Hypoxia and hyperglycaemia determine why some endometrial tumours fail to respond to metformin.

BACKGROUND: High expression of Ki67, a proliferation marker, is associated with reduced endometrial cancer-specific survival. Pre-surgical metformin reduces tumour Ki-67 expression in some women with endometrial cancer. Metformin's anti-cancer activity may relate to effects on cellular energy metabolism. Since tumour hypoxia and glucose availability are major cellular redox determinants, we evaluated their role in endometrial cancer response to metformin. METHODS: Endometrial cancer biopsies from women treated with pre-surgical metformin were tested for the hypoxia markers, HIF-1α and CA-9. Endometrial cancer cell lines were treated with metformin in variable glucose concentrations in normoxia or hypoxia and cell viability, mitochondrial biogenesis, function and energy metabolism were assessed. RESULTS: In women treated with metformin (n = 28), Ki-67 response was lower in hypoxic tumours. Metformin showed minimal cytostatic effects towards Ishikawa and HEC1A cells in conventional medium (25 mM glucose). In low glucose (5.5 mM), a dose-dependent cytostatic effect was observed in normoxia but attenuated in hypoxia. Tumours treated with metformin showed increased mitochondrial mass (n = 25), while in cultured cells metformin decreased mitochondrial function. Metformin targets mitochondrial respiration, however, in hypoxic, high glucose conditions, there was a switch to glycolytic metabolism and decreased metformin response. CONCLUSIONS: Understanding the metabolic adaptations of endometrial tumours may identify patients likely to derive clinical benefit from metformin.

Cancer stem cells (CSCs): metabolic strategies for their identification and eradication.

Phenotypic and functional heterogeneity is one of the most relevant features of cancer cells within different tumor types and is responsible for treatment failure. Cancer stem cells (CSCs) are a population of cells with stem cell-like properties that are considered to be the root cause of tumor heterogeneity, because of their ability to generate the full repertoire of cancer cell types. Moreover, CSCs have been invoked as the main drivers of metastatic dissemination and therapeutic resistance. As such, targeting CSCs may be a useful strategy to improve the effectiveness of classical anticancer therapies. Recently, metabolism has been considered as a relevant player in CSC biology, and indeed, oncogenic alterations trigger the metabolite-driven dissemination of CSCs. More interestingly, the action of metabolic pathways in CSC maintenance might not be merely a consequence of genomic alterations. Indeed, certain metabotypic phenotypes may play a causative role in maintaining the stem traits, acting as an orchestrator of stemness. Here, we review the current studies on the metabolic features of CSCs, focusing on the biochemical energy pathways involved in CSC maintenance and propagation. We provide a detailed overview of the plastic metabolic behavior of CSCs in response to microenvironment changes, genetic aberrations, and pharmacological stressors. In addition, we describe the potential of comprehensive metabolic approaches to identify and selectively eradicate CSCs, together with the possibility to 'force' CSCs within certain metabolic dependences, in order to effectively target such metabolic biochemical inflexibilities. Finally, we focus on targeting mitochondria to halt CSC dissemination and effectively eradicate cancer.

Bergamot natural products eradicate cancer stem cells (CSCs) by targeting mevalonate, Rho-GDI-signalling and mitochondrial metabolism.

Here, we show that a 2:1 mixture of Brutieridin and Melitidin, termed "BMF", has a statin-like properties, which blocks the action of the rate-limiting enzyme for mevalonate biosynthesis, namely HMGR (3-hydroxy-3-methylglutaryl-CoA-reductase). Moreover, our results indicate that BMF functionally inhibits several key characteristics of CSCs. More specifically, BMF effectively i) reduced ALDH activity, ii) blocked mammosphere formation and iii) inhibited the activation of CSC-associated signalling pathways (STAT1/3, Notch and Wnt/beta-catenin) targeting Rho-GDI-signalling. In addition, BMF metabolically inhibited mitochondrial respiration (OXPHOS) and fatty acid oxidation (FAO). Importantly, BMF did not show the same toxic side-effects in normal fibroblasts that were observed with statins. Lastly, we show that high expression of the mRNA species encoding HMGR is associated with poor clinical outcome in breast cancer patients, providing a potential companion diagnostic for BMF-directed personalized therapy.

GPER mediates the angiocrine actions induced by IGF1 through the HIF-1α/VEGF pathway in the breast tumor microenvironment.

