Historical Perspectives

The development of knowledge about the biochemistry and cell biology of cancer comes from a number of disciplines. Some of this knowledge has come from research initiated a century or more ago. There has been a flow of information about genetics into a knowledge base about cancer, starting with Gregor Mendel and the discovery of the principle of inherited traits and leading through Theodor Boveri's work on the chromosomal mode of heredity and chromosomal damage in malignant cells1 to Avery's discovery of DNA as the hereditary principle,2 Watson and Crick's determination of the structure of DNA,3 the human genome project, DNA microarrays, and proteomics. Not only has this information provided a clearer picture of the carcinogenic process, it has also provided better diagnostic approaches and new therapeutic targets for anticancer therapies.

Once cell culture techniques were developed4 it became possible to test which genes are involved in malignant transformation and progression. This field of research led to the discovery of oncogenes5 and tumor suppressor genes.6 Hereditary studies led to the two-hit theory7 and the concept of the hereditary nature of some cancers.8 Chromosomal staining techniques enabled Nowell and Hungerford9 and Rowley10 to identify chromosomal translocation as a tumor initiating event.

Studies in yeast produced the concept of cell cycle checkpoints,11 and investigations with

C. elegans found genes involved in apoptosis.12 The cell cycle began to be studied in great detail in lower organisms, and organisms such as clams, yeast, and fruit flies have contributed greatly to our understanding of the cell cycle events.13

The findings that simple molecules like cyclic AMP could direct a whole panoply of cellular functions14 led to the discovery of signal trans-duction pathways, which are now becoming favored molecular targets for anticancer drug discovery.

Much of what we originally knew about the biochemical differences between normal and malignant cells, however, was discovered in their patterns of enzymatic activity. In the 1920s, Warburg studied glycolysis in a wide variety of human and animal tumors and found that there was a general trend toward an increased rate of glycolysis in tumor cells.15 He noted that when normal tissue slices were incubated in a nutrient medium containing glucose, but without oxygen, there was a high rate of lactic acid production (anaerobic glycolysis); however, if they were incubated with oxygen, lactic acid production virtually stopped. The rate of lactic acid production was higher in tumor tissue slices in the absence of oxygen than in normal tissues, and the presence of oxygen slowed, but did not eliminate, lactic acid formation in the tumor slices. Warburg concluded that cancer cells have an irreversible injury to their respiratory mechanism, which increases the rate of lactic acid production even in the presence of oxygen (aerobic glyco-lysis). He regarded the persistence of this type of glycolysis as the crucial biochemical lesion in neoplastic transformation. This old idea still has some credence in that there are hypoxic areas in the core of tumors, where anaerobic metabolism predominates. This has clinical implications because hypoxic cells do not respond as well to certain anticancer drugs or radiation therapy. The ability of lactate and pyruvate, end points of glycolysis, to enhance tumor progression appears to be mediated by the activation of hypoxia in-ducible factor-1 (HIF-1).16 In addition to increased activity of enzymes of the glycolytic pathway, such as hexokinase, phosphofructokinase, and pyruvate kinase in cancer cells, hypoxia is also a common feature of many human solid cancers. These effects have been linked to tumorprogression, metastasis, and multidrug resistance.17 Interestingly, oncogenes such as ras, src, and myc enhance aerobic glycolysis by increasing the expression of glucose transporters and glycolytic enzymes (reviewed in Reference 16).

Cancer cells react to hypoxic conditions by up-regulating expression of HIF-1, which is a transcription factor that in turn up-regulates expression of genes involved in glycolysis, glucose transport (GLUT-1), angiogenesis (VEGF), cell survival, and erythropoiesis. HIF-1 expression has been observed in cancers of the brain, breast, colon, lung, ovary, and prostate and their metastases but not in the corresponding normal tissues. Its expression in tumors correlates with poor prognosis.

