Cigarette smoke promotes IL-6-dependent lung cancer migration and osteolytic bone metastasis

Abstract

Lung cancer stands as a major contributor to cancer-related fatalities globally, with cigarette smoke playing a pivotal role in its development and metastasis. Cigarette smoke is also recognized as a risk factor for bone loss disorders like osteoporosis. However, the association between cigarette smoke and another bone loss disorder, lung cancer osteolytic bone metastasis, remains largely uncertain. Our Gene Set Enrichment Analysis (GSEA) indicated that smokers among lung cancer patients exhibited higher expression levels of bone turnover gene sets. Both The Cancer Genome Atlas (TCGA) database and our clinic samples demonstrated elevated expression of the osteolytic factor IL-6 in ever-smokers with bone metastasis among lung cancer patients. Our cellular experiments revealed that benzo[α]pyrene (B[α]P) and cigarette smoke extract (CSE) promoted IL-6 production and cell migration in lung cancer. Activation of the PI3K, Akt, and NF-κB signaling pathways was involved in cigarette smoke-augmented IL-6-dependent migration. Additionally, cigarette smoke lung cancer-secreted IL-6 promoted osteoclast formation. Importantly, blocking IL-6 abolished cigarette smoke-facilitated lung cancer osteolytic bone metastasis in vivo. Our findings provide evidence that cigarette smoke is a risk factor for osteolytic bone metastasis. Thus, inhibiting IL-6 may be a valuable therapeutic strategy for managing osteolytic bone metastasis in lung cancer patients who smoke.

Keywords: Bone metastasis; Cigarette smoke; IL-6; Lung cancer; Osteoclast.

Figure 1

Association of IL-6 levels with bone metastases from lung cancer in smokers. (A) GSEA-based pathway analysis included bone remodeling, bone resorption, and bone formation gene sets linked with lung cancer in smokers and nonsmokers obtained from the BioCarta database. (B) Kaplan-Meier analysis determined levels of IL-6 (low expression n=215, high expression n=605) and RANKL (low expression n=310, high expression n=510) and overall survival rates of patients with lung cancer. (C) The IL-6 mRNA expression in indicated lung cancer cells was examined by qPCR. (D) The GEO and TCGA database revealed IL-6 expression in never-smoker and ever-smoker lung cancer samples, as well as in LUAD and LUSC tissue samples. (E) IHC staining of IL-6 in lung cancer patients with or without bone metastasis (n=7 in each group). *p < 0.05 versus the parental group.

Figure 2

Cigarette smoke promotes IL-6-dependent lung cancer migration. (A) The cell migration ability in indicated cells was examined. (B-D) Lung cancer cells were transfected with IL-6 shRNA, and IL-6 expression and cell migration were examined by ELISA, qPCR and migration assay. *p < 0.05 versus the parental group; #p < 0.05 versus the control-shRNA group.

Figure 3

PI3K and Akt pathways regulate cigarette smoke-induced IL-6 expression and cell migration in lung cancer. (A&B) IPA pathway enrichment figure showing pathways in the GSE31210 database that were markedly elevated. (C) The phosphorylation of p85 and Akt in indicated cells was examined by western blot. (D-I) Cells were pretreated with wortmannin and Akti or transfected with p85 and Akt siRNA, and IL-6 expression and cell migration were examined by ELISA, qPCR and migration assay. *p < 0.05 versus the parental group; #p < 0.05 versus the control-siRNA group.

Figure 4

NF-κB transactivation is involved in cigarette smoke-promoted IL-6-dependent cell migration in lung cancer. (A) The phosphorylation of p65 in indicated cells was examined by western blot. (B-G) Cells were pretreated with PDTC or transfected with p65 siRNA, and IL-6 expression and cell migration were examined. (H&I) Cells were pretreated with wortmannin and Akti or transfected with p85 and Akt siRNA, and NF-κB luciferase activity was examined. (J&K) The immunofluorescence staining of p65 in indicated cells was examined. *p < 0.05 versus the parental group; #p < 0.05 versus the control-siRNA group.

Figure 5

Cigarette smoke lung cancer-secreted IL-6 promotes osteoclast formation. (A-C) RAW 264.7 cells were treated with lung cancer CM with or without IL-6 antibody (1 µg/ml) for 7 days, the mature osteoclast was determined by TRAP staining. (D&E) qPCR and Western blot analysis results showing integrin β3, MMP9, CTSK, and DC-STAMP expression in RAW 264.7 cells treated with lung cancer CM for 7 days. *p < 0.05 versus the parental group; #p < 0.05 versus the A549^B[α]P or A549^CSE CM group.

Figure 6

Blocking IL-6 inhibits cigarette smoke-induced lung cancer metastasis to bone in vivo. (A&B) Representative bioluminescent images of bone metastasis at 8 weeks after caudal artery injections of indicated lung cancer cell lines into nude mice. (C) Mouse body weights were measured weekly after caudal artery. *p < 0.05 versus the parental group; #p < 0.05 versus the A549^B[α]P or A549^CSE group.

Figure 7

Blocking IL-6 abolishes cigarette smoke-promoted lung cancer osteolytic bone metastasis. (A-C) Representative bioluminescent and X-ray images of bone erosion in each study group. (D&E) Representative H&E and immunohistochemical images of IL-6 in bone specimens from mice. (F) The IL-6 mRNA expression in blood from mice was examined by qPCR using human IL-6 primers. (G&H) Representative TRAP-positive staining images of mouse leg bones. *p < 0.05 versus the parental group; #p < 0.05 versus the A549^B[α]P or A549^CSE group.

Figure 8

Schematic diagram summarizing the mechanisms by which cigarette smoke promotes IL-6-dependnet lung cancer osteolytic bone metastasis. Cigarette smoke promotes IL-6-dependent lung cancer migration through PI3K, Akt and NF-κB signaling pathways. The IL-6 secreted by metastatic lung cancers enhances osteoclastogenesis, leading to osteolytic bone metastasis.