Cabozantinib-induced osteoblast secretome promotes survival and migration of metastatic prostate cancer cells in bone

Abstract

Therapies that target cancer cells may have unexpected effects on the tumor microenvironment that affects therapy outcomes or render therapy resistance. Prostate cancer (PCa) bone metastasis is uniquely associated with osteoblastic bone lesions and treatment with cabozantinib, a VEGFR-2 and MET inhibitor, leads to a reduction in number and/or intensity of lesions on bone scans. However, resistance to cabozantinib therapy inevitably occurs. We examined the effect of cabozantinib on osteoblast differentiation and secretion in the context of therapy resistance. We showed that primary mouse osteoblasts express VEGFR2 and MET and cabozantinib treatment decreased osteoblast proliferation but enhanced their differentiation. A genome-wide analysis of transcriptional responses of osteoblasts to cabozantinib identified a set of genes accounting for inhibition of proliferation and stimulation of differentiation, and a spectrum of secreted proteins induced by cabozantinib, including pappalysin, IGFBP2, WNT 16, and DKK1. We determined that these proteins were upregulated in the conditioned medium of cabozantinib-treated osteoblasts (CBZ-CM) compared to control CM. Treatment of C4-2B4 or PC3-mm2 PCa cells with CBZ-CM increased the anchorage-independent growth and migration of these PCa cells compared to cells treated with control CM. These results suggest that the effect of cabozantinib on the tumor microenvironment may increase tumor cell survival and cause therapy resistance.

Keywords: anchorage-independent growth; cabozantinib; migration; osteoblast; secretome.

Figure 1. Effect of cabozantinib on osteoblast proliferation and differentiation

(A) Real-time RT-PCR for the expression of osteocalcin, VEGFR2, and MET in the undifferentiated (D0) and differentiated (D24) osteoblasts; (B) Effects of cabozantinib on osteoblast proliferation measured by cell counting; (C) Effects of cabozantinib on osteoblast alkaline phosphatase activity during the time course of osteoblast differentiation; (D) Effects of cabozantinib on osteocalcin protein secretion measured by ELISA; and (E) Effects of cabozantinib on osteoblast mineralization measured by Alizarin Red S staining and (F) von Kossa staining. *, p<0.05, **, p<0.01, ***, p<0.001.

Figure 2. Pathways affected by cabozantinib treatments using ingenuity pathway analysis (IPA)

(A) Pathways that may be affected by genes up-regulated by cabozantinib; (B) Pathways that may be affected by genes down-regulated by cabozantinib; (C) (i) Cabozantinib regulated genes that are involved in “differentiation of osteoblastic-lineage cells” and “inhibition of osteoblast proliferation” based on IPA; (ii) fold of change of up-regulated genes and down-regulated genes related to inhibition of osteoblast differentiation. (iii) fold of change of up-regulated genes related to inhibition of osteoblast quantity.

Figure 3. Real-time RT-PCR for mRNA levels of cabozantinib regulated genes involved in “differentiation of osteoblastic-lineage cells” and “inhibition of osteoblast proliferation”

(A) Real-time RT-PCR for the mRNAs of genes involved in increase of osteoblast differentiation; (B) Real-time RT-PCR for the mRNAs of genes involved in the inhibition of osteoblast proliferation. *, p<0.05.

Figure 4. Secretory proteins upregulated by cabozantinib treatment

(A) Secretory proteins that may be involved in “prostate cancer” and “invasion of tumor cells” from IPA. (B) Real-time RT-PCR for the mRNA levels of several secretory proteins in the RNAs prepared from D24 osteoblasts with or without cabozantinib treatment. *, p<0.05, **, p<0.01.

Figure 5. Effects of cabozantinib on PAPPA and IGFBP2 protein expression

(A) Left panel, Western blots for PAPPA in the conditioned medium from osteoblasts at various time points of differentiation. Right panel, quantification of the intensity of PAPPA in the Western blots. (B) Left panel, Western blots for PAPPA in the conditioned medium of osteoblasts treated with or without cabozantinib. Right panel, quantification of the intensity of PAPPA in the Western blots showed significant increase of PAPPA by cabozantinib treatment compared to control. (C) Left panel, Western blots for the expression of IGFBP2 in the conditioned medium from osteoblasts at various time points of differentiation. IGFBP2 is a protein with an apparent molecular mass of around 30 kDa and it could be proteolyzed to 17 and 12 kDa fragments. Right panel, quantification of the intensity of total IGFBP2 in the Western blots showed IGFBP2 expression was increased after differentiation for 12 days and remained increased throughout the time course of differentiation. (D) Left panel, Western blots for IGFBP2 in the conditioned medium of osteoblasts treated with or without cabozantinib. Right panel, quantification of the intensity of total IGFBP2 in the Western blots showed significant increase of IGFBP2 by cabozantinib treatment compared to control.

Figure 6. Effect of cabozantinib on WNT16, DKK1, and OPG expression

ELISA of (A) WNT 16, (B) DKK1, (C) OPG in the conditioned medium from osteoblasts with or without cabozantinib treatments. *, p<0.05, **, p<0.01.

Figure 7. Effect of conditioned medium from cabozantinib-treated osteoblasts on anchorage-independent growth and migration of C4-2B4 or PC3-mm2 prostate cancer cells

(A) Soft agar colony assay showed CBZ CM significantly increase C4-2B4 colonies compared to control CM. (B) CBZ CM increased the migration of C4-2B4 cells compared to control CM in Transwell migration assay. (C) Diagram illustrates effects of cabozantinib on osteoblasts and cabozantinib-induced secreted factors may promote survival and migration of PCa cells. **, p<0.01.