A preliminary study of the role of extracellular -5′- nucleotidase in breast cancer stem cells and epithelial-mesenchymal transition

Tumor stem cell theory may well explain a variety of malignant behaviors of tumors. Cells undergoing epithelial-mesenchymal transition (EMT) share many characteristics with tumor stem cells. Our previous studies showed that extracellular -5'- nucleotidase (CD73), one of the important surface markers of mesenchymal stem cells, may promote growth and metastasis of breast cancer cells both in vivo and in vitro. In this study, we assessed breast cancer stem cell (BCSC) markers [acetaldehyde dehydrogenase (ALDH)⁺ and CD44⁺CD24^?] in various breast cancer cell lines with flow cytometry after overexpression (by lentivirus infection) or suppression (by siRNA interference) of CD73. We measured CD73 expression in breast cancer mammospheres with real-time PCR and western blots. Finally, we examined the expression of CD73 and EMT markers in different breast cancer cell lines, as well as in mammary cells (MCF10A) that underwent EMT induced by transforming growth factor beta (TGF-β). We found that CD73 positively correlated with ALDH⁺ or CD44⁺CD24^? subsets of breast cancer cells. CD73 was expressed more in breast cancer mammospheres than in adherent cells. CD73 and mesenchymal marker expression was higher in breast cancer cells with more malignant features, while CD73 was lower in low malignant breast cancer cells with higher epithelial markers. Furthermore, CD73 expression increased during the process of TGF-β-induced EMT. Our results indicate that CD73 may play an important role in BCSCs.     

RASSF1C oncogene elicits amoeboid invasion,cancer stemness,and extracellular vesicle release via a SRC/Rho axis

Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces, and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here, we show that the novel oncogene RASSF1C drives mesenchymal‐to‐amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC‐mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that RASSF1C‐induced amoeboid cells display increased expression of cancer stem‐like markers such as CD133, ALDH1, and Nanog, and are accompanied by higher invasive potential in vitro and in vivo. Further, RASSF1C‐induced amoeboid cells employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C‐driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early‐stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification.     

Proteomic identification of glycosylphosphatidylinositol anchor-dependent membrane proteins elevated in breast carcinoma

The glycosylphosphatidylinositol (GPI) anchor is a lipid and glycan modification added to the C terminus of certain proteins in the endoplasmic reticulum by the activity of a multiple subunit enzyme complex known as the GPI transamidase (GPIT). Several subunits of GPIT have increased expression levels in breast carcinoma. In an effort to identify GPI-anchored proteins and understand the possible role of these proteins in breast cancer progression, we employed a combination of strategies. First, alpha toxin from Clostridium septicum was used to capture GPI-anchored proteins from human breast cancer tissues, cells, and serum for proteomic analysis. We also expressed short interfering RNAs targeting the expression of the GPAA1 and PIGT subunits of GPIT in breast cancer cell lines to identify proteins in which membrane localization is dependent on GPI anchor addition. Comparative membrane proteomics using nano-ESI-RPLC-MS/MS led to the discovery of several new potential diagnostic and therapeutic targets for breast cancer. Furthermore, we provide evidence that increased levels of GPI anchor addition in malignant breast epithelial cells promotes the dedifferentiation of malignant breast epithelial cells in part by increasing the levels of cell surface markers associated with mesenchymal stem cells.     

Identification of single chain antibodies to breast cancer stem cells using phage display

Recent evidence suggests that most malignancies are driven by “cancer stem cells” sharing the signature characteristics of adult stem cells: the ability to self renew and to differentiate. Furthermore these cells are thought to be quiescent, infrequently dividing cells with a natural resistance to chemotherapeutic agents. These studies theorize that therapies, which effectively treat the majority of tumor cells but ‘miss’ the stem cell population, will fail, while therapies directed at stern cells can potentially eradicate tumors. In breast cancer, researchers have isolated ‘breast cancer stem cells’ capable of recreating the tumor in vivo and in vitro. Generated new tumors contained both additional numbers of cancer stem cells and diverse mixed populations of cells present in the initial tumor, supporting the intriguing self‐renewal and differentiation characteristics. In the present study, an antibody phage library has been used to search for phage displayed‐single chain antibodies (scFv) with selective affinity to specific targets on breast cancer stem cells. We demonstrate evidence of two clones binding specifically to a cancer stem cell population isolated from the SUMl59 breast cancer cell line. These clones had selective affinity for cancer stem cells and they were able to select cancer stem cells among a large population of non‐stem cancer cells in paraffin‐embedded sections. The applicability of these clones to paraffin sections and frozen tissue specimens made them good candidates to be used as diagnostic and prognostic markers in breast cancer patient samples taking into consideration the cancer stern cell concept in tumor biology. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Intra-Tumour Signalling Entropy Determines Clinical Outcome in Breast and Lung Cancer

