Pathways to Cancer

Part 2. The Lives of Cell

by Ian Magrath

The Tarantula nebula, a neighbor of our galaxy, showing a cluster of massive stars (Hodge 31, bottom right), several of which have erupted into supernovae. The material spewed across vast distances of space has compressed the nebula gas into sheets and filaments (top left) and spawned new stars (center). Image from the Hubble telescope; credit: the Hubble Heritage Team; Aura/STScI/NASA.

The ability of lenses to magnify has been known since at least the 5th century B.C.E., as attested to by Aristophanes' reference to a burning glass in his play The Cloud. But it was not until the early 17th century that the simple notion of magnifying an already magnified image was conceived and multiple lenses were combined in a single instrument. Thus were born, almost simultaneously, the telescope and the microscope, which extended human vision to both the very far and the very small, thereby provoking a host of new ideas about the natural world in which we live and the universe beyond. The telescope, in the capable hands of Galileo, allowed confirmation of Copernicus' heliocentric theory of the solar system and eventually, after some four centuries of development, has penetrated the outer reaches of the visible universe, enabling astronomers to see galaxies as they were when the universe was young. The microscope led immediately to the discovery of microorganisms, spermatazoa and blood cells by Leeuwenhoek, and to the confirmation of the "germ” theory of disease. Better instruments enabled Robert Brown, in 1833, to discover cell nuclei in plants, and led, a few years later, to the independent theories of Schleiden and Schwann, who proposed that animals and plants are made of up of cells and their products (e.g., wood, shell and bone). Schleiden and Schwann both proposed that each individual cell had the potential to give rise to an entire organism - a theory which was confirmed in 1997 when Wilmut and colleagues reported that a sheep, Dolly, had been cloned by inserting the nucleus of an adult mammary gland cell into an enucleated ovum. More sophisticated microscopes and their descendants, using sub-atomic particles instead of light, led to the visualization of molecules and atoms and eventually to an ability to create and track (in cloud chambers) whole families of new subatomic particles. Physicists had finally achieved, in a manner of speaking, the alchemists' dream of transforming base metal into gold, an accomplishment aided greatly by the insights of a particularly extraordinary alchemist, Isaac Newton, who showed, perhaps more than any other, that unraveling the secrets of the alma mundi (the soul of the world) was tantamount to revealing those of the alma universalis (the universal soul).

It was also in the 5th century B.C.E. that the Greek philosopher, Democritus, building on the ideas of his teacher, Leucippus, proposed that matter is not infinitely divisible, but made up of elementary particles that he called atomos. Democritis postulated the existence of a "void” or vacuum between the material atoms, and suggested that objects are perceptible through "emanations” (eidola), leading to awareness and thereby, thought. These ideas, although entirely speculative, were embryonic forms of modern scientific concepts of the vacuum of space, electromagnetic radiation (the visible wavelengths of which we call light), and the role of both eye and brain in vision, although we still have little understanding of the physical basis of consciousness. Modern atomic theory was developed in the context of chemical reactions by John Dalton in the early 19th century, but it was another hundred years before Rutherford was able to provide the first scientific evidence that atoms consist of a tiny nucleus surrounded by electrons. While the idea that matter is not infinitely divisible was hotly contested (e.g., by Aristotle), nobody, until Max Planck, at the dawn of the 20th century, ever conceived that energy too, is not infinitely divisible, but rather, is comprised of individual packets of energy, or quanta. This counter-intuitive notion was the key to the conceptual leaps in the understanding of both matter and energy, which, as shown by Einstein's equations and subsequent experimental evidence, are inter-convertible and therefore different forms of the same thing. In the last 100 years, progress in the physical sciences has contributed greatly to the biological sciences by providing new instruments with which to probe and analyze the complex molecular interactions that govern the lives of cells in health and disease. Such instruments, along with novel biochemical techniques, have made possible the essentially final mapping of the human genome (published within the last few months), an achievement comparable in its significance to the complete mapping of the world, but achieved, from start to finish, in a tiny fraction of the time. This momentous step will help speed up the process of understanding how the approximately 25,000 genes revealed by the map (arranged along two intertwined strands of deoxyribonucleic acid, or DNA, some two meters in length and miraculously packaged into the nuclei of cells invisible to the human eye) direct the development of a human being from a fertilized ovum; regulate the interactions of the estimated 1014 cells in the microenvironments (tissues and organs) that comprise the human body; and govern the behavior of people living in the macroenvironments that exist on Earth.

