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

BACKGROUND: Subsets 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. METHODS: Cells 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. RESULTS: In both cancer biopsies and carcinoma cell lines a subset of CD44(high) 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 CD44(high) 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 CD44(high) cells showing increased clonogenicity, and a similar pattern of G2-block associated with apoptotic resistance. CONCLUSIONS: These 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.

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.

GLI1 confers profound phenotypic changes upon LNCaP prostate cancer cells that include the acquisition of a hormone independent state.

The GLI (GLI1/GLI2) transcription factors have been implicated in the development and progression of prostate cancer although our understanding of how they actually contribute to the biology of these common tumours is limited. We observed that GLI reporter activity was higher in normal (PNT-2) and tumourigenic (DU145 and PC-3) androgen-independent cells compared to androgen-dependent LNCaP prostate cancer cells and, accordingly, GLI mRNA levels were also elevated. Ectopic expression of GLI1 or the constitutively active ΔNGLI2 mutant induced a distinct cobblestone-like morphology in LNCaP cells that, regarding the former, correlated with increased GLI2 as well as expression of the basal/stem-like markers CD44, ß1-integrin, ΔNp63 and BMI1, and decreased expression of the luminal marker AR (androgen receptor). LNCaP-GLI1 cells were viable in the presence of the AR inhibitor bicalutamide and gene expression profiling revealed that the transcriptome of LNCaP-GLI1 cells was significantly closer to DU145 and PC-3 cells than to control LNCaP-pBP (empty vector) cells, as well as identifying LCN2/NGAL as a highly induced transcript which is associated with hormone independence in breast and prostate cancer. Functionally, LNCaP-GLI1 cells displayed greater clonal growth and were more invasive than control cells but they did not form colonies in soft agar or prostaspheres in suspension suggesting that they do not possess inherent stem cell properties. Moreover, targeted suppression of GLI1 or GLI2 with siRNA did not reverse the transformed phenotype of LNCaP-GLI1 cells nor did double GLI1/GLI2 knockdowns activate AR expression in DU145 or PC-3 cells. As such, early targeting of the GLI oncoproteins may hinder progression to a hormone independent state but a more detailed understanding of the mechanisms that maintain this phenotype is required to determine if their inhibition will enhance the efficacy of anti-hormonal therapy through the induction of a luminal phenotype and increased dependency upon AR function.

Stem cell patterns in cell lines derived from head and neck squamous cell carcinoma.

The initiation, growth, recurrence and metastasis of solid tumours, including squamous cell carcinoma of the head and neck region, have been related to the behaviour of a small subpopulation of 'tumour-initiating' cells. Cells with stem cell characteristics have also been identified in cell lines derived from cancers and the aim of the present work was to extend examination of such cells. Established cell lines were examined for their patterns of colony morphologies and staining, the presence of a Hoechst dye-excluding 'side population', expression of the putative stem cell markers CD44, CD133 and CD29, and their ability to grow as 'cancer spheroids'. Two cell lines, CaLH2 and CaLH3, recently generated from HNSCC tumour biopsies, were similarly examined. All cell lines showed a holoclone/meroclone/paraclone series of colony morphologies and cell sorting indicated that CD44 marker expression was related to clonogenicity. FACS analysis after exposure to Hoechst dye indicated that the CA1, H357 and UK1 cell lines contain a dye-excluding 'side population', a property associated with stem-like subpopulations. When held in suspension, all cell lines formed spheroids that could be re-passaged. These observations indicate that cell lines derived from HNSCC contain cells with stem cell properties and that such cell lines may provide experimental systems relevant to the behaviour of stem cells present in the tumours of origin and to their responses to therapy.