A crucial requirement for Hedgehog signaling in small cell lung cancer

Abstract

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer for which there is no effective treatment. Using a mouse model in which deletion of Rb1 and Trp53 in the lung epithelium of adult mice induces SCLC, we found that the Hedgehog signaling pathway is activated in SCLC cells independently of the lung microenvironment. Constitutive activation of the Hedgehog signaling molecule Smoothened (Smo) promoted the clonogenicity of human SCLC in vitro and the initiation and progression of mouse SCLC in vivo. Reciprocally, deletion of Smo in Rb1 and Trp53-mutant lung epithelial cells strongly suppressed SCLC initiation and progression in mice. Furthermore, pharmacological blockade of Hedgehog signaling inhibited the growth of mouse and human SCLC, most notably following chemotherapy. These findings show a crucial cell-intrinsic role for Hedgehog signaling in the development and maintenance of SCLC and identify Hedgehog pathway inhibition as a therapeutic strategy to slow the progression of disease and delay cancer recurrence in individuals with SCLC.

Figure 1

Cell-autonomous activation of the Hedgehog pathway in mouse SCLC. (a) X-gal staining and immunostaining for Ki-67 and the neuroendocrine marker synaptophysin (Syp) of lung tumors from Rb1^lox/lox; Trp53^lox/lox; Ptch1^lacZ/+ mice infected with Ad-Cre. Arrowheads point to a LacZ+Ki-67⁻Syp⁻ stromal cell. (b) Quantification of X-gal staining in Syp⁺ cells, lesions and tumors. ND, not detected. 20 μm 10 μm (c) Shown at the top left and in the center are X-gal staining and immunostaining for the neuroendocrine Syp marker CGRP of a mouse SCLC sphere in culture. The asterisk indicates a stromal cell. Shown at the top right is an X-gal–stained subcutaneous allograft derived from Rb1-Trp53 mutant Ptch1^lacZ/+ tumor cells (the arrow indicates the skin of the recipient mouse). Shown at the bottom is an X-gal staining of representative clones (1 and 2) derived from parental Rb1-Trp53–mutant Ptch1^lacZ/+ SCLC cells. (d) RT-PCR analysis for Shh, Ptch1, Smo and Gli1 in two subclones (1 and 2) each from two independent parental SCLC cell lines (1 and 2). We used Gapdh as a loading control. (e) Luciferase activity in Shh-LIGHT2 reporter cells co-cultured with mouse SCLC cells (n ≥ 3). We used conditioned media from either 293 cells (Con-CM) or 293 cells secreting active Sonic hedgehog (ShhN-CM) as controls. Data are relative to Con-CM values. (f) Shown at the top is Sonic hedgehog (Shh) immunostaining (brown) on sections of mouse SCLC (Tu); the arrow indicates normal airway epithelial cells, the arrowhead indicates tumor-associated stromal cells and the counterstain used was hematoxylin (Hem). At the bottom, immunostaining for polyglutamylated tubulin (Tub, red) marks the primary cilium in a SCLC sphere (left), a single cell (inset) and a primary tumor (right). PNEC/NEB, pulmonary neuroendocrine cells including neuroepithelial bodies. Mean ± s.e.m. are shown. *P < 0.01, **P < 0.001.

Figure 2

Constitutive Hedgehog signaling is sufficient to promote SCLC in mice. (a) Cell viability for two mouse SCLC cell lines (mSCLC1 and mSCLC2) treated with conditioned media from 293 cells (Con-CM) or 293 cells secreting active N-terminal Sonic hedgehog (ShhN-CM) for 4 days (n ≥ 3). (b) Quantitative RT-PCR analysis for Gli1 levels after 24 h of treatment (n ≥ 3). (c) Strategy to constitutively activate Smo (SmoM2) in Rb1-Trp53 mutant lung cells. (d) Whole-mount images of tumors (Tu) and immunostaining for synaptophysin (Syp) (red) counterstained with DAPI (blue). (e) We quantified tumor number and area in mice from both genotypes (n = 8 for Rb1-Trp53 and n = 9 for Rb1-Trp53-SmoM2 mice). (f) Quantification of cell proliferation and cell death by immunostaining for phospho histone 3 (PH3) and cleaved caspase 3 (CC3) in tumors. Mean ± s.e.m. are shown. NS, not significant. *P < 0.01, **P < 0.001.

Figure 3

Hedgehog pathway activity is necessary for the growth of mouse SCLC cells. (a,b) Cell viability (after 4 d) (a) and RT-quantitative PCR analysis of Gli1 levels (after 24 h) (b) for three independent SCLC cell lines treated with HPI-1 (10 μM) or vehicle control (Con) (n = 3). (c) The treatment protocol of mice having tumors with vehicle (n = 4 mice), cyclopamine (Cyc) (n = 4) or cisplatin (Cis) (n = 2). The arrow indicates the experimental treatment, and the arrowhead indicates the vehicle. (d) Quantification of PH3⁺ and CC3+ cells on tumor sections. (e) The experimental strategy to test the effects of deleting Smo in Rb1-Trp53-Rbl2 mutant lung cells. (f) Lung sections from mice infected with Ad-Cre and aged for 6 months before analysis. The counterstain is H&E, and the tumors appear dark purple. (g) Synaptophysin (Syp) immunostaining on tumors (Tu) counterstained with DAPI (blue). (h) Tumor numbers and tumor area in mutant mice (n = 6 for Rb1-Trp53-Rbl2 mice with wild-type Smo and n = 3 for Rb1-Trp53-Rbl2 mice with mutated Smo). (i) Quantification of cell proliferation and apoptotic cell death by immunostaining for phospho histone 3 (PH3) and cleaved caspase 3 (CC3) in tumors. Mean ± s.e.m. are shown. NS, not significant. *P < 0.01.

Figure 4

Hedgehog signaling is crucial for the growth of chemoresistant human SCLC cells. (a) Colony formation in chemonaive and chemosurviving LX22CL cells assessed by serial passage in methycellulose (n = 4). The clonal capacity following each treatment is shown relative to its respective control, to which we assigned a value of 1. Treatments and matching controls were as follows: infection with adenovirally expressed SmoM2 (AdSmoM2) or adenovirally expressed βGal (AdβGal) (green diamond); 0.2 μg ml⁻¹ of recombinant human Sonic hedgehog or vehicle (green square); 100 nM NVPLDE225 or vehicle (orange triangle); transfection with a vector expressing shRNA molecules targeting human Smo or control shRNA (red triangle); or 5 μg ml⁻¹ of the Hedgehog-neutralizing monoclonal antibody 5E1 or mouse IgG1 (brown circle). (b) Smo localization (green) in the primary cilia (AcT, red) of chemoresistant LX22CL cells counterstained with DAPI. (c) Smo expression in the primary cilia of chemoresistant LX22CL cells treated in vitro with recombinant Sonic hedgehog (rShh) (1 mg ml–1), the Smo agonist SAG (200 nM), cyclopamine (Cyc, 3 μM) or NVP-LDE225 200 nM (n = 3). (d) We treated nude mice subcutaneously implanted with LX22 tumors with vehicle (control, white square), NVP-LDE225 (80 mg per kg per day once a day, orange triangle), etoposide (12 mg per kg per day intraperitoneally on days 1, 2, 3 and 15 after the start of treatment) and carboplatin (E/C) (60 mg per kg per day intravenously on days 1, 8 and 15 after the start of treatment) alone (green diamond) or followed by NVP-LDE225 (red square) as indicated in the figure. The tumor volume (n = 8, two independent experiments) is shown. Mean ± s.e.m. are shown. **P < 0.001 compared to E/C then vehicle.