The cancer stem cell hypothesis, that a small population of tumour cells are responsible for tumorigenesis and cancer progression, is becoming widely accepted and recent evidence has suggested a prognostic and predictive role for such cells. Intra-tumour heterogeneity, the diversity of the cancer cell population within the tumour of an individual patient, is related to cancer stem cells and is also considered a potential prognostic indicator in oncology. The measurement of cancer stem cell abundance and intra-tumour heterogeneity in a clinically relevant manner however, currently presents a challenge. Here we propose signalling entropy, a measure of signalling pathway promiscuity derived from a sample’s genome-wide gene expression profile, as an estimate of the stemness of a tumour sample. By considering over 500 mixtures of diverse cellular expression profiles, we reveal that signalling entropy also associates with intra-tumour heterogeneity. By analysing 3668 breast cancer and 1692 lung adenocarcinoma samples, we further demonstrate that signalling entropy correlates negatively with survival, outperforming leading clinical gene expression based prognostic tools. Signalling entropy is found to be a general prognostic measure, valid in different breast cancer clinical subgroups, as well as within stage I lung adenocarcinoma. We find that its prognostic power is driven by genes involved in cancer stem cells and treatment resistance. In summary, by approximating both stemness and intra-tumour heterogeneity, signalling entropy provides a powerful prognostic measure across different epithelial cancers.     

Doxycycline inhibits the cancer stem cell phenotype and epithelial-to-mesenchymal transition in breast cancer

Experimental evidence suggest that breast tumors originate from breast cancer stem cells (BCSCs), and that mitochondrial biogenesis is essential for the anchorage-independent clonal expansion and survival of CSCs, thus rendering mitochondria a significant target for novel treatment approaches. One of the recognized side effects of the FDA-approved drug, doxycycline is the inhibition of mitochondrial biogenesis. Here we investigate the mechanism by which doxycycline exerts its inhibitory effects on the properties of breast cancer cells and BCSCs, such as mammosphere forming efficiency, invasion, migration, apoptosis, the expression of stem cell markers and epithelial-to-mesenchymal transition (EMT) related markers of breast cancer cells. In addition, we explored whether autophagy plays a role in the inhibitory effect of doxycycline on breast cancer cells. We find that doxycyline can inhibit the viability and proliferation of breast cancer cells and BCSCs, decrease mammosphere forming efficiency, migration and invasion, and EMT of breast cancer cells. Expression of stem cell factors Oct4, Sox2, Nanog and CD44 were also significantly downregulated after doxycycline treatment. Moreover, doxycycline could down-regulate the expression of the autophagy marker LC-3BI and LC-3BII, suggesting that inhibiting autophagy may be responsible in part for the observed effects on proliferation, EMT and stem cell markers. The potent inhibition of EMT and cancer stem-like characteristics in breast cancer cells by doxycycline treatment suggests that this drug can be repurposed as an anti-cancer drug in the treatment of breast cancer patients in the clinic.     

Endothelial induced EMT in breast epithelial cells with stem cell properties

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.     

A putative human breast stem cell population is enriched for steroid receptor-positive cells

Breast epithelial stem cells are thought to be the primary targets in the etiology of breast cancer. Since breast cancers mostly express estrogen and progesterone receptor (ERalpha and PR), we examined the biology of these ERalpha/PR-positive cells and their relationship to stem cells in normal human breast epithelium. We employed several complementary approaches to identify putative stem cell markers, to characterise an isolated stem cell population and to relate these to cells expressing the steroid receptors ERalpha and PR. Using DNA radiolabelling in human tissue implanted into athymic nude mice, a population of label-retaining cells were shown to be enriched for the putative stem cell markers p21(CIP1) and Msi-1, the human homolog of Drosophila Musashi. Steroid receptor-positive cells were found to co-express these stem cell markers together with cytokeratin 19, another putative stem cell marker in the breast. Human breast epithelial cells with Hoechst dye-effluxing "side population" (SP) properties characteristic of mammary stem cells in mice were demonstrated to be undifferentiated "intermediate" cells by lack of expression of myoepithelial and luminal apical membrane markers. These SP cells were 6-fold enriched for ERalpha-positive cells and expressed several fold higher levels of the ERalpha, p21(CIP1) and Msi1 genes than non-SP cells. In contrast to non-SP cells, SP cells formed branching structures in matrigel which included cells of both luminal and myoepithelial lineages. The data suggest a model where scattered steroid receptor-positive cells are stem cells that self-renew through asymmetric cell division and generate patches of transit amplifying and differentiated cells.     

