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Keywords:

  • breast cancer;
  • ADAMs;
  • ADAM-9, HER-2/neu;
  • metastasis

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The ADAMs (a disintegrin and metalloprotease) are membrane proteins containing both protease and adhesion domains and thus may be potentially important in cancer invasion and metastasis. The aim of our study was to investigate the distribution and potential clinical significance of ADAM-9 in breast cancer. ADAM-9 expression was measured using both reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. ADAM-9 mRNA was expressed more frequently in both breast carcinomas (72/110, 66%) and fibroadenomas (21/38, 55%) compared to normal breast tissue (6/25, 24%) (p = 0.0004, p = 0.028, respectively). Multiple forms of ADAM-9 protein were detected by Western blotting, i.e., at 124, 84 and 48 kDa under reducing conditions and at 115, 76, 55, 52 and 46 kDa under nonreducing conditions. The 84 and 55 kDa forms were detected more frequently in the primary cancers compared to normal breast tissue (p < 0.0001, p = 0.0002, respectively). In addition, relative levels of the 84 kDa mature form were significantly higher in the primary cancers than in the fibroadenomas (p = 0.003), while the reverse was found for the 124 kDa precursor form (p = 0.026). In the carcinomas, the 84 kDa form of ADAM-9 protein was expressed at higher levels in node-positive than node-negative cancers (p = 0.05) and correlated positively with HER-2/neu protein levels (r = 0.313, p = 0.016). This is the first report to describe expression of any ADAM in a large number of human carcinomas. © 2003 Wiley-Liss, Inc.

The process of cancer invasion and metastasis is a multistep event that involves angiogenesis, local invasion, cell migration, intravasation, extravasation and growth at a secondary site (for review, see reference 1). Although multiple genes have been implicated in cancer dissemination, among the best characterised are those encoding matrix degrading proteases and adhesion proteins (for review, see reference 2). Proteases such as urokinase plasminogen activator (uPA) and specific matrix metalloproteinases (MMPs) degrade or remodel the extracellular matrix (ECM) allowing cancer cells to invade locally and ultimately form distant metastases.3 Proteases may also promote metastasis by releasing or activating factors [e.g., fibroblast growth factor-2 (FGF-2), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF)], which enhance cell growth, cell migration and angiogenesis.4, 5 Consistent with their role in metastasis, high levels of multiple proteases have been associated with adverse prognosis in different malignancies (for review, see reference 6).

As with proteases, adhesion molecules are also involved at multiple stages during invasion and metastasis.2, 7 In the initial stages of the metastatic pathway, cells must detach themselves from their neighbouring cells and adhere to the basement membrane. As the invading cell migrates through the extracellular matrix (ECM), the leading edge undergoes consecutive cycles of adhesion and de-adhesion. In the circulation, tumour cells adhere to both blood cells and vascular endothelial cells. Adhesion is mediated by transmembrane proteins, such as integrins, cadherins and selectins.2 Alterations in the expression of these proteins, especially cadherins (e.g., cadherin E)8, 9, 10 and integrins11, 12 are frequently found in malignancy.

As both proteases and adhesion proteins are causally involved in metastasis, it might be expected that molecules possessing both a protease and an adhesion domain would also be involved in this process. A family of molecules containing such domains are the ADAMs (for a disintegrin and metalloprotease domain) or MDC proteins (for metalloprotease/disintegrin/cysteine-rich proteins).

The ADAMs are high molecular weight transmembrane glycoproteins possessing some or all of the following domains: a signal sequence, a prodomain, a metalloprotease domain, a disintegrin domain (i.e., binds to integrins), a cysteine-rich region, an epidermal growth factor (EGF)-like repeat, a membrane-spanning region and a cytoplasmic tail (for review, see references13, 14, 15). To date, 33 different ADAMs have been identified (www.people.virginia.edu/∼jw7g/table_of_the_ADAMs.html). A number of these such as ADAM-9, 10, 12 and 17 have been shown to possess protease activity,16 while ADAM-9, 12, 15 and 23 were shown to be involved in cell adhesion.14, 15