Interest in tumor metabolism has been stimulated once again by modern techniques such as position emission tomography (PET), sensitive mass spectrometry (MS), and high-resolution nuclear magnetic resonance spectroscopy (NMR). PET uses fluorine-18 labeled fluorodeoxyglucose (FdG) to detect tissue regions of high glucose uptake, which is indicative of up-regulated glycolysis and increased metabolic rate. FdG PET imaging has shown that most primary and metastatic human cancers have increased glucose uptake.17 This finding is indicative of a ''glycolytic switch'' in cancer cells and may be a precursor of tumor angiogenesis and metastasis.17

NMR and MS can now be used to measure mestatic profiles of cancer cells and the metabolic phenotype of tissues and organs. This so-called science of ''metabolomics'' can provide metabolic biomarkers of tumors such as pro duction of the end products of glycolysis, lipid levels indicative of cell membrane turnover, and alterations in amino acids and nucleotide levels.18

Since mitochondria contain the enzymatic cascades for oxidative metabolism, it has been suggested that damage to mitochondria may be involved in the disruptions of oxidative metabolism seen in malignant tumors. Mutations of mitochondrial DNA (mtDNA) has been observed in a variety of human cancers, including bladder, head and neck, lung,18 and ovarian19 cancers. Interestingly, in the bladder cancers, the mutation hot spots were primarily in a nicotinamide adenine dinucleotide dehydrogenase subunit, a key component of the electron transfer machinery. This suggests a mechanism for the alterations in oxidative metabolism seen in malignant cells. Because mitochondrial DNA is exposed to high levels of reactive oxygen species generated during oxidative phosphorylation, it is not surprising that mtDNA is highly susceptible to mutational events. The mutational rate of mtDNA has been estimated to be 10 times higher than that of nuclear DNA.19 Mitochondria also play a key role in apoptosis (see section on apoptosis below), and alterations in those mitochondria-mediated events are seen in cancer cells.

In the early 1950s, Greenstein formulated the ''convergence hypothesis" of cancer, which states thattheenzymaticactivityofmalignantneoplasms tends to converge to a common pattern.20 Although he recognized some exceptions to this rule, he considered the generalization, based mostly on repeatedly transplanted tumor models, to be valid. It is now more fully appreciated that even though cancer cells do have some commonly increased metabolic pathways, such as those involved in nucleic acid synthesis, there is tremendous biochemical heterogeneity among malignant neoplasms, and that there are many fairly well-differentiated cancers that do not have the common enzymatic alterations he suggested. Thus, cancers do not have a universally uniform malignant phenotype as exemplified by their enzyme patterns.

On the basis of work of about 60 years ago, which evolved from studies on the production of hepatic cancer by feeding aminoazo dyes, the Millers advanced the ''deletion hypothesis'' of cancer.21 This hypothesis was based on the ob servation that a carcinogenic aminoazo dye cova-lently bound liver proteins in animals undergoing carcinogenesis, whereas little or no dye binding occurred with the protein of tumors induced by the dye. They suggested that carcinogenesis resulted from ''a permanent alteration or loss of protein essential for the control of growth.''

About 10 years later, Potter suggested that the proteins lost during carcinogenesis may be involved in feedback control of enzyme systems required for cell division,22 and he proposed the ''feedbackdeletion hypothesis.''23 In this hypothesis, Potter postulated that ''repressors'' crucial to the regulation of genes involved in cell proliferation are lost or inactivated by the action of oncogenic agents on the cell, either by interacting with DNA to block repressor gene transcription or by reacting directly with repressor proteins and inactivating them. This prediction anticipated the discovery of tumor suppressor proteins, such as p53 and RB, by about 25 years.