Ubiquitin ligase CHIP suppresses cancer stem cell properties in a population of breast cancer cells

Cancer stem cells (CSCs) have several distinctive characteristics, including high metastatic potential, tumor-initiating potential, and properties that resemble normal stem cells such as self-renewal, differentiation, and drug efflux. Because of these characteristics, CSC is regarded to be responsible for cancer progression and patient prognosis. In our previous study, we showed that a ubiquitin E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP) suppressed breast cancer malignancy. Moreover, a recent clinical study reported that CHIP expression levels were associated with favorable prognostic parameters of patients with breast cancer. Here we show that CHIP suppresses CSC properties in a population of breast cancer cells. CHIP depletion resulted in an increased proportion of CSCs among breast cancers when using several assays to assess CSC properties. From our results, we propose that inhibition of CSC properties may be one of the functions of CHIP as a suppressor of cancer progression.     

Uncoupling of the ERα regulated morphological phenotype from the cancer stem cell phenotype in human breast cancer cell lines

The CD24^low/−CD44⁺EpCAM⁺ phenotype is associated with breast cancer initiating cells. To investigate if these putative breast cancer stem cell markers are regulated by estrogen receptor alpha (ERα) we have determined the expression levels of EpCAM, CD44 and CD24 in several well characterized breast cancer cell lines. The expression levels of the three adhesion proteins were quantitatively different in the cell lines but the composite CD24^low/−CD44⁺EpCAM⁺ breast cancer stem cell phenotype was shown to exist as a small fraction, between 0.1% and 1.2%, in all breast cancer cell lines tested. Experimental silencing of ERα resulted in a reduced epithelial appearance and partial reduction of CD24 mRNA, while levels of CD44 and EpCAM were unaltered. Moreover, knockdown of ERα led to a change in the morphology of the cells similar to the epithelial to mesenchymal transition phenotype and was associated with decreased E-cadherin expression. Our findings offer new insights into the regulation of the breast cancer stem cell phenotype by ERα and suggest that treatments targeting the breast cancer stem cell adhesion molecules and the ERα pathway may be complementary.     

Estrogen receptor alpha mediates epithelial to mesenchymal transition,expression of specific matrix effectors and functional properties of breast cancer cells

The 17β-estradiol (E2)/estrogen receptor alpha (ERα) signaling pathway is one of the most important pathways in hormone-dependent breast cancer. E2 plays pivotal roles in cancer cell growth, survival, and architecture as well as in gene expression regulatory mechanisms. In this study, we established stably transfected MCF-7 cells by knocking down the ERα gene (designated as MCF-7/SP10 + cells), using specific shRNA lentiviral particles, and compared them with the control cells (MCF-7/c). Interestingly, ERα silencing in MCF-7 cells strongly induced cellular phenotypic changes accompanied by significant changes in gene and protein expression of several markers typical of epithelial to mesenchymal transition (EMT). Notably, these cells exhibited enhanced cell proliferation, migration and invasion. Moreover, ERα suppression strongly affected the gene and protein expression of EGFR and HER2 receptor tyrosine kinases, and various extracellular matrix (ECM) effectors, including matrix metalloproteinases and their endogenous inhibitors (MMPs/TIMPs) and components of the plasminogen activation system. The action caused by E2 in MCF-7/c cells in the expression of HER2, MT1-MMP, MMP1, MMP9, uPA, tPA, and PAI-1 was abolished in MCF-7/SP10 + cells lacking ERα. These data suggested a regulatory role for the E2/ERα pathway in respect to the composition and activity of the extracellular proteolytic molecular network. Notably, loss of ERα promoted breast cancer cell migration and invasion by inducing changes in the expression levels of certain matrix macromolecules (especially uPA, tPA, PAI-1) through the EGFR–ERK signaling pathway.In conclusion, loss of ERα in breast cancer cells results in a potent EMT characterized by striking changes in the expression profile of specific matrix macromolecules highlighting the potential nodal role of matrix effectors in breast cancer endocrine resistance.     

