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Redifferentiation therapeutic strategies in cancer
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Redifferentiation therapeutic strategies in cancer Q2
Mariano Bizzarri1,2, Alessandro Giuliani3, Alessandra Cucina4,5 and Mirko Minini1,4
1 Q3 2 Department of Experimental Medicine, Sapienza University of Rome, 00161 Rome, Italy Systems Biology Group Lab, Sapienza University, Rome, Italy 3 Istituto Superiore di Sanità, 00161 Rome, Italy 4 Department of Surgery ‘Pietro Valdoni’, Sapienza University of Rome, 00161 Rome, Italy 5 Azienda Policlinico Umberto I, 00161 Rome, Italy
The widely recognized problems of pharmacological strategies based on killing cancer cells demand a rethink of therapeutic approaches. Tumor reversion strategies that aim to shift cancer cells to a healthy differentiated state are a promising alternative. Although many studies have firmly demonstrated the possibility of reverting cancer to a normal differentiated state, we are still unable (with the exception of retinoic acid in a form of leukemia) to revert cancer cells to a stable differentiated healthy state. Here, we review the main biological bases of redifferentiation strategies and provide a description of the most promising research avenues.
Introduction: crucial issues of anticancer treatments Q4 Concerns about the efficacy of solid cancer treatments are on
the increase, given that the clinical benefits achieved over the past 30 years through pharmacological interventions, notwithstanding recent attempts to establish a ‘personalized’ targetbased approach, are both limited and controversial [1]. Indeed, both industry and oncologists frequently overestimate the benefit of new treatments [2]. In principle, the current therapeutic strategy aims to promote cancer cell death through direct and indirect mechanisms, by launching an all-out attack against cancer cells, as exhorted by the National Cancer Institute’s War on Cancer in 1971. Yet, this approach suffers from intrinsic, insurmountable limits: (i) conventional treatments cannot selectively target cancer cells and spare normal cells, despite intensive research conducted to identify specific and selective targets; and (ii) tumors display an astonishing genomic heterogeneity, which is subject further to endless changes across time and is under pressure from external stimuli, thus preventing the identification of true, ‘causative’,
Corresponding author: Bizzarri, M. ([email protected]) 1359-6446/ã 2020 Published by Elsevier Ltd. https://doi.org/10.1016/j.drudis.2020.01.021
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genomic targets [3]. Therefore, we have to ask whether treatment models based mainly on cancer cell killing represent the only feasible approach. In other words, could other strategies be planned? Over the past few decades, an alternative approach aiming to manage cancer by promoting the differentiation of malignant, highly dedifferentiated tumor cells, thus provoking ‘tumor reversion’, has been gaining momentum [4]. Tumor reversion is an old concept, first suggested at the beginning of the 1960s [5]. The first report describing the phenomenon of tumor reversion can be traced back to 1907. This study referred to an ovary teratoma evolving spontaneously through differentiation toward a normal epithelial phenotype [6]. Similar findings have also been recorded in plants, fish, and other organisms. These studies demonstrated that, when tumor cells are placed within a ‘normal’ morphogenetic milieu, they can not only be ‘reprogrammed’, thus acquiring de novo a healthy phenotype, but can also be incorporated into a complex organism, behaving like native cells. Further research investigated the relation between tumor reversion and chromosomal abnormalities, gene mutations, and gene alterations in their genetic regulatory networks (GNRs), confirming that the
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phenotypic reversion can be achieved despite the huge number of genome alterations present in cancerous cells [7]. However, only during the past two decades has it been firmly ascertained that adult somatic cells, either normal or pathological, can be efficiently ‘reprogrammed’, recovering the status of induced pluripotent stem cells (iPSCs), and then be redirected to acquire a different phenotype [8]. This achievement provides the proof of principle that differentiation is not irreversible, as previously though,- but instead represents a plastic adaptive condition, ultimately subject to reversion when properly stimulated by biophysical factors [9]. Experimental studies in support of such evidence are increasing (Fig. 1), even if investigations performed so far have not allowed the identification of the exact mechanism through which tumor reversion happens and whether the ‘reverted’ phenotype loses its ‘malignant’ features efficiently and/or permanently.
Mechanisms of tumor reversion Several studies suggest that tumor initiation and progression are dependent on the cell–microenvironment crosstalk, occurring within the morphogenetic field. Any relevant perturbation of this complex network is transmitted across the cytoskeleton (CSK) to
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Myo-ins induce reversion of breast cancer (52)
RA idduces reversion of nasopharyngeal carcinoma (28)
the nucleoskeleton (NSK), through which the perturbation is transduced to the chromatin and genome, thus influencing gene expression patterns as well as many epigenetic and enzymatic processes [10] (Fig. 2). Conversely, changes in the biophysical properties of the cell microenvironment can enact the reverse process, thus ‘reverting’ the malignant phenotype. Identification of the crucial points of this transition would help to unveil those factors that have a driver role in triggering the reversion program. This approach could open the way to a pharmacological strategy aimed at reverting tumor phenotypes by the modulation of such driving factors. Scientists have explored different research areas to identify the specific conditions needed to ‘dedifferentiate’ cancer cells. As a result, reversion and differentiation of cancer cells has been obtained, both in vitro and in vivo, by the use of embryo and/or egg extracts [11,12] or by modifying the 3D microenvironment [13] in which tumor cells are cultured. A more specific target-based approach is the use of molecular factors (vitamins A and D, butyrate, and Inositol) that have been shown to deprive cancer cells of their malignant features by inducing a specific differentiating process, eventually culminating, at least partially, in the reversal of the neoplastic phenotype.
