| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Cancer Research United Kingdom Translational Oncology Laboratory, Barts and The London, Queen Mary’s School of Medicine and Dentistry, London EC1M 6BQ, [M. J. G., F. R. B.], and Oxford BioMedica United Kingdom Ltd., Oxford OX4 4GA [S. N.], United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human tumors, including carcinomas of the breast (9), ovary (3), prostate (10) and lung (11), may express ETs. Most reports focus on ET-1, which may act as an autocrine and paracrine growth factor in ovarian tumors (3, 12, 13). Treatment of human melanoma tumors grown in nude mice with an ET-RB antagonist slows tumor growth (14), whereas ET-RA antagonists slow growth of murine colorectal tumors (15).
Solid tumors contain regions of hypoxia caused either by failure of the tumor vasculature to develop or destruction of the vasculature when necrosis occurs, and this may modulate the expression of a variety of genes (16). Hypoxia modulates ET expression in several cell types e.g., ET-1 is regulated by hypoxia, either positively or negatively, in endothelial cells depending upon source (17, 18). ET receptor expression may also be regulated by hypoxia e.g., the number of ET-RBs on cultured astrocytes increases during transient hypoxia, but the ET-RA does not increase (19). Tumor hypoxia may affect ET expression: ET-2 mRNA is increased by hypoxia in squamous carcinoma cells (20). However, ET-1 production is decreased by hypoxia in colon adenocarcinoma and prostate carcinoma cells (21).
In this paper, we have studied ET expression in human breast carcinomas, which contain regions of hypoxia (22), and the links between hypoxia and ETs in a murine breast cancer model. HTH-K is a murine model of breast carcinoma derived from a spontaneous tumor in a colony of BALB/c c-neu transgenic mice and established as a transplantable tumor and cell line (23, 24). HTH-K forms comedo-type, metastatic, and highly angiogenic breast carcinomas that share several features with the equivalent human malignancy.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cell Culture.
HTH-K cells were cultured as described previously (23). Several human breast tumor cell lines (MCF-7, BT20, BT474, and MDAMB468) were cultured similarly. For hypoxic experiments, cells were grown to semiconfluence, and the medium was replaced before hypoxic culture. Cells were incubated in a Heto-Holten (Camberley, United Kingdom) hypoxic incubator (0.1% O2, 5% CO2, 37°C).
HTH-K Tumor and NITP Treatment.
The tumor was grown in female BALB/c mice as described previously (23). The tumor was excised when 
1 cm2. Normal breast tissue was taken at the same time from the tumor-bearing mouse. The tissue was snap frozen in liquid nitrogen or placed in 10% formol saline.
NITP [supplied by Oxford BioMedica (United Kingdom) Ltd.] is a marker of tumor hypoxia. Two h before the tumors were excised, sonicated NITP mixture (7 mg of NITP, 50 µl of DMSO, and 0.45 ml peanut oil) was injected i.p. (0.2 ml/mouse).
Treatment of HTH-K Tumors with ET Receptor Antagonists.
HTH-K cells were injected s.c. into 24 mice. After 3 days, tumors were injected intratumorally with an ET-RA-specific antagonist (25–27) or an ET-RB-specific antagonist BQ-788 (28) from American Peptide Co., Inc., (50 µg/mouse/day in 0.1 ml of PBS; eight mice/group) for 5 days. Control groups were injected with PBS only. Tumor size was measured using calipers. Twenty-four h after the final injection, tumors were removed and fixed in 10% formal saline.
IHC.
Paraffin sections of tumors were examined for reduced NITP using a rabbit antitheophylline antibody (Sigma-Aldrich Co. Ltd., St. Louis, MO) or for mature ET using a rabbit antimouse ET antibody (Oncogene Research Products, Cambridge, MA), which binds all ET isoforms (ET-1, ET-2, and ET-3). An appropriate biotinylated secondary antibody was used. The sections were probed with streptavidin-peroxidase and developed using 3,3'-diaminobenzidine substrate followed by counterstaining with Harris’s hematoxylin.
