Molecular mechanisms involved in tumorigenesis and their surgical implications

MOLECULAR BIOLOGY

Molecular Mechanisms Involved in Tumorigenesis and Their Surgical Implications

Timothy Weiner, MD, William G. &rice, MD, Chapel Hill, North Carolina

Our understanding of normal and malignant cell growth has rapidly advanced in the last two decades as new technologies have accelerated the ability to resolve the underlying molecular mechanisms. The speed and complexity of this advance, as well as the inadequate and confused nomenclature of molecular oncology, has made this field difficult to follow. Nevertheless, many future therapies will be targeted to a genomic level and clinicians will he called upon to com- municate with basic scientists in implementing them. To facilitate the surgeon’s part in this dia- logue, we present a broad review of the molecular genetics of cancer. The selected bibliography contains many review articles by leading re- searchers in this field. Particular emphasis has been placed on the themes that are emerging, such as oncogenic mechanisms, tumor suppressor genes, signal transduction, the multistage concept of carcinogenesis, and molecular diag- nostice and therapies.

I n 1911, Rous demonstrated that a cell-free extract iso- lated by filtering chicken sarcomas could reproduce the

tumor when introduced into normal birds.’ Subsequent work identified the transforming agent as a retrovirus that became known as the Rous sarcoma virus (RSV); other cancer-inducing viruses were thereafter identified in a variety of animals. When investigators initially failed to iden- tify human tumor-causing viruses, retroviruses became laboratory curiosities while attention was directed at hereditary and carcinogenic causes of cancer. With the advent of molecular bicrtechnology in the 197Os, Bishop returned to RSV and were able to isolate a discrete sequence from the viral genome that could transform normal fibroblasts into malignant cells.* They designated this the “SK” gene, and following a hypothesis proposed by Huebner and Tadaro, believed it to be a covert viral gene that became activated in host cells to cause tumor formation.3 To, their surprise, they observed src-like sequences in normal chicken cells as well as in the normal cells of several other vertebrate species. &c-like sequences were also seen to occur in the human genome. This led to the speculation that a normal STC gene (c-(for cellular+) was present in normal cells as a so-called “proto-oncogene.” At some point in evolutionary history, it had been acquired by RSV as a genetic hitchhiker and altered to a tumorigenic form

From the Department of Surgery and The Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina, 27599.

Requests for reprints should be addressed to William G. Cance, MD, 3010 Old Clinic Building, CB#7210, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.

Manuscript received June 1, 1993, and accepted in revised form October 1, 1993.

(v-(for viral)src), and upon reintroduction into host cells it acted as a cancer-causing “oncogene.” Although the viral form of the gene, V-SK, appeared to have relevance only to the formation of tumors in chickens, altered src gene products have been seen in a variety of animal and human cancers. The theory behind this observation, moreover, has had a revolutionary impact on cancer research. The idea that specific alterations in a limited set of discrete DNA sequences (ie, causing the activation of otherwise normal proto-oncogenes to oncogenes) are conserved from cell to progeny cell, represented the point of convergence for research in oncogenic viruses, environmental carcinogens, and hereditary cancers. While the search for other retrovi- ml oncogenes has discovered a further 20 or so sequences capable of tumor formation in animals, it is their concep- tual impact on our understanding of the molecular genetics of cancer that has been profound. By viral activation4 as well as a variety of other mechanisms, the normal cellular genome can be changed in ways that allow for un- controlled and uncoordinated cell growm5-*

THE GENETIC THEORY OF CANCER It is now believed that the different agents of tumor for-

mation (chemical, viral, radiation, etc.) cause genetic alterations that affect a limited number of critical control points which are intimately involved in the inter- and intracellular signal pathways of cell growth and differentiation.9+10 Under normal conditions these pathways process signals relating to diverse, but crucial, functions such as cell division, cell differentiation, cell senescence, and cell adhesion and migration (Figure 1). When this communi- cation system is disrupted at different and multiple levels, a cancer cell can separate from its normal milieu and be- gin to achieve independence from its host organism. In other words, it is not that silent “cancer genes” somehow become activated, but rather that normally active genes which subserve important cell functions become function- ally altered in ways that derange cellular growth. This would explain why only a limited number of oncogenes have been discovered and why the same oncogenes occur in such a diversity of cancers. These oncogenic changes are thereafter carried in a genetically stable way through cell divisions, so that at some point in its evolution a tumor becomes a monoclonal cell population which has successfully evaded normal growth control. Subsequent se- lection pressures would then select various cells with more successful malignant and metastatic characteristics so that eventually the tumor would appear heterogeneous in its phenotypic make-up. These additional genetic changes, such as the ability to evade normal host immune surveillance, would allow for the successful establishment of tumor progenitor cells at distant sites.

