CaSR Induces Osteoclast Differentiation and Promotes Bone Metastasis in Lung Adenocarcinoma

Abstract

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.

Predictive and Prognostic Biomarkers for Lung Cancer Bone Metastasis and Their Therapeutic Value

“…Its expression is low or absent in normal lung tissue, whereas it is significantly higher in lung cancer tissue (173). Moreover, Lian et al (174) demonstrated that the expression of CASR in 120 cancer tissues with bone metastasis was significantly higher than that recorded in non-metastatic lung cancer tissues. Another study found that the increase of CASR was the result of PTHrP and NF-kB upregulation, which was also positively correlated with bone metastasis in lung cancer (173,175,176).…”

The pathogenesis of bone metastasis in solid tumors: a review

“…(73). In addition to growth factors, tumor cells respond to calcium ions (via CaSR) released in the process of bone resorption (74).…”

“…It stimulates osteoclasts directly and indirectly via osteoblasts ( 73 ). In addition to growth factors, tumor cells respond to calcium ions (via CaSR) released in the process of bone resorption ( 74 ).…”

Signaling Pathways in Osteoclast-driven Bone Metastasis: A mini-review

“…In the context of bone diseases, OCs are thought to be involved in several bone disorders, such as osteoporosis, Paget's disease, rheumatoid arthritis, and especially cancer metastasis [12]. Over few decades, there have been lots of considerable efforts on elucidation of the regulatory roles of OCs in metastatic diseases that originate from multiple myeloma, prostate cancer, breast cancer, and/or lung cancer [13,14]. In fact, many animal and clinical researches have indicated that OCs play a central role in strengthening the occurrence and progression of bone metastasis through a mechanism underlying the release of factors such as interleukin (IL)-1 and transforming growth factor-beta (TGF-β) that stimulate proliferation, differentiation and growth of metastatic cancer cells.…”