Thrombin cleavage of osteopontin initiates osteopontin's tumor-promoting activity

Abstract

Background: Osteopontin (OPN) is a multifunctional proinflammatory matricellular protein overexpressed in multiple human cancers and associated with tumor progression and metastases. Thrombin cleavage of OPN reveals a cryptic binding site for α₄ β₁ and α₉ β₁ integrins.

Methods: Thrombin cleavage-resistant OPN_R153A knock-in (OPN-KI) mice were generated and compared to OPN deficient mice (OPN-KO) and wild type (WT) mice in their ability to support growth of melanoma cells. Flow cytometry was used to analyze tumor infiltrating leukocytes.

Results: OPN-KI mice engineered with a thrombin cleavage-resistant OPN had reduced B16 melanoma growth and fewer pulmonary metastases than WT mice. The tumor suppression phenotype of the OPN-KI mouse was identical to that observed in OPN-KO mice and was replicated in WT mice by pharmacologic inhibition of thrombin with dabigatran. Tumors isolated from OPN-KI mice had increased tumor-associated macrophages with an altered activation phenotype. Immunodeficient OPN-KI mice (NOG-OPN-KI) or macrophage-depleted OPN-KI mice did not exhibit the tumor suppression phenotype. As B16 cells do not express OPN, thrombin-cleaved fragments of host OPN suppress host antitumor immune response by functionally modulating the tumor-associated macrophages. YUMM3.1 cells, which express OPN, showed less tumor suppression in the OPN-KI and OPN-KO mice than B16 cells, but its growth was suppressed by dabigatran similar to B16 cells.

Conclusions: Thrombin cleavage of OPN, derived from the host and the tumor, initiates OPN's tumor-promoting activity in vivo.

Keywords: B16 melanoma; metastasis; osteopontin; thrombin inhibitor; tumor-associated macrophages.

Figure 1:. Suppression of B16 tumor growth and metastasis in OPN-KO and OPN-KI mice.

A. Model of OPN cleavages and its functional modulation by thrombin and carboxypeptidase N or B2. OPN-FL=OPN-full length; OPN-R=OPN-Arg; OPN-L=OPN-Leu. DC=dendritic cells. B. Time course (min) of thrombin treatment (10 nM) of E. coli produced GST-OPN-FL and GST-OPN-FL_R153A analyzed by SDS-PAGE. FL=full length, NTF=N-terminal fragment, CTF=C-terminal fragment. C. Daily determination of B16 tumor volume grown on WT (blue), OPN-KI (green) and OPN-KO (red) mice. ⁺⁺: p<0.01 WT vs. OPN-KI, ⁺⁺⁺: p<0.001 WT vs. OPN-KI, ⁺⁺⁺: p<0.0001 WT vs. OPN-KI, *: p<0.05 WT vs. OPN-KO, **: p<0.01 WT vs. OPN-KO, ****: p<0.0001 WT vs. OPN-KO. D. B16 tumor weight after sacrifice. *: p<0.05 WT vs. OPN-KI, **: p<0.01 WT vs. OPN-KO. E. Lungs from mice sacrificed 15 days after tumor injection i.v. F. Number of visible metastatic lung nodules in mice sacrificed 13 days after tumor injection i.v. G. Melanin content of lungs in mice sacrificed 13 days after tumor injection i.v. . All data are shown as mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey’s multiple comparison test. **: p<0.01 WT vs. OPN-KI or OPN-KO, ****: p<0.0001 WT vs. OPN-KI or OPN-KO.

Figure 2:. Inhibition of thrombin in WT phenocopies the OPN-KI phenotype.

A. Daily determination of B16 tumor growth implanted on the flank of WT mice either fed on DE-containing chow (WT (DE) open circles) or control chow (WT (Control) closed circles). B. B16 tumor weights after sacrifice in OPN-KI mice either on DE-containing chow (OPN-KI (DE) open triangles) or control chow (OPN-KI (Control) closed triangles). C. B16 tumor volumes after sacrifice. D. B16 tumor weights after sacrifice. E. aPTT determined from blood collected at the time of sacrifice. In A – E, data are shown as mean ± SEM. Statistical significance was calculated by Student’s t-test comparing each genotype with and without DE in the chow. *: p<0.05 Control vs. DE, **: p<0.01 Control vs. DE, ****: p<0.0001 Control vs. DE F. Correlation of aPTT prolongation and tumor weights in WT mice treated with DE. G. Number of visible nodules counted in the lungs of mice sacrificed 13 days after inoculation in WT, OPN-KI and OPN-KO mice fed either DE or control chow in the metastasis model. H. Melanin content of lungs at 13 days after inoculation. In G and H, data are shown as mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey’s multiple comparison test. *: p<0.05 WT vs. any other group. I. Weight of YUMM3.1 tumors sacrificed 23 days after inoculation in WT, OPN-KI and OPN-KO mice fed either DE or control chow in the flank model. Statistical significance was calculated by Student’s t-test comparing each genotype with and without DE in the chow. **: p<0.01 Control vs. DE.

