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Cell Hierarchy, Metabolic Flexibility and Systems Approaches to Cancer Treatment Patries M. Herst and Michael V. Berridge* Malaghan Institute of Medical Research, PO Box 7060, Wellington 6242, New Zealand Abstract: The proliferative cancer cell paradigm that has driven cancer drug development for the past 50 years has failed to generate treatments that cure most metastatic adult cancers. This view is supported not only by cumulative experience with conventional cytotoxic anticancer drugs, but also by the application of highly-targeted anticancer compounds against, for example, BCR-ABL in CML and mutant BRAF in metastatic melanoma. Such drugs often send their respective cancers into complete molecular remission but fail to effect cures because a small population of quiescent or slowly selfrenewing cancer cells that are drug and radiation resistant survive treatment indefinitely. This review explores the grounds for an emerging cancer paradigm that views cancer as a disorganized tissue with hierarchical cellular compartments in which the boudaries are less well-defined than in normal tissues with plasticity controlled by epigenetic changes mediated by the local microenvironment. Increased metabolic flexibility and adaptability give cancer cells an additional survival advantage that may be able to be targeted with drugs like metformin. Combining approaches that target the increased metabolic flexibility of cancer cells as well as ablating rapidly-proliferating cells and self-renewing cancer stem cells in individual cancers are needed to address the holy grail of cancer cure.
Keywords: Cancer paradigms, cell hierarchy, drug targeting, metabolic flexibility, stem cells, tissue organization. INTRODUCTION Although biomedical research has contributed to greatly improved health outcomes in cardiovascular and infectious diseases over the last 50 years, the impact of research on overall cancer incidence and mortality has been less dramatic [1]. Nevertheless, a significant decrease in age-adjusted mortality is seen in cancers such as childhood leukemias, stomach, uterine and colorectal cancer, and more recently, lung and prostate cancer in men and breast cancer in women [2]. Progress with other cancers such as pancreatic and ovarian cancer and adult leukemias has lagged behind with the overall burden of cancer remaining high and cures with metastatic cancer rare. This review highlights the proliferative cell paradigm that has shaped the field of cancer research over the last 50 years. We contend that an emergent paradigm of cancer as a disorganized, evolving, hierarchical tissue better explains our current understanding of cancer. This view introduces additional hallmarks of cancer, including the presence of self-renewing stem cell-like cells, plasticity, metabolic flexibility and evolutionary issues that will need to be addressed if the ultimate goal of curing metastatic cancer is to be addressed. CANCER AS A DISEASE OF UNCONTROLLED CELL PROLIFERATION The proliferative paradigm views cancer as a clonal expansion of genetically-altered cells that proliferate in an uncontrolled manner, “fending for themselves” and competing *Address correspondence to this author at the Malaghan Institute of Medical Research, PO Box 7060, Wellington 6242, New Zealand; Tel: 64 4 499 6914; Fax: 64 4 499 6915; E-mail:
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for oxygen and nutrients with their neighbours. This viewpoint has been driven largely by in vitro approaches involving isolated cancer cell lines grown under optimal nutrient conditions and oxygen tension that favour maximum cell proliferation rates. The cancer cell line model, epitomized by the NCI60 cancer cell panel [3, 4], represents a snapshot of an immortalized individual cell in the evolution of a particular cancer, and has been a primary driver of research into the molecular and cellular nature of cancer as well as cancer drug development. Cancer cell lines are cultivated in isolation from other cell types and under conditions that do not appropriately reflect their physiological cellular and tissue microenvironment. In this model, all cells in the tumor are often assumed to be comparable and able to proliferate rapidly under ideal conditions. Shotgun Therapy Under the proliferative paradigm, therapeutic strategies have focussed on killing as many rapidly-dividing cancer cells as possible, resulting in tumor regression, or a slowing of tumor progression and increased disease-free survival times. In general, these treatments are not specific for cancer cells but rely on greater numbers of tumor cells being in cycle at any one time relative to other body tissues. However, these more traditional treatments can result in serious side effects, such as bone marrow depression, desquamation, mucositis, alopecia, etc. Radiation therapy and many conservative chemotherapies that target cellular metabolism (flurouracil, methotrexate, mercaptopurine), cell cycle progression (vinca alkaloids, taxanes and etoposide), DNA replication (alkylating agents and topoisomerase inhibitors), and anti-tumour antibiotics (doxorubicin, daunorubicin, mitomy© 2013 Bentham Science Publishers
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cin) and other cytotoxic drugs such as cisplatin and carboplatin often fail to achieve complete remission in metastatic cancer. Other compounds that compromise glycolytic metabolism such as 2-deoxy-D-glucose are currently in early phase clinical trials as a monotherapy, are likely to be too non-specific to generate clinically useful anti-tumor responses. Similar approaches using insulin and IGF-1 receptor blockade to reduce energy supply [5] have to date shown limited potential while compounds like dichloroacetate which inhibits pyruvate dehydrogenase kinase and 3bromopyruvate which inhibits hexokinase II have show potential with some tumors [6, 7].
