11. Mitochondria: The Ultimate Tumor Suppressor

Mitochondrial Suppression of Tumorigenicity

All other characteristics of cancer arise either directly or indirectly from insufficient respiration. It is also clear that genomic instability and the vast numbers of gene and chromosome defects seen in tumor cells can arise as secondary consequences of protracted respiratory insufficiency. Genome instability is linked to mitochondrial dysfunction through the retrograde signaling system. If all cancer arises from mitochondrial dysfunction, then replacement of damaged mitochondria with normal mitochondria should prevent cancer. In other words, mitochondria producing sufficient respiration should suppress tumor growth regardless of the numbers and types of mutations or aneuploidy present.

Energy derived from substrate level phosphorylation (including the Warburg effect and amino acid fermentation) will persist in the presence of insufficient respiration. Up-regulation of oncogenes and down regulation of tumor suppressor genes is necessary to maintain fermentation when mitochondria fail to produce sufficient energy through respiration. While the mutator phenotype of cancer can be linked to impaired mitochondrial function, substantial evidence also exists showing that normal mitochondrial function suppresses tumorigenesis.

If respiratory insufficiency is the origin of cancer, then tumor nuclei should not induce malignancy when placed in cytoplasm containing respiration competent normal mitochondria. Alternatively, if mitochondrial dysfunction is the origin of cancer, normal nuclei should be unable to prevent tumorigenesis when placed into the tumor cytoplasm.

Normal Mitochondria Suppress Tumorigenesis in Cybrids

Tumorigenicity can be suppressed when cytoplasm from enucleated normal cells is fused with nucleated tumor cells to form cybrids. Cybrids contain a single nucleus and mixtures of cytoplasm from two different cells.

The findings and conclusions of Israel and Schaeffer that cytoplasmic factors suppress tumorigenicity were also strongly supported by the findings of Shay and Werbin. These investigators also discussed the various factors that could influence the success or failure of cybrid experiments designed to uncover cytoplasmic suppressors of tumorigenicity. These factors included, (i) the relative amounts of tumorigenic and nontumorigenic cytoplasm in cybrids; (ii) the time interval that cybrids are passaged prior to testing their tumorigenicity; (iii) whether mutagenesis with carcinogens was used to introduce genetic markers on the cells; and (iv) the specific cell combinations used. They were not surprised that some investigators could obtain varying results if the various factors were not monitored carefully.

• They concluded “if tumor cell mitochondria are defective, as Warburg postulated, then suppression could result from the introduction of mitochondria from normal cells into cybrids”

The findings from the Jonasson and Harris study were remarkable for several reasons:

• First, their findings were consistent with those of many other cybrid studies indicating that something in normal cytoplasm suppresses tumorigenicity in malignant cells.
• Second, no human chromosome or nuclear genetic material was responsible for the suppressive effect.
• Finally, radiation could destroy the cytoplasmic factor responsible for tumor suppression. This last fact is consistent with Warburg findings that radiation destroys mitochondrial respiration.

Surprisingly, Jonasson and Harris excluded a mitochondrial origin in preference to a centrosome origin for the suppression effect. This decision was based on the findings of others who showed that no human mitochondrial DNA or proteins were found in human mouse cybrids. However, new studies in transmissible cancers show that tumor mitochondria can integrate with normal mitochondria in some tumors.

Paul Saxon and colleagues showed that the microcell transfer of chromosome 11 could suppress tumorigenicity in HeLa cells. They concluded that chromosome 11 contained a tumor suppressor gene. These findings are interesting and also suggest an interaction between chromosome 11 and the mitochondria. It is possible that a gene on chromosome 11 facilitates mitochondrial respiration thus suppressing tumorigenicity in the HeLa cells. It is also interesting that neuroblastoma and Wilms tumor are associated with defects on chromosome 11.

Evidence From rho0 Cells

Singh and coworkers also provided evidence for the role of mitochondria in the suppression of tumorigenicity by showing that exogenous transfer of wild-type mitochondria to cells with depleted mitochondria DNA (rho0 cells) could reverse the altered expression of the APE1 DNA repair protein and the tumorigenic phenotype. The efficiency of APE1-mediated DNA repair is dependent on the sufficiency of mitochondrial respiration. The rho0 cells have deficient respiration because they lack mtDNA, which is necessary for normal respiration. Consequently, transfer of normal mtDNA to rho0 cells will restore respiration, turn off the RTG response, and prevent genomic instability.

The authors concluded that mtDNA mutations play an important role in the etiology of prostate cancer and that cancer can be best defined as a type of mitochondrial disease.

Normal Mitochondria Suppress Tumorigenesis In Vivo

It is also well documented that nuclei from cancer cells can be reprogrammed to form normal tissues when transplanted into normal cytoplasm despite the continued presence of the tumor-associated genomic defects in the cells of the derived tissues.

Herpes viruses have an intimate attachment with mitochondria that causes dysfunctional respiration. Hence, the suppression of tumorigenesis in the Lucke frog tumor is likely due to the replacement of virus-damaged mitochondria with normal mitochondria.

Normal Mouse Cytoplasm Suppresses Tumorigenic Phenotypes

Nuclei from mouse medulloblastoma (a brain tumor thought to arise from cerebellar granule cells) could direct normal development when transplanted into enucleated somatic cells. These investigators concluded that somatic nuclear transfer into normal cytoplasm suppressed the tumorigenic phenotype. Moreover, transplanted medulloblastoma nuclei gave rise to post-implantation embryos that underwent tissue differentiation and early stages of organogenesis. Remarkably, no malignancies were observed in any of the recipient mice, and normal proliferation control was observed in cultured blastocysts.

The findings from the Lucke frog and mouse medulloblastoma experiments were further supported from the work of Konrad Hochedlinger, Rudy Jaenisch and colleagues at MIT. These investigators showed that the nuclei of many cancer cells including pancreatic cancer and melanoma were able to support preimplantation mouse development into normal-appearing blastocysts without signs of abnormal proliferation.

• They also showed that normal blastocysts could be formed from p53 –/– breast cancer cells, and that normal blastocysts and embryonic cell lines could be formed from melanoma nuclei. These investigators concluded that the oocyte environment suppressed the malignant phenotype of the various tumor types and that tumor nuclei could direct normal development in early mouse embryos. The oocyte cytoplasm would, of course, contain normal mitochondria. It is the respiratory competent normal mitochondria that would suppress tumorigenicity. The tumor nuclei direct normal development as long as normal mitochondria are present in the cytoplasm.
• These studies also showed that embryonic stem (ES) cells derived from one of the cloned melanoma cells were able to differentiate into most if not all somatic cell lineages in chimeras including fibroblasts, lymphocytes, and melanocytes. Remarkably, normal development occurred despite the persistence of severe chromosomal changes and mutations documented by array-comparative genome hybridization (CHG).

Enhanced Differentiation and Suppressed Tumorigenicity in the Liver Microenvironment

Grisham and colleagues reported that two aneuploid liver tumor cell lines, which formed aggressive tumors when grown subcutaneously, did not form tumors when grown in the liver. The tumors became morphologically differentiated when they were transplanted and grown in the liver. It is likely the normal mitochondria in the fused cell hybrids are responsible for enhanced differentiation and suppressed tumorigenicity.

Respiration is required for the emergence and maintenance of differentiation, while loss of respiration leads to glycolysis, dedifferentiation, and unbridled proliferation.

Summary of Nuclear-Cytoplasmic Transfer Experiments

According to Warburg’s theory, it would be expected that the presence of normal mitochondria in tumor cells would restore the cellular redox status, turn off the mitochondrial stress response, and reduce or eliminate the need for fermentation to maintain viability. In rephrasing, normal mitochondrial function maintains the differentiated state thereby suppressing carcinogenesis, whereas dysfunctional mitochondria can enhance dedifferentiation thereby facilitating carcinogenesis.

(a) Normal cells beget normal cells. (b) Tumor cells beget tumor cells. (c) Delivery of a tumor cell nucleus into a normal cell cytoplasm begets normal cells despite the persistence of tumor-associated genomic abnormalities. (d) Delivery of a normal cell nucleus into a tumor cell cytoplasm begets tumor cells or dead cells, but not normal cells. The results show that nuclear genomic defects alone cannot cause tumors and that normal mitochondria can suppress tumorigenesis.

