Adrian Biddle

Cancer stem cells in squamous cell carcinoma switch between two distinct phenotypes that are preferentially migratory or proliferative

Epithelial-to-mesenchymal transition (EMT) is an important driver of tumor invasion and metastasis, which causes many cancer deaths. Cancer stem cells (CSC) that maintain and initiate tumors have also been implicated in invasion and metastasis, but whether EMT is an important contributor to CSC function is unclear. In this study, we investigated whether a population of CSCs that have undergone EMT (EMT CSCs) exists in squamous cell carcinoma (SCC). We also determined whether a separate population of CSCs that retain epithelial characteristics (non-EMT CSCs) is also present. Our studies revealed that self-renewing CSCs in SCC include two biologically-distinct phenotypes. One phenotype, termed CD44(high)ESA(high), was proliferative and retained epithelial characteristics (non-EMT CSCs), whereas the other phenotype, termed CD44(high)ESA(low), was migratory and had mesenchymal traits characteristic of EMT CSCs. We found that non-EMT and EMT CSCs could switch their epithelial or mesenchymal traits to reconstitute the cellular heterogeneity which was characteristic of CSCs. However, the ability of EMT CSCs to switch to non-EMT character was restricted to cells that were also ALDH1(+), implying that only ALDH1(+) EMT cells had the ability to seed a new epithelial tumor. Taken together, our findings highlight the identification of two distinct CSC phenotypes and suggest a need to define therapeutic targets that can eradicate both of these variants to achieve effective SCC treatment.

Cancer stem cells and EMT in carcinoma

The majority of deaths from carcinoma are caused by secondary growths that result from tumour invasion and metastasis. The importance of epithelial-to-mesenchymal transition (EMT) as a driver of invasion and metastasis is increasingly recognised, and recent evidence has highlighted a link between EMT and the cancer stem cells that initiate and maintain tumours and have also been implicated in invasion and metastasis. Here, we review cancer stem cells and their link with EMT, and explore the importance of this link in metastasis and therapeutic resistance of tumours. We also discuss new evidence from our laboratory demonstrating that cancer stem cells display a remarkable phenotypic plasticity that enables them to switch between an epithelial phenotype that drives tumour growth and an EMT phenotype that drives metastasis. As successful therapies must eradicate cancer stem cells in all their guises, the identification of sub-types of cancer stem cells that display therapeutic resistance and phenotypic plasticity has important implications for the future design of therapeutic strategies. The ability to assay the responses of different cancer stem cell phenotypes in vitro holds promise for the rapid development of a new generation of targeted therapies that fulfil this objective.

Pre‐culturing human adipose tissue mesenchymal stem cells under hypoxia increases their adipogenic and osteogenic differentiation potentials

Hypoxia is an important factor in many aspects of stem‐cell biology including their viability, proliferation, differentiation and migration. We evaluated whether low oxygen level (2%) affected human adipose tissue mesenchymal stem‐cell (hAT‐MSC) phenotype, population growth, viability, apoptosis, necrosis and their adipogenic and osteogenic differentiation potential.

Normal and malignant epithelial cells with stem-like properties have an extended G2 cell cycle phase that is associated with apoptotic resistance.

BackgroundSubsets of cells with stem-like properties have been previously isolated from human epithelial cancers and their resistance to apoptosis-inducing stimuli has been related to carcinoma recurrence and treatment failure. The aim of this study was to investigate the mechanisms of resistance to apoptosis-inducing agents of cells with stem-like properties in both normal and malignant human epithelia.MethodsCells isolated from fresh human head and neck carcinomas (n = 11), cell lines derived from head and neck, prostate and breast human carcinomas (n = 7), and from normal human oral mucosa (n = 5), were exposed to various apoptosis-inducing stimuli (UV, Tumour Necrosis Factor, Cisplatin, Etoposide, and Neocarzinostatin). Flow cytometry for CD44 and epithelial-specific antigen (ESA) expression, colony morphology, tumour sphere formation and rapid adherence assays were used to identify the subset of cells with stem-like properties. Apoptosis, cell cycle and expression of various cell cycle checkpoint proteins were assessed (Western Blot, qPCR). The role of G2-checkpoint regulators Chk1 and Chk2 was investigated by use of debromohymenialdisine (DBH) and siRNA.ResultsIn both cancer biopsies and carcinoma cell lines a subset of CD44high cells showed increased clonogenicity, a significantly lower rate of apoptosis, and a significantly higher proportion of cells in the G2-phase of the cell cycle. An inverse correlation between the percentage of cells in G2-phase and the rate of apoptosis was found. Pulse-chase with iododeoxyuridine (IdU) demonstrated that CD44high carcinoma cells spent longer time in G2, even in un-treated controls. These cells expressed higher levels of G2 checkpoint proteins, and their release from G2 with BDH or Chk1 siRNA increased their rate of apoptosis. Low passage cultures of normal keratinocytes were also found to contain a subset of CD44high cells showing increased clonogenicity, and a similar pattern of G2-block associated with apoptotic resistance.ConclusionsThese data indicate that both normal and malignant human epithelial cells with stem-like properties show greater resistance to apoptosis associated with extended G2 cell cycle phase, and that this property is not a consequence of neoplastic transformation. Targeting G2 checkpoint proteins releases these cells from the G2-block and makes them more prone to apoptosis, implying an opportunity for improved therapeutic approaches.

