p53 mutants cooperate with HIF-1 in transcriptional regulation of extracellular matrix components to promote tumor progression

Abstract

Mutations in the TP53 gene and microenvironmentally driven activation of hypoxia-inducible factor-1 (HIF-1) typically occur in later stages of tumorigenesis. An ongoing challenge is the identification of molecular determinants of advanced cancer pathogenesis to design alternative last-line therapeutic options. Here, we report that p53 mutants influence the tumor microenvironment by cooperating with HIF-1 to promote cancer progression. We demonstrate that in non-small cell lung cancer (NSCLC), p53 mutants exert a gain-of-function (GOF) effect on HIF-1, thus regulating a selective gene expression signature involved in protumorigenic functions. Hypoxia-mediated activation of HIF-1 leads to the formation of a p53 mutant/HIF-1 complex that physically binds the SWI/SNF chromatin remodeling complex, promoting expression of a selective subset of hypoxia-responsive genes. Depletion of p53 mutants impairs the HIF-mediated up-regulation of extracellular matrix (ECM) components, including type VIIa1 collagen and laminin-γ2, thus affecting tumorigenic potential of NSCLC cells in vitro and in mouse models in vivo. Analysis of surgically resected human NSCLC revealed that expression of this ECM gene signature was highly correlated with hypoxic tumors exclusively in patients carrying p53 mutations and was associated with poor prognosis. Our data reveal a GOF effect of p53 mutants in hypoxic tumors and suggest synergistic activities of p53 and HIF-1. These findings have important implications for cancer progression and might provide innovative last-line treatment options for advanced NSCLC.

Keywords: HIF; SWI/SNF; chromatin architecture; microenvironment; p53.

Fig. 1.

Hypoxia-driven HIF-1 promotes the binding of p53 mutants to genomic DNA. (A) Kaplan–Meier plot indicating the overall survival estimates for patients with NSCLC presenting a mutation in the TP53 gene and activation of hypoxia (measured as a signature, including 10 different hypoxia-responsive genes). Datasets: The Cancer Genome Atlas (TCGA) lung adenocarcinoma (LUAD) and METABRIC breast cancer. The P value is indicated in the panel. “Others” indicates all of the samples not included in the hypoxia/mut-p53 groups (samples not presenting concurrent high signature and p53 mutant status). The table displays the median survival (months) of the patients comprising the different subgroups. (B and C) Western blot analysis of subcellular fractions (nuclear-soluble and chromatin-bound fractions) of NCI-H1155 and NCI-H23 cells treated with 1% oxygen for 4 h. Histograms show the ratio of Chr-bound/Nucl protein contents calculated via densitometry of Western blots. Chr, chromatin-bound fraction; Nucl, soluble nuclear fraction; MW, molecular weight (molecular mass); Nx, normoxia. (D) Western blot analysis of subcellular fractions of NCI-H1155 and NCI-H23 cells treated with 1% oxygen for 4 h following HIF-1α depletion using siRNA. CTR, control. (E) Western blot analysis of nuclear-soluble and chromatin-bound fractions of NCI-H23 cells overexpressing the HA-tagged HIF-1α PA mutant. The histogram shows the ratio of chromatin-bound/nuclear protein contents calculated via densitometry of Western blots. Error bars (in B, C, and E) indicate the SD of three densitometry measurements. (F) Coimmunoprecipitation of endogenous HIF-1α with p53 mutants (p53 R273H from NCI-H1155 cells and p53 R246I from NCI-H23 cells) in cells treated with 250 μM CoCl₂ for 4 h. IP, immunoprecipitation. (G) Coimmunoprecipitation assay of NCI-H1299 cells treated with 800 μM CoCl₂ and expressing HA-p53 R273H and different HIF-1α fragments, including the Myc-tagged N-terminal region HIF-1α, the Myc-tagged central domain HIF-1α ^401–608, and the C-terminal region HIF-1α^610–826. (H) Coimmunoprecipitation assay of NCI-H1299 cells treated with 800 μM CoCl₂ and expressing Myc-HIF-1α and different fragments of HA-Flag–tagged p53 R273H DBD and HA-Flag–tagged p53 C-terminal (C-term) domain.

