by Gaetana Tonti
“Tanny” presented this topic on the META-Health Conference 2013 in Egypt. This is her full thesis:
Metastasis derives from the Greek META: beyond, and STASIS: to place, and is used to describe the spread of cancer cells from the primary site to another adjacent organ/part of the body. In order to do this, cancer cells from the first site need to move and invade the secondary site. There has been some debate about the existence of cancerous cells circulating in peripheral blood, especially in the META-medicine world, and thus in this report I would like to give an overview of the metastatic process and propose a possible META-meaning for this phenomenon.
CIRCULATING AND DISSEMIMATED CANCER CELLS
There are many scientifically proven evidences about the existence of circulating tumor cells (CTCs), the first one dating back to 1869, when CTCs which were detected in the blood of an autopsy of a patient who died from cancer in 1869. These cells were thought to represent tumor cells in transit, some of which resulted in metastases (1). At present, attempts to study CTCs are limited by their rarity, with concentrations as low as one CTC per billion circulating hematopoietic cells. However, by applying highly sensitive and specific immunocytochemical and molecular methods, it is possible to detect disseminated tumor cells (DTC) in bone marrow and CTCs in peripheral blood in the background of millions of normal cells; these data are the first steps to unravel the metastatic cascade and understand its bio-logical significance.
Main characteristics of circulating tumor cells
The term CTC encompasses all types of cells, which are considered as foreign entities in the blood having some cancerous characters. Among CTC subpopulations, cancer stem cells have to be taken into account (2). CTCs are tumor cells present in the peripheral blood, even though in very low number: this can be due to the fact that the environment in the bloodstream is highly unfavorable to tumor cells due to physical forces, presence of immune cells and death upon detachment (anoikis), which contributes to metastatic insufficiency (3). CTCs have been found in many different carcinomas but are not present in healthy subjects or patients with benign diseases (1, 2). It has been hypothesized that CTCs may be key actors in the metastatic process (4). They have been detected months to years after complete removal of the primary tumors, indicating that these cells might circulate between different metastatic sites . The development of disseminated diseases has been traditionally viewed as a sequential rather than concurrent process, i.e. the disease initially occurs at the primary site, followed by local growth with eventual dissemination to distant sites (5). However, emerging data is challenging this theory (6, 7). In fact, the beginning of metastasis may be a relatively early event in tumor biology, evidencing the need to understand its bio-logical meaning and the significance of CTCs. In this respect it has been found that phenotypic variation exists between the primary tumor and CTCs; however CTCs share common characteristics described below.
Detection of CTCs
CTCs are generally defined as nucleated cells and DAPI positive (specific agent binding to DNA), staining positively for cytokeratin or epithelial cell adhesion molecule (EpCAM), and negatively for CD45 (8). Specifically, an antibody to the surface epCAM identifies cells of epithelial origin circulating within the blood. Additionally, cytokeratin antibodies further distinguish CTCs as those that are not white blood cells (i.e. – CD45 negative) and select for carcinomas (i.e. – anti-CK8, anti-CK18, anti-CK19)(9). Also monoclonal antibodies against various surface adhesion molecules and growth factor receptors are applied for the detection of carcinoma cells. These antibodies bind to the tumor cells which can then be visualized directly or indirectly by fluorescent dyes or colorimetrically. However, all these studies are hampered by the fact that these cells can exhibit features distinct from their corresponding primary tumors. For the moment, Cell Search system is the only method approved by FDA for the identification of CTCs (10).
Disseminated Tumor Cells
DTCs are tumor cells present in the bone marrow. Several studies have shown that patients with DTCs at the time of diagnosis have larger tumors, higher histologic grade (signifying worse prognosis) , and a higher incidence of lymph-node metastasis, distance metastasis, and cancer-related death versus those patients without (11, 12). DTCs can be already detected at early stages of tumor progression in regional lymph nodes and in bone marrow of cancer patients using highly sensitive detection methods (13). Interestingly, bone marrow has emerged as a common homing organ for metastatic epithelial tumor cells, independent of the primary tumor site and the pattern of overt metastases (14). This suggests that the involvement of bone marrow is tightly related to the presence of various primary tumors suggesting a causal link and a specific meaning for the presence of DTCs. In fact, DTCs in bone marrow have been detected in all solid tumor types suggesting that the bone marrow might be a preferred reservoir for blood-borne DTCs. The bone marrow environment may allow these cells to persist over many years and to disseminate into other organs. However, it cannot be excluded that bone marrow is simply a convenient indicator organ and that early DTCs are also present in other organs such as lung or liver, which are less easily accessible than bone marrow. Thus far unknown environmental and internal factors can promote the recirculation of DTCs from the bone marrow niche into other distant organs such as liver or lungs. Several reports indicated that DTCs might be genomically heterogeneous and can disseminate in a less-progressed genomic state and might acquire genomic alterations typical for fully metastatic cells later (15-17). Thus, DTCs may evolve independently from the primary tumor and accumulate more genomic aberrations after their homing in the bone marrow and other distant organs. A significant fraction of DTCs might never develop into overt metastases but remain in a ‘‘dormant’’ state. Several studies found DTCs in bone marrow even many years after surgery and adjuvant therapy. These results demonstrate that DTCs can reside in a prolonged latent state (referred to as dormancy) before they grow out into overt skeleton metastases (18). To understand this peculiar stage together with the identification of the founder cells of overt metastases (‘‘metastatic stem cells’’) are some of the most important and challenging areas of basic research on the biology of DTCs. A META-point of view could explain these different phenomena explaining not only the reasons for the existence of CTCs and DTCs but also explaining the stage of dormancy and why bone marrow and other organs are the site of metastases.
Detection of DTCs
The development of rare cell detection techniques allows now the reliable detection of DTCs in bone marrow of cancer patients years before the occurrence of distant overt metastases signals. DTCs are rare with only 10–20 cells among millions of bone marrow cells. In general, there are 2 different methods to screen bone marrow aspirates for DTCs, namely cytologic/cytometric (antibody-based) and molecular approaches (19). Among the cytologic methods that allow isolation and enumeration of individual cells, immunocytochemistry is the most widely used approach (19) . Because of the absence of tumor-specific target antigens, most frequently antibodies against various epithelium-specific antigens such as cytoskeleton-associated cytokeratins, surface adhesion molecules or growth factor receptors are applied for the detection of carcinoma cells (20). The main advantage of cytologic methods is the opportunity to combine immunostaining with the morphology of the cells so that both cell size and shape as well as the nucleusplasma relation might be estimated and illegitimate expression of the protein of interest in bone marrow cells can be excluded as far as possible. The detection of DTCs in bone marrow is still not part of the routine tumor staging in the clinical practice, but emerging data anticipate a future role of DTC detection for risk stratification and therapeutic monitoring of breast cancer patients (21, 22). Besides immunocytochemical methods, very sensitive nucleic acid-based techniques now enable the detection of DTCs also at the single cell level. The main advantage of these methods is the nearly unlimited availability of primers for almost every gene of interest. Although numerous genetic alterations have been described in breast cancer cells, heterogeneity is enormous, so that at present no universally applicable DNA marker exists for the primary screening of a wide range of DTCs (23). Therefore, measurement of epithelium-specific or more organ-specific mRNA species such as cytokeratin 19 or mammaglobin mRNA by RT-PCR has been proven as promising approach to detect DTCs in bone marrow samples (24, 25). Because of the absence of tumor-specific markers, the main drawback of using surrogate tissue-specific markers, however, are false-positive results due to illegitimate low-level transcription of epithelial or breast tissue-specific genes in normal cells (26, 27).
It has become more and more accepted during the last 2 decades that bone marrow is a common homing and surviving organ for cancer cells from many organs (28). These cells are likely to escape from the host immune system in a dormant state until internal and/or external signals might enable them to move and grow out to overt metastases at different organs (29-31) (Fig. 1). Thus far, little is known about the dormant cells and the conditions required—environmental factors (e.g., immune system, dietary changes…..stress, emotional traumas???….. repair of damaged tissues in a regeneration phase??? )—for their ‘‘awakening’’ from the dormant or quiescent phase into the dynamic phase of metastasis formation. According to conventional science the steady-state regulating dormancy might be disturbed by both changes in the DTCs (e.g., additional mutations) and the surrounding microenvironment (e.g., decrease in immune surveillance or increased angiogenetic potential (32, 33). But what if a more bio-logical and meaningful reason existed for the explanation of this phenomenon?!
A BRIEF OVERVIEW ON META-STASIS
Metastasis is a ‘hidden’ event, which happens inside the body and is difficult to examine. It has been calculated that approximately 1 × 106 CTCs/g of tumour tissue are released daily into the circulation, but less than 0.1 % of CTCs will successfully settle in secondary organs (34). Thus, it has been suggested that tumour cells may disseminate early in the cancer’s history, and in a far less progressed genomic state than thought before, acquiring genomic aberrations typical of metastatic cells thereafter. In this way the idea that the precursors of metastasis are derived from the most advanced clone within the primary tumour is being challenged (35). During invasion, tumor cells lose cell–cell adhesion, gain mobility, and leave the site of the primary tumor to invade adjacent tissues. In intravasation, tumor cells penetrate through the endothelial barrier and enter the systemic circulation. In extravasation, cells that survive the anchorage-independent growth conditions in the bloodstream attach to vessels at distant sites and leave the bloodstream. Finally, in metastatic colonization, tumor cells form macrometastases in the new host environment (13, 36). (Fig. 1)
A huge amount of data suggests that epithelial-mesenchymal transition (EMT) is a critical early event for the invasion and metastasis of many carcinomas (38, 39). In this EMT process, epithelial cells acquire fibroblast-like properties and show reduced intercellular adhesion and increased motility (40). In specific, one of the main characteristics of EMT is the loss of E-cadherin expression, an important feature of the epithelial phenotype (41). E-cadherin is a cell-cell adhesion molecule that participates in homotypic, calcium-dependent interactions to form epithelial adherent junctions. Loss of E-cadherin expression is consistently observed at sites of EMT during development and cancer, and the E-cadherin expression level is often inversely correlated with the tumor grade and stage (42, 43). Several transcription factors have been implicated in the transcriptional repression of E-cadherin, including zinc finger proteins of the Snail/Slug family, Twist (44, 45) δEF1/ZEB1, SIP1, and the basic helix-loop-helix factor E12/E47. These repressors can also act as molecular triggers of the EMT program by repressing a subset of common gene that encode cadherins, claudins, cytokines, integrins, mucins, plakophilin, occluding, and zonula occludens (ZO) proteins to promote EMT (46). Strikingly, all of these transcriptional repressors are best known for their roles in early embryogenesis. EMT is a dynamic process and is triggered by stimuli that emanate from microenvironments, including extracellular matrix (such as collagen and hyaluronic acid) and many secreted soluble factors. During EMT, epithelial cells lose cell-cell junctions and polarity, leading to a more migratory fibroblast-like “mesenchmymal” cell phenotype.
Interestingly (expecially from a META- point of view) EMT is a process vital for morphogenesis during embryonic development and was first recognized as a feature of embryogenesis in the early 1980s. In particular, during gastrulation in Drosophila flies and mammals, cells migrate from an epithelial-like structure to spatially reorganize one of the three main embryonic layers, the mesoderm (47, 48). EMT is essential not only for many morphogenetic events – such as gastrulation and organogenesis in embryonic development – but also in tissue remodeling, fibrosis and wound healing, and heart development (49). The involvement of the same transcription factors in cancer pathogenesis exemplifies a situation in which cancer cells opportunistically co-opt cell biological programs that are normally operative during early embryogenesis (and perhaps during certain types of wound healing). Because expression of these transcription factors by embryonic cells is provoked by contextual signals that these cells receive from their surroundings, it is likely that such signals act similarly during cancer pathogenesis to trigger expression of these EMT-inducing transcription factors in cancer cells (48-50).
EMT also generates properties of stem cells, such as self-renewal (50, 51), which will be discussed later. In this manner, EMT enables cancer cells not only to disseminate from a primary tumor but also to form the macroscopic metastases with self-renewal capability.
New views on Metastatic steps
During the formation of a primary tumor, premalignant cells acquire a series of genetic and epigenetic changes that enable them to proliferate in the absence of growth factors, to resist proapoptotic stimuli, and to stimulate angiogenesis (52). Many of the cellular phenotypes associated with the earlier steps of this cascade, such as motility and invasiveness, do not arise as purely cell-autonomous processes. Instead, recruited cells present in the environment, called stromal cells, seem to play key roles in the acquisition of these traits by carcinoma cells, and adaptation of cancer cells to these signals, rather than selection, seems to be key to initiating these phenotypes (53). This point is extremely interesting and important, because cancer formation was mainly thought to be dependent on cell mutations, while now the concept that cancer development is linked to external/environmental signals is strongly becoming alive. In this respect, one of the main features of cancer transformation is the EMT, which involves losing some (but not all) epithelial markers and acquiring concomitant expression of some (but not all) mesenchymal markers. Importantly, the EMT is reversible, and carcinoma cells that have acquired a mesenchymal phenotype can revert to an epithelial state via a mesenchymal-epithelial transition (54). The question of whether invasive and metastatic behavior is acquired as a consequence of selection or adaptation can best be addressed by studying the state of the EMT program in a variety of tumors, posing the question of whether the EMT program is turned on reversibly in tumor cells or is constitutively activated in such cells. If the EMT is reversibly activated in carcinoma cells, then the case for adaptation is strongly supported; conversely, if the EMT program is activated irreversibly (constitutively), then the case for selection is favored. Several recent data demonstrate that the EMT program in cancer cells can be triggered through signals that they receive from their microenvironment. Hence, cancer cells bearing multiple mutated genomes need not sustain additional mutations to activate this program, doing so instead in response to contextual signals that they receive from nearby tumor-associated stromal cells. A variety of extracellular signals can contribute to triggering the EMT in the context of neoplasia; included here are the signals conveyed by hepatocyte growth factor (55), tumor necrosis factor-a (56), Notch (57), Hedgehog (58), Wnts and platelet-derived growth factor (59). These reports suggest that an EMT is usually triggered by a convergence of several of these signals. (Because the EMT is used to effect morphogenetic steps in a variety of tissue sites during embryogenesis, it seems unlikely that a common, invariant set of heterotypic signals is responsible for inducing this program in all types of tumor cells). Implicit in these models is an important assumption—specifically that such EMT-inducing signals do not originate in the cancer cells themselves, and therefore do not form a self-initiating autocrine signaling loop. Instead, they seem to originate in other cell types (eg. those forming the stroma). This concept that microenvironmental signals are responsible for activating the EMT holds a number of important further implications. Because the putative inductive signals are likely to act only over short distances, it seems likely that only a subset of cells within an epithelial cell island will experience these signals and undergo an EMT (60). The EMT can be clearly observed to affect a cell layer one to two cells deep that is directly apposed to the stroma; cells lying more internally in epithelial cells islands maintain their characteristic epithelial morphology and continue to express epithelial markers. The fact that contextual signals trigger the EMT suggests its transience and reversibility: when cancer cells within a primary tumor disseminate to distant sites within the body, they will no longer encounter the mix of contextual signals that they experienced in the primary tumor, specifically the signals released by the activated stroma. In the absence of an ongoing flux of these inductive signals, the disseminated cancer cells may then revert via a mesenchymal-epithelial transition, to the epithelial phenotype of their ancestors (60).
This means that for some reason the surrounding stroma becomes ‘‘activated,’’ and it can release cancer and EMT-inducing signals. This scenario represents a clear example of the acquisition of highly malignant cell biological traits through an adaptive mechanism, arguing strongly for adaptation/response as the prime driving force that underlies the acquisition of highly malignant traits by neoplastic cells.
CANCER STEM CELL
These changes in tumor stroma may contribute to the acquisition of stem cell-like phenotypes, a more malignant behavior and invasive ability by cancer cells. Stem cells sit at the top of the developmental hierarchy, having the ability to self-renew and give rise to all the cell lineages in corresponding tissues. Stem cells divide to produce two daughter cells. One daughter remains a stem cell (self-renewal). The other daughter becomes a progenitor cell that undergoes expansion and further differentiation into mature cells (Fig. 2). Stem cells have the highest potential for proliferation and a much longer life span compared with their progeny and therefore have a greater opportunity to accumulate genetic mutations (61).The cancer stem cell concept suggests that tumors arise from a small subpopulation of stem cells; in particular, it has also been assumed that the founder cells of overt metastases might be stem cells disseminated from the primary tumor to the distant metastatic site (62, 63).
Regarding the concept of cancer stem cells it is again interesting to notice the importance of the environment. It has been proposed that one of the differences between normal stem cells and cancer stem cells is their degree of dependence on the stem cell niche, a specialized microenvironment in which stem cells reside. The stem cell niche in adult somatic tissues plays an essential role in maintaining stem cells or preventing tumorigenesis by providing inhibitory signals for both proliferation and differentiation. The niche is a physical anchoring site for stem cells, and adhesion molecules are involved in the interaction between stem cells and the niche and between stem cells and the extracellular matrix. It is composed of a group of cells in a special location that functions to maintain stem cell characteristics and generates extrinsic factors that control stem cell number, proliferation, and fate determination. Many developmental regulatory signal molecules, including hh, Wnts, bone morphogenetic proteins (BMP), fibroblast growth factors, and Notch, have been shown to play roles in controlling stem cell self-renewal and in regulating lineage fate in different systems. Normally, the niche maintains stem cells primarily in a quiescent state by providing signals that inhibit cell proliferation and growth (as evidenced by the ability of stem cells to retain bromodeoxyuridine labeling for a relatively long period of time) (64). Only upon receipt of a stimulating signal does the stem cell become activated to divide and proliferate. Therefore, stem cell proliferation depends on dynamic niche signaling. Maintaining a balance between the proliferation signal and antiproliferation signal is the key to homeostatic regulation of stem cells, allowing stem cells to undergo self-renewal while supporting ongoing tissue regeneration (63, 65). Cancer stem cells may thus arise from a microenvironment with dominant growth promoting signals rather than growth-inhibiting signals.
So, the balance between proliferation-inhibiting and proliferation-promoting signals is the key to homeostatic regulation of stem cell maintenance versus tissue regeneration and tumorigenesis. From a conventional point of view, cancer stem cells may arise from intrinsic mutations, leading to self-sufficient cell proliferation, and may also involve deregulation or alteration of the niche by dominant proliferation-promoting signals, thus from the environment. Within established tumors, the great majority of the cancer cells cannot sustain the lesion nor establish it elsewhere in the body. Only a few cells within the tumor, the cancer stem cells, are tumorigenic and possess the metastatic phenotype (63, 66). It seems as if mutated progenitor cells have acquired the ability to self-renew, a feature thought to be specific to stem cells, and to undergo unlimited growth as cancer cells. In this sense, cancer may prove to be more of a stem cell disease than previously suspected (61, 66).
THE IMPORTANCE OF THE ENVIRONMENT
We can clearly notice that cancer cell behaviors are not regulated by a linear series of commands, but rather by networks of molecular interactions that involve positive and negative reinforcement.