Sub-Sets of Cancer Stem Cells Differ Intrinsically in Their Patterns of Oxygen Metabolism

The glycolytic response of hypoxic cells is primarily mediated by the hypoxia inducible factor alpha (HIF-1α) but even in the presence of abundant oxygen tumours typically show high rates of glycolysis. Higher levels of HIF-1α in tumours are associated with a poorer prognosis and up-regulation of markers of epithelial mesenchymal transition (EMT) due to HIF-1α actions. We have recently shown that EMT occurs within the CD44high cancer stem cell (CSC) fraction and that epithelial and EMT CSCs are distinguished by high and low ESA expression, respectively. We here show that hypoxia induces a marked shift of the CSC fraction towards EMT leading to altered cell morphology, an increased proportion of CD44high/ESAlow cells, patterns of gene expression typical of EMT, and enhanced sphere-forming ability. The size of EMT fractions returned to control levels in normoxia indicating a reversible process. Surprisingly, however, even under normoxic conditions a fraction of EMT CSCs was present and maintained high levels of HIF-1α, apparently due to actions of cytokines such as TNFα. Functionally, this EMT CSC fraction showed decreased mitochondrial mass and membrane potential, consumed far less oxygen per cell, and produced markedly reduced levels of reactive oxygen species (ROS). These differences in the patterns of oxygen metabolism of sub-fractions of tumour cells provide an explanation for the general therapeutic resistance of CSCs and for the even greater resistance of EMT CSCs. They also identify potential mechanisms for manipulation of CSCs.

Hypoxia increases Sca-1/CD44 co-expression in murine mesenchymal stem cells and enhances their adipogenic differentiation potential

Mesenchymal stem cells (MSCs) are usually cultured under normoxic conditions (21% oxygen). However, in vivo, the physiological “niches” for MSCs have a much lower oxygen tension. Because of their plasticity, stem cells are particularly sensitive to their environments, and oxygen tension is one developmentally important stimulus in stem cell biology and plays a role in the intricate balance between cellular proliferation and commitment towards differentiation. Therefore, we investigated here the effect of hypoxia (2% oxygen) on murine adipose tissue (AT) MSC proliferation and adipogenic differentiation. AT cells were obtained from the omental fat and AT-MSCs were selected for their ability to attach to the plastic dishes, and were grown under normoxic and hypoxic conditions. Prior exposure of MSCs to hypoxia led to a significant reduction of ex vivo expansion time, with significantly increased numbers of Sca-1+ as well as Sca-1+/CD44+double-positive cells. Under low oxygen culture conditions, the AT-MSC number markedly increased and their adipogenic differentiation potential was reduced. Notably, the hypoxia-mediated inhibition of adipogenic differentiation was reversible: AT-MSCs pre-exposed to hypoxia when switched to normoxic conditions exhibited significantly higher adipogenic differentiation capacity compared to their pre-exposed normoxic-cultured counterparts. Accordingly, the expression of adipocyte-specific genes, peroxisome proliferator activated receptor γ (Pparγ), lipoprotein lipase (Lpl) and fatty acid binding protein 4 (Fabp4) were significantly enhanced in hypoxia pre-exposed AT-MSCs. In conclusion, pre-culturing MSCs under hypoxic culture conditions may represent a strategy to enhance MSC production, enrichment and adipogenic differentiation.

CD44 Staining of Cancer Stem-Like Cells Is Influenced by Down-Regulation of CD44 Variant Isoforms and Up-Regulation of the Standard CD44 Isoform in the Population of Cells That Have Undergone Epithelial-to-Mesenchymal Transition

CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44high cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44high population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44high population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44high population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that consideration be given to use of multiple cancer stem-like cell markers and suitable procedures for cell isolation in order that the correct populations are assayed.