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women, hormone therapy is usually preceded by hormonal inactivation of the ovaries by inhibiting the hypothalamicpituitary-ovarian axis [19].
Targeted Chemotherapy More recently, a better understanding of cancer cell and molecular biology has led to the development of targeted therapies that are much safer than untargeted therapies. Targeted therapies are designed to inhibit key components of vital proliferative signaling pathways that are altered or over expressed in cancer, switching the cell from a highly proliferative state to a quiescent state and actively inducing apoptosis. Although this approach has been spectacularly successful with Gleevec which targets the BCR-ABL oncogene in CML [8-10], and PLX4032 which targets BRAFV600E in malignant melanoma [11] and is currently in Phase II and III clinical trials as a single agent (see Fig. (1)). However, failure to achieve cures and the inevitable development of drug resistance has taken some of the gloss off these new, targeted therapies. Thus, continuous drug exposure is required with Gleevec for sustained leukemia-free status [9, 12], and with PLX4032 for disease control and progression-free survival. These results indicate that a small population of quiescent cancer cells survive drug treatment for a year or more, and that a more pervasive population of proliferating drugresistant cancer cells with additional mutations evolves over time. With Gleevec, modelling studies have indicated that the quiescent population consists of leukemic stem cells [12] and that blast crisis likely arises from drug-resistant leukemic progenitors [13]. With PLX4032, although clinical trials are still in progress, a similar explanation involving melanoma stem cells is consistent with the results. Despite the high degree of specificity of PLX4032 and its closely related analogue PLX4720 for the mutated form of BRAF [14], trans-activation of dimeric cellular RAF appears to be a limitation [15, 16] leading to basal cell carcinoma and squamous cell carcinoma in 23% of patients. Hormone Therapy Another form of targeted chemotherapy exploits the fact that some cancers depend on hormones such as estrogen for continued proliferation. Estrogen binds to estrogen receptors (ER), activating a signalling cascade that leads to cell division. Until recently, the competitive estrogen inhibitor tamoxifen was considered the gold standard in treatment of ER+ breast cancer in postmenopausal women. Third generation aromatase inhibitors such as letrozole, which inhibit estrogen production in peripheral adipose tissues, have been shown to be at least as effective in postmenopausal women with lower toxicity levels [17, 18]. In premenopausal
Fig. (1). FDG-PET scan images of a patient with metastatic melanoma before (A) and after (B) treatment with BRAF inhibitor. Therapy consisted of 15 days treatment with 960mg of the BRAFV600E inhibitor, PLX4032, given orally twice daily as a single agent. (with permission from the Molecular Imaging Department of the Peter MacCallum Cancer Centre, Melbourne).
