Prognostic significance of matrix metalloproteinases-1, -2, -7 and -13 and tissue

inhibitors of metalloproteinases-1, -2, -3 and -4 in colorectal cancer

Marja Hilska1*, Peter J. Roberts1, Yrj€o U. Collan2, Veli Jukka O. Laine2, Jyrki K€ossi3, Pirkko Hirsim€aki2,Otto Rahkonen4 and Matti Laato1,4

1Department of Surgery, Turku University Central Hospital, Turku, Finland2Department of Pathology, University of Turku, Turku, Finland3Department of Surgery, P€aij€at-H€ame Central Hospital, Lahti, Finland4Department of Medical Biochemistry, University of Turku, Turku, Finland

Strong expression of many matrix metalloproteinases (MMPs)has been related to poor survival of colorectal cancer (CRC)patients. The expression of tissue inhibitors of metalloprotei-nases (TIMPs) has been associated with both a beneficial and apoor outcome and there is thus a need to further clarify the sig-nificance of MMPs and TIMPs in CRC. The prognostic signifi-cance of 4 MMPs and TIMPs in CRC was evaluated. Formalin-fixed, paraffin-embedded tissue arrayed samples of 351 patientswith primary colon or rectal cancer of Dukes’ stages A-D wereselected for immunohistochemical staining of MMP-1, -2, -7 and-13, and TIMP-1, -2, -3 and -4. High expression of MMP-2 inthe malignant epithelium as well as in the surrounding stromawas associated with reduced survival of colon cancer patients.Strong epithelial and stromal cytoplasmic staining of TIMP-3was associated with a longer survival in rectal cancer patients,and here the interobserver variation for evaluating the degreeof staining was lower than for epithelial staining. Strong stromalcytoplasmic staining of TIMP-4 predicted longer survival of rec-tal cancer patients. Multivariate analysis showed that stromalcytoplasmic TIMP-3 staining was the only marker of independ-ent prognostic value. MMP-2 might be a useful prognosticmarker in colon cancer, and TIMP-3 and TIMP-4 in rectal can-cer, but the findings associated with stromal staining should beinterpreted with some caution. Different biologic behavior ordifferent genetic development may explain the differences be-tween colon and rectal cancers regarding the expression ofMMP-2, TIMP-3 and TIMP-4.' 2007 Wiley-Liss, Inc.

Key words: colorectal cancer; matrix metalloproteinases; tissueinhibitors of metalloproteinases; prognosis; survival

Matrix metalloproteinases (MMPs) are a large family of zinc-dependent neutral endopeptidases that play an important role inthe degradation of all matrix components crucial for malignant tu-mor growth, invasion and metastasis.1,2 Metalloproteinases areinhibited by tissue inhibitors (TIMPs) which are secreted proteins.These bioactive substances are specific inhibitors of MMPs thatbind to enzymatically active MMPs at a 1:1 molar stoichiometrythus inhibiting proteolysis.3 The role of TIMPs for the homeostasisof the extracellular matrix is critical and may inhibit or stimulatetumorigenesis.4

Many studies have shown that the expression of several MMPsis enhanced in a number of malignancies, including colorectalcancer (CRC), but the relation between the expression of MMPsand overall patient survival is not clear.5,6 Enhanced expression ofMMP-1, -7 and -13 has been reported to be associated with metas-tasis and poor survival of patients with CRC.7–9 Tissue concentra-tions of MMP-1, MMP-2 and TIMP-1 are increased in CRC com-pared to healthy colorectal tissue. This has been related to the roleof these endogenous substances in cancer progression.2 Healthytissue may contain high levels of TIMP-210 and the levels ofTIMP-3 mRNA are regionally increased in moderately and poorlydifferentiated CRC.11 On the other hand, TIMP-3 protein levelsmay decrease progressively as the stage of CRC advances.12 Theexact role of TIMP-4 in CRC is unknown.

In our study, the data of 351 patients were analyzed and cancertissue specimens from the tumors were examined to clarify theprognostic significance of the expression of MMP-1, -2, -7 and-13 and their tissue inhibitors TIMP-1, -2, -3 and -4 in patientsoperated on for CRC. Any differences between colon and rectalcancers were of special interest.

Material and methods

Tissue samples

The tissue samples were obtained from the archives of theDepartment of Pathology of the Turku University Central Hospi-tal. The data of 351 CRC patients who had undergone bowelresection in 1981–1990 at the Turku University Central Hospitalwere included in the study. Of the patients, 49 had Dukes A, 199Dukes B, 42 Dukes C and 61 Dukes D tumors. All pertinent clini-cal and histopathologic data of the patients were collected fromthe patients’ case records and stored in a computer database. Theclinicopathologic data of the patients are shown in Table I and thesurvival curves according to the modified Dukes’ classification areshown in Figure 1.

Tissue microarray

Archival paraffin-embedded CRC tissue samples were used toobtain tissue microarray (TMA) blocks for immunohistochemicalstaining. Areas 1 mm in diameter were chosen and marked in he-matoxylin and eosin-stained 4-lm-thin surface sections of theblocks under light microscopy. For carcinomas, invasive areas ofmalignant tumor of the lowest degree of differentiation or thehighest number of mitoses and of highest cellular atypia were cho-sen. Wide and homogenous areas (compatible with cellular atypiaand poor differentiation) were preferred. To avoid contamination,areas at least 2 mm apart from normal tissue or adenoma werechosen. Necrotic and autolytic areas and areas rich in stromalreaction were avoided. For tumors producing abundant intra- orextracellular mucin, invasive areas with a high number of epithe-lial cells were chosen. Representative areas of cancer mucosawhere marked by a pathologist (VJOL) on slides stained with he-matoxylin and eosin from selected paraffin blocks and a cylinderof tissue 1 mm in diameter was cut with a TMA instrument(Beecher Instruments, Sun Prairie, WI) into a new paraffin block.Serial 4-lm sections were then cut from the TMA paraffin blocks.The sections were mounted on ChemMateTM Capillary Gap PlusSlides (Grey) by DAKO. Normal colorectal mucosa also was

Grant sponsor: Special Government Funding (EVO) allocated to TurkuUniversity Central Hospital.*Correspondence to: Department of Surgery, Turku University Central

Hospital, Kiinamyllynkatu 4-8, 20520 Turku, Finland.Fax:1358-2-3132284. E-mail: marja.hilska@pp.inet.fiReceived 13 September 2006; Accepted after revision 2 February 2007DOI 10.1002/ijc.22747Published online 23 April 2007 in Wiley InterScience (www.interscience.

wiley.com).

Int. J. Cancer: 121, 714–723 (2007)' 2007 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

marked by the pathologist (VJOL) and obtained from tumorblocks adjacent to but at least 2 mm apart from malignant neoplas-tic areas of the section. If available, another sample was obtainedfrom normal colorectal mucosa at either of the resection marginsfrom the surgical specimen. Lymphatic follicles and hyperplastic

and inflamed areas were avoided. To obtain enough mucosa forthe TMA, tangentially cut areas were discarded.

Immunohistochemical staining

Stainings for MMP-1, MMP-2, MMP-7 and TIMP-2 were per-formed using a Techmate 500 immunostaining machine and a per-oxidase/diaminobenzidine (DAB) multilink detection kit (DAKO,Glostrup, Denmark) which is based on an indirect streptavidin-biotin method. Briefly, the sections were first deparaffinized inxylene (3 times for 5 min) and rehydrated through a graded seriesof ethanol after which they were rinsed briefly in Tris-bufferedsaline. For antigen retrieval, the slides were incubated in a micro-wave oven in 10 mmol/L citrate buffer (pH 6.0), but for MMP-7such pretreatment was not required. The dilutions used for MMP-1, MMP-2, MMP-7 and TIMP-2 detection were 1:250, 1:300,1:250 and 1:50, respectively. Sections were counterstained withMayer’s hematoxylin and eosin. After staining, the sections weredehydrated in ethanol, cleared in xylene and covered with Moun-tex and cover-slips.

The TMAs for MMP-13, TIMP-1, TIMP-3 and TIMP-4 werehand-stained. For TIMP-3 and TIMP-4 immunostainings, tissuesections were pretreated by boiling in microwave oven in 10mmol/L citrate buffer (pH 6.0) for 10 min as recommended bythe supplier (NeoMarkers, Fremont, CA), and for MMP-13 andTIMP-1 stainings the tissue sections were boiled in microwaveoven for 20 min. The antibody dilutions used for MMP-13,TIMP-1, TIMP-3 and TIMP-4 detection were 1:50, 1:20, 1:400and 1:2000, respectively, and were incubated at 4�C overnight.The reactivity of the antibodies was visualized using the avidin-biotin method as recommended by the supplier (Histostain-PlusKit, Zymed, South San Fransisco, CA). A brown color was devel-oped with diaminobenzidine (DAB-Plus Kit, Zymed, South San

TABLE I – UNIVARIATE ANALYSIS OF VARIABLES RELATED TO TUMOR AND PATIENT: ALL CRC PATIENTS

Variable Category/cutoff value

NPercentageof patientsin categories

5-yearsurvival (%)

p value

Age <65 years 155 44 59 0.47�65 years 196 56 58

Gender Male 162 46 52 0.033Female 189 54 64

Modified Dukes’ stage A 49 14 90 <0.0001B 199 57 67C 42 12 48D 61 17 13

Histologic differentiation Good 4 1 75 0.721

Moderate 284 81 58Poor 61 17 59Mucinous 2 1 0

Tumor site Colon 233 66 61 0.28Rectum 118 34 53

T stage 1 12 3 92 <0.00012 39 11 903 203 58 594 97 28 40

Amount of mucin 0–45% 322 92 59 0.62�50% 29 8 55

Urgency of operation Elective 301 86 62 0.0007Emergency 50 14 38

MMP-2 epithelial expression <0.65 44 13 77 0.011�0.65 307 87 56

MMP-2 stromal expression <0.65 45 13 76 0.014�0.65 306 87 56

TIMP-3 epithelial cytoplasmic expression <1.25 291 83 56 0.017�1.25 60 17 72

TIMP-3 epithelial nuclear expression <1% 106 30 67 0.02�1% 245 70 55

TIMP-3 stromal cytoplasmic expression <0.1 94 27 47 0.02�0.1 257 73 63

TIMP-3 stromal nuclear expression <25% 315 90 60 0.047�25% 36 10 44

1Excluding mucinous tumors due to small sample size.

FIGURE 1 – Kaplan–Meier survival curves of all CRC patients (n 5351) by modified Dukes’ stage.

715MMPs AND TIMPs IN COLORECTAL CANCER

Fransisco, CA), and the sections were counterstained with hema-toxylin and mounted (improved Gurr Aquamount, BDH Labora-tory Supplies, Poole, UK). The specificity of the immunoreactionswas controlled by omitting the primary antibody, by using samedilution of nonimmune sera, or—for TIMP-4—by incubating theantibody overnight at 4�C with an excess of blocking peptide(NeoMarkers, Fremont, CA). The specificity of the antibodies hasbeen tested.13–17

Evaluation of all TMA stainings was done with a Zeiss lightmicroscope at 103 objective magnification and 103 ocular mag-nification. The one who made these evaluations (MH) wasunaware of tumor grade, stage or clinical outcome. Some stainingswere randomly reevaluated by an independent pathologist (YUC).The following interobserver correlation coefficients were obtainedafter adjustment for the intraclass correlation coefficient (ICC), i.e.differences in mean level of staining: epithelial cytoplasmic stain-ing of MMP-2 0.88, epithelial cytoplasmic staining of TIMP-30.70 and epithelial nuclear staining of TIMP-3 0.76. The evalua-tion of stromal staining was less reproducible; the ICC for MMP-2

was 0.62, for TIMP-3 0.25 and for stromal nuclear TIMP-3 stain-ing 0.50.

Antibodies

The monoclonal mouse anti-human antibody used for MMP-1staining was supplied by Oncogene, Boston, MA (code IM35L,clone 41-IE5), and that used for MMP-2 staining by OncogeneResearch, Calbiochem, San Diego, CA (code IM33L, clone 42-5D11). The antibody used for MMP-7 staining was supplied byNeoMarkers, Fremont, CA (code MS-813-P0, clone ID2). Theantibody for MMP-13 detection was a monoclonal mouse anti-body supplied by NeoMarkers, Fremont, CA (code VIIIA2, cloneMS-825-P). The antibodies for TIMP-2 detection (code RB-1488-PO, clone MS-1485), TIMP-3 detection (code RB-1541, Ab-1)and TIMP-4 detection (code RB-1542-PO, LOT 1542P108), wererabbit polyclonal antibodies supplied by NeoMarkers, Fremont,CA. The antibody for TIMP-1 was a mouse anti-human antibody(code MAB 13429, LOT 23061106) supplied by Chemicon Inter-national, Temecula, CA.

FIGURE 2 – Staining patterns of MMPs-1, -2, -7 and -13 in normal and cancerepithelium of a Dukes B sigmoid cancersample. This patient had a poor outcome.Staining of normal colorectal epitheliumfor MMP-1 (a), MMP-2 (c), MMP-7 (e)and MMP-13 (g). Staining of cancer epi-thelium for MMP-1 (b), MMP-2 (d),MMP-7 (f) and MMP-13 (h).

716 HILSKA ET AL.

Evaluation of staining positivity

MMPs and TIMPs in cancer cell cytoplasm. The staining in-tensity of the epithelial cytoplasm was estimated on a scale from 0to 31 to a precision of 0.05. The intensity groups were defined asfollows:

31 5 � 50–100% of the cytoplasm of the epithelial cell wasstained with strong intensity,

21 5 only part of the cytoplasm was stained with moderate orstrong intensity (usually due to mucin goblet) and stainingwith strong intensity was seen in �15–50% of the cyto-plasm of the cell or �15–100% of the cytoplasm wasstained with moderate intensity,

11 5 5–15% of the cytoplasm of the cell was stained withmoderate or strong intensity or �15–100% of the cyto-plasm was weakly stained,

0 5 less than 5% of the cytoplasm was stained.

Strong staining intensity was defined as intensive dark browncolor, weak intensity as light brownish staining. Moderate stainingwas the intensity between strong and weak staining intensities.

A weighted staining score both for malignant epithelium as wellas for cancerous stromal tissue was counted. The staining scorewas then counted by adding the fraction of cells staining at the in-tensity 11 to the fraction of cells staining at the intensity 21 mul-tiplied by 2. Finally, the fraction of cells staining at the intensity31 multiplied by 3 was added. The fraction of cells at the inten-sity 0 did not influence the staining index.18 In cases where stain-ing was very weak, half of the area of this just discernible stainingwas scored as intensity 11 and half as 0. When the staining inten-sity was intermediate between 2 intensity classes, half of the areawas classified into the higher intensity class and half of the areainto the lower class.

TIMP-3 and TIMP-4 in nuclei of malignant epithelial anddesmoplastic stromal cells. Nuclear staining of cancerous epithelial

FIGURE 3 – Staining patterns of TIMPs-1, -2, -3 and -4 in normal and cancer epi-thelium of a Dukes B sigmoid cancersample. This patient had a poor outcome.Staining of normal colorectal epitheliumfor TIMP-1 (a), TIMP-2 (c), TIMP-3 (e)and TIMP-4 (g). Staining of cancer epi-thelium for TIMP-1 (b), TIMP-2 (d),TIMP-3 (f) and TIMP-4 (h).

717MMPs AND TIMPs IN COLORECTAL CANCER

cells was evaluated as the percentage of positive nuclei of the totalnumber of epithelial nuclei observed. Clear brown nuclear stainingwas regarded as positive but nuclear staining intensity was notseparately evaluated. Nuclear staining of stromal cells was eval-uated as in cancer cells.

MMPs and TIMPs in cytoplasm of desmoplastic stromalcells. The staining intensity of stromal cells was evaluated by thesame principles as epithelial cells on a scale from 0 to 31. Stain-ing of MMP-7 and MMP-13 in stromal cells was considered to beso weak that it was not evaluated.

TIMP-1 was scored as negative or positive. Staining wasregarded positive if in at least 1% of the stromal cells moderate orstrong TIMP-1 staining could be seen or if at least 5% of the cellshad weak staining.

Statistical analyses

An optimal cutoff value was identified by Kaplan–Meier’s uni-variate analysis by analyzing the scores of each marker by dividingthe material in 2 parts with every possible cutoff point to find the

lowest significant p value for the differences between the groupsin survival analyses. The cancer-specific survival curves were esti-mated by the Kaplan–Meier product-limit method. A log-rank testwas used to assess differences between life table data. The Cox’sproportional hazard model was used to determine which factorswere most significantly independently associated with survival.19

For all statistical analyses, differences between the groups wereconsidered significant at p values less than 0.05. All statisticalanalyses were performed using the SAS System for Windows, ver-sion 9.1 (SAS Institute, Cary, NC).

Results

General observations

Normal epithelium. The staining patterns of all MMPs andTIMPs in the normal colorectal epithelium are shown in Figures 2a,2c, 2e and 2g, and 3a, 3c, 3e and 3g, respectively. The epithelialcytoplasm stained occasionally for MMP-1 and MMP-7, but sincethere was strong variation in the level of sectioning no uniformpicture emerged. In the lamina propria, the plasma cells, part of

TABLE II – UNIVARIATE ANALYSIS OF VARIABLES RELATED TO TUMOR AND PATIENT: COLON CANCER AND RECTAL CANCER PATIENTS

VariableCategory/cutoff value

Colon cancer Rectal cancer

N5-year

survival (%) p value N5-year

survival (%) p value

Age <65 yrs 107 61 0.94 48 56 0.47�65 yrs 126 61 70 51

Gender Male 97 59 0.66 65 42 0.0042Female 136 63 53 68

Modified Dukes’ stage A 19 100 <0.0001 30 83 <0.0001B 138 73 61 52C 31 52 11 36D 45 13 16 13

Histologic differentiation Good 4 75 0.481 0 – 0.61

Moderate 178 62 106 53Poor 50 58 11 64Mucinous 1 0 1 0

T stage 1 2 100 <0.0001 10 90 0.00022 18 100 21 813 140 64 63 494 73 45 24 25

Amount of mucin 0–45% 208 61 0.87 114 54 0.15�50% 25 60 4 25

Urgency of operation Elective 184 67 0.0002 117 54 0.29Emergency 49 39 1 0

MMP-2 epithelial expression <0.65 36 83 0.0049 8 50 0.83�0.65 197 57 110 54

MMP-2 stromal expression <0.65 31 81 0.023 14 64 0.3�0.65 202 58 104 52

TIMP-3 epithelial cytoplasmic expression <1.25 191 59 0.17 100 49 0.033�1.25 42 69 18 78

TIMP-3 stromal cytoplasmic expression <0.1 59 56 0.41 35 31 0.0068�0.1 174 63 83 63

TIMP-4 stromal cytoplasmic expression <0.05 42 57 0.59 28 36 0.037�0.05 191 62 90 59

1Excluding mucinous tumors due to small sample size.

TABLE III – CANCER CELL STAINING: MMPS AND TIMPS (351 CRC SAMPLES)

Cancer cell cytoplasm Cancer nuclei

Antigen Negativesamples (%)

Meanscore

Medianscore (range)

cutoff p value Negativesamples (%)

Meanscore

Medianscore (range)

cutoff p value

MMP-1 12 (3.4) 1.28 1.3 (0–2.35) 1.0 0.11 n.d. n.d. n.d.MMP-2 5 (1.4) 1.28 1.3 (0–2.9) 0.65 0.011 n.d. n.d. n.d.MMP-7 57 (16.2) 0.58 0.55 (0–2.6) 0.5 0.4 n.d. n.d. n.d.MMP-13 100 (28.5) 0.53 0.35 (0–2.0) 0.1 0.21 n.d. n.d. n.d.TIMP-1 73 (20.8) 0.51 0.4 (0–1.6) 0.15 0.55 n.d. n.d. n.d.TIMP-2 1 (0.3) 1.71 1.7 (0–2.9) 1.40 0.37 n.d. n.d. n.d.TIMP-3 3 (0.9) 0.94 1 (0–2) 1.25 0.017 106 (30.2) 11% 2% (0–75%) 1% 0.02TIMP-4 28 (8.0) 0.77 0.95 (0–2.0) 0.75 0.3 34 (9.7) 31% 15% (0–95%) 5% 0.27

n.d., not defined.

718 HILSKA ET AL.

the lymphocytes and digitating macrophages stained likewiseoccasionally. Epithelial cytoplasmic staining was more uniformwith MMP-2 and TIMP-2, and ranged from weak to moderate forMMP-2 and from weak to strong for TIMP-2. The normal mucosawas negative for MMP-13. Only in a few areas, weak occasionalcytoplasmic staining could be seen. Normal epithelium was nega-tive for TIMP-1. For TIMP-3 and TIMP-4, staining was notalways uniform. Both nuclear and cytoplasmic epithelial stainingcould be observed in fibroblasts, lymphocytes, plasma cells andmacrophages. The mucin goblets were not stained.

Malignant epithelium. The staining patterns of MMP-1, -2 and-7 as well as TIMP-2 varied from diffuse to granular in the cyto-plasm. Usually, staining appeared uniform, and only occasionalsamples showed dramatic variation in staining intensity from cellto cell. The staining pattern of MMP-13 and TIMP-1 was diffuseand located in the cytoplasm. The staining pattern of TIMP-3 and-4 was both cytoplasmic and nuclear in epithelial and stromalcells. However, nuclear staining was generally clearer in TIMP-4

samples. The staining of markers with nonsignificant results arenot shown.

Staining of MMPs and TIMPs. The results of MMP and TIMPstaining in all CRC samples, colon cancer samples and rectal can-cer samples are shown in Tables I and II. The proportion of nega-tive samples, the mean and median scores of MMP and TIMPstaining in cancer cells and in stromal cells, are shown in Tables IIIand IV, respectively. Representative staining patterns of the 4MMPs in a Dukes B colon cancer patient who had a poor prognosisare shown in Figures 2b, 2d, 2f and 2h, and the corresponding stain-ing patterns from TIMP-1 to TIMP-4 in Figures 3b, 3d, 3f and 3h.The survival curves of patients who exhibited different marker pat-terns are shown in Figures 4–6, respectively. Overall, strong stain-ing of MMP-2 was associated with an unfavorable prognosis andstrong staining of both TIMP-3 and TIMP-4 was associated with amore favorable prognosis.

Multivariate analysis. The variables that were of statistical sig-nificance by univariate analysis were entered into Cox’s multivariate

TABLE IV – STROMAL CELL STAINING OF MMPS AND TIMPS (351 CRC SAMPLES)

Stromal cell cytoplasm Stromal cell nuclei

Antigen Negativesamples (%)

Meanscore

Medianscore (range)

cutoff p value Negativesamples (%)

Mean score Medianscore (range)

cutoff p value

MMP-1 21 (6.0) 0.57 0.55 (0-2.9) 0.25 0.15 n.d. n.d. n.d.MMP-2 4 (1.1) 1.14 1.15 (0–2.05) 0.65 0.014 n.d. n.d. n.d.MMP-7 n.d. n.d. n.d. n.d. n.d. n.d.MMP-13 n.d. n.d. n.d. n.d. n.d. n.d.TIMP-1 219 (62.4) n.d. n.d. n.d. n.d. n.d.TIMP-2 2 (0.6) 0.84 0.9 (0–1.8) 0.85 0.15 n.d. n.d. n.d.TIMP-3 34 (9.7) 0.23 0.15 (0–1) 0.1 0.02 47 (13.4) 8% 2% (0–75%) 25% 0.047TIMP-4 70 (19.9) 0.27 0.15 (0–1.3) 0.05 0.078 32 (9.1) 22% 15% (0–95%) 2% 0.11

n.d., not defined.

FIGURE 4 – Kaplan–Meier survival curves by MMP-2 staining: epithelium (a–c), stroma (d–f). Epithelial MMP-2 staining for all CRC patientsamples (a), colon cancer patient samples (b) and rectal cancer patient samples (c). Stromal MMP-2 staining in all CRC patient samples (d),colon cancer patient samples (e) and rectal cancer patient samples (f). A high staining level correlated with an unfavorable prognosis.

719MMPs AND TIMPs IN COLORECTAL CANCER

regression analysis. The T-stage was left out from the multivariateanalysis due to strong colinearity with Dukes’ stage. The results ofmultivariate analysis in all CRC patients, in colon cancer patients,and in rectal cancer patients are shown in Tables V–VII. It turnedout that the modified Dukes’ stage, the urgency of operation andthe stromal cytoplasmic TIMP-3 expression were significantlyassociated with 5-year survival in all CRC patients (Table V). Inthe group of colon cancer patients, only modified Dukes’ stageand urgency of operation were independent prognostic factors (Ta-ble VI), while rectal cancer patients had the modified Dukes’ stageand stromal cytoplasmic TIMP-3 expression as significant prog-nostic variables (Table VII).

Discussion

Many associations between prognosis and tumor markers werefound. The most significant finding in our study was the associa-tion between high TIMP-3 expression and a better outcome of rec-tal cancer patients. Both epithelial and stromal cytoplasmic TIMP-3 expression was related to a favorable outcome. By multivariateanalysis, only stromal cytoplasmic TIMP-3 turned out to carry in-dependent prognostic value. Overexpression of TIMP-3 has beenassociated with inhibition of invasion and induction of apoptoticcell death of cancer cells; thus TIMP-3 may play a role for genetherapy in many cancer types.20 TIMP-3 inhibits tumor formationin nude mice.21 According to Baker et al.20 and Bian et al.21 celldeath induced by TIMP-3 is not explained by its MMP inhibitoryactivity. In our study, TIMP-3 expression was present both in can-cer epithelium and stroma, whereas Airola et al.22 found TIMP-3mRNA expression only in stromal cells of intestinal cancers. Lateron, however, TIMP-3 expression has been discovered also in CRCepithelium.23

Another remarkable observation in our study was that epithelialand stromal expression of MMP-2 is significantly related to theprognosis of colon cancer patients. Strong expression of MMP-2secreted by tumor cells of e.g. brain, breast, gastric, ovarian andurothelial cancers has been associated with a poor prognosis.24–31

The association of elevated MMP-2 levels in colorectal tumor tis-sue with advanced tumor stage has been reported previously,32,33

but it has not been definitely proven that high MMP-2 expressionpredicts poor patient outcome. Our results regarding MMP-2expression in tumor epithelium and tumor stroma suggest thatMMP-2 may well have a role as a prognostic marker of the sur-vival of colon cancer patients. However, the amount of the activeform of MMP-2 is not clear, since the antibody recognizes simi-larly active and latent enzyme.

To our knowledge, the prognostic significance of TIMP-4 hasnot been evaluated previously in CRC. We found that high cyto-plasmic stromal cell TIMP-4 staining predicts prolonged sur-vival of patients with rectal cancer. In breast cancer, overexpres-sion of TIMP-4 associated with inhibition of tumor growth hasbeen reported34 whereas in endometrial cancer, overexpressionof TIMP-4 has been associated with invasion.35 In cervical can-cer,36 high TIMP-4 expression has been associated withadvanced disease stage. TIMP-4 counteracts the effect of MMP-2 by regulating its (gelatinase A) activity through efficient inhi-bition of MT1-MMP-mediated activation.37 In the present study,stromal cytoplasmic TIMP-4 expression was associated with abetter prognosis of rectal but not colon cancer patients, whereasMMP-2 expression was of prognostic value for colon cancerpatients.

During cancer invasion, the lamina densa of the basementmembrane (BM) and the lamina fibroreticularis (the outer layer ofthe BM) have to be degraded. Collagen IV is the structural back-bone of the lamina densa and is degraded primarily by MMP-

FIGURE 5 – Kaplan–Meier survival curves by stromal cytoplasmic TIMP-3 staining: all CRC patients (a), colon cancer patients (b) and rectalcancer patients (c). Survival curves of all CRC patients according to epithelial cytoplasmic TIMP-3 expression (d), epithelial nuclear TIMP-3expression (e) and stromal nuclear TIMP-3 expression (f). A high cytoplasmic staining level correlated with a favorable prognosis.

720 HILSKA ET AL.

238,39 and MMP-9.40 Types I and III collagen of the lamina fibror-eticularis are degraded mainly by MMP-1 and MMP-13.2,7 Incancer tissue, type IV collagen yields a positive, albeit thin andirregular immunoreaction, or the immunoreaction may be absent.The staining patterns of types I and III collagen around cancerousglands follows the staining pattern of type IV collagen.41 In ourstudy, poor survival was significantly associated with strongMMP-2 expression, but not with strong MMP-1 or MMP-13expression. The MMP-1 antibody might not have been specificfor the active form of enzyme. Earlier, Murray et al.7 showed thathigh MMP-1 expression was associated with poor prognosis, butonly 16% of the tumors were immunoreactive. High MMP-1expression has been related to hematogenous metastasis,42 andincreasing depth of invasion and metastasis43 in CRC. Likewise,weak MMP-1 expression has been related to a better prognosis ofpatients with advanced CRC.44 However, also our colon cancerpatients with high MMP-1 expression showed a tendency towards

worse prognosis. Expression of MMP-13 has been associatedwith poor prognosis in breast cancer45 and CRC.9 However, ourstudy could not confirm the notion that strong MMP-13 expres-sion would be significantly associated with survival of CRCpatients.

Matrilysin-1 (MMP-7) is overexpressed in about 80% of humanCRCs46,47 and it plays an important role for the invasive and meta-static potential of cancer cells.48 MMP-7 expression has also beenreported as being associated with distant metastasis and poor out-come.49–51 In the present study, there was an inverse tendencybetween prognosis and expression of MMP-7 in rectal cancerpatients. Like MMP-2 and MMP-9, MMP-7 can degrade type IVcollagen and thus it would be expected to correlate with metastasisand poor outcome, but this was not the case in our study. One ex-planation for this might be that MMP-7 is evenly expressed in all

TABLE VI – MULTIVARIATE COX’S PROPORTIONAL HAZARDS MODEL:COLON CANCER PATIENTS

Variable HR 95% HRconfidence limits

p value for5-year survival

Modified Dukes’ stage <0.0001A vs. B 0.000 (0.000)C vs. B 1.78 (0.9723.30)D vs. B 7.95 (4.86213.012)

Urgency of operation,emergency vs. elective

2.53 (1.6024.0) <0.0001

MMP-2 epithelialexpression, high vs. low

1.48 (0.6123.56) 0.38

MMP-2 stromalexpression, high vs. low

1.96 (0.8424.57) 0.12

HR, hazards ratio.