As a protease, ADAM-9 has been shown to degrade a number of substrates including fibronectin, β-casein, gelatin, tumour necrosis factor-α (TNF-α), p75 TNF receptor and c-kit ligand-1 (KL-1).17, 18 It was also shown to cause shedding of soluble heparin-binding epidermal growth factor (HB-EGF),19 a potent mitogen for a number of different cell types.20 As well as its role in proteolysis, ADAM-9 has been implicated in cell adhesion. ADAM-9 was found to adhere to the α6β1 integrin and enhanced cell migration on laminin, a component of basement membrane.21 In another study, ADAM-9 was shown to bind to the αvβ5 integrin on myeloma cells.22 The ability of ADAM-9 to degrade specific ECM substrates such as fibronectin, release growth factor stimulants such as HB-EGF and function in cell adhesion suggests that this protein may play a role in cancer progression.

The aim of this investigation was therefore to compare the expression of ADAM-9 in breast cancer with that in both benign breast tumours (i.e., fibroadenomas) and normal breast tissue. In the carcinomas, we also related expression of ADAM-9 to histopathological characteristics. In addition, a preliminary analysis was carried out on ADAM-9 expression in a small number of nonbreast tissues.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Patients and preparation of samples

Table I summarises the characteristics of the 110 breast cancers analysed. Table II lists the nonbreast tissues investigated. Normal breast tissue included tissue remote from primary carcinoma (n = 9), remote from fibroadenoma (n = 11) and 4 reduction mammoplasties. As similar expression levels of ADAM-9 were found in these 3 types of normal breast tissue, their values were combined. Following surgical resection and pathological assessment, tissues were snap-frozen in liquid nitrogen and then stored at −80°C.

Table I. Pathological Features and Hormone Receptor Status of 110 Primary Breast Carcinomas Assayed for ADAM-9
 Patients
nPercent
  • 1

    All tumours were infiltrating ductal or lobular carcinomas. Ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS) were excluded.

  • 2

    Cut-off point = 200 fmol/G of wet weight tissue.

  • 3

    Cut-off point = 1000 fmol/G of wet weight tissue.

Tumour size (cm)  
 ≤ 23128
 > 25449
 > 51514
 Unknown109
Nodal status  
 Positive5247.5
 Negative5146.5
 Unknown76
Histology type1  
 Ductal8678
 Lobular1413
 Ductal & Lobular54.5
 Unknown54.5
Estrogen receptor status2  
 Positive7265
 Negative3532
 Unknown33
Progesterone receptor status3  
 Positive4238
 Negative4339
 Unknown2523
Age  
 ≤ 504743
 >506357
Table II. Distribution of ADAM-9 mRNA in a Range of Non-Breast Normal and Malignant Unpaired Tissues1
TissuenNumber positive
  • 1

    Liver tumours are secondary cancers, i.e., colonic metastases, whereas colon and prostate cancers are primary carcinomas.

Normal Colon31
Colon Cancer21
Normal Liver41
Liver Cancer54
Normal Prostate21
Prostate Cancer22
Vein40
Aneurysm64
Thyroid76
Skin20
Gallbladder41
Muscle41
Ileum21
Parotid11

Tissue homogenisation was carried out using a Braun Mikro Dismembrator (Braun, Melsungen, Germany). Part of the powder was extracted with 50 mM Tris-HCl (pH 7.4) containing 1 mM monothioglycerol (2 ml/100 mg sample), followed by centrifugation at 13,000 rpm for 20 min at 4°C. The supernatants were assayed for estrogen receptor (ER) and progesterone receptor (PR) using enzyme-linked immunosorbant assay (ELISA) (Abbott Diagnostics, North Chicago, IL). The cut-off point for ER was 200 fmol/G wet weight tissue, while the cut-off for PR was 1,000 fmol/G wet weight tissue. HER-2/neu was also assayed by ELISA (Oncogene Research Products, Cambridge, MA). The cut-off for HER-2/neu positivity was 650 fmol/mg protein.

For Western blotting, part of the powdered tumour sample was resuspended in 50 mM Tris-HCl (pH 7.4) containing protease inhibitors [1 mM benzamidine-HCl, 1 μM trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) and 10 mM ethylene diamine tetraacetic acid (EDTA) (Sigma)] (2 ml/100 mg sample), aspirated through a 19-gauge needle, vortexed and agitated at 4°C for 20 min. Protein concentrations were determined using the bicinchoninic acid (BCA) method (Pierce, Rockford, IL). The residual powder was extracted for total RNA using the guanidinium thiocyanate method.23 All samples were stored at −80°C until required.

