The matrix metalloproteinase stromelysin-1 acts as a natural mammary tumor promoter

Abstract

Extracellular matrix-degrading matrix metalloproteinases (MMPs) are invariably upregulated in epithelial cancers and are key agonists in angiogenesis, invasion and metastasis. Yet most MMPs are secreted not by the cancer cells themselves, but by stromal cells within and around the tumor mass. Because the stromal environment can influence tumor formation, and because MMPs can alter this environment, MMPs may also contribute to the initial stages of cancer development. Several recent studies in MMP-overexpressing and MMP-deficient mice support this possibility, but have required carcinogens or pre-existing oncogenic mutations to initiate tumorigenesis. Here we review the spontaneous development of premalignant and malignant lesions in the mammary glands of transgenic mice that express an autoactivating form of MMP-3/stromelysin-1 under the control of the whey acidic protein gene promoter. These changes were absent in nontransgenic littermates and were quenched by co-expression of a human tissue inhibitor of metalloproteinases-1 (TIMP-1) transgene. Thus by altering the cellular microenvironment, stromelysin-1 can act as a natural tumor promoter and enhance cancer susceptibility.

Figure 1

Masson's trichrome-stained mammary gland sections from (a) nontransgenic and (b – d) WAP-Str1 transgenic mice. The normal nontransgenic gland contains relatively few resting ducts surrounded by scant periductal collagen and embedded in an adipose stroma, whereas each transgenic gland exhibits extensive accumulation of blue-stained collagen (fibrosis) and few residual adipocytes. (b) This gland from a 7-month-old transgenic mouse contains numerous collapsed alveolar structures and extensive periglandular fibrosis. (c) A large dilated duct containing proteinaceous secretory material and hyperplastic alveolar epithelial cells with secretory vacuolization are apparent in this gland even though this 16-month-old transgenic mouse had never been pregnant. (d) This section from a 10-month-old transgenic mouse contains secretory hyperplastic epithelial cells and fibrosis adjacent to a secretory adenocarcinoma. Scale bar, 150 μm

Figure 2

Carmine-stained wholemount (a,b) and H&E-stained paraffin section (c – e) of an abdominal (#4) mammary gland with diffuse hyperplasia (hp), fibrosis (fi) and lymphocytic infiltration (ly) from a 15-month-old parous WAP-Str1 mouse sacrificed 4 months after its pups were removed. The hyperplastic branches indicated by the arrow in (a) are outlined in (c) and are shown at higher magnification in (b and d). These sparse and disproportionately short secondary branches terminate in relatively well-developed lobuloalveolar structures and are surrounded by multilocular adipocytes (asterisk). The boxed area to the left of the central lymph node (LN) in c is enlarged in e and shows three hyperplastic areas, each with a distinct histologic appearance. Dilated (ectatic) primary ducts (du) containing considerable amounts of residual secretory material are also evident throughout the gland. Scale bars, 5 mm (a,c), 500 μm (b,d,e)

Figure 3

Histologic appearance of hyperplastic alveolar nodules (HANs) from 23- (a), 16- (b), 24- (c) and 12-month-old (d – f) virgin WAP-Str1 transgenic mice as seen by wholemount (a – c), H&E (d), anti-cytokeratin-8 immunoperoxidase (e) and anti-smooth muscle actin immunoperoxidase (f) staining. The multiple alveolar structures are composed of an internal layer of cytokeratin-8-positive luminal epithelial cells with lipid vacuolization (e) surrounded by a single smooth muscle actin-positive myoepithelial cell layer (f). Adjacent normal areas (nl) contain mostly adipocytes and sparse ducts with the same bilayered luminal and myoepithelial cell staining, but without secretory vacuoles. LN, lymph node. Scale bars, 2 mm (a – c), 100 μm (d – f)