Fig. 2.

p53 mutants cooperate with HIF-1 in transcriptional regulation of ECM components. (A) Venn diagram of gene expression profiling analysis representing the distribution of genes differentially regulated by p53 R273H in NCI-H1155 cells treated with normoxia or 1% oxygen (8 h). (B) Pathway clustering analysis of 301 genes differentially regulated by p53 R273H in NCI-H1155 cells treated with 1% oxygen. Pathways of major interest are highlighted in red. (C) RT-qPCR analysis of genes encoding ECM components differentially regulated by the p53 mutants and hypoxia in NCI-H1155 cells. CTR, control. *P < 0.05, **P < 0.01, ***P < 0.001; paired two-tailed t test. Error bars indicate SD of independent biological replicates (n = 3). (D) Western blot analysis of Lam-γ2 and type VII collagen in whole-cell extracts of NCI-H1155 cells following knockdown of p53 R273H and exposure to 1% oxygen for 24 h. MW, molecular weight (molecular mass); Nx, normoxia. (E) ChIP-qPCR analysis of CoCl₂-treated NCI-H1155 cells for the binding of HIF-1α to response elements in the proximity of the collagen VIIa1 and collagen XIIIa1 genomic loci. (F) ChIP-qPCR analysis of CoCl₂-treated NCI-H1155 cells following mutant p53 silencing by siRNA for the binding of HIF-1α to response elements in the proximity of the collagen VIIa1 and collagen XIIIa1 genomic loci. The response element in the VEGFA and p21 genomic loci represent the positive and negative controls, respectively, for HIF-1α binding. (G) ChIP-qPCR analysis of CoCl₂-treated or untreated NCI-H1155 cells for the binding of p53 R273H to response elements in the proximity of the collagen VIIa1 genomic locus. CoCl₂-treated NCI-H1155 has been subjected to 4 h with 250 μM CoCl₂ and then collected and processed, along with the untreated cells, for ChIP assay. The Mll1 transcription starting site (TSS)-proximal peak and the p21 promoter region represent HIF-1–independent positive and negative controls for p53 mutant binding, respectively. Error bars indicate the SD of technical replicates, and the histogram shows representative ChIP-qPCR assays of four (or more) independent biological replicates. (H) ChIP/re-ChIP analysis for the binding of p53 R273H to a response element in the proximity of the collagen VIIa1 genomic locus performed on NCI-H1155 cells treated with 250 μM CoCl₂ and subjected to two subsequent immunoprecipitations (IP; as indicated in the panel). The Mll1 TSS-proximal peak represents a HIF-1–independent positive control for p53 mutant binding. Error bars indicate the SE of three biological replicates. (I) ChIP-qPCR assay for the binding of p53 R273H to response element in the proximity of the collagen VIIa1 genomic locus in NCI-H1155 cells following HIF-1α silencing of cells and CoCl₂ treatment. NCI-H1155 cells have been transfected with siRNA control or HIF-1α for 48 h. CoCl₂-treated NCI-H1155 cells have been treated in the last 4 h of transfection with 250 μM CoCl₂ and then, along with untreated cells, collected, processed, and subjected to immunoprecipitation with p53 DO-I antibody. The Mll1 TSS-proximal pick represents a HIF-1–independent positive control for p53 mutant binding. Error bars indicate the SE of three biological replicates. *P < 0.05, paired two-tailed t test.

Fig. 3.