RNA Extraction.
Frozen tissue was homogenized in a mill under liquid nitrogen (Glen Creston, Stanmore, United Kingdom). Tri-Reagent (Sigma-Aldrich Co. Ltd.) was added to the homogenized tissue. For cell lines, Tri-Reagent was added to the adherent cells. Total RNA was prepared according to the manufacturer’s instructions, and the isolated RNA was DNase treated.
RT-PCR.
Total RNA was reverse transcribed using the Ready-To-Go kit from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ). One µl of cDNA was amplified by 35 rounds of PCR, and products were sequenced to confirm identity. Previously published RT-PCR primers were used or primers were designed from murine/human mRNA database sequences (Table 1).
|
Enzyme Immunoassay.
Conditioned medium was tested for the presence of mature ET-2 peptide using enzyme immunoassay kits from Peninsula Laboratories Europe, Ltd. (Merseyside, United Kingdom).
Western Blotting.
Cell extract (10 µg) was run on a SDS 10% acrylamide gel and transferred to a nylon membrane. The membrane was probed using a sheep anti-ET-RA polyclonal antibody (Alexis Biochemicals, San Diego, CA) or a sheep anti-ET-RB polyclonal antibody from Calbiochem- Novabiochem Corporation (San Diego, CA; 20 µg/ml in 5% milk powder PBS-T, overnight at 4°C). After washing with PBS-T, the membrane was incubated in 5% milk powder PBS-T containing an horseradish peroxidase-conjugated antisheep secondary antibody (1/5000 dilution in 5% milk powder PBS-T; room temperature for 1 h). The secondary antibody was detected using the Western Blot Chemiluminescence Reagent Plus kit (NEN Life Science Products, Boston, MA). Protein concentration equivalence was confirmed after probing by amido black staining.
Analysis of Apoptosis.
Cells were analyzed for the presence of histone-associated DNA fragments using a cell death detection ELISA from Roche Molecular Biologicals (Mannheim, Germany).
Statistical Analysis.
Results were tested for statistical significance using ANOVA or Students t test with InStat Version 2.01 software.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
|
The HTH-K Tumor Model Has Hypoxic Regions That Colocalize with ET Expression.
Because the murine breast and HTH-K tumor tissues had similar mRNA expression profiles of ET compared with equivalent human tissues, we used the HTH-K model to analyze the relationship between hypoxia and ETs in breast carcinomas. IHC was used to analyze the distribution of hypoxia and ET peptides in paraffin sections of HTH-K tumors from mice that had been treated with the hypoxia marker NITP. Serial sections were stained for reduced-NITP and for ET; the ET antibody recognizes all ET isoforms. Similar to human tumors (22), staining for reduced-NITP showed the presence of focal areas of hypoxia within the HTH-K tumor, particularly in areas adjacent to necrotic lesions (Fig. 2B); mature ET colocalized with these areas of hypoxia.
HTH-K Cell Line mRNA Expression of ET-2 and ET-RAs and ET-RBs Is Increased by Hypoxia.
ET-2 mRNA was tumor specific and ET expression was associated with areas of hypoxia in the HTH-K tumor. Using Northern blotting, we analyzed the effect of in vitro hypoxia (0.1% oxygen) on the HTH-K cell line expression of ET-2 and its receptors. mRNA levels were normalized to rRNA levels (18S and 28S) as hypoxia can affect housekeeping gene expression and, indeed, HTH-K ß-actin mRNA was reduced compared with rRNA during hypoxia (Fig. 3a).
|
ET-1 and ET-3 mRNA expression by HTH-K cells were low, and hypoxia did not increase these mRNAs to levels detectable by Northern blotting (data not shown).
ET-2 Peptide and Receptor Synthesis Is Increased by Hypoxia.