Biochemical studies of cancer were also aided by the so-called minimal-deviation hepatomas developed by Morris and colleagues.24 These tumors were originally induced in rats by feeding them the carcinogens fluorenylphthalamic acid, fluorenylacetamide compounds, or trime-thylaniline. These hepatocellular carcinomas are transplantable in an inbred host strain of rats and have a variety of growth rates and degrees of differentiation. They range from slowly growing, well-differentiated, karyotypically normal cells to rapidly growing, poorly differentiated, poly-ploid cells. All these tumors are malignant and eventually kill the host. The term ''minimal deviation" was coined by Potter23 to convey the idea that some of these neoplasms differ only slightly from normal hepatic parenchymal cells. The hypothesis was that if the biochemical lesions present in the most minimally deviated neoplasm could be identified, the crucial changes defining the malignant phenotype could be determined. As Weinhouse25 indicated, studies of these tumors greatly advanced our knowledge of the biochemical characteristics of the malignant phenotype, and they have ruled out many secondary or nonspecific changes that relate more to tissue growth rate than to malignancy.

The extensive biochemical analyses of the Morris minimal-deviation hepatomas led Weber to formulate the ''molecular correlation concept''

of cancer, which states that ''the biochemical strategy of the genome in neoplasia could be identified by elucidation of the pattern of gene expression as revealed in the activity, concentration, and isozyme aspects of key enzymes and their linking with neoplastic transformation and progression.''26 Weber proposed three general types of biochemical alterations associated with malignancy: (1) transformation-linked alterations that correlate with the events of malignant transformation and that are probably altered in the same direction in all malignant cells; (2) progression-linked alterations that correlate with tumor growth rate, invasiveness, and metastatic protential; and (3) coincidental alterations that are secondary events and do not correlate strictly with transformation or progression. Weber maintained that key enzymes, that is enzymes involved in the regulation ofrate and direction of flux of competing synthetic and catabolic pathways, would be the enzymes most likely to be altered in the malignant process. In contrast, ''non-key'' enzymes, that is, enzymes that are not rate limiting and do not regulate reversible equilibrium reactions, would be oflesser importance. As one would expect, a number of enzyme activities that Weber and others have found to be altered in malignant cells are those involved in nucleic acid synthesis and catabolism. In general, the key enzymes in the de novo and salvage pathways of purine and pyrimidine biosynthesis are increased and the opposing catabolic enzymes are decreased during malignant transformation and tumor progression. Weber noted that the degree of neoplasia was related to the concentrations of certain regulators of key metabolic pathways. The question of why anaplastic, rapidly growing tumors tend to be biochemically alike, whereas more well-differentiated tumors display a vast array of phenotypic characteristics, was approached by Knox.27 He thought that the vast bulk of biochemical components in tumor tissues are ''normal,'' in the sense that they are produced by certain specialized adult normal cells or by normal cells at some stage of their differentiation. In cancer cells, it is the combination and proporations of these normal components that are abnormal. The biochemical diversity of cancer cells, then, would depend on the cell of origin of the neoplasm and its degree of neoplasticity.22 All too frequently, even now, in the histopathologic or biochemical characterization of cancer, a biochemical component that is present or absent or increased or decreased is not considered in relation to the particular cell of origin of a tumor, its differentiation state, or its degree of neoplasticity.

Taken together, the data on enzyme patterns of cancer cells indicate that undifferentiated, highly malignant cells tend to resemble one another and fetal tissues more than their adult normal counterpart cells, whereas well-differentiated tumors tend to resemble their cell of origin more than other tumors. Of course, between these two extremes several levels of neoplastic gradation occur, leading to the vast biochemical heterogeneity of tumors. This heterogeneity also exists for tumors of the same tissue type arising in different patients or even in the same patient at different stages of the disease.

The fact that more undifferentiated tumors tend to converge to a more fetal-like state is evidenced by a frequently observed production of oncodevelopmental gene products. A number of cancer cell characteristics, such as invasiveness and "metastasis," are also seen in embryonic tissue. For example, the developing trophoblast invades the uterine wall during the implantation step of embryonic development. During organogenesis, embryonic cells dissociate themselves from the surrounding cells and migrate to new locations, a process not unlike metastasis.

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