Research ArticleExperimental Studies

The Impact of Angio-associated Migratory Cell Protein (AAMP) on Breast Cancer Cells In Vitro and Its Clinical Significance

YUKUN YIN, ANDREW J. SANDERS and WEN G. JIANG
Anticancer Research April 2013, 33 (4) 1499-1509;

Abstract

Background/Aim: Angio-associated migratory cell protein (AAMP), which belongs to the immunoglobulin superfamily, was found to be expressed in different human cell lines and exhibited a predominant cytosolic localization in epithelial cells. Previous studies show that the specific gene product is functional in cell migration and angiogenesis and can also be used as a marker of poor prognosis in invasive gastrointestinal stromal tumours and ductal carcinoma in situ (DCIS) of the breast. However, the cellular role of AAMP in breast cancer is still unclear. The aim of the current study was to provide new insights into the implication of AAMP in breast cancer. Materials and Methods: We knocked-down the expression of AAMP through transfection of MCF-7 and MDA-MB-231 breast cancer cells with a hammerhead ribozyme transgene (MCF-7AAMPrib and MDA-MB-231AAMPrib) and examined the impact on cell function using in vitro assays. Additionally, AAMP expression was examined in a cohort of breast specimens (normal, n=28; cancer, n=102) using quantitative-real-time polymerase chain reaction (Q-PCR) and immunohistochemical methods. Results: AAMP knock-down dramatically reduced cell adhesion and cell growth of MCF-7 cells (p<0.05), and suppressed cell invasion of MDA-MB-231 cells (p<0.05). Increased expression of AAMP in breast cancer was observed compared with that in normal tissues (p<0.05). High levels of AAMP transcripts were associated with disease progression, metastasis, and poor prognosis of the patients. Disease-free and overall survival time of patients with lower levels of AAMP were significantly longer compared to those of patients with high levels (p<0.05). Conclusion: AAMP has a significant influence on the biological functions of breast cancer cells and its high expression correlates with poor prognosis and metastasis.

Angio-associated migratory cell protein (AAMP), a 52-kDa protein initially isolated from a human melanoma cell line during a search for motility-associated cell surface proteins (1), is extensively expressed in different types of endothelial cells and aortic smooth-muscle cells (human and bovine), human melanoma cells, activated T-lymphocytes, renal proximal tubular cells, prostate and breast carcinoma cells, dermal fibroblasts, glomerular mesangial cells, benign mammary cells, and rat myocytes (1-7). The expression of AAMP was found to be increased in invasive gastrointestinal stromal tumours (8) and in ductal carcinoma in situ (DCIS) of the breast with necrosis, where it is considered to be a marker of poor prognosis (9).

The AAMP protein contains two immunoglobulin-like domains, the WD40 repeat motif, and a heparin-binding consensus sequence (1). The immunoglobulin superfamily relatives include proteins that either are known or are suspected to play a role in cell adhesion (8, 10, 11). Some immunoglobulin superfamily members are multifunctional and participate in both cell binding and signalling (8, 12). The WD40 repeat motif found in β-transducin and other proteins is speculated to represent a general protein-protein recognition/binding site (13). The AAMP-derived peptide, P189, contains a heparin-binding domain and mediates heparin-sensitive cell adhesion (1, 2, 14). A previous study identified a direct interaction of AAMP with nucleotide-binding oligomerization domain containing-2 (NOD2), but declared that Ig domains in AAMP could not be confirmed (6). Therefore, AAMP sequence homologies indicate that it may play a role in cell adhesion, migration and innate immune response.

Functional studies show that anti-recombinant AAMP (anti-rAAMP) inhibits tubule formation by endothelial cells cultured on Matrigel (3). Anti-rAAMP also inhibits endothelial and smooth-muscle cell motility (11, 14, 15). It has also been found that the extracellular form of AAMP plays a positive role in angiogenesis and can be regulated by astrocytes, and has led to the hypothesis that the regulation of extracellular AAMP in endothelial cells by astrocytes may aid in the angiogenesis of the nervous system (14). The peptide P189, derived from AAMP was also found to possess the ability to bind and cluster MCF-7 breast cancer cells and human A2058 melanoma cells (5).

