The cancer-associated, gain-of-function TP53 variant P152Lp53 activates multiple signaling pathways implicated in tumorigenesis

Abstract

TP53 is the most frequently mutated tumor suppressor gene in many cancers, yet biochemical characterization of several of its reported mutations with probable biological significance have not been accomplished enough. Specifically, missense mutations in TP53 can contribute to tumorigenesis through gain-of-function of biochemical and biological properties that stimulate tumor growth. Here, we identified a relatively rare mutation leading to a proline to leucine substitution (P152L) in TP53 at the very end of its DNA-binding domain (DBD) in a sample from an Indian oral cancer patient. Although the P152Lp53 DBD alone bound to DNA, the full-length protein completely lacked binding ability at its cognate DNA motifs. Interestingly, P152Lp53 could efficiently tetramerize, and the mutation had only a limited impact on the structure and stability of full-length p53. Significantly, when we expressed this variant in a TP53-null cell line, it induced cell motility, proliferation, and invasion compared with a vector-only control. Also, enhanced tumorigenic potential was observed when P152Lp53-expressing cells were xenografted into nude mice. Investigating the effects of P152Lp53 expression on cellular pathways, we found that it is associated with up-regulation of several pathways, including cell-cell and cell-extracellular matrix signaling, epidermal growth factor receptor signaling, and Rho-GTPase signaling, commonly active in tumorigenesis and metastasis. Taken together, our findings provide a detailed account of the biochemical and cellular alterations associated with the cancer-associated P152Lp53 variant and establish it as a gain-of-function TP53 variant.

Keywords: DNA-binding protein; P152Lp53; carcinogenesis; gain-of-function mutant; gene expression; p53 tetramerization; transcriptional regulator; tumor cell biology; tumor suppressor gene.

Figure 1.

Identification of P152Lp53 mutation in a patient sample and prevalence of somatic P152Lp53 missense mutations in human cancers. a, immunohistochemistry for mutant p53 by PAb240 antibody (indicated by →) on an oral cancer sample; b, sequence chromatogram showing C > T substitution at 152 position; c, location of Pro-152 (shown in pink) in 3D structure of the p53 core domain bound to DNA visualized by PyMOL (molecular visualization software); d, multiple sequence alignment of human TP53 protein sequence across various organisms. The Pro-152 residue is indicated by a black arrow. e, prevalence of missense mutations in a stretch of 150–155 amino acids of p53 analyzed from COSMIC database version 86; f, bar plot representation of number and frequency of various missense mutations present at TP53 (Pro-152) loci out of 111 samples of available data at COSMIC. The percentage frequency of various amino acid substitutions are shown above the bars. g, bar plot representation of number and frequency distribution of P152Lp53 mutation occurrence over various cancer tissue types out of 111 samples of P152L mutations. CNS, central nervous system, others: adrenal gland, breast, hematopoietic and lymphoid tissue, liver, lung, pancreas, skin, soft tissue, and stomach tissue.

Figure 2.

Biochemical characterization of P152Lp53. a, EMSA showing the abrogation of the DNA-binding property of recombinant full-length P152Lp53; b, EMSA showing the DNA binding property of DBD of WT and P152Lp53 (protein loading is shown in Coomassie-stained SDS-PAGE gel image); c, ChIP-qPCR to determine the occupancy of Wtp53 and P152Lp53 at p53 responsive gene promoters. The mean relative fold-change of p53 occupancy over IgG ± S.D. have been plotted (n = 2). d, tetramer formation assay by glutaraldehyde cross-linking resolved in 3–12% gradient SDS-PAGE followed by Western blotting. e, equilibrium GuHCl denaturation of wildtype (○) and P152L (●) p53. The fitted curve is to a two-state equation. Each data point is an average of three independent biological replicates.

Figure 3.

Gain-of-function properties of P152Lp53 in H1299 cells. a, wound-healing assay showing the migration rate of the H1299 cell line stably expressing P152Lp53 compared with pCMV10 vector control (n = 2); b, colony formation assay of pCMV10 Ctrl and the P152Lp53 stable cell line to assess cell proliferation (n = 3); c, Transwell assay showing increased invasive potential of cells expressing P152Lp53 (n = 3); d, non-orthotopic nude mice showing enhanced tumor formation ability of H1299 cells stably expressing P152Lp53 injected in the right flank region; e, tumor extracted after the 38th day of injection showing the difference in size of the tumor of P152Lp53 mice as compared with vector control mice; f, statistical analysis of weight and volume of the tumor extracted from three pCMV10 Ctrl and P152Lp53 mice.

Figure 4.

Selection of enriched pathways and genes and their involvement in various tumorigenic pathways. a, Onco, tumor suppressor, and dual role of DEG and their enriched pathway networks showing various pathways in metastasis and proliferation. b, hierarchical clustering of DEG, GSEA, and Reactome FI enrichment and other comparisons. DEG up-regulated (log2FC) and in TSG/ONCO data sets (TSG/ONCO genes), the ONCO genes and the literature reported P53 up-regulated genes as shown in red. DEG down-regulated (log2FC) and in TSG/ONCO data sets (TSG/ONCO genes), the TSG (tumor suppressor) genes, and the literature reported P53 down-regulated genes as shown in blue. The black lines are the genes selected for ECM, Rho-GTPase, and EGFR pathways, which mostly fall in the densely clustered region of the genes that are involved in various P53-regulated mechanisms.

Figure 5.

Network of pathways and genes induced by P152Lp53 expression. RNA-seq analysis of the tumor (P152Lp53 versus pCMV10 Ctrl) showing the pathways and genes up-regulated (fold-change ≥ 1.5, p value ≥ 0.05). Some of the boundary genes (fold-change ≥ 1.41 but <1.5) are also considered in the pathway network.

Figure 6.

Validation of RNA-Seq analysis by qPCR. a, selected up-regulated pathways and genes (with fold-change (log2) and p value) from RNA-Seq analysis. Verification of b, cell-cell/cell-ECM signaling pathway; c, EGFR signaling pathway; and d, Rho-GTPase signaling pathway was performed by qPCR. Data are plotted as the relative fold-change increase in genes of P152Lp53 tumor over pCMV10 vector control tumor (n = 2). Individual fold-change values are indicated above the bar. All error bars are calculated using standard deviation.

Figure 7.

Summarized cartoon representation of the biochemical and functional characterization of P152L p53.