Differentiation arrest by autologously replicating DNA loops formed along differentiation pathway: An hypothesis of carcinogenesis

Medical Hypotheses (1996) 47, 129-135

© Pearson ProfessionalLtd 1996

Differentiation Arrest by Autologously Replicating DNA Loops Formed Along Differentiation Pathway: An Hypothesis of Carcinogenesis BAO-HUA XUE Department of Medicine, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153, USA (Tel: (+1) 708-327-3126)

Abstract - - The current hypothesis attempts to explain tumor development from the perspective of deoxyribonucleic acid structural changes rather than mutational alterations of single or multiple genes. The hypothesis postulates that stable deoxyribonucleic acid loops capable of autologous replication, translation and expression cause cell-differentiation arrest and contribute to the carcinogenesis and various abnormal biological behaviors of tumor. The formation of deoxyribonucleic acid loops at particular steps along the differentiation pathway determines tumor phenotype, grade and behavior. The outcome of deoxyribonucleic acid loop-formation in a cell is highly affected by the differentiation signals imposed by the cell's differentiation microenvironment which is considered as a very important regulatory factor during tumor development in this hypothesis. The incompatibility of adhesion molecules between tumor cells and surrounding normal cells is proposed in this hypothesis as a major reason for separation of tumor cells from primary lesions and thus metastasis. This hypothesis also postulates that tumor invasion is caused by the expression of proteins related to the transient invasive phenotype of normal cells in physiologic process that is controlled by the genes within autologous deoxyribonucleic acid loops.

Introduction The view that tumor represents aberrant cell differentiation is widely held. The use of terms such as 'dedifferentiation' and 'oncofetal gene expression' attest to the prevailing view that malignant cells result from an alteration in cell differentiation. It has been observed for many years that, both at histological and molecular levels, tumor cells resemble poorly differentiated

embryonic tissues in many aspects. Accordingly, tumor is graded from well-differentiated to very poorly differentiated. This tumor grading is used as a gauge of tumor' s degree of malignancy. Our understanding of normal cell differentiation process and its regulation has been greatly improved in the past decade due to the notable progress in the field of cell-cell and cell-matrix interactions and communications (1). It is now understood that both

Date received 4 August 1995 Date accepted5 February 1996 129

130 cells and their surrounding microenvironment (cells, matrix, soluble factors) must be included to understand the initiation and maintenance of cell differentiation (2). A cell's behavior is controlled not only by its developmental lineage, but also by neighboring cells and extracellular matrix (ECM) (3). All these intriguing new observations and concepts of cell differentiation have inspired us to reconsider our way of viewing the fundamental issue of tumor-carcinogenesis. The hypothesis proposed here is an attempt to develop an experimental concept of carcinogenesis from the perspective of the recent discoveries of cellular differentiation as well as the development in the understanding of gene regulation.

Hypothesis: Carcinogenesis results from the formation of autologously replicating deoxyribonucleic acid loops at various stages along the differentiation pathway In normal growth and development, cells become irreversibly more specialized or 'committed' to a particular development lineage, or pathway of differentiation. Recent findings indicate that interactions and information exchange between cells and their surrounding cells and cell matrix play an important role in this process (1-5). Normal differentiation involves a broad repertoire of cell adhesion molecules and ECM which interact with complementary specific receptors on other cells. The alterations in receptors, induced by ligand binding, in turn cause rearrangement of the cytoskeletal network, and an intracellular cascade of signal transduction, leading to changes in gene expression. Novel cell adhesion molecules and ECM are thus produced which act in a hierarchical fashion and exact higher and higher degrees of stringency to achieve full functional differentiation (4). At the same time, the regulation of cell differentiation is interrelated with the action of other regulators of cellular function, such as growth factors and hormones. Normally, cell differentiation is tightly coupled to proliferation, i.e. terminally differentiated cell continually lost, to be replaced by newly proliferated and differentiated cells. In contrast, tumor cells manifest an apparent uncoupling of differentiation and proliferation, with the resulting accumulation of cells that have not attained terminal differentiation. It is believed that tumor cells, unlike normal cells which differentiate in a linear pathway from less differentiated to fully differentiated cells and from highly proliferative to less proliferative cells, represent stabilization of a specific phase of differentiation pathway which is only transiently attained in normal cells. In other words, tumor cells

