Hacking hexokinase halts tumor growth

It has been increasingly noted that malignant cells often evolve aberrant bioenergetic activities to benefit their proliferation and survival. One of the most drastically altered metabolic pathways in malignant cells is glycolysis. As first documented by Otto Warburg nearly eight decades ago, compared to non-malignant cells, cancerous cells exhibit higher glycolytic activity even when oxygen is abundant.¹ This phenomenon has been referred to as “Warburg effect,” or “aerobic glycolysis,” which serves as the foundation for the positron emission tomography (PET) that is widely used in clinical cancer diagnosis.² The use of PET has in turn confirmed the “Warburg effect” as a universal property of cancer cells.

Warburg originally attributed the higher glycolytic activity in cancer cells to mitochondrial deficiency. Recent studies gradually reveal that other factors including hypoxia, redox stress, oncogene activation, as well as loss of tumor suppressor genes can all contribute to the high rate of glycolysis.³ An abnormal glucose metabolism fulfills the bioenergetic and biosynthesis needs of cancer cells. It is tightly associated with cell death evasion, cell proliferation, angiogenesis and metastasis of various cancers. Targeting the glycolysis pathway thus provides a reasonable therapeutic opportunity.

Glycolysis is controlled by glucose transporters (GLUTs) and a series of glycolytic enzymes. Among these, hexokinase (HK), phosphofructokinase (PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and lactate dehydrogenase (LDH) have been explored as anti-cancer therapeutic targets (Table 1). Hexokinase controls the first rate-limiting step of glycolysis by converting glucose into glucose-6-phosphate (G6P). There are four isoforms of HK in mammals, among which hexokinase II (HKII) is the major form that has been found upregulated in various cancer cells. In addition to its glucose phosphorylation activity, HKII is capable of binding to the voltage-dependent anion channel (VDAC) on the mitochondrial outer membrane through its N-terminal residues.⁴ The binding of HKII to VDAC places it in close proximity to the mitochondria, which is believed to allow efficient use of mitochondria-generated ATP by HKII, thus enhancing the glycolytic flux. Moreover, the binding of HKII to VDAC has been found to stabilize the mitochondrial membrane and prevent the release of apoptogenic factors, such as cytochrome C and AIF, from the mitochondrial intermembrane space. Disrupting interactions between HKII and mitochondria has been shown to lead to cell growth arrest and death.⁵ This may result from decreased glycolysis and ATP supply, as well as the destabilization of the mitochondrial outer membrane. Thus, in order for HKII to fully protect the cell from cell death, contributions from both its kinase activity and its binding to mitochondria are required. Disrupting only one of these two functions may not be able to completely abolish HKII’s pro-survival effect in tumor cells.⁶

Table 1.

The potential therapeutic targets of glycolytic pathway and their inhibitors

Targets	Inhibitors	Possible mechanisms	Clinical trails
HK	2DG and derivates	Compete with glucose for the GLUTs and HK; disrupt the association between HKII and mitochondria.	Phase I/II7
	3BA and derivates	Disrupt the association between HKII and mitochondria; inhibit HK kinase activity. Also inhibit GAPDH	Preclinical8–10
	Lonidamine	Disrupts the association between HKII and mitochondria. Also inhibit the lactate transport.	Phase III19
	Methyl jasmonate	Disrupts the association between HKII and mitochondria; does not inhibit the HKII activity.	Preclinical11
PFK	3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO)	Small molecule blocks the PFK/FB3enzyme	Preclinical20
GAPDH	Koningic acid; Methylglyoxal	GAPDH enzyme inhibitors	Preclinical21,22
LDH	Oxamate; shRNA	Metabolic inhibitor; Gene silencer	Preclinical14,23

Several glycolytic inhibitors target HKII. 2-deoxy-glucose (2DG), an analog of glucose, functions as a competitor of glucose for entering the cell via GLUTs and phosphorylation by HKs. The phosphorylated product, 2DG-6-phosphate (2DG-6P), can not be processed further through the glycolysis pathway. 2DG-6P accumulates in the cells and disrupts the binding of HKII to the mitochondria without inhibiting HKII kinase activity.⁷ 3-bromo-pyruvate (3BA), an analog of pyruvate, functions as a protein alkylating agent. It has a strong ATP depletion ability by disrupting the HKII association with mitochondria and inhibiting its kinase activity.^8–10 Methyl jasmonate (MJ), a plant lipid derivative, which functions as a signaling molecule in the stress response of plants, disrupts the interaction between HKII and VDAC without inhibiting the kinase activity.¹¹ These pharmacological inhibitors have been shown to have antitumor activity by inhibiting glycolysis, depleting ATP, inhibiting cell cycle progression, and inducing cell death. However, their molecular target specificity and non-specific inhibition of all isoforms of HKs may lead to unwanted side effects when systemically used in patients. There is a need to develop agents specifically targeting the HKII of cancer cells.

RNA interference (RNAi) technology is based on the sequence-dependent degradation of mRNA to inhibit specific gene expression.¹² RNAi-based agents have recently been developed as a new therapeutic strategy for inhibiting specific gene targets. Two major RNAi methods are used in mammalian systems: small interfering RNA (siRNA) that is formed through the annealing of two complementary RNA oligos, and short-hairpin RNA (shRNA) that is a single-stranded RNA molecule able to form a hairpin structure and function as a pro-siRNA molecule. Preclinical studies have shown the inhibition of growth and survival of tumor cells using RNAi techniques to knockdown oncogenes or tumor-promoting genes. These include growth and angiogenic factors or their receptors, telomerase, viral oncogenes (papillomavirus E6 and E7), BCR-ABL, Ras, as well as the anti-apoptotic Bcl-2 family proteins.¹³ Along the glycolysis pathway, knockdown of LDH-A using shRNA compromised the ability of tumors cells to proliferate under hypoxic conditions, and suppressed tumorigenicity in nude mice.¹⁴

High HK activity has been found in colorectal tumor tissues,^15,16 and high HKII expression was detected in several colon cancer cell lines.¹⁷ This prompted Liang and colleagues, in this issue of Cancer Biology Therapy, to test the effects of knocking down HKII using shRNA in LoVo cells, a colon cancer cell line. Indeed, they find that the downregulation of HK II expression significantly suppressed LoVo cell cloning efficiency in cell culture, and tumor growth in mouse xenografts. These observations correlated with decreased ATP content, decreased cell cycle progression, and increased spontaneous apoptosis. This is the first preclinical study that uses an RNAi technique to decrease HKII expression in an attempt to suppress tumor cell growth and survival. Their work provides proof-of-principle evidence in support of targeting glycolysis in cancer cells, and brings up an encouraging case of the RNAi technique in a cancer therapeutic application.¹⁸

It should be noted that in this study, both cell cycle arrest and apoptosis did not occur to an extent that cause a complete cell cycle arrest, or massive cell death. This raises an issue concerning the effectiveness of knocking down HKII as an anti-cancer therapy. A few considerations should be taken. One is that due to the nature of this study, which is designed to select for cell clones with stable HKII knockdown, only the clones retaining cell viability and growth ability could have been established. Moreover, because the RNAi process is mediated by several biochemical reactions that need ATP, a profound shutdown of the energy production will prevent RNAi itself from working efficiently. This may cause a dilemma when targeting the cellular bioenergetic machinery, which may be the reason why a complete knockdown of HKII could not be reached. Nevertheless, it needs to be cautioned that approaches that target cell metabolism will lead to bioenergetically inert tumor cells. While this tumor static effect can certainly be beneficial to the patients, it may introduce a potential hazard as the bioenergetically inert tumor cells may become more resistant to other anti-cancer agents during adjuvant treatments. This may also provide the tumor cells with a chance to accumulate pro-malignancy mutations. A better understanding of biology of bioenergetically inactive tumor cells will help to design safer and more effective strategies to target cancer cell metabolism.