Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells.

INTRODUCTION: The identification of potential breast cancer stem cells is of importance as the characteristics of stem cells suggest that they are resistant to conventional forms of therapy. Several techniques have been proposed to isolate or enrich for tumorigenic breast cancer stem cells, including (a) culture of cells in non-adherent non-differentiating conditions to form mammospheres and (b) sorting of the cells by their surface phenotype (expression of CD24 and CD44). METHODS: We have cultured metastatic cells found in pleural effusions from breast cancer patients in non-adherent conditions without serum to form mammospheres. Dissociated cells from these mammospheres were used to determine the tumorigenicity of these cultures. Expression of CD24 and CD44 on uncultured cells and mammospheres derived from the pleural effusions was documented. RESULTS: We found that the majority (20/27) of the pleural effusions tested contained cells capable of forming mammospheres of varying sizes that could be passaged. After dissociation and plating with serum onto adherent dishes, the cells can differentiate, as determined by the increased expression of cytokeratins and MUC1. Analysis of surface expression of CD24 and CD44 on uncultured cells from 21 of the samples showed that the cells from some samples separated into two populations, but some did not. The proportion of cells that could be considered CD44+/CD24low/- was highly variable and did not appear to correlate with the ability to form the larger mammospheres. Of eight pleural effusion mammospheres tested in severe combined immunodeficiency disease (SCID) mice, four were found to induce tumours when only 5,000 or fewer cells were injected, whereas the same number of uncultured cells did not form tumours. The ability to induce tumours appeared to correlate with the ability to produce the larger mammospheres. Uncultured cells from a highly tumorigenic sample (PE14) were uniformly negative for surface expression of both CD24 and CD44. CONCLUSION: This paper shows, for the first time, that mammosphere culture of pleural effusions enriches for cells capable of inducing tumours in SCID mice. The data suggest that mammosphere culture of these metastatic cells could provide a highly appropriate model for studying the sensitivity of the tumorigenic 'stem' cells to therapeutic agents and for further characterisation of the tumour-inducing subpopulation of breast cancer cells.

Endothelins induce CCR7 expression by breast tumor cells via endothelin receptor A and hypoxia-inducible factor-1.

Endothelin expression is increased in breast tumors and is associated with invasion and metastasis, whereas CCR7 expression by breast tumor cells may have a role in the organ specificity of breast cancer spread. In this article, we have analyzed whether endothelins influence breast tumor cell expression of the chemokine receptor CCR7. Stimulation of human breast tumor cell lines with endothelins increased cell surface expression of CCR7 via endothelin receptor A. The iron chelators desferrioxamine and cobalt chloride, which induce hypoxia-inducible factor (HIF)-mediated transcription, also increased CCR7 expression; transfection of a dominant-negative version of the HIF regulatory subunit, HIF-1alpha, into MCF-7 cells abolished CCR7 induction by endothelins, indicating that increased expression is due to HIF-1 stabilization. Endothelin stimulation promoted invasion toward the CCR7 ligands CCL19 and CCL21. Endothelin-mediated chemokine-independent invasion itself is dependent on CCR7 activity and could be abolished using a CCR7-neutralizing monoclonal antibody. In human breast carcinomas, mRNA expression of endothelins correlated with the level of CCR7 expression, both of which were associated with the presence of lymph node metastases. Expression of the CCR7 ligands CCL19 and CCL21 was also higher in breast cancer patients with lymph node involvement compared with those without, but expression of these chemokines did not correlate with endothelin expression. These data show that CCR7 may be regulated by the breast tumor microenvironment and further support the use of endothelin receptor antagonists in the treatment of invasive and metastatic breast cancer.

In vivo loss of expression of argininosuccinate synthetase in malignant pleural mesothelioma is a biomarker for susceptibility to arginine depletion.