MEDICALHYPOTHESES

arrest at a loop status along the differentiation pathway. As long as the tumor cells stay in the loop, they will proliferate indefinitely yet retain their un-fully differentiated status. Is it possible that the loop status of differentiation of tumors is actually the manifestation of the formation of a stable structural deoxyribonucleic acid (DNA) loop? It is well known that many carcinogens are DNA cross-linkers. And circular DNA is a natural structural form of genomic DNA. Thus, it is possible that a stable loop or a circular DNA structure would be formed in mammalian cells as a result of DNA cross-linking during carcinogenesis. Circular DNA are widely found in bacteria which share a most essential character with tumor cells. Both bacteria and tumor cells can divide in an unlimited fashion while maintaining a stable phenotype provided they are supplied with proper nutrients. Studies have demonstrated that DNA within the eucaryotic nucleus is organized in the form of supercoiled loops which are motile with respect to their nuclear matrix anchorage sites during DNA replication (7). These DNA loops are likely to define functional units as well as topological domains, contributing to the regulation of both gene expression and DNA replication (8,9). In tumor cells, cross-linking of DNA by carcinogens may lead to the stabilization of DNA loops that transiently form in normal cells and consequently to the continuous expression of genes within the loop and thus to the disorders of cell functions. Several experiments revealed that most tumor samples contain autologously replicating DNA loops (10). These DNA loops often contain amplified genes such as the C-myc oncogene in the human leukemic cell line HL-60 and the colon carcinoma cell line COLO 320 (11); the human multidrug resistance gene, mdr-1, in human KB carcinoma cell line (12); the EGFR gene in the human colorectal cancer cell line DiFi (13) and the N-myc gene in primary neuroblastomas (14). Although we still know very little about the other DNA information within these DNA loops and the exact relationship between these autologously replicating DNA loops and tumor development, it is a reasonable suggestion that stable replicating DNA loops contribute to the tumor development. Tumors represent clonal outgrowth of cells that replicate independently of appropriate stimuli. An autologously replicating DNA loop is probably a reasonable explanation to the autologous growth of tumor. And circularized DNA might be a mechanism to account for the stable and constant expression of proteins that are related to tumor development. As shown in the Figure, we propose that whether tumor is well-differentiated or poorly differentiated depends on where the loop is formed along the differentiation pathway. If the loop is formed in the very early phase

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Schematicpresentationof the relationshipbetweendifferentiation arrest and tumor development.In our hypothesis,we postulatethat differentiationarrest is causedby the formationof autologouslyreplicating DNA loops capable of constanttranslatingand expressingembryonicproteins that are highlyrelated to tumor development.Tumorgrade and its biological behavior is determinedby where the DNA loop is formedalong the differentiationpathway. Figure

of the differentiation pathway, tumor will show the characteristics of a very poorly differentiated tumor. Conversely, if the loop is formed in the late phase of the differentiation p~thway, tumors will exhibit benign characteristics akin to normal tissue. We also propose that the products of genes contained in these DNA loops are several functionally related gene regulatory proteins that in turn control the expression of other proteins of specific differentiation stages. In summary, we hypothesize that carcinogenesis results from autologous replicating DNA loop formation along the differentiation pathway. Tumor development is the outcome and manifestation of this differentiation arrest phenomenon which allows tumor cells to continuously replicate, translate and express proteins within the loop that direct uncontrolled proliferation, metastasis and invasion.

Differentiation microenvironment is an important regulatory factor in tumor development Whether or not the DNA loop formation will finally result in the outcome of tumor development could be affected by many humeral and cellular factors, i.e. hormone regulation or immune surveillance. In this hypothesis, we will specifically look into the possible effects of normal cell differentiation process on the carcinogenesis and tumor development. Our understanding of cell differentiation process has been greatly improved in recent years. Besides humeral factors, much attention has been given to the