AAMP was identified as a binding partner of NOD2 through co-immunoprecipitation studies, using human embryonic kidney cells (HEK293T), and was found to have functional implication in NOD2-mediated signalling, playing a negative regulatory role in NOD2-mediated nuclear factor-kappa B (NF-κB) pathways (6, 7). Studies have demonstrated that AAMP causes the translocation and activation of ras homolog gene family, member A (RhoA) in smooth muscle and endothelial cells. Activated RhoA subsequently interacts with its effector rho-kinase (ROCK), generating the driving force for smooth muscle cells to migrate and to divide, leading to re-stenosis and atherosclerosis (11). AAMP has been identified as a novel interacting partner of both thromboxane A2 receptor-alpha (TPα) and -beta (TPβ) through an interaction dependent on common and unique sequences within their carboxyl-terminal tail domains. The identification of a specific interaction between TPα/β and AAMP is likely to have substantial functional implications for vascular pathologies in which they are both implicated (7).

Previous studies have shown that AAMP plays a role in angiogenesis, one of the most important mechanisms of tumour development and metastasis, and is involved in signal transduction related to the cellular processes of adhesion, migration and proliferation. AAMP is found to be expressed in many tumour tissues and cell lines. Together, these data suggest that AAMP may play a role in tumourigenesis, tumour development and/or spread. However, the potential contribution of AAMP to breast carcinogenesis and tumour progression has not been directly investigated. In this study, we examined the expression of AAMP in human breast cancer specimens and cell lines. An AAMP knockdown cell model using hammerhead ribozymes was used to study the function of AAMP in vitro.

Materials and Methods

Human breast specimens. A total of 130 breast samples were obtained from patients with breast cancer (28 were background normal breast tissues, and 102 were breast cancer tissues). These tissues were collected immediately after mastectomy and snap-frozen in liquid nitrogen, following approval of a local Ethical Committee. A number of background normal mammary tissue samples were obtained from non-cancerous regions from the same breast cancer patients. The pathologist verified normal background and cancer specimens and confirmed that the background samples were free from tumour deposits. The median follow-up for the cohort was 120 months. The relevant information is provided in Table I.

View this table:
Table I.

Transcript levels of angio-associated migratory cell protein (AAMP) in breast cancer.

Immunohistochemical staining of AAMP. The Avidin-Biotin Complex (ABC) method of immunohistochemistry staining was used to assess the protein expression of AAMP in tissue sections. Paraffin sections of mammary tissues (18 paired normal and 18 matched tumour tissues, as well as dissected tumour tissues) were cut at a thickness of 6 μm. The sections were first dewaxed using a series of gradient alcohol washes. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 15 min before washes. Sections were boiled using microwaves in antigen retrieval solution (pH 6.0) to retrieve antigen. After three washes in tris-buffered salin (TBS), the samples were blocked in TBS containing horse at room temperature for 30 min. The primary antibody (used at dilution of 1:100), second antibody and Avidin biotin complex (Vector Laboratories Inc., Burlingame, USA) was added after every three TBS washes and successively incubated for 30 min, 30 min and 45 min respectively. Diaminobenzidine chromogen (DAB; Vector Laboratories Inc.) was then added to the sections which were then incubated in the dark for 5 min. Sections were subsequently counter-stained in Gill's haematoxylin and dehydrated in ascending grades of methanol before clearing in xylene and mounting under a coverslip. Monoclonal mouse anti-AAMP (111-211) was obtained from ABNOVA (Abnova Gmbh, Heidelberg, Germany), and anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-32233), was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The peroxidase-conjugated anti-mouse antibody was purchased from Sigma-Aldrich Ltd. (Poole, Dorset, UK).

View this table:
Table II.

Primers used for reverse transcription-polymerase chain reaction (RT-PCR) and quantitative-polymerase chain reaction (Q-PCR) in the present study.

Cell culture. Breast cancer cell lines MDA-MB-231, MCF-7 and ZR-751 were purchased from the European Collection of Animal Cell Cultures (Salisbury, UK). A highly invasive cell line, MDA-MB-231, which is oestrogen receptor alpha (ERα)-negative and ERβ-positive, and a weakly-invasive cell line, MCF-7, that is ERα- and ERβ-positive, were chosen for functional assays. These two cell lines are both from ductal carcinoma, which is the main tumour histological type of breast cancer and are also the most studied in recent research. The cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 with L-glutamine medium (Sigma, Poole, Dorset, UK), supplemented with antibiotics and 10% foetal calf serum (Sigma), and incubated at 37.0°C in an atmosphere of 5% CO2 and 95% humidity.

