CDO, an Hh-Coreceptor, Mediates Lung Cancer Cell Proliferation and Tumorigenicity through Hedgehog Signaling

<div><p>Hedgehog (Hh) signaling plays essential roles in various developmental processes, and its aberrant regulation results in genetic disorders or malignancies in various tissues. Hyperactivation of Hh signaling is associated with lung cancer development, and there have been extensive efforts to investigate how to control Hh signaling pathway and regulate cancer cell proliferation. In this study we investigated a role of CDO, an Hh co-receptor, in non-small cell lung cancer (NSCLC). Inhibition of Hh signaling by SANT-1 or siCDO in lung cancer cells reduced proliferation and tumorigenicity, along with the decrease in the expression of the Hh components. Histological analysis with NSCLC mouse tissue demonstrated that CDO was expressed in advanced grade of the cancer, and precisely co-localized with GLI1. These data suggest that CDO is required for proliferation and survival of lung cancer cells via Hh signaling.</p></div

Hh signaling, which is associated with lung cancer cell proliferation was inhibited by CDO depletion.

<p>A. Real-time qRT-PCR for the expression levels of CDO and Hh signaling components (SHH, PTCH1 and GLI1) in BEAS-2B and NSCLC cell lines (A549, H1299, H460 and H520). Each expression level was normalized to the level of 18S rRNA. The relative amount of each component in NSCLCs was determined as the amount of each in BEAS-2B was set to 1.0. B. The number of viable cells was determined by cell counting at indicated time in A549, H1299, H460 and H520 treated with or without 50 µM SANT1. C. Cell viability was assessed by MTT assay at indicated time in A549, H1299, H460 and H520 treated with or without 50 µM SANT1. The absorbance was measured at 595 nm. D. RT-PCR for CDO in A549, H1299 and H460 transfected with the scrambled siRNA or siCDO #1. E. Real-time qRT-PCR for Hh signaling components in A549, H1299 and H460 transfected with the scrambled siRNA or siCDO. The expression level of each component was normalized to the level of 18S rRNA. The relative amount of each component in CDO-depleted NSCLCs was determined as the amount of each in the control cells was set to 1.0 (red line). All the values represent means of at least triplicate determinations ±1 SD. *<i>p</i><0.05 and **<i>p</i><0.01.</p

Knockdown of CDO in NSCLCs showed the reduction of <i>in vitro</i> and <i>in vivo</i> tumorigenesis.

<p>A. Colony formation of the control or CDO knockdown A549, H1299, H460 and H520 in soft agar. B. Quantitative analysis of the experiment described in A. Colony numbers per field were determined by ImageJ. The values represent means of triplicate determinations ±1 SD. *<i>p</i><0.05 and ** <i>p</i> <0.01. C. Representative tumor growth 50 days after subcutaneous injection of nude mice with the control or shCDO-treated A549 cells. D. The tumor volume of the nude mice with the control A549 (n = 8) or CDO-depleted A549 (n = 10) described in C. Means are shown as error bars. E. Tumors from the nude mice injected with the control A549 or CDO-depleted A549 cells shown in C.</p

CDO expression was colocalized with GLI1 in high-grade of NSCLCs.

<p>Confocal immunofluorescence detection of CDO (red) and GLI1 (green) in grade-2, -3, and -4 lung tumor tissues from <i>LSL-K-ras ^G12D</i> model. Cell nuclei were visualized by DAPI (blue) and the phase contrast images are shown. Scale bar indicates 50 µm.</p