role of cell-cell and cell-matrix interactions and intercommunications. It is now believed that cell adhesion mechanisms play major roles in vital processes such as cell differentiation, embryogenesis, tissue and organ pattern formation, and maintenance of specific tissue architecture (4,5,15,16). Cellular differentiation during embryonic development is very dependent upon the interaction between adhesion molecules on cell surface and the corresponding ligands on neighboring cells or ECM that are in direct contact These interactions serve as instructions, leading cells to the differentiation and expression of new adhesion molecules which in turn initiate further differentiation instructions and so on until the cells achieve full differentiation. During this process, both cell functions and cell adhesion molecules become more and more restricted and tissue specific, with each adhesion molecule having a unique spatial and temporal pattern of expression (6). Blocking adhesion molecules will inhibit differentiation process (17,18), and result in malformation of tissue structure and disorder of cell functions (19,20). Thus, in a sense, cell differentiation is an instructive process. Whether a cell differentiates or not, and in which direction it will differentiate depends heavily on what instructions it receives from surrounding environment both through cell-cell and cell-ECM interactions as well as from humeral factors which also may be tissue and developmental specific. We may call the differentiation status of the cells and ECM, as well as differentiation related humeral factors that directly surround and are in contact with a particular cell, the differentiation microenviron-

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ment of that cell. During differentiation process, each cell differentiates synchronously with its differentiation microenvironment, for a cell in such a microenvironment will continuously receive differentiation instructions imposed by its differentiation microenvironment until fully differentiated. Since we consider that carcinogenesis is a process of cell differentiation arrest, we believe the differentiation microenvironment will have critical effect on this process. Experiments have indicated that adhesion molecules expressed on tumor cells are differentiation related (21). Tumors tend to express adhesion molecules associated with undifferentiated precursor cells. For example, the serological analysis of human melanoma has shown that antigens expressed on malignant melanoma cells reflect the differentiation state of normal cell counterpart at the same stage of differentiation (22). So, in terms of the differentiation microenvironment, tumor cells are most comparable to embryonic cells or regenerating cells of the corresponding differentiation degree. Our hypothesis postulates that, if tumor cells were put into such a differentiation microenvironment, they would receive proper instructions from surrounding cells via adhesion molecules and their receptor's interactions, and thus could be instructed to further differentiate to more differentiated or even normal cells. Several observations strongly suggest this hypothesis may be true. Many of these examples seem puzzling and not worthy of attention at first glance, but when viewed together they merit serious consideration (23). • Only 3 in 100 000 neonates develop tumors during the first month of life. • Malignant tumors are rarely found in spontaneously aborted embryos or fetuses. • Attempts to induce cancer in early stage animal embryos by irradiation or transplacental chemical carcinogenesis have been unsuccessful. However, animals become susceptible to carcinogenesis at later stage of gestation. • Embryonic cells can easily be transformed when removed from the embryonic environment. • Malignant cells frequently lose their malignant characteristics when they are transplanted into embryo. • Spontaneous regression of tumors is not an uncommon finding. However, most spontaneous regression occur before the age of 2 years. • It has been shown that in amphia, which are capable of regenerating whole limbs, that transplantation of a malignant tumor to a regenerating limb, or amputation of a limb distally of a chemically induced tumor, in many cases leads to differentiation and spontaneous regression of the tumor.

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• So-called embryonic tumors are tumors that in humans arise mainly during the first four years of life and are comprised of immature tissues. However, in spite of their embryonic appearance, the most common embryonic tumors in humans Wilms' tumor, neuroblastoma, and retinoblastoma - arise in organs which form during late stage of embryogenesis. These organs partly retain their embryonic appearance until the end of the gestation. It thus seems that in spite of the embryonic histology of these tumors, they probably develop at the end of the gestation or soon after birth. All of the above evidence strongly suggests that, in embryonic and regenerating tissues, cells are highly protected against tumor development. Our hypothesis is that, in such a differentiation microenvironment, cells continuously receive differentiation signals or instructions from their differentiation microenvironment, which prevent them from differentiation arrest. Under the situation of carcinogenesis, this differentiation microenvironment can induce tumor cells to escape from DNA loops and to further differentiate into more differentiated cells or even fully differentiated normal cells. As the aging process proceeds and cells gradually lose their ability to proliferate and differentiate, differentiation arrest will be more likely to occur. This may be one of the important reasons why the incidence of cancer rises steeply as a function of age.