Generation of AAMP knock-down in breast cancer cell lines. Hammerhead ribozyme transgenes were designed, based on the secondary structure of AAMP and predicted using the Zuker RNA mFold program (16) to target the expression of human AAMP. The ribozymes were generated and inserted into the pEF6/V5-His-TOPO plasmid vector in accordance with the manufacturer's instructions (Invitrogen, Paisley, UK). Following verification of correctly-orientated inserts, plasmids were amplified in bacteria before being extracted using a Gen Elute plasmid extraction kit (Sigma). Plasmids containing the AAMP ribozyme transgenes or empty pEF6 control plasmids were transfected into MCF-7 and MDA-MB-231 cells using electroporation as described previously (17-19). Cells were subjected to a selection period in the presence of blasticidn (5 μg/ml) before being subsequently cultured in maintenance medium (0.5 μg/ml blasticidin) and the wild-type cells were cultured in normal media. Transfected cells were routinely tested to confirm knock-down of AAMP expression using reverse transcription-polymerase chain reaction (RT-PCR) or western blotting. Cells were respectively labelled as MCF-7AAMPrib and MCF-7pEF6, MDA-MB-231AAMPrib and MDA-MB-231pEF6, MCF-7WT and MDA-MB-231WT.

RNA extraction and RT-PCR. RNA was extracted from cells using the TRI-reagent (Sigma). Reverse transcription was carried out to generate a cDNA template from the extracted RNA, using the iScript™ cDNA synthesis kit (Bio-Rad, Hercules CA, USA). PCR conditions were: denaturing at 94°C for 40 s, annealing at 55°C for 40 s and extension at 72°C for 60 s. PCR was conducted over 32-36 cycles and consisted of an initial denaturing step (94°C, 5 min) and a final extension step (72°C, 10 min) before a final hold at 4°C. PCR products were subsequently separated electrophoretically on an agarose gel, stained and visualised. Primer sequences are provided in Table II.

Figure 1.
Figure 1.

Angio-associated migratory cell protein (AAMP) expression in human breast tissues and breast cancer cell lines. A: Polymerase chain reaction (PCR) analysis of AAMP expression in a panel of breast cancer cell lines. B: Quantitative PCR analysis of AAMP expression in human breast tissues, showing significantly higher levels of AAMP in breast tumour tissues than normal background tissues.

Quantitative-polymerase chain reaction (Q-PCR). AAMP transcript levels present in the breast cancer and control specimens and cell lines (shown as copies/μl based on internal standard) were assessed using real-time Q-PCR, as previously reported (18). Briefly, the number of AAMP transcripts in these samples were detected and quantified by the iCycler IQ system. Transcript copy numbers were obtained based on an internal standard and normalized against GAPDH levels in the same samples. Conditions for Q-PCR were: 95°C for 15 min, followed by 60 cycles at 95°C for 20 s, 55°C for 30 s and 72°C for 20 s.

SDS-PAGE and western blotting. Proteins of control and transfected cells were obtained following lysis with a protein lysis buffer containing 0.5% sodium dodecyl sulphate (SDS), 1% Triton X-100, 2 mM CaCl2, 100 mg/ml phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml aprotinin and, 10 mM sodium orthovanadate. Samples were quantified using a Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Hemel Hempstead, Hertfordshire, UK) and standardized amounts of each sample were separated on a 10% acrylamide gel. The proteins were blotted onto a nitrocellulose membrane (Santa Cruz Biotechnology) before being probed with the specific primary antibodies (AAMP and GAPDH) at a concentration of 1:70, and specific peroxidase-conjugated secondary antibodies at a dilution of 1:350 in accordance with the SnapID protocol (Millipore, Watford, UK). Protein bands were documented using a gel documentation system (UVITech, Cambridge, UK).

In vitro growth assay. Cell growth was measured using an in vitro cell growth assay. Briefly, 2,000 cells/well were seeded into triplicate 96-well plates (corresponding to day 1, day 3, and day 5). Following the appropriate incubation period, the cells were fixed in 4% formaldehyde (v/v) and stained with 0.5% (w/v) crystal violet. The crystal violet stain was subsequently extracted using 10% acetic acid (v/v) and cell density was determined by measuring the absorbance at a wavelength of 540 nm using an ELx800 spectrophotometer (Bio-Tek Instruments Inc., Winooski, VT, USA).