BACKGROUND: The G protein estrogen receptor GPER/GPR30 mediates estrogen action in breast cancer cells as well as in breast cancer-associated fibroblasts (CAFs), which are key components of microenvironment driving tumor progression. GPER is a transcriptional target of hypoxia inducible factor 1 alpha (HIF-1α) and activates VEGF expression and angiogenesis in hypoxic breast tumor microenvironment. Furthermore, IGF1/IGF1R signaling, which has angiogenic effects, has been shown to activate GPER in breast cancer cells. METHODS: We analyzed gene expression data from published studies representing almost 5000 breast cancer patients to investigate whether GPER and IGF1 signaling establish an angiocrine gene signature in breast cancer patients. Next, we used GPER-positive but estrogen receptor (ER)-negative primary CAF cells derived from patient breast tumours and SKBR3 breast cancer cells to investigate the role of GPER in the regulation of VEGF expression and angiogenesis triggered by IGF1. We performed gene expression and promoter studies, western blotting and immunofluorescence analysis, gene silencing strategies and endothelial tube formation assays to evaluate the involvement of the HIF-1α/GPER/VEGF signaling in the biological responses to IGF1. RESULTS: We first determined that GPER is co-expressed with IGF1R and with the vessel marker CD34 in human breast tumors (n = 4972). Next, we determined that IGF1/IGF1R signaling engages the ERK1/2 and AKT transduction pathways to induce the expression of HIF-1α and its targets GPER and VEGF. We found that a functional cooperation between HIF-1α and GPER is essential for the transcriptional activation of VEGF induced by IGF1. Finally, using conditioned medium from CAFs and SKBR3 cells stimulated with IGF1, we established that HIF-1α and GPER are both required for VEGF-induced human vascular endothelial cell tube formation. CONCLUSIONS: These findings shed new light on the essential role played by GPER in IGF1/IGF1R signaling that induces breast tumor angiogenesis. Targeting the multifaceted interactions between cancer cells and tumor microenvironment involving both GPCRs and growth factor receptors has potential in future combination anticancer therapies.

G Protein-Coupled Receptors at the Crossroad between Physiologic and Pathologic Angiogenesis: Old Paradigms and Emerging Concepts.

G protein-coupled receptors (GPCRs) have been implicated in transmitting signals across the extra- and intra-cellular compartments, thus allowing environmental stimuli to elicit critical biological responses. As GPCRs can be activated by an extensive range of factors including hormones, neurotransmitters, phospholipids and other stimuli, their involvement in a plethora of physiological functions is not surprising. Aberrant GPCR signaling has been regarded as a major contributor to diverse pathologic conditions, such as inflammatory, cardiovascular and neoplastic diseases. In this regard, solid tumors have been demonstrated to activate an angiogenic program that relies on GPCR action to support cancer growth and metastatic dissemination. Therefore, the manipulation of aberrant GPCR signaling could represent a promising target in anticancer therapy. Here, we highlight the GPCR-mediated angiogenic function focusing on the molecular mechanisms and transduction effectors driving the patho-physiological vasculogenesis. Specifically, we describe evidence for the role of heptahelic receptors and associated G proteins in promoting angiogenic responses in pathologic conditions, especially tumor angiogenesis and progression. Likewise, we discuss opportunities to manipulate aberrant GPCR-mediated angiogenic signaling for therapeutic benefit using innovative GPCR-targeted and patient-tailored pharmacological strategies.

Cancer stem cell metabolism.

Cancer is now viewed as a stem cell disease. There is still no consensus on the metabolic characteristics of cancer stem cells, with several studies indicating that they are mainly glycolytic and others pointing instead to mitochondrial metabolism as their principal source of energy. Cancer stem cells also seem to adapt their metabolism to microenvironmental changes by conveniently shifting energy production from one pathway to another, or by acquiring intermediate metabolic phenotypes. Determining the role of cancer stem cell metabolism in carcinogenesis has become a major focus in cancer research, and substantial efforts are conducted towards discovering clinical targets.

Catabolic cancer-associated fibroblasts transfer energy and biomass to anabolic cancer cells, fueling tumor growth.