2014 miRNA induce tumor
2011 Metabolic reprogramming with butyrate (37)
2010 2003
RA induces reversion of neuroblastoma (29)
differentiation (57)
Butyrate as HDAC inhibitor (39)
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HDAC inhibitors induce differentiation of breast cancer (30) Differentiation of APL cells with arsenic acid (19)
In vivo RA yields complete remission of APL (16) Vitamin D inhibits cancer growth (45)
1988 1983 1980
Differentiation of APL cells with RA (15)
Differentiation of teratocarcinoma by RA (14)
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FIGURE 1
Milestones in the drug-differentiating treatment of cancer. The most representative steps are depicted from the first report (differentiation of teratocarcinoma with retinoic acid), to the most recent ones (role of miRNAs in promoting tumor reversion). Relevant references are provided in brackets [14–16,19,28,29,30,37,39,45,52,57]. Abbreviations: APL, acute promyelocytic leukemia; HDAC, histone deacetylase; Myo-Ins, myo-inositol; RA, retinoic acid. 2
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Reprogramming genes
Integrin linked kinases
Oct4, Nanog, Klf4, c-Myc, Sox2
MAPK ERK1-2
WNT
TGFβ
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Microenvironmental factors
EMT Inhibition
ErbB
Cytoskeleton factors Cofilin, actin, ecadherin
Demethylation Epigenetic remodeling
Metabolic reversion
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FIGURE 2
Principal mechanisms involved in promoting cancer cell differentiation. Differentiation processes enacted by several small molecules are likely to involve several tightly intertwined mechanisms, whereas others occur at a later stage. Chromatin and epigenetic remodeling have a pivotal role, mostly by interfering with methylation processes, and eventually allowing for a reframing of the gene expression pattern. Chromatin remodeling is dependent on both molecular [histone deacetylases (HDACs) and methylation enzyme inhibition] and biophysical (architecture of the nucleo- and/or cytoskeleton) cues. Together, these factors modulate several pathways, especially those involved in the cell–microenvironment crosstalk, ultimately antagonizing the epithelial–mesenchymal transition (EMT). Inhibition of EMT is a mandatory step during epithelial differentiation, which ultimately results in relevant metabolic and shape changes. For definitions of abbreviations, please see the main text.
Reversion through differentiation A specific area of investigation is represented by those studies dealing with tumor reversion by the action of differentiating factors. Initially, this concept emerged from the observation that hormones or cytokines can promote differentiation ex vivo, thereby irreversibly changing the phenotype of cancer cells. Its hallmark success has been the treatment of acute promyelocytic leukemia (APL), a condition that is now curable by a combination of retinoic acid (RA) and arsenic. However, other factors that trigger differentiation in a variety of primary tumour cells have been recently identified, suggesting that ‘redifferentiation’ is not restricted solely to hematological malignancies. However, the
‘differentiating’ property is displayed not only by retinoids, but also by an array of small molecules.
Retinoic acid and hematological malignancies RA, which was initially demonstrated to promote reversion of teratocarcinoma [14], was later recognized to induce a nearcomplete differentiation of leukemogenic cells in APL through the induction of terminal differentiation into granulocytes [15], which are then digested by stromal macrophages, allowing rapid APL clearance and complete remission in most patients [16]. Mechanistically, RA binds to the RA receptor-a (RARa) portion of the promyelocytic leukemia protein (PML)-RARa fusion www.drugdiscoverytoday.com
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protein, promoting the dissociation of several co-repressors, which is required for epigenetic modifications. Subtle changes in the methylation–demethylation balance can reactivate silenced targets, therefore resuming differentiation, which can lead in turn to partial or complete clinical remission. RA acts on both leukemia cells and cells of the hematological niche (i.e., the specific microenvironment supporting the accrual and the proliferation of blood cells). The loss of RAR from the cells of the niche fosters the autonomous proliferation of precancerous cells, because of the severe distortion occurring in the microenvironment architecture, along with the emergence of an inflammatory-like milieu [17]. Overall, these data have laid the foundations for the clinical use of differentiation therapy through which suppression of malignancy can bypass genetic abnormalities that give rise to malignancy. However, recent research [18] has cast on doubt this simplified scenario. For instance, arsenic acid, which does not activate RARadependent transcription, but binds to the PML moiety of the PML–RARa fusion protein, can induce remission in up to 70% of patients with APL [19]. Both arsenic and RA induce differentiation, albeit by different mechanisms that can eventually synergize when the two compounds are simultaneously administered. However, a high rate of stable remissions and/or cures are achieved only by using arsenic acid, which appears to promote the more efficient degradation of PML–RARa, leading to reactivation of p53 [20]. The p53-PML checkpoint is a crucial target in APL [21], and resistance to differentiating treatments is likely the result of the lack of p53 activation [22]. Moreover, arsenic acid and RA might improve p53 signaling by inhibiting mouse double minute 2 homolog (MDM2), the physiological antagonist of p53, through degradation of cytoplasmic nucleophosmin 1 (NPM1) and reformation of PML nuclear bodies [23]. Expression of the mutant oncoprotein NPM1 in the cytoplasm is a common but understudied event in several cancers, and blunts p53 activity through stabilization of MDM2 function, disorganization of the nucleolus, and destabilization of the tumour suppressor ARF. Other molecular factors, including cAMP [24], as well as biophysical factors, such as hypoxia, can synergize with RA in inducing differentiation and clinical remission of hematological cancers. For example, vitamin D3 can promote differentiation, slowing down overall AML progression when added to primary AML blasts [25]. Conversely, differentiating activity of RA is not restricted to either APL cells or hematopoietic cells, because RA can act through RARamodulated transcription independently from PML–RARa and induce normal cell differentiation by regulating stem cell fate [26].
Differentiation in solid cancers There is consensus on the fact that solid tumors display a deregulation of multiple pathways and, thus, are more ‘complex’ than hematological malignancies [27]. Furthermore, assessing differentiation state is usually difficult in solid cancers, given that laboratory cultures of solid tumors do not reflect the in vivo condition (whereas it appears that ‘liquid’ cultures of leukemogenic cells can better mimic the physiological milieu) and consensus on differentiation parameters (morphological shape, key markers) is still lacking. Yet, over the past few decades, several studies have questioned such assumptions. For instance, RA was shown to reactivate 4
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IKKA, a nuclear factor (NF)-kB kinase subunit, promoting the differentiation of nasopharyngeal carcinoma [28] as well as neuroblastoma cells [29]. Many other differentiating factors, including the peroxisome proliferator-activated receptor gamma (PPAR-g) and histone deacetylases (HDACs), cAMP, sodium butyrate, and cytokines, have been identified as initiating and enhancing factors during the tumor reversion that occurs in different cancer types [30]. This suggests the existence of differentiation trajectories shared (at least in part) by different tissue types.
cAMP Selective modulation of PKA isozymes using cAMP-elevating agents induces growth inhibition in a variety of cancer models through the induction of apoptosis and/or cell cycle arrest [31]. By contrast, protein kinase A (PKA) activation induced by cAMP promotes mesenchymal-to-epithelial transition (MET) of transformed mesenchymal-like breast cells [32], whereas activation of the cAMP/ cAMP response element-binding protein (CREB) signaling pathway promotes the differentiation and regression of glioblastoma multiforme [33]. Differentiation into normal astrocytes can also be triggered by restoring normal oxidative phosphorylation in glioblastoma cells treated with cAMP [34]. A similar effect has been noted in breast cancer cells, in which recovery of the oxidative mitochondrial metabolism induced by morphogenetic factors from eggs as instrumental in driving reversion of the tumor phenotype [35]. Unfortunately, despite their potent antiproliferative effects in many cancer cells, substances that increase intracellular cAMP have severe usage limitations because of their high cytotoxicity. To surmount these problems, there have been attempts to increase cAMP levels with physiological endocrine factors, such as leptin, which do not show relevant adverse effects [36].
Butyrate Differentiation through rewiring of glycolytic (‘Warburg-like’) glucose metabolism can also be achieved with butyrate [37], although the mechanisms of action involved remain unclear. Indeed, butyrate modulates the expression of miRNAs [38] as well as DNA methylation [39], acting mostly as HDAC inhibitor (HDACi), leading to hyperacetylation of histones [40]. Stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades, as mirrored by an analysis of the butyrate-responsive proteome that uncovered several integrated cellular processes and pathways [41], including inhibition of aromatase synthesis in breast tissue [42]. These studies outline that differentiation is a delicate, multistep process that could be enhanced by adding differentiating factors at carefully selected key points. Unfortunately, butyrate had received little research interest until recently because of its poor bioavailability (as a result of its quick catabolism by the colonic epithelium, short half-life, and first-pass hepatic clearance) and the consequent huge doses (>grams) needed to achieve therapeutic concentrations in vivo [43]. However, as suggested by some recent investigations, optimization of the route and length of administration of butyrate or use of butyrate prodrugs could help reduce these impediments [44].
Vitamin D The differentiating capacity of vitamin D is even more complex than that of butyrate. Differentiating activity of calcitriol
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Myo-Inositol Inositol was recently demonstrated to induce differentiation in breast cancer cells, principally by counteracting EMT and fostering tumor reversion toward a normal, epithelial phenotype. In the wake of studies carried out on the anticarcinogenic action of inositol hexakisphosphate (InsP6), preliminary reports highlighted the involvement of myo-inositol (myo-Ins) in carcinogenesis and its supportive role in cancer treatment, by interfering with several crucial pathways [51], including phosphoinositide-3kinase–protein kinase B (PI3K)/AKT and Wnt/b-catenin. Even more exciting are findings suggesting that myo-Ins triggers a specific tumor reversion program. Indeed, breast cancer cells treated in vitro with myo-Ins showed increased E-cadherin, downregulation of metalloproteinase-9, and redistribution of b-catenin behind the cell membrane, whereas the motility and invading capacity of the cells were severely inhibited [52]. Those changes were associated with a significant downregulation of PI3K/Akt activity, leading to a decrease in downstream signaling effectors: NF-kB and cyclo-oxygenase (COX)-2. Moreover, myo-Ins inhibits presenilin-1 (PSEN1), thus reducing Notch 1 release, which reduces Snail family transcriptional repressor 1 (SNAI1) levels, a crucial marker of EMT. All these effects partly result from the
profound CSK remodeling that occurs in inositol-treated cells. Finally, myo-Ins was also shown to counteract the precancerous transformation induced by TGF-b in normal breast cells, by antagonizing the inflammatory pathway that drives cells adopting a mesenchymal phenotype [53]. Overall, these data indicate that myo-Ins inhibits the principal molecular pathway supporting EMT in cancer cells. In addition, it has been demonstrated that increased biosynthesis of myo-Ins might have detrimental effects on cancer cells because overexpression of ISYNA1, which drives the intracellular biosynthesis of inositol, increases myo-Ins levels in HCT116 colon cancer cells and suppresses tumor cell growth, mostly through reactivation of p53 activity [54]. Those data suggest that myo-Ins exerts a pivotal inhibitory control on two crucial pathways, the PI3K/Akt and the PSEN1 pathways, both of them known to participate in modulating apoptosis, proliferation, and phenotypic differentiation.
Discussion Basic insights provided by molecular in vitro studies have translated to clinical experimentation with promising results. Differentiation induced with RA and arsenic, as well as treatments based on HDACi or vitamin D, has been obtained in both xenograft mouse models and patients. Although induction of differentiation provides objective benefits, at least for hematological malignancies, it generally only provides a short remission, unless when associated with chemotherapy (even at low doses). To date, no differentiation-inducing agent has shown an effect comparable to that of RA in the treatment of APL. One reason is that, unusually, APL is a case in which malignancy is principally (even if not exclusively) supported by a single, crucial gene defect. Reversal of this pathway is sufficient to trigger the differentiation of APL. However, it is likely that solid tumors encompass a wider array of altered structures and pathways. Hence, focusing on a monotarget strategy is probably inadequate in inducing the phenotypic reprogramming of cancers. Therefore, less consistent clinical results have been achieved with butyrate and inositol, although well-designed and specific clinical trials are still warranted. Yet, we could overcome such shortcomings by recognizing the specific limitations suffered by studies already carried out. First, differentiation effects could involve not only cancer cells, but also stromal cells, as aptly epitomized by the key role of the niche in the self-renewal of hematopoietic malignancies. This implies that we have to reassess our investigations by considering the 3D context in which differentiation happens. Experiments have been conducted mostly on 2D-based culture models. However, given the amount of evidence accumulated for the role of biophysical constraints acting across a 3D architecture, it is arguable that many biological processes, including tumor reversion, are significantly influenced by the 3D context to which the cancerous cell population belongs [55]. Therefore, it is mandatory that future studies should be performed on 3D models, aiming at ascertaining how changes in the 3D structure modulate cell fate commitment under the effect of differentiating factors. Second, it is likely that reprogramming of cancerous cells would require more than a single factor to achieve a complete ‘reversion’. Indeed, the association of RA and arsenic enables the successful management of hematological malignancies, with a high rate of www.drugdiscoverytoday.com
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[1,25(OH)2D3], the active form of vitamin D, was noted during the early 1980s, when its anticancer effects were suggested by epidemiological and in vitro studies [45]. From the early 1980s onwards, the antiproliferative effects of 1,25(OH)2D3 have been explored in a variety of cancer cell lines, including all major solid tumors and leukemia. Even if vitamin D does not achieve complete reversion of the neoplastic phenotype, secosteroids hormone can efficiently promote the differentiation of cancer cells, eventually inhibiting their proliferating capacity or fostering their removal through sustained apoptosis [46]. Vitamin D exerts several pleiotropic effects on various cellular and microenvironment targets, through both receptor-dependent and receptor-independent pathways. Vitamin D also modulates cell adhesion by interfering with the interplay between E-cadherin and the Wnt pathway, finally reverting MET [47]. Genome-wide transcriptomic screens have revealed that vitamin D3 interacts with several targets, although significant differences have been reported because of context-dependent effects. Moreover, the possibility that lipophilic vitamins, A and D, can mutually cooperate in modulating differentiation requires in-depth exploration. Indeed, combinations of vitamin D derivatives and retinoids exhibit cooperative effects on differentiation in established leukemia cell lines, such as HL-60, U937, and NB4 [48]. Furthermore, vitamin D compounds, although not able to induce apoptosis when used by alone, potentiate apoptosis induced by RA in HL-60 cells and differentially regulate the expression of the apoptosis-related gene products Bcl-2 and Bax. It is relevant that the mechanisms involved in regulating differentiation and apoptosis by these agents are mediated through the interactions of the nuclear receptors for vitamin D (VDR), retinoids (RAR), and 9cis-RA (RXR), which are able to form homo- or heterodimeric complexes and transcriptionally activate or repress target gene expression [49]. As usually observed during tumor reversion experiments, the differentiation prompted by vitamin D implies a mandatory inhibition of the epithelial–mesenchymal transition (EMT), namely by antagonizing the proinflammatory and procarcinogenic activity of transforming growth factor (TGF)-b [50].
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clinical, stable remissions. Thus, the association of several ‘differentiating’ factors, at least in principle, could enable the modulation of several crucial targets, according to the ‘network polypharmacology’ approach, as recently advocated by several researchers [56]. This perspective can be properly appreciated only if ‘differentiation’ is considered in terms of its epigenetic aspects. Any reprogramming implies by definition an epigenetic remodeling of the chromatin and the GRN. Although a miscellaneous group of different ‘raw’ compounds has been demonstrated to exert such an effect, evidence is mounting that epigenetic reprogramming is mechanistically sustained by the modulation of specific miRNAs. MicroRNAs are a class of small endogenous noncoding RNA molecules that regulate post-transcriptional events, and could be considered both targets of, and actors in, differentiation therapy [57]. Upregulation or silencing of some tissue-specific miRNAs can serve as enhancers of the differentiation of the solid malignancies to their original tissue types [58]. Moreover, miRNAs can also efficiently target the cluster of cancer stem cells (CSCs) that have a crucial role in ensuring drug resistance to conventional anticancer treatments. In fact, some miRNAs function by blocking CSC-associated pathways, thus forcing CSC maturation and finally ‘rebooting’ the differentiation state of tumors [59,60]. These assumptions has been vindicated by studies in which breast cancer reversion is obtained by using differentiating factors, including some still unidentified miRNAs, extracted from zebrafish embryos [61]. Overall, those data support the idea that miRNAs can effectively ‘restore’ order in the global genomic function by handcuffing genomic abnormalities, thus promoting efficient tumor reversion. Finally, the crucial key events that should be triggered to enact both differentiation and cancer removal still need to be investigated in detail. EMT is among those pathways, but other processes should be explored, namely by focusing on the specific cellular and microenvironmental context in which they occur. Again, this requires an investigation performed using 3D models. This approach helps to correlating parameters belonging to different system levels: from the microscopic (molecular and genetic factors) to the mesoscopic (cells and tissues), placing them all in relationships with those factors (i.e., differentiating prodrugs) believed to trigger the tumor reversion process, based on a framework provided by quantum mechanics [62]. According to this system-level approach, recent studies have investigated how rewiring of the metabolomic fingerprint could be instrumental in promoting overall phenotype reprogramming, both in normal and cancerous cells [63,64]. Several core fluxes, including aerobic glycolysis, de novo lipid biosynthesis, and glutamine-dependent anaplerosis, form a mandatory platform supporting the proliferation and/or invasiveness of diverse cancer cell types [65]. Yet, manipulation of cancer metabolism, through nutrient deprivation [66] or interference with metabolic key pathways [67], can
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promote cancer differentiation by inducing a metabolic shift from glycolysis to oxidative phosphorylation (thus counteracting the ‘Warburg effect’). This process entails the activation of cAMP and te mitochondrial biogenesis, which in turn leads to metabolic reprogramming and tumor differentiation [68]. These findings prompted researchers to investigate the usefulness of a range of natural compounds that have been shown to modulate cAMP and AMP kinase (AMPK), two key hubs within the metabolic network [69]. A further relevant area of study is represented by the modulation of cholesterol metabolism, given that some cholesterol metabolites can trigger phenotype differentiation and cancer inhibition. The relationship between cholesterol metabolism and cancer is complex and controversial results have been obtained in trying to modulate this lipid [70]. Yet, by favoring a cholesterol metabolic shift toward the production of dendrogenin A, it was possible to efficiently promote cancer cell redifferentiation both in vitro and in vivo and improve survival in animal models [71]. Furthermore, promising results have been obtained by associating nitric oxide with protease inhibitors, triggering cancer cell differentiation, while modulating autophagy [72]. This area of research should be explored in more detail, because it could help to establish a mechanistic link between diet and cancer management, thus fostering a reappraisal of the already known correlation between food intake and cancer risk.