We next studied whether mature ET-2 peptide synthesis and release were increased by hypoxia using an enzyme immunoassay. Hypoxia consistently increased the level of ET-2 peptide in the medium of cultured HTH-K cells. In one representative experiment, ET-2 peptide increased 12-fold in the hypoxic medium compared with the normoxic medium (normoxia 4 ± 2.7 pg/ml, hypoxia 48 ± 11.3, n = 8, P < 0.0005; Fig. 3d).
Hypoxia also resulted in increased ET receptor protein synthesis. HTH-K cells were incubated either normoxically or hypoxically for 24 h, and Western blotting of the cell extracts showed that protein levels of ET-RA and ET-RB were increased by hypoxia (Fig. 3e).
Up-Regulation of ET-2 mRNA Is in a HIF-dependent Manner.
To further characterize ET-2 induction by hypoxia, we analyzed the time course of ET-2 mRNA up-regulation by HTH-K cells and incubated cells with inhibitors or activators of the transcription factor HIF-1.
HTH-K cells were incubated from 0 to 24 h in 0.1% oxygen. By 3 h, ET-2 mRNA had increased significantly compared with the normoxic control (Fig. 4, a and b).
|
ET-2 Signals via ET-RB to Increase HTH-K Cell Survival during Hypoxia.
We next analyzed the effects of increased ET-2 synthesis on hypoxic survival of HTH-K cells using antagonists of the ET receptors. Semiconfluent HTH-K cells (40,000 cells/10 cm2 well) were incubated in low-serum medium (2% FCS) under hypoxic conditions for 72 h. During hypoxia in low-serum conditions, HTH-K cells are viable for 3–4 days but there is little, if any, proliferation (our unpublished data).
During hypoxic incubation, cells were treated with the ET-RA-specific antagonist BQ-123 (Refs. 25–27; 100 ng/ml) or the ET-RB-specific antagonist BQ-788 (Ref. 28; 100 ng/ml). The number of viable cells recovered was significantly decreased by the presence of ET-RB inhibitor BQ-788 (Fig. 5a). Microscopically, cells treated with BQ-788 were smaller and had condensed nuclei (data not shown). The ET-RA antagonist BQ-123 had no significant effect on hypoxic cell survival. The effects of BQ-788 were concentration dependent, showing a typical dose-response curve between 0 and 1000 ng/ml (Fig. 5b).
|
Addition of ET-2 peptide also increased survival of the human breast tumor cell lines during hypoxia, whereas BQ-788 decreased the number of viable cells recovered. Of all of the cell lines tested, MCF-7 cells showed the greatest and most consistent response to ET-2 or BQ-788 (Fig. 5d). All cells treated with BQ-788 were smaller and had condensed nuclei (data not shown).
To analyze whether the effect of ET-2 was hypoxia- specific, we repeated the experiments under normoxic conditions. Neither ET-2 peptide or receptor antagonists had any significant effect on growth or viability of HTH-K cells grown in normoxic conditions (data not shown).
BQ-788 Increases Apoptosis of HTH-K Cells during Hypoxia.
HTH-K cells cultured in DMEM medium supplemented with 2% FCS were treated with 100 ng/ml BQ-123 or BQ-788 for 24 h under hypoxic conditions. Culture medium was removed, and cells were analyzed for apoptosis using a cell death detection ELISA that detects histone-associated DNA fragments. Incubation of HTH-K cells under hypoxia with BQ-788 increased the level of apoptosis compared with untreated hypoxic cells (Fig. 5e). The ET-RA antagonist, however, had no effect on the levels of apoptosis during hypoxia. Neither antagonist affected apoptosis in normoxic cells (data not shown).
An ET-RB Antagonist Increases HTH-K Tumor Necrosis in Vivo.