12. Abnormalities in Growth Control, Telomerase Activity, Apoptosis, and Angiogenesis Linked to Mitochondrial Dysfunction

Growth Signaling Abnormalities and Limitless Replicative Potential

A central concept in linking abnormalities of growth signaling and replicative potential to impaired energy metabolism is in recognizing that proliferation, rather than quiescence, is the default state of both microorganisms and metazoans. The default state of the cell is the condition under which cells are found when they are freed from any active control. Respiring cells in mature organ systems are largely quiescent because their replicative potential is under negative control through the action of normal mitochondrial function. Respiration maintains differentiation and quiescence. In addition, tumor suppressor genes such as p53 and the retinoblastoma protein, pRB, can also help to maintain quiescence.

• As p53 function is linked to cellular respiration, prolonged respiratory insufficiency will gradually reduce p53 function, thus inactivating the negative control of p53 and other tumor suppressor genes on cell proliferation.
• A persistent impairment in respiratory function will trigger the RTG response, which is necessary for upregulating fermentation pathways needed to maintain the GATP for viability. The RTG response will activate numerous oncogenes such as MYC, Ras, HIF-1α, Akt, and m-Tor, which are required to sustain fermentation as the prime energy source in the presence of insufficient respiration.
• Glucose and glutamine become the main energy substrates for driving fermentation through glycolysis and TCA cycle substrate-level phosphorylation. Salvadore Moncada and colleagues showed how glucose and glutamine are linked to the cell cycle and proliferation. This is important, as glucose and glutamine are the main energy metabolites needed for fermentation and tumor growth.

In addition to facilitating the metabolism of glucose and glutamine through fermentation and substrate-level phosphorylation, MYC and Ras also stimulate cell proliferation. Part of this mechanism also includes inactivation of pRB, the function of which is dependent on mitochondrial activities and the cellular redox state. Disruption of the pRB signaling pathway will contribute to cell proliferation and neoplasia. Cell proliferation is linked to fermentation, whereas quiescence is linked to respiration.

• Unlike normal cells, which engage respiration following proliferation, tumor cells remain dependent on glucose and glutamine for fermentation because their OxPhos is insufficient to maintain homeostasis. Hence, the growth signaling abnormalities and limitless replicative potential of tumor cells can be linked directly to the requirements of fermentation energy, which originates ultimately from impaired or insufficient respiration.

The greater the loss of mitochondrial function, the greater the induction of the RTG response and the greater the longevity (bud production) in yeast. As mitochondrial energy efficiency declines with age, fermentation energy becomes necessary to compensate for insufficient respiration. A reliance on fermentation becomes essential if a cell is to remain alive. A greater dependency on substrate-level phosphorylation will induce oncogene expression and unbridled proliferation, which could underlie in part the enhanced longevity in yeast. When this process occurs in mammalian cells, however, the phenomenon is referred to as neoplasia or new growth.

Linking Telomerase Activity to Cellular Energy and Cancer

Telomerase is a ribonucleoprotein enzyme complex associated with cellular immortality through telomere maintenance. Telomerase is activated in about 90% of human cancers, suggesting a role in tumorigenesis. Evidence suggests that mitochondrial dysfunction could underlie the relocation of telomerase from the mitochondria, where it seems to have a protective role, to the nucleus, where it maintains telomere integrity necessary for limitless replicative potential. Interestingly, telomerase activity is high during early embryonic development when cell proliferation is high, but telomerase activity is low in adult tissues where most cells are differentiated and quiescent.

Telomerase activity is high in normal cells or tumor cells that primarily use fermentation for energy, but the activity is low or nonexistent in nontumorigenic differentiated cells that primarily use OxPhos for energy. These findings suggest that the energy state of the cells dictates the activity level of telomerase. Elevated telomerase activity in tumor cells would therefore be an effect rather than a cause of cancer.

Evasion of Programmed Cell Death (Apoptosis)

Damage to mitochondrial energy production is one type of insult that can trigger the apoptotic cascade, which ultimately involves release of mitochondrial cytochrome c, activation of intracellular caspases, and death. In contrast to normal cells, acquired resistance to apoptosis is a hallmark of most types of cancer cells. The evasion of apoptosis is a predictable physiological response for tumor cells that primarily use fermentation and substrate-level phosphorylation for energy production following respiratory damage during the protracted process of carcinogenesis. Only those cells capable of making the gradual energy transition from respiration to fermentation in response to respiratory insufficiency will be able to evade apoptosis. Cells unable to make this energy transition will die and thus never become tumor cells.

Numerous findings indicate that the genes and signaling pathways needed to upregulate and sustain fermentation are themselves antiapoptotic. For example, sustained glycolysis or glutamine fermentation requires participation of mTOR, MYC, Ras, HIF-1a, and the IGF-1/PI3K/Akt signaling pathways.

• The upregulation of these genes and pathways together with inactivation of tumor suppressor genes such as p53, which is required to initiate apoptosis, will disengage the apoptotic signaling cascade thus preventing programmed cell death.
• Abnormalities in the outer mitochondrial membrane and inner membrane potential can also induce expression of known antiapoptotic genes (Bcl2 and Ccl-X L). Tumor cells will continue to evade apoptosis as long as they have access to glucose and glutamine, which are required to maintain fermentation energy. Glycolytic tumor cells, however, can readily express a robust apoptotic phenotype if their glucose supply is targeted.

Sustained Vascularity (Angiogenesis)

Angiogenesis is required for most tumors to grow beyond an approximate size of 0.2–2.0 mm. This is necessary to provide the tumor with essential energy nutrients, including glucose and glutamine, and to remove toxic tumor waste products such as lactic acid and ammonia.

In addition to its role in upregulating glycolysis in response to hypoxia, HIF-1α is also the main transcription factor for vascular endothelial growth factor (VEGF), which stimulates angiogenesis. HIF-1α is part of the IGF-1/PI3K/Akt signaling pathway that also indirectly influences the expression of βfibroblast growth factor (FGF), another key angiogenesis growth factor.

The sustained vascularity of tumors can be linked mechanistically to the metabolic requirements of fermentation and substrate-level phosphorylation necessary for tumor cell survival.

13. Metastasis

Metastasis Overview

Metastasis is the term used to describe the spread of cancer cells from the primary tumor to surrounding tissues and to distant organs and is the primary cause of cancer morbidity and mortality. It is estimated that metastasis is responsible for about 90% of cancer deaths. Although systemic metastasis is responsible for 90% of cancer deaths, most research in cancer does not involve metastasis in the in vivo state.

In order to complete the metastatic cascade, cancer cells must detach from the primary tumor, intravasate into the circulatory and lymphatic systems, evade immune attack, extravasate at distant capillary beds, and invade and proliferate in distant organs. Metastatic cells also establish a microenvironment that facilitates angiogenesis and proliferation, resulting in macroscopic, malignant secondary tumors.

The key phenotype of metastasis is that the tumor cells spread naturally from the primary tumor site to secondary locations. Nevertheless, numerous investigators use intravenous tumor cell injection models to study metastasis. While these models can provide information on tumor cell survival in circulation, it is not clear if this information is relevant to survival of naturally metastatic tumor cells.

Cellular Origin of Metastasis

Epithelial to Mesenchymal Transition (EMT):

• The epithelial to mesenchymal transition (EMT) posits that metastatic cells arise from either epithelial stem cells or differentiated epithelial cells through a stepwise accumulation of gene mutations that eventually transform the epithelial cell into a tumor cell with mesenchymal features. This idea comes from findings that many cancers arise in epithelial tissues where abnormalities in cell–cell and cell–matrix interactions occur during tumor progression. Eventually, neoplastic cells emerge that appear as mesenchymal cells, which lack cell–cell adhesion, are dysmorphic in shape, and eventually spread to distant organs.
• Recent studies also suggest that misplaced (ectopic) co-expression of only two genes might be all that is necessary to facilitate EMT in some gliomas, though the process is highly complex. However, considerable controversy surrounds the EMT hypothesis of metastasis, as EMT is not often detected in tumor pathological preparations. The EMT is primarily considered a phenomenon of the in vitro environment. It remains debatable whether this in vitro model of metastasis has an in vivo counterpart.
• The recapitulation of epithelial characteristics at distant secondary sites is referred to as the mesenchymal–epithelial transition (MET) and is thought to involve a reversal of the changes responsible for the EMT. No explanation has appeared on how the genomic instability and multiple point mutations and chromosomal rearrangements responsible for the neoplastic mesenchymal phenotype could be reversed or suppressed when the tumor cells recapitulate the epithelial phenotype at distant sites.
• The massive complexity associated with the EMT hypothesis is largely man-made, especially in attempting to describe the phenomenon as a gene-driven process. If one looks closely, many of the gene expression profiles observed in metastatic cancers are similar to those associated with the function of macrophages or other fusogenic cells of the immune system. Many gene changes associated with EMT can also be found in most benign tumors. 