Accumulated evidence strongly suggests that direct growth stimulation plays a crucial role in the development

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SIGNAL TRANSDUCTION PATHWAY (via protein phosphorylalion)

ALTERATION IN CELLULAR STRUCTURE OR FUNCTION

and progression of cancer but, more recently, attention has focused on elements that restrain growth.” These elements, commonly known as “anti-0ncogenes” or “tumor suppressor genes,” are a group of genes and gene products whose loss or inactivation releases a cell from the constraints that normally hold growth in check. As in the case of Rb’* and ~53,‘~ they may be gene products that control progression through the cell cycle. Or like the “deleted in colon carcinoma” (DCC) gene, I4 they may be involved in cellular adhesion and through their loss allow cells to supersede the contact inhibition that typifies normal tissues. In other words, derangements in both stimulation and restraints are involved in altering the careful counterbalance of normal cell growth. A simple analogy to this concept would be of an automobile out of control; this could be a result of either a stuck accelerator or brakes that do not work.

A MODEL OF CARCINOGENESIS Cancers appear to arise as a result of multiple changes in

the cellular genome. In the case of pediatric retinoblastoma, a tumor develops as a result of inactivation of both copies (alleles) of a retinoblastoma-susceptibility gene, called Rb.‘* In inherited retinoblastoma, a parental chromosome already lacking a copy of Rb has been passed to the child and all that remains is for some event to “hit” the other Rb copy. When this occurs, tumor formation begins, and these tumors are seen earlier in life, bilaterally, and with a familial association. This is in contrast to the sporadic form of the disease that occurs unilaterally, later in childhood, and without a clear family history. The natural history of this form suggests that the child inherited two normal Rb copies and that two subsequent mutations were necessary to cause the tumor. The idea that tumors that occur very early in life, such as retinoblastoma, require fewer genetic events after birth than tumors that occur later in life was

MECHANISMS IN TI’~OKICFYF,PlS

Figure 1. Schematic representation of inter- and intracellular signaling events that result in cellular changes. In this figure, a growth factor achvates its cog- nate receptor and generates an amplii fied signal within the cell. Thereafter a variety of effects, including ion channel changes, ion fluxes, and signal transduction through secondary messenger proteins can occur. These effects either directly alter the cell or can stirn- ulate the transcription of genes whose protein products cause alterations. Disruption of these pathways is be lieved to promote the pathogenesis and progression of cancer.

originally put forth by Knudson.‘5 His theory offered a statistical analysis of the age of onset and number of tumors per individual and predicted that pediatric cancers would require fewer genetic hits to achieve a malignant form than would adult cancers (Figure 2). Recent work on colorectal cancer supports this idea and has suggested that multiple molecular events are necessary to convert colonic mucosa to an invasive carcinoma, and that certain of these events correspond to pathological stages of the disease. This model of accumulating hits has been worked out in detail by Fearon and Vogelstein’s group, who exploited the avail- ability of colonoscopic samples to catalog the molecular changes occurring as mucosa progressed to polyps and on to carcinomas.t6 Their model supports an earlier theory of carcinogenesis in which multiple chemical carcinogens, some classified as initiators and some as promoters, were necessary to induce tumor formation in animalsI It also paralleled in vitro studies, in which it was necessary to introduce at least two oncogenes into a normal cell in culture before it manifested a transformed phenotype.18 These ob- servations have confirmed that cancer is a multistage process that requires an accumulation of changes in a variety of cell functions. Until they are perturbed, these functions are vital to the normal behavior of the cell.