PURPOSE: Malignant pleural mesothelioma (MPM) is an increasing health burden on many societies worldwide and, being generally resistant to conventional treatment, has a poor prognosis with a median survival of <1 year. Novel therapies based on the biology of this tumor seek to activate a proapoptotic cellular pathway. In this study, we investigated the expression and biological significance of argininosuccinate synthetase (AS), a rate-limiting enzyme in arginine production. EXPERIMENTAL DESIGN: Initially, we documented down-regulation of AS mRNA in three of seven MPM cell lines and absence of AS protein in four of seven MPM cell lines. We confirmed that the 9q34 locus, the site of the AS gene, was intact using a 1-Mb comparative genomic hybridization array; however, there was aberrant promoter CpG methylation in cell lines lacking AS expression, consistent with epigenetic regulation of transcription. To investigate the use of AS negativity as a therapeutic target, arginine was removed from the culture medium of the MPM cell lines. RESULTS: In keeping with the cell line data, 63% (52 of 82) of patients had tumors displaying reduced or absent AS protein, as assessed using a tissue microarray. Cell viability declined markedly in the AS-negative cell lines 2591 and MSTO but not in the AS-positive cell line, 28. This response was apparent by day 4 and maintained by day 9 in vitro. Arginine depletion induced BAX conformation change and mitochondrial inner membrane depolarization selectively in AS-negative MPM cells. CONCLUSIONS: In summary, we have identified AS negativity as a frequent event in MPM in vivo, leading to susceptibility to cytotoxicity following restriction of arginine. A phase II clinical trial is planned to evaluate the role of arginine depletion in patients with AS-negative MPM.

Expression and regulation of tumor necrosis factor alpha in normal and malignant ovarian epithelium.

Epidemiologic studies implicate inflammatory stimuli in the development of ovarian cancer. The proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and both its receptors (TNFRI and TNFRII) are expressed in biopsies of this malignancy. Here, we tested the hypothesis that TNF-alpha is a regulator of the proinflammatory microenvironment of ovarian cancer. A cancer profiling array showed higher expression of TNF-alpha in ovarian tumors compared with normal ovarian tissue, and cultured ovarian cancer cells expressed up to 1,000 times more TNF-alpha mRNA than cultured normal ovarian surface epithelial cells; TNF-alpha protein was only detected in the supernatant of tumor cell cultures. Treatment with TNF-alpha induced TNF-alpha mRNA via TNFRI in both malignant and normal cells with evidence for enhanced TNF-alpha mRNA stability in tumor cells. TNF-alpha induced TNF-alpha protein in an autocrine fashion in tumor but not in normal ovarian surface epithelial cells. The TNF-alpha neutralizing antibody infliximab reduced the constitutive levels of TNF-alpha mRNA in tumor cell lines capable of autocrine TNF-alpha production. Apart from TNF-alpha mRNA expression, several other proinflammatory cytokines were constitutively expressed in malignant and normal ovarian surface epithelial cells, including interleukin (IL)-1alpha, IL-6, CCL2, CXCL8, and M-CSF. TNF-alpha treatment further induced these cytokines with de novo transcription of IL-6 mRNA contrasting with the increased stability of CCL2 mRNA. RNA interference directed against TNF-alpha was highly effective in abolishing constitutive IL-6 production by ovarian tumor cells. In summary, we show that TNF-alpha is differentially regulated in ovarian cancer cells compared with untransformed cells and modulates production of several cytokines that may promote ovarian tumorigenesis. Infliximab treatment may have a role in suppressing the TNF-alpha-driven inflammatory response associated with ovarian cancer.

A role for endothelin-2 and its receptors in breast tumor cell invasion.

We have studied the role of endothelins (ET-1, ET-2 and ET-3) and ET receptors (ET-RA and ET-RB) in the invasive capacity of breast tumor cells, which express ET-1 and ET-2 as well as ET-RA and ET-RB. Of five human breast tumor cell lines tested, all expressed mRNAs for ET-1, ET-2, and ET-RB. ET-RA mRNA was expressed by four of five tumor cell lines. Breast tumor cells migrated toward ET-1 and ET-2 but not toward ET-3. Chemotaxis involved signaling via both receptors, and a pertussis toxin-sensitive p42/p44 mitogen-activated protein kinase (MAPK)-mediated pathway that could be inhibited by MAPK kinase (MEK)1/2 antagonists. Chemotaxis toward ETs did not involve p38 or stress-activated protein kinase (SAPK)/Jun N-terminal kinase (JNK) and was not inhibited by hypoxia. Incubation of tumor cells with ET-2 also increased chemotaxis toward the chemokines CXCL12 and CCL21. As well as inducing chemotaxis of tumor cells, ET-1 and ET-2 increased tumor cell invasion through Matrigel. Furthermore, stimulation of macrophage/tumor cell cocultures with ETs led to increased matrix metalloproteinase (MMP)-2 and -9 production by macrophages and a marked increase in invasion of tumor cells. Antagonism of either ET-RA or ET-RB decreased the invasion seen in ET-stimulated cocultures, as did a broad-spectrum MMP inhibitor. Immunohistochemical staining of human breast tumor sections showed increased ET and ET receptor protein expression by tumor cells in invasive ductal carcinoma compared with normal breast tissue or ductal carcinoma in situ. Furthermore, tumor cell ET and receptor expression was stronger at the invasive margin of invasive ductal carcinomas, in the lymphovascular space, and in lymph node metastases. ET expression often colocalized with ET-RB expression in all neoplastic tissue indicating a possible autocrine action of ETs. We suggest that expression of ETs and their receptors by human breast tumors, particularly in conjunction with a high macrophage infiltrate, may have a role in the progression of breast cancer and the invasion of tumor cells.