RT-PCR

In a final volume of 20 μl, 1 μg of total RNA was reverse transcribed into cDNA. The reaction mixture contained 0.5 mM of each deoxynucleotide triphosphates (dNTP), 10 mg/ml of Oligo(dT)12–18, 10 mM dithiothreitol (DTT), 50 mM Tris-HCl (pH 8.3), 75 mM potassium chloride (KCl) and 3 mM magnesium chloride (MgCl2) (Promega, Madison, WI). The reaction mix was incubated for 5 min at 70°C to remove secondary DNA structures, centrifuged and cooled on ice. Eight units (U) of recombinant human placental ribonuclease inhibitor (Promega) and 200 U of Moloney murine leukaemia virus reverse transcriptase (Gibco BRL, Gaithersburg, MD) were then added followed by incubation for a further 60 min at 37°C. Finally, the samples were heated for 5 min at 65°C and then stored at −20°C until required for PCR amplification. Amplification of cDNA was carried out using the primers outlined below. ADAM-9: (designed by PrimerSelect software for the Macintosh) sense, 5′ CCT CGG GGA CCC TTC GTG T 3′ antisense, 5′ ATC CCA TAA CTC GCA TTC TCT AAA 3′ GAPDH:24, 25 sense, 5′ CCA CCC ATG GCA AAT TCC ATG GCA 3′ antisense, 5′ TCT AGA CGG CAG GTC AGG TCC ACC 3′

PCR was performed in 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton®X-100, 0.2 mM of each dNTP (Promega), 20 pmol each of upstream and downstream ADAM-9 primer (Genosys, Pampisford, UK), 2 μl of cDNA, 1 mM MgCl2 and 2.5 U of Taq DNA polymerase (Promega) in a final volume of 50 μl. All PCR reactions were performed in an automated thermocycler (MJ Research, Watertown, MA). The amplification conditions for the ADAM-9 primer sets were as follows: a denaturation step for 2 min at 94°C, followed by 45 sec at 94°C, 45 sec at 56°C and 1.5 min at 72°C for 36 cycles, followed by 10 min at 72°C. Using these conditions, amplification products were obtained in the exponential phase. Following amplification, 10 μl of PCR product were run on a 2% agarose gel in Tris Acetate EDTA (TAE). The gels were stained by ethidium bromide and visualised under UV light. As a control, PCR with primers specific for the housekeeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH), was carried out on each sample. RNA isolated from MDA-MB-231 breast cancer cells was used as a positive control for ADAM-9. Negative controls included (a) omission of reverse transcriptase and (b) replacement of cDNA by water. The ADAM-9 PCR products were confirmed by direct sequencing using ABI Prism 310 technology.

Western blot analysis

Polyclonal peptide antibodies to ADAM-9 protein (cytoplasmic domain: amino acids 753–762: VPRHVSPVTP) were raised in rabbits against a conjugate coupled to keyhole limpet hemocyanin (KLH) through a C-terminal cysteine residue. Rabbits were immunised twice with the peptide-KLH conjugate at an interval of 3 months. IgG fractions were purified using protein A Sepharose column. Antisera were tested for ADAM-9 immunoreactivity against cellular extracts from COS-7 cell clones bearing an ADAM-9 transgene.

Briefly, COS7 cells were grown in Dulbecco's modified Eagles medium with 10% foetal calf serum (FCS) at 37°C, 5% CO2. Transient transfections of pCIneo-ADAM-9 plasmid DNA were performed by DEAE-dextran dimethylsulphoxide shock with inclusion of 5% FCS in the transfection medium (method exactly as in reference.26 Cells were transferred to serum-free Dulbecco's medium and allowed to grow for approximately 48 hr. Cells were harvested and total cell lysates prepared for Western blotting. The precursor and mature forms of ADAM-9 were detected in the COS-7 cell extracts and migrated with molecular masses of 116 and 79 kDa, respectively (Fig. 1).