Immunotherapy A better understanding of innate and adaptive immunity, which is variably active in cancer patients, has resulted in successful applications in cancer vaccination, diagnosis and treatment. For example, monoclonal antibodies such as Trastuzumab (Herceptin), Rituximab (CD20), Bevacizumab (Avastin) and Cetuximab (Erbitux) are now used widely in cancer treatment [20, 21] while vaccines against Hepatitis B and HPV (Gardasil) are also in common use. In addition to inhibiting signalling cascades that lead to cell proliferation, the presence of monoclonal antibodies on cancer cells, results in opsonization and activation of the innate immune system and activation of the complement cascade leading to cell death and phagocytosis. In general, immune therapies are highly specific, avoiding the serious side effects often seen in more traditional therapies. Recently, adoptive cell therapy (ACT) using patient-derived tumor-specific T cells [22], and vaccines that enhance recognition and destruction of cancer cells by the patients’ own immune cells [23-25] have been trialled clinically. However, the efficacy of these therapeutic vaccines in curing metastatic disease has been quite variable [26] with some trials of selected patients responding well and others closing early due to poor response rates. Most cancer patients that enrol in vaccination trials have late stage disease and will have had chemotherapy and/or radiation therapy with consequent immunosuppression. Combining relatively tumor-specific targeted chemotherapies with a high safety margin together with active
Cell Hierarchy, Metabolic Flexibility and Systems Approaches to Cancer Treatment
therapeutic vaccination may offer synergies not previously possible with drugs that have inherent cytotoxic activity. Differentiation Therapy The introduction in the 1990s of differentiation therapy (all-trans retinoic acid: ATRA) in combination with cytotoxic chemotherapy for acute promyelocytic leukemia (APL) has seen very poor prognosis (2yr disease-free survival of 20-30%) improve to a 95% initial remission rate in newly diagnosed patients [27]. PML is caused by at (15;17) (q22;q21) chromosomal translocation, which produces a chimeric fusion protein that is unable to activate retinoic acid response elements, thus blocking differentiation [28]. ATRA binds to the chimeric fusion protein, causing de-repression of the retinoic acid response elements and targeting the protein for proteosomal degradation [28, 29]. Although targeted therapies specifically kill their target cancer cells or cause them to differentiate, the inherent plasticity and genomic instability of cancer cells can ultimately circumvent the target in question. Enzyme isoforms, new mutations, alternative pathways and epigenetic changes can all overcome the inhibitory effects leading to loss of tumor control. These temporal changes are set on an already highly complex background of genetic and epigenetic changes occurring in tumors as demonstrated by genome wide approaches [30-34]. CANCER AS A DISORGANISED TISSUE A shift in the predominant cancer paradigm of a rogue cell to a disorganized tissue may give better insight into tumor initiation and progression and pave the way for novel anticancer strategies. Instead of viewing cancer as a collection of rogue cells, the entire tumour is regarded as a “disorganised tissue” containing different cell phenotypes in a hierarchical structure that is similar to that of its equivalent healthy tissue
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counterpart (see Fig. (2)). This proposal is an extension of the cancer stem cell model [35, 36] that posits that cancer, like normal tissues, is underpinned by a small homeostatically-regulated population of slowly self-renewing cells that give rise to rapidly-proliferating progenitors with the potential to differentiate along several lineages into functional tissue cells with a finite lifespan. The extent to which the cancer stem cell model applies to cancer is presently a topic of hot debate [37-42]. The hemopoietic tissue is a good example of a tissue that consists of three distinct compartments. The smallest compartment contains tissue-specific self-renewing stem cells (hemocytoblasts) that divide infrequently. Stem cells give rise to cells that proliferate rapidly (transit-amplifying myeloid and lymphoid progenitors). Over time, the proliferating cells differentiate into a third compartment of nonproliferating tissue cells responsible for tissue function (e.g. erythrocytes, platelets, granulocytes, monocytic cells and lymphocytes). Each compartment has well-defined characteristics that set it apart from the other compartments. The relative proportions of the three compartments may differ in a tissue-specific manner. Interestingly, recent research suggests that the tissue-compartment model may be more plastic in normal tissues than previously assumed. Several research groups have shown that normal tissue cells can change their gene expression profile in response to changes in their environment in an atypical manner [43-45]. Tissue stem cells themselves may be plastic with respect to their self-renewal capacity and stem cell-like characteristics may overlap with progenitor cells along a continuum [43]. Cancer Stem Cells The hierarchical cellular composition of tumors is less well defined than that of normal tissues, with self-renewing cancer stem cells being a hot topic of current debate. Evi-
Fig. (2). Model of compartmentalization of cell phenotypes within normal and cancerous tissues. In normal tissues, cells are highly organized and exhibit lower plasticity with respect to their phenotype and gene expression profile than cancer cells. The cancer stem cells compartment of tumors is often proportionally larger and can be derived from stem cells, rapidly-proliferating progenitor cells and occasionally from more differentiated cells. The cancer stem cell population gives rise to all the different cell phenotypes that comprise the tumor. However the defining characteristics of each compartment may be shared to some extent with other compartments. This gives tumors a level of plasticity that is not seen in normal tissues, allowing them to adapt rapidly to changes in their microenvironment. *Blast crisis is the aggressive phase of myelogenous leukemia and is evidenced by an increased number of immature white blood cells in the circulating blood.