E Unibus Plurum

Taoists believe that the world of ten thousand things we inhabit evolved from an ultimate single reality (the Tao, equivalent to Brahman, in Hindu, or the void in Buddist philosophies) which continues to permeate and energize the entire web of differentiated being we experience today.This perspective is similar to that of modern cosmologists, who continue to seek a unified field theory in which the four fundamental forces of nature and the numerous sub-atomic particles currently recognized, existed originally as a single force immanent in the high energy universe that came into being immediately after the big bang. Neither physicists nor Taoists deny the present existence of a multiplicity of things, but rather recognize their interrelatedness, and their emergence or differentiation from the original unity. This evolutionary process appears to have been both self-realizing and self-organizing and also exquisitely dependent, with respect to the structure of the universe, the chemical characteristics of the elements and the emergence of life on earth, on the values of the fundamental constants of nature, such as the relative masses of protons and electrons and the relative strengths of the four fundamental forces.

The Creation of Elements

There is general agreement among cosmologists that immediately (i.e., prior to 10^-35 seconds) after the big bang, the universe was extremely hot - above 10²⁸ degrees Kelvin! Extremely rapid cooling, due to the massive expansion caused by the big bang and subsequent inflation, led to the differentiation of primordial energy into a plasma of quarks and gluons, which existed for perhaps 10 microseconds after the big bang. After 10 milliseconds, the temperature would have dropped to 100 billion degrees, leading to the emergence of high energy particles, including electrons and baryons (protons and neutrons), the latter comprised of quark triplets bound together by the strong nuclear force mediated by gluons. In the next three minutes or so, further cooling (to about a billion degrees) permitted protons and neutrons to combine to form the nuclei of the lighter elements, but some three hundred thousand years would pass before the temperature dropped sufficiently (to perhaps three thousand degrees) to permit electrons, under the influence of the electromagnetic force carried by photons, to stably associate with nuclei to form neutral atoms.

Galaxies and stars probably began to form a few hundred million years after the big bang, as a consequence of minute differences in the distribution of energy (quantum fluctuations), prior to its condensation into matter. Over time, in regions of sufficient density, the predominant element, hydrogen, began to aggregate under the influence of gravity. In large enough aggregations, destined to become stars, the gravitational force was sufficient to cause hydrogen nuclei (protons) to fuse, forming helium nuclei (with the aid of the weak nuclear force) and releasing electromagnetic energy, including light, in the process. In more massive stars, then and now, more powerful gravitational fields drive the hydrogen cycle more rapidly, converting all of the hydrogen to helium after only a few hundred million years. At very high temperatures, carbon-12 can be formed via fusion of thee helium-4 nuclei and energy produced by a carbon fusion cycle. Elements beyond carbon (i.e., of higher atomic weight) can also be made by successive nuclear fusions, but fusion releases energy only up to the formation of iron-56. Building elements of higher atomic weight requires energy. Thus, as iron accumulates energy production is sharply cut back, gravity is unopposed by the energy pressure, and gravitational collapse occurs, causing electrons to fuse to protons in the star's core, and the formation of a massive nucleus composed almost exclusively of neutrons. If this process is sufficiently rapid the result is a cataclysmic thermonuclear explosion, or supernova, in which the mantle of the star and its contained elements are expelled into space, leaving behind the dense core as a neutron star. Elements of higher atomic weight than iron-56 are probably produced in supernovae when existing elements capture high energy neutrons and convert some into protons via the weak nuclear force. Hydrogen is also recreated in supernovae, via fission of elements made in the course of millions of years, and from this, new stars can form, but the stellar dust is now also rich in carbon, oxygen and nitrogen and may even contain carbohydrates and amino acids - the building blocks of proteins. Matter of this kind, containing the full range of elements, may form solar systems, in which the central region of the aggregated matter forms a star, and outer regions condense into various satellites, including planets. Our own solar system was formed in this way some 5 billion years ago.

Recombination and the Evolution of Complexity

The successive stages of differentiation that have occurred since the big bang permit corresponding increases in complexity, not only through the emergence of new entities, but through their recombination. Thus, each atom is comprised of only three types of subatomic particle (up and down quarks, and electrons), but combinations of protons and neutrons form the atomic nuclei of the 92 naturally occurring elements. These, in turn, combine, according to their chemical properties, (determined by the number of protons in the nucleus, which is identical to the number of surrounding electrons), to create an enormous number of different molecules. Among the elements, none has as broad a spectrum of compounds as carbon, which accounts for approximately 75% of the 7 million known chemical compounds, and although, today, many carbon compounds can be synthesized, the bulk of them are associated with living organisms - hence carbon chemistry is known as organic chemistry. The ability of carbon to combine with itself and produce a variety of rings, chains and branched molecule, accounts for its broad range of compounds and also creates the possibility of storing large quantities of information within the molecules - in DNA, for example. Here, the sequence of four organic bases arranged along its length determines the sequence of amino acids in proteins made according to the DNA blueprint. The latter also contains various "punctuation marks” which signify the beginnings and ends of polypeptide chains, and sequences involved in the regulation of the expression of the gene (i.e., translation of the encoded information into a protein). Proteins are organized into interdigitating molecular pathways, which are ultimately responsible for the wide range of structures and functions that comprise living systems.