Stem Cell Origin of Metastasis:

• Most tissues contain cells in semi-differentiated states that can replace dead or damaged cells due to natural wear and tear. These undifferentiated or semi-differentiated cells are often referred to as tissue stem cells and are considered by many to be the origin of metastatic cancers.
• Observations that tumor cells express characteristics of undifferentiated stem cells come from the fact that embryonic stem cells and tumor cells largely use anaerobic energy (fermentation) for metabolism.
• As stem cells are known for their ability to proliferate and migrate during tissue morphogenesis and differentiation, it was reasonable to assume that genetic damage to stem cells could give rise to metastatic cancers in various tissues. However, many tumor cells with stem cell properties do not express systemic metastasis. Indeed, many of the chemically induced brain tumors Seyfried developed in mice express stem cell properties but do not display extensive invasion or metastasis. Most of these tumors also express several of the Hanahan–Weinberg cancer hallmarks. However, only those tumor cells that expressed characteristics of macrophages showed systemic metastasis.
• While metastatic cancers can express properties of stem cells, expression of stem cell properties alone is not synonymous with expression of distant invasion and metastasis. Tumors derived from hematopoietic stem cells, however, may be an exception. Hematopoietic stem cells can give rise to myeloid cells, which we consider the cellular origin of most metastatic cancers.
• According to his hypothesis, metastatic cancers arise from respiratory insufficiency in hematopoietic stem cells or their lineage descendants, for example, macrophages or lymphocytes. Chronic hypoxia in the inflamed microenvironment can permanently damage mitochondrial respiration in activated macrophages.

Myeloid Cells as the Origin of Metastasis:

• Neoplastic brain macrophages (microglia) could also represent the most highly invasive cells in glioblastoma. The myeloid cell origin of metastasis proposes that metastatic cancer cells arise from myeloid cells regardless of tissue origin.
• Myeloid cells are already mesenchymal cells and would therefore not require the complicated genetic mechanisms proposed for the EMT in order to metastasize. Macrophages arise from the myeloid lineage and have long been considered the origin of human metastatic cancer. Macrophages can fuse with epithelial cells within the inflamed microenvironment, thus manifesting properties of both the epithelial cell and macrophage in the fusion hybrids.
• According to Seyfried’s hypothesis, it is hematopoietic stem cells themselves or their lineage descendants that become the metastatic cells either through direct transformation in the inflamed microenvironment or through their fusion with neoplastic tumor cells.
• The macrophages present in tumors are generally referred to as tumor-associated macrophages (TAMs) and often comprise significant numbers of the inflammatory cell infiltrate in tumors. TAMs also establish the premetastatic niche, while enhancing tumor inflammation and angiogenesis. The properties of TAM facilitate tumor development and progression. While some TAMs are certainly part of the stroma, they recently reviewed evidence showing that human tumors also contain neoplastic cells with macrophage properties.
• Seyfried suggests that rodent tissues respond to tumor implants as if they were an acute infection or wound. This would involve invasion of TAM and activation of local macrophages. It is also possible that fusion hybrids would form between tumor cells and host macrophages, but this might not cause damage to the macrophage mitochondria.
• In contrast to the acute situation in mice, neoplastic transformation is a protracted process in humans. In other words, murine myeloid cells respond acutely to the tumor implant, whereas human myeloid cells in the inflamed microenvironment respond chronically to the tumor initiating insult. It is not really clear why highly metastatic carcinomas, similar to those seen in humans, are rarely found in experimental rodent tumors. There are, however, some exceptions.

Macrophages and Metastasis

Macrophages are among the most versatile cells of the body with respect to their ability to migrate, to change shape, and to secrete growth factors and cytokines. These macrophage behaviors are also the recognized behaviors of metastatic cells. Macrophages manifest two distinct polarization phenotypes: the classically activated (M1 phenotype) and the alternatively activated (M2 phenotype). Macrophages acquire the M1 phenotype in response to proinflammatory molecules and release inflammatory cytokines, reactive oxygen species, and nitric oxide. In contrast, macrophages acquire the M2 phenotype in response to anti-inflammatory molecules such as IL-4, IL-13, and IL-10 and to apoptotic cells. M2 macrophages promote tissue remodeling and repair but are immunosuppressive and poor antigen presenters. Although the M1 and M2 macrophages play distinct roles during tumor initiation and malignant progression, macrophage–epithelial cell fusions can involve either activation state.

M1 macrophages facilitate the early stages of tumorigenesis through the creation of an inflammatory microenvironment that can produce nuclear and mitochondrial damage. However, TAM can also undergo a phenotypic switch to the M2 phenotype during tumor progression. The TAM population comprising M2 macrophages scavenge cellular debris, promote tumor growth, and enhance angiogenesis. M2 macrophages also fuse with tumor cells and are considered facilitators of metastasis.

Several human metastatic cancers express multiple molecular and behavioral characteristics of macrophages, including phagocytosis, cell–cell fusion, and antigen expression. Tarin also considers the expression of osteopontin (OPN) and CD44 as important in the regulatory gene group/network associated with metastasis. This is interesting as there is strong evidence that both OPN and CD44 are expressed in monocytes and macrophages under various physiological and pathological states.

Macrophages express most hallmarks of metastatic tumor cells when responding to tissue injury or disease. For example, monocytes (derived from hematopoietic bone marrow cells) extravasate from the vasculature and are recruited to the wound via cytokines released from the damaged tissue. Within the wound, monocytes differentiate into alternatively activated macrophages and dendritic cells where they release a variety of proangiogenic molecules, including vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor. M2 macrophages also actively phagocytize dead cells and cellular debris. Occasionally, macrophages undergo homotypic fusion resulting in multinucleated giant cells with increased phagocytic capacity. Following these wound healing activities, macrophages intravasate back into the circulation where they travel to the lymph nodes to participate in the immune response.

Phagocytosis: A Shared Behavior of Macrophages and Metastatic Cells

Phagocytosis involves the engulfment and ingestion of extracellular material and is a specialized behavior of M2 macrophages and other professional phagocytes. This process is essential for maintaining tissue homeostasis by clearing apoptotic cells, cellular debris, and invading pathogens. Like M2 macrophages, many malignant tumor cells are phagocytic both in vitro and in vivo.

Fais and colleagues provided dramatic evidence of tumor cell phagocytosis in showing how malignant melanoma cells eat T-cells. This is remarkable as T-cells are thought to target and kill tumor cells.

There is also evidence that some tumor cells can eat NK cells. If macrophage-derived metastatic cells can eat T-cells and possibly NK cells, then it is possible that immune therapies involving these cells might not be effective for long-term management of some metastatic cancers.

Melanocytes are the resident macrophages of the skin. Expression of cathepsins B and D are elevated in the phagocytic melanoma cells just as they are in malignant melanomas. These tumor cell phagocytic/cannibalistic behaviors are not to be confused with autophagy; a cellular self-digestion process often associated with starvation conditions. Many human cancers and some murine cancers can phagocytize other tumor cells, erythrocytes, leukocytes, platelets, dead cells, as well as extracellular particles. Hence, the characteristics of phagocytosis appear similar in resident skin macrophages and in malignant melanoma.

Phagocytic Cancers:

• While extracranial metastasis of central nervous system tumors is not common, many gliomas, especially glioblastoma multiforme, are highly metastatic if the tumor cells gain access to extra-neural tissue. Several investigators have documented the metastatic behavior of malignant brain cancers, especially GBM.
• Extracranial metastasis of brain tumors portends an extremely poor survival, with the vast majority of patients surviving <6 months from the diagnosis of metastatic GBM disease. The widely held view that metastasis does not occur for GBM should be reevaluated. Many GBM patients often die before detection of systemic metastasis. A take-home message is that GBM patients should not donate their organs.
• While it might be difficult to prove a myeloid origin of invasive GBM cells, substantial evidence shows that subpopulations of neoplastic GBM cells display the phagocytic behavior of macrophages/microglia. As microglia are the resident macrophages of the brain, they considered that some of the cells in those tumors could arise from neoplastic microglia/macrophages. Human GBM contains mixtures of numerous neoplastic cell types, many of which have mesenchymal properties, do not express glial fibrillary acidic protein (GFAP), and are of unknown origin.
• According to his hypothesis, many of the neoplastic mesenchymal cells seen in GBM arise from transformed macrophages or microglia that fuse with neoplastic stem cells. Such hybrid cells would represent the most invasive neoplastic cells within the tumor. It also appears that bevacizumab (Avastin) selects the most invasive cells within GBM. This could account for why tumor recurrence following bevacizumab therapy is universally fatal.
• Phagocytic behaviors have been reported for many human cancers including skin, breast, lymphoma, lung, brain, ovarian, pancreatic, renal, endometrial, rhabdomyosarcoma, myeloma, fibrosarcoma, and bladder. For most of these tumors, the phagocytic phenotype was restricted primarily to those cells that were also highly invasive and metastatic. Hence, the most potentially deadly cells within tumors are those with macrophage properties.
• Numerous phagocytic tumor cells were identified within metastatic breast cancer lesions and were not observed within the primary tumor of the same patient. This is similar to the appearance of phagocytic signet-ring cells observed in secondary metastatic lesions in other breast cancer patients. Additionally, the number of phagocytic tumor cells present within the tumor stroma correlates with breast cancer malignancy and grade. Hence, phagocytosis is a common macrophage phenotype seen in many metastatic human cancers.