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dence for the cancer stem cell concept was first introduced more than a decade ago by Bonnet and Dick for acute leukemia [35]. The presence of cancer stem cells has been reported in many solid tumors [40, 46-51]. Cancer stem cells are often referred to as tumor-initiating cells and are thought to be responsible for loss of local control and the appearance of distant metastases. Considering tumors as disorganized tissue structures with hierarchical cellular composition will require that we reconsider anticancer strategies currently in vogue. While cytotoxic drugs and targeted therapies directed against rapidlyproliferating tumor cells will continue to play a vital role in cancer treatment, cancer cure will require a more holistic approach that also eliminates quiescent cancer stem cells. Because the signaling pathways of these self-renewing cells may be distinct from those of progenitor cells to which they give rise (see above), and from their normal stem cell counterparts, new approaches, targeting these differences may address this Archilles’ heel of cancer treatment. Self-renewal may not be the exclusive prerogative of cancer stem cells but may be shared with other cancer cells that have lost their capacity to differentiate along multiple lineages. Alternatively, the definition of cancer stem cells could be more broadly defined as any cancer cell that has retained the ability to self-renew under specific conditions. This raises questions about the validity of highly immunocompromised xenotransplantation models that are widely used to investigate the tumor-initiating potential of human tumors [39] some of which may overestimate the frequency of self-renewing cells. Tumor Cell Plasticity and the Microenvironment With increased knowledge of cancer cell biology has come the realization that individual cancer cells within a tumor experience vastly different microenvironments. Although normal tissue cells may be able to adapt to their environment by changing their gene expression profiles to a greater extent than previously thought, cancer cells appear to exhibit much greater plasticity. Unstable genomes combined with defective DNA repair mechanisms, make cancer cells more susceptible to DNA damage. Epigenetic changes contribute significantly to the heterogeneity of the cancer cell population and tumor progression. Fluctuating nutrient and oxygen levels are common challenges for most cancer cells in growing tumors that often rely on disorganized blood supply and a poorly developed lymphatic system [52]. Steep oxygen gradients in the tissue adjacent to blood vessels result in near-zero partial pressures of oxygen (pO2) at distances as short as 100μm from the nearest blood vessel [53-55]. The development of prodrugs that reversibly release highly cytotoxic compounds under hypoxic conditions is a promising anticancer strategy that may overcome the inherent resistance of cells in hypoxic regions in solid tumors [56, 57], while anaerobic microbial spores that germinate under hypoxic conditions are also being explored in preclinical models [57]. CANCER CELL ENERGY METABOLISM Different microenvironments favour different pathways of energy metabolism. Cells in hypoxic areas close to the
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tumor core are glycolytic whereas cells closer to the arterial microvasculature use mitochondrial electron transport (MET) and oxidative phosphorylation (OXPHOS) [52]. Superimposed on this is the observation that the most aggressive tumors are also the most glycolytic [58-60]. In addition, there is ongoing discussion about the reversibility of the OXPHOS to glycolysis shift [60, 61]. Maintaining High Glycolytic Rates The ability to sustain increased glycolytic rates to ensure adequate ATP production under hypoxic conditions, mediated by HIF-1 stabilization resulting in the downstream transcription of hypoxia-inducible genes [62, 63] is associated with a number of advantages and challenges. Shifting towards a purely glycolytic metabolism under hypoxic conditions may give cells a proliferative advantage over cells that use glycolysis minimally [64]. In addition, glycolytic metabolism reduces the production of reactive oxygen species (ROS) that primarily occurs at respiratory complexes I and III, but also at complex II [65, 66]. This generates resistance to therapies that rely on increased ROS production in the mitochondria such as radiation and some types of chemotherapy [58] Growth factors and oncogenes have been shown to increase cellular glucose uptake by increasing expression [6772] and activity [73-78] of glucose transporters and glycolytic enzymes [70, 79]. Increased glucose uptake by solid tumors can be visualized by positron emission tomography (PET) using [18F]-2-fluoro-2-deoxy-D-glucose (FDG), a competitive substrate analogue of glucose (see Fig. (1)) [8082]. FDG-PET scans showing extensive glucose uptake in solid tumors have been correlated with increased Glut1 expression as well as increased hexokinase II expression [83] and poor patient survival [72, 79, 84]. Maintaining Internal Redox Status Another major challenge that highly glycolytic cells face is to maintain NADH/NAD+ ratios conducive to sustained glycolysis. Increased NADH production during glycolysis and mitochondrial TCA cycle activity, combined with a lack of NADH recycling in the absence of mitochondrial electron transport results in intracellular reductive stress [85]. The accepted dogma is that, in the absence of mitochondrial electron transport, reoxidation of NADH occurs mainly via lactate dehydrogenase. Indeed, highly aggressive glycolytic cancers are known to produce substantial amounts of lactate [86-89]. Plasma Membrane Electron Transport (PMET) Plasma membrane electron transport (PMET) is an alternative NADH oxidation system that maintains intracellular redox homeostasis [85, 90-94] by transporting electrons from intracellular NADH to extracellular electron acceptors, such as oxygen. A major PMET pathway in mammalian cells involves a multi-component system that reduces the water soluble tetrazolium dye, WST-1, to its formazan in the presence of an obligate intermediate electron acceptor, 1methoxyphenazine methylsulphate (1mPMS) (see Fig. (3)), [85, 90, 93, 95-97].
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Fig. (3). Diagram of major NADH-recycling pathways. One PMET pathway is responsible for facilitated reduction of WST-1. Electrons from intracellular NADH are transported via an NADH:oxidoreductase (such as NQO1) via plasma membrane ubiquinone and a surface NADH-oxidase to 1mPMS, which in turn reduces WST-1 to its soluble formazan at the cell surface in two single electron transfer steps (reviewed by [97]).
PMET activity is closely linked to metabolic activity and intracellular NADH flux. Rapidly proliferating cells display high WST-1/PMS reduction rates compared with quiescent cells, and dye reduction is strongly affected by metabolic inhibitors such as 2-deoxy-D-glucose and iodoacetamide [90, 96]. Inhibition of MET by the mitochondrial poisons, myxothiazol, potassium cyanide and rotenone, increases intracellular NADH and thus PMET activity. Conversely, the MET uncoupler, FCCP (carbonyl cyanide para-trifluoromethoxy phenylhydrazone), decreases intracellular NADH and inhibits PMET activity in mitochondrially competent cells [90, 93, 96, 98]. Mitochondrial NADH levels are intimately linked with PMET via the malate-aspartate shuttle because the shuttle inhibitor, aminooxyacetic acid, extensively inhibits extracellular WST-1 reduction [99]. In addition to reducing WST-1/PMS, this major PMET pathway can also use oxygen as a terminal electron acceptor (see Fig. (3)), a process we have referred to as “cell surface respiration” because it consumes oxygen to support glycolytic ATP production [93, 96]. Cell surface oxygen consumption is also dependent on intracellular NADH flux and severely inhibited by cell-impermeable NADH and NADPH which block a cell surface NADH-oxidase, also referred to as ECTO-NOX [100, 101], but not NAD+. Interestingly, oxygen and PMS were shown to compete for electrons through PMET at the level of the surface oxidase, with a 3.54.5-fold increase in WST-1/PMS reduction rates under anaerobic conditions [96]. WST-1/mPMS reduction by cycling tumour cell lines (e.g. HeLa) is similar to that of the fully-activated human respiratory burst of neutrophils [90]. If cell surface oxygen consumption (inhibited by extracellular NADH) is taken as a measure of physiological PMET activity, the rate of cell sur-
face oxygen consumption in e.g. D2SC/1 cells, a dendritic cell line is about 5 times that of mitochondrial O2 consumption in the same line (8.9 pmol O2 s-1106cells-1 compared with 2.5 pmol O2 s-1106cells-1), and about 8 times that of mitochondrial oxygen consumption in J774 macrophages (4.9 pmol O2 s-1106cells-1 compared with 0.6 pmol O2 s-1106cells1 ). This is similar to the oxygen consumed by OXPHOS in HL60 cells (10.6 pmol O2 s-1106cells-1) which extensively use mitochondrial respiration [93]. Given that 4 electrons reduce O2 to water in both systems, this should be a direct comparison of the electrons used. On this basis, we contend that PMET, which has been largely ignored in terms of cellular bioenergetics, can contribute extensively to NADH oxidation in some cell types. Physiologically, proliferating cells are thought to be glycolytic and therefore across the whole body, the physiological contribution of PMET could be considerable, e.g. in immune responses, in cellular homeostasis and in wound repair where rapid cell proliferation pertains. The physiological