In vitro Matrigel invasion assay (20). A 24-well plate was set up together with trans-well inserts containing 8.0 μm pores (Becton Dickinson Labware, NJ, USA) that had been previously coated with 50 μg/insert of Matrigel Matrix Basement Membrane (BD Biosciences, Oxford, UK). A total of 15,000 cells were seeded into the trans-well inserts and incubated for three days. After the incubation, cells which had invaded through the artificial basement membrane to the underside of the trans-well insert were fixed, stained and counted.

In vitro Matrigel adhesion assay (20). A 96-well plate was pre-coated with 5 μg of Matrigel per well. Subsequently, 45,000 cells were seeded into each well. After 45 min of incubation, non-adherent cells were removed by vigorous washing with balanced salt solution (BSS). Adherent cells that remained were subsequently fixed, stained and counted.

Electric cell-substrate impedance sensing (ECIS) based cellular motility assay. The ECIS instrument, together with the 96W1E array, (Applied Biophysics Inc, NJ, USA) was used in the current study to analyze migratory rates of control and transfected cell lines (18). After stabilizing the array, identical numbers of MDA-MB-231wt, MDA-MB-231pEF6, MDA-MB-231AAMPrib, MCF-7wt, MCF-7pEF6, and MCF-7AAMPrib (200,000 per well) were seeded into the array wells in 300 μl of medium. After 10 h, when a confluent monolayer had formed in the wells, the monolayer was electrically wounded at 6 V for 30 s. Subsequently, the impedance and resistance change within the array wells were recorded for a period of up to 20 h.

Statistical analysis. Experimental procedures were repeated independently as least three times. Statistical analysis was undertaken using the Minitab statistical software package (version 14; Minitab Ltd, Coventry, UK). Non-normally distributed data were assessed using the Mann Whitney test (median±SEM), whereas the two-sample t-test was used for normally distributed data (mean±SEM). Kaplan Meier survival analysis was conducted using PSAW (version 18; SPSS. Chicago, IL, USA). Differences were considered to be statistically significant at p<0.05.

Figure 2.
Figure 2.

Immunohistochemical staining of angio-associated migratory cell protein (AAMP) in human breast tissues. Left panel: the AAMP protein was found to be highly stained in breast cancer tissues; right panel: staining of AAMP in normal background breast tissue was found to be relatively low compared to levels seen in the cancerous tissue.

Results

The expression of AAMP in breast cancer cell lines and human breast tissues. The presence of AAMP was examined in three human breast cancer cell lines through RT-PCR. AAMP was expressed in the breast cell lines (MCF-7, MDA-MB-231 and ZR-75-1) (Figure 1A). To investigate the biological function of AAMP in breast cancer, two of the examined cell lines were chosen for knock-down studies. We also quantified AAMP transcript levels in the breast cohort specimens using real-time quantitative PCR and discovered a significant increase in the levels of AAMP present in tumour tissues compared to normal background tissues (tumour, n=102, 10.9±1.4 copies/μl; background, n=28, 1.1±0.2 copies/μl; p=0.0077) (all values are displayed as median AAMP transcript copies/μl of cDNA from 50 ng total RNA, Figure 1B).

Immunohistochemical staining of human breast tissue specimens. To assess the expression pattern of AAMP at the protein level, we performed immunohistochemical staining analysis of AAMP expression in the human breast cancer tissue sections (n=18 pairs). Using a specific monoclonal antibody against AAMP. AAMP was detected in the cytoplasm, cell membrane and extracellular matrix. Upon the analysis of breast cancer tissues, the level of AAMP expression was found to be enhanced in comparison to that seen in the normal background tissues (Figure 2).

Correlation of AAMP expression with histological type, grade, tumour-node-metastasis (TNM) staging and ER status. To assess the relation of AAMP expression with disease progression, AAMP transcript levels in breast cancer samples were analyzed against histological type, histological grade, TNM staging and ER status (Table I). There were no statistical differences among the levels of AAMP transcripts in ductal, lobular and other types of breast cancer (Figure 3A). In relation to the histological grade of tumour tissues, the AAMP expression was positively correlated with tumour differentiation, and higher levels of AAMP were seen in the well-differentiated grade 1 tumours compared with the moderately-differentiated grade 2 tumours and the poorly-differentiated grade 3 tumours (p=0.0054 vs. grade 1; Figure 3B).

Figure 3.
Figure 3.

