the cellular environment.[17]

Clinical significance[edit]


HK2 is highly expressed in several cancers, including breast cancer and colon cancer.[9][15][18] Its role in coupling ATP from oxidative phosphorylation to the rate-limiting step of glycolysis may help drive the tumor cells’ growth.[15] Notably, inhibition of HK2 has demonstrably improved the effectiveness of anticancer drugs.,[18] Thus, HK2 stands as a promising therapeutic target, though considering its ubiquitous expression and crucial role in energy metabolism, a reduction rather than complete inhibition of its activity should be pursued.[15][18]

Non-insulin-dependent diabetes mellitus[edit]

A study on non-insulin-dependent diabetes mellitus (NIDDM) revealed low basal G6P levels in NIDDM patients that failed to increase with the addition of insulin. One possible cause is decreased phosphorylation of glucose due to a defect in HK2, which was confirmed in further experiments. However, the study could not establish any links between NIDDM and mutations in the HK2 gene, indicating that the defect may lie in HK2 regulation.[10]


HK2 is known to interact with:


Interactive pathway map[edit]

Click on genes, proteins and metabolites below to link to respective articles. [§ 1]



|{{{bSize}}}px|alt=Glycolysis and Gluconeogenesis edit]]

Glycolysis and Gluconeogenesis edit

1 Jump up
The interactive pathway map can be edited at WikiPathways: “GlycolysisGluconeogenesis_WP534″.

See also[edit]


Mitochondria portal






1^ Jump up to:
a b c GRCh38: Ensembl release 89: ENSG00000159399Ensembl, May 2017

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a b c GRCm38: Ensembl release 89: ENSMUSG00000000628Ensembl, May 2017

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“Mouse PubMed Reference:”.

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Lehto M, Xiang K, Stoffel M, Espinosa R, Groop LC, Le Beau MM, Bell GI (Dec 1993). “Human hexokinase II: localization of the polymorphic gene to chromosome 2″. Diabetologia. 36 (12): 1299–302. doi:10.1007/BF00400809. PMID 8307259.

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a b “Entrez Gene: HK2 hexokinase 2″.

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Murakami K, Kanno H, Tancabelic J, Fujii H (2002). “Gene expression and biological significance of hexokinase in erythroid cells”. Acta Haematologica. 108 (4): 204–9. doi:10.1159/000065656. PMID 12432216.

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a b c d e f g h i j Okatsu K, Iemura S, Koyano F, Go E, Kimura M, Natsume T, Tanaka K, Matsuda N (Nov 2012). “Mitochondrial hexokinase HKI is a novel substrate of the Parkin ubiquitin ligase”. Biochemical and Biophysical Research Communications. 428 (1): 197–202. doi:10.1016/j.bbrc.2012.10.041. PMID 23068103.

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a b c d Schindler A, Foley E (Dec 2013). “Hexokinase 1 blocks apoptotic signals at the mitochondria”. Cellular Signalling. 25 (12): 2685–92. doi:10.1016/j.cellsig.2013.08.035. PMID 24018046.

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a b c d e f g h i j k l m n Printz RL, Osawa H, Ardehali H, Koch S, Granner DK (Feb 1997). “Hexokinase II gene: structure, regulation and promoter organization”. Biochemical Society Transactions. 25 (1): 107–12. doi:10.1042/bst0250107. PMID 9056853.

11^ Jump up to:
a b c d e Ahn KJ, Kim J, Yun M, Park JH, Lee JD (Jun 2009). “Enzymatic properties of the N- and C-terminal halves of human hexokinase II”. BMB Reports. 42 (6): 350–5. doi:10.5483/bmbrep.2009.42.6.350. PMID 19558793.

12^ Jump up to:
a b Aleshin AE, Zeng C, Bourenkov GP, Bartunik HD, Fromm HJ, Honzatko RB (Jan 1998). “The mechanism of regulation of hexokinase: new insights from the crystal structure of recombinant human brain hexokinase complexed with glucose and glucose-6-phosphate”. Structure. 6 (1): 39–50. doi:10.1016/s0969-2126(98)00006-9. PMID 9493266.

13 Jump up
Cárdenas, ML; Cornish-Bowden, A; Ureta, T (5 March 1998). “Evolution and regulatory role of the hexokinases”. Biochimica et Biophysica Acta. 1401 (3): 242–64. doi:10.1016/s0167-4889(97)00150-x. PMID 9540816.

14 Jump up
Shan D, Mount D, Moore S, Haroutunian V, Meador-Woodruff JH, McCullumsmith RE (Apr 2014). “Abnormal partitioning of hexokinase 1 suggests disruption of a glutamate transport protein complex in schizophrenia”. Schizophrenia Research. 154 (1–3): 1–13. doi:10.1016/j.schres.2014.01.028. PMC 4151500. PMID 24560881.