Fibroblasts are the most abundant "non-cancerous" cells in tumors. However, it remains largely unknown how these cancer-associated fibroblasts (CAFs) promote tumor growth and metastasis, driving chemotherapy resistance and poor clinical outcome. This review summarizes new findings on CAF signaling pathways and their emerging metabolic phenotypes that promote tumor growth. Although it is well established that altered cancer metabolism enhances tumor growth, little is known about the role of fibroblast metabolism in tumor growth. New studies reveal that metabolic coupling occurs between catabolic fibroblasts and anabolic cancer cells, in many types of human tumors, including breast, prostate, and head & neck cancers, as well as lymphomas. These catabolic phenotypes observed in CAFs are secondary to a ROS-induced metabolic stress response. Mechanistically, this occurs via HIF1-alpha and NFκB signaling, driving oxidative stress, autophagy, glycolysis and senescence in stromal fibroblasts. These catabolic CAFs then create a nutrient-rich microenvironment, to metabolically support tumor growth, via the local stromal generation of mitochondrial fuels (lactate, ketone bodies, fatty acids, glutamine, and other amino acids). New biomarkers of this catabolic CAF phenotype (such as caveolin-1 (Cav-1) and MCT4), which are reversible upon treatment with anti-oxidants, are strong predictors of poor clinical outcome in various types of human cancers. How cancer cells metabolically reprogram fibroblasts can also help us to understand the effects of cancer cells at an organismal level, explaining para-neoplastic phenomena, such as cancer cachexia. In conclusion, cancer should be viewed more as a systemic disease, that engages the host-organism in various forms of energy-transfer and metabolic co-operation, across a whole-body "ecosystem".

Deficiency of hyccin, a newly identified membrane protein, causes hypomyelination and congenital cataract.

We describe a new autosomal recessive white matter disorder ('hypomyelination and congenital cataract') characterized by hypomyelination of the central and peripheral nervous system, progressive neurological impairment and congenital cataract. We identified mutations in five affected families, resulting in a deficiency of hyccin, a newly identified 521-amino acid membrane protein. Our study highlights the essential role of hyccin in central and peripheral myelination.

Cav1 suppresses tumor growth and metastasis in a murine model of cutaneous SCC through modulation of MAPK/AP-1 activation.

Caveolin-1 (Cav1) is a scaffolding protein that serves to regulate the activity of several signaling molecules. Its loss has been implicated in the pathogenesis of several types of cancer, but its role in the development and progression of cutaneous squamous cell carcinoma (cSCC) remains largely unexplored. Herein, we use the keratinocyte cell line PAM212, a murine model of cSCC, to determine the function of Cav1 in skin tumor biology. We first show that Cav1 overexpression decreases cell and tumor growth, whereas Cav1 knockdown increases these attributes in PAM212 cells. In addition, Cav1 knockdown increases the invasive ability and incidence of spontaneous lymph node metastasis. Finally, we demonstrate that Cav1 knockdown increases extracellular signaling-related kinase 1/2 mitogen-activated protein kinase/activator protein-1 pathway activation. We attribute the growth and invasive advantage conferred by Cav1 knockdown to increased expression of activator protein-1 transcriptional targets, including cyclin D1 and keratin 18, which show inverse expression in PAM212 based on the expression level of Cav1. In summary, we demonstrate that loss of Cav1 affects several characteristics associated with aggressive human skin tumors and that this protein may be an important modulator of tumor growth and invasion in cSCC.

Mitophagy and cancer: role of BNIP3/BNIP3L as energetic drivers of stemness features, ATP production, proliferation, and cell migration.

Mitophagy is a selective form of autophagy which permits the removal of dysfunctional or excess mitochondria. This occurs as an adaptative response to physiological stressors, such as hypoxia, nutrient deprivation, or DNA damage. Mitophagy is promoted by specific mitochondrial outer membrane receptors, among which are BNIP3 and BNIP3L. The role of mitophagy in cancer is being widely studied, and more specifically in the maintenance of cancer stem cell (CSC) properties, such as self-renewal. Given that CSCs are responsible for treatment failure and metastatic capacity, targeting mitophagy could be an interesting approach for CSC elimination. Herein, we describe a new model system to enrich sub-populations of cancer cells with high basal levels of mitophagy, based on the functional transcriptional activity of BNIP3 and BNIP3L. Briefly, we employed a BNIP3(L)-promoter-eGFP-reporter system to isolate cancer cells with high BNIP3/BNIP3L transcriptional activity by flow cytometry (FACS). The model was validated by using complementary lysosomal and mitophagy-specific probes, as well as the mitochondrially-targeted red fluorescent protein (RFP), namely mt-Keima. High BNIP3/BNIP3L transcriptional activity was accompanied by increases in i) BNIP3/BNIP3L protein levels, ii) lysosomal mass, and iii) basal mitophagy activity. Furthermore, cancer cells with increased BNIP3/BNIP3L transcriptional activity exhibited CSC features, such as greater mammosphere-forming ability and high CD44 levels. To further explore the model, we also analysed other stemness characteristics in MCF7 and MDA-MB-231 breast cancer cell lines, directly demonstrating that BNIP3(L)-high cells were more metabolically active, proliferative, migratory, and drug-resistant, with elevated anti-oxidant capacity. Therefore, high levels of basal mitophagy appear to enhance CSC features.