Quantitative-polymerase chain reaction (Q-PCR) analysis of angio-associated migratory cell protein (AAMP) expression in human breast cancer tissues. AAMP expression was quantified and examined alongside patients' clinical pathological data. A: No significant differences in AAMP expression were seen in connection with histological type. B: Reduced levels of AAMP were observed in grade 3 tissues compared to grade 1 tissues. C: AAMP levels were found to be higher in the later compared to early TNM stages, although this did not reach statistical significance. D: AAMP was found to be more highly expressed in oestrogen receptor-β (ERβ)-negative samples than in ERβ-positive samples and did not demonstrate any differential expression between the ERα-positive and -negative samples.

The expression of AAMP was found to be increased in advanced breast cancer according to TNM stage grouping, in comparison with that of TNM1 and TNM2, AAMP expression was enhanced in TNM3 and TNM4 tissues, although these differences were not found to be significant (Figure 3C). Significant differences were also seen in AAMP expression between patients with ERβ-positive tumours and those ERβ-negative tumours. However, no significant differences were observed between patients with ERα-positive tumours and those with ERα-negative tumours (p= 0.24; Figure 3D).

Prognostic relevance and clinical outcomes as related to AAMP in breast cancer. The prognostic potential of AAMP expression was firstly examined in accordance with the Nottingham prognostic index (NPI) of the patients. The NPI 1-group (NPI score <3.4; n=49), NPI 2-group (NPI score=3.4-5.4; n=36), and NPI 3-group (NPI score >5.4; n=14) represent patients with good, moderate, and poor prognosis, respectively. Our data showed that there were no statistical differences among different NPI groups (Figure 4A). In regard to the clinical outcomes, AAMP transcript levels seemed to be increased in patients with poor prognosis, including those with local recurrence, metastases and those who died of breast cancer (p=0.137) compared with that of patients who remained disease-free (Figure 4B and C). It was found that patients with lower AAMP transcript levels had a longer overall survival (138.7±4.7 months; 95% confidence interval=129.6-147.8 months; p=0.01) compared with those with high levels (90.9±23.4 months; 95% confidence interval=45.1-136.6 months; Figure 4D). It is interesting to note that patients who developed local recurrence and who died of breast cancer (134.0±5.2 months; 95% confidence interval=123.9-144.2 months; p=0.005) exhibited significantly high AAMP levels compared with those who remained disease-free (78.9±22.1 months; 95% confidence interval=35.6-122.1 months; Figure 4E).

Figure 4.
Figure 4.

The relationship between angio-associated migratory cell protein (AAMP) expression and prognostic clinical outcome. A: AAMP expression tended to be reduced in higher nottingham prognostic index (NPI)-staged samples than in comparison to the lower NPI stages, although this was not significant. B: In relation to survival status, significantly higher levels of AAMP were found in samples from patients who had died from breast cancer compared to those remaining disease-free. C: Elevated levels of AAMP, although not significant, were observed in patients who were considered to have a poorer prognosis that those remaining disease-free. D and E: Kaplan-Meier curves showing that patients with higher levels of AAMP had significantly reduced overall survival (D; p=0.01) and disease-free survival (E; p=0.005) than patients with lower AAMP expression.

Figure 5.
Figure 5.

Knock-down of angio-associated migratory cell protein (AAMP) in breast cancer cell lines. Knock-down of AAMP in MCF-7 and MDA-MB-231 breast cancer cell lines was confirmed at the transcript level using quantitative polymerase chain reaction (Q-PCR) (A and B) and at the protein level, using western blot analysis (C and D), in comparison to the respective control cell lines.

Manipulation of AAMP expression by ribozyme transgene. Q-PCR demonstrated that AAMP mRNA expression was successfully knocked-down in the breast cancer cell lines that had been transfected with the AAMP ribozyme transgene (MCF-7AAMPrib and MDA-MB-231AAMPrib), with dramatically reduced levels of AAMP observed in comparison to the level of expression in wild-type cells (MCF-7WT and MDA-MB-231WT) and in empty-plasmid control cells (MCF-7pEF6 and MDA-MB-231pEF6) (Figure 5A and B). Additionally, western blotting was used to probe for the AAMP protein. Similar to the trends seen at the mRNA level, transfection with the AAMP ribozyme transgene was able to bring about a reduction in AAMP protein levels in both cell lines in comparison to the respective empty-plasmid control cells (Figure 5C and D).

