Wnt and Hedgehog are critical mediators of cigarette smoke-induced lung cancer

Abstract

Background: Lung cancer is the leading cause of cancer death in the world, and greater than 90% of lung cancers are cigarette smoke-related. Current treatment options are inadequate, because the molecular basis of cigarette-induced lung cancer is poorly understood.

Methodology/principal findings: Here, we show that human primary or immortalized bronchial epithelial cells exposed to cigarette smoke for eight days in culture rapidly proliferate, show anchorage-independent growth, and form tumors in nude mice. Using this model of the early stages of smoke-induced tumorigenesis, we examined the molecular changes leading to lung cancer. We observed that the embryonic signaling pathways mediated by Hedgehog and Wnt are activated by smoke. Pharmacological inhibition of these pathways blocked the transformed phenotype.

Conclusions/significance: These experiments provide a model in which the early stages of smoke-induced tumorigenesis can be elicited, and should permit us to identify molecular changes driving this process. Results obtained so far indicate that smoke-induced lung tumors are driven by activation of two embryonic regulatory pathways, Hedgehog (Hh) and Wnt. Based on the current and emerging availability of drugs to inhibit Hh and Wnt signaling, it is possible that an understanding of the role of Hh and Wnt in lung cancer pathogenesis will lead to the development of new therapies.

Figure 1

Chronic smoke exposure induces toxicity and changes associated with cellular transformation. A: Toxicity caused by smoke exposure. Shown are cultured BEAS-2B cells after 0, 4 and 8 days of growth in medium containing smoke extract. B: Proliferation assay on cell lines cultured for 1–5 days, derived from CSE-exposed cells at 4 time points (0,3,7 and 8 days). Error bars represent±s.e.m. *P<0.001. C: Cell-substrate adhesion assays on plastic, placental collagen and fibronectin using cultured BEAS-2B cells after the specified time point in days of growth in medium containing smoke extract. Assays were analyzed after 60 minutes in culture. Error bars represent s.e.m. n = 4 *P<0.001. D: Cell migration assays using established cell lines after 24 h in culture. Error bars represent±s.e.m. n = 4 *P<0.001. E: Actin cytoskeleton of cell lines B0 and B8 imaged by fluorescent microscopy using Alexa Fluor 594 phalloidin. Scale bars represent 20 µm.

Figure 2

Chronic smoke exposure induces growth in soft agar and tumor formation in nude mice. A: Anchorage independence was assayed by the ability of CSE-exposed cells to grow in soft agar. B0 or B8 cells (1×10³) were grown in soft agar for 15 days before photography. Quantification of colonies is shown on the right. Results are representative of 5 experiments. B: Tumor formation in nude mice, 4 weeks after inoculation with B0 or B8 cells. Arrows indicate large tumor masses formed in mice injected with B8 cells. C: Section of a representative tumor from a mouse inoculated with B8 cells stained with H and E. D: Images of soft agar assays using HBE cells after no smoke exposure (P0) or recovered HBE cells after 8 days of CSE exposure (P8). Quantification of colonies is shown on the right. Results are representative of 5 experiments. E: Tumor formation in nude mice, 4 weeks after inoculation with P0 and P8 cells. Arrows indicate large tumor masses formed in mice injected with P8 cells. F: Section of a representative tumor from a mouse inoculated with P8 cells stained with H and E. Scale bars represent 50 µm.

Figure 3

Chronic smoke exposure induces the Wnt and Hedgehog signaling pathways A: Wnt signaling assay of smoke exposed Beas-2b cell populations after transfection with TOPFLASH/FOPFLASH luciferase reporter constructs. B: Beas-2b cells were transfected with Gli-TK or TK alone to assay Hh signaling. C: Notch signaling assay in Beas-2b cells using HES luciferase reporter. D, E and F: Same as in A, B and C, using HBE cell populations. Error bars represent±s.e.m. n = 6 *P<0.001.

Figure 4

Wnt and Hedgehog signaling are linked to phenotypic changes induced by chronic smoke exposure. A: Immunostaining shows increased levels of β-catenin (green) in the nucleus of B8 cells compared to B0 cells and B: in the nucleus of P8 cells as compared to P0. C: Immunostaining shows increased expression of Gli (red) in the nucleus of B8 cells compared to B0 cells and D: in P8 cells as compared to P0. Scale bar represents 20 µm.

Figure 5

Inhibitors of Hh and Wnt signaling blocked anchorage-independent growth and cell hyperproliferation of CSE exposed cells. A: The ability of chronic smoke exposed cells (B8), or B: (P8), to grow in soft agar for 15 days was assayed after treatment with tomatidine, cyclopamine, sulindac, NS398, GSI or vehicle control. Quantification of colonies is shown on the right. Results are representative of 5 experiments. C: B0 and B8 cells in culture after treatment with tomatidine, cyclopamine, sulindac or vehicle control.

Figure 6

Inhibition of Wnt and Hedgehog signaling reduces tumor size in nude mice A: Effect of Wnt and Hh inhibitors on Wnt signaling in B0 vs. B8 cells using TOPFLASH/FOPFLASH luciferase reporter constructs. B: Effect of Wnt and Hh inhibitors on Hh signaling in B0 vs. B8 cells transfected with Gli-TK or TK alone. C: Representative tumors excised from mice after no treatment (Con) or after 15 days of treatment with drugs as indicated. Graphs show comparison of tumor volume (cm³) and weight (gm). Veh is vehicle alone. Error bars represent±s.e.m. n = 5 *P<0.001. D: H and E staining of representative tumors formed in mice injected with B8 cells and treated with drugs as indicated.