importance of cell surface oxygen consumption is also suggested by the inability of mitochondrial gene knockout HL60 o cells to grow and survive in the absence of oxygen [96]. This is significant because it suggests that LDH activity alone may be insufficient to sustain the glycolytic rates necessary for growth and long-term survival under anaerobic conditions in this cell type. However, it cannot be excluded that these cells may have a mandatory requirement for oxygen other than to support the bioenergetic oxidation of excess NADH. Cell surface oxygen consumption is not restricted to o cell lines but also occurs to some extent in other tumor cell lines that are mitochondrially competent [61, 93]. Because a highly glycolytic energy metabolism has been associated with increased tumor aggressiveness and inva-
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siveness in humans [58-60, 102], we hypothesized that o cells would follow this pattern. Indeed, o MCF12A breast cancer cells were demonstrated to facilitate anchorageindependent cell growth in soft agar, increased migration in matrigel, and tumor growth in SCID mice [103]. In contrast, we showed in a B16 metastatic melanoma model that o cells exhibited delayed subcutaneous tumor growth and failed to form lung metastases [104]. The delayed growth observed in o cells may be due to different gene expression profiles between o and parental cells. In support of this, Hoek and colleagues demonstrated that highly proliferative/non-invasive and slowly proliferating/highly invasive melanoma cells have distinctive gene expression profiles. Fast proliferating melanoma cells expressed high levels of the microphthalmiaassociated transcription factor (Mitf), a lineage survival factor required for proliferation in melanoma. Mitf+ cell lines, derived from non-invasive fast proliferating metastatic melanoma tumors, proliferated faster in vitro and produced tumors in athymic nude mice several weeks sooner than Mitfmelanoma cell lines derived from invasive tumors. Both phenotypes produced tumors that contained both Mitf+ and Mitf- cells, with fast proliferating Mitf+ cells concentrated in the tumor periphery [105]. These results indicate that the expression profile of individual tumor cells may change, depending on their microenvironment. Lack of metastatic potential in o cells has also been observed in T47D human breast cancer cells [106] and in B16 melanoma cells [104] and may be associated with inability to produce sufficient ROS to activate signaling pathways. Involvement of ROS signaling in growth control in vivo has been shown in several other cell types [103, 107] In general, compounds that affect PMET also affect cancer cell survival [85, 108, 109]. One explanation for this could be that blocking electron transport through PMET interferes with membrane ubiquinone recycling, destabilizing the redox status of the cell membrane. This may stimulate acid sphingomyelinase activity, resulting in the conversion of sphingomyelin to ceramide [110]. Involvement of a plasma membrane redox mechanism in the activation of acid sphingomyelinase, and the resultant formation of ceramideenriched membrane islands leading to apoptosis, has recently been postulated by Dumitru and Gulbins [111]. Therapies that target or involve MET and OXPHOS have little effect on highly glycolytic cancer cells, leaving residual live cells to repopulate the tumour. Although it is widely assumed that cancer stem cells are highly glycolytic because of their resistance to radiation and other ROS-inducing chemotherapies, this remains to be validated experimentally. The reliance of all glycolytic cancer cells on PMET for their continued survival poses the question of its suitability as a target for anticancer drug development strategies [85]. The challenge for novel anticancer drug strategies that target PMET will be to discover or make drugs that specifically locate to the plasma membrane without entering the cell, thus minimizing side effects. Metabolic Flexibility in Cancer Cells Tumour cells adapt to acute changes in their microenvironment by adjusting their bioenergetic status [52, 112] and this will apply to self-renewing cancer stem cells and progenitor cells during tumor vascularization and metastasis. In