In Vitro Investigation of Therapy-Induced Senescence and Senescence Escape in Breast Cancer Cells Using Novel Flow Cytometry-Based Methods.

Although cellular senescence was originally defined as an irreversible form of cell cycle arrest, in therapy-induced senescence models, the emergence of proliferative senescence-escaped cancer cells has been reported by several groups, challenging the definition of senescence. Indeed, senescence-escaped cancer cells may contribute to resistance to cancer treatment. Here, to study senescence escape and isolate senescence-escaped cells, we developed novel flow cytometry-based methods using the proliferation marker Ki-67 and CellTrace CFSE live-staining. We investigated the role of a novel senescence marker (DPP4/CD26) and a senolytic drug (azithromycin) on the senescence-escaping ability of MCF-7 and MDA-MB-231 breast cancer cells. Our results show that the expression of DPP4/CD26 is significantly increased in both senescent MCF-7 and MDA-MB-231 cells. While not essential for senescence induction, DPP4/CD26 contributed to promoting senescence escape in MCF-7 cells but not in MDA-MB-231 cells. Our results also confirmed the potential senolytic effect of azithromycin in senescent cancer cells. Importantly, the combination of azithromycin and a DPP4 inhibitor (sitagliptin) demonstrated a synergistic effect in senescent MCF-7 cells and reduced the number of senescence-escaped cells. Although further research is needed, our results and novel methods could contribute to the investigation of the mechanisms of senescence escape and the identification of potential therapeutic targets. Indeed, DPP4/CD26 could be a promising marker and a novel target to potentially decrease senescence escape in cancer.

Nutrient restriction and radiation therapy for cancer treatment: when less is more.

Calorie restriction (CR), or a diet modification aiming to reduce the total intake of calories by 20%-40%, has been shown to increase longevity across multiple species. Recently, there has been growing interest in investigating the potential role of CR as a treatment intervention for age-related diseases, such as cancer, because an increasing body of literature has demonstrated a metabolic component to both carcinogenesis and tumor progression. In fact, many of the molecular pathways that are altered with CR are also known to be altered in cancer. Therefore, manipulation of these pathways using CR can render cancer cells, and most notably breast cancer cells, more susceptible to standard cytotoxic treatment with radiation and chemotherapy. In this review article we demonstrate the laboratory and clinical evidence that exists for CR and show compelling evidence through the molecular pathways CR induces about how it may be used as a treatment in tandem with radiation therapy to improve our rates of disease control.

Caveolin-1 and accelerated host aging in the breast tumor microenvironment: chemoprevention with rapamycin, an mTOR inhibitor and anti-aging drug.

Increasing chronological age is the most significant risk factor for human cancer development. To examine the effects of host aging on mammary tumor growth, we used caveolin (Cav)-1 knockout mice as a bona fide model of accelerated host aging. Mammary tumor cells were orthotopically implanted into these distinct microenvironments (Cav-1(+/+) versus Cav-1(-/-) age-matched young female mice). Mammary tumors grown in a Cav-1-deficient tumor microenvironment have an increased stromal content, with vimentin-positive myofibroblasts (a marker associated with oxidative stress) that are also positive for S6-kinase activation (a marker associated with aging). Mammary tumors grown in a Cav-1-deficient tumor microenvironment were more than fivefold larger than tumors grown in a wild-type microenvironment. Thus, a Cav-1-deficient tumor microenvironment provides a fertile soil for breast cancer tumor growth. Interestingly, the mammary tumor-promoting effects of a Cav-1-deficient microenvironment were estrogen and progesterone independent. In this context, chemoprevention was achieved by using the mammalian target of rapamycin (mTOR) inhibitor and anti-aging drug, rapamycin. Systemic rapamycin treatment of mammary tumors grown in a Cav-1-deficient microenvironment significantly inhibited their tumor growth, decreased their stromal content, and reduced the levels of both vimentin and phospho-S6 in Cav-1-deficient cancer-associated fibroblasts. Since stromal loss of Cav-1 is a marker of a lethal tumor microenvironment in breast tumors, these high-risk patients might benefit from treatment with mTOR inhibitors, such as rapamycin or other rapamycin-related compounds (rapalogues).