The Emergence of Cells

In the case of the planet Earth, as with the universe as a whole, the early conditions were very different from those that exist today. As the planet cooled, a tempestuous climate resulted in the generation of a variety of organic molecules, some of which must have had the ability to self-replicate. Whether carbon compounds present in interstellar dust were important to this process is unknown. Nor is it clear how self-replicating pre-biotic molecules evolved into primitive life forms within a mere half a billion years or so after the formation of the planet. Life, as we know it, first appears in the fossil record in the context of another self-realizing and self-organizing system - the cell. Cells are characterized by a lipid membrane which separates internal from external cellular environments and provides protection for the cell's molecularly coded information (DNA) as well as the protein products made in the cell's synthetic machinery. Some proteins (called receptors) are expressed in the cell membrane where they can detect molecules in the external world and respond by initiating pre-programmed reactions. Other membrane or secreted molecules can influence the behavior of other cells through binding to their receptors. Finally, cells are able to replicate themselves and their contained information.

Cancer develops in a stepwise process through the accumulation of multiple mutations associated with successive increments in "invasiveness” or malignant potential. The earlier in this process that interventions are made, the more successful they are likely to be.

The earliest microfossils, dating to almost 3.5 billion years ago, already demonstrate the existence of single-celled organisms called prokaryotes, i.e., consisting of a simple cell type in which the single loop of DNA is not enclosed within a cell nucleus. Many species of Archaea, one of the two prokaryotic domains, are able to survive in extreme conditions, e.g., boiling water and acid environments. Some use hydrogen gas or sulfur as their energy source. They and their predecessors were capable of flourishing in the harsh environments of the kind which must have abounded on the young planet at a time when its atmosphere consisted of methane and ammonia, but no oxygen. The other group of prokaryotes, Eubacteria, also evolved into species able to survive in a broad range of environments by developing relevant metabolic processes. The cyanobacteria, for example, are able to use sunlight as a source of energy, trapping it in pigments such as rhodopsin and chlorophyll, and using it to convert carbon dioxide and water into glucose, with release of oxygen. In the course of a further half billion years such photosynthetic bacteria began to create the atomospheric oxygen essential to the evolution of plants and animals. But oxygen is highly reactive and capable of damaging DNA – a problem overcome when some bacteria began to use oxygen as an energy source. These aerobic bacteria collaborated with other "single celled" organisms (probably Archeans), in some cases living within cells larger than themselves (so called endosymbiosis) in a mutually beneficial relationship. Photosynthetic bacteria also shared their metabolic skills through endosymbiosis, and the permanent fusion of prokaryotes is the probable origin of both mitochondria and chloroplasts, responsible for aerobic respiration and photosynthesis respectively, in the more complex eukaryotic cells which evolved some two billion years ago. The first such cells - algae and protozoans - were also "single celled" organisms, but much larger than prokaryotes. Retaining the most basic genes (e.g., for protein production), which had evolved in prokaryotes, eukaryotic cells expanded the genetic repertoire, and enclosed the now much longer DNA strands in a second, protective nuclear membrane, coiling them up into multiple chromosomes which allow the DNA to be packaged in the smallest possible volume while ensuring efficient replication and, in multicellular organisms, differential gene expression. Eukaryotic cells also contain a number of cell organelles, e.g., membrane structures involved in manufacturing proteins via RNA intermediates, and a cytoskeleton that gives shape to cells and allows movement.

Multicellular plants and animals doubtless evolved (perhaps a billion years ago) from variable aggregations of identical single cells (colonies) which permanently fused, allowing cells to become specialized, via differentiation, creating tissues and organs, and in the process, organisms able to function as individuals. Cell specialization inevitably resulted in restrictions on the right of abode of cells in each tissue (microenvironment), and also meant that replication of the entire organism had to be concentrated in special cells (germ cells) that retained totipotentiality. Thereafter, evolution would require the death of individual organisms. Moreover, horizontal exchange of genetic material, possible in prokaryotic cells, even of different species, is essentially prohibited in eukaryotic cells (except by viruses). In multicellular organisms, however, cooperative genetic exchange can occur within a single species via the process of sexual reproduction (requiring germ cell fusion) - and a new form of cooperation is possible; the association of individuals in communities, which may include specialized members.

