MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis

Abstract

The matrix metalloproteinase MMP-9/gelatinase B is upregulated in angiogenic dysplasias and invasive cancers of the epidermis in a mouse model of multi-stage tumorigenesis elicited by HPV16 oncogenes. Transgenic mice lacking MMP-9 show reduced keratinocyte hyperproliferation at all neoplastic stages and a decreased incidence of invasive tumors. Yet those carcinomas that do arise in the absence of MMP-9 exhibit a greater loss of keratinocyte differentiation, indicative of a more aggressive and higher grade tumor. Notably, MMP-9 is predominantly expressed in neutrophils, macrophages, and mast cells, rather than in oncogene-positive neoplastic cells. Chimeric mice expressing MMP-9 only in cells of hematopoietic origin, produced by bone marrow transplantation, reconstitute the MMP-9-dependent contributions to squamous carcinogenesis. Thus, inflammatory cells can be coconspirators in carcinogenesis.

Figure 1. MMP-2 and MMP-9 Are Activated during Epithelial Carcinogenesis in HPV16 Transgenic Mice

Gelatin-substrate zymography of skin biopsies from ear skin of negative litter mates (norm) and K14-HPV16 transgenic mice with hyperplastic (hyp), dysplastic (dys) and Grade II SCCs. Arrows indicate location of enzymatic activity corresponding to pro- and active forms of MMP-9 and MMP-2. Molecular weight markers (kDa) are shown at left.

Figure 2. HPV16/MMP-9 –/– Mice Develop Fewer, but More Malignant, SCCs

(A) Percentage of SCC-free transgenic mice in three cohorts: HPV16/MMP-9 +/+, n = 133 (FVB/n N14-N20; yellow triangles), HPV16/MMP-9 +/–, n = 76 (FVB/n N4; pink squares), and HPV16/MMP-9 –/–, n = 137 (FVB/n N4; blue diamonds). Both HPV16/MMP-9 +/+ and HPV16/MMP-9 +/– mice exhibit an overall SCC incidence of ~50% (50% and 47% respectively). In contrast, the incidence of SCCs in HPV16/MMP-9 –/– mice was reduced, to ~25% (Fisher's Exact Test, p = 0.0004). (B) Immunostaining of intermediate filaments (brown staining) reveals degree of keratinocyte differentiation retained in various grades of SCC. Well-differentiated Grade I SCCs exhibit hallmark keratin pearl structures (asterisks) and express the suprabasal keratin K10 while not expressing the simple keratin K8. Grade II SCCs appear less differentiated and express both K10 and K8. In contrast, poorly differentiated Grade III SCCs lose terminal differentiation capacity, do not express K10, while maintaining expression of K8. Grade IV SCCs attain a spindle cell morphology, exhibit limited K10 expression and primarily express the mesenchymal intermediate filament protein, vimentin. Bar: 50 μm. (C) A shift in tumor grade in MMP-9 –/– mice. SCCs were collected from the three cohorts of mice shown in panel (A) (HPV16/MMP-9 +/+, 133 mice, 67 mice with invasive SCCs, 80 SCCs total; HPV16/MMP-9 +/–, 76 mice, 35 mice with invasive SCCs, 43 SCCs total; HPV16/MMP-9 –/–, 137 mice, 37 mice with invasive SCCs, 42 SCCs total) and graded based on the criteria in panel (B). Both MMP-9-sufficient/HPV16 (+/+ and +/–) cohorts exhibited a similar spectrum of SCC grades, with no incidence of Grade IV SCCs. In contrast, MMP-9-deficient (–/–) transgenic mice exhibited an altered spectrum of SCCs biased toward less differentiated, more malignant cancers (p < 0.0001, Wilcoxon Score for Variable Grade).

Figure 3. Reduced Epithelial Hyperproliferation at All Stages of Carcinogenesis in HPV16/MMP-9 –/– Mice

Percentage of bromodeoxyuridine (BrdU)-positive keratinocytes in age-matched normal FVB/n negative littermates (-LM; [1], MMP-9 –/– [1]), and HPV16/MMP-9 +/– and HPV16/MMP-9 –/– mice at 1 and 2 months [2], 3 and 4 months [3], greater than 5 months of age [4], and from Grade II SCCs [5]. BrdU-positive keratinocytes were counted from a minimum of five high power (40×) fields from five mice per age group. Results are shown as mean percentages, ± standard error of the mean. P values (unpaired, nonparametric Mann-Whitney) for the respective groups are 0.66 [1], 0.11 [2], 0.73 [3], 0.03 [4], and 0.06 [5], respectively.