Tumor metastasis and invasion is the result of differentiation arrest Several essential steps are now believed to be involved in the process of tumor metastasis (24). Tumor cells initially separate from other cells in the primary tumor lesions, then enter and arrest in the vasculature at secondary site and pass through vessel wall into the tissue and establish new metastatic colonies. Each step requires dynamic adhesive interactions involving specific adhesion molecules and receptors. Enormous complexity is likely to exist in these steps, since each cell often interacts with multiple cells and with each of a number of ECMs via a variety of adhesion mechanisms, each of which can be regulated independently or co-operatively. Adhesion molecules on both tumor and normal host cells are differentiation related. Expression of normal adhesion molecules on tumor cells are often downregulated. It has been observed that the expression of normal adhesion molecules is inversely correlated with the loss of differentiation of tumor cell (15,2527), i.e. well-differentiated tumor cells usually ex-

DIFFERENTIATION ARREST BY AUTOLOGOUSLYREPLICATING DNA LOOPS FORMED ALONG DIFFERENTIATION PATHWAY

press near normal levels of adhesion molecules, while poorly differentiated tumor cells are almost absent of normal adhesion molecules. At the same time, some other adhesion molecules on tumor cells, especially those of embryonic stages, such as CEA, are up regulated. In our hypothesis, we propose that the down-regulation of normal adhesion molecules and up-regulation of embryonic related adhesion molecules is the consequence of DNA loop formation and differentiation arrest. For normal cells, during embryogenesis, morphogenesis and differentiation, extensive coordination and delicate balance of cell-cell and cell-matrix interactions are involved (5,6,15,28,29). These cell-cell and cell-matrix interactions involve a large group of cell adhesion molecules and their ligands that are stage and tissue specific during development processes, i.e. cells express different complements of adhesion molecules at different periods of cell differentiation; cells of different tissues also express different sets of adhesion molecules. It is now understood that the initial separation of tumor cells from other cells is the key step in the metastatic process which is considered to be caused by decreased function of cell-cell adhesion molecules, leading to easier cell separation. To explain the decreased cell adhesion, both tumor cells and surrounding normal cells and matrix should be included. As for tumor cells, poorly differentiated tumor cells, almost absent of normal adhesion molecules, are easily dissociated from normal tissues and anchored to new sites which may have receptors for alternative adhesion molecules on tumor cells. Well-differentiated tumor cells, expressing high levels of normal adhesion molecules, have less tendency to dissociate from primary lesion and cause metastasis. It has been noticed that the expression of normal adhesion molecules on tumor cells is inversely correlated with the lymph node metastasis (15,25-27). On the other hand, as for normal host environment, cell adhesion molecules also change during development and differentiation. An un-fully differentiated normal tissue, such as embryonic and regenerating tissues, may share many adhesion molecules with tumor cells of the corresponding stage of differentiation. Thus, they may adhere with each other more firmly, metastasis is less likely going to happen. More importantly, as we have stated before, as a result of adhesive interactions with normal cells, tumor cells could be driven by their differentiation microenvironment to further differentiate. Therefore, separation and thus metastasis of tumor cells depends at least in part on the compatibility between tumor cells and surrounding normal cells of host in terms of differentiation degree and thus the expression of adhesion molecules.

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It is very interesting to note that the invasive behavior of tumor cells is also shared by normal cell types and occurs to a limited degree in other physiologic conditions (30). For example, trophoblasts invade the endometrial stroma and blood vessels to establish contact with the circulation during embryogenesis (5). In addition to the important role of adhesion molecules, several ECM-degrading proteinases, mainly plasminogen activators and metalloproteinases (MMPs), are clearly required during this process. Recent studies have demonstrated that these proteinases are also important components of the invasive phenotype of many tumors (31). The extraordinary similarity between these processes when observed at the molecular level indicated that the invasive ability of tumor cells may result from the loss of control over the expression of the invasive phenotype observed in normal cells during differentiation and embryogenesis. This is another extraordinary example of how closely tumor development parallels normal differentiation processes and embryogenesis. We postulate that tumor development including metastasis and invasion is the result of continuous expression of embryonic proteins and loss of expression of normal proteins resulting from differentiation arrest caused by DNA loop formation.