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Figure 1. Western blot analysis of untransfected COS7 cells (lane 1) and COS7 cells expressing ADAM-9 (lane 2). The sizes in kDa are indicated to the left of the blot. ADAM-9 protein forms are labelled on the right.

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Immediately before use, samples were diluted in sodium dodecyl sulphate (SDS) sample buffer containing 50 mM Tris-HCl (pH 6.8), 2% SDS, 8% glycerol, 5% 2-mercaptoethanol and 0.01% bromophenol blue, and heated at 95°C for 4 min. Thirty micrograms of total protein was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) using Mini-Gel equipment supplied by Bio-Rad Laboratories, Ltd. (Hertfordshire, UK). Molecular weight (MW) markers (SeeBlue Pre-Stained Standard; Novex Experimental Technology, San Diego, CA) and a positive control (30 μg of cell lysate from the MDA-MB-231 breast cancer cell line) were included on every gel. After electrophoresis, proteins were transferred to nitrocellulose membranes (Sigma) using a semi-dry blotting apparatus (Atto). After transfer, blots were immersed in a blocking buffer of 5% Marvel (instant dried skimmed milk, Tesco Ireland) in Tris-buffered saline (TBS) containing 0.05% Triton®X-100 (Sigma) (TBS-T) for 1 hr at room temperature. Primary ADAM-9 anti-serum was diluted 1 in 1,000 (2.5 μg/ml) in TBS-T and incubated with the blots overnight at 4°C with gentle shaking. Blots were then washed 3 times for 10 min in TBS-T and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody [goat anti-rabbit IgG (Sigma), diluted 1/3,000 in blocking solution] for 1 hr at room temperature. The wash step was as before except an additional 10 min wash in TBS was added. HRP was detected using enhanced chemiluminescence (ECL) reagent (Luminol, Santa Cruz). Chemiluminescence was detected by autoradiography using X-ray film (Fuji).

Analysis of β-actin expression was carried out on the same blot using a mouse monoclonal anti-β-actin antibody (Sigma), as previously described.27 The intensity of the resulting bands was semi-quantified by scanning densitometric analysis (EagleEye™, Stratagene UK), with normalisation of ADAM-9 protein against the “house-keeping” protein, β-actin.

A number of control experiments were also carried out, i.e., specificity of ADAM-9 bands were confirmed using (a) blocking peptide, corresponding to amino acids VPRHVSPVTP, at 30-fold concentration excess of the primary antibody, (b) control IgG, i.e., immunoglobulin fraction of serum from healthy nonimmunised rabbits (Dako Co., Carpinteria, CA) and (c) omission of the primary antibody.

Cell culture

MDA-MB-231 breast cancer cells [American Type Culture Collection (ATCC), Manassas, VA] were cultured in Leibovitz's L-15 medium, supplemented with 10% (w/v) heat-inactivated FCS, 2 mM L-glutamine, 20 mM HEPES, 50 unit/ml penicillin, 50 μg/ml streptomycin and 2 μg/ml fungizone (amphotericin B) (Gibco BRL) and maintained in 5% CO2 (v/v) at 37°C. Cells were routinely cultured for at least 2 passages and were transferred to serum-free medium for 24 hr, when they reached 70–80% confluence. Adherent cells were detached using trypsin-EDTA solution [0.25% (w/v) trypsin with 0.02% EDTA in Hanks Balanced Salt Solution (HBSS) with 20 mM HEPES (Gibco BRL)] and centrifuged at 2,000 rpm for 4 min. The resulting pellet was washed twice with phosphate-buffered saline (PBS). RNA and protein were harvested as previously described and stored at −80°C until required.

Statistical analysis

The Spearman Rank correlation (for continuous variables) and the Mann-Whitney U test (for categorical/nominal variables) were used to evaluate the strength of associations between the various parameters. The frequency of ADAM-9 expression in each category was analysed using the chi-square (χ2) test. The parametric Student's t-test for paired data was used to compare ADAM-9 expression in different tissue types from the same patient, e.g., nodal metastasis and breast carcinoma tissue. p-values below 0.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

ADAM-9 mRNA expression in normal, benign and malignant breast tissue

A representative ethidium bromide stained agarose gel illustrating ADAM-9 RT-PCR products in normal, benign and malignant breast tissue is shown in Figure 2. ADAM-9 mRNA was expressed more frequently in both the fibroadenomas (21/38, 55%) and primary breast cancers (72/110, 66%) than in normal breast tissue (6/25, 24%) (χ2 = 4.83, p = 0.0281; χ2 = 12.71, p = 0.0004, respectively). The proportion of samples positive for ADAM-9 was not significantly different when fibroadenomas were compared to cancers.