Figure 4. MMP-9 in Neoplastic Tissue Is Present in Reactive Stromal Cells

(A–C) In situ hybridization analysis of neoplastic tissue from normal and HPV16 transgenic skin for expression of MMP-9 mRNA. Dark-field photographs of ear-tissue sections from normal nontransgenic ear skin (A), HPV16 dysplastic ear skin (B), and a Grade II SCC (C). The epidermal (e), dermal (d), cartilagenous (c), malignant tumor keratinocytes (t), and tumor stroma (s) regions of the tissues are indicated. The epithelial basement membrane is marked with a dashed line. Bar: 50 μm (A); 100 μm (B and C). (D–I) Immunolocalization of MMP-9 (brown staining) in normal and HPV16 transgenic skin sections counterstained with methyl green from normal nontransgenic ear (D), dysplastic ear skin (E), and a Grade II SCC (F). High power microscopy reveals presence of MMP-9 in mast cell granules in dysplasias (G, arrows), consistent with our previous observations (Coussens et al., 1999). In addition, MMP-9 is found in neutrophils with characteristic bilobed nuclei in both dysplasias and carcinomas (H, arrows). In tumors, MMP-9 is also found in macrophages (I, arrow) with characteristic membranous folds and finger-like projections, as well as in the ECM (F, arrowheads). The epidermal (e), dermal (d), cartilagenous (c), malignant tumor keratinocytes (t), tumor stroma (s), and capillaries (*) are indicated. The epithelial basement membrane is marked with a dashed line. Bar: 50 μm (D); 75 μm (E); 20 μm (F); 10 μm (G); 7 μm (H, I).

Figure 5. Restoration of MMP-9 Activity in MMP-9-Deficient/HPV16 Mice by Bone Marrow Transplantation

(A) Scheme of bone marrow transplantation (BMT) protocol where 1-month-old mice (HPV16/MMP-9 +/–, HPV16/MMP-9 –/–) were lethally irradiated (7.5 Gy) and transplanted with BM-d cells (1 × 10⁶) from nontransgenic donor mice (MMP-9 +/+ or MMP-9 –/–). Each cohort contained a minimum of ten animals. Ear biopsies were taken at 8 and 16 weeks post-BMT when animals were 3 and 5 months of age. All animals were studied either until tumors appeared, or animals developed health-compromising phenotypes, or until 12 months of age. (B) Gelatin-substrate zymography of BM-d cells (1 × 10⁶) from MMP-9 +/+ (+/+, lane 1) and MMP-9 –/– (–/–, lane 2) donor mice. An arrow indicates enzymatic activity corresponding to proMMP-9. (C) Gelatin-substrate zymography of tissue biopsies from HPV16/MMP-9 –/– mice reconstituted with either wild-type (+/+) or MMP-9-deficient (–/–) BM-d cells (1 × 10⁶) at 8 and 16 weeks post-BMT. Presence of enzymatic activity corresponding to pro- and active MMP-9, pro- and active MMP-2, as well as an uncharacterized gelatinase are indicated. Molecular weight markers (kDa) are shown at right.

Figure 6. MMP-9 from Bone Marrow-Derived Cells Is Sufficient to Restore Wild-Type Neoplastic Phenotype in HPV16/MMP-9 –/– Mice

(A) Percentage of PCNA-positive keratinocytes present in Grade I and/or Grade II SCCs from nonirradiated, nontransplanted, HPV16/MMP-9 +/+ and –/– mice, and from HPV16/MMP-9 +/– and –/– mice that were lethally irradiated and transplanted with either wild-type (+/+) or MMP-9-deficient (–/–) BM-d cells. PCNA-positive keratinocytes were counted from a minimum of five high power (40×) fields/tumor analyzed. Results shown are derived from 5 SCCs each from nonirradiated, nontransplanted HPV16/MMP-9 +/– [46.6% ± 1.9] and –/– [32.3% ± 2.3] mice, 5 SCCs from irradiated/transplanted HPV16/MMP-9 +/–, BMT +/+ [45.6% ± 5.0] and HPV16/MMP-9 –/–, BMT –/– [45.9% ± 4.6] mice, and from the 1 SCC-bearing HPV16/MMP-9 –/–, BMT –/– [29.9% ± 5.1] mouse (p < 0.05, ANOVA [one-way analysis of variance]). Results are shown as mean percentages, ± standard error of the mean. (B) Keratinocyte proliferation, as measured by immunoreactivity for proliferating cell nuclear antigen (PCNA), in Grade II SCCs from four cohorts of mice: (a) HPV16/MMP-9 +/– (b) HPV16/MMP-9 –/–, (c) HPV16/MMP-9 –/–, irradiated and transplanted with MMP-9 +/+ BM-d cells, and (d) HPV16/MMP-9 –/–, irradiated and transplanted with MMP-9 –/– BM-d cells. Note in (a) that proliferation is heterogeneous throughout the malignant clusters, whereas in (b), proliferation is restricted to within 3–4 cell layers proximal to stroma (s). Transplantation of MMP-9 +/+ BM-d cells restores the characteristic pattern of proliferation to keratinocytes (c). Bar: 100 μm (a–d). (C) Incidence of SCCs in animals from BMT study (HPV16/MMP-9 +/–, BMT +/+,n = 10 mice, 7 with SCCs; HPV16/MMP-9 –/–, BMT +/+, n = 10 mice, 5 with SCCs; HPV16/MMP-9 –/–, BMT –/–, n = 11 mice, 1 with an SCC) was determined in comparison to nonirradiated, nontransplanted transgenic animals (HPV16/MMP-9 +/+, n = 133, 67 mice with SCCs; HPV16/MMP-9 –/–, n = 137 mice, 37 with SCC).