The Role of Oxidative Metabolism in Tumorigenesis and Drug Resistance.

Aerobic glycolysis, i.e., non-oxidative glycolysis occurring under aerobic conditions (the so-called Warburg effect) is now recognized as a hallmark of cancer. However, evidence increasingly indicates that upregulated oxidative metabolism is also pivotal in tumorigenesis. In this article, we discuss factors that upregulate oxidative metabolism in tumor cells. These factors are associated with tumor cell-intrinsic and -extrinsic stimuli including antitumor drugs, requirements related to the different steps of tumorigenesis (initiation and acquisition of cancer stem-like cell functions, primary tumor growth, quiescence, metastatic dissemination), factors related to the phenotypic changes of tumor cells (e.g., autophagy and epithelial-mesenchymal transition), and particular metabolic requirements of proliferating tumor cells. In this context, we also discuss drug resistance associated with upregulated oxidative metabolism. We conclude by proposing a model whereby these factors, either individually or in combination, promote upregulation of oxidative metabolism. In the following, we address some mechanistic aspects that underlie the upregulation of oxidative metabolism and discuss the consequences on tumor prognosis. In the conclusion section of this article, we discuss possible therapeutic implications of the knowledge gathered in this field over the years.

Immune checkpoints between epithelial-mesenchymal transition and autophagy: A conflicting triangle.

Inhibitory immune checkpoint (ICP) molecules are pivotal in inhibiting innate and acquired antitumor immune responses, a mechanism frequently exploited by cancer cells to evade host immunity. These evasion strategies contribute to the complexity of cancer progression and therapeutic resistance. For this reason, ICP molecules have become targets for antitumor drugs, particularly monoclonal antibodies, collectively referred to as immune checkpoint inhibitors (ICI), that counteract such cancer-associated immune suppression and restore antitumor immune responses. Over the last decade, however, it has become clear that tumor cell-associated ICPs can also induce tumor cell-intrinsic effects, in particular epithelial-mesenchymal transition (EMT) and macroautophagy (hereafter autophagy). Both of these processes have profound implications for cancer metastasis and drug responsiveness. This article reviews the positive or negative cross-talk that tumor cell-associated ICPs undergo with autophagy and EMT. We discuss that tumor cell-associated ICPs are upregulated in response to the same stimuli that induce EMT. Moreover, ICPs themselves, when overexpressed, become an EMT-inducing stimulus. As regards the cross-talk with autophagy, ICPs have been shown to either stimulate or inhibit autophagy, while autophagy itself can either up- or downregulate the expression of ICPs. This dynamic equilibrium also extends to the autophagy-apoptosis axis, further emphasizing the complexities of cellular responses. Eventually, we delve into the intricate balance between autophagy and apoptosis, elucidating its role in the broader interplay of cellular dynamics influenced by ICPs. In the final part of this article, we speculate about the driving forces underlying the contradictory outcomes of the reciprocal, inhibitory, or stimulatory effects between ICPs, EMT, and autophagy. A conclusive identification of these driving forces may allow to achieve improved antitumor effects when using combinations of ICIs and compounds acting on EMT and/or autophagy. Prospectively, this may translate into increased and/or broadened therapeutic efficacy compared to what is currently achieved with ICI-based clinical protocols.

On the Role of Glycolysis in Early Tumorigenesis-Permissive and Executioner Effects.

Reprogramming energy production from mitochondrial respiration to glycolysis is now considered a hallmark of cancer. When tumors grow beyond a certain size they give rise to changes in their microenvironment (e.g., hypoxia, mechanical stress) that are conducive to the upregulation of glycolysis. Over the years, however, it has become clear that glycolysis can also associate with the earliest steps of tumorigenesis. Thus, many of the oncoproteins most commonly involved in tumor initiation and progression upregulate glycolysis. Moreover, in recent years, considerable evidence has been reported suggesting that upregulated glycolysis itself, through its enzymes and/or metabolites, may play a causative role in tumorigenesis, either by acting itself as an oncogenic stimulus or by facilitating the appearance of oncogenic mutations. In fact, several changes induced by upregulated glycolysis have been shown to be involved in tumor initiation and early tumorigenesis: glycolysis-induced chromatin remodeling, inhibition of premature senescence and induction of proliferation, effects on DNA repair, O-linked N-acetylglucosamine modification of target proteins, antiapoptotic effects, induction of epithelial-mesenchymal transition or autophagy, and induction of angiogenesis. In this article we summarize the evidence that upregulated glycolysis is involved in tumor initiation and, in the following, we propose a mechanistic model aimed at explaining how upregulated glycolysis may play such a role.

