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, 113 (12), 1735-43

Overexpression of Major CDKN3 Transcripts Is Associated With Poor Survival in Lung Adenocarcinoma

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Overexpression of Major CDKN3 Transcripts Is Associated With Poor Survival in Lung Adenocarcinoma

Chao Fan et al. Br J Cancer.

Abstract

Background: The cyclin-dependent kinase inhibitor 3 (CDKN3) has been perceived as a tumour suppressor. Paradoxically, CDKN3 is often overexpressed in human cancer. It was unclear if CDKN3 overexpression is linked to alternative splicing variants or mutations that produce dominant-negative CDKN3.

Methods: We analysed CDKN3 expression and its association with patient survival in three cohorts of lung adenocarcinoma. We also examined CDKN3 mutations in the Cancer Genome Atlas (TCGA) and the Moffitt Cancer Center's Total Cancer Care (TCC) projects. CDKN3 transcripts were further analysed in a panel of cell lines and lung adenocarcinoma tissues. CDKN3 mRNA and protein levels in different cell cycle phases were examined.

Results: CDKN3 is overexpressed in non small cell lung cancer. High CDKN3 expression is associated with poor overall survival in lung adenocarcinoma. Two CDKN3 transcripts were detected in all samples. These CDKN3 transcripts represent the full length CDKN3 mRNA and a normal transcript lacking exon 2, which encodes an out of frame 23-amino acid peptide with little homology to CDKN3. CDKN3 mutations were found to be very rare. CDKN3 mRNA and protein were elevated during the mitosis phase of cell cycle.

Conclusions: CDKN3 overexpression is prognostic of poor overall survival in lung adenocarcinoma. CDKN3 overexpression in lung adenocarcinoma is not attributed to alternative splicing or mutation but is likely due to increased mitotic activity, arguing against CDKN3 as a tumour suppressor.

Figures

Figure 1
Figure 1
CDKN3 expression is elevated in NSCLC. (A) CDKN3 expression in the GSE19188 data set of NSCLC tumour and available adjacent normal samples (Engel et al, 2013). LUAD, lung adenocarcinoma. LULC, lung large cell carcinoma. LUSC, lung squamous cell carcinoma. Gene expression was determined via microarray for CDKN3 using probe 209714_s_at. The horizontal lines are the mean+s.d. of each group of samples. (B) CDKN3 expression in LUAD, LUSC and matched normal tissues from TCGA. The normal group is the combined data from the matched normal of LUAD and LUSC. The difference between the matched normal of LUAD and LUSC was not statistically significance.
Figure 2
Figure 2
Expression of CDKN3 mRNA and protein in lung cell lines. (A) RNA was isolated from indicated cells and the relative amounts of CDKN3 mRNA were determined by RT–qPCR using primers CK2/CK2R. The CDKN3 mRNA level in hTBE/v cells was arbitrary set as 1. (B) HEK293 cells were transfected with empty or CDKN3-encoding lentiviral vector (plasmid). Cell lysates from non-transfected and transfected cells were analysed with antibodies 2H10 or actin. NS, a non-specific reactive band. (C) Cell lysates from indicated cells were analysed by immunoblotting with CDKN3 antibody 2H10.
Figure 3
Figure 3
CDKN3 expression is highly prognostic in three lung adenocarcinoma cohorts. CDKN3 mRNA level was correlated with patient outcome by dichotomizing patients into low and high expression groups based on the median expression value. The overall survival of the two CDKN3 groups was compared by Kaplan–Meier estimate and two-sided log-rank test. CDKN3 mRNA and patient survival data were from (A) MCLA, (B, C) TCGA, (D) Moffitt's Lung SPORE 422.
Figure 4
Figure 4
Schematic presentation of CDKN3 transcripts, exon boundaries and PCR primers. The CDKN3 mRNA (Genebank NM_005192.3) is shown. Heavy box indicates the protein coding sequence. Exons 1–8 are according to the notation of Genebank NM_005192.3. The amino acid sequence encoded by the short (exon 2 skip) splicing variant is shown and aligned with the long, CDKN3-encoding transcript. fs, frame shift. Also shown are locations of three pairs of PCR primers.
Figure 5
Figure 5
CDKN3 expression is elevated in M phase. (A and B, left panels) HeLa and A549 cells were subjected to double thymidine blockage. At the indicated time after release from the second thymidine block, cell lysates were collected and analysed by immunoblotting with indicated antibodies. (A and B, right panels, and C) Cell lysates from unsynchronized (U), serum-deprived (G0/1), double thymidine-blocked (S) and nocodazole block (M) cells were analysed by immunoblotting with indicated antibodies. (D) Cells were treated as in C. RNA was isolated and analysed by RT–qPCR using CK-T/CK-R primers. The data were averages from two experiments. The relative amount of CDKN3 mRNA in the serum-deprived cells from each cell line was set as 1.

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