Insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) has been shown to induce cellular senescence or apoptosis of breast and prostate cancer cell lines in vitro. To examine whether IGFBP-rP1 acts as a tumor-suppressive protein in vivo, we established two model systems. Expression of IGFBP-rP1 in the human bladder carcinoma cell line EJ-1 was blocked by RNA interference. Human colon cancer cell line DLD-1, which did not express endogenous IGFBP-rP1, was transfected with an IGFBP-rP1 expression vector. When injected intraperitoneally or subcutaneously into nude mice, the IGFBP-rP1-expressing EJ-1 and DLD-1 cell lines grew poorly, whereas the IGFBP-rP1 non-producers grew rapidly and produced large tumors. In monolayer culture the IGFBP-rP1 producers and nonproducers grew similarly in each model, whereas in soft agar culture the former produced far less colonies than the latter. The IGFBP-rP1 producers had IGFBP-rP1 bound to the cell surface, and adhered more efficiently to fibronectin and laminin-5 than the respective non-producers. Expression of IGFBP-rP1 did not affect the efficiency of insulin signaling. These results demonstrate that IGFBP-rP1 strongly suppresses tumor growth by an insulin-independent or insulin-like growth factor-independent mechanism. Cell surface IGFBP-rP1 may reduce the anchorage-independent growth ability, leading to the marked loss of tumorigenicity. (Cancer Sci 2007; 98: 1055-1063)
Objective: Explore the mechanism of CaSR's involvement in bone metastasis in lung adenocarcinoma. Methods: Immunohistochemistry (IHC) was used to detect the expression of calcium-sensing receptor (CaSR) in 120 cases of lung adenocarcinoma with bone metastasis. Stably transfected cell lines with CaSR overexpression and knockdown based on A549 cells were constructed. The expression of CaSR was verified by western blot and qPCR. The proliferation and migration abilities of A549 cells were tested using cholecystokinin-8 (CCK-8) and Transwell assays, respectively. Western blotting was used to detect the expression of matrix metalloproteinases MMP2, MMP9, CaSR, and NF-κB. The supernatant from each cell culture group was collected as a conditional co-culture solution to study the induction of osteoclast precursor cells and osteoblasts. Western blot and qPCR were used to validate the expression of bone matrix degradation-related enzymes cathepsin K and hormone calcitonin receptor (CTR) and osteoblast-induced osteoclast maturation and differentiation enzyme receptor activator of nuclear factor-κB ligand (RANKL), macrophage colony-stimulating factor (M-CSF), osteoprotegerin (OPG), and PTHrP. Immunofluorescent staining was used to detect F-actin ring formation and osteocalcin expression. Western blot results for NF-κB expression identified a regulatory relationship between NF-κB and CaSR. Results: CaSR expression in lung cancer tissues was significantly higher than that in adjacent and normal lung tissues. The expression of CaSR in lung cancer tissues with bone metastasis was higher than that in non-metastatic lung cancer tissues. The proliferation and migration ability of A549 cells increased significantly with overexpressed CaSR. The co-culture solution directly induced osteoclast precursor cells and the expression of bone matrix degradation-related enzymes significantly increased. Osteoblasts were significantly inhibited and osteoblast-induced osteoclast maturation and differentiation enzymes were significantly downregulated. It was found that the expression of NF-κB and PTHrP increased when CaSR was overexpressed. Osteoclast differentiation factor expression was also significantly increased, which directly induces osteoclast differentiation and maturation. These results were reversed when CaSR was knocked down. Liu et al. CaSR Osteoclast Differentiation Bone Metastasis Conclusions: CaSR can positively regulate NF-κB and PTHrP expression in A549 cells with a high metastatic potential, thereby promoting osteoclast differentiation and maturation, and facilitating the occurrence and development of bone metastasis in lung adenocarcinoma.
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