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the longer term, this metabolic flexibility may involve epigenetic and genetic reprogramming, particularly during clonal evolution to an invasive phenotype. Transcriptional flexibility of metastatic melanoma cells between proliferative and invasive states has recently been described [105, 113], with Mitf expression shown to be a key feature of proliferating melanoma cells. In comparison to normal cells, breast cancer cells show distinct bioenergetic adaptations in response to microenvironmental changes such as glycemic and hypoxic stress [114] while the observation that metformin and compounds like parthenolide selectively target stem cells [115, 116] supports the view that cancer stem cells have altered bioenergetic pathways. Metabolic flexibility in cancer may involve similar mechanisms to those that occur during embryonic development. Other examples of metabolic flexibility in normal physiological situations are encountered during hypoxia and insulin signalling [117] where oxidative phosphorylation is transiently suppressed and glycolysis enhanced by AMP-activated protein kinase, hypoxia-inducible factors and mTOR. Overall, there appears to be little evidence to support the view that tumor cells become hard-wired to glycolytic metabolism (aerobic glycolysis or “the Warburg effect”). Oxygen concentrations are seldom limiting for mitochondrial oxidative phosphorylation, even in the most hypoxic regions of tumors [118], and as discussed, many tumor cell lines use their mitochondria for energy production purposes, at least to some extent. With the exception of mitochondrial geneknockout o cells, few cell lines are purely glycolytic [61, 87, 93, 118]. Furthermore, de Groof et al. have shown that Ras-transformed fibroblasts exhibit increased OXPHOS activtiy followed by development of a glycolytic Warburg phenotype [119], while transformation of human mesenchymal stem cells increases their OXPHOS dependence [120]. Therefore, at least with those tumor cell types investigated, a model of metabolic flexibility may be more applicable than a model of metabolic switching. Metabolic flexibility means that the bioenergetic status of the cancer cell is governed by tumor cell type, its proliferative and self-renewal status as well as by microenvironmental factors. Increased glucose uptake and PET imaging of some primary and many metastatic tumors could be explained by relatively small changes in energy metabolism, given the increased cell proliferation in solid tumors and glucose transporter activation (see above). Whether slowly self-renewing cancer stem cells are supported by a “safe” glycolytic metabolism that generates fewer reactive oxygen species and is used by rapidly proliferating cells to meet their anabolic energy needs [64], or by a less “safe” catabolic mitochondrial metabolism that is used by non-dividing functional cells and accounts for more than 80% of energy requirements, remains a topic of considerable interest. TARGETING CANCER STEM CELLS It is becoming increasingly clear that novel cancer strategies will be needed to eradicate cancer stem cells, as even a small number of these cells can cause tumor recurrence and metastasis (see section on targeted chemotherapy). It must be noted here however that there will always be a need for therapies that target rapidly-proliferating cells in solid tu-
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mors as these cause morbidity and mortality because they encroach and put pressure on vital structures, such as the brain, and compromise their blood supply. The current status of strategies that target cancer stem cells has recently been reviewed [121]. Approaches include the use of monoclonal antibodies and activated immune cells, employing oncolytic viruses, reversing drug resistance, interfering with cancer stem cell function and inducing differentiation. In addition, high-throughput screening strategies have identified small molecules that selectively inhibit cancer stem cells [122]. In 2005, Guzman and colleagues showed that the NF-B inhibitor sesquiterpene lactone, parthenolide, induced apoptosis in progenitor cells and also stem cells in AML and blast crisis CML [123]. Subsequent studies demonstrated selective effects of parthenolide on tumor-initiating cells in breast and prostate cancer [124, 125] by complex mechanisms involving NFkB and signalling pathways used by tumor-initiating cells. In another study, Guzman and colleagues showed that TDZD-8, originally developed as a non-competitive GSK-3 inhibitor, selectively killed leukemic stem and progenitor cells [126]. More recently, the tamoxifen analogue, DPPE/Tesmilifene, was shown to preferentially kill breast cancer tumor-initiating cells and to synergize with doxorubicin to completely eradicate tumorigenic cells [127]. Finally, metformin, a drug that is widely used to treat type-2 diabetes has been shown to reduce the risk of breast cancer and other cancers [128-130]. Metformin activates AMP-kinase via indirect effects on mitochondrial complex I [131], but also exhibits AMP-kinase independent mechanisms of action via Rag GTPase-dependent inhibition of mTORC1 activation [132]. Recently Hirsch et al. showed that low dose metformin selectively targets breast cancer stem cells and synergises with doxorubicin in blocking tumor growth and prolonging tumor remission [115]. The