Notch3 promotes prostate cancer-induced bone lesion development via MMP-3

Abstract

Prostate cancer metastases primarily localize in the bone where they induce a unique osteoblastic response. Elevated Notch activity is associated with high-grade disease and metastasis. To address how Notch affects prostate cancer bone lesions, we manipulated Notch expression in mouse tibia xenografts and monitored tumor growth, lesion phenotype, and the bone microenvironment. Prostate cancer cell lines that induce mixed osteoblastic lesions in bone expressed 5-6 times more Notch3, than tumor cells that produce osteolytic lesions. Expression of active Notch3 (NICD3) in osteolytic tumors reduced osteolytic lesion area and enhanced osteoblastogenesis, while loss of Notch3 in osteoblastic tumors enhanced osteolytic lesion area and decreased osteoblastogensis. This was accompanied by a respective decrease and increase in the number of active osteoclasts and osteoblasts at the tumor-bone interface, without any effect on tumor proliferation. Conditioned medium from NICD3-expressing cells enhanced osteoblast differentiation and proliferation in vitro, while simultaneously inhibiting osteoclastogenesis. MMP-3 was specifically elevated and secreted by NICD3-expressing tumors, and inhibition of MMP-3 rescued the NICD3-induced osteoblastic phenotypes. Clinical osteoblastic bone metastasis samples had higher levels of Notch3 and MMP-3 compared with patient matched visceral metastases or osteolytic metastasis samples. We identified a Notch3-MMP-3 axis in human prostate cancer bone metastases that contributes to osteoblastic lesion formation by blocking osteoclast differentiation, while also contributing to osteoblastogenesis. These studies define a new role for Notch3 in manipulating the tumor microenvironment in bone metastases.

Figure 1.. NICD3 inhibits osteolytic lesions.

Levels of (A) Notch1 and (B) Notch3 mRNA in tumor cell lines. Log₂-fold change relative to PC3 cells. Levels of (C) Notch1 and (D) Notch3 measured by immunoblotting. Tubulin is loading control. (E,G) PC3 cells harboring Tet-inducible (E) NICD3 or (G) Notch3 shRNA (shN3) injected into tibiae of mice treated with sucrose (Suc) or doxycycline (Dox). X-rayed lytic lesion area quantified. (F,H) Tibiae from (E) and (G) were stained with H&E (top, bottom panel of F), anti-Notch3 (2nd panel), TRAP (3rd panel), and number of osteoclasts and osteoblasts (OB) quantified. T=tumor; B=bone; arrows indicate examples of an osteoclast or osteoblast. Error bars are S.E.M, n≥11; *0.01≤p≤0.05; **0.001≤p<0.01; ***p<0.001.

Figure 2.. Inhibiting Notch3 promotes osteolytic lesions.

(A-B) 22Rv1 or (C-F) C4–2B cells harboring doxycycline-induced Notch3 shRNA (shN3) injected into tibiae of mice treated with sucrose (Suc) or doxycycline (Dox). (A, C) X-rayed lytic lesion area quantified. (B, D) Tibiae were stained with H& E (top, bottom panel of D), anti-Notch3 (2nd panel), TRAP (3rd panel). (E) Number of osteoclasts quantified. (F) Number of osteoblasts (OB) quantified. T=tumor; B=bone; arrows indicate examples of an osteoclast or osteoblast. Error bars are S.E.M, n≥7; *0.01≤p≤0.05.

Figure 3.. Notch3 promotes the expression of MMP-3.

(A) Tibiae from mice injected with PC3-NICD3, PC3-shN3, or C4–2B-shN3 cells treated with sucrose (Suc) or doxycycline (Dox) were stained with human-specific MMP-3 antibody. T=tumor (B-D) Levels of human-specific MMP-3 mRNA from tibiae in (A) assessed by qRT-PCR. Expressed Log₂ fold relative to sucrose controls. (E) Relative levels of MMP-3 in conditioned medium (CM) from PC3-NICD3 cells treated with vehicle (Ctrl) or doxycycline (Dox) assessed by ELISA. Error bars are S.E.M, n≥4; *0.01≤p≤0.05.