HIF-1/mutant p53 directly controls transcription of ECM components involving the SWI/SNF chromatin remodeling complex. (A) Coimmunoprecipitation assay performed by immunoprecipitating mutant p53 in untreated or CoCl₂-treated (for 4 h with 250 μM) NCI-H1155 cells and immunoblotting for p53, the SWI/SNF subunits (BAF155 and Brg1), HIF-1α, and the control GAPDH (4 h at 1% O₂). IP, immunoprecipitation; MW, molecular weight (molecular mass); Unt, untreated. (B) Western blot analysis of subcellular fractions (nuclear-soluble and chromatin-bound fractions) of NCI-H1155 treated with a time course (0–24 h) of 1% oxygen. Chr, chromatin-bound fraction; Nucl, soluble nuclear fraction. (C) RT-qPCR analysis of genes encoding ECM components following silencing of SWI/SNF ATPase subunit BRM and Brg1 in hypoxic NCI-H1155 cells (24 h at 1% O₂). CTR, control. *P < 0.05, **P < 0.01; paired two-tailed t test. Error bars indicate the SE of independent biological replicates (n = 3). (D) ChIP-qPCR assay for the binding of the SWI/SNF ATPase subunit Brg1 in NCI-H1155 cells treated for 4 h with 250 μM CoCl₂. VEGFR2 (−150 bp) and VEGFR2 (+30 Kb) represent positive and negative controls, respectively, of Brg1 binding. Error bars indicate the SE of technical replicates, and the histogram shows representative ChIP-qPCR assays of three independent biological replicates. (E) ChIP-qPCR analysis of CoCl₂-treated NCI-H1155 cells following concurrent Brg1 and BRM silencing by siRNA for the binding of mutant p53 to response elements in the proximity of the collagen VIIa1 and collagen XIIIa1 genomic loci. Error bars indicate the SE of independent biological replicates (n = 3). (F) ChIP-qPCR analysis of CoCl₂-treated NCI-H1155 cells following concurrent Brg1 and BRM silencing by siRNA for the binding of HIF-1α to response elements in the proximity of the collagen VIIa1 and collagen XIIIa1 genomic loci. Error bars indicate the SE of independent biological replicates (n = 3). (G) Chromatin was digested with MNase, and mononucleosome-sized DNA fragments were isolated. qPCR was performed on the promoter regions of the hypoxic-responsive genes Lam-γ2, type VII collagen, and VEGFA. Error bars represent the SE of three independent experiments. *P < 0.05, paired two-tailed t test. (H) ChIP-qPCR assay assessing the content of histone H2A.Z at the collagen VIIa1 genomic locus in p53-depleted NCI-H1155 cells following 16 h of hypoxia (at 1% O₂). Error bars indicate the SE of technical replicates, and the histogram shows representative ChIP-qPCR assays of three independent biological replicates.

Fig. 4.

Type VIIa1 collagen and laminin-γ2 promote a p53 mutant/HIF-1–dependent proinvasive phenotype. (A and B) p53 mutants affect hypoxia-mediated migration and invasion of NCI-H1155 and NCI-H23 NSCLC cells, and overexpression of type VIIa1 collagen and laminin-γ2 reverts this impairment. CTR, control; pCMV6, empty vector-transfected control cells. *P < 0.05, **P < 0.02; two-tailed paired t test. Error bars indicate the SE of independent biological replicates (n = 5 for NCI-H1155, n = 3 for NCI-H23). (C) Conditioned media (CM) from corresponding p53 mutant silenced cells treated for 24 h with 1% oxygen alter the migration and invasion capacity of NCI-H1155 and NCI-H23 NSCLC cells. *P < 0.05, **P < 0.01; two-tailed paired t test. Error bars indicate the SE of independent biological replicates (n = 4 for NCI-H1155, n = 3 for NCI-H23). (D) Western blot analysis of NCI-H1155 cells stably overexpressing type VIIa1 collagen or laminin-γ2 displays hyperactivation of pAKT T308. EV, empty vector; MW, molecular weight (molecular mass). (E) Proliferation doublings of NCI-H1155 cells stably overexpressing type VIIa1 collagen or laminin-γ2. *P < 0.05, **P < 0.01; paired two-tailed t test. Error bars indicate the SE of independent biological replicates (n = 4).

Fig. 5.

Type VIIa1 collagen and laminin-γ2 promote the p53 mutant/HIF-1–dependent protumorigenic phenotype in vivo. (A and B) NCI-H1155 cells stably overexpressing type VIIa1 collagen or laminin-γ2 injected s.c. into immunocompromised mice grow faster than empty vector-transfected cells. (A) Columns indicate distribution of tumor weight. Gray lines indicate the average of the distribution. P values are indicated in the corresponding panel (unpaired two-tailed t test; n = 4 for NCI-H1155 cells, NCI-H1155 CollVIIa1 and Lam-γ2). CTR, control. (B) Photographs of tumors after dissection. Full photography of tumors after dissection is shown in SI Appendix, Fig. S9E. (C) Composite images of mice and fluorescent tumor signals (mCherry) were acquired a day before the end point using an IVIS Spectrum In Vivo Imaging System (PerkinElmer). (D–F) Genetic deletion of p53 R273H by inducible CRISPR/Cas9 in NCI-H1155 cells significantly impairs tumor growth after s.c. transplantation in immunocompromised mice, and overexpression of type VIIa1 collagen or laminin-γ2 reverts this effect. NCI-H1155 Tet-On guide RNA (gRNA) p53 cells (parental, type VIIa1 collagen-overexpressing, or laminin-γ2–overexpressing) were treated for 72 h with 2 μg/mL doxycycline and exposed for the last 24 h before injection with 1% oxygen. (D) Columns indicate distribution of tumor weight. Gray lines indicate average of the distribution. P values are indicated in the corresponding panel (two-tailed unpaired t test; n = 4 for p53 R273H^+/+ and p53 R273H^−/− laminin-γ2 groups, n = 5 for p53 R273H^−/− and p53 R273H^−/− type VII collagen groups). (E) Representative hematoxylin/eosin images of xenograft tumors from F. (Scale bars: Left, 2 mm; Right, 500 μm.) (F) Photographs of tumors after dissection. Full photography of tumors after dissection is shown in SI Appendix, Fig. S9H.

Fig. 6.

p53 mutants and HIF-1 synergistically act in human NSCLC. (A and B) Correlation analysis of mRNA expression levels in surgically resected human NSCLC specimens based on RT-qPCR in blue samples expressing wt p53 and in red samples expressing mutant p53. P values are indicated in the corresponding panel (wt p53, n = 22; p53 mutant, n = 23). R, Pearson correlation coefficient; RQ, relative quantification. (C, Left) Representative images of the immunohistochemical staining for CAIX and Lam-γ2 on the human lung adenocarcinoma tissue microarray. (C, Right) Pie chart reports the median distribution of Lam-γ2 staining in the 110 cases analyzed. (Scale bar: 100 μm.) (D) Correlation between expression of CAIX and Lam-γ2 (Upper) and the correlation between histological invasion and CAIX or Lam-γ2 (Lower) are shown. Correlation coefficients have been calculated as Spearman’s rho. P values are indicated in the corresponding panel. Computation has been performed on tumor cores (All, n = 210; p53 wt, n = 111; p53 mut, n = 99). pos, positive. (E and F) Correlations between the expression of HIF-1 targets and our genes of interest, Col VIIa1 and Lam-γ2, were computed in two cohorts of a human NSCLC dataset (GSE36471) stratified according to p53 status (n = 292; Pearson correlation coefficients and P values are indicated in the corresponding panel). ALDOA, Aldolase A. (G) Kaplan–Meier plots indicating the overall survival estimates for patients with NSCLC expressing high levels of Col VIIa1 and Lam-γ2. P values are indicated in the corresponding panel. TCGA, The Cancer Genome Atlas.