Matrix metalloproteinases contribute distinct roles in neuroendocrine prostate carcinogenesis, metastasis, and angiogenesis progression

Abstract

Prostate cancer is the leading form of cancer in men. Prostate tumors often contain neuroendocrine differentiation, which correlates with androgen-independent progression and poor prognosis. Matrix metalloproteinases (MMP), a family of enzymes that remodel the microenvironment, are associated with tumorigenesis and metastasis. To evaluate MMPs during metastatic prostatic neuroendocrine cancer development, we used transgenic mice expressing SV40 large T antigen in their prostatic neuroendocrine cells, under the control of transcriptional regulatory elements from the mouse cryptdin-2 gene (CR2-TAg). These mice have a stereotypical pattern of tumorigenesis and metastasis. MMP-2, MMP-7, and MMP-9 activities increased concurrently with the transition to invasive metastatic carcinoma, but they were expressed in different prostatic cell types: stromal, luminal epithelium, and macrophages, respectively. CR2-TAg mice treated with AG3340/Prinomastat, an MMP inhibitor that blocks activity of MMP-2, MMP-9, MMP-13, and MMP-14, had reduced tumor burden. CR2-TAg animals were crossed to mice homozygous for null alleles of MMP-2, MMP-7, or MMP-9 genes. At 24 weeks CR2-TAg; MMP-2(-/-) mice showed reduced tumor burden, prolonged survival, decreased lung metastasis, and decreased blood vessel density, whereas deficiencies in MMP-7 or MMP-9 did not influence tumor growth or survival. Mice deficient for MMP-7 had reduced endothelial area coverage and decreased vessel size, and mice lacking MMP-9 had increased numbers of invasive foci and increased perivascular invasion, as well as decreased tumor blood vessel size. Together, these results suggest distinct contributions by MMPs to the progression of aggressive prostate tumor and to helping tumors cleverly find alternative routes to malignant progression.

Figure 1. MMP activity and localization in normal prostate and CR2-TAg tumors

A, Detected fluorescence (pmol) following activation of MMPSense probe using tomography. B, Gelatin zymography of ventral and dorsolateral prostates, seminal vesicle, and liver collected from CR2-TAg transgenic mice and normal littermates at 16 and 24 weeks. Western blot analysis of MMP-7 activity in normal prostate and CR2-TAg tumorigenic prostate over time. Samples were collected at 9, 12, 16 and 24 weeks. Graphs represent the expression of MMP-7 normalized to actin staining. C, Immunohistochemistry of MMP-2 (a-c), MMP-7 (d-f), and MMP-9 (g-i) in CR2-TAg tumors at 24 weeks of age. Arrows, positively stained cells. The box in a is enlarged in b. D, Co-localization of MMP-2 (red) and F4/80 (green) (top panel), MMP-2 (green) and smooth muscle actin (SMA; red) (middle panel), and MMP-9 and F4/80 (green) (bottom panel) by immunofluorescence in CR2-TAg tumor stroma at 24 weeks. Scale bars, for C, 100 μm in a, b, d, e, g, and i; 40 μm in c, f, and h; for D, 50 μm.

Figure 2. Tumors in CR2-TAg mice deficient for MMPs

A, Left graph, CR2-TAg mice treated with MMP inhibitor AG3340 have decreased tumor weight (grams) compared to uninjected nontransgenic (wild type) mice or to wild type untreated mice. Shapes, prostate weight from one mouse. Lines, median prostate weight per cohort. Right graph, CR2-TAg;MMP-2^-/- mice have reduced tumor burden compared to wild type or to heterozygotes. B, H&E staining of representative tissue sections from CR2-TAg tumors at 24 weeks. AG3340 arrow, high grade PIN; Arrowheads, glands. Other arrows, neuroendocrine rosettes. C, H&Es of CR2-TAg;MMP-9^+/- (left) and CR2-TAg;MMP-9^-/- (right) at 16 weeks. Graph, Quantification of invasive foci as a percentage of total number of glands per section. D, Survival curve for CR2-TAg;MMP-2^-/- mice. MMP-2 deficient mice have delayed survival compared to wild type. Shapes, censored subjects.