Figure 3:. Different OPN cleavage products produce different responses from B16 cells.

The effects of OPN fragments on B16 cell adhesion (A), growth (B), migration (C and apoptosis (D) were determined. Data are shown as mean ± SEM. Statistical significance was calculated by one-way ANOVA comparing all OPN fragments to albumin (negative control) followed by Dunnett’s multiple comparison test. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.001. OPN-FL=OPN full length, OPN-R=OPN-Arg, OPN-L=OPN-Leu, OPN-CTF=OPN-C-terminal fragment. Subscript RAA denotes substitution of the RGD sequence by RAA in that OPN fragment.

Figure 4:. B16 growth in immune-deficient mice is not affected by OPN status.

A. Daily determination of B16 tumor volume grown on NOG-WT (blue circles), NOG-OPN-KI (green triangles) and NOG-OPN-KO (red squares) mice in the flank model. B. B16 tumor weights after sacrifice in NOG-WT, NOG-OPN-KI and NOG-OPN-KO mice in the flank model. C. Number of visible metastatic lung nodules counted in the lungs of mice sacrificed 13 days after tumor injection i.v. Dotted line represents maximum number of nodules that could be counted. D. Melanin content of lungs. All data are shown as mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey’s multiple comparison test.

Figure 5:. B16 tumors from OPN-KI and OPN-KO mice contain more macrophages.

A.H&E staining of tumor sections from WT, OPN-KI and OPN-KO mice. B. Tumor sections from WT, OPN-KI and OPN-KO mice stained with anti-F4/80 antibody. C. Tumor sections from WT, OPN-KI and OPN-KO mice stained with anti-CD3 antibody. Scale bars = 100nm. D. The percentage of F4/80⁺ cells present in tumors from OPN-KI, OPN-KO and WT mice determined by flow cytometry. All data are shown as mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey’s multiple comparison test. **: p<0.01 WT vs. OPN-KI.

Figure 6:. Changes in macrophages in tumors from OPN-KI and OPN-KO mice compared to WT mice.

Infiltrating macrophages (A), M1 macrophages (B), M2 macrophages (C and D), Macrophages with new phenotype (E), neutrophils (F), B-cells (G) and T-cells (H) were determined by flow cytometry in tumor samples from the flank model in WT, OPN-KI and OPN-KO mice. All data are shown as mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey’s multiple comparison test. The experiment (n=5) shown is representative of two independent experiments. Model of thrombin +/− CPN/CPB2 cleavage(s) of OPN and their effect on macrophages, tumor growth and metastasis. (I). Cleaved OPN fragments affect the infiltration and composition of macrophages in the tumor maintaining tumor-promoting M2 macrophages (green cells) in WT mice. These OPN fragments are absent in either OPN KO or thrombin-resistant OPN-KI mice, leading to decrease in M2 macrophages and replacement by macrophages with a different activation phenotype (red cells) leading to tumor suppression.

Figure 7:. Depletion of macrophages by clodronate reverses the B16 tumor suppression phenotype in OPN-KI mice.

A. WT mice were treated with either clodronate (open circles) or control (closed circles) liposomes and the tumor volumes measured daily. B. OPN-KI mice were treated with either clodronate (open triangles) or control (closed triangles) liposomes and the tumor volumes measured daily. C. WT or OPN-KI mice were treated with either clodronate or control liposomes and the tumor volumes measured after sacrifice. D. WT or OPN-KI mice treated with either clodronate or control liposomes and the tumor weights measured after sacrifice. The percentage of F4/80⁺ cells was determined in the bone marrow (E), blood (F) and tumors (G) from WT or OPN-KI mice treated with either clodronate or control liposomes 14 days after inoculation with B16 cells. All data are shown as mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey’s multiple comparison test. *: p<0.05 control vs. clodronate, **: p<0.01 p<0.05 control vs. clodronate, ***: p<0.001 p<0.05 control vs. clodronate, ****: p<0.001 p<0.05 control vs. clodronate. H. Production of PGE₂ by RAW cells in response to OPN fragments (BSA control subtracted). Data are shown as mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey’s post-hoc test. **: p<0.01 vs. OPN-R, ***: p<0.001 vs. OPN-R, ****: p<0.001 vs. OPN-R, #: p<0.05 vs. OPN-R_RAA. Subscript RAA denotes substitution of the RGD sequence by RAA in that OPN fragment.