Cancer stands as one of the most heterogeneous human diseases. It can be caused by many factors- most of which are still unknown or only partially understood. Cancer can originate in any tissue regardless of embryological origin and can spread between tissues. The degree of malignancy and metastasis of cancer is highly variable, as are the effect on human mortality and the physical symptoms it presents in patients. The Encyclopedia Britannica states that 1 of every three people in developed countries will develop some form cancer during their lifetime. Cancer is also the leading cause of death for adults between the ages of 24 and 65 according to the Center for Disease Control and the National Center for Health Statistics’ provisional data for 1990. The heterogeneity and omnipotence of cancer has made the diagnosis and treatment paramount to both research and medicine.
Surprisingly, the most universal similarity found in the majority of cancers, both animal and human, is a not a product of the recent molecular genetics revolution. Its discovery actually predates the elucidation of the structure of DNA by more than 20 years. As early as 1927, Warburg observed that cancers possessed a remarkable ability to sustain high rates of anaerobic glycolysis (1). Anaerobic glycolysis utilizes glucose to produce lactate, while aerobic glycolysis produces pyruvate, which enters the tricarboxylic acid cycle (TCA); ultimately, the latter produces energy via oxidative phosphorylation. The most pernicious types of cancer not only metastasize to other tissues, but also exhibit a high growth rate. Rapidly proliferating cells require energy and carbon sources for synthesis, as well. Energy production in some cancer cells has been found to be over 400-fold higher than the energy demands of biosynthesis(2). It is widely supposed that the by-product of such exaggerated metabolics supplies cancers with precursors for protein, nucleic acids and structural components for division and further growth. As heterogeneity is the rule of thumb for cancer, there are some exceptions to the hyper-glycolytic state.
Warburg thought the hyper-glycolytic state was characteristic of all cancers, and to his credit, this was true for all of the cancers he studied. The Warburg effect, so named after its founder, is used to describe the high rates of anaerobic glycolysis. Although the Warburg effect is not present in all cancers, it is the most common phenotype in rapidly-growing cancers(2-23). Highly de-differentiated cancer cells usually have a high glycolytic rate, and thus a high growth rate. The opposite is true for cancer cells which have not de-differentiated (e.g. they remain similar to the non-cancerous parental cells). Warburg’s correct assumption that the glycolytic rate was a measurement of malignancy stands with only a few exceptions. The extremely malignant cells of the Morris hepatoma have normal rates of glycolysis and a very slow growth rate(24). Warburg’s observations lead him to the incorrect assumption that cancer was in part the result of the loss of the cells ability to use oxygen(1). This idea has been proven untrue for many years and by many researchers, yet the validity of the hyper-glycolytic phenomena in rapidly growing cancers remains(25).
The subject of this review will focus on mechanisms utilized by cancer cells to maintain the hyper-glycolytic state. It is necessary to examine aspects of metabolism to present a coherent review on this subject. Because glycolysis is central to cellular metabolism, certain enzymes in the pathway are tightly regulated. This review will focus on 3 key enzymes: hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK). As a short prodrome, the normal action and regulation of these enzymes will be discussed under ‘Regulation of Glycolysis’. The body of the review will attempt to cover the vast array of alterations in the regulation of these glycolytic enzymes in cancer cells. The author has chosen to use the term ‘de-regulation’ to denote alterations in enzyme regulation in cancer cells. This is not to suggest that cancer cell’s enzymes are not regulated, rather that they are controlled differently than those of normal cells.
In order to efficiently cover this topic, the types of de-regulation have been classified into several groups. The author began this research by posing a simple question: how could a cancer cell increase its glycolytic rate in light of the tight regulation of the enzymes in the pathway? Each group will attempt to provide an answer to this question. For instance, one might imagine the simplest way to increase glycolysis is to synthesize more enzymes in the pathway. Thus, the first group- entitled quantitative de-regulation- investigates the over-production of enzymes in cancer cells. Another way to increase glycolysis would be to alter the mechanisms of enzyme control to prevent inhibition and/or promote stimulation. The second group, entitled qualitative de-regulation will look at alterations in the quality of enzymes, which effects the regulation of the enzyme. The later group will include alteration of modifiers, subunit composition and gene transcription. The information in the review is presented from the eyes of a fascinated researcher and is not meant to applaud cancer’s ingenious methods of de-regulation. The author hopes to highlight cancer’s weaknesses for a future eulogy, and wishes to dedicate this paper to the past, present and future victims of cancer.