Functional Heterogeneity within the CD44 High Human Breast Cancer Stem Cell–Like Compartment Reveals a Gene Signature Predictive of Distant Metastasis

The CD44^hi compartment in human breast cancer is enriched in tumor-initiating cells; however, the functional heterogeneity within this subpopulation remains poorly defined. We used a triple-negative breast cancer cell line with a known bilineage phenotype to isolate and clone CD44^hi single cells that exhibited mesenchymal/basal B and luminal/basal A features, respectively. Herein, we demonstrate in this and other triple-negative breast cancer cell lines that, rather than CD44^hi/CD24⁻ mesenchymal-like basal B cells, the CD44^hi/CD24^lo epithelioid basal A cells retained classic cancer stem cell features, such as tumor-initiating capacity in vivo, mammosphere formation and resistance to standard chemotherapy. These results complement previous findings using oncogene-transformed normal mammary cells showing that only cell clones with a mesenchymal phenotype exhibit cancer stem cell features. Further, we performed comparative quantitative proteomic and gene array analyses of these cells and identified potential novel markers of breast cancer cells with tumor-initiating features, such as lipolysis-stimulated lipoprotein receptor (LSR), RAB25, S100A14 and mucin 1 (MUC1), as well as a novel 31-gene signature capable of predicting distant metastasis in cohorts of estrogen receptor–negative human breast cancers. These findings strongly favor functional heterogeneity in the breast cancer cell compartment and hold promise for further refinements of prognostic marker profiling. Our work confirms that, in addition to cancer stem cells with mesenchymal-like morphology, those tumor-initiating cells with epithelial-like morphology should also be the focus of drug development.     

Delta/notch-like epidermal growth factor-related receptor promotes stemness to facilitate breast cancer progression

DNER, Delta/Notch-like epidermal growth factor (EGF)-related receptor, is a neuron-specific transmembrane protein carrying extracellular EGF-like repeats. The function of DNER in breast cancer has not been evaluated. The present study demonstrates that the expression of DNER in breast cancer tissue is significantly higher than its expression in breast benign disease and is associated with poor recurrence-free survival (RFS) of breast cancer patients. It demonstrated that DNER could enhance the proliferation and metastasis of breast cancer cells in vitro and significantly increases tumor growth in vivo. Our study uncovered that DNER can promote breast cancer cells proliferation and metastasis by activating Girdin/PI3K/AKT signaling and subsequently regulating several key genes involving the characters of cancer stem cells. Taken together, DNER promotes breast cancer growth and metastasis, which provided a theoretical basis for future applications of DNER inhibitors in the treatment of breast cancer.     

Isolation and characterization of human mammary stem cells

Since stem cells are present throughout the lifetime of an organism, it is thought that they may accumulate mutations, eventually leading to cancer. In the breast, tumours are predominantly oestrogen and progesterone receptor-positive (ERalpha/PR+). We therefore studied the biology of ERalpha/PR-positive cells and their relationship to stem cells in normal human mammary epithelium. We demonstrated that ERalpha/PR-positive cells co-express the putative stem cell markers p21(CIP1/WAF1), cytokeratin (CK) 19 and Musashi-1 when examined using dual label immunofluorescence on tissue sections. Next, we isolated a Hoechst dye-effluxing 'side population' (SP) from the epithelium using flow cytometry and demonstrated them to be undifferentiated cells by lack of expression of myoepithelial and luminal cell-specific antigens such as CALLA and MUC1. Epithelial SP cells were shown to be enriched for the putative stem cell markers p21(CIP1/WAF1), Musashi-1 and ERalpha/PR-positive cells. Lastly, SP cells, compared to non-SP, were highly enriched for the capacity to produce colonies containing multiple lineages in 3D basement membrane (Matrigel) culture. We conclude that breast stem cells include two populations: a primitive ERalpha/PR-negative stem cell necessary for development and a shorter term ERalpha/PR-positive stem cell necessary for adult tissue homeostasis during menstrual cycling. We speculate these two basic stem cell types may therefore be the cells of origin for ERalpha-positive and -negative breast tumours.