Having shown that ET-2 can act as a protective factor in vitro and that BQ-788 reduces hypoxic survival of HTH-K cells, we examined the effect of ET receptor antagonists in vivo. Paraffin sections of tumors treated with either BQ-123 or BQ-788 were stained and analyzed by Gordon Stamp, Professor of Histopathology, Transgenic Pathology Unit, Cancer Research United Kingdom, who was blinded to the treatment and control groups. Treatment of established HTH-K tumors with BQ-788 for 5 days led to an increase in the extent and development of necrosis (Fig. 6a) in all tumors compared with control-treated tumors. The extent of necrosis was calculated by grading 20 random fields/tumor as necrotic or nonnecrotic tumor tissue. Tumors treated with BQ-788 were significantly more necrotic than either control or BQ-123 treated tumors (Fig. 6b). Treatment with the ET-RA antagonist BQ-123 did not produce any gross morphological changes compared with control tumors. However, treatment for 5 days with either the B-receptor antagonist BQ-788 or the A-receptor antagonist BQ-123 led to a significantly (P < 0.05) decreased tumor volume compared with control tumors (Fig. 6c).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The presence of mature ET peptides colocalized with regions of hypoxia in the HTH-K tumor in vivo, and hypoxia increased ET-2 and ET-RB expression (mRNA and peptide or protein) by HTH-K cells in vitro. In the human c-neu-positive tumors, ET staining was strongest in prenecrotic regions or areas of tumor cells bordering necrotic lesions; such areas are likely to be hypoxic (34). In contrast, in the c-neu-negative carcinomas, discreet tumor islands that stained strongly for ET could be seen. There may be a greater deal of vascularity in c-neu-positive tumors (35), and the tumor islands appear essentially avascular and, hence, are also likely to be hypoxic (22). ET-2 mRNA was consistently found in these tumors, whereas expression of the other ET occurred less often, and so it is likely that the positive staining of tumor cells is because of ET-2 production. However, it cannot be ruled out that some tumors may express ET-1 or ET-3 as well as ET-2.
IHC of the normal breast and benign fibroadenomas showed no positive ET staining in these tissues, whereas all tumors examined produced positive staining. In contrast, ET mRNAs could be detected by RT-PCR in all tissues. This may be because of different rates of ET mRNA/peptide production and degradation but is more likely because of the extreme sensitivity of RT-PCR; similarly, ET-1 mRNA could be detected in HTH-K cells by RT-PCR, but Northern blotting showed that ET-1 mRNA expression was very low.
Induction of ET-2 mRNA by desferrioxamine and cobalt (30) and partial inhibition of hypoxic ET-2 induction by diphenylene iodonium (31) indicate that up-regulation of ET-2 mRNA during hypoxia is HIF-1 dependent. This is supported by the time course of ET-2 mRNA induction: ET-2 mRNA had not increased within 1 h of hypoxia but was induced within 3 h, which is similar to many HIF-1-dependent genes (16). This is the first evidence of the involvement of HIF-1 in hypoxic expression of ET-2 mRNA. No hypoxically responsive elements have yet been described in the promoter of murine or human ET-2. The promoter of the ET-1 gene has a HIF-1 binding site, the hypoxia response element, which is bound by HIF-1 during hypoxia in microvascular endothelial cells (36). However, it is worth noting that ET-1 mRNA can be down-regulated during hypoxia (18).
Adding ET-2 peptide to the culture medium modestly reduces the tumor cell death associated with hypoxia. It should be noted that the cells have increased ET-2 production in response to hypoxia, and hence the effect of additional ET-2 might be expected to be small. This effect was mediated via the ET-RB receptor as only treatment with an ET-RB antagonist (28) in vitro led to increased cell death during hypoxia; a similar ET-RA antagonist (25–27) had no effect. Increased cell death caused by the ET-RB antagonist was associated with increased histone-associated DNA fragments indicating increased apoptosis. ET-1 has been shown to protect astrocytes from hypoxic/ischemic injury (37) and has previously been suggested to have a paracrine action in breast tumors (12), but this is the first time that a protective autocrine action of ET-2 has been proposed.
