In vivo evidence that the stromelysin-3 metalloproteinase contributes in a paracrine manner to epithelial cell malignancy

Abstract

Stromelysin-3 (ST3; Basset, P., J.P. Bellocq, C. Wolf, I. Stoll, P. Hutin, J.M. Limacher, O.L. Podhajcer, M.P. Chenard, M.C. Rio, P. Chambon. 1990. Nature. 348:699-704) is a matrix metalloproteinase (MMP) expressed in mesenchymal cells located close to epithelial cells, during physiological and pathological tissue remodeling processes. In human carcinomas, high ST3 levels are associated with a poor clinical outcome, suggesting that ST3 plays a role during malignant processes. In this study we report the ST3 gene inactivation by homologous recombination. Although ST3 null mice (ST3-/-) were fertile and did not exhibit obvious alterations in appearance and behavior, the lack of ST3 altered malignant processes. Thus, the suppression of ST3 results in a decreased 7, 12-dimethylbenzanthracene-induced tumorigenesis in ST3-/- mice. Moreover, ST3-/- fibroblasts have lost the capacity to promote implantation of MCF7 human malignant epithelial cells in nude mice (P < 0.008). Finally, we show that this ST3 paracrine function requires extracellular matrix (ECM)-associated growth factors. Altogether, these findings give evidence that ST3 promotes, in a paracrine manner, homing of malignant epithelial cells, a key process for both primary tumors and metastases. Therefore, ST3 represents an appropriate target for specific MMP inhibitor(s) in future therapeutical approaches directed against the stromal compartment of human carcinomas.

Figure 1

Targeted disruption of the mouse ST3 gene. (A) Structure of the mouse ST3 gene. (B) Structure of the mouse ST3 targeting construct. (C) Targeted mouse ST3 gene. Closed boxes represent exons. The P probe is indicated by an horizontal bar. Restriction sites: B, BamHI; B2, BglII; E, EcoRI; N, NsiI; S, SpeI; letter in parentheses indicates nonfunctional restriction sites; B* indicates a BamHI site that was generated during genomic library construction (see Materials and Methods). (D) Southern blot analysis of SpeI-digested genomic DNA extracted from tail of ST3^+/+ (lane 1), ST3^+/− (lane 2) and ST3^−/− (lane 3) mice, and hybridized with the P probe. The wild-type (8.2 kb) and recombinant (2.4 kb) mouse ST3 DNA sizes are indicated.

Figure 2

Northern blot analysis of mouse ST3 expression in 2 d postpartum involuting uterus of ST3^+/+, ST3^+/− and ST3^−/− mice. Each lane contained 10 μg of total RNA extracted from uterus of ST3^+/+, ST3^+/− or ST3^−/− mice. Lanes 1 and 2, uterus of virgin ST3^+/+ and ST3^−/− mice were devoid of ST3 expression and exhibited a low level of Gel A expression. Lanes 3–5, ST3 transcripts were detected in ST3^+/+ (lane 3) and ST3^+/− (lane 4), but not in ST3^−/− (lane 5) involuting uterus. Gel A expression was detected in the three types of mice. The 36B4 probe (Masiakowski et al., 1982) was used as internal control. Autoradiography was for 16 h.

Figure 3

Whole mount RNA in situ hybridization of limb buds of ST3^+/+ and ST3^−/− 14.5 d post-coitum mouse embryos. Photographs of whole mount in situ hybridization of ST3^+/+ (A) and ST3^−/− (B) limb buds. ST3 mRNA was observed in the interdigital parts of the limb of ST3^+/+ (A) but not in that of ST3^−/− (B) mice. No morphological differences were observed between limb buds from ST3^+/+ and ST3^−/− mice. Bar, 500 μm.

Figure 4

Western blot analysis of conditioned media (48 h) from F2ST3^+/+ and F1ST3^−/− 15 d post-coitum embryonic fibroblasts. F2ST3^+/+ and F1ST3^−/− embryonic fibroblasts (C57BL/6J/ 129/Svj) were grown in serum-free medium and in the presence or absence of TPA (10 ng/ml), as indicated. 100-fold concentrated (80% ammonium sulfate) culture media (10 μg of total proteins) conditioned by F1ST3^−/− (lanes 2 and 3) or F2ST3^+/+ (lanes 4 and 5) embryonic fibroblasts were loaded in each lane. Purified mature mouse ST3 was used as control (lane 1). Protein species were revealed using monoclonal antibody 5ST-4C10 against the ST3 catalytic domain (Santavicca et al., 1995) followed by enhanced chemiluminescence detection (ECL kit; Dupont-NEN, Boston, MA). The positions of 66- and 45-kD molecular mass markers are indicated.

Figure 5

Analysis of the effect of ST3^+/+ and ST3^−/− embryonic fibroblasts on MCF7 human breast cancer cell tumorigenicity in nude mice. (A) Tumor latency: number of days after cell injection necessary to obtain tumors (volume >80 mm³) at 50 and 100% injection sites. Each fibroblast culture (8 × 10⁵ cells) was subcutaneously coinjected with MCF7 cells (2 × 10⁵ cells) into four nude mice (BALB/c nu/nu). The fibroblast genetic background and ST3 status were as indicated. The results presented are representative of one out of 3 independent experiments. (B) Kinetics of tumor volumes measured from 10 to 40 d after injection of MCF7 cells alone (control) or in the presence of F2ST3^+/+, F9ST3^+/+, F1ST3^−/−, or F6ST3^−/− fibroblasts. Each point represents the mean of four individual values (error bars).

Figure 6

Analysis of the effect of the ECM-associated growth factors on the capacity of ST3^+/+ fibroblasts to promote MCF7 cell tumorigenicity. Experimental conditions are as described in the Fig. 5 legend. Tumor incidence after subcutaneous injection of MCF7 cells either alone (Control), or together with F2ST3^+/+ or F1ST3^−/− fibroblasts, in presence of growth factors depleted (M ⁻) or undepleted (M ⁺) matrigel.