Endothelins in breast tumour cell invasion.

Endothelins are a family of small, structurally related, vasoactive peptides that have a great number of physiological roles in many tissues. The 'endothelin axis' consists of three 21 amino acid peptides (ET-1, ET-2 and ET-3), two G-protein-coupled receptors (ET-RA and ET-RB), and two activating peptidases or endothelin-converting enzymes (ECE-1 and ECE-2). There is increased expression of the endothelin axis in invasive breast cancer compared to the normal breast or non-invasive neoplastic tissue. Endothelin expression is associated with invading regions of tumours in patient biopsies and is more common in tumours with high histological grade and lymphovascular invasion, and there is increased systemic endothelin in patients with lymph node metastases compared to those without lymph node involvement. Stimulation of breast tumour cell lines with endothelins leads to an invasive phenotype in vitro. Over-expression of the endothelins and their receptors is insufficient to induce an invasive phenotype in benign cells, yet expression by tumour cells leads to markedly increased invasive ability indicating that endothelins act in concert with other factors--both autocrine and paracrine--including cytokines, matrix metalloproteinases and the activation of tumour-associated macrophages. The association between endothelins, poor prognosis and invasion may mean that the endothelin axis is a valid therapeutic target for the treatment of invasive breast cancer. This review summarises our current knowledge of endothelins in breast cancer invasion and discusses the potential further directions of such research as well as the possibility of anti-endothelin-based therapy of breast cancer.

Expression of endothelins and their receptors promotes an invasive phenotype of breast tumor cells but is insufficient to induce invasion in benign cells.

There is increased staining of endothelins (ET-1, -2, and -3) and receptors (ET-RA and -RB) in invasive breast tumors compared to nonneoplastic tissue, and ETs stimulate MCF-7 cell invasion in vitro. We analyzed ETstimulation of benign and transformed mammary epithelial cells, and whether expression of ETs is sufficient to induce invasiveness. In breast cancer patient serum, ET-1 was increased in those patients with lymph node metastases compared to those with no lymph node involvement; ETs, however, had no mitogenic effect on breast tumor cell lines in vitro. The benign mammary epithelial cell line, hTERT-HME1, and the poorly invasive breast tumor cell line MCF-7 secreted low levels of ET-1, while the invasive cell lines SKBR3 and MDAMB231 secreted high levels. Expression of the ETs and receptors by the cell lines broadly correlated with their in vitro invasiveness; overexpression of ETs in MCF-7 cells increased basal invasion. ET-mediated invasion involved both receptors and a calcium influx to induce a pertussis toxin-sensitive MAPK pathway. MMP-14 activity was induced via ET-RA in an autocrine manner. In contrast to transformed cells, ET stimulation or overexpression did not induce an invasive phenotype in benign cells. Benign cells do not respond to ETs, and ET expression is not sufficient to induce invasion; however, the level of ET production by tumor cells correlates with their invasiveness, and increasing expression of the ET axis promotes breast tumor cell invasion via both receptors, while MMP-14 is induced via ET-RA.

Endothelin-2 is a hypoxia-induced autocrine survival factor for breast tumor cells.

Endothelins (ETs) are a group of vasoactive peptides (ET-1, ET-2 and ET-3) produced by many cell types that bind to G-protein-linked transmembrane receptors, ET-A receptors (ET-RAs) and ET-B receptors (ET-RBs). These peptides are expressed in several human tumors, including carcinomas of the breast, and have a mitogenic effect in ovarian cancer cell lines. We investigated ET expression in infiltrating ductal carcinomas (IDCs) of the breast and the relationship between ET and hypoxia. ET staining was increased in human grade II IDC samples compared with normal breast tissue. ET-2 and ET-RB mRNA expression were absent in the majority of normal human breast samples (1 of 5 and 0 of 5, respectively) but was present in the majority of IDC tested (13 of 15 and 12 of 15, respectively). In a murine breast cancer model, HTH-K, ET-2, and ET-RB mRNA were detected in tumor but not normal breast tissue, and ET expression colocalized with areas of hypoxia. In vitro, ET-2, ET-RA, and ET-RB mRNA were increased by incubating HTH-K cells in hypoxia (0.1% oxygen) for 24 h. Hypoxia also up-regulated ET-2 mRNA in several human breast tumor cell lines. ET-2 mRNA increased within 3 h in a hypoxia-inducible factor 1-dependent manner. The ET-RB antagonist BQ-788 increased in hypoxia-associated apoptosis of breast tumor cells in vitro. These effects could be reversed by addition of ET-2 peptide. Intratumoral injection of BQ-788 led to an increase in the development and extent of necrosis within the HTH-K tumor and a decrease in the rate of tumor growth. The ET-RA antagonist, BQ-123, also led to a decrease in tumor growth but without a concomitant increase in necrosis. We propose that modulation of ET-2 production via the hypoxia-inducible factor 1 transcription factor and autocrine signaling via ET-RB is a novel mechanism by which tumor cells can withstand hypoxic stress. Treatment of breast carcinomas with ET receptor antagonists may have a therapeutic benefit.

Genetically modified macrophages expressing hypoxia regulated cytochrome P450 and P450 reductase for the treatment of cancer.

This study describes a combined gene and cell therapy based on the genetic modification of primary human macrophages, as a treatment for cancer. Here, we have utilised the tumour-infiltrating properties of macrophages as vehicles to deliver a gene encoding a prodrug-activating enzyme such as human cytochrome P450 2B6 (CYP2B6) inside tumours followed by killing the tumour cells with the prodrug cyclophosphamide (CPA). Macrophages were transduced with an adenoviral vector that expresses human cytochrome CYP2B6 via a synthetic hypoxia responsive promoter (OBHRE) and with human P450 reductase (P450R), via the CMV promoter. In the presence of CPA, these genetically modified macrophages showed increased cytotoxicity against various tumour cell lines compared to untransduced macrophages or macrophages transduced with CYP2B6 alone. In human ovarian carcinoma xenograft models, the median survival of mice treated with genetically modified macrophages plus CPA increased up to two-fold compared to the survival of mice treated with untransduced macrophages and CPA. Genetically modified autologous macrophages may be a feasible therapeutic option for the treatment of some solid tumours, such as ovarian cancer.

Stromal endothelin B receptor-deficiency inhibits breast cancer growth and metastasis.

The endothelin (ET) axis, often deregulated in cancers, is a promising target for anticancer strategies. Whereas previous investigations have focused mostly on ET action in malignant cells, we chose a model allowing separate assessment of the effects of ETs and their receptors ET(A)R and ET(B)R in the tumor cells and the stromal compartment, which is increasingly recognized as a key player in cancer progression. In homozygous spotting lethal rats (sl/sl), a model of constitutive ET(B)R deficiency, we showed significant reduction of growth and metastasis of MAT B III rat mammary adenocarcinoma cells overexpressing ET(A)R and ET-1 but negative for ET(B)R. Lack of stromal ET(B)R expression did not influence angiogenesis. However, it was correlated with diminished infiltration by tumor-associated macrophages and with reduced production of tumor necrosis factor-alpha, both known as powerful promoters of tumor progression. These effects were almost completely abolished in transgenic sl/sl rats, wherein ET(B)R function is restored by expression of an intact ET(B)R transgene. This shows that tumor growth and metastasis are critically dependent on ET(B)R function in cells of the microenvironment and suggests that successful ETR antagonist therapy should also target the stromal component of ET signaling

Sialyl-Lewis(x) on P-selectin glycoprotein ligand-1 is regulated during differentiation and maturation of dendritic cells: a mechanism involving the glycosyltransferases C2GnT1 and ST3Gal I.