mechanistic basis for this selectivity was not addressed. Targeting stem cell-like cells in tumors and in metastatic disease is at a very early stage. The largely quiescent nature and variable occurance of these cells in solid tumors and leukemias, with their likely drug and radiation resistance and enhanced DNA repair capabilities, presents a tough challenge for cancer cell and molecular biologists and drug developers. Exploiting the power of the immune system to seek out and destroy cancer stem cells in individual patients is an attractive strategy because the immune system 1) is blind to the proliferative status of the tumor cell 2) is not affected by multidrug resistance mechanisms, 3) chooses the most appropriate molecular targets based on ability of antigen presenting cells to activate T-cells in the context of MHC, 4) is capable of seeking out and destroying subpopulations of cells within tumors and 5) to date, has been shown to be remarkably safe, at least in the case of dendritic cell immunotherpy. On the negative side, 1) cytotoxic drugs and radiation are immunosuppressive and therefore cannot be used concurrently with cell-based immunotherapy approaches, 2) tumors suppress immune responses through a variety of mechanisms including regulatory T-cells and suppressor myeloid cells and these suppressive effects will need to be addressed if cancer stem cell immunotherapy is to succeed in the clinic, 3) biasing immune responses towards a minor
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population of tumor cells presents considerable challenges, 4) immune system homeostasis will need to be reestablished to avoid potential uncontrolled autoimmune responses. The future of cancer stem cell targeting may lie with a combination of highly-targeted tumor-specific cytoxic drugs like gleevec and PLX4032, which would not be expected to be immunosuppressive but which do not seem to destroy cancer stem cells, and active cellular immunotherapy against these tumor stem cells. CONCLUDING REMARKS The interaction between individual cancer cells and their ever-changing microenvironment may be more complicated than previously thought. Cancer cells are affected by metabolic by-products and signaling molecules produced by neighboring cancer cells, stromal cells, fibroblasts and immune cells that infiltrate the tumor. This, in addition to steep oxygen and nutrient gradients, results in the formation of highly specialised niches, which are subject to extensive fluctuation over time. Distinct cell phenotypes within a tumor occupy different niches at different times and will respond to changes in their microenvironment in a manner that reflects their individual flexibility leading to complex adaptive responses. Tumor adaptability may involve changes in gene expression profiles that are much more fluid than previously predicted [43-45, 105]. Those that subscribe to the “cancer as a disorganised tissue” paradigm would contend that the ability to change gene expression profiles is limited by the tissue compartment to which the cell belongs. Those who see cancer as a “collection of individual rogue cells” would argue that each individual cell evolves clonally to adjust to its changing environment and can acquire stem celllike properties to repopulate the tumor and generate metastases. However, regardless of the prevailing paradigm, tumor cells adapt to their fluctuating microenvironment very successfully. These adaptive strategies should be taken into account when designing anti-cancer treatments. It is becoming increasingly apparent that the ultimate solution to cancer cure will be multifactorial and will embrace not only surgery, combinations of conventional cytotoxic drugs, radiotherapy, and highly-targeted drugs with low toxicity, but also novel strategies that ablate residual populations of quiescent cells capable of regenerating the cancer. Such approaches will include new compounds that target infrequently self-renewing tumor-initiating cells, compounds that modify energy metabolism and others that block stress adaptation pathways such as plasma membrane electron transport. The choice of therapeutic combinations chosen will be increasingly informed by detailed cellular and molecular knowledge about the particular cancer and about the individual patient involved. CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS The authors acknowledge the support of the New Zealand Breast Cancer Research Trust, Genesis Oncology Trust, the
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Child Health Research Foundation, the Melanoma Research Alliance and the Department of Radiation Therapy, University of Otago.
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Received: April 19, 2010
Revised: July 15, 2010
Accepted: September 17, 2010
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