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Figure 2. Ethidium bromide stained agarose gel illustrating RT-PCR products for ADAM-9 and GAPDH. Carcinoma, primary breast carcinomas; FA, fibroadenomas; normal, normal breast tissue. Positive and negative controls were included in all experiments. RNA isolated from MDA-MB-231 breast cancer cells was used as a positive control for ADAM-9. The negative control shown here is one in which the template was omitted from the PCR reaction. MW denotes 100 bp molecular weight marker. The most intense marker, which migrates at 600 bp, is indicated above.

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Relationship between ADAM-9 mRNA and prognostic factors for breast cancer

No significant relationship was found between frequency of expression of ADAM-9 mRNA and either tumour size, tumour grade, nodal status, histological type, ER status or patient age. However, ADAM-9 mRNA was detected in a greater proportion of PR-negative cancers (34/43, 79%) compared to PR-positive cancers (24/42, 57%) (χ2 = 3.77, p = 0.05).

ADAM-9 protein expression in normal, benign and malignant breast tissue

Electrophoresis under reducing conditions.

Figure 3 illustrates a representative blot of ADAM-9 protein expression in primary carcinomas, fibroadenomas and normal breast tissue, following electrophoresis under reducing conditions. In total, 4 different bands were detected, migrating with molecular masses of 124, 84, 60 and 48 kDa. Reactivity of the 124 and 48 kDa bands was inhibited, while the intensity of the 84 kDa band was significantly reduced, in the presence of excess blocking peptide. In addition none of these proteins were visible following immunoblotting with control IgG (Fig. 3c), which is further evidence for their specificity. The intense band at 60 kDa appeared to be nonspecific as staining was not inhibited in the presence of excess blocking peptide and it was also detected in the presence of control IgG (Fig. 3c). A similar band was also found with human serum suggesting that the 60 kDa band may be derived from serum IgG contaminating the tumour samples (results not shown).

thumbnail image

Figure 3. Western blots, following electrophoresis under reducing conditions, of (a) ADAM-9 and (b) β-actin. Carcinoma, primary breast carcinomas; normal, normal breast tissues. Levels of ADAM-9 were semi-quantified by scanning densitometric analysis, with normalisation of ADAM-9 protein against the “house-keeping” protein, β-actin. (c) Specificity of ADAM-9 bands were confirmed using a blocking peptide, corresponding to amino acids VPRHVSPVTP, at 30-fold concentration excess of the primary antibody and control IgG, i.e., immunoglobulin fraction of serum from healthy nonimmunised rabbits (Dako). PBC, primary breast carcinoma; FA, fibroadenoma; pos, positive control. The positive control used was a cell lysate from the breast cancer cell line MDA-MB-231. MW denotes molecular weight marker and the sizes in kDa are indicated to the left of the blots. ADAM-9 protein forms, i.e., 124, 85 and 48 kDa, are labelled on the right. The nonspecific 60 kDa band is also indicated. Thirty micrograms of protein was loaded for each tissue sample.

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Table III summarises the frequency of expression and the relative levels of the ADAM-9 124, 84 and 48 kDa forms, in normal breast tissue, fibroadenomas and primary breast carcinomas. There was no significant difference in the frequency of expression of the ADAM-9 precursor form (124 kDa) between the different types of breast tissue examined. However, relative levels of the 124 kDa protein were significantly higher in the fibroadenomas relative to the cancers (p = 0.0255; Mann-Whitney U test) (Fig. 4).