To fulfil their function as APCs, dendritic cells (DC) and their precursors need to travel from blood to the peripheral tissues and, upon activation, migrate from tissues to draining lymph nodes. Because O-glycans play a role in T cell trafficking, we investigated the O-glycosylation profile of human monocyte-derived DC. Sialyl-Lewis(x) (sLe(x)), a glycan involved in extravasation via selectin binding, was found to be expressed exclusively on P-selectin glycoprotein ligand-1 in monocytes and immature DC. However, sLe(x) was lost from mature DC even though these cells retained expression of P-selectin glycoprotein ligand-1. Maturation of DC led to a rapid change in the expression of glycosyltransferases involved in O-linked glycosylation. A down-regulation of C2GnT1 mRNA and enzymatic activity was observed with a concurrent up-regulation of ST3Gal I and ST6GalNAc II mRNA resulting in a loss of the core 2 structures required for sLe(x) expression as a P-selectin ligand. Interestingly, the early regulation of these glycosyltransferases was mediated by PGE(2), which is known to be required for human DC migration. The pattern of O-glycosylation seen in mature cells was very similar to that expressed by naive T cells, which home to lymph nodes. Our data show that the regulation of O-glycosylation controls sLe(x) expression, and also suggest that O-glycans may have a function in DC migration.

Aberrant regulation of argininosuccinate synthetase by TNF-alpha in human epithelial ovarian cancer.

The pro-inflammatory cytokine, tumour necrosis factor-alpha, TNF-alpha, is dysregulated in malignant compared with normal ovarian surface epithelium (OSE). Several epidemiological studies have associated inflammation with ovarian tumorigenesis, with TNF-alpha playing a key role in modulating invasion, angiogenesis and metastasis. Here, we show that TNF-alpha also induces expression of arate-limiting enzyme in arginine synthesis, argininosuccinate synthetase (AS), thereby linking inflammation with several arginine-dependent metabolic pathways, implicated in accelerated carcinogenesis and tumour progression. Having identified AS mRNA induction in TNF-alpha-treated IGROV-1 ovarian cancer cells, using RNA-arbitrarily primed-PCR, we then observed differential regulation of AS mRNA and protein in malignant, compared with normal, OSE cells. A cDNA cancer profiling array with matched normal ovarian and ovarian tumour samples revealed increased expression of AS mRNA in the latter. Moreover, AS protein co-localised with TNF-alpha in ovarian cancer cells, with significantly higher levels of AS in malignant compared with normal ovarian tissue. Increased co-expression of AS and TNF-alpha mRNA was also observed in 2 other epithelial tumours, non-small cell lung and stomach cancer, compared with normal corresponding tissues. In summary, high levels of AS expression, which may be required for several arginine-dependent processes in cancer, including the production of nitric oxide, proline, pyrimidines and polyamines, is regulated by TNF-alpha and may provide an important molecular pathway linking inflammation and metabolism to ovarian tumorigenesis.

Endothelin-2 is a macrophage chemoattractant: implications for macrophage distribution in tumors.

Endothelins (ET-1, ET-2 and ET-3) are 21-amino acid vasoactive peptides that bind to G-protein-linked transmembrane receptors, ET-RA and ET-RB. As well as modulating vasoconstriction, endothelins regulate growth in several cell types and may also affect differentiation, inflammation and angiogenesis. Both macrophages and endothelins are found in areas of hypoxia in solid tumors and ET-2 expression may be modulated by hypoxia in some tumors. As the peptide structure of mature endothelins is similar to that of CXC chemokines, we asked if endothelins contribute to control of macrophage distribution in tumors. We found that ET-2 is a chemoattractant for macrophages and THP-1 monocytic cells, but not for freshly isolated monocytes. The chemotactic response to ET-2 shows a typical bell-shaped response curve. Experiments with endothelin receptor antagonists showed that migration to ET-2 is mediated via the ET-RB receptor. Moreover, monocytes do not express ET-RB. Chemotaxis towards ET-2 is via the MAPK pathway: p44 and p42 are phosphorylated when THP-1 cells are stimulated with ET-2, and the MAPKK inhibitor PD98059 stops chemotaxis. As with 'classical' chemokines, migration toET-2 is also inhibited by hypoxia and by pertussis toxin. As well as its chemotactic properties, ET-2 leads to activation of macrophages. In human breast tumors that express ET-2, endothelins and ET-RB expressing macrophages often co-localized. While shorter than 'classical' chemokines, ET-2 shares a similar peptide sequence with chemokines and may signal via a similar receptor and MAPK-mediated pathway. Furthermore, ET-2 expression by tumors may modulate the behavior of macrophages such that activated cells accumulate in areas of hypoxia.