Figure 3. Metastasis to lung and liver in MMP deficient backgrounds

A, Graphs of percentage of mice that have macro (solid black) or micro (stripes) metastases to the lung in CR2-TAg mice deficient for MMP-2, -7, and -9. B, Representative lung metastases in CR2-TAg mice with the indicated MMP deficiencies. C, Graphs of percentage of mice that have macro or micro metastases to the liver in CR2-TAg mice deficient for MMP-2, -7, and -9. D, Pictures of representative liver metastases in CR2-TAg mice with the indicated MMP deficiencies. Dotted lines, metastatic nodules within lung and liver tissue harvested from animals of the indicated genotype. Squares with dotted lines, regions enlarged in insets. Scale bars, 500 μm for MMP-2^+/+ and 100 μm for inserts.

Figure 4. Mice deficient for MMP-2 have decreased blood vessel density in tumor regions

Tissues from mice with the indicated genotypes were collected at 24 weeks and stained by immunofluorescence with markers of vasculature (CD31) and pericytes (SMA) and analyzed by morphometric analysis. A, Quantification of vessel density based on immunofluorescence staining using CD31 antibodies. MMP-2 deficient tumors have reduced vessel density (number of CD31⁺ vessels per 100,000 pixels). B, Quantification of mature vessel density (CD31⁺SMA⁺) based on immunofluorescence staining using CD31 and smooth muscle actin (SMA) antibodies. C, Immunofluorescence of vessels stained with antibodies to CD31 (green) and SMA (red) in tumors from CR2-TAg;MMP-2^+/+ (top row) and CR2-TAg;MMP-2^-/- (bottom row) prostate tumors. Arrows: CD31⁺SMA^- vessels. Scale bar: 100 μm.

Figure 5. CR2-Tag mice deficient for MMP-7 have decreased endothelial area and decreased vessel size in regions of the prostate with tumor

Tissues from mice with the indicated genotypes were collected at 24 weeks, stained by immunofluorescence with markers of endothelium (CD31) and pericytes (SMA), and scored within the tumor region by morphometric analysis. Shapes, one per mouse, Line, mean. A, Quantification of vessel density (CD31⁺ per 100,000 pixels). Quantification of mature vessel density (CD31⁺SMA⁺ per 100,000 pixels). Quantification of endothelial area in MMP-7 deficient mice. Quantification of average size of CD31⁺ vessels (pixels). B, Immunofluorescence of vessels stained with Hoechst (left two panels) and CD31 (right two panels) in tumors from CR2-TAg;MMP-7^+/- (upper two panels) or CR2-TAg;MMP-7^-/-. Arrows, CD31⁺ vessels.

Figure 6. Mice deficient for MMP-9 have increased perivascular invasion, increased stromal vasculature density, and decreased vessel size in tumor regions

A, Quantification of infiltrative and perivascular invasion at 24 weeks of age. Tumor cells escaped from the tumor into the stroma were scored as cells that were (perivascular) or were not (infiltrative) associated with the vasculature. Shapes, one per mouse. Line, mean. B, Immunofluorescence staining of tumor cells and blood vessels at tumor-stroma border at 24 weeks using SV40 TAg (red) and CD31 (green) antibodies, respectively. Arrows, tumor cells not associated with vessels (left image, CR2-TAg;MMP-9^+/-) or associated with vessels (right image, CR2-Tag;MMP-9^-/-). C, Quantification of average vessel size of CD31⁺ vessels at 24 weeks. Line, mean. MMP-9 deficient tumors have reduced vessel size. D, Immunofluorescence staining of tumor vasculature at 24 weeks using CD31 (green) and SMA (red) antibodies. Arrows, vasculature.