Truncating mutations of PPM1D are found in blood DNA samples of lung cancer patients

Abstract

Background: PPM1D (WIP1) negatively regulates by dephosphorylation many proteins including p53 tumour suppressor. The truncating mutations (nonsense and frameshift) in exon 6 of PPM1D were found recently in blood cells of patients with breast, ovarian or colorectal cancer. These mutants code for gain-of-function PPM1D with retained phosphatase activity. Their significance in carcinogenesis is unknown.

Methods: The exon 6 of PPM1D was sequenced in blood DNA of 543 non-small-cell lung cancer patients (NSCLC). The functional significance of selected PPM1D alterations (Arg458X, Lys469Glu) was compared with the wild-type gene and examined by recombinant DNA techniques, immunoblotting and luciferase reporter assays.

Results: The frameshift mutations were found in five NSCLC patients (5/543; 0.92%), all of them had squamous cell carcinomas (5/328; 1.5%). All patients with the mutations were exposed, before the blood collection, to the DNA damaging agents as a part of chemotherapeutic regimen. Functional tests demonstrated that truncating mutation Arg458X causes enhancement of dephosphorylation activity of PPM1D toward serine 15 of p53, whereas Lys469Glu version is equivalent to the wild-type. Neither version of PPM1D (wild-type, Arg458X, Lys469Glu) significantly modulated the ability of p53 to transactivate promoters of the examined p53-target genes (BAX and MDM2).

Conclusions: The truncating mutations of PPM1D are present in blood DNA of NSCLC patients at frequency similar to percentage determined for ovarian cancer patients. Our findings raise a question if the detected lesions are a result of chemotherapy.

Figure 1

Sequencing electropherograms of PPM1D showing localisation of frameshift mutations found in DNA samples isolated from blood of NSCLC patients. The strings of letters illustrate superimposed sequences that start from the site of deletion (arrows).

Figure 2

Sequencing electropherograms of PPM1D from genomic DNA samples (upper panels) and from bacterial clones (lower panels) derived from cloned PCR product of amplified exon 6 of case 304 whose genomic electropherogram showed evidence of putative mutation.

Figure 3

Expression of total p53, p53 phosphorylated on Ser15 and expression of p21, MDM2 and WIP1 proteins in NCI-H1299 cells. The level of HSC70 is a loading control. This cell line does not express endogenous p53. The lysate in lane 1 is from cells transfected with empty vector. Other cells were co-transfected with vector expressing p53 and either empty vector or vector expressing various forms of WIP1 protein (Mut458—Arg458X, Mut469—Lys469Glu) whose molecular weight can be estimated by comparison with markers shown on the left. The lysates were prepared after 48-h transfection.

Figure 4

Expression of total p53, p53 phosphorylated on Ser15 and expression of p21 in SAOS-2 cells. The lysates in lanes 1 and 6 are from cells transfected with empty vector. Other cells were co-transfected with vector expressing p53 and either empty vector or vector expressing various forms of WIP1 protein. After 24-h transfection, the cells were either mock-treated or incubated with 1 μM camptothecin for 24 h.

Figure 5

The fold change of the normalised firefly luciferase activity (NFLA) in NCI-H1299 cells. Normalised firefly luciferase activity was calculated by dividing firefly luciferase activity by the Renilla sp. luciferase activity. The cells were co-transfected as described in the Materials and Methods with reporter vectors (MDM2 or BAX) with or without the pC53-SN3 (p53-plasmid) and the vectors expressing various forms of WIP1. The NFLA value in cells transfected with reporter vector and empty plasmid was set as 1. The graph shows means and standard deviations from three independent experiments performed in triplicate. The expression of either wild-type (WT) or mutant WIP1 does not significantly modulate the strong stimulatory effect of p53 on MDM2 or BAX promoters (P>0.05 by Student t-test).