Switching between different cell fates that are critical for cancer (e.g., growth, differentiation, apoptosis, motility) can be triggered in normal and transformed cells, as well as stem cells, by changes in extracellular matrix (ECM) structure and cell shape distortion (67-76), ‘non-specific’
chemical solvents and electrical ion flows (77-85) that influence multiple gene activities,
as well as by distinct molecular factors or specific gene mutations in epithelial or mesenchymal
cells. In this respect cancer can be viewed as a disease of development: it results from loss of the normal controls that direct cells to assemble into tissues, and that hold tissues within their organ confines (86-91). In addition, provocative experiments first carried out over thirty five years ago show that certain cancers differentiate and normalize their growth when combined with normal mesenchyme, other embryonic tissues, or with ECMs that are deposited as a result of interactions between these tissues (reviewed in 92). Some human malignant carcinomas also induce host stroma to be tumorigenic in nude mice (93). Thus, cancer is a developmental disease that involves dysfunction of multi-cellular inductive interactions; it does not result from unregulated growth of a single cell type. These observations raise the possibility that the production of cancer stem cells, epithelial-mesenchymal transitions, increased angiogenesis and unrestrained cell growth that drive cancer formation may result from deregulation of the tissue microenvironment. For example, normal breast epithelium can be induced to progress through hyperplasia and to transform into cancerous tissue by constitutively overexpressing the ECM-degrading enzyme, stromelysin, in transgenic mice. Importantly, these cells that were transformed as a result of changes of tissue structure also exhibit genomic abnormalities (92, 94). This concept that structural or mechanical changes in the tissue microenvironment may actively contribute to tumor formation is supported by early experiments in the cancer research field, which showed that implanting a rigid piece of metal or plastic can trigger cancer formation in animals, whereas tumors do not form when the same material is introduced as a powder (95). Thus, changes in ECM and tissue structure appear to have the potential to be as carcinogenic as oncogenic chemicals, viruses, radiation and gene mutations. It is therefore helpful to view cancer progression in the context of embryological development taking a different direction, or having a different meaning. Interestingly, although stable adult epithelial tissues do not normally exhibit major changes in ECM structure or undergo active changes in form, ultrastructural changes in the basement membrane do occur during early phases of cancer formation, prior to development of a palpable tumor (96, 97). These structural alterations include the appearance of basement membrane gaps, thickening and reduplication, as well as loosening of basal cells from one another and from neighboring connective tissue (98). If cancer is a reversible process, then the entire anti-cancer paradigm will change. There are many studies in experimental systems to suggest that cancers can be induced to become quiescent, differentiate, die or form completely normal tissues, if provided with the correct set of complex signals, as conveyed by embryonic tissues or other
microenvironmental cues (99). The finding that cancer formation can be promoted or accelerated by altering the mechanics or structure of the ECM (96, 98), or by chemically altering the connective tissue stroma (99, 100), suggests that physical cues conveyed by stroma (mesenchyme) may be equally important. Similarly, few investigators are aware of the critical role that electrical potentials play in normal tissue and organ regeneration, that membrane potentials are altered in tumor cells, and that certain cancers can be induced to stop growing and differentiate by normalizing their bioelectrical properties (99-101). Studies carried out in the field of Mechanical Biology over the past twenty years have confirmed that cells can be switched between phenotypes critical for neoplastic transformation, including growth, differentiation, motility and apoptosis in the presence of soluble mitogens by mechanically distorting cells and altering cytoskeletal structure. For example, epithelial and endothelial cells generally proliferate on ECM substrates that resist cell traction forces generated in the actin cytoskeleton and promote cell distortion (spreading) (102, 103). In contrast, these cells round and undergo apoptosis on ECM substrates that fail to bear these loads, whereas they differentiate and express tissue-specific functions when cultured on substrates that maintain an intermediate degree of shape distortion (reviewed in 92).<7p>
A NEW META-PERSPECTIVE ON CANCER
As can be seen, several observations are leading to a new hypothesis for cancer mechanism (104). A very important one states that cancer appears only on those multicellular organisms with complicated wound healing capacities, that wounds considered as risk factors can be identified in all cancers in clinics and finally, that oncogene activation appears not only in cancer, but also in normal physiology and noncancer pathology processes. On these bases a very challenging hypothesis has been proposed: that cancer is a natural wound healing related process, which includes oncogene activations, cytokine secretions, stem cell recruitment differentiation, and tissue remodeling. Wounds activate oncogenes of some cells and the latter secrete cytokines to recruit stem cells to heal the wounds. However, if the cause of the wound or if the wound persists, such as under the persists the continuous wound healing process will lead to a clinical cancer mass. There is no system in nature to stop or reverse the wound healing process in the middle stage when the wound exists. The outcome of the cancer mechanism is either healing the wound or exhausting the whole system (death). The logic of this cancer mechanism is consistent with the rationales of the other physiological metabolisms in the body—for survival (104). The hypothesis can be used to interpret and guide cancer prevention, recurrence, metastasis, in vitro and in vivo studies, and personalized treatments. Supporting this hypothesis are various paradoxes related to the mutation theory, such as a slower proliferation rate of cancer cells than that of normal cells, silent metastatic tumors, normal tissue formation by injecting teratocarcinoma cells into normal blastocysts, cancer formation by transplanting normal murine ovary tissue into the spleen, and spontaneous regression of cancers (105). If oncogenes are defined as the genes that exist in the normal cells (106) and can transform normal cells into cancer cells when over-expressed (or tumor suppressor genes in opposite ways) (106), at the molecular level, many oncogenes (if not all) found in cancers are also found to be active in the early wound-healing process to proliferate repair cells. The tumor suppressor genes that are inactivated in cancer are found to be inactive in the early wound healing and active again in the late wound healing process to stop the repair cell proliferation. These oncogene activities in the wound healing indicate that oncogene mechanisms also play important roles in the wound healing. If cancer is an outcome of oncogene over-expression, it is possible that a cancer cell is an assembly of activated oncogenes which plays a role to help heal the wound. The wound signaling molecules cause over-expression of the existing oncogenes (and some other genes), leading to the changes of certain chromosomes and cancer cell phenotypes (106), while all the normal cell oncogene (not limited to oncogenes) activities responding to wounds are just a part of the cell’s natural metabolism for survival. Therefore, a new cancer theory can be logically speculated: cancer formation is a natural process that organisms have used in wound healing. The following scenario of wound–oncogene–wound healing (WOWH) is described for the cancer mechanisms in mammals. When defined wounds occur in mammals, the body starts the complicated, inflammatory, and stem cell involved wound healing . Molecules such as growth factors, cytokines, and other proteins from the cells in the wound area interrupt the balance of normal molecular metabolism, leading to the activation of corresponding oncogenes and inducing cancerization in some cells (stem cells or actively dividing cells). The cells with activated oncogenes can secrete molecules to recruit stem cells, stimulate stem cell proliferation, and enhance cell differentiation to repair the wound. Oncogenes are activated in the early stages of the wound and tumor suppressor genes are activated in the late stages or the healed wound. Mostly, the wound is healed after above efforts. Oncogenes are deactivated and tumor suppressor genes are activated, then the metabolism reverts to normal. However, if the wound conditions are still persistent this WOWH mechanism will not stop. Oncogenes will be activated continuously and more cancer cells (over-activation of the oncogenes transformed normal cells into malignant cells (107, 108) are divided to secrete more molecules for wound healing, forming a clinical cancer mass. After the wound is healed, the molecules of a healed environment initiate cancer cell differentiation or apoptosis. Subsequently, the clinical cancer mass will be gone. However, if a small clinical cancer cannot heal the wound, the cancer mass will grow large and some part of the cancer itself will be necrotic (inducing inflammation—a new wound) due to the lack of nutrients and oxygen. Both the original and new wounds will induce more cancerization and lead to a positive feedback loop until the wounds are healed or the whole system is exhausted; if wounds persist the cancer mass will grow larger and larger without a natural mechanism to stop the positive feedback loop in our system until the whole system becomes exhausted (death). The logic that normal cells control themselves on developments, wound healing, and ensuring a homeostasis after the process is understandable (109). However, if wound causes and wounds persist, then what possible mechanisms could nature use to heal the wound? Continue fighting? Reverse the process? Facing the persistent wounds, natural selection seems to have no better choice in logic besides fighting the wounds until the system becomes exhausted since the chances of the natural regression of late-stage cancer do exist. More importantly, breaking this positive feedback loop at some point, e.g., at late stage of a cancer, means all the molecular responses to the wound must be stopped or reversed, leading the logical problems for survival—no response or reversal responses to wounded and aging cells in membranes, skin, liver, blood cells, and the other fast metabolized tissues. The commonalities of wound healing and cancer indicate two possibilities: One is that the process approaches the wound incorrectly, leading to wounds that do not heal, or cancer (110, 111). The other is that nature developed a process to heal the wound—the WOWH mechanism accompanied the wound all the time . The first possibility fits the situation of the positive feedback loop described above. However, if the oncogene activations are investigated on a broader scale, such as in pregnancy (112), embryonic development (113), bone and teeth development (114), as well as wound healing , the commonalities of the those oncogene activities indicate the molecules with the same functions exist in both normal and cancer states, rather than a mistake programming de novo in cancer. Gene mutations were not only in cancer cells (at a higher rate), but also in benign hyperplasia or precancerous diseases (at a lower rate) (115), indicating that gene mutations in cancer should be the result of the tissue adaptations rather than the causes of a cancer. Since cancer only appears in species with relatively complicated wound healing, (116), this indicates that cancer is not from a simple random mutation or a programming mistake. The cancer mysteries above and applications below can be understood better and united together only when cancer is considered as an active wound healing tissue. Destroying cancer cells without healing the underlying wounds will allow for an eventual recurrence since the cancer mechanism developed by nature cannot be destroyed in a live mammal. This model leads to a new concept of metastasis. In the conventional view, metastasis has been considered as cancer cell migration and proliferation from the primary site to a distant tissue (117); however, it is not clear why the metastasis did not occur in many cancer cell lines in animal models (118-121). Based on the idea that cancer cells are present to repair a wound, a META explanation of metastasis is that cancer cells in the primary tumor site can “sense” the presence of wounds at distant sites and these cancer cells can migrate through the circulation in order to heal the secondary wound. At this point it might be interesting to define the term “wound”: is it only a physical wound?! But from a META-perspective we know that there is never a physical wound without an emotional/spiritual wound. In this respect it might be interesting to point out which are the most common sites of metastasis. If we consider the 3 most common cancer in the world (breast, prostate and colon) it is amazing (or maybe not really for META-scientists!!!) to notice that the most common sites for secondary tumors are the lungs, followed by the bones (with the vertebrae showing the highest frequency) . The higher occurency of metastases and secondary tumors in these sites can be easily explained. Regarding the lungs it can be argued that when a patient hears from a doctor that he has a cancer, he can experience a fear of death, a diagnostic shock and so there will be a proliferation of the lung cells in order to have an increased breathing. This happens in the stress phase, in order to provide a survival mechanism (cell plus for brain stem issues, with bio-logical meaning in the first phase). The diagnosis may be that of a benign tumor or of a malignant carcinoma, according to the mass of the conflict shock. Many people can also feel worthless, incapable, not able to sustain the situation, and in this case the bone tissue will react. The event of bone metastasis occurs in the stress phase. As discussed previously, metastatic cells invade the bone marrow, and these cells, even though bearing some characteristics of the primary tumor from which they derive, are un-differentiated, as it has been postulated that they derive from a cancer stem cell. When these metastatic cells invade the bone marrow they pick up some feature of the main bone cell types, which are osteoblasts (physiologically devoted to bone reconstruction) or osteclasts (which mediate bone resorption). It may be interesting to highlight that metastatic cells in bone marrow show osteoclast proliferation with hypertrophy, which explains why metastasis is an event happening in the stress phase (cell minus of cerebral medulla). Following the resolution, then there will be calcification of cells and the bone will be stronger that before (bio-logical meaning is in cell increase and strengthening of the bone at the end of the regeneration phase). But why the cancer cells from the primary tumor migrate to the bone marrow? According to the WOWH theory they might sense on emotional wound/stress that hits the core of who we are, the sense that lives gives to us or that we give to life… the inmost, the essential part of who we are… the marrow.
In conclusion on one side these data show how the view of what cancer and metastasis are, is changing from the conventional scientific point of view, getting closer to a META-perspective, and on the other side these data may help to understand in more detail from a META-point of view the events associated with cancer initiation, progression and meta-stasis.
1.Clin.CancerRes. 10, 6897–6904.). Kagan M, Howard D, Bendele T, et al: A sample preparation and analysis system for identification of circulating tumor cells. J Clin Ligand Assay 2002, 25:104-110.
2.Kagan M, Diamandis EP, Fritsche HA, et al: Tumor markers: physiology, pathobiology, technology and clinical applications. Washington, D.C.: AACC Press; 2002, 405-409.].
3.[Trepel M, Arap W, Pasqualini R: In vivo phage display and vascular heterogeneity: implications for targeted medicine. Curr Opin Chem Biol 2002, 6:399-404.].
4.Ashworth,T.R. A case of cancer in which cells similar to those in the tumor were seen in the blood after death. Aus.Med.J. 1869: 14, 146–149
5.Guislaine Barri`ere, Michel Tartary, and Michel Rigaud . Epithelial Mesenchymal Transition: A New Insight into the Detection of Circulating Tumor Cells. Int. Schol. Res. Net. ISRN Oncol. Volume 2012: doi:10.5402/2012/382010)
6.[Mego M, Mani SA, Cristofanilli M: Molecular mechanisms of metastasis -clinical applications. 2010, 7(12):693-701
7.Kim MY, Oskarsson T, Acharyya S, Nguyen DX, Zhang XH-F, Norton L, Massague J: Tumor self-seeding by circulating cancer cells. Cell 2009, 139:1315-1326.].
8.Allard,W.J.,Matera,J.,Miller,M.C., Repollet,M.,Connelly,M.C.,Rao, C., etal.(2004)
9.[De Giorgi U, Valero V, Rohren E, Mego M, Doyle GV, Miller MC, Ueno NT, Handy BC, Reuben JM, Macapinlac HA, Hortobagyi GN, Cristofanilli M: Circulating tumor cells and bone metastases as detected by FDG-PET/CT in patients with metastatic breast cancer. Annals of Oncol 2010, 21:33-39.]
10.(Identification and enumeration of circulating tumor cells in the cerebrospinal fluid of breast cancer patients with central nervous system metastases, Patel et al.; Oncotarget, 2011;2: 752-760)
11.[S. Braun, K. Pantel, P.M¨uller et al., Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer,” New England Journal of Medicine, vol. 342, no. 8, pp. 525–533, 2000.
12.S. Braun, F. D. Vogl, B. Naume et al., “A pooled analysis of bone marrow micrometastasis in breast cancer,” New England Journal of Medicine, vol. 353, no. 8, pp. 793–802, 2005.].
13.Pantel K, Brakenhoff RH. Dissecting the metastatic cascade. Nat Rev Cancer 2004;4:448^56.).
14.[Pantel K, Cote RJ, Fodstad O: Detection and clinical importance of micrometastatic disease. J Natl Cancer Inst 1999; 91: 1113–1124.]
15.Klein CA, BlankensteinTJ, Schmidt-Kittler O, et al. Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer. Lancet 2002;360:683^9.
16.Schmidt-Kittler O, RaggT, Daskalakis A, et al. From latent disseminated cells to overt metastasis: genetic analysis of systemic breast cancer progression. Proc Natl Acad Sci US A 2003;100:7737^ 42.
17.Gangnus R, Langer S, Breit E, Pantel K, Speicher MR. Genomic profiling of viable and proliferative micrometastatic cells from early-stage breast cancer patients. Clin Cancer Res 2004;10:3457^64.).
18.JanniW, Rack B, Schindlbeck C, et al. The persistence of isolated tumor cells in bone marrow from WiedswangG , Borgen E, Karesen R, et al. Isolated tumor cells in bone marrow three years after diagnosis in disease-free breast cancer patients predict unfavorable clinical outcome. Clin Cancer Res 2004;10: 5342^8.).
19.[Lacroix M: Significance, detection and markers of disseminated breast cancer cells. Endocr Relat Cancer 2006; 13: 1033–1067. 16 Wolfle U, Muller V, Pantel K: Disseminated tumor cells in breast cancer: detection, characterization and clinical relevance. Future Oncol 2006; 2: 553–561.]
20.[Wolfle U, Muller V, Pantel K: Disseminated tumor cells in breast cancer: detection, characterization and clinical relevance. Future Oncol 2006; 2: 553–561.]
21.[Braun S, Kentenich C, Janni W, Hepp F, de Waal J, Willgeroth F, Sommer H, Pantel K: Lack of effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high-risk breast cancer patients. J Clin Oncol 2000; 18: 80–86
22.Janni W, Rack B, Schindlbeck C, Strobl B, Rjosk D, Braun S, Sommer H, Pantel K, Gerber B, Friese K: The persistence of isolated tumor cells in bone marrow from patients with breast carcinoma predicts an increased risk for recurrence. Cancer 2005; 103: 884– 891.]
23.[Ring A, Smith IE, Dowsett M: Circulating tumour cells in breast cancer. Lancet Oncol 2004; 5: 79–88.
24.[Luchtenborg M, Sinnett HD, Cross NC, Coombes RC: Response of circulating tumor cells to systemic therapy in patients with metastatic breast cancer: comparison of quantitative polymerase chain reaction and immunocytochemical techniques. J Clin Oncol 2000; 18: 1432–1439.
25.Berois N, Varangot M, Aizen B, Estrugo R, Zarantonelli L, Fernandez P, Krygier G, Simonet F, Barrios E, Muse I, Osinaga E: Molecular detection of cancer cells in bone marrow and peripheral blood of patients with operable breast cancer: comparison of CK19, MUC1 and CEA usingRT-PCR. Eur J Cancer 2000; 36: 717–723.]
26.[Ballestrero A, Garuti A, Bertolotto M, Rocco I, Boy D, Nencioni A, Ottonello L, Patrone F: Effect of different cytokines on mammaglobin and maspin gene expression in normal leukocytes: possible relevance to the assays for the detection of micrometastatic breast cancer. Br J Cancer 2005; 92: 1948–1952
27.Ring A, Smith IE, Dowsett M: Circulating tumour cells in breast cancer. Lancet Oncol 2004; 5: 79–88.]
28.[Pantel K, Woelfle U: Micrometastasis in breast cancer and other solid tumors. J Biol Regul Homeost Agents 2004; 18: 120–125.]
29.[Allan AL, Vantyghem SA, Tuck AB, Chambers AF: Tumor dormancy and cancer stem cells: implications for the biology and treatment of breast cancer metastasis. Breast Dis 2006; 26: 87–98.
30.Alix-Panabieres C, Muller V, Pantel K: Current status in human breast cancer micrometastasis.Curr Opin Oncol 2007; 19: 558–563
31. Pantel K, Schlimok G, Kutter D, Schaller G, Genz T, Wiebecke B, Backmann R, Funke I, Riethmuller G: Frequent down-regulation of major histocompatibility class I antigen expression on individual micrometastatic carcinoma cells. Cancer Res 1991; 51: 4712–4715.] .
32.Naumov GN, Bender E, Zurakowski D, et al. A model of human tumor dormancy: an angiogenic switch from the non angiogenic phenotype. J Natl Cancer Inst 2006;98:316
33.Marches R, Scheuermann R, Uhr J. Cancer dormancy: from mice to man. Cell Cycle 2006;5: 1772
34.Dawood and Cristofanilli, 2007
35.Schmidt-Kittler et al., 2003
36.Chambers AF, Groom AC, MacDonald IC. Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer 2002;2:563–572
37.Clin Cancer Res 2005;11:8534–8553. Thompson EW, Newgreen DF, Tarin D. Carcinoma invasion and metastasis: a role for epithelialmesenchymal transition?
38.Cardiff RD. Epithelial to mesenchymal transition tumors: fallacious or snail’s pace? Clin Cancer Res 2005;11:8534–8553
39.Thompson EW, Newgreen DF, Tarin D. Carcinoma invasion and metastasis: a role for epithelialmesenchymal transition? Cancer Res 2005;65:5991–5995
40.Huber MA, Kraut N, Beug H. Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr Opin Cell Biol 2005;17:548–558
41.Kang Y, Massague J. Epithelial-mesenchymal transitions: twist in development and metastasis. Cell 2004;118:277–279].
42.Cowin P, Rowlands TM, Hatsell SJ. Cadherins and catenins in breast cancer. Curr Opin Cell Biol 2005;17:499–508
43.Junghans D, Haas IG, Kemler R. Mammalian cadherins and protocadherins: about cell death, synapses and processing. Curr Opin Cell Biol 2005;17:446–452
44.Karreth F, Tuveson DA. Twist induces an epithelial-mesenchymal transition to facilitate tumor metastasis. Cancer Biol Ther 2004;3:1058–1059.
45.Vernon AE, LaBonne C. Tumor metastasis: a new twist on epithelial-mesenchymal transitions. Curr Biol 2004:R719–R721
46.Huber MA, Kraut N, Beug H. Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr Opin Cell Biol 2005;17:548–558
47.Thiery JP. Epithelial-mesenchymal transitions in development and pathologies. Curr Opin Cell Biol 2003;15:740–746
48.[Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 2006;7:131–142
49.Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2002;2:442–454
50.Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, et al. The epithelialmesenchymal transition generates cells with properties of stem cells. Cell 2008;133:704–715.
51.Mani SA, Yang J, Brooks M, Schwaninger G, Zhou A, Miura N, Kutok JL, et al Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers. Proc Natl Acad Sci USA 007;104:10069–10074].
52.Hanahan D, Weinberg RA. The hallmarks of cancer.Cell 2000;100:57–70.).
53.Condeelis J, Pollard JW. Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell 2006;124:263–6
54.Chaffer CL, Brennan JP, Slavin JL, Blick T, Thompson EW, Williams ED. Mesenchymal-to-epithelial transition facilitates bladder cancer metastasis: role of fibroblast growth factor receptor-2. Cancer Res 2006;66:11271–8.).
55.Savagner P, Yamada KM, Thiery JP. The zinc-finger protein slug causes desmosome dissociation, an initial and necessary step for growth factor-induced epithelial-mesenchymal transition. J Cell Biol 1997;137:1403–19.
56.Bates RC, Mercurio AM. Tumor necrosis factor-a stimulates the epithelial-to-mesenchymal transition of human colonic organoids.Mol Biol Cell 2003;14:1790–800.
57.Zavadil J, Cermak L, Soto-Nieves N, Bottinger EP. Integration of TGF-h/Smad and Jagged1/Notch signaling in epithelial-to-mesenchymal transition. EMBO J 2004;23:1155–65
58.Karhadkar SS, Bova GS, Abdallah N, et al. Hedgehog signalling in prostate regeneration, neoplasia and metastasis. Nature 2004;431:707–12
59.Yook JI, Li XY, Ota I, et al. A Wnt-Axin2-3h cascade regulates Snail1activity in breast cancer cells. Nat Cell Biol 2006;8:1398–406.),
60.Brabletz T, Jung A, Reu S, et al. Variable h-catenin expression in colorectal cancers indicates tumor progression driven by the tumor environment. Proc Natl Acad Sci U S A 2001;98:10356–61.
61.Wicha MS, Liu S, Dontu G. Cancer stem cells: an old idea — a paradigm shift. Cancer Res 2006;66: 1883^90;
62.Liu R,WangX , Chen GY, et al. The prognostic role of a gene signature fromtumorigenic breast-cancer cells. NEngl JMed 2007;356:217^26
63.Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001;414: 105–11
64.Pardal R, Clarke MF, Morrison SJ. Applying the principles of stem-cell biology to cancer. Nat Rev Cancer 2003;3:895–902
65.Tumbar T, Guasch G, Greco V, et al. Defining the epithelial stem cell niche in skin. Science 2004;303: 359–63.).
66.He XC, Zhang J, Li L. Cellular and molecular regulation of hematopoietic and intestinal stem cell behavior. Ann N Y Acad Sci 2005;1049:28–38
67.Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol CellBiol 2006;7:131–42
68.Krishnamachary B, Zagzag D, Nagasawa H, et al. Hypoxia-inducible factor-1-dependent repression of E-cadherin in von Hippel-Lindau tumor suppressor-null renal cell carcinoma mediated by TCF3, ZFHX1A, and ZFHX1B. Cancer Res 2006;66:2725–31
69.(Maestro R, Dei Tos AP, Hamamori Y, et al. Twist is a potential oncogene that inhibits apoptosis. Genes Dev 1999;13:2207–17.
70.Valsesia-Wittmann S, Magdeleine M, Dupasquier S, et al. Oncogenic cooperation between H-Twist and N-Myc overrides failsafe programs in cancer cells. Cancer Cell 2004;6:625–30.
71.kajita M, McClinic KN, Wade PA. Aberrant expression of the transcription factors snail and slug alters the response to genotoxic stress. Mol Cell Biol 2004;24:7559–66.).
72.R. Kalluri and R. A. Weinberg, “The basics of epithelial-mesenchymal transition,” Journal of Clinical Investigation, vol. 119, no. 6, pp. 1420–1428, 2009.
73.M.W. Klymkowsky and P. Savagner, “Epithelial-mesenchymal transition: a cancer researcher’s conceptual friend and foe,” American Journal of Pathology, vol. 174, no. 5, pp. 1588–1593, 2009.
74.D. Hanahan and R. A. Weinberg, “The hallmarks of cancer,” Cell, vol. 100, no. 1, pp. 57–70, 2000.
75.A. F. Chambers, A. C. Groom, and I. C. MacDonald, “Dissemination and growth of cancer cells in metastatic sites,” Nature Reviews Cancer, vol.. 2, no. 8, pp. 563–572, 2002
76.ADAPTATION VERSUS SEPECTION: THE ORIGINS OF METASTATIC BEHAVIOUR; CANCER RES, 2007;67:11476-11480, Christina Scheel, Tamer Onder, Antoine Karnoub, et al.
77.Singhvi R, Kumar A, Lopez GP, Stephanopoulos GN, Wang DI, et al. Engineering cell shape andfunction. Science 1994;264:696–698. [PubMed: 8171320]
78.Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE. Geometric control of cell life and death.Science 1997;276:1425–1428. [PubMed: 9162012]
79.Parker KK, Brock AL, Brangwynne C, Mannix RJ, Wang N, et al. Directional control of lamellipodiaextension by constraining cell shape and orienting cell tractional forces. FASEB J 2002;16:1195–1204. [PubMed: 12153987]
80.Paszek MJ, Zahir N, Johnson KR, Lakins JN, Rozenberg GI, et al. Tensional homeostasis and themalignant phenotype. Cancer Cell 2005;8:241–254. [PubMed: 16169468]Ingber Page 10 Semin Cancer Biol. Author manuscript; available in PMC 2009 October 1.
81.19. McBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS. Cell shape, cytoskeletal tension, andRhoA regulate stem cell lineage commitment. Dev Cell 2004;6:483–495. [PubMed: 15068789]
82. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification.Cell 2006;126:677–689. [PubMed: 16923388]
83.Huang S, Eichler G, Bar-Yam Y, Ingber DE. Cell fates as high-dimensional attractor states of a complex gene regulatory network. Phys Rev Lett 2005;94:128701. [PubMed: 15903968]
84.Hellmann P, Grummer R, Schirrmacher K, Rook M, Traub O, Winterhager E. Transfection with different connexin genes alters growth and differentiation of human choriocarcinoma cells. Exp CellRes 1999;246:480–490. [PubMed: 9925764]
85. Jones SM, Ribera AB. Overexpression of a potassium channel gene perturbs neural differentiation. J Neurosci 1994;14:2789–2799. [PubMed: 8182440]
86.Strobl JS, Wonderlin WF, Flynn DC. Mitogenic signal transduction in human breast cancer cells. Gen Pharmacol 1995;26:1643–1649. [PubMed: 8745151]
87.Rich IN, Worthington-White D, Garden OA, Musk P. Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na(+)/H(+) exchanger. Blood 2000;95:1427–1434. [PubMed: 10666221
88.Clark WH Jr. The nature of cancer: morphogenesis and progressive (self)-disorganization in neoplastic development and progression. Acta Oncol 1995;34:3–21. [PubMed: 7865232]
89.Huang S, Ingber DE. The structural and mechanical complexity of cell-growth control. Nat Cell Biol 1999;1:E131–138. [PubMed: 10559956]
90.Ingber DE. Cancer as a disease of epithelial-mesenchymal interactions and extracellular matrix regulation. Differentiation 2002;70:547–560. [PubMed: 12492496]
91. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001; 414: 105-111.
92.Ingber DE, Can cancer be reversed by engineering the tumour microenvironment? Semin cancer Biol 2008; 18: 356-364.
93.Goldenberg DM, Pavia RA. Malignant potential of murine stromal cells after transplantation of human tumors into nude mice. Science 1981;212:65–67. [PubMed: 7209521]
94.Sternlicht MD, Bissell MJ, Werb Z. The matrix metalloproteinase stromelysin-1 acts as a natural mammary tumor promoter. Oncogene 2000;19:1102–1113. [PubMed: 10713697]
95.Bischoff F, Bryson G. Carcinogenesis through Solid State Surfaces. Prog Exp Tumor Res 1964;14:85–133. [PubMed: 14317768]
96.Lu S, Huang M, Kobayashi Y, Komiyama A, Li X, et al. Alterations of basement membrane in diisopropanolnitrosamine-induced carcinogenesis of the rat thyroid gland: an immunohistochemical study. Virchows Arch 2000;436:595–601. [PubMed: 10917175]
97.Li SC, Chen GF, Chan PS, Choi HL, Ho SM, Chan FL. Altered expression of extracellular matrix and proteinases in Noble rat prostate gland after long-term treatment with sex steroids. Prostate 2001;49:58–71. [PubMed: 11550211]
98.Paszek MJ, Zahir N, Johnson KR, Lakins JN, Rozenberg GI, et al. Tensional homeostasis and themalignant phenotype. Cancer Cell 2005;8:241–254. [PubMed: 16169468]
99.Maffini MV, Soto AM, Calabro JM, Ucci AA, Sonnenschein C. The stroma as a crucial target in rat mammary gland carcinogenesis. J Cell Sci 2004;117:1495–1502. [PubMed: 14996910]
100.Levin M, Thorlin T, Robinson KR, Nogi T, Mercola M. Asymmetries in H+/K+-ATPase and cell membrane potentials comprise a very early step in left-right patterning. Cell 2002;111:77–89. [PubMed: 12372302]
101.Levin M. Bioelectromagnetics in morphogenesis. Bioelectromagnetics 2003;24:295–315. [PubMed: 12820288]
102.Folkman J, Moscona A. Role of cell shape in growth control. Nature 1978;273:345–349. [PubMed: 661946]
103.Nelson CM, Jean RP, Tan JL, Liu WF, Sniadecki NJ, et al. Emergent patterns of growth controlled by multicellular form and mechanics. Proc Natl Acad Sci U S A 2005;102:11594–11599. [PubMed:16049098]
104.Cancer Metastasis Rev (2012) 31:247–268. A new hypothesis for the cancer mechanism Xiaolong Meng & Jie Zhong & Shuying Liu & Mollianne Murray & Ana M. Gonzalez-Angulo
105.Baker, S. G., & Kramer, B. S. (2007). Paradoxes in carcinogenesis: new opportunities for research directions. BMC Cancer, 7, 151.
106.Zhou, Y., Ma, B. G., & Zhang, H. Y. (2007). Human oncogene tissue-specific expression level significantly correlates with sequence compositional features. FEBS Letters, 581, 4361–4365.
107.Haber, M., & Stewart, B. W. (1985). Oncogenes. A possible role for cancer genes in human malignant disease. The Medical Journal of Australia, 142(7), 402–406.
108.Mitsushita, J., David Lambeth, J., & Kamata, T. (2004). The superoxide-generating oxidase Nox1 Is functionally required for Ras oncogene transformation. Cancer Research, 64,3580–3585
109.Todd, R., &Wong, D. T. (1999). Oncogenes. Anticancer Research, 19(6A), 4729–4746.
110.Hanahan, D., & Weinberg, R. A. (2011). Hallmarks of cancer: the next generation. Cell, 144(5), 646–674.
111.Riss, J., Khanna, C., Koo, S., Chandramouli, G. V. R., Yang, H. H., Hu, Y., Kleiner, D. E., Rosenwald, A., Schaefer, C. F., Ben-Sasson, S. A., Yang, L., Powell, J., Kane, D.W., Star, R. A., Aprelikova, O., Bauer, K.,Vasselli, J. R.,Maranchie, J.K., Kohn, K.W., Buetow, K. H., Marston Linehan,W.,Weinstein, J. N., Lee, M. P., Klausner, R. D., & Carl Barrett, J. (2006). Cancers as wounds that do not heal: differences and similarities between renal regeneration/repair and renal cell carcinoma. Cancer Research, 66, 7216–7224.
112.Dvorak, H. F. (1986). Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. The New England Journal of Medicine, 315, 1650–1659.
113.Quenby, S., Brazeau, C., Drakeley, A., Lewis-Jones, D. I., & Vince, G. (1998). Oncogene and tumour suppressor gene products during trophoblast differentiation in the first trimester. Molecular Human Reproduction, 4, 477–481.
114.Quenby, S. M., Gazvani, M. R., Brazeau, C., Neilson, J., Lewis- Jones, D. I., & Vince, G. (1999). Oncogenes and tumour suppressor genes in first trimester human fetal gonadal development. Molecular Human Reproduction, 5(8), 737–741.
115.Wagner, E. F. (2002). Functions of AP1 (Fos/Jun) in bone development. Annals of the Rheumatic Diseases, 61, 40.
116.Vogelstein, B., Fearon, E. R., Hamilton, S. R., Kern, S. E., Preisinger, A. C., Leppert, M., Nakamura, Y., White, R., Smits, A. M., & Bos, J. L. (1988). Genetic alterations during colorectaltumor development. The New England Journal of Medicine, 319 (9), 525–532.
117.Harding, C., Pompei, F., Lee, E. E., & Wilson, R. (2008). Cancer suppression at old age. Cancer Research, 68, 4465–4478.
118.Paris, S., & Sesboue, R. (2004). Metastasis models: the green fluorescent revolution? Carcinogenesis, 25(12), 2285–2292.
119.Enomoto, T., Oda, T., Aoyagi, Y., Sugiura, S., Nakajima, M., Satake, M., Noguchi, M., & Ohkohchi, N. (2006). Consistent liver metastases in a rat model by portal injection of microencapsulated cancer cells. Cancer Research, 66(23), 11131–11139.
120.Weiss, L., Mayhew, E., Rapp, D. G., & Holmes, J. C. (1982). Metastatic inefficiency in mice bearing B16 melanomas. British Journal of Cancer, 45(1), 44–53.
121.Nathoo, N., Chahlavi, A., Barnett, G. H., & Toms, S. A. (2005). Pathobiology of brain metastases. Journal of Clinical Pathology, 58(3), 237–242.