Antibiotics that target mitochondria extend lifespan in C. elegans.

Aging is a continuous degenerative process caused by a progressive decline of cell and tissue functions in an organism. It is induced by the accumulation of damage that affects normal cellular processes, ultimately leading to cell death. It has been speculated for many years that mitochondria play a key role in the aging process. In the aim of characterizing the implications of mitochondria in aging, here we used Caenorhabditis elegans (C. elegans) as an organismal model treated a panel of mitochondrial inhibitors and assessed for survival. In our study, we assessed survival by evaluating worm lifespan, and we assessed aging markers by evaluating the pharyngeal muscle contraction, the accumulation of lipofuscin pigment and ATP levels. Our results show that treatment of worms with either doxycycline, azithromycin (inhibitors of the small and the large mitochondrial ribosomes, respectively), or a combination of both, significantly extended median lifespan of C. elegans, enhanced their pharyngeal pumping rate, reduced their lipofuscin content and their energy consumption (ATP levels), as compared to control untreated worms, suggesting an aging-abrogating effect for these drugs. Similarly, DPI, an inhibitor of mitochondrial complex I and II, was capable of prolonging the median lifespan of treated worms. On the other hand, subjecting worms to vitamin C, a pro-oxidant, failed to extend C. elegans lifespan and upregulated its energy consumption, revealing an increase in ATP level. Therefore, our longevity study reveals that mitochondrial inhibitors (i.e., mitochondria-targeting antibiotics) could abrogate aging and extend lifespan in C. elegans.

New insights into cholesterol-mediated ERRα activation in breast cancer progression and pro-tumoral microenvironment orchestration.

Breast cancer remains the greatest cause of cancer-related death in women worldwide. Its aggressiveness and progression derive from intricate processes that occur simultaneously both within the tumour itself and in the neighbouring cells that make up its microenvironment. The aim of the present work was firstly to study how elevated cholesterol levels increase tumour aggressiveness. Herein, we demonstrate that cholesterol, by activating ERRα pathway, promotes epithelium-mesenchymal transition (EMT) in breast cancer cells (MCF-7 and MDA-MB-231) as well as the release of pro-inflammatory factors able to orchestrate the tumour microenvironment. A further objective of this work was to study the close symbiosis between tumour cells and the microenvironment. Our results allow us to highlight, for the first time, that breast cancer cells exposed to high cholesterol levels promote (a) greater macrophages infiltration with induction of an M2 phenotype, (b) angiogenesis and endothelial branching, as well as (c) a cancer-associated fibroblasts (CAFs) phenotype. The effects observed could be due to direct activation of the ERRα pathway by high cholesterol levels, since the simultaneous inhibition of this pathway subverts such effects. Overall, these findings enable us to identify the cholesterol-ERRα synergy as an interesting target for breast cancer treatment.

FoxO3a Drives the Metabolic Reprogramming in Tamoxifen-Resistant Breast Cancer Cells Restoring Tamoxifen Sensitivity.

Tamoxifen-resistant breast cancer cells (TamR-BCCs) are characterized by an enhanced metabolic phenotype compared to tamoxifen-sensitive cells. FoxO3a is an important modulator of cell metabolism, and its deregulation has been involved in the acquisition of tamoxifen resistance. Therefore, tetracycline-inducible FoxO3a was overexpressed in TamR-BCCs (TamR/TetOn-AAA), which, together with their control cell line (TamR/TetOn-V), were subjected to seahorse metabolic assays and proteomic analysis. FoxO3a was able to counteract the increased oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) observed in TamR by reducing their energetic activity and glycolytic rate. FoxO3a caused glucose accumulation, very likely by reducing LDH activity and mitigated TamR biosynthetic needs by reducing G6PDH activity and hindering NADPH production via the pentose phosphate pathway (PPP). Proteomic analysis revealed a FoxO3a-dependent marked decrease in the expression of LDH as well as of several enzymes involved in carbohydrate metabolism (e.g., Aldolase A, LDHA and phosphofructokinase) and the analysis of cBioPortal datasets of BC patients evidenced a significant inverse correlation of these proteins and FoxO3a. Interestingly, FoxO3a also increased mitochondrial biogenesis despite reducing mitochondrial functionality by triggering ROS production. Based on these findings, FoxO3a inducing/activating drugs could represent promising tools to be exploited in the management of patients who are refractory to antiestrogen therapy.