ONCOGENIC ALTERATIONS At this point it is important to reiterate that when talking

about changes in genes, we are really referring to their functional effect on the proteins the genes code for. As dis- cussed, these proteins participate in a variety of essential cell functions. They are receptors on the cell surface (eg, the epidermal growth factor receptor). as well as the down- stream molecules (eg, the src proto-oncogene product) that transduce signals from these receptors toward the nu- cleus.i9 They are proteins that bind to DNA (eg, the myc

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GENETIC “HITS”

SECOND Rb ALLELE INACTIVATED

RETINOBLASTOMA

HYPERPLASTIC MUCOSA

proto-oncogene product) in response to these signals and effect the transcription of appropriate genes. Finally, they are the products of these activated genes that can alter the structure and function of the cell. Viewed in this way, it becomes more apparent how an inappropriately activated receptor for example,20 or an altered intracellular signal molecule,21 or a DNA transcription factor2* can supersede the cell’s normal behavior and set the stage for its transformation to a malignant phenotype. As these changes ac- cumulate within a cell, either by the activation of genes that enhance growth or the inactivation of genes that sup- press growth, the cell moves toward a state independent of its normal context. This again describes a multistep mechanism of cancer that begins to tie together many diverse areas of cancer research and has become a central theme in understanding cancer.23

As was evident before these basic principles were theo- rized, cancer develops in response to many different stimuli including viruses, chemical carcinogens, and ionizing radiation. These stimuli, as well as random errors introduced in normal DNA replication and repair, cause alterations in the genome. Many of these changes are silent, in that they do not impact on the cells routine functioning. If, however, they occur in a limited set of genes whose products have important regulatory functions, that cell, as well as its progeny, are a step closer to becoming tumorigenic. The specific genetic changes that occur can be of several types (Figure 3). As mentioned, they may be mutations or deletions that disrupt the normal activity of the protein product. Or they may result in the overexpression of an otherwise normal protein such that its function is inappropriately amplified. 24 Chromosomal rearrangements may occur that delete critical control genes or place genes of low expression under the control of gene regulatory se-

Figure 2. Models of multistage carcinogenesis in which a sequence of ge netic changes (“hits”) lead to tumor formation and increasing malignant potential. Cancers that occur later in life are believed to require a greater number of such changes.

quences that cause high expression.25 Regardless of the stimuli that introduce the change, or even the type of change itself, the common result is an incremental loss in the tightly regulated circuits within and between cells that are the hallmark of normal tissues.

MECHANISMS OF TRANSFORMATION Beyond gene mutation and amplification or chromoso-

mal rearrangement, how do the multistage changes that have been postulated actually affect the cell? In other words, from a mechanistic standpoint, what is occurring to and within a cell to cause it to become malignant? Although a comprehensive answer to this is as yet unclear, patterns are emerging. These represent a potpourri of the- oretical mechanisms, some or all of which may be oper- ating in concert or at varying times to yield a cell capable of enhanced growth, de-differentiation, more rapid division, invasion, generation of a blood supply, avoidance of the immune system, intravascular spread, and the successful implantation in distant tissues (Figure 4). For con- ceptual purposes, the following mechanisms can be broadly grouped into those which initiate cellular transformation and those which promote the transformed cell to become more malignant. Regardless of how they are classified, these mechanisms probably act in a synergistic and overlapping fashion and the final malignant clone is that cell that has achieved the ideal dynamic balance of these various mechanisms.

Cell cycle enhancement: The general idea behind this mechanism of carcinogenesis is that genetic changes have occurred that allow a quiescent cell to regain its ability to divide or allow an actively dividing cell to cycle more rapidly.26 In this scenario, a differentiated, nondividing cell reenters the cell cycle, perhaps by changes in growth-


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inhibitory tumor suppressor genes such as Rb or p53.“*** Equally possible are changes in normally quiet signal pathways that were once used for appropriate cell growth and division and which become reactivated. The result is cell division out of context and without normal controls, or at a rate that prevents repair of DNA replication errors.

