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Comparative Study
, 7 (12), 1058-64

Expression of Axl in Lung Adenocarcinoma and Correlation With Tumor Progression

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Comparative Study

Expression of Axl in Lung Adenocarcinoma and Correlation With Tumor Progression

Yi-Shing Shieh et al. Neoplasia.

Abstract

We used the Transwell system to select highly invasive cell lines from minimally invasive parent cells, and we compared gene expression in paired cell lines with high and low invasive potentials. Axl was relatively overexpressed in the highly invasive cell lines when compared with their minimally invasive counterparts. However, there is only limited information about the role of Axl in cancer invasion. The biologic function of Axl in tumor invasion was investigated by overexpression of full-length Axl in minimally invasive cells and by siRNA knockdown of Axl expression in highly invasive cells. Overexpression of Axl in minimally invasive cells increased their invasiveness. siRNA reduced cell invasiveness as Axl was downregulated in highly invasive cells. We further investigated the protein expression of Axl by immunohistochemistry and its correlation with clinicopathologic features. Data from a study of 58 patient specimens showed that Axl immunoreactivity was statistically significant with respect to lymph node status (P < .0001) and the patient's clinical stage (P < .0001). Our results demonstrate that Axl protein kinase seems to play an important role in the invasion and progression of lung cancer.

Figures

Figure 1
Figure 1
Establishment of cell lines with high and low invasion ability. (A) Lung cancer CL1-0, bladder cancer HT1197, and ovary cancer A59 cells were subjected to repeated selection procedures and denoted CL1-5, A59-4, and HT1197-4, respectively. Their invasion abilities were evaluated using Matrigel-coated Transwell membranes. (B) Gene expression analysis revealed that Axl expression was significantly different in highly and minimally invasive cell lines.
Figure 2
Figure 2
Axl expression is relevant to cell invasion ability. Several cell lines from different tissues (colon, lung, bladder, and breast) were investigated for their invasion abilities (A). Axl RNA expression (B) and Axl protein levels (C) were also analyzed relative to their invasiveness.
Figure 3
Figure 3
Double-stranded siRNA/Axl downregulation of Axl expression accompanied the reduction of cell invasion ability. The highly invasive cell line CL1-5 was transfected with siRNA/Axl. Total RNA was extracted and analyzed by RT-PCR using primers specific to Axl and GAPDH (A). Cells collected on the indicated day (RNAi-3, 3 days; RNAi-7, 7 days) were lysed, and the expression of Axl was analyzed by Western blot analysis using anti-Axl or anti-tubulin antibodies (B). Invasion assays showed reduced cell invasiveness due to Axl knockdown compared to control (mock) cells (C).
Figure 4
Figure 4
Axl transfection of minimally invasive cell lines resulted in increased cell invasion in minimally invasive cell lines. CL1-0/Axl-p (a mixed population and stable clone), CL1-0-Axl-10, and CL1-0-Axl-15 showed increased Axl RNA (A) and protein expressions (B) compared to control cells CL1-0 and CL1-0-neo (vector alone). Cell invasiveness was also increased in Axl-transfected cells compared to the control cells (C).
Figure 5
Figure 5
Axl expression in vascular (A) and cancer tissues with negative (B) and positive staining (C).

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