Conclusion

The progress in understanding tumor development has been slow. Although many hypotheses have been put forward, they have tended to treat different disorders of tumors as separate events. As a matter of fact, it is recognized that different disorders of tumor cells are closely related. Thus the underlying mechanisms of these disorders presumably are highly linked. Their outcomes are probably the manifestation of the disordered DNA replication, translation and expression of a group of genes. This hypothesis is an attempt to explain different aspects of carcinogenesis in a unified fashion. We believe carcinogenesis leads to the formation of stable DNA loops capable of autologous replication, translation and expression of a group of functionally related genes. The phenotypic character as well as biological behavior of tumors is determined by the genes contained within the loop, i.e. where the loop is formed along the differentiation pathway. The possible effects of the differentiation microenvironment as an important regulatory mechanism on the outcome and development of tumor were also proposed in this hypothesis. It is the intention of this hypothesis, by providing a different view of carcinogenesis, to inspire new ideas and discussions. First of all, we hope that people will

134 b e c o m e m o r e i n t e r e s t e d in r e s e a r c h e s o n the p o s s i b l e role o f D N A structural a b e r r a t i o n in c a r c i n o g e n e s i s in t h e c l o s e c o n t e x t o f b i o l o g i c a l d e v e l o p m e n t a n d cell d i f f e r e n t i a t i o n . It is n o w w i d e l y a c c e p t e d t h a t t u m o r development involves abnormality of several relevant g e n e s that c a u s e d i f f e r e n t b i o l o g i c a l disorders. It s e e m s m o r e r e a s o n a b l e a n d logical to a c c o u n t t h e s e s e v e r a l a b n o r m a l i t i e s b y o n e m e c h a n i s m in t h e u n i f i e d m a n n e r r a t h e r t h a n to e x p l a i n t h e m as s e v e r a l u n r e l a t e d e v e n t s , for v a r i o u s a b n o r m a l b i o l o g i c a l diso r d e r s o f t u m o r are so h i g h l y related. W e b e l i e v e t h a t it m i g h t b e m o r e l o g i c a l to e x p l a i n t u m o r d e v e l o p m e n t f r o m the p e r s p e c t i v e o f D N A structural c h a n g e s r a t h e r t h a n m u t a t i o n a l a l t e r a t i o n s o f single or m u l t i p l e genes. S e c o n d l y , w e also h o p e that m o r e a t t e n t i o n b e p a i d to t h e p o s s i b l e r e l a t i o n s h i p b e t w e e n s t r u c t u r e a n d f u n c t i o n o f D N A . N a t u r e h a s p r o v i d e d us w i t h two D N A structures - l i n e a r a n d circular. T u m o r d e v e l o p m e n t c o u l d b e the d i s a s t r o u s o u t c o m e o f the interc h a n g e b e t w e e n t h e s e t w o f o r m s o f D N A structures. H i s t o r y o f s c i e n c e h a s r e p e t i t i v e l y p r o v e d t h a t different functions always have their own representative of structure. T h e e n d l e s s p r o l i f e r a t i o n o f t u m o r cells c o u l d b e the f u n c t i o n a l r e p r e s e n t a t i o n o f c i r c u l a r D N A w h i c h h a s n o end. T h i r d l y , t e s t i n g o f this h y p o thesis m a y p r o v i d e us w i t h n e w a l t e r n a t i v e s to h a n d l e for the t h e r a p e u t i c a n d p r e v e n t i v e p u r p o s e s . F o r e x a m p l e , m e d i c i n e s c o u l d b e s e a r c h e d or i n v e n t e d to b l o c k the v i c i o u s circle o f t u m o r p r o l i f e r a t i o n b y b r e a k i n g t h e D N A l o o p s or b y i n d u c i n g t u m o r cells to e s c a p e f r o m t h e D N A loops. F i n a l l y , w e feel t h a t m u c h m o r e k n o w l e d g e o f cell d i f f e r e n t i a t i o n a n d e m b r y o n i c d e v e l o p m e n t as well as t h e i r r e g u l a t i o n will b e n e e d e d to fully u n d e r s t a n d t h e c a r c i n o g e n e s i s a n d t u m o r d e v e l o p m e n t d u e to the close r e l a t i o n s h i p between them.

Acknowledgement The author would like to thank Dr David J. Peace for helpful discussions and critical reading of the manuscript.

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