Role of cholesterol in the development and progression of breast cancer.

Diet and obesity are important risk factors for cancer development. Many studies have suggested an important role for several dietary nutrients in the progression and development of breast cancer. However, few studies have specifically addressed the role of components of a Western diet as important factors involved in breast cancer initiation and progression. The present study examined the role of cholesterol in the regulation of tumor progression in a mouse model of mammary tumor formation. The results suggest that cholesterol accelerates and enhances tumor formation. In addition, tumors were more aggressive, and tumor angiogenesis was enhanced. Metabolism of cholesterol was also examined in this mouse model. It was observed that plasma cholesterol levels were reduced during tumor development but not prior to its initiation. These data provide new evidence for an increased utilization of cholesterol by tumors and for its role in tumor formation. Taken together, these results imply that an increase in plasma cholesterol levels accelerates the development of tumors and exacerbates their aggressiveness.

Identification of natural products and FDA-approved drugs for targeting cancer stem cell (CSC) propagation.

Here, we report the identification of key compounds that effectively inhibit the anchorage-independent growth and propagation of cancer stem cells (CSCs), as determined via screening using MCF7 cells, a human breast adenocarcinoma cell line. More specifically, we employed the mammosphere assay as an experimental format, which involves the generation of 3D spheroid cultures, using low-attachment plates. These positive hit compounds can be divided into 5 categories: 1) dietary supplements (quercetin and glucosamine); 2) FDA-approved drugs (carvedilol and ciprofloxacin); 3) natural products (aloe emodin, aloin, tannic acid, chlorophyllin copper salt, azelaic acid and adipic acid); 4) flavours (citral and limonene); and 5) vitamins (nicotinamide and nicotinic acid). In addition, for the compounds quercetin, glucosamine and carvedilol, we further assessed their metabolic action, using the Seahorse to conduct metabolic flux analysis. Our results indicate that these treatments can affect glycolytic flux and suppress oxidative mitochondrial metabolism (OXPHOS). Therefore, quercetin, glucosamine and carvedilol can reprogram the metabolic phenotype of breast cancer cells. Despite having diverse chemical structures, these compounds all interfere with mitochondrial metabolism. As these compounds halt CSCs propagation, ultimately, they may have therapeutic potential.

SOX2-high cancer cells exhibit an aggressive phenotype, with increases in stemness, proliferation and invasion, as well as higher metabolic activity and ATP production.

Cancer stem cells (CSCs) are responsible for cancer recurrence, treatment failure and metastatic dissemination. As such, the elimination of CSCs represents one of the most important approaches for the future of cancer treatment. Among other properties, CSCs show the activation of particular cell signalling pathways and the over-expression of certain transcription factors, such as SOX2. Herein, we describe a new model system to isolate stem-like cancer cells, based on the functional transcriptional activity of SOX2. Briefly, we employed a SOX2-enhancer-GFP-reporter system to isolate cancer cells with high SOX2 transcriptional activity by FACS sorting. The over-expression of SOX2 in this sub-population was validated by Western blot analysis and flow cytometry. SOX2-high cancer cells showed CSCs features, such as greater mammosphere forming ability, validating that this sub-population was enriched in CSCs. To further explore the model, we analysed other stemness characteristics in MCF7 and MDA-MB-231 breast cancer cell lines, corroborating that SOX2-high cells were more metabolically active, proliferative, migratory, invasive, and drug-resistant. SOX2-high MDA-MB-231 cells also showed a loss of E-cadherin expression, and increased Vimentin expression, consistent with an epithelial-mesenchymal transition (EMT). Therefore, endogenous SOX2 transcriptional activity and protein levels are mechanistically linked to aggressive phenotypic behaviours and energy production in CSCs.