The lung-enriched p53 mutants V157F and R158L/P regulate a gain of function transcriptome in lung cancer

Abstract

Lung cancer is the leading cause of cancer-related deaths in the USA, and alterations in the tumor suppressor gene TP53 are the most frequent somatic mutation among all histologic subtypes of lung cancer. Mutations in TP53 frequently result in a protein that exhibits not only loss of tumor suppressor capability but also oncogenic gain-of-function (GOF). The canonical p53 hotspot mutants R175H and R273H, for example, confer upon tumors a metastatic phenotype in murine models of mutant p53. To the best of our knowledge, GOF phenotypes of the less often studied V157, R158 and A159 mutants-which occur with higher frequency in lung cancer compared with other solid tumors-have not been defined. In this study, we aimed to define whether the lung mutants are simply equivalent to full loss of the p53 locus, or whether they additionally acquire the ability to drive new downstream effector pathways. Using a publicly available human lung cancer dataset, we characterized patients with V157, R158 and A159 p53 mutations. In addition, we show here that cell lines with mutant p53-V157F, p53-R158L and p53-R158P exhibit a loss of expression of canonical wild-type p53 target genes. Furthermore, these lung-enriched p53 mutants regulate genes not previously linked to p53 function including PLAU. Paradoxically, mutant p53 represses genes associated with increased cell viability, migration and invasion. These findings collectively represent the first demonstration that lung-enriched p53 mutations at V157 and R158 regulate a novel transcriptome in human lung cancer cells and may confer de novo function.

Figure 1.

Somatic mutations in TP53 exhibit a specific mutational pattern in lung cancers. (A) The TP53 gene is shown here, with transactivation domain (TAD), DNA-binding domain (DBD) and tetramerization domain (TD) labeled. Top, number of mutations at each amino acid out of 5461 total somatic missense mutations in non-lung cancers in the cBioPortal for Cancer Genomics (34,35). Bottom, number of mutations at each amino acid out of 505 total somatic missense mutations in the Pan-Lung Cancer study (4). (B) Oncoprint (34,35) showing cancer and genetic alteration type for the Pan-Lung Cancer study (4), with a TP53 somatic mutation rate of 68%. (C) Frequency of mutations at TP53 hotspots (unshaded) and lung-specific amino acid residues (shaded in blue) among non-lung versus lung cancers.

Figure 2.

Lung cancers exhibit single nucleotide mutations in TP53 hotspots in patterns distinct from non-lung cancers. Graphs depict the frequency of nucleotide transversions (G>T, G>C, C>G, C>A, T>A, T>G) and transitions (G>A, C>T) among somatic missense mutations from non-lung cancers in the cBioPortal for Cancer Genomics and from the Pan-Lung Cancer study (4,34,35).

Figure 3.

Human lung cancer cell lines with endogenous mutant p53 exhibit loss of expression of canonical wild-type p53 target genes. (A) qRT–PCR for p53 mRNA expression after depletion via transfection of siRNA targeting p53, with and without cisplatin treatment (10 uM for 24 h) in A549 (wtp53), H2087 (mutp53(V157F)), H441 (mutp53(R158L)), and H2110 (mutp53(R158P)) cells. (B) qRT–PCR was performed for canonical target genes of wtp53 in RNA isolated from A549, H441, and H2110 cell lines. (C) Western blot for p53 and p21 protein expression with and without cisplatin treatment.

Figure 4.

The p53 mutants V157F, R158L and R158P exhibit differential expression of genes associated with cell migration and invasion. (A) Western blot of cell lysates. (B) qRT–PCR for mRNA expression of TP53 in cells expressing endogenous mutant p53 or depleted of p53. (C) Overlap of differentially expressed genes identified among siCtrl versus sip53 comparisons within each cell line. (D) Heatmap of 53 differentially expressed genes among all three mutant alleles (V157F, R158L and R158P). (E). mRNA expression, in representative experiments, of enriched genes identified by RNA-sequencing. mRNA expression values are normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and A549 siCtrl condition. (F) Selected top biological functions significantly altered by mutant p53 depletion in H2087, H441 and H2110 cells. (G) Selected top signaling pathways significantly altered by mutant p53 depletion in H2087 cells.

Figure 5.

V157F mutp53 impedes migration of lung cancer cells. Cells harboring endogenous wild-type or lung-enriched mutp53 was transfected with siRNA targeting p53. (A). Cell proliferation, measured by MTT assay, in A549 (wild-typet), H2087 (V157F), H441 (R158L) and H2110 (R158P) cells. (B and C) Cell migration in a wound healing assay in A549 and H2087 cells.

Figure 6.

uPA protein expression is mutp53-dependent. Protein expression of uPA encoded by the RNA-seq identified gene PLAU, was assessed in p53-depleted and control conditions. (A) Western blot of A549, H2087, H441, H2110 and H2009 cells transfected with negative control or siRNA directed against p53 (25 nM). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown as a loading control. (B) qRT–PCR was performed to evaluate mRNA expression of PLAU after depletion of p53 by siRNA. mRNA expression values are normalized to GAPDH and siCtrl condition within each cell line. (C) Protein and conditioned media were harvested from cells after overnight incubation in reduced-serum conditions. Nitrocellulose membranes were stained with Ponceau S dye to visualize protein bands for loading control. Protein expression of pro-uPA (49 kDa) and its cleaved and activated forms B-chain (33 kDa) and A-chain (19 kDa) are shown in cell lysates and conditioned media. (D) Protein and E. mRNA expression of uPA (PLAU) following depletion of V157F mutp53 by siRNA.