Lung tumourigenesis in a conditional Cul4A transgenic mouse model

Abstract

Cullin4A (Cul4A) is a scaffold protein that assembles cullin-RING ubiquitin ligase (E3) complexes and regulates many cellular events, including cell survival, development, growth and cell cycle control. Our previous study suggested that Cul4A is oncogenic in vitro, but its oncogenic role in vivo has not been studied. Here, we used a Cul4A transgenic mouse model to study the potential oncogenic role of Cul4A in lung tumour development. After Cul4A over-expression was induced in the lungs for 32 weeks, atypical epithelial cells were observed. After 40 weeks, lung tumours were visible and were characterized as grade I or II adenocarcinomas. Immunohistochemistry (IHC) revealed decreased levels of Cul4A-associated proteins p21(CIP1) and tumour suppressor p19(ARF) in the lung tumours, suggesting that Cul4A regulated their expression in these tumours. Increased levels of p27(KIP1) and p16(INK4a) were also detected in these tumours. Moreover, the protein level of DNA replication licensing factor CDT1 was decreased. Genomic instability in the lung tumours was further analysed by the results from pericentrin protein expression and array comparative genomic hybridization analysis. Furthermore, knocking down Cul4A expression in lung cancer H2170 cells increased their sensitivity to the chemotherapy drug cisplatin in vitro, suggesting that Cul4A over-expression is associated with cisplatin resistance in the cancer cells. Our findings indicate that Cul4A is oncogenic in vivo, and this Cul4A mouse model is a tool in understanding the mechanisms of Cul4A in human cancers and for testing experimental therapies targeting Cul4A.

Keywords: Ad-Cre; Cul4A; cell cycle; non-small cell lung cancer; transgenic mouse models.

Figure 1

Morphology of Cul4A transgenic mouse lungs. (A) A summary of lung morphology after Cul4A overexpression was induced. (B) Alveolar structure is normal in Ad-GFP induced mouse lung at 32 weeks post-induction, whereas (C) the Ad-Cre induced mouse lung at 40 weeks showed alveolar edema (arrow). (D) Lymphocyte aggregations (arrows) near respiratory epithelium were present in Ad-Cre induced mouse lungs at 24 weeks, and (E) the aggregation was enlarged at 40 weeks (arrow). Atypical epithelial cells (arrows) were observed in Ad-Cre induced mouse lungs (F) at 32 weeks (arrow) and (G) 40 weeks (arrow). Cell nuclear size and cell size were enlarged in the atypical epithelial cells compared to the nearby respiratory epithelium. Images were captured at 20× magnification. (Scale bar: 50μm)

Figure 2

Lung tumors in the Ad-Cre induced Cul4A transgenic mouse. (A) A summary of nine tumors (T1-T9) isolated from the mouse lungs. Samples with unknown tumor grade or tumor size were labeled as NA. (B,C) Tumors were identified in Ad-Cre induced mouse lungs (arrow) while the majority of the lung surface remained smooth. (Scale bar: 5mm) (B) The lung tumor observed at 48 weeks post-induction was characterized as Grade I adenocarcinoma with (D) well-differentiated tumor cells (Grade I, T2). (C) The lung tumor observed at 64 weeks post-induction was characterized as Grade II adenocarcinoma with (E) moderately differentiated tumor cells (Grade II, T5). Images were captured at 20× magnification. (Scale bar: 50μm)

Figure 3

Tumor-associated changes in the lungs of Ad-Cre induced Cul4A transgenic mice. (A) Groups of tumor cells were observed outside of the tumor barrier (arrows) (20× magnification; Scale bar: 50μm). (B) Tumor cells were observed in the lymphatic channels (10× magnification; Scale bar: 100μm). (C) A tumor island (arrow) was observed near the main tumor (10× magnification; Scale bar: 100μm). (D) Lymphocyte aggregations (arrows) were observed surrounding the developed adenocarcinoma (10× magnification; Scale bar: 100μm).

Figure 4

IHC analysis of Ad-Cre induced Cul4A mouse lung tumors and control lungs. Samples were immunostained with antibody specific to (A) p21^CIP1, (C) PCNA, (D) p14/p19^ARF, (E) p16^INK4a, (F) p27^KIP1. Expression of each protein was analyzed in lung tumors from Ad-Cre induced mouse lungs (main figure) and in the control Ad-GFP induced mouse lungs (inset). Images were captured at 20× magnification. (Scale bar: 50μm) (B) Western blotting of p21^CIP1 in primary MEFs co-transfected with HA-p21^CIP1-pcDNA3 plasmid and either Cul4A siRNA or control siRNA.

Figure 5

Genomic instability in lung tumors from the Cul4A transgenic mice. Expression of (A) CDT1 and (C) pericentrin was analyzed by IHC in tumor samples from Ad-Cre induced mouse lungs (main figure) and in the control Ad-GFP induced mouse lungs (inset). Images were captured at 20× magnification (Scale bar: 50μm). (B) Western blotting of CDT1 in primary MEFs transfected with either Cul4A siRNA or control siRNA. CDT1 expression was quantitated and normalized to β-actin expression. (D) Percentage of tumors with DNA copy number alterations at each locus across the genome was analyzed with aCGH in six tumors that overexpressed Cul4A.

Figure 6

Cul4A copy number in lung cancer cell lines and inhibition effects of cisplatin on cellular viability. (A) Cul4A expression was scored from moderate (++) to high (+++) in six lung cancer cell lines after IHC analysis. Among these cell lines, H2170 had the highest copy number of Cul4A measured using FISH. H2170 cells were challenged with (B) 1nM siRNA or (C) 100 nM siRNA before treatment with increasing concentrations of cisplatin (Cis) (0.03 μM to 300 μM). Cellular viability was measured after 72 hrs, and data points represent the relative cell numbers after the treatment (percentage to control). (D) IC₅₀ was calculated based on the cellular viability curve. H2170 cells treated with 0.1% DMSO were used as a control and the percentage of cells in this group was set as 100%. Data points of Cul4A siRNA (solid circle with dotted line), control siRNA (solid triangle with dashed line), and cisplatin only (solid square with solid line) were presented. The assay was done in triplicate and bars indicate SD.

Figure 7

Schematic explanation of potential mechanisms of initiation and progression of lung tumor in Ad-Cre induced Cul4A transgenic mice. Lungs from the transgenic mice appear to be normal without tumor until Cul4A overexpression was induced by Ad-Cre. Lesions were observed in the lungs in which Cul4A was overexpressed, including alveolar edema, lymphocyte aggregation, and atypical epithelial cells. Decreased levels of p21^CIP1, p19^ARF and CDT1 proteins and increased levels of PCNA, p27^KIP1, p16^INK4a, and pericentrin proteins contributed to cell-cycle defects and genomic instability, resulting in tumor development in the lungs in which Cul4A was overexpressed.