SignaI transduction and transcription: The multistep signal pathways that relay messages into the interior of a cell and that initiate the transcription of genes are very likely sites of disruption that induce abnormal growth. Dysfunctional receptors or second-messenger proteins can inappropriately trigger or amplify signals that result in genes being transcribed out of their normal context. lg The alteration of DNA transcription factors themselves can also cause the chronic and inappropriate gene transcription that results in abnormal structural or functional changes in cells?2

The autocrine/paracrine mechanism: This model is es- sentially one of growth enhancement in which a cell or cell population gains the ability to co-express growth factors and their receptors in ways that give them growth-selective advantages over normal cell~.~~~ This mechanism has been observed in tumors that co-express the plateletderived growth factor (PDGF) and its receptor, and may represent a means whereby the tumor cells enhance their own growth by self-stimulation with PDGF.3O

Angiogenesis: At a critical size, tumors become depen- dent on the production of new blood vessels to feed their further growth. It appears that several families of growth factors, such as the fibroblast growth factors and the

Figure 3. General scheme of genetic changes that can transform cells. In 0 and (B1 mutations, such as point mu tations or deletions caused by viruses, radiation, or chemicals, can alter the DNA coding for important regulatory proteins. (C) Chromosomal transloca tions, such as those seen in Burktis lymphoma, can occur and place genes like the mvc transcriotion factor adia cent to highly active’gene promoters like the immunogk)bulin promoter. tD1 Amplification of DNA regions, such as the neu gene in breast cancer cells, can result in the overproduction of their protein products. (El Loss of both aC leles of a tumor suppressor gene such as Rb or p53 can deregulate cell growth.

PDGFs, are induced by malignant cells to stimulate angiogenesis. 31 Inhibition of such angiogenic factors is a promising area of therapy.

Invasion and metastasis: The processes that underlie the ability for a cell or cell population to successfully in- vade through the extracellular matrix, avoid destruction by the immune system, survive in a vascular compartment, sequester in a foreign tissue, and then grow there are clearly complex and certainly involve a multitude of genetic and phenotypic changes. Numerous genes concerned with cy- toskeletal construction, extracellular adhesion, proteolytic invasion, cell motility, immune cloaking, and so forth must be altered or activated. 32 Although exceedingly compli- cated, the phenomenon of metastasis may offer several loopholes that could be exploited as therapies. Such treatments would have an enormous impact on the successful surgical treatment of cancer before it becomes widely disseminated.

IMPLICATIONS FOR THE SURGEON To the research scientist, cancer provides a set of mu-

tated reagents with which they can understand the various components involved in normal cell growth. To the clini- cian, science offers a scheme for understanding the disease state and potentially highlighting points at which it can be interrupted. Therefore, the surgeon’s interest in understanding the advances in molecular oncology are es- sentially twofold: does it offer prognostic markers to aid in clinical decision-making, and does it offer adjuvant therapies that will improve patient survival?

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STIMULATION

As with biochemical or pathological markers, the search for reliable molecular markers has been limited by the heterogeneity of cancer as a disease and by the logistical dif- ficulty of arranging adequate clinical studies. Several pre- liminary studies, however, support the idea that molecular diagnostics will be useful in stratifying and selecting sub- groups of patients for surgical and postoperative treatments. For example, in children with neuroblastoma and in patients with small-cell lung cancer, amplification of the N-myc gene has been observed in the clinically more ag- gressive tumors. 33,34 It has been suggested that children with high-grade, high myc-copy number neuroblastomas undergo a complete resection of their tumors to improve survival, while low myc-copy tumors could be managed with subtotal resections. 35 More recently, overexpression of the trkA gene, which codes for a nerve growth factor receptor, was associated with more differentiated neuroblastomas, and when seen without myc amplification, was highly predictive for improved survival.36 In breast cancer, much attention has focused on the overexpression of the oncogene HER2/neu, which codes for a truncated form of the epidermal growth factor receptor.37 While its over- all prognostic capabilities in the general population remain uncertain, it has been seen that HER2/neu overexpression is highly predictive for a subset of women with node-positive cancer who will benefit from high-dose adjuvant chemotherapy.38 Even more profound in its prognostic implications will be the identification of the breast cancer susceptibility gene (BRCAl) that is thought to be on chromosome 17q. 3g The recent identification of the gene for familial adenomatous polyposis40 offers the possibility of screening for alterations that may promote malignant de- generation in this disease. The eventual identification and characterization of the presumed colon cancer susceptibility gene, FCC, on chromosome 2 will likewise have broad

Figure 4. Summary diagram of cell functions that may be altered by ge netic changes and result in the development and proliferation of tumor cells.

implications for early colorectal cancer screening.41 As useful markers are discovered, new technologies may facilitate rapid, inexpensive screening and postoperative surveillance. For example, with a DNA-amplification tech- nique called the polymerase chain reaction (PCR) and an appropriate tumor-specific gene sequence, tumor cells in the blood or at surgical anastomoses can be identified early in recurrence.42

Like the search for markers, the ability to effect therapies through genetic manipulations has proven to be frus- trating, particularly in the transition from the laboratory to the clinic. Attempts to interrupt the growth of tumor cells have employed a variety of methods and targets including a cell’s DNA, RNA, and protein components, while other approaches have tried to induce phenotypic alterations that would make tumor cells more susceptible to immunogenic killing. One strategy involves replacing or activating genes that have been lost during the oncogenic transformation. As with nononcologic diseases such as cystic fibrosis, gene therapy for cancer hinges on obtaining a vector that can successfully target selected cells and incorporate the de- sired genetic material. 43.44 Recently, an adenoviral shuttle has been used to successfully deliver a marker gene into specific rat neural cells.45 This represents an initial barrier that has been cleared, and it is hoped that such viral vectors may perhaps prove capable of replacing deleted or mutated genes in premalignant tumors or in cohorts such as those with Li-Fraumeni cancer-susceptibility syndrome where a high rate of somatic ~53 gene alterations are seen.

Viral vectors may also make possible therapies that at- tenuate the overexpression of genes associated with cancers. This “antisense” therapy is based on the idea that the translation of single-stranded messenger RNA into protein can be blocked by a complementary, or antisense, strand of nucleotides to that particular RNA. One group has used


a K-rus antisense retroviral construct to infect lung tumor cells, which carry a high percentage of K-ras mutations.46 They observed a fivefold attenuation of tumor cell proliferation in vitro and suggest that this could be a therapy for tumors accessible from the tracheobronchial tree. In an in viva system, DNA episomes that expressed antisense insulin-like growth factor I (IGFI) were transfected into rat glioma cells and these cells were then injected subcuta- neously or in the vicinity of glioblastomas in rats.47 Glioblastoma are believed to produce IGFI as part of an autocrine loop, and this antisense blocking produced a re- gression of tumors in the rats.

Therapies can also be targeted at the protein products of genes associated with cancers, and in the case of mem- brane-associated proteins this represents a very attractive potential approach. These proteins tend to be more phys- ically accessible and are often involved in transformation- related processes such as growth factor/receptor autocrine stimulation, immunogenicity, endothelial cell angiogenesis, adhesion and invasion, and so forth. Attempts have been made to disrupt these ligand/receptor interactions with chemicals such as suramin4* or recombinant toxins49 and monoclonal antibodies to growth factors or growth- factor receptors.s0 More recently, therapeutic drugs have been designed that target intracellular signal transduction proteins in an effort to cause changes much like cy- closporine’s effect on T-cell transcription.51 Related approaches have been made to stimulate the immune system to reject tumor cells by an enhanced induction of tumor- specific antigens within those cells”? or by a generalized immune response against the tumor with individually pre- pared recombinant vaccines.“’

Nevertheless, despite a multitude of novel approaches, most of these therapies as yet remain experimental or clinically untested. Investigations into the details of cell biology continue in the hopes that better treatments based on molecular manipulations will be forthcoming. As leaders in the multidisciplinary approach to cancer therapies, sur- geons must remain abreast of this evolving literature to ef- fectively test and use these treatments as they emerge.

SUMMARY With rapid advances in research technologies, there has

been a convergence in many areas of biomedical investi- gation toward a unifying view of normal and abnormal cell growth. Underscoring this view is the idea that the structure and function of cells is tightly regulated by their genetic make-up and when this is altered by external factors or replicative errors, cancers can develop. These genetic alterations effect changes through the proteins for which they code and these proteins frequently are involved at critical points of intracellular signaling pathways that control growth and differentiation. When a critical number of alterations has accumulated, a cell or cell population can es- cape the constraints of normal growth and become tmns- formed. Further genetic changes confer other malignant properties until a population of cells arises that is capable of proliferating independently and at the expense of the host organism. The multiplicity and variety of these changes may account for the phenotypic heterogeneity of cancers and their requirement for multidisciplinary treat-

merits. Effective therapies will depend on unclersrand~ng the intricacies of these processes to find sites w here I he L i--

cious circle of malignant growth can be nrtcrru~ted.

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Molecular mechanisms involved in tumorigenesis and their surgical implications