Generation of Biodiversity

Ultimately, the potential for variability in cell types resides in the informational content of DNA. While it may be surprising that a human genome consists of only some 25,000 genes, the degree of possible complexity is enormously increased by the fact that various modules or domains of proteins are able to assume three-dimensional shapes that enable them to complex with and often modify other proteins (enzymatic activity), such that the proteome, or total protein expression pattern, is much larger than would be predicted by the size of the genome. This is further amplified by the differential expression of genes in different cellular contexts (including during the process of differentiation), resulting in enormous numbers of expression patterns in different cells, and in the same cell in different circumstances. The evolutionary process has been driven by modification of the genetic information, both by small progressive changes in sequence and by duplication of entire genes and re-use of modular elements in new contexts. Clearly, the permissible degree of genetic change must be finely tuned in order that new species emerge in the context of different or changing environments, while ensuring that existing species have stability within biological time-frames. This has led to the evolution of multiple mechanisms to protect the integrity of the information encoded in DNA, including protection against chemical damage as well as repair mechanisms for damaged DNA, or errors that occur during DNA replication. As a last resort, cells with irreparable damage are eliminated through the activation of molecular pathways that lead to cell death (hence the term programmed cell death, or apoptosis). Apoptosis is also essential for "fine tuning" structural relationships during embryological development and cell differentiation that occurs throughout life. In some circumstances, it can be activated via receptors in the cell membrane. Interestingly, some of the apoptotic pathways involve mitochondria, derived from the original endosymbiotic prokaryotes.

Genetic Pathways to Cancer Pathway Involved Consequence of Abnormality Examples of Genes Involved DNA protection Increased rate of mutation CYPp450, GSTM1 DNA repair Persistence of mutations MMR, NER, BER Genome stability Gross chromosomal alterations BRCA1, BLM, ATM Apoptosis in presence of damaged DNA Survival of cells with gross DNA damage PARP, p53, p73, MDM Cell cycle (DNA replication) Inappropriate cell division Rb, p16, CDK4, cyclin D1 Differentiation Differentiation block Notch, STATS Apoptosis and differentiation Prolonged lifespan BCL2, BAX, APAF1 Receptor-induced apoptosis Failure to activate apoptotic pathways when stimulated to do so FAS, TRAIL, TNF Growth factor Proliferation SIS, KIT, FGF Receptor for growth factor Proliferation with or without a signal HER2/neu, FMS Signal Transduction Proliferation in absence of signal PI3K, APC, SMADS Master control gene Altered regulation of many genes MYC, NFKB Production of enzymes able to cleave basement membranes Inappropriate ability to enter and leave blood vessels, i.e., to migrate HGF, MET, MMPs Production of factors that stimulate endothelial cells Formation of new blood vessels able to supply cancer cells HIF1,VGEF, PDGF Cell senescence genes Allows cell to survive longer Telomerase Table 1.  Multiple pathways must be affected to cause cancer. Some genes (referred to as tumor suppressor genes) are inactivated e.g., DNA repair genes. Others (referred to as oncogenes) – e.g., those involved in cell division, are over- or inappropriately expressed. Sometimes, pathways active during embryogenesis, e.g., those related to cell migration, are reactivated. Genetic changes in cancer cells can also provide tumor markers or tumorspecific (or relatively specific) therapeutic targets, which are beginning to be exploited.

Cancer Cells

Invasive cancer is a disease process that, to a degree, may be seen as a negative consequence of the evolutionary need for information resident in DNA to undergo change and thus to allow for Darwinian ecological adaptation and the evolution of species. In multicellular organisms, genetic changes (mutations) must occur in germ cells if they are to influence the characteristics of the offspring. In cancer, however, the genetic changes occur in somatic cells. Not all such changes lead to cancer. Some may be inconsequential, others may lead to the death of the cell, e.g, via apoptosis. The most dangerous are those which impair the efficiency of pathways that normally protect the integrity of DNA (including apoptotic pathways) such that genetic changes are more likely to occur, or persist. Some of the genetic changes must also lead to relevant alterations in cell behavior, including inappropriate cell proliferation and prolonged lifespan. Altered proliferation can be associated with mutations in growth factors, their receptors, the molecular pathways that transduce the signals from receptors, or the cell cycle genes and their inhibitors, that regulate the actual process of cell division. Altered proliferation and lifespan are frequently coupled to a failure to differentiate with retention of self-renewal properties, leading to abnormal accumulation of the genetically modified cells and their progeny. Cancer cells may also develop the ability to cross barriers that obstruct normal cells, e.g., through the inappropriate expression of particular enzymes, and to survive in microenvironments hostile to their normal counterpart cells; the ability to enter blood vessels, travel to other sites and proliferate there are the hallmarks of metastasis. In order to grow above a minimal size, however, and hence to be harmful, most cancer cells need to coerce nearby cells to cooperate with them in providing their essential needs, and in particular to create new blood vessels (angiogenesis) that can bring them essential nutrients and oxygen.