Expression of Betapapillomavirus Oncogenes Increases the Number of Keratinocytes with Stem Cell-Like Properties

ABSTRACT Human papillomaviruses (HPV) of genus Betapapillomavirus (betaPV) are associated with nonmelanoma skin cancer development in epidermodysplasia verruciformis (EV) and immunosuppressed patients. Epidemiological and molecular studies suggest a carcinogenic activity of betaPV during early stages of cancer development. Since viral oncoproteins delay and perturb keratinocyte differentiation, they may have the capacity to either retain or confer a “stem cell-like” state on oncogene-expressing cells. The aim of this study was to determine (i) whether betaPV alters the expression of cell surface markers, such as CD44 and epithelial cell adhesion molecule (EpCAM), that have been associated with epithelial stemness, and (ii) whether this confers functional stem cell-like properties to human cutaneous keratinocytes. Fluorescence-activated cell sorter (FACS) analysis revealed an increase in the number of cells with high CD44 and EpCAM expression in keratinocyte cultures expressing HPV type 8 (HPV8) oncogenes E2, E6, and E7. Particularly through E7 expression, a distinct increase in clonogenicity and in the formation and size of tumor spheres was observed, accompanied by reduction of the epithelial differentiation marker Calgranulin B. These stem cell-like properties could be attributed to the pool of CD44high EpCAMhigh cells, which was increased within the E7 cultures of HPV5, -8, and -20. Enhanced EpCAM levels were present in organotypic skin cultures of primary keratinocytes expressing E7 of the oncogenic HPV types HPV5, -8, and -16 and in clinical samples from EV patients. In conclusion, our data show that betaPV may increase the number of stem cell-like cells present during early carcinogenesis and thus enable the persistence and accumulation of DNA damage necessary to generate malignant stem cells.

Maintenance of stem cell self‐renewal in head and neck cancers requires actions of GSK3β influenced by CD44 and RHAMM

Cells sorted from head and neck cancers on the basis of their high expression of CD44 have high potency for tumor initiation. These cells are also involved in epithelial to mesenchymal transition (EMT) and we have previously reported that cancer stem cells (CSCs) exist as two biologically distinct phenotypes. Both phenotypes are CD44high but one is also ESAhigh and maintains epithelial characteristics, the other is ESAlow, has mesenchymal characteristics and is migratory. Examining CD44‐regulated signal pathways in these cells we show that CD44, and also RHAMM, act to inhibit phosphorylation of glycogen synthase kinase 3β (GSK3β). We show that inhibitory phosphorylation reduces the formation of both “tumor spheres” and “holoclone” colonies, functional indicators of stemness. GSK3β inhibition also reduces the expression of stem cell markers such as Oct4, Sox2, and Nanog and upregulates expression of the differentiation markers Calgranulin B and Involucrin in the CD44high/ESAhigh cell fraction. Transition of CSCs out of EMT and back to the epithelial CSC phenotype is induced by GSK3β knockdown. These results indicate that GSK3β plays a central role in determining and maintaining the phenotypes and behavior of CSCs in vitro and are likely to be involved in controlling the growth and spread of tumors in vivo.

Phenotypic Plasticity Determines Cancer Stem Cell Therapeutic Resistance in Oral Squamous Cell Carcinoma.

Cancer stem cells (CSCs) drive tumour spread and therapeutic resistance, and can undergo epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) to switch between epithelial and post-EMT sub-populations. Examining oral squamous cell carcinoma (OSCC), we now show that increased phenotypic plasticity, the ability to undergo EMT/MET, underlies increased CSC therapeutic resistance within both the epithelial and post-EMT sub-populations. The post-EMT CSCs that possess plasticity exhibit particularly enhanced therapeutic resistance and are defined by a CD44highEpCAMlow/− CD24+ cell surface marker profile. Treatment with TGFβ and retinoic acid (RA) enabled enrichment of this sub-population for therapeutic testing, through which the endoplasmic reticulum (ER) stressor and autophagy inhibitor Thapsigargin was shown to selectively target these cells. Demonstration of the link between phenotypic plasticity and therapeutic resistance, and development of an in vitro method for enrichment of a highly resistant CSC sub-population, provides an opportunity for the development of improved chemotherapeutic agents that can eliminate CSCs.