Figure 4

Histologic appearance of a florid papillary hyperplasia (intraductal papillomatosis) in the mammary gland of a 2-year-old virgin WAP-Str1 transgenic mouse as seen by wholemount (a), H&E (b,c), anti-smooth muscle actin (d) and anti-cytokeratin-8 (e) staining. The area outlined in (b) is shown at higher magnification in (c – e). The small, basophilic cells within the papillary projections (me) are smooth muscle actin-positive (d) and cytokeratin-8-negative (e), indicating the abnormal, internal presence of myoepithelial cells. Although small focal collections of lymphocytes were present (not shown), fibrosis was not observed and the far ends of the gland were essentially normal. Scale bars, 2 mm (a,b), 200 μm (c – e)

Figure 5

Histologic appearance of moderately well-differentiated mammary adenocarcinomas from 15- (a,b), 19- (c – e) and 23-month-old (f – h) WAP-Str1 transgenic mice as seen after wholemount (a), H&E (b,c,f), anti-cytokeratin-8 (d,g), anti-smooth muscle actin (e) and anti-vimentin (h) staining. (a,b) The tumor at right contains cystic spaces and necrotic debris, and sits adjacent to a diffuse lactational-like hyperplasia (asterisk) and a lymph node (LN) that is surrounded by muscle. The strip of connective tissue and skeletal muscle (arrow) is indicated for orientation. (c – e) This complex tumor (at right in each panel) contains numerous cystic spaces and tumor cell nests composed of mixed small and large cell populations. The small cells are smooth muscle actin-positive (e) and have small nuclei with a dense chromatin structure. The larger cells are cytokeratin-8 positive (d), have larger nuclei with a more open chromatin structure, and exhibit distinct intercellular bridges. The tumor is surrounded by a fibrotic stroma, atypical papillary lesions (c) and areas of secretory hyperplasia (asterisk). (f – h) These serial sections show a papillary adenocarcinoma with large cytokeratin-positive tumor cells, numerous mitotic figures, and an abundant vimentin-positive stroma (st). Scale bars, 2 mm (a,b), 300 μm (c), 200 μm (d,e), 50 μm (f – h)

Figure 6

Histologic appearance of poorly-differentiated mammary tumors and a tumor-derived cell line from WAP-Str1 transgenic mice. (a) This H&E-stained area of primary mammary cancer from a 16-month-old virgin mouse contains spindle-shaped cells (left) and polygonal, epithelial-like tumor cells at lower right. (b,c) These two lung metastases from the tumor in (a) also contain spindled and polygonal cancer cells and stain positive for both cytokeratins (c) and vimentin (not shown). (d – f) The tumor cell line (TCL-1) established from the tumor in (a) exhibits a spindle-cell morphology and cytokeratin (d) and vimentin (e) immunoreactivity by dual immunocytochemistry and DAPI counterstaining (f). (g – i) TCL-1-derived tumors in immunocompromised mice show a pure spindle-cell morphology and numerous mitotic figures by H&E (g) and continue to stain positive for both cytokeratins (h) and vimentin (i). (j – l) Serial sections of this carcinosarcoma from a 17-month-old virgin transgenic mouse reveal a mixed cellular morphology by H&E (j) with polygonal carcinomatous cells that are cytokeratin-positive (k) and spindle-shaped sarcomatous cells that are vimentin-positive (l). Scale bars 100 μm (a,c), 200 μm (b), 50 μm (d – i), 75 μm (j – l)

Figure 7

A hypothetical model of how Str1 may affect cellular behavior via the β-catenin/LEF signal transduction pathway. Following E-cadherin cleavage by Str1 or another metalloproteinase, free cytosolic β-catenin pools are increased. In the absence of Wnt signaling, glycogen synthase kinase 3β (GST-3β) phosphorylates β-catenin, thus targeting it for association with the adenomatous polyposis coli (APC) gene product and axin, ubiquitination, and proteosomal degradation. Alternatively, unphosphorylated β-catenin enters the nucleus where it interacts with LEF/TCF transcription factors, thus regulating the transcription of genes containing functional LEF recognition sites. A number of potential target genes are shown, although only c-myc, cyclin-D1 and matrilysin (MMP-7) have been so far shown to respond to β-catenin/LEF transactivation. Molecules indicated in red have been shown to play a causal role in cancer development. Frz, Frizzled family Wnt/Wg receptor; Dsh, Disheveled family or other GSK inhibitor; Wg, Wingless; ms, mouse