Figure 1. Chromosomes from a cell line derived from prostate cancer, demonstrated by spectral karyotyping, a technique in which chromosome-specific probes are used and the data analyzed by computer, which assigns each chromosome a single color. SKY readily detects the extensive exchanges (translocations) among chromosomes that have occurred as a result of genetic mutations causing genomic instability. Reproduced by courtesy of Meena Augustus, Ph.D., Avalon Pharmaceuticals Inc., Germantown, MD, USA.

Modifications of so many pathways (Table 1) do not occur all at once. Cancer develops in a stepwise process through the accumulation of multiple mutations. Some cells are partially transformed and may, in certain circumstances, be detected as abnormal cellular accumulations, e.g., polyps, or so-called carcinoma-in-situ, in their tissue of origin. Not all of these pre-malignant lesions develop the necessary genetic changes to become invasive cancers within the lifetime of the individual. Other cancers may be detected prior to deactivation of molecular pathways that would prevent them from spreading beyond their tissue or organ of origin (so called "early stage” cancers). It is clear why early detection is critically important. More advanced cancers are much more difficult to treat both because they are more widespread, and also because the multiple genetic changes required to permit them to spread, after which local therapy is ineffective, also make them more resistant to chemotherapy. The malignant behavior of cancer cells arises from their ability to grossly disrupt the organization of their tissue or organ of origin, and/or to illegitimately invade other tissues with impunity where they compete with the rightful occupants. Negative effects may be due to pure physical encroachment (sometimes leading to compression of vital structures) as cancer cells continue to accumulate, or to the indirect effects of the biochemical products of cancer cells on normal cells - either at a local level, or at the level of the whole patient.

Genetic changes leading to cancer may involve modifications in the base sequence of specific genes or in their regulatory elements, alterations in gene expression caused by changes in chromosomal proteins or chemical changes (e.g., methylation) in DNA, by amplification or deletion of regions of DNA, or by gross structural changes in chromosomes, such as translocations (Figure 1). Ultimately, all such changes result in an intrinsically altered pattern of gene expression (Figure 2) and/or protein function (some genes being over expressed or inappropriately expressed, others under expressed, or inactivated), as well as the failure to create appropriate expression patterns in response to external stimuli, particularly those associated with cell proliferation, differentiation and survival. Gross structural changes in DNA may lead to the creation of a new fusion gene through the interchange of pieces of chromosomes in which the chromosomal breakpoints are within the involved gene sequences. Sometimes, the equivalent of a genetic change may be provided by a viral gene. Rarely, mutation in the germline (usually in genes involved in maintaining the integrity of the genome) may predispose family members, from then on, to cancer.

Figure 2. Profiling techniques enable the expression of large numbers of genes to be examined simultaneously. Here, each row represents a different gene, and each column, messenger RNA (corresponding to the expressed genes) extracted from a sample of a specific normal or tumor cell type (e.g., GC = germinal center cells, BL = Burkitt's lymphoma). The degree of expression of each gene is represented by a color (red, highest, green, lowest). Reproduced by courtesy of U. Klein and R. Dalla-Favera, Columbia University, New York.

A Work in Progres?

Life evolved only after billions of years of preparation in a vast universe consisting of hundreds of billions of galaxies each containing hundreds of billions of stars. Here, the chemical elements were built, step by step, permitting the formation of at least one planet in which conditions were appropriate for the creation of the complex molecules that regulate the lives of cells. Billions of years of subsequent evolution, involving an enormous degree of cooperation, led eventually to brain cells that were able to unravel the remarkable history of the universe and to learn that cancer is a process in which the information stored in cells is corrupted, such that the normal regulatory systems have gone awry and cells function selfishly rather than to the benefit of the individual. Perhaps the accumulation and use of human knowledge will prove to be a continuation of the process of self-realization that began some 15 billion years ago, leading to a reduction in human inequities, environmental damage and other ills of the world (including diseases, such as cancer). Alternatively, through too great an emphasis on competition and conquest rather than cooperation and compassion (a kind of cancer community?) progress in understanding may simply herald a cataclysmic end - to human history, at least. One or other of these alternatives is likely to prevail before the end of the present century.