Tumorigenic potential of extracellular matrix metalloproteinase inducer

Abstract

Extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein present on the cancer cell plasma membrane, enhances fibroblast synthesis of matrix metalloproteinases (MMPs). The demonstration that peritumoral fibroblasts synthesize most of the MMPs in human tumors rather than the cancer cells themselves has ignited interest in the role of EMMPRIN in tumor dissemination. In this report we have demonstrated a role for EMMPRIN in cancer progression. Human MDA-MB-436 breast cancer cells, which are tumorigenic but slow growing in vivo, were transfected with EMMPRIN cDNA and injected orthotopically into mammary tissue of female NCr nu/nu mice. Green fluorescent protein was used to visualize metastases. In three experiments, breast cancer cell clones transfected with EMMPRIN cDNA were considerably more tumorigenic and invasive than plasmid-transfected cancer cells. Increased gelatinase A and gelatinase B expression (demonstrated by in situ hybridization and gelatin substrate zymography) was demonstrated in EMMPRIN-enhanced tumors. In contrast to de novo breast cancers in humans, human tumors transplanted into mice elicited minimal stromal or inflammatory cell reactions. Based on these experimental studies and our previous demonstration that EMMPRIN is prominently displayed in human cancer tissue, we propose that EMMPRIN plays an important role in cancer progression by increasing synthesis of MMPs.

Figure 1.

A: Schematic illustration of the EMMPRIN/GFP plasmid. A 1.6-kb cDNA representing the entire EMMPRIN sequence was placed at an EcoR 1 site under the control of the CMV promoter in pcDNA 3. GFP cDNA was inserted along with an upstream CMV promoter into the EMMPRIN expression vector between NotI and XhoI sites. A polyadenylation (PA) signal was placed downstream. B: Northern blot analysis of EMMPRIN. Approximately 20 μg of total cellular RNA from plasmid alone-transfected, GFP-transfected, and EMMPRIN/GFP-transfected MDA-MB-436 breast cancer cells (from experiment 3) was size fractionated in a 1% denaturing agarose gel, transferred to a nylon membrane, and incubated with 1.7 kb of ³²P-radiolabeled EMMPRIN cDNA as a probe. Blots were analyzed by autoradiography. A single 1.7-kb mRNA transcript corresponding to the known EMMPRIN band was detected at ∼20× greater intensity in EMMPRIN/GFP-transfected cells as compared to plasmid alone or GFP-transfected cells.

Figure 2.

A: MDA-MB-436 breast cancer cells transfected with EMMPRIN/GFP cDNA resulted in enhanced rate of tumor growth after tumor cell implantation into the mammary fat pad of nude mice as compared to GFP-transfected cells. The tumorigenicity of transfected cells was assessed by weekly measurement of tumor size. The data represent the mean ± SE observed in 10 animals in each group injected with 1 × 10 transfected cancer cells. The numbers associated with each symbol refer to the number of mice alive at each time point (ie, at week 8, three mice in the EMMPRIN/GFP group had large tumors and were sacrificed, hence the number seven is listed). B: GFP-transfected tumors are readily visible under fluorescent light. EMMPRIN/GFP-transfected MDA-MB-436 breast cancer cells were injected into the mammary tissue of a female NCr nu/nu mouse. Eight weeks later, the mouse was sacrificed and extensive green colored metastatic tumors in the peritoneum, liver, spleen, and mediastinum were visible under fluorescent light (left photo). The photo on the right demonstrates the same tumors visualized by bright light (arrowheads identify tumors). C: Comparison of gelatinases secreted by MDA-MB-436 cells cultivated in serum-free medium and extracts of nude mouse tumors. Spent serum-free conditioned medium from primary cells cultivated for 18 hours with vehicle (medium), PMA, and thrombin (left) and tumor cell extracts (right) were assessed by gelatin substrate zymography. Protein concentration (15 μg/well) of tissue samples was equalized within each group. Conditioned medium and tumor extracts from the EMMPRIN/GFP group displayed more gelatinolytic activity than did the GFP-alone group of mice. The displayed extract from the GFP-alone tumor is from the largest tumor (1.4 cm 3 ) in this group of mice. The intensity of tumor gelatinolytic activity demonstrated in each group of mice did not correlate with tumor size (data not shown). Molecular weights were calculated using protein standards. The conditioned medium of HT-1080 cells was used to confirm the molecular weight of human gelatinase A and gelatinase B (data not shown).

Figure 3.

In situ hybridization of primary tumors from mice injected with EMMPRIN/GFP and GFP-transfected MDA-MB-436 breast cancer cells. Serial sections from tumor tissue (panels 1–4, and 9–12) and surrounding nonmalignant tissue (panels 5–8 and 13–16) were examined. Panels 1 and 9 represent H&E staining of cancer tissues from EMMPRIN/GFP and GFP tumors, respectively; panels 5 and 13 represent H&E staining of non malignant mammary tissues (tumor cells not identified) adjacent to the primary EMMPRIN/GFP and GFP tumors, respectively. Cells in the primary tumor mass from mice injected with EMMPRIN/GFP-transfected cells revealed widely distributed, specific staining with EMMPRIN, gelatinase A (GLA), and gelatinase B (GLB) antisense riboprobes (panels 2–4, respectively). Minimal cell staining for EMMPRIN, gelatinase A, and gelatinase B was seen in cancer cells from GFP-transfected MDA-MB-436 cells (panels 10–12). Nonmalignant tissues adjacent to the primary tumors from EMMPRIN/GFP mice demonstrated focal staining for EMMPRIN, gelatinase A, and gelatinase B in mammary ducts (D) and myocytes (M) (panels 6–8: arrows identify EMMPRIN-expressing cells). Nonmalignant tissue from GFP mice revealed no discernable staining for EMMPRIN, gelatinase A, or gelatinase B (panels 14–16). The insets in panels 1–4 display higher magnifications of mRNA-stained tumor cells; prominent nuclear staining is noted. Comparable cells (but minimally stained) in the GFP-transfected tumors are demonstrated in panels 10–12.