Metastatic Behavior of the RAW 264.7 Mouse Macrophages:

• RAW 264.7 cells are considered a normal mouse macrophage cell line and are widely used to study a broad range of macrophage properties. RAW 264.7 cells were transformed with Abelson leukemia virus and derived from BALB/c mice. It is known that viruses damage mitochondrial function.
• He used the RAW 264.7 cells as a control cell line for his metastatic VM-M2 and VM-M3 metastatic cancer cells. Using fluorescent microspheres, he found that the phagocytic activity of the metastatic VM-M2 and VM-M3 tumor cells was similar to that of the RAW 264.7 macrophage cell line. Not only were there similarities between the RAW cells and the metastatic VM tumor cells for phagocytic behavior, but these cells also were similar in their morphology, gene expression, and lipid composition.
• He then asked his students to implant the RAW 264.7 cells subcutaneously into the flanks of immunodeficient BALB/SCID mice to confirm that they did not form tumors. The RAW 264.7 cells not only formed tumors, but the cells from these tumors also metastasized throughout the mice. They discovered that the RAW 264.7 macrophage cell line is highly metastatic following intracerebral and subcutaneous transplantation into SCID mice.
• The metastatic behaviors of the RAW cells also appear similar to the metastatic behaviors of the tumors described by Kerbel et al. Like the VMM2 and VM-M3 tumor cell lines, the RAW 264.7 cells express little ganglioside GM3 and metastasize to multiple organ systems (liver, spleen, kidney, lung, and brain) when grown subcutaneously outside the brain. Ganglioside GM3 inhibits angiogenesis and blocks tumor cell invasion.
• These findings provided direct evidence showing that cells with macrophage properties can give rise to metastatic cancer regardless of how the cells are classified.
• Moreover, their findings suggest that a single mechanism was responsible for the metastatic phenotype regardless of cell origin. They think that this mechanism involves fusion hybridization followed by or commensurate with mitochondrial damage and respiratory insufficiency.

Fusogenicity: A Shared Behavior of Macrophages and Metastatic Cells:

• Fusogenicity is the ability of a cell to fuse with another cell through the merging of their plasma membranes. This process can arise in vitro as is seen with the formation of antibody-producing hybridomas. However, fusion in human cells is a highly regulated process that is essential for fertilization (sperm and egg) and skeletal muscle (myoblasts), and placenta (trophoblast) formation. Outside of these developmental processes, cell-to-cell fusion is normally restricted to differentiated cells of myeloid origin. During differentiation, subsets of macrophages fuse with each other to form multinucleated osteoclasts in bone or multinucleated giant cells in response to foreign bodies. Osteoclasts and giant cells have increased cell volume that facilitates the engulfment of large extracellular materials.
• Macrophages are also thought to fuse with damaged somatic cells during the process of tissue repair. In addition to homotypic fusion, macrophages are known to undergo heterotypic fusion with tumor cells.
• Wong and coworkers described how macrophages fuse with tumor epithelial cells. Besides inflammation, radiation also increases the fusion hybrid process. Is it possible that decreased long-term survival in some irradiated cancer patients results from enhanced production of macrophage–epithelial fusion hybrids? They have stated that the human brain should rarely if ever be irradiated.
• Fusion between neoplastic tumor cells and myeloid cells, with subsequent nuclear fusion, could produce new phenotypes in the absence of new mutations, as the hybrids would express genetic and functional traits of both parental cells. These neoplastic hybrids would have the ability of macrophages to intravasate, extravasate, and migrate to distant organs, while also possessing the unlimited proliferative potential of the cancer cells. Since myeloid cells are part of the immune system, it would be easy to see how tumor hybrids would also be able to evade immune surveillance.

Fusogenic Cancers:

• Fusogenic tumor cells are found in a wide variety of cancer types including melanoma, breast, renal, liver, gall bladder, lymphoma, and brain. Tumor cell hybrids can form either in vitro or in vivo from fusions between two tumor cells or between a tumor cell and a normal somatic cell.
• Artificial fusions of human monocytes and mouse melanoma cells revealed that the resulting hybrids expressed both human and mouse genes. Other investigators also showed that the macrophage-specific antigens F4/80 and Mac-1 were expressed in murine Meth A sarcoma cells after spontaneous in vivo fusion with host cells. Interestingly, latex bead phagocytosis was also expressed in the Meth A sarcoma–host cell fusion hybrids. Since these fusion hybrids expressed genotypes and phenotypes of both parental cells, it appears that the nonmetastatic tumor cells could acquire an invasive/metastatic phenotype without new mutations.
• TAMs promote tumor progression through the release of cytokines and proangiogenic and prometastatic molecules. However, the fusion of cancer cells with tissue macrophages could also accelerate tumor progression. Fusion among tumor cells in human solid tumors is difficult to detect. Several reports provide evidence for fusions between tumor cells and myeloid cells in human bone marrow transplant (BMT) recipients. Such fusions would accelerate tumor progression.
• It is important to recognize that both radiation therapy and immunosuppression can increase the incidence of metastatic cancers. DNA analysis of micro-dissected metastatic cells from a child diagnosed with renal cell carcinoma after a BMT revealed DNA from both the BMT donor and the recipient in the metastatic cells. Bone marrow and tumor cell hybrids were also identified in a female, who developed renal carcinoma after receiving a BMT from a male donor. These reports provided genetic evidence that spontaneous fusions can occur between human myeloid cells and tumor cells. It should not be surprising that the radiation therapy given to many cancer patients might actually exacerbate the formation of metastases and disease progression.
• Macrophage–macrophage fusions could also induce aneuploidy in the fused hybrids. The numerous in vitro studies and in vivo reports suggest that myeloid hybrids could be responsible for the metastatic progression of numerous cancers. Multinucleated giant cells, a signature of hybrid formation, are frequently seen in human cancers, suggesting that cell fusions are not rare events. Regardless of the mechanism, metastatic cells express numerous behaviors of mesenchymal/myeloid cells and, if exploited, could generate novel therapeutic strategies for managing metastatic cancers.

Myeloid Biomarkers Expressed in Tumor Cells:

• Since TAMs are often correlated with a poor patient prognosis, tumor biopsies are frequently evaluated for macrophage markers. The macrophage antigen-expressing cells within the tumor stroma are usually classified as TAMs. However, several reports show that macrophage-specific antigens and biomarkers are also expressed on a wide variety of human cancer cells.
• Ruff and Pert demonstrated that several macrophage antigens (CD26, C3bi, and CD11b) were expressed on tumor cells from small cell lung carcinoma (SCLC). Levels of expression were comparable to that seen in the monocyte controls. It is important to note that the macrophage antigens were also expressed in the cultured tumor cells themselves. This tumor cell expression was confirmed from in vivo tissue preparations. This eliminated the possibility that the antigen expression was derived from TAMs. These investigators concluded that the SCLC tumor cells in their specimens were not of lung epithelial origin but rather were of “myeloid origin.” A malignant transformation of recruited myeloid cells, from smoking-related tissue damage, was offered as an explanation for the origin of tumor cells with myeloid/macrophage properties.
• It is also possible that the myeloid properties of the SCLC were derived from fusions of macrophages and neoplastic lung epithelial cells.
• Besides SCLC, myeloid-associated antigens (CD14 and CD11b) were also expressed in five metastatic breast cancer cell lines. None of the breast cancer cell lines, however, expressed markers for B- or T-cells. The authors suggested that common antigen sharing between different cell types could be related to common cellular interactions. Further evidence for a mesenchymal origin of metastatic cancer comes from tissue microarray analysis of 127 breast cancer patients. The CD163 macrophage scavenger receptor was expressed on the tumor cells of 48% of the patients, while MAC387 macrophage marker was expressed on the tumor cells of 14% of the patients.
• As in breast cancer patients, CD163 was expressed on tumor cells in many patients with rectal cancer. CD163 expression was found in 31% of the rectal tumors from patients in the preoperative irradiation group but was expressed in only 17% in the non-irradiation group. Prognosis was also worse for those patients with CD163-positive cancer cells than in those patients with CD163-negative cancer cells. Inflammation and radiation are known to enhance formation of macrophage–epithelial cell fusion hybrids.
  • Maniecki et al. presented findings at the 2011 meeting of the Amer. Assoc. Cancer Res. (AACR) showing that expression of CD163 could be a common phenotype of many metastatic cancers arising from heterotypic cell fusions between tumor cells and macrophages. The findings in these metastatic cancers are consistent with the origin of metastatic cells from macrophage fusion hybrid cells, which are increased by radiation and inflammation.
• Although radiation therapy can help some cancer patients, radiation therapy will also enhance mitochondrial damage and fusion hybridization, thus potentially making the disease much worse. These findings are consistent with the role of radiation in inducing tumor cell–macrophage fusions and in exacerbating the metastatic properties of some cancers.
• It also appears that some antiangiogenic drugs such as bevacizumab and cediranib actually increase the number of invasive cells with macrophage properties in brain tumors. In light of the findings presented here, he suggests that these drugs select for invasive tumor cells with macrophage properties. This would not be beneficial to patients. Viewed together, these studies demonstrate that macrophage antigens, which are associated with enhanced metastasis and poor prognosis, are expressed on the tumor cells of patients with breast, bladder, rectal, and brain cancers.

Cathepsins, Ezrin, and E-Cadherin:

• Macrophages express high levels of lysosomal-enriched cathepsins, which facilitate the digestion of proteins ingested following phagocytosis or pinocytosis. This is interesting since lysosomal cathepsins D and B are viewed as prognostic factors in cancer patients. Indeed, a high content of these enzymes in tumors of the head and neck, breast, brain, colon, or endometrium was considered a sign for high malignancy, high metastasis, and overall poor prognosis.
• Activated macrophages also express ezrin as part of a protein complex with radixin and moesin. The ezrin–radixin–moesin is a family of molecules that play essential roles in tissue remodeling by linking the cell surface with the actin cytoskeleton and facilitating signal-transduction pathways. There is increasing awareness that ezrin is also expressed in metastatic cancer cells, suggesting an important role in metastatic phenotype of cancer cells.
• The transition from the EMT is associated with downregulation of the cell adhesion molecule, E-cadherin. It is important to recognize that E-cadherin is either unexpressed or expressed in low levels in macrophages.

Anemia and Increased Hepcidin in Metastatic Cancer:

• Iron-deficient anemia is a comorbid trait in many patients with metastatic cancers. Hepcidin is a key regulator of iron metabolism and plasma iron levels by controlling the efflux of iron from enterocytes, hepatocytes, and macrophages and by internalizing and degrading the iron exporter, ferroportin. Chris Tselepis, Douglas Ward, and colleagues suggested that hepcidin contributes to the systemic anemia in colorectal cancer patients by acting at the level of the macrophage.
• Activated macrophages express IL-6, which induces expression of hepcidin. Macrophages are the major cell type responsible for systemic iron recycling. The findings of Ward et al. are therefore consistent with their hypothesis that metastatic cancer is a disease of myeloid cells, especially macrophages. Many characteristics of metastatic cancers can be explained once it becomes recognized that metastatic cancer is a macrophage metabolic disease. Hence, iron-deficient anemia should not be unexpected for metastatic cancers derived from transformed macrophages or macrophage fusion hybrids.

Carcinoma of Unknown Primary Origin

Carcinoma of unknown primary (CUP) origin is a systemic metastatic disease without an identifiable primary tumor and is often associated with poor prognosis. Approximately 5% of all newly diagnosed cancers are classified as CUP. These cancers are often classified as adenocarcinomas, squamous cell carcinomas, poorly differentiated carcinoma, and neuro-endocrine carcinomas. It is thought that these cancers metastasize before the primary tumor has had time to develop into a macroscopic lesion. Signet-ring cells were found in some CUP, indicating that subsets of these cancers exhibit phagocytic behavior like other metastatic cancers. Aneuploidy was identified in 70% of CUP adenocarcinomas but was not found in about 30% of the tumors. Aneuploidy can arise in part from cell fusion events. Survival was better in patients with aneuploid tumors than with diploid tumors, showing that patients with diploid tumors do not have a more favorable prognosis. This is interesting and is consistent with findings that aneuploidy actually slows cell growth. Owing to their high aggressiveness, they suggested that some CUPs could arise from macrophage fusion hybrids.

Many Metastatic Cancers Express Multiple Macrophage Properties

Many phagocytic or fusogenic tumors also express myeloid antigens, further supporting a myeloid origin of these metastatic cancers. It is important to mention that the myeloid properties are expressed in the tumor cells themselves and should not be confused with myeloid properties expressed in TAM, which are also present in the tumors but are not part of the neoplastic cell population.

The macrophage cell fusion explanation of metastasis does not require the induction and reversion of extremely complicated gene regulatory systems.

Linking Metastasis to Mitochondrial Dysfunction

When permanent respiratory damage occurs in cells of myeloid origin, including hematopoietic stem cells and their fusion hybrids, metastasis would be a potential outcome. Numerous studies indicate that mitochondria from a broad range of metastatic cancers are abnormal and incapable of generating energy through normal respiration.

Mitochondrial damage can arise in any cell within the inflammatory microenvironment of the incipient tumor, including TAM, homotypic fusion hybrids of hematopoietic cells, or heterotypic fusion hybrids of macrophages and neoplastic epithelial cells. The end result would be cells with metastatic potential. Although metastatic cells will differ in their morphology from one organ system to the next, they all suffer from insufficient respiration. The origin of metastatic cancer from myeloid cells and fusion hybrids can explain the substantial morphological and genetic diversity seen among different tumor types. It is clear that metastasis can arise in macrophage fusion hybrids that sustain irreversible mitochondrial damage.

What About the Tumor Suppressive Effects of Mitochondria?

• Although normal mitochondria would initially suppress tumorigenicity in fused hybrids, persistent inflammation in the microenvironment will eventually damage the majority of mitochondria in the fused hybrids, thus initiating the path to metastasis. As macrophages also evolved to survive in hypoxic and inflammatory environments, considerable time would likely be required to damage mitochondria in the fusion hybrids from these environments. However, progressive damage to mitochondria in fusion hybrids would be unlikely in nonhypoxic or noninflammatory microenvironments. It is also noteworthy that radiation exposure would not only enhance fusion hybrid formation but would also damage respiration. It should not be surprising why long-term survival is reduced or why more aggressive tumors recur in many patients that receive radiation to treat their cancers.
• As respiration is responsible for maintaining genomic stability and the differentiated state, respiratory insufficiency will eventually induce the default state of unbridled proliferation. If this occurs in cells of myeloid origin such as macrophages, then emergence of cells with enhanced metastatic potential would be a predicted outcome. Macrophages are genetically programmed to exist in the circulation and to enter and exit tissues.

Revisiting the “Seed and Soil” Hypothesis of Metastasis

Metastatic tumor cells do not invade distant organs randomly, but invade in a nonrandom pattern with lung, liver, and bone as primary sites of metastases.

Macrophages enter and engraft tissues in a nonrandom manner. Macrophages are genetically programmed to exist in circulation and to preferentially enter various tissues during wound healing and the replacement of resident myeloid cells. Some macrophage populations in the liver are regularly replaced with bone marrow-derived monocytic cells, whereas other macrophage populations are more permanent and require fewer turnovers. It is reasonable to assume that metastatic cancer cells derived from macrophages or fusions of monocytic cells with epithelial cells will also preferentially home to those tissues that naturally require regular replacement of resident macrophages.

Macrophage turnover should be greater in tissues such as liver and lung where the degree of bacterial exposure and the wear and tear on the resident macrophage populations is considerable. This could explain why these organs are a preferred soil of many metastatic cancer cells. Bone marrow should also be a common target of metastatic cells because this site is the origin of the hematopoietic stem cells, which give rise to myeloid cells. Liver, lung, and bone are also preferential sites for metastatic spread for the VM mouse tumor cells.