Although ET-2 results in an increased number of cells surviving hypoxic stress and addition of BQ-788 leads to increased apoptosis, the potential mechanisms of ET-2- mediated cell survival are yet to be elucidated. Hypoxia induces caspase-dependent apoptosis in some cell types (38), and ET-2, signaling via ET-RB, may inhibit caspase activation. Several other potential mechanisms of preventing apoptosis exist such as increased production of antiapoptotic cytokines (39), enhanced glucose uptake (40), and blocking of apoptosis signaling (41). Hypoxia may also cause microsatellite instability in tumor cells (42), which may induce apoptosis (43); ET-2 may modulate mismatch repair genes (44) such that either the damage is repaired or the cell is not prompted to apoptose.
These data indicated that ET-2 is acting via ET-RB to protect cells from hypoxia, therefore we treated the HTH-K tumor in vivo with ET receptor antagonists. BQ-788 led to an increased extent and development of necrosis within the tumor compared with untreated tumors or tumors treated with BQ-123. This increase in tumor necrosis may be because of the ET-RB antagonists inhibiting the tumor cells ability to withstand intratumoral hypoxic stress similar to the situation in vitro. BQ-788 also led to a decreased tumor volume after 5 days of treatment compared with control tumors.
However, although an ET-RA-specific inhibitor had no effect on hypoxic cell survival in vitro and did not increase necrosis in vivo, hypoxia increased the production of ET-RA in vitro and led to decreased tumor volume in vivo. The role of ET-RA up-regulation and presumed signaling is yet to be elucidated but may have a role in another of the ETs’ potential functions distinct from the ET-2/ET-RB protective function. However, it is also likely that the receptor antagonist is affecting cells distinct from the tumor cells, particularly the cells of the vasculature.
Furthermore, it is likely that cells other than the breast epithelial cells or the tumor cells may contribute to ET expression. ET-1 mRNA, which could be detected in the HTH-K tumor but was expressed at very low levels by the HTH-K tumor cells in vitro, may originate from cells distinct from the tumor cells. Indeed, endothelial cells may express all ETs, and ET-1 can be regulated by hypoxia both positively and negatively in these cells (17, 18). It is likely that hypoxia also contributes to the modulation of ET gene expression in these and other cells within the tumor. Moreover, ET-2 released by tumor cells is likely to act on other cells within the tumor further modulating tumor biology, e.g., ETs can stimulate release of proinflammatory cytokines from macrophages (45), and ET-2 may modulate the distribution of macrophages within tumor (46).
In summary, we have shown that ETs are increased in human IDC of the breast compared with normal breast or benign tissue and that these tumors express ET-2 and ET-RB. In our model of breast carcinoma, HTH-K, expression of ET-2 and its receptors is rapidly induced by hypoxia in a HIF-1-dependent manner. ET-2 protects breast tumor cells from hypoxic injury via the ET-RB receptor in an autocrine loop. ET-2 may therefore protect tumor cells against hypoxia within the tumor in vivo. Antagonism of the ET-RB receptor in vivo leads to increased tumor necrosis as well as delayed tumor growth and may therefore be a therapeutic target for the treatment of breast cancer.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 To whom requests for reprints should be addressed, at Cancer Research United Kingdom, Translational Oncology Laboratory, Barts and The London, Queen Mary’s School of Medicine and Dentistry, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, United Kingdom. Phone: 00-44-20-7882-5796; Fax: 00-44-20-7882-6110; E-mail: grimshaw{at}cancer.org.uk
3 Present address: Oxford BioMedica (UK) Ltd., Medawar Centre, The Oxford Science Park, Oxford OX4 4GA, United Kingdom.
4 The abbreviations used are: ET, endothelin; ET-RA, ET-A receptor; ET-RB, ET-B receptor; NITP, nitroimidazole-L-yl-butyl theophylline; IDC, infiltrating ductal carcinoma; IHC, immunohistochemistry; RT-PCR, reverse transcription-PCR; PBS-T, PBS with 0.1% Tween; HIF, hypoxia-inducible factor; DPI, diphenylene iodonium.
Received 5/22/02; revised 9/13/02; accepted 10/ 4/02.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  C. Guruvayoorappan Tumor Versus Tumor-Associated Macrophages: How Hot is the Link? Integr Cancer Ther, June 1, 2008; 7(2): 90 - 95. [Abstract] [PDF]
|
|
|
|
 |
|
 G. M Tse, B. Chaiwun, K.-M. Lau, R. Scolyer, C S. Lee, R. Z Karim, T. C Putti, B. K Law, P. C Lui, and P. H. Tan Endothelin-1 expression correlates with atypical histological features in mammary phyllodes tumours J. Clin. Pathol., September 1, 2007; 60(9): 1051 - 1056. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M J Grimshaw Endothelins and hypoxia-inducible factor in cancer Endocr. Relat. Cancer, June 1, 2007; 14(2): 233 - 244. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Wilson, J. Burchell, and M. J. Grimshaw Endothelins Induce CCR7 Expression by Breast Tumor Cells via Endothelin Receptor A and Hypoxia-Inducible Factor-1 Cancer Res., December 15, 2006; 66(24): 11802 - 11807. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Hearnes, D. J. Mays, K. L. Schavolt, L. Tang, X. Jiang, and J. A. Pietenpol Chromatin Immunoprecipitation-Based Screen To Identify Functional Genomic Binding Sites for Sequence-Specific Transactivators Mol. Cell. Biol., November 15, 2005; 25(22): 10148 - 10158. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Takizawa, T. Uchide, J. Adur, T. Kozakai, E. Kotake-Nara, J. Quan, and K. Saida Differential expression of endothelin-2 along the mouse intestinal tract J. Mol. Endocrinol., October 1, 2005; 35(2): 201 - 209. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Z. Olenyuk, G.-J. Zhang, J. M. Klco, N. G. Nickols, W. G. Kaelin Jr., and P. B. Dervan Inhibition of vascular endothelial growth factor with a sequence-specific hypoxia response element antagonist PNAS, November 30, 2004; 101(48): 16768 - 16773. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Murdoch, A. Giannoudis, and C. E. Lewis Mechanisms regulating the recruitment of macrophages into hypoxic areas of tumors and other ischemic tissues Blood, October 15, 2004; 104(8): 2224 - 2234. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Grimshaw, T. Hagemann, A. Ayhan, C. E. Gillett, C. Binder, and F. R. Balkwill A Role for Endothelin-2 and Its Receptors in Breast Tumor Cell Invasion Cancer Res., April 1, 2004; 64(7): 2461 - 2468. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Wulfing, C. Kersting, J. Tio, R.-J. Fischer, C. Wulfing, C. Poremba, R. Diallo, W. Bocker, and L. Kiesel Endothelin-1-, Endothelin-A-, and Endothelin-B-Receptor Expression Is Correlated with Vascular Endothelial Growth Factor Expression and Angiogenesis in Breast Cancer Clin. Cancer Res., April 1, 2004; 10(7): 2393 - 2400. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
4). ET-2 was significantly increased by hypoxia (P < 0.0001), as was ET-RA (P < 0.01) and ET-RB (P < 0.05). ET-1 and ET-3 mRNA were undetectable by Northern blotting. c, Northern blot of ET-2 mRNA expression by MDAMB468 cells under normoxia and hypoxia. d, ET-2 peptide release by HTH-K cells cultured under hypoxic conditions for 24 h. One representative experiment of three is shown. Hypoxia significantly increased the amount of mature ET-2 peptide released into the medium (P < 0.0005). e, Western analysis of ET-RA and ET-RB protein expression by HTH-K cells cultured for 24 h under either normoxia (N) or hypoxia (H). The blots were probed for ß-actin protein as a control. Shown is one representative experiment (n = 3).