Figure 4.. NICD3 inhibits osteoclastogenesis and promotes osteoblastogenesis.

(A-C) Expression of mouse-specific mRNA isolated in vivo from tibiae of mice injected with PC3-NICD3 cells and treated with sucrose (Suc) or doxycycline (Dox): (A) IL-10, (B) OPG/RANKL ratio, and (C) bone sialoprotein (BSP), alkaline phosphatase (ALP), or osteocalcin (OCN). Expressed Log₂ fold relative to sucrose controls. (D-H) Bone marrow-derived osteoblasts differentiated in the presence of conditioned medium (CM) from PC3-NICD3 cells treated with doxycycline (Dox) or vehicle (Ctrl). (D) Colonies were stained for ALP or crystal violet (CV). (E) Colonies were stained for Alizarin red or crystal violet (CV). (F) Expression of mouse-specific mRNA from treated osteoblast cultures: osterix (OX), bone sialoprotein (BSP), or osteocalcin (OCN). Expressed Log₂ fold relative to vehicle controls. (G) MTT assay of treated osteoblast cultures. (H) Levels of Cyclin A, D, E and tubulin (Tub) from treated osteoblast cultures assessed by immunoblotting. (I-J) Bone marrow-derived osteoclasts differentiated in the presence of conditioned medium (CM) from doxycycline (Dox) or vehicle-treated (Ctrl) PC3-NICD3 cells. (I) TRAP+ cells with 2 ≥ nuclei quantified. (J) Expression of mouse-specific mRNA from osteoclast cultures: MMP9, cathepsin K (CathK), calcitonin receptor (CR), or DC-Stamp. (K-M) Bone marrow-derived osteoclasts differentiated in the presence of conditioned medium (CM) from (K) 22Rv1-shN3 (Notch3 shRNA), (L) C4–2B-shN3, and (M) C4–2B-NICD3 expressing cells. Percentage of TRAP+ cells quantified. Error bars are S.E.M, n=3; *0.01≤p≤0.05; **0.001≤p<0.01; ***p<0.001.

Figure 5.. Notch3 promotes osteoblastic lesion development in an MMP-3-dependent manner.

(A) Bone marrow-derived osteoclasts differentiated in the presence of 25 ng/ml recombinant human MMP-3 (rMMP3). Percentage of TRAP+ cells with 2 ≥ nuclei quantified. (B-C) Bone marrow-derived osteoblasts differentiated in the presence of recombinant MMP-3 (rMMP3) and (B) immunostained for ALP or (C) proliferation measured by MTT assay. (D-F) Tet-inducible PC3-NICD3 cells engineered to stably express MMP-3 shRNA (PC3-NICD3-shMMP3). (D) Bone marrow-derived osteoclasts or (E) osteoblasts differentiated in the presence of conditioned medium (CM) from doxycycline-treated (Dox) or vehicle-treated (Ctrl) PC3-NICD3-vector or PC3-NICD3-shMMP3 cells. (D) Percentage of TRAP+ cells quantified. (E) MTT assay of treated osteoblasts. (F) Tet-inducible PC3-NICD3-vector and PC3-NICD3-shMMP3 injected into tibiae of mice treated with doxycycline (Dox) or sucrose (Suc). Lytic lesion area on X-ray quantified. (G) Tibial tumors from (F) immunostained (IHC) with Notch3 or MMP-3 antibodies. Error bars are S.E.M, n≥8; *0.01≤ p≤0.05; **0.001≤p<0.01.

Figure 6.. Notch3 and MMP-3 expression is elevated in bone metastases.

Tissue microarrays (A) TMA-170 and (B) UWTMA79 probed for Notch3 and MMP-3 expression by IHC. Levels of expression were compared between patient-matched visceral and bone metastases. (C) Frequency of Notch3 and MMP-3 co-expression across all samples. (D) UWTMA79 bone metastases were assessed for lytic (n=18) versus osteoblastic (n=42) lesions. Association of elevated Notch3 or MMP-3 levels with lesion type was assessed. Error bars are S.E.M; *0.01≤ p≤0.05.