Because the metastatic cells express insufficient respiration with compensatory fermentation, these cells will enter their default state of proliferation, as would any neoplastic cell. In addition to those organs receiving high macrophage turnover, macrophages also target sites of inflammation and injury. This is interesting in light of findings showing that metastatic cancer cells from lung and breast can appear in the mouth following recent tooth extraction or along needle tracts following biopsy. An unhealed wound is an ideal “soil” for macrophage infiltration. This phenomenon is referred to as inflammatory oncotaxis and can explain in part the seed and soil hypothesis. If metastasis were a metabolic disease of myeloid cells, then the appearance of metastatic cells in recent tooth extraction or wounds would not be unexpected.

Revisiting the Mesenchymal Epithelial Transition (MET)

In contrast to the EMT, the MET involves proliferation and expression of epithelial characteristics following extravasation, invasion, and proliferation at distant cites. The MET is considered a reversibility of the EMT. How is it possible that a series of somewhat random somatic mutations orchestrate the sophisticated series of behaviors associated with the EMT and then have most of these behaviors reversed during the MET? An origin from myeloid cells provides a more credible explanation of metastasis.

Metastatic cells arising from myeloid cell fusions would retain the genetic architecture necessary for entering and exiting the circulation at recognized sites. It is not necessary to construct complicated mutation-based regulatory systems to explain these phenomena. Macrophages naturally enter and exit the circulatory and lymphatic systems. The circulatory system is not a “hostile” environment for cells in the macrophage lineage. These cells also express the cell-surface adhesion molecules (selectins) necessary for extravasation at designated organs. They already express the batteries of metalloproteases necessary for degradation of basement membranes and invasion. When these capabilities occur together with impaired respiration, dysregulated proliferation would be an expected outcome. While these properties certainly implicate myeloid cells as the origin of metastatic cells, the fusogenic properties of myeloid cells can also explain how metastatic cells can recapitulate the epithelial characteristics of the primary tumor at secondary growth sites.

Wong and colleagues showed how macrophage/epithelial cell hybrids could recapitulate phenotypes of epithelial cells, while retaining the properties of macrophages. It is clear that phenotypes of epithelial cells and macrophages can be maintained in fusion hybrids of macrophages and intestinal epithelial tumor cells. Moreover, these characteristics are passed on to daughter cells through somatic inheritance.

Fusions of activated macrophages with epithelial cells in the primary tumor microenvironment will bestow the capability of the fused cells to degrade basement membranes, to enter and exit the circulatory and lymphatic systems, and to recapitulate the epithelial characteristics of the primary tumor at distant secondary sites. The dysregulated growth at secondary sites is the consequence of damaged respiration in these cells. Hence, the origin of metastatic cells from macrophage fusion hybrids with dysfunctional mitochondria can explain the phenomenon of metastasis.

Genetic Heterogeneity in Cancer Metastasis

Considerable genetic heterogeneity is observed by comparing tumor tissue from primary growth sites with tissue from distant metastases. Genetic heterogeneity is seen not only between patients with similar tumor histopathology but also for the tumors growing at different sites within the same patient.

Almost every type of genetic heterogeneity imaginable from point mutations to major genomic rearrangements can be found in metastatic and highly invasive cancers, including those from breast, brain, and pancreas. The mostly nonuniform distribution of mutations in these tumors is consistent with findings that each neoplastic cell within a given tumor can have a profile of changes uniquely different from any other cell within the tumor. Moreover, if the spread of metastatic cells to some organs (such as liver and lung) occurs earlier than spread to other organs, it is possible that genetic heterogeneity would be greater in these organs than in organs that receive metastatic cells later in the disease progression. This is expected if the number of divisions is greater for tumor cells that arrive earlier in these organs than for tumor cells that arrive later in other organs. This could explain why genomic heterogeneity is more diverse in some organs than in other organs or in the primary tumor.

The richness is the likely consequence of damaged respiration in populations of fusion hybrids that differ from each other in genetic architecture. A nonuniform or random distribution of mutations arises from the migration of these hybrid cells to other organs. The driver of the metastatic phenomenon is not a gene but is respiratory insufficiency in macrophages or their fusion hybrids. The gene mutations arise as downstream epiphenomena.

Fusion hybrid hypothesis of cancer cell metastasis. According to their hypothesis, metastatic cancer cells arise following fusion hybridization between neoplastic epithelial cells and myeloid cells (macrophages). Macrophages are known to invade in situ carcinoma as if it were an unhealed wound. This creates a protracted inflammatory microenvironment leading to fusion hybridization between the neoplastic epithelial cell and the macrophage. Fusion hybridization can explain the phenomenon of EMT without invoking new mutations. Inflammation damages mitochondria leading to enhanced fermentation and acidification of the microenvironment. Mitochondrial damage becomes the driver for the neoplastic transformation of the epithelial cell and of the fusion hybrids. As macrophages are already mesenchymal cells that naturally possess the capability to enter (intravasate) and exit (extravasate) the circulation, the neoplastic fusion hybrid will behave as a rogue macrophage. The fusogenic properties of macrophage cells can also explain how metastatic cells can recapitulate the epithelial characteristics of the primary tumor at secondary micrometastatic growth sites. This process can explain the phenomenon of MET without invoking a mutation suppression mechanism.

Transmissible Metastatic Cancers

The best known is the canine transmissible venereal tumors and the Tasmanian devil tumor disease (DFTD). These tumors will often spread from the primary site of contact to distant organs. The metastatic behavior of these transmissible tumors is basically the same as that seen for the nontransmissible human metastatic cancers.

Murchison et al. have recently shown that DFTD originated from cells expressing Schwann cell and epithelial characteristics. It is important to mention that hematopoietic bone marrow cells can elaborate Schwann cell-like phenotypes in injury conditions.

Like all cancers, mitochondrial dysfunction and respiratory insufficiency would be the expected driver phenotype of these transmissible cancers. However, Tasmanian devils living in the western part of the island are resistant to the disease. It appears that the resistance results from a unique DNA polymorphism in the mitochondria of these animals. This is interesting in light of findings showing that transmissible cancers will occasionally acquire mitochondria from the host.

The Absence of Metastases in Crown-Gall Plant Tumors

Crown-gall tumors arise from bacterial infections that enter damaged areas of the plant leading to plant cell proliferation. The mechanisms by which bacteria induce crown-gall disease in plants are similar to those by which viruses induce tumors in animals. Defects in mitochondrial morphology and energy metabolism were later described in crown-gall tumors.

Crown-gall tumors express four of the Hanahan and Weinberg hallmarks of cancer, that is, self-sufficiency in growth signals, insensitivity to growth inhibitory (antigrowth) signals, evasion of programmed cell death (apoptosis), and limitless replicative potential. However, these tumors do not express invasion or metastasis. With the exception of metastasis and invasion, the abnormalities in growth and physiology are similar in crown-gall disease and in animal tumors.

The crown-gall tumors do not metastasize because they do not have macrophages or myeloid cells as part of their immune system. The findings in crown-gall are also consistent with Tarin’s hypothesis, “that metastasis cannot occur until an organism has evolved the genes for lymphocyte trafficking.”

Chapter Summary

Seyfried proposes that the metastatic mesenchymal phenotype arises primarily from respiratory damage in macrophages or in epithelial–macrophage fusion hybrids. Inflammation and radiation damage enhances hybridization, while also damaging mitochondrial function over time. It is my opinion that the myeloid origin of metastasis is the most compelling explanation for the origin of metastasis and tumor progression.

14. Mitochondrial Respiratory Dysfunction and the Extrachromosomal Origin of Cancer

He discussed in Chapters 7 and 8, amino acid fermentation and anaerobic respiration in tumor mitochondria can give the appearance that aerobic respiration is normal when, in fact, it is not. In Chapter 13, he discussed how mitochondrial dysfunction can account for the phenomenon of metastasis in macrophage fusion hybrids and, in Chapters 7 and 8, how amino acid fermentation might simulate OxPhos. This evidence more strongly supports cancer as a metabolic disease than as a genetic disease.

Connecting the Links

Any unspecific condition that damages a cell’s respiratory capacity, but is not severe enough to kill the cell, can potentially initiate the path to a malignant cancer. Reduced respiratory capacity could arise from damage to any mitochondrial protein, lipid, or mtDNA. Some of the many unspecific conditions that can damage a cell’s respiratory capacity thus initiating carcinogenesis include inflammation, carcinogens, radiation (ionizing or ultraviolet), intermittent hypoxia, rare germline mutations, viral infections, and age.

Inflammation has long been recognized in the initiation and promotion of cancer. Inflammation produces ROS and elevates TGF-β, which damages mitochondria while disrupting tissue morphogenetic fields.

Besides producing mutations, carcinogens also produce ROS. Carcinogens, in addition to causing mutations, disrupt OxPhos and cause permanent injury to mitochondria. It is this effect of the carcinogen on mitochondrial energy production rather than its mutagenic effect that primarily initiates cancer. It is unfortunate that the Ames tests focused only on the mutagenic effects of carcinogens rather than on the mitochondrial damaging effects of these compounds.

Radiation not only causes mutations, but also injures mitochondria. Radiation causes necrotic cell death and inflammation. It is the production of ROS and the injurious effect of radiation on OxPhos that causes cancer. While radiation can certainly kill cancer cells, radiation can also initiate cancer through its effect on the mitochondrial energy production.

Similar to inflammation, hypoxia produces high levels of ROS in the microenvironment, which will damage mitochondrial respiratory capacity thus facilitating cancer initiation and progression.

The accumulation of ROS with age damages mitochondrial respiratory energy production. If mitochondrial damage underlies the origin of cancer according to his central hypothesis, then it is predictable that cancer risk should increase with age.

Rare germline mutations increase cancer risk through a direct effect on mitochondrial function. Hence, the plethora of nonspecific factors known to increase the risk of cancer can all be linked to the disease through their protracted and deleterious effects on mitochondrial function, which leads to respiratory insufficiency.

Addressing the Oncogenic Paradox

Chronic injury to the structure and function of mitochondria, which impairs respiration, will activate the mitochondrial RTG response within the damaged cell. The RTG response is an epigenetic system that upregulates those genes needed to derive energy through fermentation. Fermentation involves SLP through glycolysis in the cytoplasm and through amino acid fermentation in the mitochondria. Uncorrected mitochondrial damage will require continuous compensatory energy through fermentation involving SLP in order to maintain the GATP of approximately −56 kJ/mol. This standard energy of ATP hydrolysis is essential for cell viability. This ATP hydrolysis remains mostly constant regardless of whether the ATP is synthesized through respiration or fermentation.

Although fermentation energy can temporally compensate for disruptions to respiration in order to maintain cell viability, persistent energy production through fermentation can compromise cellular differentiation. Tumor cells require energy production through fermentation because their mitochondrial respiration is insufficient to maintain energy homeostasis. If their respiration was sufficient, fermentation would not persist. Confusion arises from amino acid fermentation, which can simulate properties of normal respiration. Cancer cells appear to respire while also fermenting glucose (aerobic glycolysis). Hence, tumor cells differ from normal cells because they generate a significant amount of energy through fermentation.

Tumor progression is linked to irreversible respiratory damage with fermentation becoming the permanent compensatory energy source for the tumor cells. The change in shape of mitochondrial cristae from convoluted- to smooth illustrates the transition from respiration to fermentation. Persistent and cumulative mitochondrial damage underlies the initiation and progression of cancer.

It is only when fermentation compensates for the greater part of total cellular energy production that tumor progression becomes irreversible according to their model. Tumor progression can be reversed, however, as long as some functional mitochondrial respiration remains. Mitochondrial enhancement therapies can help restore impaired respiration.

In addition to the fermentation of glucose to lactate, which is needed to drive glycolysis, many cancer cells might also ferment glutamine in the mitochondria. It is the fermentation of glucose and glutamine that primarily drives tumor progression and makes tumor cells unresponsive to most conventional therapies.

Most of the gene changes associated with tumor progression arise as direct or indirect consequences of insufficient respiration and elevated fermentation. Oncogene upregulation coupled with tumor-suppressor-gene downregulation becomes necessary in order to increase those metabolic pathways needed for fermentation. If the oncogenes needed to drive cellular fermentation are not expressed then the cell will die from energy failure. Oncogene expression is essential to maintain cell viability following respiratory damage.

Succinate produced through mitochondrial glutamine fermentation could be responsible, in part, for stabilization of Hif-1α. Hif-1α is required for maintaining elevated glucose uptake and glycolysis. Respiratory injury becomes the driver for the gene regulatory changes needed for increasing compensatory energy production through fermentation. Insufficient respiration drives oncogene expression, not the reverse.

As DNA repair mechanisms are dependent on the efficiency of respiratory energy production, the continued impairment of respiration will gradually undermine nuclear genome integrity leading to a mutator phenotype and the plethora of somatic mutations identified in tumor cells. Specifically, the integrity of the nuclear genome is dependent on normal cellular respiration. When cellular respiration becomes compromised, genomic instability increases. Activation of oncogenes, inactivation of tumor-suppressor genes, and aneuploidy will be the natural consequences of protracted mitochondrial dysfunction. These gene abnormalities will contribute to accumulative mitochondrial dysfunction while also enhancing those energy pathways needed to upregulate and sustain fermentation energy. The greater the dependency on fermentation and SLP over time, the greater will be the degree of malignancy.

As respiration is necessary for maintaining cellular differentiation, loss of respiration leads to dedifferentiation and a return to the default state of proliferation. Szent-Gyorgyi considered this cellular state as that which existed in the α-period in the history of life on the planet.

• To make life perennial, the living systems, in this period, had to proliferate as fast as conditions permitted. Energy for this proliferation had to be produced by fermentation so that the α-period could also be called the fermentative period of unbridled proliferation.

The first three cancer hallmarks are consequences of the cell’s return to its mode of existence during the α-period. This would naturally involve increased aerobic glycolysis and resistance to apoptosis. A large number of fermenting cells will also produce an excess of lactate and succinate. This would naturally produce an acidic microenvironment. Angiogenesis is a natural response to wound healing and to the metabolic state in the tumor microenvironment. All of these cancer hallmarks arise as a consequence of insufficient respiration and tumor cell fermentation.

According to the recent commentary of Lazebnik, all hallmarks of cancer with the exception of invasion and metastasis can be found in benign tumors or nonmetastatic cancers. Seyfried also mentioned in Chapter 13 that four of the five hallmarks of cancer are also found in the crown-gall tumors of plants. In contrast to animal cancers, crown-gall tumors do not invade or develop metastases. Hence, it is the hallmark of invasion and metastasis that primarily makes cancer the deadly disease that it is.

As an alternative to the EMT explanation of metastasis, Seyfried showed in Chapter 13 how macrophage fusion hybridization with neoplastic epithelial cells can logically account for all characteristics of the metastatic cascade. Many of the gene expression profiles observed in metastatic cancers are similar to those associated with the function of macrophages or other fusogenic cells of the immune system. Damaged respiration in these fusion hybrids can account for the invasive and metastatic properties found in cancer cells.

Is Cancer Many Diseases or a Singular Disease of Energy Metabolism?

If all cancer cells suffer from respiratory insufficiency, then respiratory insufficiency becomes the central hallmark of the disease. The current view of cancer as a hodgepodge of many diseases is fundamentally inaccurate in view of the central defect of the disease. Cancer appears as many diseases only if viewed from its histological appearance and from its genomic changes. Seyfried considers the histological appearance and gene expression profiles of cancer cells as “red herrings.” When viewed in the light of energy metabolism, cancer is a singular disease of respiratory insufficiency.

15. Nothing in Cancer Biology Makes Sense Except in the Light of Evolution

Revisiting Growth Advantage of Tumor Cells, Mutations, and Evolution

John Cairns has proposed that carcinogens induce mutations in genes that further enhance mutagenesis. These mutations were considered to produce cells with greater fitness and adaptability than normal cells. According to Cairns’ view, natural selection becomes a liability during cancer progression by selecting dangerous mutations that confer an increased survival advantage on a cell.

• Fitness and survival are generally less for humans that inherit deleterious mutations than for humans that do not inherit these mutations. While a subtle genomic change might enhance the survival of cells under certain stressful conditions, large numbers of deletions, duplications, broken chromosomes, and aneuploidy cannot be considered favorable traits with respect to cell strength and viability.

According to Seyfried’s view, cancer cells proliferate and survive not because of their genomic instability, but because of their respiratory insufficiency. Respiratory insufficiency enhances fermentation, destabilizes the genome, and causes entry into the default state of unbridled proliferation. Unlike mammalian embryos, which would abort if they expressed the types of mutations found in cancer cells, cancer cells survive and grow with these mutations. The mutations are not lethal and are tolerated because the cancer cells depend more on fermentation than on respiration for energy. Glycolysis-derived pyruvate also enhances the p-glycoprotein activity. The p-glycoprotein is responsible for pumping toxic drugs out of cells, and, when activated, makes tumor cells resistant to most chemotherapy. This is often referred to as the multidrug resistance (MDR)phenotype, which is glycolysis dependent. Hence, this aspect of cancer cell drug resistance arises as a consequence of the glycolytic phenotype.

• The accumulated mutations arise as downstream effects of inadequate respiration coupled with compensatory fermentation. As nuclear genomic repair mechanisms are dependent on the integrity of mitochondrial respiration, mutations and genomic instability are the expected consequences of protracted energy transition from respiration to fermentation. The mutations arise following mitochondrial damage and are largely irrelevant to the origin of the disease despite reports to the contrary. Mutations could, however, contribute to cancer progression and irreversibility of the disease.

Aborted development, rather than neoplasia, arises from the epigenetic reprogramming of tumor nuclei. That normal tissues can be derived from the nuclei of cancer cells provides compelling evidence against the notion that somatic mutations are the origin of cancer or drive the disease. There is no conceivable explanation in Darwin’s doctrine that could account for the enhanced survival of organisms that express multiple types and kinds of deleterious mutations.

Cells adapted to ferment glucose and glutamine can survive better in hypoxic environments than cells that require respiration for survival. Adaptation to fermentation is the consequence of protracted damage to respiration during the initiation and progression of cancers. Fermentation energy is primal energy. Fermentation is linked to unbridled proliferation. Somatic mutations do not drive this process, but arise as the result of the process.

Also, somatic mutations and aneuploidy reduce rather than enhance the rate of tumor growth. Support of the inhibitory effects of mutations on tumor growth comes from the well-documented findings that glioma progression is generally slower in patients with chromosome 1p/19q codeletions, promoter hypermethylation of the O6-methylguanine methyltransferase (MGMT) gene, or mutations in the gene for isocitrate dehydrogenase 1 (IDH1).

Work from the Amon and the Compton groups shows that aneuploidy retards cell growth. These findings are also inconsistent with the view that mutations enhance the fitness and growth advantage of tumor cells. The paradox is resolved once cancer becomes recognized as a mitochondrial metabolic disease rather than as a genetic disease.

Normal cells of any tissue follow their genetically scripted program for the differentiated state. Rapid growth is generally not part of this scripted program. If, however, growth becomes necessary for tissue repair, then the normal cells can grow fast. The dysregulated growth of tumor cells arises as a consequence of their abnormal metabolic state involving enhanced fermentation. Fermentation is linked to unbridled proliferation. Unbridled proliferation is the default state of metazoan cells when released from active negative control.

It is now recognized that Ras oncogenes damage the mitochondria to induce cell senescence. The transition from respiration to fermentation allows tumor cells to circumvent the Ras-induced senescence checkpoint. This also allows the tumor cells to survive despite high production of oxidative stress. The origin of Ras-induced oncogenic transformation is damaged mitochondria. Nuclear genome integrity unravels as a result of protracted respiratory dysfunction.

Unbridled proliferation existed during the oxygen-sparse α period of species evolution. This was also a highly reduced state where the ancient pathways of fermentation predominated in driving cell physiology. The appearance of oxygen gave rise to the oxidized state and the emergence of respiration. The emergence of respiration facilitated greater complexity in biological systems.

Respiration largely maintains the differentiated state of metazoan cells. Irreversible damage to respiration, coincident with a rise in fermentation, would unlock the toolkit of preexisting adaptations needed to survive in low oxygen environments.

Tumor Cell Fitness in Light of the Evolutionary Theory of Rick Potts

Potts, a paleoanthropologist at the Smithsonian Institution, has suggested that the evolutionary success of our species has been due largely to the germ line inheritance of traits that have bestowed adaptive versatility. Adaptability was defined in terms of (i) the ability of an organism to persist through major environmental shifts, (ii) to spread to new habitats, and (iii) to respond in novel ways to its surroundings. These characteristics were honed over millions of years and have enabled humans to adapt rapidly to abrupt changes in the physical environment including changes in moisture, temperature, food resources, and so on. The adaptability to abrupt environmental change is a property of the genome, which was selected for in order to ensure survival under environmental extremes.

The success in dealing with environmental stress and disease is therefore dependent on the integrated action of all cells in the organism. Further, this integrated action depends on the flexibility of each cell’s genome, which responds to both internal and external signals according to the needs of the organism. More specifically, only those cells that possess flexibility in nutrient utilization will be able to survive under nutrient stress. Environmental forcing has therefore selected those genomes that are most capable of adapting to change in order to maintain metabolic homeostasis.

According to Darwin and Potts, mutations that bestow a selective advantage are those that will enhance survival under environmental stress. If the multiple pathogenic mutations, chromosomal rearrangements, and mitochondrial abnormalities confer a fitness or survival advantage to tumor cells, then survival under environmental stress should be better in tumor cells than in normal cells. This is not what actually happens when the hypothesis is tested.

• When mice or people with tumors are placed under energy stress using dietary energy reduction (DER), many tumor cells die while normal cells survive. Indeed, the health and vitality of the normal cells improves with time under DER while the tumor cells die from apoptotic death.
• This also explains why cell death is generally greater in tumor cells than in normal cells following exposure to toxic radiation and cancer drugs. In contrast to DER, radiation and toxic drugs run the risk of creating tumor cells that are highly resistant to drugs and radiation. This comes in large part from the damage to respiration in bystander precancerous cells. These cells are often those that eventually become heavily dependent on fermentation for energy.
• Genomic defects will disrupt metabolic flexibility under energy stress, thus inhibiting adaptability.

Because tumor cells ferment rather than respire, they are dependent on the availability of fermentable fuels (glucose and glutamine). Normal cells shift metabolism from glucose to ketone bodies and fats when placed under energy stress. This is dependent on genomic stability.

Ketone bodies and fats are nonfermentable fuels in mammalian cells. Tumor cells have difficulty in using ketone bodies and fats for fuel when glucose is reduced. Because tumor cells lack genomic stability, they are less able than normal cells to adapt to changes in the metabolic environment. The survival of such cells will be counterproductive for the survival of society and will be eliminated for the good of society.

Cancer cells survive and multiply only in physiological environments that provide fuels necessary for fermentation through substrate-level phosphorylation. If these fuels become restricted, tumor cells will have difficulty in surviving and growing regardless of their complement of genomic changes. Multiple genetic defects will reduce genomic flexibility, thus increasing the likelihood of cell death under environmental stress. Regardless of when or how genomic defects become involved in the initiation or progression of tumors, these defects can be exploited for the destruction or management of the tumor.

Cancer Development and Lamarckian Inheritance

Lamarck’s theory of evolution, involving the use and disuse of organs and the inheritance of acquired characters, can better explain the origin and progression of cancer than Darwin’s views. According to Lamarck, the environment produces changes in biological structures. Through adaptation and differential use, these changes lead to modifications in the structures. The modifications would then be passed on to successive generations as acquired traits.

The degree of use or disuse has shaped biological evolution along with the inheritance of acquired adaptability. Lamarck’s ideas could also accommodate a dominant role for epigenetics and horizontal gene transfer, as factors that could facilitate progression. Epigenetic mechanisms in the form of cell fusion and horizontal gene transfer also contribute to cancer progression and metastasis.

Considering the dynamic behavior of mitochondria involving regular fusions and fissions, abnormalities in mitochondrial structure and function can be rapidly disseminated throughout the cellular mitochondrial network and passed along to daughter cells somatically through cytoplasmic inheritance. The degree of mitochondrial respiratory function becomes progressively less with each cell division as adaptability to fermentation increases.

The most malignant cancer cells would sustain the near-complete replacement of their respiration with fermentation. This process could be viewed as Lamarckian inheritance. Although Lamarck considered the inheritance of acquired characteristics as enhancing biological complexity and perfection, the opposite effect would occur in adapting his evolutionary concepts to cancer progression. More specifically, a Lamarckian view can account for the escalation situation of biological chaos and the nonuniform accumulation of mutations seen during cancer progression.

Can Teleology Explain Cancer?

Teleology involves design with a purpose and is the cornerstone of arguments involving intelligent design or creationism. A teleological explanation for complicated phenomena assumes that the system examined has an intended purpose and was designed to accomplish that purpose. Evolution operates without purpose, but rather by genetic chance and environmental necessity.

Cybernetic-type diagrams, which are sometimes used to describe the complexity of cancer signaling systems, can also be considered as teleological explanations. Although teleological explanations might appear appealing on the surface, they muddle mechanistic explanations for the phenomena under investigation. Intentions and purpose are not assumed in mechanistic explanations. Descriptions of cancer cells as being motivated, choosing to do something, or having an agenda are examples of teleology. It is unlikely that teleological explanations will provide much insight into the origin or progression of cancer.