Hexokinase (HK) is responsible for phosphorylating hexoses. There are four different isozymes produced in mammals (I-IV) (26). The numbers represent corresponding electrophoretic mobilities such that IV is the most mobile. The IV isozyme is also called glucokinase, although the specificity for all of the isozymes for glucose is identical(26). However, there are some tissue-specific differences in the isozyme expression. Glucokinase (IV) is produced in hepatic and b -cell pancreatic tissue. It has a high Km for hexoses, thus there must be a high concentration of the hexose for high activity. By contrast, HK I and II are produced by extra-hepatic tissue, and have a low Km for hexoses. The evolutionary significance of this adaptation is evident in that extra-hepatic tissue uses hexoses, namely glucose, for energy production. Whereas hepatic tissue can produce glucose in times of fasting, and store it as glycogen after eating. Thus, the high Km of HK IV prevents the liver from removing glucose needed for extra-hepatic tissue. Another difference which exists between isozymes is regulation. HK I and II are inhibited by glucose-6-phosphate by feedback. By contrast, HK IV is not inhibited in this manner. For this review, it is important to remember that HK IV is normally expressed in hepatic tissue only.
Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. There are three subunits of PFK: muscle-type (M), liver-type (L) and a plalete-type (P)(23). As was the case for HK, PFK also exhibits tissue-specific patterns of expression(23). PFK is inhibited by ATP and citrate and stimulated by AMP and fructose-2,6-bisphosphate. The later is produced by a tandem enzyme called phosphofructokinase-II, and represents an important regulation step in glycolysis. When level of fructose-6-phosphate are high, PFKII will make fructose-2,6-bisphosphate, which stimulates PFK. Thus, PFK is regulated by the energy charge and the glycolytic intermediates in the cell.
Pyruvate Kinase (PK) catalyzes the formation of pyruvate from phosphenolpyruvate. There are several different isozymes present, and some evidence for tissue-specific expression(27). PK is stimulated in a feed-forward mechanism by fructose-1,6-bisphosphate, which is the product of the reaction catalyzed by PFK. One important fact to mention is that PK produces ATP, whereas both HK and PFK use ATP. Anaerobic glycolysis further reduces pyruvate to lactate, thus the energy produced by PK is very important in cancer metabolism.
Evident from above, it is easy to see that these enzymes are tightly controlled in the normal cell. Further there exists hormonal regulation of glycolysis. Because the topic of this review is centered towards the intra-cellular alterations, hormonal regulation will be discussed as needed. There are many points of control in glycolysis to regulate the rate at which glucose is used. These general aspects of regulation provide cellular control of energy utilization. As a cell becomes transformed to a cancer cell specific changes occur which disrupt native regulation. With this is mind, the question of how de-regulation occurs in cancer may be addressed.
One strategy to increase the rate of glycolysis is to simply increase the number of enzymes in the pathway. At first glance this may seem to be the easiest and thus the most common approach to resolve the energy demands of cancer cells. However, the blatant over-expression of wild-type glycolytic enzymes was not found to be a common approach of cancer cells. Although no detailed research was found to explain why this approach is uncommon, there are several likely causes. First, all of these glycolytic enzymes are highly regulated in normal cells as previously discussed. Although synthesizing more wild-type enzyme would increase the number, all of the de novo enzymes retain the ability to be regulated. Thus, it would be counter-productive for a cancer cell to waste the energy required for synthesis if the enzyme was subsequently inhibited by allosteric modifiers or by phosphorylation. By contrast, if the number of enzymes were doubled in a cell, you would half the effect of a constant number of allosteric modifiers. However, when the energy demands for cancer are far greater than that of normal tissue, the law of diminishing return obviates wild-type hyper-expression as an effective means of increasing glycolytic flux. In some tumor lines the rates of energy production can be over 400 fold higher than required for anabolic processes (2) , suggesting that changes in the quantity of glycolytic enzymes are important, but not sufficient, for the growth of the tumor.
However, it is possible for cancer cells to achieve a high glycolytic flux by altering the amount of enzyme. In order to discuss this approach, it is imperative to understand that the effect of the type of enzyme is more important than the concentration of the enzyme. Thus, it is the over-production of an atypical enzyme instead of the wild type which turns the futile process into a highly beneficial method to answer the energy needs of rapidly proliferating cells. For instance, the hyper-expression of PK mRNA has been found in a number of colorectal cancers(15). In many tumors the uptake of glucose is completely unrestricted (11) , suggesting that number of glucose transporters is increased in cancer. The glucose is then phosphorylated by HK. This enzyme provides a prime example of the qualitative de-regulation in the rat AS-30D hepatoma cell line. Based on previous research findings that HKII is hyper-expressed in many cancer cells, Rempel et al. found this phenotype to be due in part to gene amplification (21). Interestingly, this specific type of HKII has several characteristics that make it ideal for quantitative de-regulation. From nucleotide sequence and amino acid determination, the HKII involved was different from wild type rat HK’s (28). In the same study, the mutant HKII (mHKII) was found to be composed of atypical subunits that are immunologically distinct when compared to the HKIV (glucokinase) normally found in rat hepatocytes and wild type HKII in rat muscle cells. Further, the catalytic activity of HK in cultured cell lines is directly correlated to glycolytic flux (29), suggesting the increase of HK is sufficient to promote the hyper-glycolytic state of cancer cells. Another atypical feature is the cellular location of the mHKII. Although other forms of enzyme translocation will be discussed later, they involve subunit anomalies covered in the qualitative section of this review. Researchers found that in the same cell line the mHKII was bound to the mitochondrial membrane (28) and that it has preferential access to the ATP generated by the mitochondria (3). These reports suggest that the effect of the aberrant location and/or ATP source may remove the inhibitory effect of glucose-6-phosphate on the mHKII. Similar findings regarding the cellular location of HKII variants have been reported in the Novikoff ascites tumor line derived from human liver (30). In both cell lines HKII is not produced by or bound to either normal rat (28) or human (30) heptocyte mitochondria.
Recent research can also help shed light on some of the first data gathered on the action of HK. As early as 1976, it was known that the binding of Ehrlich ascites carcinoma HK to mitochondrial and endoplasmic membranes was stimulated by glucose-6-phosphate(31). At the time, there were many speculations on the effect this enhanced binding might have on the glycolytic flux. However, it is now known that both the location (30) and the amount (32) of HK are very important in supply of needs of rapidly proliferating cells. Further, the finding that the hyper-expression of HK mRNA is present in cells lines derived from extra-hepatic tumors (33) emphasizes the importance of HK in tumor proliferation.
These data emphasize the importance of several factors effecting quantitative de-regulation. First, the futile hyper-expression wild type glycolytic genes is not characteristic of metastases in general. Both of the cell lines mentioned are but a small sample of the many types of cancer. Thus, they may serve more of a reference to the general heterogeneity in energy utilization in cancer rather than the rule. Secondly, if quantitative de-regulation is present, it is accompanied by some degree of qualitative de-regulation that alters the ability of the enzyme to be regulated in a controlled manner. For instance, the gene for HK in the AS-30D cell line was reported to be present in ca. 4 extra copies on the same chromosome (21) and each gene copy was that of the mutant HKII, not wild type HKII. Lastly, no evidence could be found which suggests that aberrant hyper-expression of mHKII was sufficient or necessary for transformation in hepatocytes. Thus, the gene amplification of HKII is not the cause of metastases per se, but the effect of multiple factors on the glycolytic flux in cancer cells.
By far the most efficient way to de-regulate an enzyme is by change some quality inherent in the wild type protein. This review will focus on several ways this can occur. First, mutations in the gene coding for the enzyme will be discussed. These glycolytic enzyme mutation cover a variety of changes in the protein amino acid composition which in turn serves to release the mutant protein from normal regulation. Secondly, mutations in genes whose products either allow the over-expression of or effect the regulation on glycolytic enzymes. The former group contains enzymes which code for trans-acting factors that regulate gene expression, while the later includes kinases which activate or deactivate the glycolytic enzymes. The final mechanism discussed is qualitative changes which effect the subunit composition of the enzyme. Because most of the glycolytic enzymes contain both regulatory and catalytic subunits, the composition of subunits is a powerful tool in the de-regulation of glycolysis.
As previously discussed, the random expression of wild type genes is not very effective in stimulating the hyper-glycolytic state of proliferating cells unless it is accompanied by a mutation in the gene that renders the over-expressed protein insensitive to innate regulation. The mutant HK found in the c37 mouse hepatoma cell line was found to have a total of 32 different amino acids from nucleic acid sequence analysis than the normal glucokinase found in liver (28). In this same report, Arora et al. found that the HK found a N-terminal portion on the hepatoma HK, which was normally present in the rat brain HK. Although the exact effect of each of these mutations is still unknown, it is very easy to speculate they may either enhance the binding of HK to the mitochondrial membrane or de-sensitize the mutant HK to the normal negative regulation of glucose-6-phosphate. The mutation of genes distant to glycolysis can also effect the rate of glycolysis. In a human cancer cell line, researchers found that PFK-2 was constituitively expressed (6). This causes the increase in fructose-2,6-bisphosphate concentration which in turn activates PFK-1. Although the regulation of the PFK-2 was not researched by Chesney et al., this presents an interesting mechanism in activating glycolysis.
The quality of genes whose products control glycolytic enzymes is another method of activating glycolysis. The activation of PFK-2 was previously shown to effect glycolysis. Data from studies on PFK-2 suggest that an novel transacting gene product is sufficient to activate PFK-2 (14). This would have the same effect as over-expressing PFK as discussed above. Further, another novel trans-acting factor was shown to be responsible for HK over-expression in the rat ascites hepatoma cell line (32). It is possible to activate glycolysis by changing proteins which control the expression of glycolytic enzyme. Genetic mutation may accomplish the same task on a protein level rather than the genetic level. For instance, the oncogenic human papillomavirus encodes a small number of genes, but one of those, E7, is a oncoprotein which binds type M2 pyruvate kinase (34). The effect of the binding causes the tetrameric form of PK to disassociate to its dimeric form which lowers the enzymes affinity for phosphoenolpyruvate (PEP). Thus, glycolytic intermediates can be used for synthesis of DNA or proteins for cell division. The same effect has been found by the expression of the src-ongene, however by another mechanism. The oncoprotein from src was found to phosphorylate PK, which led to the disassociation and decrease in PEP affinity (10). Thus, genetic modification, or rather mutations, can effect the regulation of glycolytic enzymes in the progression of tumor growth.
Under genetic modification, transcriptional modifications can also effect the regulation of glycolytic enzymes. One well studied form of transcriptional modification involves the effect of hypoxic conditions. Genes which are regulated by oxygen concentration are called hypoxia-inducible genes. Hypoxic conditions are known to effect glycolysis (35). The abundance of oxygen is a determining factor in aerobic versus anaerobic respiration, however the effects discussed herein are those of transcription and not of substrate availability. The glycolytic enzymes regulated by hypoxia possess regions in their promoters which can be bound by trans-acting factors called hypoxia-inducible factors (HIF) to induce transcription. Some of the glycolytic enzyme promoters also possess glucose responsive elements that regulate transcription in a similar way as HIF-1. HIF-1 was one of the factors which is made in response to hypoxia (36). This factor is responsible for binding the promoter elements in glycolytic genes causing their transcription. Hypoxia is critical condition in cancer due to the fact that rapidly proliferating cells found in a tumor are in constant need of oxygen and nutrients to fuel their growth. Thus, the simple growth of a tumor causes hypoxia which can stimulate glycolysis.
HIF-1 is known to effect several critical enzymes in glycolysis. First, it induces the expression of GLUT3, which is a type glucose transporter found in certain tissues (37). In doing so, the GLUT3 would transport more glucose into the cell for energy needs. HIF-1 also causes the increase in PK mRNA levels (38). Further studies have shown that hypoxia causes an increase in type M2 PK mRNA without out an increase in HIF-1 mRNA (15). These data suggest that the effect of HIF-1 is not caused by transcription but by translation or by a manner directly related to PK transcription without the involvement of HIF-1. Another gene effected by this factor is the type L PK. Research found that the c-Myc oncogenic transcription factor stimulates HIF-1 (7) and glucose responsive elements in the type L PK (39). Thus, the induction of HIF-1 during hypoxia causes the up-regulation of key enzymes in glycolysis.
Another transcriptional regulator studied in cancer is p53. Wild type p53 is a trans-acting factor that reduces the expression of gene needed for proliferation (40). In the cell cycle, p53 acts as a policeman halting the progression of the cycle when growth is not required. In cancer cells, p53 may be mutated in such a way that the cell cycle is allowed to progress without regulation. Although there is no evidence that p53 mRNA is increased in hypoxia (15), p53 can effect glycolysis in other ways. There are two p53 binding motifs present in the HKII gene in the AS-30D hepatoma cell line, and binding of p53 is sufficient to cause an increase in the expression of HKII (41, 42). In this cell line, mutated p53 causes the expression of HKII which in turn leads to the expression of more HK. Thus, mutation of p53 reverses its normal repression of HKII. There are several mutants of p53 which have been classified in cancer cell lines. One such mutant was found to be sensitive to temperature in its regulatory action on the cell cycle (43). Therefore, mutation of a transcriptional regulator may effect genes involved in cell cycle regulation and glycolysis. Because both the de-regulation of cell cycle and activation of glycolysis are important in cancer growth, p53 serves as a gene which can control aspects of both.
As mentioned earlier, glucose response elements (GRE) are present in the promoters of key glycolytic enzymes. One type of GRE is sensitive not to a trans-acting protein, but to glucose itself. In a set of experiments, researchers found that the type L PK is transcriptionally activated by glucose in both cancer cells and in normal tissue (44). However, in the cancer cells the activation did not require insulin and was not blocked by cAMP (45). These data suggest that a mutation in the GRE for glucose obviated the need for insulin to cause induction and blocked repression by cAMP which is normally found in non-metastatic tissue. This presents another de-regulation mechanism which causes the increase in PK synthesis regardless of insulin and insensitive to glucagon-mediated cAMP repression.
The final type of qualitative de-regulation involves the subunit composition of glycolytic enzymes. Previously, it was found that activity and regulation of PFK is dependent on subunit composition (46). Not surprisingly, PFK found in cancer cells are frequently altered in subunit type. In ascites tumors, the C type PFK subunit is more predominate than the L type found in normal tissue (22). In the same study, the ascites tumor PFK was found to be insensitive to frutose-1,6-bisphosphate activation and to phosphoenolpyruvate inhibition. Similarly, the PFK expressed in human gliomas are less sensitive to citrate inhibition and more sensitive to fructose-2,6-bisphosphate stimulation. Conversely, the L type PFK was more predominate in human gliomas (47). The PFK isozyme isolated from human gliomas, although uncharacterized, were found to be less sensitive to ATP and citrate inhibition than the PFK isozyme in normal astrocytes (9). These examples show how subunit composition can effect the regulation of PFK. Similar results have been found with PK. The type M2 PK gene produced at high levels in tumors is subject to de-regulation by several types of oncogens (10). The production of the type M2 PK is so constant that researchers have suggested that level of this isoenzyme corresponds to the degree of malignancy (48). In studies on human brain tumors, PK regulation has also been implemented in glycolytic regulation. Specifically, the type K PK is phosphorylated in a number of tumors studied causing the deactivation of the enzyme (49, 50).
There is a myriad of examples of how de-regulation of glycolysis occurs in cancer cells. This paper presents some of the most outstanding forms of de-regulation. It is evident that a key to curing cancer is finding how to prevent high and sustained rates of anaerobic respiration from occurring in the cancer cell. The side-effects of this treatment may be as pernicious as those for chemotherapy. However, there have been some attempts to use characteristics of cancer cells to their disadvantage. The HKII is hyper-expressed in several forms of cancer (see above). Researchers have used the promoter of HK II to achieve tumor-specific killing (51). This was achieved by linking the HK II promoter to a gene which, when expressed, made the cancer cell extremely sensitive to anti-tumor agent. Transfection of tumor cells with this construct and administration of the agent resulted in the reduction of live tumor cells by 54% (51). Thus, this method in addition to other therapy may help in cancer treatment.
From the research, there are several approaches to cancer therapy which may be effective. The ability to decrease the expression of these enzymes would effectively regulated glycolysis. There are bacterial models which use the expression of anti-sense RNA to do just that. By transfecting cancer cells with anti-sense DNA, the opposing strand of RNA would base-pair with the mRNA of the over-expressed enzyme and prevent translation. The question of how to efficiently transfect the cancer cells still remains a problem. However, the promoter of HKII could be used for anti-sense mRNA from HK, PFK or PK. Until effective targeting systems are developed these ideas will have to wait.
The heterogeneity of cancer precludes one therapy for all cancer. It is a rule for cancer, and subsequently it should be the rule for research. This review has attempted to shed light on the diverse nature of cancer. The vast array of alterations which occur in cells changes not only the glycolytic rate but the rate of synthesis and division. As glycolysis is the one central theme of cellular metabolism, effective control of it by gene therapy or drugs may be the key to the treatment of cancer.
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