Concluding remarks Approaches based on systems biology could be fruitful for planning future studies in this field. Indeed, it is unlikely that studies carried out according to the biased reductionist approach would be able to describe the dynamics of processes involving living objects across different scales. Moreover, according to this novel framework, we would be able to identify new clinical markers to monitor differentiation processes, both in vitro and in vivo. Clinical markers of the tumor response currently focus on the disappearance or shrinkage of the cancer mass, accordingly to the mainstream paradigm for which the only cure of cancer depends on the rate of cancer killing. Obviously, this framework in inappropriate for differentiation treatments that restores the differentiation program of tumor cells rather than killing them. Therefore, the identification of novel biomarkers of ‘tumor reversion’ has become an urgent task to properly assess the clinical benefit of differentiating treatment. We should not be discouraged by the marginal clinical benefits obtained with differentiating factors, the noticeable exception of RA-treated APL notwithstanding. Tumor reversion and differentiation therapies are still in relative infancy. However, this approach carries huge potential, given that it might not only have fewer adverse effects than conventional treatments, but also because it appears to provide real promise for the development of an effective Q5 cure for cancers. Q6
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69 Sengupta, S. and Mehta, G. (2018) Natural products as modulators of the cyclicAMP pathway: evaluation and synthesis of lead compounds. Org. Biomol. Chem. 16, 6372–6390 70 Silvente-Poirot, S. and Poirot, M. (2014) Cholesterol and cancer, in the balance. Science 343, 1445–1446 71 de Medina, P. et al. (2013) Dendrogenin A arises from cholesterol and histamine metabolism and shows cell differentiation and anti-tumour properties. Nat. Commun. 4, 1840 72 Basile, M.S. et al. (2018) Anticancer and differentiation properties of the nitric oxide derivative of lopinavir in human glioblastoma cells. Molecules 23, E2463
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Redifferentiation therapeutic strategies in cancer Q2
Mariano Bizzarri1,2, Alessandro Giuliani3, Alessandra Cucina4,5 and Mirko Minini1,4
1 Q3 2 Department of Experimental Medicine, Sapienza University of Rome, 00161 Rome, Italy Systems Biology Group Lab, Sapienza University, Rome, Italy 3 Istituto Superiore di Sanità, 00161 Rome, Italy 4 Department of Surgery ‘Pietro Valdoni’, Sapienza University of Rome, 00161 Rome, Italy 5 Azienda Policlinico Umberto I, 00161 Rome, Italy
The widely recognized problems of pharmacological strategies based on killing cancer cells demand a rethink of therapeutic approaches. Tumor reversion strategies that aim to shift cancer cells to a healthy differentiated state are a promising alternative. Although many studies have firmly demonstrated the possibility of reverting cancer to a normal differentiated state, we are still unable (with the exception of retinoic acid in a form of leukemia) to revert cancer cells to a stable differentiated healthy state. Here, we review the main biological bases of redifferentiation strategies and provide a description of the most promising research avenues.
Introduction: crucial issues of anticancer treatments Q4 Concerns about the efficacy of solid cancer treatments are on
the increase, given that the clinical benefits achieved over the past 30 years through pharmacological interventions, notwithstanding recent attempts to establish a ‘personalized’ targetbased approach, are both limited and controversial [1]. Indeed, both industry and oncologists frequently overestimate the benefit of new treatments [2]. In principle, the current therapeutic strategy aims to promote cancer cell death through direct and indirect mechanisms, by launching an all-out attack against cancer cells, as exhorted by the National Cancer Institute’s War on Cancer in 1971. Yet, this approach suffers from intrinsic, insurmountable limits: (i) conventional treatments cannot selectively target cancer cells and spare normal cells, despite intensive research conducted to identify specific and selective targets; and (ii) tumors display an astonishing genomic heterogeneity, which is subject further to endless changes across time and is under pressure from external stimuli, thus preventing the identification of true, ‘causative’,
Corresponding author: Bizzarri, M. ([email protected]) 1359-6446/ã 2020 Published by Elsevier Ltd. https://doi.org/10.1016/j.drudis.2020.01.021
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genomic targets [3]. Therefore, we have to ask whether treatment models based mainly on cancer cell killing represent the only feasible approach. In other words, could other strategies be planned? Over the past few decades, an alternative approach aiming to manage cancer by promoting the differentiation of malignant, highly dedifferentiated tumor cells, thus provoking ‘tumor reversion’, has been gaining momentum [4]. Tumor reversion is an old concept, first suggested at the beginning of the 1960s [5]. The first report describing the phenomenon of tumor reversion can be traced back to 1907. This study referred to an ovary teratoma evolving spontaneously through differentiation toward a normal epithelial phenotype [6]. Similar findings have also been recorded in plants, fish, and other organisms. These studies demonstrated that, when tumor cells are placed within a ‘normal’ morphogenetic milieu, they can not only be ‘reprogrammed’, thus acquiring de novo a healthy phenotype, but can also be incorporated into a complex organism, behaving like native cells. Further research investigated the relation between tumor reversion and chromosomal abnormalities, gene mutations, and gene alterations in their genetic regulatory networks (GNRs), confirming that the
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phenotypic reversion can be achieved despite the huge number of genome alterations present in cancerous cells [7]. However, only during the past two decades has it been firmly ascertained that adult somatic cells, either normal or pathological, can be efficiently ‘reprogrammed’, recovering the status of induced pluripotent stem cells (iPSCs), and then be redirected to acquire a different phenotype [8]. This achievement provides the proof of principle that differentiation is not irreversible, as previously though,- but instead represents a plastic adaptive condition, ultimately subject to reversion when properly stimulated by biophysical factors [9]. Experimental studies in support of such evidence are increasing (Fig. 1), even if investigations performed so far have not allowed the identification of the exact mechanism through which tumor reversion happens and whether the ‘reverted’ phenotype loses its ‘malignant’ features efficiently and/or permanently.
Mechanisms of tumor reversion Several studies suggest that tumor initiation and progression are dependent on the cell–microenvironment crosstalk, occurring within the morphogenetic field. Any relevant perturbation of this complex network is transmitted across the cytoskeleton (CSK) to
2016
Myo-ins induce reversion of breast cancer (52)
RA idduces reversion of nasopharyngeal carcinoma (28)
the nucleoskeleton (NSK), through which the perturbation is transduced to the chromatin and genome, thus influencing gene expression patterns as well as many epigenetic and enzymatic processes [10] (Fig. 2). Conversely, changes in the biophysical properties of the cell microenvironment can enact the reverse process, thus ‘reverting’ the malignant phenotype. Identification of the crucial points of this transition would help to unveil those factors that have a driver role in triggering the reversion program. This approach could open the way to a pharmacological strategy aimed at reverting tumor phenotypes by the modulation of such driving factors. Scientists have explored different research areas to identify the specific conditions needed to ‘dedifferentiate’ cancer cells. As a result, reversion and differentiation of cancer cells has been obtained, both in vitro and in vivo, by the use of embryo and/or egg extracts [11,12] or by modifying the 3D microenvironment [13] in which tumor cells are cultured. A more specific target-based approach is the use of molecular factors (vitamins A and D, butyrate, and Inositol) that have been shown to deprive cancer cells of their malignant features by inducing a specific differentiating process, eventually culminating, at least partially, in the reversal of the neoplastic phenotype.
2014 miRNA induce tumor
2011 Metabolic reprogramming with butyrate (37)
2010 2003
RA induces reversion of neuroblastoma (29)
differentiation (57)
Butyrate as HDAC inhibitor (39)
1999 1998 1997
HDAC inhibitors induce differentiation of breast cancer (30) Differentiation of APL cells with arsenic acid (19)
In vivo RA yields complete remission of APL (16) Vitamin D inhibits cancer growth (45)
1988 1983 1980
Differentiation of APL cells with RA (15)
Differentiation of teratocarcinoma by RA (14)
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FIGURE 1
Milestones in the drug-differentiating treatment of cancer. The most representative steps are depicted from the first report (differentiation of teratocarcinoma with retinoic acid), to the most recent ones (role of miRNAs in promoting tumor reversion). Relevant references are provided in brackets [14–16,19,28,29,30,37,39,45,52,57]. Abbreviations: APL, acute promyelocytic leukemia; HDAC, histone deacetylase; Myo-Ins, myo-inositol; RA, retinoic acid. 2
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Reprogramming genes
Integrin linked kinases
Oct4, Nanog, Klf4, c-Myc, Sox2
MAPK ERK1-2
WNT
TGFβ
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Microenvironmental factors
EMT Inhibition
ErbB
Cytoskeleton factors Cofilin, actin, ecadherin
Demethylation Epigenetic remodeling
Metabolic reversion
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FIGURE 2
Principal mechanisms involved in promoting cancer cell differentiation. Differentiation processes enacted by several small molecules are likely to involve several tightly intertwined mechanisms, whereas others occur at a later stage. Chromatin and epigenetic remodeling have a pivotal role, mostly by interfering with methylation processes, and eventually allowing for a reframing of the gene expression pattern. Chromatin remodeling is dependent on both molecular [histone deacetylases (HDACs) and methylation enzyme inhibition] and biophysical (architecture of the nucleo- and/or cytoskeleton) cues. Together, these factors modulate several pathways, especially those involved in the cell–microenvironment crosstalk, ultimately antagonizing the epithelial–mesenchymal transition (EMT). Inhibition of EMT is a mandatory step during epithelial differentiation, which ultimately results in relevant metabolic and shape changes. For definitions of abbreviations, please see the main text.
Reversion through differentiation A specific area of investigation is represented by those studies dealing with tumor reversion by the action of differentiating factors. Initially, this concept emerged from the observation that hormones or cytokines can promote differentiation ex vivo, thereby irreversibly changing the phenotype of cancer cells. Its hallmark success has been the treatment of acute promyelocytic leukemia (APL), a condition that is now curable by a combination of retinoic acid (RA) and arsenic. However, other factors that trigger differentiation in a variety of primary tumour cells have been recently identified, suggesting that ‘redifferentiation’ is not restricted solely to hematological malignancies. However, the
‘differentiating’ property is displayed not only by retinoids, but also by an array of small molecules.
Retinoic acid and hematological malignancies RA, which was initially demonstrated to promote reversion of teratocarcinoma [14], was later recognized to induce a nearcomplete differentiation of leukemogenic cells in APL through the induction of terminal differentiation into granulocytes [15], which are then digested by stromal macrophages, allowing rapid APL clearance and complete remission in most patients [16]. Mechanistically, RA binds to the RA receptor-a (RARa) portion of the promyelocytic leukemia protein (PML)-RARa fusion www.drugdiscoverytoday.com
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protein, promoting the dissociation of several co-repressors, which is required for epigenetic modifications. Subtle changes in the methylation–demethylation balance can reactivate silenced targets, therefore resuming differentiation, which can lead in turn to partial or complete clinical remission. RA acts on both leukemia cells and cells of the hematological niche (i.e., the specific microenvironment supporting the accrual and the proliferation of blood cells). The loss of RAR from the cells of the niche fosters the autonomous proliferation of precancerous cells, because of the severe distortion occurring in the microenvironment architecture, along with the emergence of an inflammatory-like milieu [17]. Overall, these data have laid the foundations for the clinical use of differentiation therapy through which suppression of malignancy can bypass genetic abnormalities that give rise to malignancy. However, recent research [18] has cast on doubt this simplified scenario. For instance, arsenic acid, which does not activate RARadependent transcription, but binds to the PML moiety of the PML–RARa fusion protein, can induce remission in up to 70% of patients with APL [19]. Both arsenic and RA induce differentiation, albeit by different mechanisms that can eventually synergize when the two compounds are simultaneously administered. However, a high rate of stable remissions and/or cures are achieved only by using arsenic acid, which appears to promote the more efficient degradation of PML–RARa, leading to reactivation of p53 [20]. The p53-PML checkpoint is a crucial target in APL [21], and resistance to differentiating treatments is likely the result of the lack of p53 activation [22]. Moreover, arsenic acid and RA might improve p53 signaling by inhibiting mouse double minute 2 homolog (MDM2), the physiological antagonist of p53, through degradation of cytoplasmic nucleophosmin 1 (NPM1) and reformation of PML nuclear bodies [23]. Expression of the mutant oncoprotein NPM1 in the cytoplasm is a common but understudied event in several cancers, and blunts p53 activity through stabilization of MDM2 function, disorganization of the nucleolus, and destabilization of the tumour suppressor ARF. Other molecular factors, including cAMP [24], as well as biophysical factors, such as hypoxia, can synergize with RA in inducing differentiation and clinical remission of hematological cancers. For example, vitamin D3 can promote differentiation, slowing down overall AML progression when added to primary AML blasts [25]. Conversely, differentiating activity of RA is not restricted to either APL cells or hematopoietic cells, because RA can act through RARamodulated transcription independently from PML–RARa and induce normal cell differentiation by regulating stem cell fate [26].
Differentiation in solid cancers There is consensus on the fact that solid tumors display a deregulation of multiple pathways and, thus, are more ‘complex’ than hematological malignancies [27]. Furthermore, assessing differentiation state is usually difficult in solid cancers, given that laboratory cultures of solid tumors do not reflect the in vivo condition (whereas it appears that ‘liquid’ cultures of leukemogenic cells can better mimic the physiological milieu) and consensus on differentiation parameters (morphological shape, key markers) is still lacking. Yet, over the past few decades, several studies have questioned such assumptions. For instance, RA was shown to reactivate 4
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IKKA, a nuclear factor (NF)-kB kinase subunit, promoting the differentiation of nasopharyngeal carcinoma [28] as well as neuroblastoma cells [29]. Many other differentiating factors, including the peroxisome proliferator-activated receptor gamma (PPAR-g) and histone deacetylases (HDACs), cAMP, sodium butyrate, and cytokines, have been identified as initiating and enhancing factors during the tumor reversion that occurs in different cancer types [30]. This suggests the existence of differentiation trajectories shared (at least in part) by different tissue types.
cAMP Selective modulation of PKA isozymes using cAMP-elevating agents induces growth inhibition in a variety of cancer models through the induction of apoptosis and/or cell cycle arrest [31]. By contrast, protein kinase A (PKA) activation induced by cAMP promotes mesenchymal-to-epithelial transition (MET) of transformed mesenchymal-like breast cells [32], whereas activation of the cAMP/ cAMP response element-binding protein (CREB) signaling pathway promotes the differentiation and regression of glioblastoma multiforme [33]. Differentiation into normal astrocytes can also be triggered by restoring normal oxidative phosphorylation in glioblastoma cells treated with cAMP [34]. A similar effect has been noted in breast cancer cells, in which recovery of the oxidative mitochondrial metabolism induced by morphogenetic factors from eggs as instrumental in driving reversion of the tumor phenotype [35]. Unfortunately, despite their potent antiproliferative effects in many cancer cells, substances that increase intracellular cAMP have severe usage limitations because of their high cytotoxicity. To surmount these problems, there have been attempts to increase cAMP levels with physiological endocrine factors, such as leptin, which do not show relevant adverse effects [36].
Butyrate Differentiation through rewiring of glycolytic (‘Warburg-like’) glucose metabolism can also be achieved with butyrate [37], although the mechanisms of action involved remain unclear. Indeed, butyrate modulates the expression of miRNAs [38] as well as DNA methylation [39], acting mostly as HDAC inhibitor (HDACi), leading to hyperacetylation of histones [40]. Stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades, as mirrored by an analysis of the butyrate-responsive proteome that uncovered several integrated cellular processes and pathways [41], including inhibition of aromatase synthesis in breast tissue [42]. These studies outline that differentiation is a delicate, multistep process that could be enhanced by adding differentiating factors at carefully selected key points. Unfortunately, butyrate had received little research interest until recently because of its poor bioavailability (as a result of its quick catabolism by the colonic epithelium, short half-life, and first-pass hepatic clearance) and the consequent huge doses (>grams) needed to achieve therapeutic concentrations in vivo [43]. However, as suggested by some recent investigations, optimization of the route and length of administration of butyrate or use of butyrate prodrugs could help reduce these impediments [44].
Vitamin D The differentiating capacity of vitamin D is even more complex than that of butyrate. Differentiating activity of calcitriol
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Myo-Inositol Inositol was recently demonstrated to induce differentiation in breast cancer cells, principally by counteracting EMT and fostering tumor reversion toward a normal, epithelial phenotype. In the wake of studies carried out on the anticarcinogenic action of inositol hexakisphosphate (InsP6), preliminary reports highlighted the involvement of myo-inositol (myo-Ins) in carcinogenesis and its supportive role in cancer treatment, by interfering with several crucial pathways [51], including phosphoinositide-3kinase–protein kinase B (PI3K)/AKT and Wnt/b-catenin. Even more exciting are findings suggesting that myo-Ins triggers a specific tumor reversion program. Indeed, breast cancer cells treated in vitro with myo-Ins showed increased E-cadherin, downregulation of metalloproteinase-9, and redistribution of b-catenin behind the cell membrane, whereas the motility and invading capacity of the cells were severely inhibited [52]. Those changes were associated with a significant downregulation of PI3K/Akt activity, leading to a decrease in downstream signaling effectors: NF-kB and cyclo-oxygenase (COX)-2. Moreover, myo-Ins inhibits presenilin-1 (PSEN1), thus reducing Notch 1 release, which reduces Snail family transcriptional repressor 1 (SNAI1) levels, a crucial marker of EMT. All these effects partly result from the
profound CSK remodeling that occurs in inositol-treated cells. Finally, myo-Ins was also shown to counteract the precancerous transformation induced by TGF-b in normal breast cells, by antagonizing the inflammatory pathway that drives cells adopting a mesenchymal phenotype [53]. Overall, these data indicate that myo-Ins inhibits the principal molecular pathway supporting EMT in cancer cells. In addition, it has been demonstrated that increased biosynthesis of myo-Ins might have detrimental effects on cancer cells because overexpression of ISYNA1, which drives the intracellular biosynthesis of inositol, increases myo-Ins levels in HCT116 colon cancer cells and suppresses tumor cell growth, mostly through reactivation of p53 activity [54]. Those data suggest that myo-Ins exerts a pivotal inhibitory control on two crucial pathways, the PI3K/Akt and the PSEN1 pathways, both of them known to participate in modulating apoptosis, proliferation, and phenotypic differentiation.
Discussion Basic insights provided by molecular in vitro studies have translated to clinical experimentation with promising results. Differentiation induced with RA and arsenic, as well as treatments based on HDACi or vitamin D, has been obtained in both xenograft mouse models and patients. Although induction of differentiation provides objective benefits, at least for hematological malignancies, it generally only provides a short remission, unless when associated with chemotherapy (even at low doses). To date, no differentiation-inducing agent has shown an effect comparable to that of RA in the treatment of APL. One reason is that, unusually, APL is a case in which malignancy is principally (even if not exclusively) supported by a single, crucial gene defect. Reversal of this pathway is sufficient to trigger the differentiation of APL. However, it is likely that solid tumors encompass a wider array of altered structures and pathways. Hence, focusing on a monotarget strategy is probably inadequate in inducing the phenotypic reprogramming of cancers. Therefore, less consistent clinical results have been achieved with butyrate and inositol, although well-designed and specific clinical trials are still warranted. Yet, we could overcome such shortcomings by recognizing the specific limitations suffered by studies already carried out. First, differentiation effects could involve not only cancer cells, but also stromal cells, as aptly epitomized by the key role of the niche in the self-renewal of hematopoietic malignancies. This implies that we have to reassess our investigations by considering the 3D context in which differentiation happens. Experiments have been conducted mostly on 2D-based culture models. However, given the amount of evidence accumulated for the role of biophysical constraints acting across a 3D architecture, it is arguable that many biological processes, including tumor reversion, are significantly influenced by the 3D context to which the cancerous cell population belongs [55]. Therefore, it is mandatory that future studies should be performed on 3D models, aiming at ascertaining how changes in the 3D structure modulate cell fate commitment under the effect of differentiating factors. Second, it is likely that reprogramming of cancerous cells would require more than a single factor to achieve a complete ‘reversion’. Indeed, the association of RA and arsenic enables the successful management of hematological malignancies, with a high rate of www.drugdiscoverytoday.com
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[1,25(OH)2D3], the active form of vitamin D, was noted during the early 1980s, when its anticancer effects were suggested by epidemiological and in vitro studies [45]. From the early 1980s onwards, the antiproliferative effects of 1,25(OH)2D3 have been explored in a variety of cancer cell lines, including all major solid tumors and leukemia. Even if vitamin D does not achieve complete reversion of the neoplastic phenotype, secosteroids hormone can efficiently promote the differentiation of cancer cells, eventually inhibiting their proliferating capacity or fostering their removal through sustained apoptosis [46]. Vitamin D exerts several pleiotropic effects on various cellular and microenvironment targets, through both receptor-dependent and receptor-independent pathways. Vitamin D also modulates cell adhesion by interfering with the interplay between E-cadherin and the Wnt pathway, finally reverting MET [47]. Genome-wide transcriptomic screens have revealed that vitamin D3 interacts with several targets, although significant differences have been reported because of context-dependent effects. Moreover, the possibility that lipophilic vitamins, A and D, can mutually cooperate in modulating differentiation requires in-depth exploration. Indeed, combinations of vitamin D derivatives and retinoids exhibit cooperative effects on differentiation in established leukemia cell lines, such as HL-60, U937, and NB4 [48]. Furthermore, vitamin D compounds, although not able to induce apoptosis when used by alone, potentiate apoptosis induced by RA in HL-60 cells and differentially regulate the expression of the apoptosis-related gene products Bcl-2 and Bax. It is relevant that the mechanisms involved in regulating differentiation and apoptosis by these agents are mediated through the interactions of the nuclear receptors for vitamin D (VDR), retinoids (RAR), and 9cis-RA (RXR), which are able to form homo- or heterodimeric complexes and transcriptionally activate or repress target gene expression [49]. As usually observed during tumor reversion experiments, the differentiation prompted by vitamin D implies a mandatory inhibition of the epithelial–mesenchymal transition (EMT), namely by antagonizing the proinflammatory and procarcinogenic activity of transforming growth factor (TGF)-b [50].
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clinical, stable remissions. Thus, the association of several ‘differentiating’ factors, at least in principle, could enable the modulation of several crucial targets, according to the ‘network polypharmacology’ approach, as recently advocated by several researchers [56]. This perspective can be properly appreciated only if ‘differentiation’ is considered in terms of its epigenetic aspects. Any reprogramming implies by definition an epigenetic remodeling of the chromatin and the GRN. Although a miscellaneous group of different ‘raw’ compounds has been demonstrated to exert such an effect, evidence is mounting that epigenetic reprogramming is mechanistically sustained by the modulation of specific miRNAs. MicroRNAs are a class of small endogenous noncoding RNA molecules that regulate post-transcriptional events, and could be considered both targets of, and actors in, differentiation therapy [57]. Upregulation or silencing of some tissue-specific miRNAs can serve as enhancers of the differentiation of the solid malignancies to their original tissue types [58]. Moreover, miRNAs can also efficiently target the cluster of cancer stem cells (CSCs) that have a crucial role in ensuring drug resistance to conventional anticancer treatments. In fact, some miRNAs function by blocking CSC-associated pathways, thus forcing CSC maturation and finally ‘rebooting’ the differentiation state of tumors [59,60]. These assumptions has been vindicated by studies in which breast cancer reversion is obtained by using differentiating factors, including some still unidentified miRNAs, extracted from zebrafish embryos [61]. Overall, those data support the idea that miRNAs can effectively ‘restore’ order in the global genomic function by handcuffing genomic abnormalities, thus promoting efficient tumor reversion. Finally, the crucial key events that should be triggered to enact both differentiation and cancer removal still need to be investigated in detail. EMT is among those pathways, but other processes should be explored, namely by focusing on the specific cellular and microenvironmental context in which they occur. Again, this requires an investigation performed using 3D models. This approach helps to correlating parameters belonging to different system levels: from the microscopic (molecular and genetic factors) to the mesoscopic (cells and tissues), placing them all in relationships with those factors (i.e., differentiating prodrugs) believed to trigger the tumor reversion process, based on a framework provided by quantum mechanics [62]. According to this system-level approach, recent studies have investigated how rewiring of the metabolomic fingerprint could be instrumental in promoting overall phenotype reprogramming, both in normal and cancerous cells [63,64]. Several core fluxes, including aerobic glycolysis, de novo lipid biosynthesis, and glutamine-dependent anaplerosis, form a mandatory platform supporting the proliferation and/or invasiveness of diverse cancer cell types [65]. Yet, manipulation of cancer metabolism, through nutrient deprivation [66] or interference with metabolic key pathways [67], can
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promote cancer differentiation by inducing a metabolic shift from glycolysis to oxidative phosphorylation (thus counteracting the ‘Warburg effect’). This process entails the activation of cAMP and te mitochondrial biogenesis, which in turn leads to metabolic reprogramming and tumor differentiation [68]. These findings prompted researchers to investigate the usefulness of a range of natural compounds that have been shown to modulate cAMP and AMP kinase (AMPK), two key hubs within the metabolic network [69]. A further relevant area of study is represented by the modulation of cholesterol metabolism, given that some cholesterol metabolites can trigger phenotype differentiation and cancer inhibition. The relationship between cholesterol metabolism and cancer is complex and controversial results have been obtained in trying to modulate this lipid [70]. Yet, by favoring a cholesterol metabolic shift toward the production of dendrogenin A, it was possible to efficiently promote cancer cell redifferentiation both in vitro and in vivo and improve survival in animal models [71]. Furthermore, promising results have been obtained by associating nitric oxide with protease inhibitors, triggering cancer cell differentiation, while modulating autophagy [72]. This area of research should be explored in more detail, because it could help to establish a mechanistic link between diet and cancer management, thus fostering a reappraisal of the already known correlation between food intake and cancer risk.
Concluding remarks Approaches based on systems biology could be fruitful for planning future studies in this field. Indeed, it is unlikely that studies carried out according to the biased reductionist approach would be able to describe the dynamics of processes involving living objects across different scales. Moreover, according to this novel framework, we would be able to identify new clinical markers to monitor differentiation processes, both in vitro and in vivo. Clinical markers of the tumor response currently focus on the disappearance or shrinkage of the cancer mass, accordingly to the mainstream paradigm for which the only cure of cancer depends on the rate of cancer killing. Obviously, this framework in inappropriate for differentiation treatments that restores the differentiation program of tumor cells rather than killing them. Therefore, the identification of novel biomarkers of ‘tumor reversion’ has become an urgent task to properly assess the clinical benefit of differentiating treatment. We should not be discouraged by the marginal clinical benefits obtained with differentiating factors, the noticeable exception of RA-treated APL notwithstanding. Tumor reversion and differentiation therapies are still in relative infancy. However, this approach carries huge potential, given that it might not only have fewer adverse effects than conventional treatments, but also because it appears to provide real promise for the development of an effective Q5 cure for cancers. Q6
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