15^ Jump up to:
a b c d e f g h Palmieri D, Fitzgerald D, Shreeve SM, Hua E, Bronder JL, Weil RJ, Davis S, Stark AM, Merino MJ, Kurek R, Mehdorn HM, Davis G, Steinberg SM, Meltzer PS, Aldape K, Steeg PS (Sep 2009). “Analyses of resected human brain metastases of breast cancer reveal the association between up-regulation of hexokinase 2 and poor prognosis”. Molecular Cancer Research. 7 (9): 1438–45. doi:10.1158/1541-7786.MCR-09-0234. PMC 2746883. PMID 19723875.

16 Jump up
Reid, S; Masters, C (1985). “On the developmental properties and tissue interactions of hexokinase”. Mechanisms of ageing and development. 31 (2): 197–212. doi:10.1016/s0047-6374(85)80030-0. PMID 4058069.

17^ Jump up to:
a b Wyatt, E; Wu, R; Rabeh, W; Park, HW; Ghanefar, M; Ardehali, H (3 November 2010). “Regulation and cytoprotective role of hexokinase III”. PLOS ONE. 5 (11): e13823. doi:10.1371/journal.pone.0013823. PMC 2972215. PMID 21072205.

18^ Jump up to:
a b c Peng Q, Zhou J, Zhou Q, Pan F, Zhong D, Liang H (2009). “Silencing hexokinase II gene sensitizes human colon cancer cells to 5-fluorouracil”. Hepato-Gastroenterology. 56 (90): 355–60. PMID 19579598.

Cancer Cell. 2013 Aug 12; 24(2): 213–228.

Published online 2013 Aug 1. doi:  10.1016/j.ccr.2013.06.014

PMCID: PMC3753022


PMID: 23911236

Hexokinase 2 is required for tumor initiation and maintenance and its systemic deletion is therapeutic in mouse models of cancer

Krushna C. Patra,1 Qi Wang,#1 Prashanth T. Bhaskar,#1 Luke Miller,1 Zebin Wang,1 Will Wheaton,2 Navdeep Chandel,2 Markku Laakso,3 William J. Muller,4 Eric L. Allen,5 Abhishek K. Jha,5 Gromoslaw A. Smolen,5 Michelle F. Clasquin,5 Brooks Robey,6,7 and Nissim Hay1,8,*

Author information ► Copyright and License information ► Disclaimer

This article has been corrected. See the correction in volume 24 on page 399.

See other articles in PMC that cite the published article.

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Accelerated glucose metabolism is a common feature of cancer cells. Hexokinases catalyze the first committed step of glucose metabolism. Hexokinase 2 (HK2) is expressed at high level in cancer cells, but only in a limited number of normal adult tissues. Using Hk2 conditional knockout mice, we showed that HK2 is required for tumor initiation and maintenance in mouse models of KRas-driven lung cancer, and ErbB2-driven breast cancer, despite continued HK1 expression. Similarly HK2 ablation inhibits the neoplastic phenotype of human lung and breast cancer cells in vitro and in vivo. Systemic Hk2 deletion is therapeutic in mice bearing lung tumors without adverse physiological consequences. Hk2 deletion in lung cancer cells suppressed glucose-derived ribonucleotides and impaired glutamine-derived carbon utilization in anaplerosis.

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Accelerated glucose metabolism under aerobic conditions is one of the hallmarks of cancer cells. The elevated glucose metabolism is required to provide sufficient amounts of metabolic intermediates to support anabolic processes such as nucleic acid, lipid, and protein synthesis in the rapidly dividing cancer cells (reviewed in (Lunt and Vander Heiden, 2011; Schulze and Harris, 2012)). The dependency of cancer cell proliferation on accelerated glucose metabolism distinguishes them from their normal counterparts and could render them more vulnerable to the disruption of glucose metabolism. Therefore, cancer cells could be selectively targeted by the disruption of intracellular glucose metabolism. However, it unclear whether it is feasible to inhibit enzymatic activities required for glucose metabolism, at the organism level, and to selectively target cancer cells without adverse physiological consequences. The identification of isoform-specific contributors to cancer cell glucose metabolism that could be selectively targeted to disadvantage cancer cells without compromising systemic homeostasis or corresponding metabolic functions in normal cells could make such an approach feasible.


—–Original Message—–

From: inlightenconsent <>

To: inlightenconsent <>

Sent: Sun, Oct 14, 2018 7:36 pm

Subject: Cancer Nuclear Hexokinase

YU nuclear transfer

Travis Chris






Epigenix Foundation

Published on Jun 8, 2016



Travis Christofferson, MS, Science Writer, Author, Single Cause Single Cure Foundation Travis Christofferson is a graduate of the Montana State Honors Program in molecular biology. He received the Nelson Fellowship for “outstanding undergraduate research”, and continued graduate research in bioremediation and cancer theory culminating in an M.S. in Material Engineering and Science from the South Dakota School of Mines and Technology. He is a science writer and the author of the recently released book Tripping Over the Truth: The Metabolic Theory of Cancer. The book offers a historical perspective on the reemerging metabolic theory of cancer — a theory that contends cancer is precipitated and driven by damage to mitochondria.



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