Tumor Cell Glycolysis-At the Crossroad of Epithelial-Mesenchymal Transition and Autophagy.

Upregulation of glycolysis, induction of epithelial-mesenchymal transition (EMT) and macroautophagy (hereafter autophagy), are phenotypic changes that occur in tumor cells, in response to similar stimuli, either tumor cell-autonomous or from the tumor microenvironment. Available evidence, herein reviewed, suggests that glycolysis can play a causative role in the induction of EMT and autophagy in tumor cells. Thus, glycolysis has been shown to induce EMT and either induce or inhibit autophagy. Glycolysis-induced autophagy occurs both in the presence (glucose starvation) or absence (glucose sufficiency) of metabolic stress. In order to explain these, in part, contradictory experimental observations, we propose that in the presence of stimuli, tumor cells respond by upregulating glycolysis, which will then induce EMT and inhibit autophagy. In the presence of stimuli and glucose starvation, upregulated glycolysis leads to adenosine monophosphate-activated protein kinase (AMPK) activation and autophagy induction. In the presence of stimuli and glucose sufficiency, upregulated glycolytic enzymes (e.g., aldolase or glyceraldehyde 3-phosphate dehydrogenase) or decreased levels of glycolytic metabolites (e.g., dihydroxyacetone phosphate) may mimic a situation of metabolic stress (herein referred to as "pseudostarvation"), leading, directly or indirectly, to AMPK activation and autophagy induction. We also discuss possible mechanisms, whereby glycolysis can induce a mixed mesenchymal/autophagic phenotype in tumor cells. Subsequently, we address unresolved problems in this field and possible therapeutic consequences.

Depleting Tumor Cells Expressing Immune Checkpoint Ligands-A New Approach to Combat Cancer.

Antibodies against inhibitory immune checkpoint molecules (ICPMs), referred to as immune checkpoint inhibitors (ICIs), have gained a prominent place in cancer therapy. Several ICIs in clinical use have been engineered to be devoid of effector functions because of the fear that ICIs with preserved effector functions could deplete immune cells, thereby curtailing antitumor immune responses. ICPM ligands (ICPMLs), however, are often overexpressed on a sizeable fraction of tumor cells of many tumor types and these tumor cells display an aggressive phenotype with changes typical of tumor cells undergoing an epithelial-mesenchymal transition. Moreover, immune cells expressing ICPMLs are often endowed with immunosuppressive or immune-deviated functionalities. Taken together, these observations suggest that compounds with the potential of depleting cells expressing ICPMLs may become useful tools for tumor therapy. In this article, we summarize the current state of the art of these compounds, including avelumab, which is the only ICI targeting an ICPML with preserved effector functions that has gained approval so far. We also discuss approaches allowing to obtain compounds with enhanced tumor cell-depleting potential compared to native antibodies. Eventually, we propose treatment protocols that may be applied in order to optimize the therapeutic efficacy of compounds that deplete cells expressing ICPMLs.

Glycolysis-induced drug resistance in tumors-A response to danger signals?

Tumor cells often switch from mitochondrial oxidative metabolism to glycolytic metabolism even under aerobic conditions. Tumor cell glycolysis is accompanied by several nonenzymatic activities among which induction of drug resistance has important therapeutic implications. In this article, we review the main aspects of glycolysis-induced drug resistance. We discuss the classes of antitumor drugs that are affected and the components of the glycolytic pathway (transporters, enzymes, metabolites) that are involved in the induction of drug resistance. Glycolysis-associated drug resistance occurs in response to stimuli, either cell-autonomous (e.g., oncoproteins) or deriving from the tumor microenvironment (e.g., hypoxia or pseudohypoxia, mechanical cues, etc.). Several mechanisms mediate the induction of drug resistance in response to glycolytic metabolism: inhibition of apoptosis, induction of epithelial-mesenchymal transition, induction of autophagy, inhibition of drug influx and increase of drug efflux. We suggest that drug resistance in response to glycolysis comes into play in presence of qualitative (e.g., expression of embryonic enzyme isoforms, post-translational enzyme modifications) or quantitative (e.g., overexpression of enzymes or overproduction of metabolites) alterations of glycolytic metabolism. We also discern similarities between changes occurring in tumor cells in response to stimuli inducing glycolysis-associated drug resistance and those occurring in cells of the innate immune system in response to danger signals and that have been referred to as danger-associated metabolic modifications. Eventually, we briefly address that also mitochondrial oxidative metabolism may induce drug resistance and discuss the therapeutic implications deriving from the fact that the main energy-generating metabolic pathways may be both at the origin of antitumor drug resistance.

The tumor-promoting effects of the adaptive immune system: a cause of hyperprogressive disease in cancer?

Adaptive antitumor immune responses, either cellular or humoral, aim at eliminating tumor cells expressing the cognate antigens. There are some instances, however, where these same immune responses have tumor-promoting effects. These effects can lead to the expansion of antigen-negative tumor cells, tumor cell proliferation and tumor growth, metastatic dissemination, resistance to antitumor therapy and apoptotic stimuli, acquisition of tumor-initiating potential and activation of various forms of survival mechanisms. We describe the basic mechanisms that underlie tumor-promoting adaptive immune responses and try to identify the variables that induce the switching of a tumor-inhibitory, cellular or humoral immune response, into a tumor-promoting one. We suggest that tumor-promoting adaptive immune responses may be at the origin of at least a fraction of hyperprogressive diseases (HPD) that are observed in cancer patients during therapy with immune checkpoint inhibitors (ICI) and, less frequently, with single-agent chemotherapy. We also propose the use of non-invasive biomarkers allowing to predict which patients may undergo HPD during ICI and other forms of antitumor therapy. Eventually, we suggest possibilities of therapeutic intervention allowing to inhibit tumor-promoting adaptive immune responses.

Effect of acetylsalicylic acid on inflamed adipose tissue. Insulin resistance and hepatic steatosis in a mouse model of diet-induced obesity.

AIMS: Obesity represents a global health problem. Excessive caloric intake promotes the release of inflammatory mediators by hypertrophic adipocytes and obesity-induced inflammation is now recognized as a risk factor for the development of several diseases, such as cardiovascular diseases, insulin resistance, type-II diabetes, liver steatosis and cancer. Since obesity causes inflammation, we tested the ability of acetylsalicylic acid (ASA), a potent anti-inflammatory drug, in counteracting this inflammatory process and in mitigating obesity-associated health complications. MAIN METHODS: Mice were fed with standard (SD) or high fat diet (HFD) for 3 months and then treated with acetylsalicylic acid for the subsequent two months. We then analyzed the metabolic and inflammatory status of their adipose and liver tissue by histological, molecular and biochemical analysis. KEY FINDINGS: Although ASA did not exert any effect on body weight, quantification of adipocyte size revealed that the drug slightly reduced adipocyte hypertrophy, however not sufficient so as to induce weight loss. Most importantly, ASA was able to improve insulin resistance. Gene expression profiles of pro- and anti-inflammatory cytokines as well as the expression of macrophage and lymphocyte markers revealed that HFD led to a marked macrophage accumulation in the adipose tissue and an increase of several pro-inflammatory cytokines, a situation almost completely reverted after ASA administration. In addition, liver steatosis caused by HFD was completely abrogated by ASA treatment. SIGNIFICANCE: ASA can efficiently ameliorate pathological conditions usually associated with obesity by inhibiting the inflammatory process occurring in the adipose tissue.

Context-Dependent Pharmacological Effects of Metformin on the Immune System.

Metformin is used for the treatment of type 2 diabetes mellitus and has shown therapeutic effects in preclinical models of other pathologies, such as cancer and autoimmune diseases. The antitumor activity of metformin is due, in part, to immunostimulatory effects. In the context of other pathologies, such as autoimmune or inflammatory diseases, metformin has immunosuppressive effects. There is evidence that the immunostimulatory effects of metformin are indirect. The immunosuppressive effects of metformin in other pathologies appear to be a direct consequence of its action on immune cells. Based on these observations we opine that the pharmacology of metformin is dependent on the pathological context which, to our knowledge, is a novel concept in pharmacology.

The Vicious Cross-Talk between Tumor Cells with an EMT Phenotype and Cells of the Immune System.

Carcinoma cells that undergo an epithelial-mesenchymal transition (EMT) and display a predominantly mesenchymal phenotype (hereafter EMT tumor cells) are associated with immune exclusion and immune deviation in the tumor microenvironment (TME). A large body of evidence has shown that EMT tumor cells and immune cells can reciprocally influence each other, with EMT cells promoting immune exclusion and deviation and immune cells promoting, under certain circumstances, the induction of EMT in tumor cells. This cross-talk between EMT tumor cells and immune cells can occur both between EMT tumor cells and cells of either the native or adaptive immune system. In this article, we review this evidence and the functional consequences of it. We also discuss some recent evidence showing that tumor cells and cells of the immune system respond to similar stimuli, activate the expression of partially overlapping gene sets, and acquire, at least in part, identical functionalities such as migration and invasion. The possible significance of these symmetrical changes in the cross-talk between EMT tumor cells and immune cells is addressed. Eventually, we also discuss possible therapeutic opportunities that may derive from disrupting this cross-talk.

Development of a direct ESI-MS method for measuring the tannin precipitation effect of proline-rich peptides and in silico studies on the proline role in tannin-protein interactions.

Tannins are a heterogeneous class of polyphenols that are present in several plants and foods. Their ability to interact and precipitate proline-rich proteins leads to different effects such as astringency or antidiarrheal activity. Thus, evaluation of the tannin content in plant extracts plays a key role in understanding their potential use as pharmaceuticals and nutraceuticals. Several methods have been proposed to study tannin-protein interactions but few of them are focused on quantification. The purpose of the present work is to set up a suitable and time efficient method able to quantify the extent of tannin protein precipitation. Bradykinin, chosen as a model, was incubated with increasing concentrations of 1,2,3,4,6-penta-O-galloyl-ß-d-glucose and tannic acid selected as reference of tannic compounds. Bradykinin not precipitated was determined by a mass spectrometer TSQ Quantum Ultra Triple Quadrupole (direct infusion analysis). The results were expressed as PC50, which is the concentration able to precipitate 50% of the protein. The type of tannin-protein interaction was evaluated also after precipitate solubilisation. The involvement of proline residues in tannin-protein interactions was confirmed by repeating the experiment using a synthesized peptide (RR-9) characterized by the same bradykinin sequence, but having proline residues replaced by glycine residues: no interaction occurred between the peptide and the tannins. Moreover, modelling studies on PGG-BK and PGG-RR-9 were performed to deeply investigate the involvement of prolines: a balance of hydrophobic and H-bond contacts stabilizes the PGG-BK cluster and the proline residues exert a crucial role thus allowing the PGG molecules to elicit a sticking effect.

Antibody-Drug Conjugates (ADC) Against Cancer Stem-Like Cells (CSC)-Is There Still Room for Optimism?

Cancer stem-like cells (CSC) represent a subpopulation of tumor cells with peculiar functionalities that distinguish them from the bulk of tumor cells, most notably their tumor-initiating potential and drug resistance. Given these properties, it appears logical that CSCs have become an important target for many pharma companies. Antibody-drug conjugates (ADC) have emerged over the last decade as one of the most promising new tools for the selective ablation of tumor cells. Three ADCs have already received regulatory approval and many others are in different phases of clinical development. Not surprisingly, also a considerable number of anti-CSC ADCs have been described in the literature and some of these have entered clinical development. Several of these ADCs, however, have yielded disappointing results in clinical studies. This is similar to the results obtained with other anti-CSC drug candidates, including native antibodies, that have been investigated in the clinic. In this article we review the anti-CSC ADCs that have been described in the literature and, in the following, we discuss reasons that may underlie the failures in clinical trials that have been observed. Possible reasons relate to the biology of CSCs themselves, including their heterogeneity, the lack of strictly CSC-specific markers, and the capacity to interconvert between CSCs and non-CSCs; second, inherent limitations of some classes of cytotoxins that have been used for the construction of ADCs; third, the inadequacy of animal models in predicting efficacy in humans. We conclude suggesting some possibilities to address these limitations.

How Tumor Cells Choose Between Epithelial-Mesenchymal Transition and Autophagy to Resist Stress-Therapeutic Implications.

Tumor cells undergo epithelial-mesenchymal transition (EMT) or macroautophagy (hereafter autophagy) in response to stressors from the microenvironment. EMT ensues when stressors act on tumor cells in the presence of nutrient sufficiency, and mechanistic target of rapamycin (mTOR) appears to be the crucial signaling node for EMT induction. Autophagy, on the other hand, is induced in the presence of nutrient deprivation and/or stressors from the microenvironment with 5' adenosine monophosphate-activated protein kinase (AMPK) playing an important, but not exclusive role, in autophagy induction. Importantly, mTOR and EMT on one hand, and AMPK and autophagy on the other hand, negatively regulate each other. Such regulation occurs at different levels and suggests that, in many instances, these two stress responses are mutually exclusive. Nevertheless, EMT and autophagy are able to interconvert and we suggest that this may depend on spatiotemporal changes in the tumor microenvironment and/or on duration/intensity of the stressor signal(s). Eventually, we propose a three-pronged therapeutic approach aimed at targeting these three major tumor cell populations. First, cytotoxic drugs that act on differentiated and proliferating tumor cells and which, per se, may promote induction of EMT or autophagy in surviving tumor cells. Second, inhibitors of mTOR in order to prevent EMT induction. Third inducers of autophagic cell death (autosis) in order to deplete tumor cells that are constitutively in an autophagic state or are induced to enter an autophagic state in response to antitumor therapy.

LC-MS/MS Identification of Species-Specific Muscle Peptides in Processed Animal Proteins.

An innovative analytical strategy has been applied to identify signature peptides able to distinguish among processed animal proteins (PAPs) derived from bovine, pig, fish, and milk products. Proteomics was first used to elucidate the proteome of each source. Starting from the identified proteins and using a funnel based approach, a set of abundant and well characterized peptides with suitable physical-chemical properties (signature peptides) and specific for each source was selected. An on-target LC-ESI-MS/MS method (MRM mode) was set up using standard peptides and was then applied to selectively identify the PAP source and also to distinguish proteins from bovine carcass and milk proteins. We believe that the method described meets the request of the European Commission which has developed a strategy for gradually lifting the "total ban" toward "species to species ban", therefore requiring official methods for species-specific discrimination in feed.

Tumor cell-associated immune checkpoint molecules - Drivers of malignancy and stemness.

Inhibitory or stimulatory immune checkpoint molecules are expressed on a sizeable fraction of tumor cells in different tumor types. It was thought that the main function of tumor cell-associated immune checkpoint molecules would be the modulation (down- or upregulation) of antitumor immune responses. In recent years, however, it has become clear that the expression of immune checkpoint molecules on tumor cells has important consequences on the biology of the tumor cells themselves. In particular, a causal relationship between the expression of these molecules and the acquisition of malignant traits has been demonstrated. Thus, immune checkpoint molecules have been shown to promote the epithelial-mesenchymal transition of tumor cells, the acquisition of tumor-initiating potential and resistance to apoptosis and antitumor drugs, as well as the propensity to disseminate and metastasize. Herein, we review this evidence, with a main focus on PD-L1, the most intensively investigated tumor cell-associated immune checkpoint molecule and for which most information is available. Then, we discuss more concisely other tumor cell-associated immune checkpoint molecules that have also been shown to induce the acquisition of malignant traits, such as PD-1, B7-H3, B7-H4, Tim-3, CD70, CD28, CD137, CD40 and CD47. Open questions in this field as well as some therapeutic approaches that can be derived from this knowledge, are also addressed.

Ultra-low Doses of Follicle Stimulating Hormone and Progesterone Attenuate the Severity of Polycystic Ovary Syndrome Features in a Hyperandrogenized Mouse Model.

BACKGROUND: Polycystic-ovary syndrome (PCOS) is a reproductive illness characterized by hyperandrogenism and anovulation. Using hyperandrogenized mice, it was demonstrated that the oral administration of incremental dose of follicle stimulating hormone (FSH) attenuated some of PCOS characteristics. This work aimed to study the effect of ultra-low doses of combined FSH and progesterone orally administered on PCOS murine model. Moreover, the effect of sequential kinetic activation of administered hormones was tested. METHODS: Thirty-two female mice were used as animal model (four groups of eight animals each). Mice were hyperandrogenized by injection of dehyidroepiandrosterone diluted in sesame oil. Control group received only oil. Simultaneously, each animal daily received per os an activated or a not-activated combination of FSH (0.44 pg) plus progesterone (0.44 pg) or saline solution as control. Serum testosterone, estradiol, progesterone and luteinizing hormone were analyzed as endocrine markers and a morphological study of antral follicle was conducted. Data were analyzed by one-way ANOVA, followed by multiple comparison test. The p<0.05 was considered significant. RESULTS: Dehyidroepiandrosterone treatment increased both estradiol and progesterone serum levels, besides testosterone, while reduced luteinizing hormone (p<0.05); histological examination revealed an increase of cystic follicles (p<0.05). Irrespective of activation, the combined FSH and progesterone treatments restored estradiol level (p>0.05 vs. control group) and reduced cystic signs in the follicles (p<0.05 vs. dehyidroepiandrosterone treatment). CONCLUSION: This study indicate that ultra-low doses of FSH and progesterone orally administrated can reduce the sternness of PCOS in the mouse model and open a route for the study of innovative approaches for PCOS treatment.

Evidence of Presynaptic Localization and Function of the c-Jun N-Terminal Kinase.

The c-Jun N-terminal kinase (JNK) is part of a stress signalling pathway strongly activated by NMDA-stimulation and involved in synaptic plasticity. Many studies have been focused on the post-synaptic mechanism of JNK action, and less is known about JNK presynaptic localization and its physiological role at this site. Here we examined whether JNK is present at the presynaptic site and its activity after presynaptic NMDA receptors stimulation. By using N-SIM Structured Super Resolution Microscopy as well as biochemical approaches, we demonstrated that presynaptic fractions contained significant amount of JNK protein and its activated form. By means of modelling design, we found that JNK, via the JBD domain, acts as a physiological effector on T-SNARE proteins; then using biochemical approaches we demonstrated the interaction between Syntaxin-1-JNK, Syntaxin-2-JNK, and Snap25-JNK. In addition, taking advance of the specific JNK inhibitor peptide, D-JNKI1, we defined JNK action on the SNARE complex formation. Finally, electrophysiological recordings confirmed the role of JNK in the presynaptic modulation of vesicle release. These data suggest that JNK-dependent phosphorylation of T-SNARE proteins may have an important functional role in synaptic plasticity.

T cell costimulation blockade blunts pressure overload-induced heart failure.

Heart failure (HF) is a leading cause of mortality. Inflammation is implicated in HF, yet clinical trials targeting pro-inflammatory cytokines in HF were unsuccessful, possibly due to redundant functions of individual cytokines. Searching for better cardiac inflammation targets, here we link T cells with HF development in a mouse model of pathological cardiac hypertrophy and in human HF patients. T cell costimulation blockade, through FDA-approved rheumatoid arthritis drug abatacept, leads to highly significant delay in progression and decreased severity of cardiac dysfunction in the mouse HF model. The therapeutic effect occurs via inhibition of activation and cardiac infiltration of T cells and macrophages, leading to reduced cardiomyocyte death. Abatacept treatment also induces production of anti-inflammatory cytokine interleukin-10 (IL-10). IL-10-deficient mice are refractive to treatment, while protection could be rescued by transfer of IL-10-sufficient B cells. These results suggest that T cell costimulation blockade might be therapeutically exploited to treat HF.

The role of autophagy in the cross-talk between epithelial-mesenchymal transitioned tumor cells and cancer stem-like cells.

Epithelial-mesenchymal transition (EMT) and cancer stem-like cells (CSC) are becoming highly relevant targets in anticancer drug discovery. A large body of evidence suggests that epithelial-mesenchymal transitioned tumor cells (EMT tumor cells) and CSCs have similar functions. There is also an overlap regarding the stimuli that can induce the generation of EMT tumor cells and CSCs. Moreover, direct evidence has been brought that EMT can give rise to CSCs. It is unclear however, whether EMT tumor cells should be considered CSCs or if they have to undergo further changes. In this article we summarize available evidence suggesting that, indeed, additional programs must be engaged and we propose that macroautophagy (hereafter, autophagy) represents a key trait distinguishing CSCs from EMT tumor cells. Thus, CSCs have often been reported to be in an autophagic state and blockade of autophagy inhibits CSCs. On the other hand, there is ample evidence showing that EMT and autophagy are distinct events. CSCs, however, represent, by themselves, a heterogeneous population. Thus, CSCs have been distinguished in predominantly non-cycling and cycling CSCs, the latter representing CSCs that self-renew and replenish the pool of differentiated tumor cells. We now suggest that the non-cycling CSC subpopulation is in an autophagic state. We propose also two models to explain the relationship between EMT tumor cells and these two major CSC subpopulations: a branching model in which EMT tumor cells can give rise to cycling or non-cycling CSCs, respectively, and a hierarchical model in which EMT tumor cells are first induced to become autophagic CSCs and, subsequently, cycling CSCs. Finally, we address the therapeutic consequences of these insights.