Table III. Distribution of ADAM-9 Protein Forms in Normal Breast Tissue, Fibroadenomas and Primary Breast Cancers1
ADAM-9 protein (kDa)Tissue typenNumber positivePercent positiveMedian level
  • 1

    The 124, 84 and 48 kDa forms were detected by Western blotting under reducing conditions, while the 55 and 52 kDa forms were only detected under non-reducing conditions. ADAM-9 protein levels are represented as arbitrary units/β-actin. As β-actin could not be visualised under non-reducing conditions, only qualitative analysis was carried out on the 55 kDa and 52 kDa proteins. ND = not determined.

124Normal breast tissue2114670.16
 Fibroadenoma2319830.63
 Primary carcinoma8572850.29
84Normal breast tissue215240
 Fibroadenoma2317741.33
 Primary carcinoma8576893.09
55Normal breast tissue19526ND
 Fibroadenoma191158 
 Primary carcinoma665076 
52Normal breast tissue19947ND
 Fibroadenoma19842 
 Primary carcinoma661320 
48Normal breast tissue2116760.30
 Fibroadenoma2317740.11
 Primary carcinoma8570820.14
thumbnail image

Figure 4. Relative levels of ADAM-9 precursor and mature protein forms, i.e., 124 and 84 kDa, respectively, in benign and malignant breast tissue. These protein forms were detected by Western blotting under reducing conditions. Protein levels are represented as arbitrary units/β-actin. Box boundaries, the 25th and 75th percentiles of the observed values; capped bars, the 10th and 90th percentiles; solid line, median. Data was analysed using the Mann-Whitney U test.

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In contrast, the 84 kDa form was detected more frequently in the primary cancers (76/85, 89%) and fibroadenomas (17/23, 74%) compared to normal breast tissue (5/21, 24%) (χ2 = 36.65, p < 0.0001; χ2 = 9.11, p = 0.0025, respectively). Consistent with these results, the 84 kDa form was also expressed at higher levels in the primary cancers and fibroadenomas compared to normal breast tissue (p < 0.0001, p = 0.0002, respectively; Mann-Whitney U test). Although there was no difference in the proportion of samples positive in the cancers and fibroadenomas, expression levels of the 84 kDa form were significantly higher in the primary cancers than the fibroadenomas (p = 0.0033; Mann-Whitney U test) (Fig. 4). In addition, we also examined ADAM-9 expression in 6 paired samples of breast carcinoma and nodal metastasis (data not shown). Significantly higher levels of the 84 kDa species were detected in the involved nodes compared to the carcinomas (p = 0.04, Student's t test).

Neither the frequency of expression nor the levels of the 48 kDa form were significantly different in the different breast tissue groups.

Electrophoresis under nonreducing conditions.

A representative blot following electrophoresis under non-reducing conditions is shown in Figure 5. Under these conditions, multiple forms of ADAM-9 were also detected, i.e., at 115, 76, 55, 52 and 46 kDa. In the absence of reducing agent, the strong band previously seen at 60 kDa appeared to migrate with a molecular mass >200 kDa. Both the 52 and 55 kDa bands detected under nonreducing conditions are likely to have been “masked” by the intense, apparently nonspecific 60 kDa band found with reducing agent present. As β-actin could not be visualised under nonreducing conditions, only qualitative analysis was carried out on the 55 kDa and 52 kDa proteins.

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Figure 5. Western blot, following electrophoresis under nonreducing conditions, of ADAM-9. Carcinoma, primary breast carcinomas; FA, fibroadenomas; normal, normal breast tissues. The positive control used was a cell lysate from the breast cancer cell line MDA-MB-231. MW denotes molecular weight marker and the sizes in kDa are indicated to the left of the blot. ADAM-9 protein forms, i.e., 115, 76, 55, 52 and 46 kDa, are labelled on the right.

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Table III summarises the frequency of expression of the ADAM-9 55 and 52 kDa forms, in normal breast tissue, fibroadenomas and primary breast carcinomas. Using the chi-square (χ2) test, the 55 kDa form was detected more frequently in the primary carcinomas (50/66, 76%) compared to normal breast tissue (5/19, 26%) (χ2 = 13.72, p = 0.0002). In contrast, the 52 kDa form was expressed more frequently in normal breast tissue (9/19, 47%) relative to the cancers (13/66, 20%) (χ2 = 4.55, p = 0.0334). No significant difference was found between carcinomas and fibroadenomas, for either the 55 kDa or 52 kDa bands.

Relationship between ADAM-9 protein and prognostic factors for breast cancer

No significant relationship was found between ADAM-9 protein expression and either tumour grade, ER levels, PR levels or patient age. However, an association was found between high expression levels of the 84 kDa form and nodal status. Using the median level as a cut-off, 65% (24 of 37) of patients with high levels of the mature form of ADAM-9 (84 kDa) had nodal metastases, compared to 40% of patients with low expression levels of the 84 kDa form (17 of 42) who were node-positive (χ2 = 3.77, p = 0.05). Furthermore, ADAM-9 protein (84 kDa) levels were also significantly higher in infiltrating ductal carcinomas compared to infiltrating lobular carcinomas (p = 0.0002; Mann-Whitney U test).

Using Spearman Rank analysis, a positive correlation was also found between relative levels of the 84 kDa form and HER-2/neu protein levels (n = 60, r = 0.313, p = 0.0163). In addition, levels of ADAM-9 protein (84 kDa) tended to be higher in HER-2/neu positive cancers compared to HER-2/neu negative cancers, although this failed to reach significance (p = 0.0967, Mann-Whitney U test).

Finally, the 52 kDa species was detected more frequently in cancers ≤ 2 cm (6/15, 40%) compared to the larger tumours (> 2 cm) (5/42, 12%) (p = 0.046; χ2 test).

Relationship between ADAM-9 mRNA and protein in primary breast carcinomas

No significant relationship was found between ADAM-9 mRNA and any form of ADAM-9 protein.

Expression of ADAM-9 mRNA in non-breast tissues

Figure 6 shows a representative ethidium bromide stained agarose gel illustrating ADAM-9 RT-PCR products from a range of normal and malignant nonbreast tissues. The number of positive samples are summarised in Table II. ADAM-9 mRNA was expressed in a wide range of human tissues examined. Although numbers were small, high frequency of expression of ADAM-9 mRNA was detected in liver cancer (4/5) and thyroid (6/7), while ADAM-9 was undetectable in venous (0/4) and skin (0/2) tissue. For those tissues, i.e., colon, liver and prostate, where both malignant and normal samples were available, ADAM-9 appeared to be expressed more frequently and at higher levels in the cancers.

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Figure 6. Ethidium bromide stained agarose gel illustrating RT-PCR products for ADAM-9 and GAPDH in a range of non-breast tissues. Positive and negative controls were included in all experiments. RNA isolated from MDA-MB-231 breast cancer cells was used as a positive control for ADAM-9. The negative control shown here is one in which the template was omitted from the PCR reaction. MW denotes 100 bp molecular weight marker.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Apart from preliminary studies on cell lines18, 28, 29 and small numbers of human cancers,30, 31, 32 little work has been carried out on the role of ADAMs in malignancy. In 1993, the ADAM-11 gene, located at chromosome 17q21.3, was reported to be somatically rearranged in 2 primary breast cancers.32 In another study, increased expression of ADAM-12 mRNA and protein was detected in 9 infiltrating ductal breast carcinomas compared to paired normal breast tissues.31 Recently, high expression levels of METH-1 (ADAMTS-1) mRNA were associated with local invasion, lymph node metastasis and poor prognosis in pancreatic cancer.33

Our study provides the first analysis of ADAM-9 expression in human breast cancer. We show that ADAM-9 mRNA was present in approximately 2/3 of primary breast cancers. ADAM-9 mRNA expression was found more frequently in both the carcinomas and fibroadenomas compared to normal breast tissue. However, frequency of expression in carcinomas and fibroadenomas was not significantly different.

With COS7 transfected cells, 2 forms of ADAM-9 protein were observed. These migrated with molecular masses of 116 and 79 kDa, respectively, and are likely to represent the precursor and active forms of ADAM-9. With the breast carcinomas however, 4 bands were detected. The 124 and 84 kDa forms are likely to correspond to the 116 and 79 kDa forms seen in the COS7 transfected cells, the minor differences in molecular mass being possibly due to different levels of glycosylation. Other authors also found that ADAM-9 migrated with molecular masses of 124 and 84 kDa.19, 34, 35 To our knowledge, the 48 kDa protein has not been previously reported. This protein may represent a processed form of ADAM-9. We should point out that in our study, in vitro proteolysis was minimised by the inclusion of protease inhibitors, i.e., EDTA, benzamidine-HCl and E-64, in the extraction buffer. As staining of the 60 kDa band was not inhibited by excess concentration of blocking peptide, this band appears to be nonspecific. Further evidence of nonspecificity was (a) detection of this band using control IgG, (b) its significantly altered mobility under nonreducing conditions, i.e., in the absence of reducing agent it migrated with a molecular mass >200 kDa, (c) a similar intense band at the same molecular mass was seen with normal human sera and (d) the large 60 kDa band was not detected in 3 different breast cancer cell lines, i.e., MDA-MB-231, BT-474 and MCF-7 (O'Shea et al., unpublished observation). The combination of all of these findings suggests that the 60 kDa band is due to serum contamination of the tumour, with what is likely to be an IgG.

Owing to the high cysteine content of ADAM-9, the precursor (124 kDa) and mature (84 kDa) forms have been reported to migrate differently in the presence and absence of reducing agent.34, 35 Thus the 46, 76 and 115 kDa forms observed in the absence of reducing agent are likely to correspond to the 48, 84 and 124 kDa proteins, respectively, seen under reducing conditions. The 52 and 55 kDa forms detected under nonreducing conditions were likely to have been “masked” by the intense 60 kDa band found in the presence of reducing agent.

The 84 and 55 kDa forms were detected more frequently and at higher levels in the primary cancers compared to normal breast tissue. In contrast, the 52 kDa form was expressed significantly more often in normal breast tissue relative to the cancers. This result suggests that differential processing or post-translational modification of ADAM-9 protein occurs in breast cancer compared to normal breast tissue. Further evidence of differential processing of ADAM-9 protein in benign and malignant breast tissue was that levels of the mature 84 kDa form were significantly higher in the primary cancers than the fibroadenomas, while the reverse was found for the 124 kDa precursor form.

As with previous studies on MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and stromelysin-3,36, 37, 38, 39 ADAM-9 mRNA levels exhibited no significant relationship with ER levels. However, an inverse relationship was observed between ADAM-9 mRNA and PR. It is of interest that an inverse relationship has also been reported between PR levels and MMP-1,40 MMP-838 and MMP-941 in breast cancer.

In our study, high levels of the 84 kDa or mature form of ADAM-9 correlated with indices of poor prognosis in breast cancer such as nodal status,42, ductal rather than lobular cancers43 and HER-2/neu overexpression.44, 45 All of these findings, when taken together, suggest that high levels of this form of ADAM-9 may correlate with adverse outcome in patients with breast cancer.

In contrast to certain ADAMs, which have a restricted tissue distribution, e.g., ADAM-22 and 23 are expressed predominantly in the brain46 and ADAM-2, 3, 18, 20, 21, 29 and 30 appear to be testis-specific,15 we have found that ADAM-9 mRNA was expressed in a wide variety of tissues (Table II). Previous analyses of mouse and human tissues have demonstrated the constitutive expression of ADAM-9 in several visceral organs, skeletal muscle and the gonads.35, 47 In our study, a high frequency of expression of ADAM-9 mRNA was detected in aneurysms and normal thyroid tissue, while ADAM-9 was undetectable in vein and skin. Although the number of specimens was small, higher frequency of expression of ADAM-9 mRNA was found in colon, liver and prostate cancers vis-à-vis the corresponding normal tissue, which is in agreement with our data for breast cancer.

In conclusion, this is the first study to investigate the expression of ADAM-9 in a large series of human cancers. Our findings suggest that there is differential processing of ADAM-9 protein in benign and malignant breast tissue. Furthermore, the increased expression levels of the 84 kDa mature form in the carcinomas correlated positively with certain prognostic factors for breast cancer, i.e., nodal status and HER-2/neu protein levels. Overexpression of ADAM-9 may contribute to degradation of the ECM and/or release of growth factors such as HB-EGF, which may in turn lead to tumour progression. Future work should, therefore, focus on investigating a potential role for ADAM-9 in malignancy, especially cancer invasion and metastasis.

REFERENCES

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES