Int. J. Cancer: 64,70-75 (1995) 0 1995 Wiley-Liss, Inc.

Publication of the International Union Against Cancer Publication de I Union lnternationale Contre le Cancer

NEOPLASTIC PROGRESSION OF HUMAN COLORECTAL CANCER IS ASSOCIATED WITH OVEREXPRESSION OF THE STROMELYSIN-3 AND BM-40ISPARC GENES Henri PORTE', Eric CHASTRE', Sophie PREVOT', Bernard NORDLINGER3, Sylvie EMPEREUR~, Paul BASE?, Pierre CHAMBON4 and Christian GEsPACH'.5 Unite' INSERM 55, Equipe Canctrogtnksse et Difftrenciation de I'EpithClium Gastro-intestinal; 2Service Central d 'Anatornie et de

Cytologie Pathologiques; Tentre de Chirurgie Digestive et Unit6 INSERM U 402, Hbpital Saint-Antoine, 184 Rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12; and 4Laboratoire de Gtnttique Moltculaire des Eucaryotes du CNRS, Unitt INSERM 184 de Biologie Moltculaire et de G6nie Gtnttique, 11 Rue Humann, 67085 Strasbouig, France.

The interaction of neoplastic cells with the extracellular matrix is a critical event for the initiation of cancer invasion and metastasis. This study was designed to evaluate the potential implication of stromelysin-3 (ST3), a newly identified member of the matrix-degrading metalloproteinase family, and of BM-401 SPARC, a glycoprotein associated with the extracellular matrix, during the progression of human colorectal cancers. We ana- lyzed the relative abundance of ST3 and BM40lSPARC tran- scripts by Northern blot, and their distribution by in situ hybridization, in normal mucosa, benign adenomas, and pri- mary colorectal adenocarcinomas and their liver metastases. The ST3 and BM-40lSPARC transcripts were overexpressed in primary colorectal cancers and their liver metastases compared to non-neoplastic mucosa. These transcripts were localized in stromal fibroblasts adjacent to the neoplastic foci. Overexpres- sion of ST3 correlated with the progression of human colorectal tumors toward local invasion and liver metastasis. Induction of these genes also occurred in diverticulitis and digestive neo- plasms such as gastric and esophageal carcinomas, o 1995 Wiley-Liss, Inc.

Colorectal tumors arise from the stepwise accumulation of genetic and epigenetic events, including the activation, deregu- lation and suppression of a series of proto-oncogenes or tumor-suppressor genes, such as Ki-ras, src, myc, APC (adeno- matous polyposis coli), p53, DCC (deleted in colon cancers), and DRA (down-regulated in adenomas) (Fearon and Vogel- stein, 1990; Schweinfest et al., 1993; Chastre et al., 1993). Thercfore, human colonic cancers develop with profoundly heterogeneous molecular alterations. However, none of these alterations has proved sufficient to induce liver metastases of colorectal adenocarcinomas (Fearon and Vogelstein, 1990). Half of the patients with colorectal cancers die of metastatic disease (Bengtson et al., 1981). At present, pathologic classifi- cations are still the best criteria for evaluating the prognosis of colorectal cancers. However, they do not allow precise evalua- tion of the risk of the subsequent recurrences or metastases (Dukes and Bussey, 1958; Astler and Coller, 1954). Identifica- tion and characterization of the molecular and cellular mecha- nisms involved in this process should provide the basis for defining potential markers and identifying patients at risk of relapse or liver metastasis.

The first steps in the invasive and metastatic cascade involve disruption of the extracellular matrix (ECM) and enhanced motility of thc neoplastic cells (Liotta et al., 1986). Deteriora- tion of the basement membrane during invasiveness is due to the expression and activation of proteolytic enzymes, eg., cathepsins, and matrix-degrading metalloproteinases (MMPs) by the tumor cells themselves, and to the recruitment of adjacent normal cells (Matrisian, 1992). The MMPs comprise the collagenases, gelatinases and stromelysins (Matrisian, 1992). A putative MMP, stromelysin-3 (ST3), was recently identified in breast carcinomas and classified on the basis of its structural homology with other MMPs (Basset et al., 1990). Although the substrate specificity of ST3 is unknown, some of

its functional properties suggest that it may belong to a new MMP subgroup (Murphy et al., 1993). ST3 constitutes a potential marker of breast cancer invasion, because this putative MMP is induced in the fibroblasts surrounding neoplastic cells, but not in the normal mammary gland (Basset et al., 1990; Hahnel et al., 1993).

Basement membranes and some ECM constituents modu- late various cellular activities, including differentiation, prolif- eration, adhesiveness, chemotaxis and MMP production (Sage and Bornstein, 1989; Sage et al., 1991; Funk and Sage, 1993; Tremble et al., 1993). The osteonectiniBM-40, also termed SPARC, is a 43-kDa glycoprotein that interacts with the ECM components in connective tissues, including collagen types I, 111, IV and V (Sage and Bornstein, 1989; Sage et al., 1991). In endothelial cells and fibroblasts, BM-40/SPARC behaves like a negative mediator of spreading, and is associated with pathophysiologic events requiring tissue remodeling and de novo formation of basement membranes (Sage and Bornstein, 1989; Sage et al., 1991). BM-40/SPARC may also regulate and coordinate both endothelial cell proliferation and migration during morphogenetic processes such as wound healing and angiogenesis (Sage and Bornstein, 1989; Sage et al., 1991; Funk and Sage, 1993; Lane et al., 1992). ST3 and BM-40/SPARC may therefore constitute critical effectors during the initial steps in the process of invasion and metastasis in colorectal neoplasia.

On the basis of the observation that ST3 and BM-40/ SPARC are 2 potential effectors of cellular remodeling and invasion, we used Northern blot analysis and in situ hybridiza- tion to analyze the relative levels of expression and cellular localization of their transcripts, in a collection of normal and neoplastic human colonic tissues at different Dukes' stages of cancerous progression.

The purpose of the study was to explore the possibility of a correlation between the levels or cellular topography of the ST3 and BM40/SPARC messages, and the Dukes' stage of the primary tumor and of liver colonization.

MATERIAL AND METHODS Tissue samples

All specimens of colorectal, gastric and esophageal cancers, diverticulitis and liver metastases were delivered in fresh condition by the Department of Surgery to the Department of Pathology (HGpital Saint-Antoine, Paris, France), between January 1991 and April 1993. We collected adjacent control tissues, located at least 10 cm from the tumor, or at the

whom correspondence and reprint requests should be ad- dressed, at: INSERM U55, HBpital Saint-Antoine, 184 Rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12. Fax: 33 1 49284694.

Received: September 9,1994 and in revised form October 4, 1994.

ST3, BM40lSPARC AND HUMAN COLORECTAL CANCER 71

resection margin, taking care to avoid necrotic areas. Tissue samples of 0.5 to 2 g were snap-frozen in liquid nitrogen and stored at -80°C until use. The relative amounts of neoplastic mucosa and stromal tissue in tumor samples were assessed after staining of the tissue section, and ranged from 15 to 20% of the resection. Colonic crypts were isolated from the intesti- nal wall at 4”C, using a solution containing 2.5 mM EDTA and 250 mM NaCl (pH 7.5).

Clinical and pathological information The clinical records of each patient and the histology of the

tumors were reviewed without prior knowledge of the results obtained in the molecular analyses. A follow-up status was obtained up to May 1993. Individual tumors were staged according to Dukes’ classification, as modified by Astler and Coller (Dukes and Bussey, 1958; Astler and Coller, 1954), as follows. Stage A, tumors confined to the mucosa and/or submucosa; Stage B, tumors which had spread into the muscle wall and/or adjacent organs, without lymph-node invasion; Stage C, as for stage B, but with regional lymph-node invasion; Stage D, invasion without the possibility of surgical removal of a macroscopic tumor from adjacent organs and/or without discovery of metastases at the time of surgery.

RNA isolation and Northem-blot analysis Frozen tissue samples were homogenized with a Polytron

(Kinematica, Littau, Switzerland) apparatus in 4.7 M gua- nidinium isothiocyanate lysis buffer (10% w/v) and ultracentri- fuged for 20 hr through a cesium chloride solution at 32,000 rpm and 20°C. After denaturation, RNA samples (15 pg) underwent electrophoresis in 1% agarose-2.2 M formaldehyde gels and were then transferred to nylon membranes (Hybond N+, Amersham, Les Ulis, France). The relative loading and quality of the RNAs were first evaluated by methylene blue staining of the ribosomal RNA bands. The membranes were hybridized overnight with the 32P-labeled probes (Megaprime, Amersham), washed twice for 15 min at room temperature in 2 x SSC, 0.1% SDS (2x SSC is a 0.3-M NaCI, 30-mM sodium citrate solution, pH 7.0) and incubated for 60 min at 56°C in 0 . 1 ~ SSC, 0.1% SDS. The ST3 and BM-40/SPARC probe domains (Basset et al., 1990; Lankat-Buttgereit et aZ., 1988) were the 1.7-kb ST3 cDNA extending from nucleotides 346 to 2,105 and the 1-kb BM-40/SPARC cDNA encompassing the entire coding sequence. The hEFl probe was the correspond- ing cDNA fragment isolated after Pst I digestion of the plasmid, a generous gift from Dr. A. Zantema (Sylvius Labora- tory, Leiden, The Netherlands). Autoradiography was per- formed at -70°C using Kodak X-OMAT AR films (Rochester, NY) and a Chronex Quanta 111 intensifying screen (Dupont de Nemours, Les Ulis, France). For each blot, 3 successive exposures were carried out for 8, 24 and 72 hr. Expression of the ST3 and BM-40/SPARC transcripts was quantified by densitometry (Lecphor Program, Biocom, Les Ulis, France), and corrected for the non-linearity of the autoradiographic signals by reference to a standard curve obtained after serial dilutions of 32P-labeled probes. The ST3 and BM40ISPARC signals were then normalized for the level of mRNA encoding human elongation factor hEF-1, that was similar to the methylene-blue staining of the rRNA. One RNA sample was included as internal control throughout the series of Northern blots. Accumulation of the transcripts was classified as unde- tectable, weak (up to 0.86 and 1.4 arbitrary units for ST-3 and BM40/SPARC, respectively), moderate (between 0.8645 and 1.4-8.5 arbitrary units) or strong (over 4.5 and 8.5 arbitrary units, respectively). The Kruskal-Wallis test was used to delineate any relationship between the Dukes’ stage of the tumors and the relative levels of the ST3 and BM-40/SPARC transcripts.

In situ hybridization Thirteen cases of primary colonic adenocarcinomas, lymph

nodes and liver metastases were analyzed by in situ hybridiza- tion. Tissues were fixed either in aqueous Bouin’s solution or in 4% buffered formamide. The deparaffinized sections (15 pm) were spread out on silane-coated slides and hybridized overnight at 37°C with the probes specific for ST3 and BM-IOISPARC genes or for the polyoma middle-T viral oncogene, which was used as a negative control and labeled with 35S by random priming (PrCvot et a/., 1989; Chastre et al., 1993). After stringent washes at room temperature (once in 50% formalin and 4 x SSC for 30 min, twice in 2 x SSC for 30 min, once in 0.1 x SSC for 15 min) and at 37°C (in 0.2 x SSC for 60 min), autoradiography was performed for 14 to 21 days, using NTB2 emulsion (Kodak, Paris, France).

RESULTS Expression of ST3 in colorectal tumors and liver metastases

The relative levels of expression of the 2.4-kb ST3 tran- scripts were analyzed in each sample by Northern blot on total RNAs (Fig. 1). Results were quantified by densitometric analysis of the autoradiographs, and normalized to hEF-1 (Table I). ST3 transcripts were detected in 50 out of 69 primary colorectal cancers (72%) and in the 20 liver metasta- ses of colorectal adenocarcinomas analyzed. In contrast, the level of ST3 transcripts was very low or undetectable in their corresponding normal control tissues, i.e., non-neoplastic colo- rectal mucosa, freshly isolated colonic epithelial crypts, and liver tissue. The levels of ST3 expression in 3 out of 12 benign adenomas (25%) and 3 out of 9 Dukes’ A (33%) did not differ significantly from the ST3 levels in control colorectal tissue. In contrast, high levels of ST3 transcripts were commonly found in stages B, C and D, and in liver metastases of colorectal adenocarcinomas ( p < 0.05, compared to controls). This in- creased accumulation of the ST3 transcripts correlated with the progression of colorectal tumors, from Dukes’ stages A to D ( p < 0.02).

ST3 transcripts were identified in 33% of the colonic specimens with diverticulitis. These positive cases corre- sponded to diverticulitis associated with processes involving local spatial remodeling of the colonic wall, i.e., one inflamma- tory pseudo-tumor and 2 cases of colonic stenosis. Among the other types of tumor investigated, ST3 transcripts were overex- pressed in 415 gastric adenocarcinomas, 3 esophageal carcino- mas, 2 liver metastases of a gastric cancer or a leiomyosarcoma, and 3 hepatocarcinomas which developed in cirrhotic liver. In contrast, ST3 mRNA was not detected in 2 hepatocarcinomas which developed in normal liver, or in 2 liver metastases of an intestinal carcinoid tumor and a pancreatic endocrine carci- noma.

Expression of BM-40ISPARC in colorectal tumors and liver metastases

As shown in Table 11, BM40/SPARC mRNA was detected in 83% of primary adenocarcinomas and 95% of liver metasta- ses from colorectal cancers. The BM40/SPARC transcripts were identified as major and minor species of 2.2 and 3kb (Fig. l), originating from the same open reading frame and distinct polyadenylation sites (Lankat-Buttgereit et a/., 1988). In con- trast to the results obtained with ST3, identification of the BM-40ISPARC transcripts was not restricted to cancer tis- sues. Low or moderate BM-40ISPARC mRNA levels were found in 42% of non-neoplastic colorectal mucosa samples and 28% of control liver tissue, but at a lower incidence than in stages B, C, or D, or in liver metastases ( p < 0.05). However, the relative expression of BM40/SPARC transcripts in colo- rectal adenocarcinomas was not significantly related to the

72 PORTE E T A L

FIGURE 1 - Northern-blot analysis of ST3, BM40/SPARC and hEF-1 in human primary colorectal tumors, liver metastases and control tissues. Total RNAs (15 kg) were extracted from colorectal adenomas, adenocarcinornas graded according to Dukes' classification, and liver metastases from colorectal adenocarcinomas, which were matched with control tissues, i.e., non-neoplastic colonic mucosa, colonic crypts and liver tissue. Samples underwent electrophoresis on 1% agarose-formaldehyde gel and were hybridized with the ST3 and BM40/SPARC probes. The 28s and 18s rRNAs were used as size markers. Hybridization of the blots with hEF-1 served as a control for RNA loading, and to normalize the relative expression of the ST3 and BM-40/SPARC transcripts. ADK adenocarcinoma.

TABLE I - RELATIVE EXPRESSION OF THE ST-3 TRANSCRIPTS IN CONTROL AND PATHOLOGIC DIGESTIVE TISSUES ____

Stromelystn 3 mRNA levels Densitometric values2 Number

of cases Undetectable' Weak Moderate Strong Positive Samples

Mean Median Range

Normal colonic crypts 5 5(100%) 0 0 0 0 0 0 - Control colorectal mucosa 36 35 977 1 3%) 0 0 1 3%) 0 0 0-0.17 Mucosa from diverticulitis 9 6 /66.:k) 2 122.2%) 1 (11.1%) 0 3 133.3%) 0.18 0 0-0.9 Benign adenomas 12 9(75%) 3(25%) 0 0 3 (25%) 0.07 0 0-0.41 Colorectal ADKs, Dukes'

B 7[26% 6 122%) 8[29%) 20 (74%) 3.69 0.86 0-35.1 C 0-34.3 D 15 1 6.6 4 26.6% 4 26.6% 6 (40%) 14 193.3;) 3.4 1.14 0-16.5

A 9 6 66.6%) 2 22.2%) 1 11.1%) 0 3 (33.3%) 0.17 0 0-1.1 ?: 5 1284) 2111.1%) 3 116.6%) i[i??k) 13 72.27 5.15 2.48

Control liver tissue 14 14(100%) 0 0 0 0 0 0 - Liver metastases (colorectal ADKs) 20 0 5 (25%) 7(35%) 8(40%) 20(100%) 5.77 2.47 0.12-44.5 Other types of digestive tumor 17 5 (29.4%) 5 (29.4%) 4 (23.5%) 3 (17.6%) 12 (70.6%) 2.15 0.18 0-13.5

'Numbers correspond to the samples found in each class of ST-3 RNA levels. Undetectable, 0; weak, between 0 and 0.86; moderate, between 0.86 and 4.5; strong, > 4.5 arbitrary densitometric units.-2Minimal and maximal values are expressed in arbitrary densitometric units. ADKs: adenocarcinomas.

Dukes' stage of the tumors. The proportion of samples which to stromal cells, presumably the fibroblasts immediately sur- overexpressed BM-40/SPARC was comparable in diverticuli- rounding the carcinomatous islets, and was undetectable in the tis and all stages of colorectal cancer. Among the samples that neoplastic cells and the corresponding normal compartment overexpressed ST3, 94% were also positive for BM-40/ (Fig. Zb, e ) . In contrast to the distribution pattern of the ST3 SPARC. signal, expression of BMdO/SPARC was not restricted to the

stromal cells in close association with the neoplastic cells. but was diffusely scattered throughout the stroma (Fig. 2h). As shown in Figure 2, f and i, the Py-MT probe produced a In situ hybridization of the ST3 and BM-40 transcripts

ST3 transcripts were detected in 7/9 colonic adenocarcino- mas analyzed by in situ hybridization. ST3 mRNA was confined

homogeneous deposit of silver grains, demonstrating the specificity of the ST3 and BM-40/SPARC signals.

73 ST3, BM40ISPARC AND HUMAN COLORECTAL CANCER

TABLE I1 - RELATIVE EXPRESSION OF THE BM40ISPARC TRANSCRIPTS IN CONTROL AND PATHOLOGIC DIGESTIVE TISSUES

BM40ISPARC mRNA levels ~~~ ~ ~ ~~ ~ ~ . .

Densitometric values2 Number Of Undetectable' Weak Moderate Strong Positive Samples

Mean Median Range

Normal colonic crypts 5 5(100%) 0 0

Benign adenomas 12 6(50%) 6150%) 0

Control colorectal mucosa 36 21 58.3% 9(25%) 6 16.6% Mucosa from diverticulitis 9 4 {44.4%{ 0 3 133.3%{

Colorectal ADKs, Dukes' A 9 4 (44.4%) 3 133.3%) 1 (11.1%)

14.8%) 3 (11.1%) 9 (33.3% 11.1%) 5 (27.7%) 6 (33.3%

B C D 15 2 (13.3%) 3 (20%) ' 6 (40%) '

Control liver tissue 14 10 71.4%) 2 (14.3%) 2 14.3%) Liver metastases (colorectal 20 1 [5%) 4 (20%) 4

- 0 0 0 0 0 15 41 6% 1.1 0 0-6.53

2(22.2%) 5 {55:5%] 10.7 1.9 0-76 0 6 (50%) 0.4 0.2 0-1.22

5 55.5%) 3.7 0.43 0-25.8 23[85% 16 3.5 0-111 16 7.1 1.77 040.6 13 86% 7.1 2.06 0-29.2

0 4 28% 0.5 0 0-2.45 1(55%) 19[95%{ 20.3 11.09 0-137

Other types of digestive tumor 17 7(41.1%) 4(23.5%) 6(35.3%) 17(100%) 17 5.16 0.16-137 ~

'Numbers correspond to the samples found in each class of BM40/SPARC mRNA levels. Undetectable, 0; weak, between 0 and 1.4; moderate, between 1.4 and 8.5; strong, > 8.5 arbitrary densitometric ~nits.-~Minimal and maximal values are expressed in arbitrary densitometric units. ADKs: adenocarcinomas.

FIGURE 2 - I n situ hybridization of the ST3 and BM-40/SPARC transcripts in a lymph-node and a liver metastasis of colorectal adenocarcinomas, and in a liver metastasis of a leiomyosarcoma. Hematoxylin staining was detected by bright-field microscopy (a, d, g), and silver grains were detected by dark-field microscopy of paraffin-embedded tissue sections, after in sztu hybridization using the 35S-labeled probes specific for ST3 (b, e), and BMdO/SPARC (h). The polyoma middle-T viral oncogene was used as a negative control (c, f i). a, b, c, lymph-node metastasis of a rectal colloid adenocarcinoma; d, e, f. liver metastasis of a leiomyosarcoma; and g, h, i, liver metastasis of a well-differentiated colonic cancer. Bar = 20 bm.

Our results indicate that the increased abundance of the ST3 transcripts is associated with the anatomopathologic tumor staging. The ST3 gene was overexpressed in most of the invasive colorectal adenocarcinomas (stages B, C and D) and

DISCUSSION

We have shown that the ST3 and BM-4OISPARC genes are overexpressed in the peritumoral stroma of human colorectal cancers.

PORTE ETAL 74

in their liver metastases. The ST3 mRNA was not observed in control tissues, and rarely detected in benign adenomas or Dukes’ stage A tumors. These results strongly suggest that ST3 is implicated in the progression of colorectal cancer, local invasion and development of metastases. Furthermore, ST3 gene overexpression was also identified in gastric adenocarci- nomas, esophageal carcinomas, hepatocarcinomas and a liver metastasis of a leiomyosarcoma in the present study, as well as in invasivc breast adenocarcinomas, squamous cancers of the neck and cutaneous basal-cell carcinomas (Basset et al., 1990; Hahnel et aL, 1993; Muller et al., 1993). Therefore, ST3 may be one of the major molecular alterations occurring during human carcinogenesis. In contrast, up-regulation of the BM-40/ SPARC gene seems to occur at relatively early, locally invasive stages, and its possible role in the pathogenesis of colorectal carcinoma warrants further study.

Another finding of this study concerns the close association between the expression of ST3 and BM-40ISPARC in primary colorectal tumors and in their liver metastases. It is noteworthy that the induction of both transcripts was not restricted to the neoplastic tissues, but also occurred in diverticulitis associated with stenosis or pseudo-tumor. Under normal conditions, high levels of ST3 and BM-40ISPARC mRNA are found in embryo limb bud, placenta and uterus, and during wound healing (Basset et al., 1990; Sage et al., 1991). This pattern of expression has been observed in tissues that underwent pro- found changes in architecture, including extensive cell prolif- eration, spreading, extracellular matrix remodeling and apop- tosis (Basset et al., 1990; Sage and Bornstein, 1989). Aberrant expression of the ST3 and BM-IOISPARC genes may play a role in the pathogenesis of human colorectal tumors, presum- ably through the remodeling of the extracellular matrix.

However, the molecular targets of ST3 and BM-40/SPARC, and their specific roles during the neoplastic process, have not yet been fully determined. BM40ISPARC modulates the spreading and proliferation of fibroblasts and endothelial cells in vitro and reduces the expression of thrombospondin, a potent inhibitor of angiogenesis (Funk and Sage, 1993; Lane et al., 1992). In addition, BM-40ISPARC induces the transcrip- tion of matrix-degrading metalloproteinases involved in the degradation of basement membranes and interstitial matrices, including 92-kDa collagenase, gelatinase and transin in fibro- blasts (Tremble et al., 1993). Thus, as far as BM-40ISPARC is concerned in cancer progression, it is likely that this extracellu- lar matrix component is involved in the control of growth and invasiveness of colorectal adenocarcinomas. A most intriguing question is the biological significance of the induction of ST3 during neoplastic and inflammatory diseases. Several lines of evidence suggest that ST3 is a proteinase that exhibits a structural homology with other members of the matrix- degrading metalloproteinase family. Expression of recombi- nant ST3 in the mouse myeloma NSO cell line made it possible to identify a C-terminally truncated 28-kDa ST3 form with weak stromelysin activity (Murphy et al., 1993). However, it is not known whether full-length ST3 possesses similar activity, and an alternative hypothesis is that full-length ST3 has specific properties for an as yet undefined substrate (Murphy et al., 1993).

Previous authors reported increased expression of proteo- lytic enzymes involved in the normal turnover of basement membranes, such as type-IV collagenase and urokinase-type plasminogen activator in stromal cells surrounding colorectal adenocarcinomas (Poulsom et al., 1992; Pyke et al., 1991 and 1993). The peculiarities of ST3 concern its ectopic induction in cancers and inflammatory diseases and its substrate specificity. In the present study, in situ hybridization revealed that ST3 gene expression was restricted to stromal cells in the vicinity of the colonic neoplastic cells, in good agreement with the localization of ST3 in breast adenocarcinomas, squamous cancers of the neck and cutaneous basal-cell carcinomas (Basset et al., 1990; Hahnel et al., 1993; Muller et al., 1993). This pattern of expression differed slightly from that of BM-40ISPARC, which was detected throughout the peritu- moral stroma. Consequently, ST3 and BM40/SPARC may be considered as paracrine stroma-derived factors necessary for the invasiveness of colorectal cancers and for metastasis. These observations emphasize the pivotal role of the interac- tions between neoplastic and stromal cells in cancer progres- sion. In this connection, it was recently shown that fibroblasts markedly enhanced tumor growth of human prostatic, breast and bladder epithelial cell lines when they were co-inoculated into nude mice (Camps et al., 1990). Stromal cells also conferred the invasive phenotype upon the rat colonic carci- noma PROb cell line in vitro (Martinet al., 1992).

Important questions not directly addressed by our study concern the molecular and cellular mechanisms responsible for generating deregulation of the ST3 and BM-IOISPARC genes in human colorectal cancers. While most attention has been paid to the role of oncogenes and tumor-suppressor genes in colon carcinogenesis, data are scarce concerning the effector systems involved in the deregulation of neoplastic cells and their related peritumoral stroma, leading to tumor growth, local invasion, and distant metastasis. Several cytokines and growth factors induce the expression of ST3 (e.g., EGF, PDGF and bFGF), and of BM-40ISPARC ( e g , TGF-6) in cultured fibroblasts (Basset et al., 1990; Wrana etal., 1991). Such factors are often produced by neoplastic cells and macrophages, and during inflammation. It is not yet known whether any of these autocrine or paracrine factors are involved in the transcrip- tional induction of the ST3 and BM-40ISPARC messages in human colon cancers. It is clearly important to define the regulatory elements of these genes more precisely, as well as the mechanisms involved in the maturation and stabilization of their transcripts in fibroblast-like cells. Follow-up of our patients will allow us to determine whether accumulation of the ST3 transcripts has a prognostic value for the development of recurrences and metachronous presentation of liver and lymph-node metastases.

ACKNOWLEDGEMENTS

We are indebted to Drs R. Parc and J.P. HervC de Sigalony, and to the staff of the Department of Surgery (H6pital St Antoine) for their collaboration. We are grateful to Dr. T. Kreig (Dermatology Clinic, University of Munich, Germany) for providing the BM-40/SPARC cDNA. We also thank Mr. Y. IssouliC for photographic reproduction.

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