TABLE VII – MULTIVARIATE COX’S PROPORTIONAL HAZARDS MODEL:RECTAL CANCER PATIENTS

Variable HR95% HRconfidence

limits

p value for5-yearsurvival

Gender, men vs. women 1.22 (0.6422.31) 0.55Modified Dukes’ stage <.0001

B vs. A 2.96 (1.1127.87)C vs. A 5.00 (1.48216.87)D vs. A 16.21 (4.92253.36)

TIMP-3 epithelial cytoplasmicexpression, high vs. low

0.89 (0.2922.70) 0.84

TIMP-3 stromal cytoplasmicexpression, high vs. low

0.43 (0.2320.81) 0.0086

TIMP-4 stromal cytoplasmicexpression, high vs. low

0.72 (0.3921.33) 0.29

HR, hazards ratio.

FIGURE 6 – Kaplan–Meier survival curves by stromal cytoplasmic TIMP-4 staining: all CRC patients (a), colon cancer patients (b) and rectalcancer patients (c). A high staining level correlated with a favorable prognosis.

TABLE V – MULTIVARIATE COX’S PROPORTIONAL HAZARDS MODEL: ALL CRC PATIENTS

Variable HR 95% HRconfidence limits

p value for5-year survival

Gender, men vs. women 1.07 (0.7621.505) 0.7Modified Dukes’ stage <0.0001B vs. A 3.49 (1.3928.76)C vs. A 5.17 (1.93213.82)D vs. A 22.65 (8.94257.38)

Urgency of operation, emergency vs. elective 2.06 (1.3423.16) 0.0011MMP-2 epithelial expression, high vs. low 1.33 (0.6822.63) 0.41MMP-2 stromal expression, high vs. low 1.55 (0.8222.93) 0.17TIMP-3 epithelial cytoplasmic expression, high vs. low 0.60 (0.3521.01) 0.052TIMP-3 epithelial nuclear expression, high vs. low 1.22 (0.8221.81) 0.33TIMP-3 stromal cytoplasmic expression, high vs. low 0.54 (0.3720.78) 0.0009TIMP-3 stromal nuclear expression, high vs. low 1.34 (0.8122.23) 0.26

HR, hazards ratio.

721MMPs AND TIMPs IN COLORECTAL CANCER

stages of cancer progression and that different MMPs act simulta-neously in cancer tissue; the exact mechanism of cooperationamong the different MMPs is not known.

In the present study, neither TIMP-1 nor TIMP-2 was statisti-cally significantly associated with survival. Strong stromal TIMP-1expression has been related to poor survival.52 TIMP-2 expressionhas been reported to occur more often in localized than in dissemi-nated tumors.53 Such associations cannot reliably be assessed inour study because it is likely that many Dukes B tumors have beenincorrectly staged, since lymph node sampling was incomplete inthe 1980s. Thus some of the tumors diagnosed as Dukes B orDukes A tumors may have, in fact, been Dukes’ stage C cancers.

In conclusion, the finding that MMP-2 and TIMP-3 are prognos-tic factors is in accordance with earlier studies linking thesemarkers to prognosis in a number of malignancies. Our resultssuggest also an important role for MMP-2 and TIMP-3 as prog-nostic markers for patients with colon and rectal cancer, respec-tively.

Because the evaluation of stromal staining is difficult, as shownby the limited reproducibility of the evaluations, our result regard-ing the prognostic value of TIMP-3 must be considered prelimi-nary. The findings need to be verified in further studies. On the

other hand, there was a good interobserver correlation regardingthe assessments of the degree of epithelial staining of MMP-2 andof TIMP-3 and this implies that these results are biologically andclinically relevant. TIMP-4 was of prognostic significance forpatients with rectal cancer. The different patterns of expression ofthese markers by tumor site may reflect differences in histopatho-logic characteristics and molecular patterns of proximal and distalcancers.54 If so, this supports the hypothesis that these malignan-cies may have different etiologies.55 Two different pathways,microsatellite instability (MSI) and chromosomal instability(CIN), are involved in the pathogenesis of CRC. Tumors originat-ing in the proximal part of the colon carry a better patient progno-sis due to a high percentage of MSI-positive lesions,56,57 whereasdistal cancers are more often initiated through the CIN pathway58

with a potentially worse prognosis. MMP-2, TIMP-3 and TIMP-4may be of future clinical value.

Acknowledgements

The authors thank Mrs. Sinikka Kollanus for technical assis-tance in preparing the samples, and Mr. Tero Vahlberg for the sta-tistical analyses.

References

1. Zucker S, Vacirca J. Role of matrix metalloproteinases (MMPs) incolorectal cancer. Cancer Metastasis Rev 2004;23:101–17.

2. Ala-aho R, K€ah€ari VM. Collagenases in cancer. Biochimie2005;87:273–86.

3. Matrisian LM. Metalloproteinases and their inhibitors in matrixremodeling. Trends Genet 1990;6:121–5.

4. Jiang Y, Goldberg ID, Shi YE. Complex roles of tissue inhibitors ofmetalloproteinases in cancer. Oncogene 2002;21:2245–52.

5. Curran S, Murray GI. Matrix metalloproteinases in tumour invasionand metastasis. J Pathol 1999;189:300–8.

6. Liabakk NB, Talbot I, Wilkinson K, Balkwill F. Matrix metalloprotei-nase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) type IVcollagenases in colorectal cancer. Cancer Res 1996;56:190–6.

7. Murray GI, Duncan ME, O’Neil P, Melvin WT, Fothergill JE. Matrixmetalloproteinase-1 is associated with poor prognosis in colorectalcancer. Nat Med 1996;2:461–2.

8. Adachi Y, Yamamoto H, Itoh F, Hinoda Y, Okada Y, Imai K. Contri-bution of matrilysin (MMP-7) to the metastatic pathway of humancolorectal cancers. Gut 1999;45:252–8.

9. Leeman MF, McKay JA, Murray GI. Matrix metalloproteinase 13 ac-tivity is associated with poor prognosis in colorectal cancer. J ClinPathol 2002;55:758–62.

10. Baker EA, Bergin FG, Leaper DJ. Matrix metalloproteinases, their tis-sue inhibitors and colorectal cancer staging. Br J Surg 2000;87:1215–21.

11. Powe DG, Brough JL, Carter GI, Bailey EM, Stetler-Stevenson WG,Turner DR, Hewitt RE. TIMP-3 mRNA is regionally increased inmoderately and poorly differentiated colorectal adenocarcinoma. Br JCancer 1997;75:1678–83.

12. Zeng ZS, Sun Y, Shu WP, Guillem JG. Tissue inhibitor of metallopro-teinase-3 is a basement membrane-associated protein that is signifi-cantly decreased in human colorectal cancer. Dis Colon Rectum2001;44:1290–6.

13. Koskivirta I, Rahkonen O, M€ayr€anp€a€a M, Pakkanen S, Husheem M,Sainio A, Hakovirta H, Laine J, Jokinen E, Vuorio E, Kovanen P,J€arvel€ainen H. Tissue inhibitor of metalloproteinases 4 (TIMP4) isinvolved in inflammatory processes of human cardiovascular pathol-ogy. Histochem Cell Biol 2006;126:335–42.

14. Liotta LA, Stetler-Stevenson WG. Metalloproteinases and cancerinvasion. Semin Cancer Biol 1990;1:99–106.

15. Woessner JF Jr. Matrix metalloproteinases and their inhibitors in con-nective tissue remodeling. FASEB J 1991;5:2145–54.

16. Johansson N, Vaalamo M, Grenman S, Hietanen S, Klemi P, Saar-ialho-Kere U, K€ah€ari VM. Collagenase-3 (MMP-13) is expressed bytumor cells in invasive vulvar squamous cell carcinomas. Am J Pathol1999;154:469–80.

17. Oksjoki S, Rahkonen O, Haarala M, Vuorio E, Anttila L. Differencesin connective tissue gene expression between normally functioning,polycystic and post-menopausal ovaries. Mol Hum Reprod 2004;10:7–14.

18. Lipponen PK, Collan Y. Simple quantitation for immunohistochemi-cal staining positivity in microscopy for histopathology routine. ActaStereol 1992;11:125–32.

19. Kronqvist P, Kuopio T, Collan Y. Morphometric grading in breastcancer: thresholds for mitotic counts. Hum Pathol 1998;29:1462–8.

20. Baker AH, George SJ, Zaltsman AB, Murphy G, Newby AC. Inhibi-tion of invasion and induction of apoptotic cell death of cancer celllines by overexpression of TIMP-3. Br J Cancer 1999;79:1347–55.

21. Bian J, Wang Y, Smith MR, Kim H, Jacobs C, Jackman J, Kung HF,Colburn NH, Sun Y. Suppression of in vivo tumor growth and induc-tion of suspension cell death by tissue inhibitor of metalloproteinases(TIMP)-3. Carcinogenesis 1996;17:1805–11.

22. Airola K, Ahonen M, Johansson N, Heikkil€a P, Kere J, K€ah€ari VM,Saarialho-Kere U. Human TIMP-3 is expressed during fetal develop-ment, hair growth cycle, and cancer progression. J Histochem Cyto-chem 1998;46:437–48.

23. Curran S, Dundas SR, Buxton J, Leeman MF, Ramsay R, Murray GI.Matrix metalloproteinase/tissue inhibitors of matrix metalloproteinasephenotype identifies poor prognosis colorectal cancers. Clin CancerRes 2004;10:8229–34.

24. J€a€alinoja J, Herva R, Korpela M, H€oyhty€a M, Turpeenniemi-HujanenT. Matrix metalloproteinase 2 (MMP-2) immunoreactive protein isassociated with poor grade and survival in brain neoplasms. J Neuro-oncol 2000;46:81–90.

25. Talvensaari-Mattila A, P€a€akk€o P, H€oyhty€a M, Blanco-Sequeiros G,Turpeenniemi-Hujanen T. Matrix metalloproteinase-2 immunoreac-tive protein: a marker of aggressiveness in breast carcinoma. Cancer1998;83:1153–62.

26. Grigioni WF, D’Errico A, Fortunato C, Fiorentino M, Mancini AM,Stetler-Stevenson WG, Sobel ME, Liotta LA, Onisto M, Garbisa S.Prognosis of gastric carcinoma revealed by interaction between tumorcells and basement membrane. Mod Pathol 1994;2:220–5.

27. Sier CF, Kubben FJ, Ganesh S, Heerding MM, Griffioen G, Hane-maaijer R, van Krieken JHJM, Lamers CBHW, Verspaget HW. Tis-sue levels of metalloproteinases MMP-2 and MMP-9 are related tothe overall survival of patients with gastric carcinoma. Br J Cancer1996;74:413–7.

28. Allgayer H, Babic R, Grutzner KU, Beyer BC, Tarabichi A, Schild-berg FW, Heiss MM. Tumor-associated proteases and inhibitors ingastric cancer: analysis of prognostic impact and individual risk prote-ase pattern. Clin Exp Metastasis 1998;16:62–73.

29. Westerlund A, Apaja-Sarkkinen M, H€oyhty€a M, Puistola U, Turpeen-niemi—Hujanen T. Gelatinase A-immunoreactive protein in ovarianlesions-prognostic value in epithelial ovarian cancer. Gynecol Oncol1999;75:91–8.

30. Kanayama H, Yokota K, Kurokawa Y, Murakami Y, Nishitani M,Kagawa S. Prognostic values of matrix metalloproteinase-2 and tissueinhibitor of metalloproteinase-2 expression in bladder cancer. Cancer1998;82:1359–66.

31. Kanda K, Takahashi M, Murakami Y, Kanayama H, Kagawa S. Therole of activated form of matrix metalloproteinase-2 in urothelialcancer. BJU Int 2000;86:553–7.

32. D’Errico A, Garbisa S, Liotta LA, Castronovo V, Stetler-StevensonWG, Grigioni WF. Augmentation of type IV collagenase, laminin re-ceptor, and Ki67 proliferation antigen associated with human colon,gastric, and breast carcinoma progression. Mod Pathol 1991;4:239–46.

722 HILSKA ET AL.

33. Levy AT, Cioce V, Sobel ME, Garbisa S, Grigioni WF, Liotta LA,Stetler-Stevenson WG. Increased expression of the Mr 72,000 type IVcollagenase in human colonic adenocarcinoma. Cancer Res 1991;51:439–44.

34. Wang M, Liu YE, Greene J, Sheng S, Fuchs A, Rosen EM, Shi YE.Inhibition of tumor growth and metastasis of human breast cancercells transfected with tissue inhibitor of metalloproteinase 4. Onco-gene 1997;14:2767–74.

35. Tunuguntla R, Ripley D, Sang QXA, Chegini N. Expression of matrixmetalloproteinase-26 and tissue inhibitors of metalloproteinasesTIMP-3 and TIMP-4 in benign endometrium and endometrial cancer.Gynecol Oncol 2003;89:453–9.

36. Lizarraga F, Espinosa M, Maldonado V, Melendez-Zajgla J. Tissueinhibitor of metalloproteinases-4 is expressed in cervical cancerpatients. Anticancer Res 2005;25:623–7.

37. Bigg HF, Morrison CJ, Butler GS, Bugoyevitch MA, Soloway PD,Overall CM. Tissue inhibitor of metalloproteinases-4 inhibits but doesnot support the activation of gelatinase A via efficient inhibition ofmembrane type 1-matrix metalloproteinase. Cancer Res 2001;61:3610–8.

38. Liotta LA, Abe S, Robey PG, Martin GR. Preferential digestion ofbasement membrane collagen by an enzyme derived from a metastaticmurine tumor. Proc Natl Acad Sci USA 1979;76:2268–72.

39. Collier IE, Smith J, Kronberger A, Bauer EA, Wilhelm SM, EisenAZ, Goldberg GI. The structure of the human skin fibroblast collage-nase gene. J Biol Chem 1988;263:10711–3.

40. Wilhelm SM, Collier IE, Marmer BL, Eisen AZ, Grant GA, GoldbergGI. SV40-transformed human lung fibroblasts secrete a 92-kDa typeIV collagenase which is identical to that secreted by normal humanmacrophages. J Biol Chem 1989;264:17213–21.

41. Hilska M, Collan Y, Peltonen J, Gullichsen R, Paajanen H, Laato M.The distribution of collagen types I, III, and IV in normal and malig-nant colorectal mucosa. Eur J Cancer 1998;164:457–64.

42. Sunami E, Tsuno N, Osada T, Saito S, Kitayama J, Tomozawa S,Tsuruo T, Shibata Y, Muto T, Nagawa H. MMP-1 is a prognosticmarker for hematogenous metastasis of colorectal cancer. Oncologist2000;5:108–14.

43. Shiozawa J, Ito M, Nakayama T, Nakashima M, Kohno S, Sekine I.Expression of matrix metalloproteinase-1 in human colorectal carci-noma. Mod Pathol 2000;13:925–33.

44. Bendardaf R, Lamlum H, Vihinen P, Ristam€aki R, Laine J, Pyrh€onenS. Low collagenase-1 (MMP-1) and MT-MMP-1 expression levelsare favourable survival markers in advanced colorectal carcinoma.Oncology 2003;65:337–46.

45. Freije JMP, Diez-Itza I, Balbin M, S�anchez LM, Blasco F, Tolivia J,L�opez-Otin C. Molecular cloning and expression of collagenase-3, a

novel human matrix metalloproteinase produced by breast carcino-mas. J Biol Chem 1994;269:16766–73.

46. Newell KJ, Witty JP, Rodgers WH, Matrisian LM. Expression andlocalization of matrix-degrading metalloproteinases during colorectaltumorigenesis. Mol Carcinog 1994;10:199–206.

47. Yoshimoto M, Itoh F, Yamamoto H, Hinoda Y, Imai K, Yachi A.Expression of MMP-7 (PUMP-1) mRNA in human colorectal cancers.Int J Cancer 1993;54:614–8.

48. Yamatoto H, Itoh F, Hinoda Y, Imai K. Suppression of matrilysin inhib-its colon cancer cell invasion in vitro. Int J Cancer 1995;61:218–22.

49. Adachi Y, Yamamoto H, Itoh F, Arimura Y, Nishi M, Endo T, ImaiK. Clinicopathologic and prognostic significance of matrilysin expres-sion at the invasive front in human colorectal cancers. Int J Cancer2001;95:290–4.

50. Masaki T, Matsuoka H, Sugiyama M, Abe N, Goto A, Sakamoto A,Atomi Y. Matrilysin (MMP-7) as a significant determinant of malig-nant potential of early invasive colorectal carcinomas. Br J Cancer2001;84:1317–21.

51. Wang WS, Chen PM, Wang HS, Liang WY, Su Y. Matrix metallopro-teinase-7 increases resistance to Fas-mediated apoptosis and is a poorprognostic factor of patients with colorectal carcinoma. Carcinogene-sis 2006;27:1113–20.

52. Joo YE, Seo KS, Kim J, Kim HS, Rew JS, Park CS, Kim SJ. Role oftissue inhibitors of metalloproteinases (TIMPs) in colorectal carci-noma. J Korean Med Sci 1999;14:417–23.

53. Ring P, Johansson K, H€oyhty€a M, Rubin K, Lindmark G. Expressionof tissue inhibitor of metalloproteinases TIMP-2 in human colorectalcancer—a predictor of tumour stage. Br J Cancer 1997;76:805–11.

54. Gervaz P, Bouzourene H, Cerottini JP, Chaubert J, Benhattar J, SecicM, Wexner S, Givel JC, Belin B. Dukes B colorectal cancer: distinctgenetic categories and clinical outcome based on proximal or distaltumor location. Dis Colon Rectum 2001;44:364–72.

55. Birkenkamp-Demtroder K, Olesen SH, Sørensen FB, Laurberg S,Laiho P, Aaltonen LA, Ørntoft TF. Differential gene expression in co-lon cancer of the caecum versus the sigmoid and rectosigmoid. Gut2005;54:374–84.

56. Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancerof the proximal colon. Science 1993;260:816–9.

57. Kim IJ, Kang HC, Park JH, Shin Y, Ku JL, Lim SB, Park SY, JungSY, Kim HK, Park JG. Development and applications of a b-cateninoligonucleotide microarray: b-catenin mutations are dominantlyfound in the proximal colon cancers with microsatellite instability.Clin Cancer Res 2003;9:2920–25.

58. Gervaz P, Bucher P, Morel P. Two colons—two cancers: paradigmshift and clinical implications. J Surg Oncol 2004;88:261–6.

723MMPs AND TIMPs IN COLORECTAL CANCER