AAMP has differing effects on cell function in MCF-7 and MDA-MB-231 cell lines. The impact of AAMP on the growth rate of MCF-7 and MDA-MB-231 was examined using an in vitro tumour cell growth assay. Contrasting results for the effects of AAMP expression on tumour cell growth were seen between the MCF-7 and MDA-MB-231 cell lines. In the MCF-7 cell line, knock-down of AAMP resulted in a significant decrease in growth rate resulting in significant differences being observed between MCF-7AAMPrib and MCF-7pEF6 cell lines after 3- and 5-day incubation periods (p=0.002 and p=0.007 respectively, Figure 6A). Strangely, the MDA-MB-231 cell line did not follow a similar trend and no significant difference in growth was seen between the MDA-MB-231AAMPrib and MDA-MB-231pEF6 cell lines over the 3- or 5-day incubation period (p=0.529, Figure 6B).

Figure 6.
Figure 6.

Angio-associated migratory cell protein (AAMP) has differential effects on cell function in MCF-7 and MDA-MB-231 breast cancer cells. Knock-down of AAMP in MCF-7 cells significantly reduced cell growth (A) and cell matrix adhesion (C) but did not have any significant impact on cell invasion (E). In contrast, knock-down of AAMP in the MDA-MB-231 cell line significantly inhibited cell invasion (F) but did not have any significant impact on cell growth (B) or cell matrix adhesion (D). Knock-down of AAMP did not significantly impact on migration rates of either cell line detected using an ECIS model system.

The capacity of MCF-7 and MDA-MB-231 breast cancer cells to adhere to an artificial Matrigel basement membrane was examined using an in vitro Matrigel adhesion assay. In the MCF-7 cell line, knock-down of AAMP also resulted in a dramatic decrease in cell matrix adhesion and a significant difference was seen between the MCF-7AAMPrib and MCF-7pEF6 cell line (p=0.0097, Figure 6C). In contrast, no significant difference in adhesive capacity was seen between MDA-MB-231 cells containing the ribozyme transgene and their respective pEF6 control cells (p=0.27, Figure 6D).

Interestingly, over a three-day incubation, MCF-7 cells showed no changes in invasiveness following AAMP knock-down (p=0.54), while targeting of AAMP in MDA-MB-231 cells resulted in a reduction of cellular invasion through the artificial Matrigel basement (p=0.017, Figure 6E and F).

No significant differences in cell motility, assessed using the ECIS model system, were apparent in either cell line following knock-down of AAMP. No significant alterations in migration rates were seen over the course of the experiment following electrical wounding of the monolayer (p>0.05; Figure 6G and H).

Discussion

AAMP, a protein that was first isolated during a search for motility-associated proteins, has been shown to be expressed in different cell types, including endothelial cells, vascular smooth muscle cells, human dermal fibroblasts, activated T-lymphocytes, and also in the MCF-7 and MDA-MB-231 breast cancer cell lines (1). Clinical studies showed that AAMP is highly-expressed in invasive gastrointestinal stromal tumours (8) and in DCIS of the breast (9). Thus, the role of AAMP in cancer has drawn scientific interest. In the present study, we report the increased expression of AAMP in breast cancer tissues and its strong expression in breast cancer cell lines. The association of AAMP with clinical outcomes of patients with breast cancer and its cellular functions was tested, further highlighting its potential as an indicator of poor prognosis in breast cancer.

In the current study, we demonstrated, using Q-PCR and also immunohistochemical analysis, that AAMP expression levels are associated with clinical aspects of breast cancer in our cohort of human breast cancer tissues. Higher transcript levels or staining intensity of AAMP was revealed in breast cancer tissues compared with background mammary tissues. The increasing expression of this gene was also seen in advanced disease according to TNM staging. The link between AAMP expression levels and ER status also suggests overexpression of AAMP is linked with poor prognosis. From the clinical outcomes, we also observed a relationship between AAMP expressions and patient prognosis, where AAMP is weakly expressed in the cancer tissues of disease-free patients compared with those who have metastases or who died from this disease. The long-term survival function showed the same trend, namely that AAMP is a cancer-enhancing gene and that high expression can lead to shorter disease-free and overall survival rates. Thus, our current study demonstrates a clinical association between AAMP expression levels and breast cancer clinical outcomes, indicating that high levels of AAMP expression are associated with poor clinical outcomes or shorter survival, and contributes to the evidence highlighting the potential of AAMP in playing a role in breast tumour metastasis and progression.

AAMP expression was also observed in MCF-7, MDA-MB-231 and ZR-751 breast cancer cell lines. To further explore the role of AAMP in breast cancer we targeted the expression of AAMP in MCF-7 and MDA-MB-231 cell lines using a ribozyme transgene system. In the present study, we demonstrated that knock-down of AAMP leads a reduced invasive potential of MDA-MB-231 cells, however, a similar trend was not observed in the MCF-7 cell line. Additionally, AAMP knock-down reduces the cell matrix adhesion of MCF-7 cells, while the same inhibition was not seen in MDA-MB-231 cells. This differential impact of AAMP knock-down between the two cell lines was also seen in the growth rate analyses, where loss of endogenous AAMP resulted in a reduced growth rate of MCF-7 cells, but did not change the growth of MDA-MB-231 cells. The potential role of AAMP in MCF-7 cells on adhesion is in agreement with other studies focusing on endothelial cell lines (1, 4), and may be related to its immunoglobulin domain (1). AAMP does not change the ability of migration in these two breast cancer cell lines, which is somewhat in contrast to the established role of AAMP in endothelial cell lines (4), smooth-muscle cell lines (11) and melanoma cells (5), as previously reported. The differential role of AAMP in the growth and invasiveness of MCF-7 and MDA-MB-231 cells is interesting and the reason for this is currently unknown. Further study is required in order to explain this phenomenon and to offer further insight into the mechanisms of action of this molecule.

Our study firstly demonstrates the functional effects of AAMP in vitro in breast cancer cell lines, indicating that AAMP may play a role in breast cancer invasion, adhesion and growth, differently influencing these traits in different cell lines. Our study also provides evidence that high levels of AAMP in clinical samples are associated with poorer prognosis. This suggests a cancer-promoting influence of AAMP in breast cancer, however, as with the in vitro data, its role and importance may be dependent on the individual cancer type.

Acknowledgements

The Authors wish to thank the Albert Hung Foundation and Cancer Research Wales for supporting this work. Dr Yin is a recipient of the China Medical Scholarship of the Cardiff University.

  • Received January 1, 2013.
  • Revision received March 1, 2013.
  • Accepted March 4, 2013.

References

  1. ↵
    1. Beckner ME,
    2. Krutzsch HC,
    3. Stracke ML,
    4. Williams ST,
    5. Gallardo JA,
    6. Liotta LA
    : Identification of a new immunoglobulin superfamily protein expressed in blood vessels with a heparin-binding consensus sequence. Cancer Res 55: 2140-2149, 1995.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Beckner ME,
    2. Krutzsch HC,
    3. Klipstein S,
    4. Williams ST,
    5. Maguire JE,
    6. Doval M,
    7. Liotta LA
    : AAMP, a newly identified protein, shares a common epitope with alpha-actinin and a fast skeletal muscle fiber protein. Exp Cell Res 225: 306-314, 1996.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Beckner ME,
    2. Liotta LA
    : AAMP, a conserved protein with immunoglobulin and WD40 domains, regulates endothelial tube formation in vitro. Lab Invest 75: 97-107, 1996.
    OpenUrlPubMed
  4. ↵
    1. Beckner ME,
    2. Peterson VA,
    3. Moul DE
    : Angio-associated migratory cell protein is expressed as an extracellular protein by blood vessel-associated mesenchymal cells. Microvasc Res 57: 347-352, 1999.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Beckner ME,
    2. Krutzsch HC,
    3. Tsokos M,
    4. Moul DE,
    5. Liotta LA
    : The aggregated form of an AAMP derived peptide behaves as a heparin sensitive cell-binding agent. Biotechnol Bioeng 54: 365-372, 1997.
    OpenUrlPubMed
  6. ↵
    1. Bielig H,
    2. Zurek B,
    3. Kutsch A,
    4. Menning M,
    5. Philpott DJ,
    6. Sansonetti PJ,
    7. Kufer TA
    : A function for AAMP in NOD2-mediated NF-κB activation. Mol Immunol 46: 2647-2654, 2009.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Reid HM,
    2. Wikstrom K,
    3. Kavanagh DJ,
    4. Mulvaney EP,
    5. Kinsella BT
    : Interaction of angio-associated migratory cell protein with the TPα and TPβ isoforms of the human thromboxane A(2) receptor. Cell Signal 23: 700-717, 2011.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Allander SV,
    2. Nupponen NN,
    3. Ringner M,
    4. Hostetter G,
    5. Maher GW,
    6. Goldberger N,
    7. Chen Y,
    8. Carpten J,
    9. Elkahloun AG,
    10. Meltzer PS
    : Gastrointestinal stromal tumors with KIT mutations exhibit a remarkably homogeneous gene expression profile. Cancer Res 61: 8624-8628, 2001.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Adeyinka A,
    2. Emberley E,
    3. Niu Y,
    4. Snell L,
    5. Murphy LC,
    6. Sowter H,
    7. Wykoff CC,
    8. Harris AL,
    9. Watson PH
    : Analysis of gene expression in ductal carcinoma in situ of the breast. Clin Cancer Res 8: 3788-3795, 2002.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Balzan S,
    2. Del Carratore R,
    3. Nicolini G,
    4. Beffy P,
    5. Lubrano V,
    6. Forini F,
    7. Iervasi G
    : Proangiogenic effect of TSH in human microvascular endothelial cells through its membrane receptor. J Clin Endocrinol Metab 97: 1763-1770, 2012.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Holvoet P,
    2. Sinnaeve P
    : Angio-associated migratory cell protein and smooth muscle cell migration in development of restenosis and atherosclerosis. J Am Coll Cardiol 52: 312-314, 2008.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Adombi CM,
    2. Lelenta M,
    3. Lamien CE,
    4. Shamaki D,
    5. Koffi YM,
    6. Traore A,
    7. Silber R,
    8. Couacy-Hymann E,
    9. Bodjo SC,
    10. Djaman JA,
    11. Luckins AG,
    12. Diallo A
    : Monkey CV1 cell line expressing the sheep-goat SLAM protein: A highly sensitive cell line for the isolation of peste des petits ruminants virus from pathological specimens. J Virol Methods 173: 306-313, 2011.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Del Carratore R,
    2. Carpi A,
    3. Beffy P,
    4. Lubrano V,
    5. Giorgetti L,
    6. Maserti BE,
    7. Carluccio MA,
    8. Simili M,
    9. Iervasi G,
    10. Balzan S
    : Itraconazole inhibits HMEC-1 angiogenesis. Biomed Pharmacother 66: 312-317, 2011.
    OpenUrlPubMed
  14. ↵
    1. Beckner ME,
    2. Jagannathan S,
    3. Peterson VA
    : Extracellular angio-associated migratory cell protein plays a positive role in angiogenesis and is regulated by astrocytes in coculture. Microvasc Res 63: 259-269, 2002.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Vogt F,
    2. Zernecke A,
    3. Beckner M,
    4. Krott N,
    5. Bosserhoff AK,
    6. Hoffmann R,
    7. Zandvoort MA,
    8. Jahnke T,
    9. Kelm M,
    10. Weber C,
    11. Blindt R
    : Blockade of angio-associated migratory cell protein inhibits smooth muscle cell migration and neointima formation in accelerated atherosclerosis. J Am Coll Cardiol 52: 302-311, 2008.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Zuker M
    : Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31: 3406-3415, 2003.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Jiang WG,
    2. Davies G,
    3. Fodstad O
    : COM-1/P8 in oestrogen regulated growth of breast cancer cells, the ER-β connection. Biochem Biophys Res Commun 330: 253-262, 2005.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Jiang WG,
    2. Martin TA,
    3. Lewis-Russell JM,
    4. Douglas-Jones A,
    5. Ye L,
    6. Mansel RE
    : Eplin-α expression in human breast cancer, the impact on cellular migration and clinical outcome. Mol Cancer 7: 71-80, 2008.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Sanders AJ,
    2. Parr C,
    3. Mason MD,
    4. Jiang WG
    : Suppression of hepatocyte growth factor activator inhibitor-1 leads to a more aggressive phenotype of prostate cancer cells in vitro. Int J Mol Med 20: 613-619, 2007.
    OpenUrlPubMed
  20. ↵
    1. Jiang WG,
    2. Hiscox S,
    3. Hallett MB,
    4. Scott C,
    5. Horrobin DF,
    6. Puntis MC
    : Inhibition of hepatocyte growth factor-induced motility and in vitro invasion of human colon cancer cells by γ-linolenic acid. Br J Cancer 71: 744-752, 1995.
    OpenUrlPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
The Impact of Angio-associated Migratory Cell Protein (AAMP) on Breast Cancer Cells In Vitro and Its Clinical Significance
YUKUN YIN, ANDREW J. SANDERS, WEN G. JIANG
Anticancer Research Apr 2013, 33 (4) 1499-1509;
Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords