Disease Markers 23 (2007) 189–196 189 IOS Press

Matrix expression patterns in luminal A type breast carcinomas

J. Decocka,∗, W. Hendrickxa,b, M. Drijkoningenb,c, H. Wildiersa,b,P.Nevenb, A. Smeetsb and R. Paridaensa,b aLaboratory for Experimental Oncology (LEO), Department of General Medical Oncology, University Hospital Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium bMultidisciplinary Breast Center, University Hospital Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium cPathology Department, University Hospital Sint Rafael,¨ Minderbroedersstraat 12, 3000 Leuven, Belgium

Abstract. Objective: Aberrant expression of individual matrix has been associated with poor prognosis in various human carcinomas. The current study aimed at defining an RNA expression profile of various MMPs in breast cancer and correlating their expression with clinicopathological parameters. Methods: The RNA expression patterns of 6 MMPs (MMP2, MMP8, MMP9, MMP10, MMP11, MMP13) were determined in 25 breast carcinomas using quantitative RT-PCR and correlated with clinicopathological parameters, including menopausal status, tumor size and grade, and lymph node involvement. Results: We observed high MMP2 levels more frequently in premenopausal than in postmenopausal women (p = 0.02). Analysis of luminal A type invasive ductal carcinomas (19/25), revealed an even stronger association of MMP2 with menopausal status (p = 0.005). Within this subgroup, we also found a correlation between MMP11 and menopausal status (p = 0.02). No correlation was found between MMP expressions and other clinicopathological parameters. In co-expression analyses MMP2-MMP10 and MMP8-MMP9 showed a weak correlation of their expression. Conclusions: Although this is a pilot study, our findings indicate that luminal A invasive ductal carcinomas commonly express high MMP2 and MMP11 levels in premenopausal breast cancer patients and suggest a co-regulation of MMP2-MMP10 and MMP8-MMP9.

Keywords: Breast cancer, matrix metalloproteinases, menopausal status, luminal A, clinicopathological parameters

1. Introduction ification of patients according to these clinicopatho- logical parameters fails to identify all patients at risk Breast cancer is one of the most common cancers as illustrated by heterogeneous clinical outcome. Re- in the world. Despite early diagnosis and administra- cent DNA microarray profiling studies on breast tumors tion of adjuvant systemic therapy, metastasis accounts have identified distinct subtypes of breast carcinomas for the majority of treatment failures and deaths. The that are associated with different clinical outcomes [1, classical prognostic parameters of breast carcinoma in- 17,18]. Using an intrinsic set of 534 , Sorlie et clude tumor size and grade, axillary lymph node in- al. analyzed the expression profiles of 115 indepen- volvement, menopausal status, steroid hormone recep- dent locally advanced breast tumor samples and cate- tor and Her2/neu status. However, prognostic strat- gorized them into five groups according to their estro- gen receptor (ER), progesterone receptor (PR) and hu- man epidermal growth factor receptor (Her2/neu) ex- ∗ Corresponding author: J. Decock, Lab for Experimental Oncolo- pression: luminal A (ER+/PR+−/Her2−), luminal B gy, University Hospital Gasthuisberg, O&N 1, Room 815, Herestraat + +− + + 49, B-3000 Leuven, Belgium. Tel.: +32 16 34 62 93; Fax: +32 16 (ER /PR /Her2 ), normal breast-like, Her2 /ER- − − + − − − 34 69 01; E-mail: [email protected]. (ER /PR /Her2 ) and basal-like (ER /PR /Her2 )

ISSN 0278-0240/07/$17.00  2007 – IOS Press and the authors. All rights reserved 190 J. Decock et al. / expression patterns in luminal A type breast carcinomas with the latter two associated with poor outcomes [18]. ed [2,6,9,15,16,19,20,23]. Hereby, we focused on the Classification of breast tumors into these distinct sub- luminal A subtype of invasive ductal carcinomas (IDC) types in combination with classical clinicopathological as this subgroup was the best represented in our study. parameters might improve prognostic accuracy. Fur- In addition, we investigated plausible co-regulation of thermore, identification of new biomarkers of invasion MMPs using co-expression analysis. and metastasis might result in a better prognostic strat- ification and lead to the development of specific thera- pies directed towards the prevention of invasion. 2. Materials and methods Tumor invasion and metastasis are the result of a multi-step process that includes basement membrane 2.1. Patients disruption, stromal infiltration of the site of origin, in- travasation, extravasation and invasion of a target or- A total of 25 patients with primary operable breast gan. All these steps require the degradation of base- cancer were included in this study. Patient character- ment membrane components and of extracellular ma- istics, including age at diagnosis and menopausal sta- trix (ECM) macromolecules which is in part medi- tus, were extracted from clinical files. Tumor size, tu- ated by the activity of the matrix metalloproteinases mor grade and lymph node status were retrieved from (MMPs). Twenty-four members of the human MMP pathology reports, while ER, PR and Her2/neu ex- family have been identified and found to play a cru- pression was determined on new immunohistochemi- cial role in tumorigenesis by ECM degradation, in- cal stained sections. Finally, all data were gathered in duction of angiogenesis, inhibition of apoptosis and of the central MBC database. All patients were newly the immune system. remodelling diagnosed at the Multidisciplinary Breast Center of the requires the synergistic action of several metallopro- University Hospital of Leuven between 1998 and 2000, teinases produced by the cancer cells or by the sur- underwent mastectomy or local wide excision of their rounding stromal cells [13]. Based on substrate speci- primary breast tumor and axillary lymph node dissec- ficity and domain organisation, MMPs are classified tion for staging and treatment. None of the patients into 5 subgroups: , matrilysins, collagenas- had received neo-adjuvant treatment, had any histo- es, stromelysins and membrane bound MMPs. MMP ry of cancer, multiple tumor foci, bilateral cancer or activity is regulated at several levels, including Paget disease. The presence of formalin-fixed paraf- expression, secretion, activation and inhibition by their fin embedded tumor tissue blocks and freshly frozen specific endogenous tissue inhibitors TIMPs [5]. Aber- tumor tissue stored at −80◦C were required for inclu- rant expression of various members of the MMP family sion. This study was conducted in the frame of the EU has been associated with a high potential of invasion Framework 6 Cancerdegradome project and its design and metastasis in a broad variety of human carcino- was approved by the Medical Ethical Committee of the mas [3,7,8,10,21,24]. University Hospital Leuven. The aim of this study was to explore the expression of a panel of MMPs in primary tumor tissue of 25 breast 2.2. Pathological assessment of tumor tissue cancer patients, using quantitative reverse transcriptase polymerase chain reaction (RT-PCR). We focused on All paraffin embedded haematoxylin-eosin (H&E) MMP2 and MMP9, MMP8 and MMP13, MMP10 and slides from the primary tumor as well as from the MMP11 as members of the 3 main MMP subclasses: axillary lymph nodes were reviewed. Tumor typing gelatinases, and stromelysins. We exam- and grading were performed according to the WHO- ined whether the RNA expressions of these 6 MMPs are classification and the Ellis and Elston grading system associated with specific clinical and pathological char- respectively. From each frozen block, one 4 µm slide acteristics, including menopausal status, tumor size, tu- was cut and stained with H&E. Only frozen samples mor grade and lymph node involvement. As we and that consist of at least 60% invasive tumor cells on H&E others have shown an association between individu- staining were selected for real time RT-PCR analysis. al aberrant MMP expressions and Her2/neu status in Immunohistochemical staining for ER, PR and Her2 breast cancer patients and as distinct breast cancer sub- was performed on 4 µm thick serial paraffin sections. types have been identified according to their ER, PR and Heat-induced epitope retrieval was carried out in a Her2/neu status, we also correlated MMP RNA expres- calibrated water bath (95–99◦C) and antibody com- sion with ER/PR/Her2 profiles of the tumors includ- plexes were visualised by Envision+ (Dako, Glostrup, J. Decock et al. / Matrix metalloproteinase expression patterns in luminal A type breast carcinomas 191

Denmark) and DAB. The primary rabbit monoclonal 2.4. RNA extraction antibodies SP1 directed against ER and SP2 directed against PR (Labvision Corporation,Fremont CA, USA) Confluent cell layers were washed twice with were applied in a dilution of 1/150 and 1/600 respec- phosphate-buffered saline before harvesting. Total tively. The primary mouse monoclonal antibody CB11 RNA was isolated from the cell lines and breast cancer (Novocastra Laboratories, Newcastle-upon-Tyne, UK) specimens using RNAbee (Campro Scientific, Berlin, directed against Her2/neu was applied in a dilution of Germany) and the SV Total RNA Isolation System 1/40. Immunostained slides were semi-quantitatively (Promega, Madison WI, USA). Approximately 100 mg evaluated. Using the Allred score for interpretation of of fresh frozen tumor tissue was homogenized in 1 ml ER and PR immunostaining, the proportion of positive RNAbee, followed by centrifugation at 14 000 rpm for tumor cell nuclei (score 0–5) and the staining intensity 10 minutes. The clear supernatant was collected into µ (score 0–3) were evaluated. Immunostaining was con- 200 l chloroform, shaken vigorously for 15 seconds, incubated for 3 minutes at room temperature and cen- sidered positive when the sum of the proportion and in- trifuged for 15 minutes at 14 000 rpm. The upper phase tensity score was 2–8, while complete absence of stain- was collected into 200 µl of 95% ethanol, mixed and ing was defined negative. The FDA-approved DAKO transferred into a spin basket assembly. The SV Total scoring system for Her2/neu immunostaining was ap- RNA Isolation System protocol was further followed plied, taking both the proportion of tumor cells with from step 7 to the end, according to the manufactur- positive membrane staining and the staining intensity er’s instructions. RNA was resuspended in nuclease into account. A score of 0 or 1 was defined negative, free water and concentrations were determined by spec- while a score of 2 or 3 was considered positive for trophotometry using a Nanodrop (Isogen Life Science, Her2/neu overexpression. Cases with Her2/neu overex- IJsselstein, The Netherlands). All RNA samples were pression were further analyzed by dual-colour Fluores- stored at −80◦C until reverse transcription. ence in situ Hybridization (FISH) in order to distinguish true HER-2/neu gene amplification from polysomy 17 2.5. Reverse transcription (PathVision, Vysis, Downers Grove, IL, USA). FISH analysis was performed manually on 4 µm thick paraf- In total, 1 µg of total RNA was reverse transcribed fin sections, according to the manufacturer’s recom- into cDNA in a 20 µl reaction volume containing mendations. Using a Zeiss Axioplan 2 epifluorescence 2 µg random hexamers (GE Healthcare, Bucking- microscope (Carl Zeiss, Germany) with appropriate fil- hamshire, UK), 200 units of Superscript II reverse ters, signals were counted in 60 nuclei of invasive tu- transcriptase, 5x first strand buffer (final concentra- mor cells in at least two distinct regions of the tissue tions 50 mM Tris-HCl pH 8.3; 75 mM KCl; 3 mM section. Tumors showing a mean Her2/ MgCl2, 0.02 mM DTT), 0.5 mM of each deoxynu- 17 ratio of more than 2 were considered amplified for cleotide triphosphate (dATP, dCTP, dGTP, dTTP) and the Her2 gene, and hence Her2 positive. 40 units of RNaseOUT recombinant ribonuclease in- hibitor (Invitrogen, Carlsbad CA, USA). Reverse tran- scription was carried out at 70◦C for 10 minutes and ◦ 2.3. Cell lines continued at 42 C for 1 hour. cDNA was stored at −80◦C until use.

Cell lines known to express the investigated MMPs 2.6. Quantitative RT-PCR were used as positive controls for real time quantitative RT-PCR. The human breast cancer cell line MDA-MB Specific primers and fluorogenic probes for MMP2, 231, human PC3 prostate adenocarcinoma cell line and MMP8, MMP9, MMP10, MMP11 and MMP13 the human G3-61 melanoma cell line were a kind gift were designed with Primer Express software (Applied of Prof. D. Edwards (University of East Anglia, Nor- Biosystems, Foster City CA, USA) and synthesized by wich UK). All cell lines were cultured in RPMI-1640 Eurogentec (Eurogentec, Seraing, Belgium). The se- medium, supplemented with 10% Fetal Bovine Serum, quences for the MMP primers and probes are given in 100 units/ml of penicillin and 100 µg/ml streptomycin Table 1. In order to prevent amplification of genomic and 0.5 µg/ml Fungizone Antimycotic at 37◦C and DNA, primers were generated with sequences within 5% CO2 (Invitrogen, Carlsbad CA, USA). different exons, close to intron-exon boundaries. PCR 192 J. Decock et al. / Matrix metalloproteinase expression patterns in luminal A type breast carcinomas

Table 1 TaqMan primer and probe sequences for the studied human MMPs Gene Genebank sequence of primers pairs and FAM-TAMRA probe∗ exon boundary amplicon accesion no. size (bp) MMP2 NM004530 TGGCGATGGATACCCCTTT exon12/exon13 83 TTCTCCCAAGGTCCATAGCTCAT CTCCTGGCTCATGCCTTCGCCC MMP9 NM004994 CCTGGGCAGATTCCAAACCT exon12/exon13 54 GCAAGTCTTCCGAGTAGTTTTGGAT CTCAAGTGGCACCACCACAACATCACC MMP8 NM002424 CACTCCCTCAAGATGACATCGA exon2/exon3 120 ACGGAGTGTGGTGATAGCATCA CAAGCAACCCTATCCAACCTACTGGACCAA MMP13 NM002427 AAATTATGGAGGAGATGCCCATT exon5/exon6 91 TCCTTGGAGTGGTCAAGACCTAA CTACAACTTGTTTCTTGTTGCTGCGCATGA MMP10 NM002425 GGACCTGGGCTTTATGGAGATAT exon9/exon10 82 CCCAGGGAGTGGCCAAGT CATCAGGCACCAATTTATTCCTCGTTGCT MMP11 NM005940 CCGCCAGATGCCTGTGA 66 CGGAGGCGCCACACAA CCTCCTTTGACGCGGTCTCCACC ∗Forward primer, reverse primer and probe are shown as upper, middle and lower sequence respectively. All sequences are written 5’-3’. reactions were carried out on the ABI Prism 7700 Se- MMP RNA expression was defined high when it was quence Detection System (Applied Biosystems, Fos- above the median expression of this specific MMP. As- ter City CA, USA). Each reaction was performed in a sociations between MMP expression (high/low) and 25 µl reaction volume, containing 5 ng cDNA (1 ng various clinicopathological variables were determined for 18S analyses), 8.33 µl qPCR Master mix (Euro- using the two-tailed Fisher’s exact test. Associations gentec, Seraing, Belgium), 100 nM probe and 200 nM among the individual MMP normalized values were each primer. Conditions for the PCR reaction were evaluated by the Spearman’s Rank correlation coeffi- 2 min at 50◦C, 10 min at 95◦C followed by 40 cycles cient. Statistical analyses were performed using the of 15 s at 95◦C and 1 min at 60◦C. The 18S ribosomal software package SPSS version 13, the level of signif- RNA (18S rRNA) gene was used as an endogenous icance being set at p  0.05. control to normalize for differences in the amount of total RNA in each sample and its expression was deter- mined with a commercially available 18S rRNA primer 3. Results and probe set (Applied Biosystems, Foster City CA, USA). To determine the relative RNA levels within the 3.1. Patient and tumor characteristics samples, standard curves for the PCR reactions were prepared. Serial cDNA dilutions of the human breast The median age at diagnosis of all patients was cancer cell line MDA-MB 231 served as positive con- 51 (range 36–79). Twelve (48%) patients were pre- trol for 18S rRNA, while dilution series of the human menopausal with a median age of 47 (range 36–51) and PC3 prostate adenocarcinoma cell line and of the hu- 13 (52%) were postmenopausal with a median age of man G3-61 melanoma cell line served as positive con- 65 (range 51–79). Small and large tumors were equal- trols for MMP2, MMP9, MMP10, MMP11, MMP13 ly distributed, 12 (48%) and 13 (52%) tumors respec- and for MMP8 respectively. tively. None of the tumors were well differentiated, 14 (56%) were moderately and 11 (44%) were poorly 2.7. Statistical analyses differentiated. No nodal involvement was found in 7 (28%) patients whereas the remaining 18 (72%) were Assumption of normality was verified using the nor- lymph node positive. Stratification of tumors into dis- mal probability plot, Shapiro-Wilk’s W test and the tinct subtypes according to ER, PR and Her2/neu status Levene’s test for homogeneity of variances. None of resulted in 19 luminal A invasive ductal carcinomas of the data were found to have a normal distribution. The no specific type, 2 basal-like carcinomas, 1 Her2+/ER- J. Decock et al. / Matrix metalloproteinase expression patterns in luminal A type breast carcinomas 193 tumor, 1 luminal A invasive lobular carcinoma of the 4. Discussion pleomorphic type and 2 luminal A invasive lobular car- cinomas of the classic type. According to the AJCC This report presents an expression profile analysis staging system, 4 patients had stage I disease, 13 had of a panel of 6 MMPs on the RNA level in 25 pri- stage II disease and 8 had stage III disease. mary invasive breast carcinomas. Using real time RT- PCR, we examined the RNA expressions of the gelati- nases MMP2 and MMP9, the collagenases MMP8 and 3.2. Association of MMP expressions and MMP13 and the stromelysins MMP10 and MMP11, clinicopathological parameters correlated our findings with classical clinicopathologi- cal parameters and explored co-expression patterns of MMP gene pairs. Within the whole study group we Table 2 summarizes the most important clinical and found significantly higher MMP2 RNA levels in tu- pathological characteristics at the time of diagnosis; in- mors from premenopausal patients than in tumors from cluding menopausal status, tumor size, tumor grade and postmenopausal patients. However, this finding is most lymph node involvement. No difference was found in probably the reflection of the result found in luminal the RNA expression levels of MMP2, MMP9, MMP8, A IDCs as these tumors represent 19 out of 25 tu- MMP13, MMP10 and MMP11 between small and large mors in our study and show the same result at an even tumors, moderately and poorly differentiated tumors or higher level of significance. Moreover, these tumors between tumors with or without lymph node involve- also showed significantly higher MMP11 expression ment. MMP2 expression was significantly correlated levels in premenopausal than in postmenopausal wom- with menopausal status (p = 0.02): 75% of tumors en. No significant associations were found between the from premenopausal women showed high MMP2 RNA RNA expressions of the other MMPs (MMP8, MMP9, levels, while this was only the case in 23% of tumors MMP10 and MMP13) and menopausal status, neither for the whole group, nor for the subgroup of luminal from postmenopausal women. As the luminal A sub- A IDC. None of the 6 MMPs studied were differential- group of invasive ductal carcinomas was the only breast ly expressed according to tumor size, tumor grade or cancer subtype that was well represented in our study lymph node involvement. population (19 of 25 tumors), we further investigated As mainly conflicting data have been reported on cor- the MMP expression profiles in these tumors (Table 3). relations between individual MMP RNA expressions Within this group of tumors the association between and clinicopathological parameters, our findings are MMP2 RNA expression and menopausal status was consistent with some reports and discordant with oth- p = even more significant ( 0.005) than in the whole ers [4,11,12,14,20,22]. This discrepancy may be partly study population with 80% of premenopausal patients explained by the use of different patient selection cri- and only 11% of postmenopausal women expressing teria or by the different analytical sensitivities of the high MMP2 levels. Furthermore, we found that in this methods used, including in situ hybridisation, north- particular tumor subtype also MMP11 RNA expression ern blotting and quantitative RT-PCR. Therefore, pru- was correlated with menopausal status (p = 0.02). dence is called for comparison of studies using various technologies. Pair-wise expression analyses of the present data in- 3.3. MMP co-expression analyses dicated a potential co-regulation of two sets of gene pairs: MMP2 and MMP10 on the one hand and MMP8 and MMP9 on the other. Hence, further investigation of The present real time RT-PCR data were also as- the underlying signalling pathways of MMP regulation sessed by co-expression analyses to look for potential seems indicated. patterns of MMP co-regulation in breast cancer (Ta- In conclusion, we observed higher MMP2 RNA ex- ble 4). These analyses revealed that 2 particular MMP pression levels in tumors from premenopausal patients gene pairs showed a weak association of their expres- than in those from postmenopausal patients. As lu- sion: MMP2 and MMP10 on the one hand, and MMP8 minal A invasive ductal carcinomas represent 19 out and MMP9 on the other. MMP11 and MMP13 RNA of 25 tumors in our study, this result was a reflection expressions were significantly correlated with both sets of the same association found in this subtype. More- of genes. over, differential expression of MMP2 in relation to 194 J. Decock et al. / Matrix metalloproteinase expression patterns in luminal A type breast carcinomas ∗ ∗ low (%) high (%) p low (%) high (%) p ∗ ∗ low (%) high (%) p low (%) high (%) p ∗ ∗ low (%) high (%) p low (%) high (%) p ∗ ∗ Table 2 Table 3 low (%) high (%) p low (%) high (%) p ∗ ∗ low (%) high (%) p low (%) high (%) p ∗ ∗ Correlations of MMP RNA expressions with clinicopathological parametes in the whole study population Correlations of MMP RNA expressions with clinicopathological parametes in the luminal A subtype of invasive ductal breast carcinomas patients low (%) high (%) p patients low (%) high (%) p 2cm13544654465446693154465446 2 cm 9 56 44 56 44 44 56 67 33 44 56 44 56 2 cm 12 50 50 1 50 50 1 50 50 1 33 67 0.11 50 50 1 50 50 1 2 cm 10 50 50 1 40 60 0.66 50 50 1 30 70 0.18 50 50 1 50 50 1 = = Fisher’s exact test, 2-tailed. Fisher’s exact test, 2-tailed. variableMenopausal status premenopausalpostmenopausal allTumor size 12< 13> 25 77 MMP2 75 23 0.02 58Note: MMP9 MMP expression 46∗ is defined 42 high if the value is above the 0.69 median. 54 42 58 MMP8 62 0.43 50 38 50 MMP13 1 54 42 46 MMP10 58 0.43 62 33 38 67 MMP11 0.11 69 31 Tumor grade moderatepoorLymph node status NegativePositive 14 57 11 7 18 43 45 43 0.69 56 55 57 57 44 0.67 43 43 45 0.69 57 56 57 55 0.67 43 44 43 0.69 45 57 57 56 0.67 55 43 57 0.69 44 50 43 45 50 1 50 55 1 57 50 64 43 55 36 1 50 45 0.24 57 50 43 36 1 50 64 50 variableMenopausal status premenopausalpostmenopausal allTumor size 10< 9 20 89 MMP2 80 11 0.005 50Note: MMP expression MMP9∗ is defined 50 high if 44 the value is above the median. 1 56 40 MMP8 60 56 0.66 40 44 MMP13 60 0.66 56 30 44 70 MMP10 0.18 20 67 80 33 MMP11 0.02 78 22 > Tumor grade moderatepoorLymph node status NegativePositive 11 64 8 4 15 36 37 50 0.37 53 63 55 50 47 1 45 0.65 37 50 55 47 63 50 45 53 1 0.65 45 37 25 53 55 63 75 0.58 47 1 50 45 50 50 47 55 50 1 1 53 50 55 50 50 45 47 50 1 0.65 53 50 37 50 47 1 63 53 J. Decock et al. / Matrix metalloproteinase expression patterns in luminal A type breast carcinomas 195

Table 4 Correlation table comparing individual MMP RNA expressions MMP2 MMP9 MMP8 MMP13 MMP10 MMP2 rs = 1 MMP9 p = 0.64 rs = 1 rs = 0.10 MMP8 p = 0.24 p = 0.03 rs = 1 rs = 0.24 rs = 0.43 MMP13 p = 0.012 p = 0.0009 p = 0.05 rs = 1 rs = 0.49 rs = 0.62 rs = 0.39 MMP10 p = 0.019 p = 0.18 p = 0.68 p = 0.0061 rs = 1 rs = 0.47 rs = 0.28 rs = 0.09 rs = 0.54 MMP11 p = 0.002 p = 0.04 p = 0.28 p = 0.0003 p = 0.002 rs = 0.58 rs = 0.42 rs = 0.22 rs = 0.73 rs = 0.58 rs = Spearman Rank Correlation coefficient. Values in bold indicate p<0.05 note: all correlations are positive, defined weak if 0

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