ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells

Abstract

Cancer stem cells (CSCs) may serve as the cellular seeds of tumor recurrence and metastasis, and they can be generated via epithelial-mesenchymal transitions (EMTs). Isolating pure populations of CSCs is difficult because EMT programs generate multiple alternative cell states, and phenotypic plasticity permits frequent interconversions between these states. Here, we used cell-surface expression of integrin β4 (ITGB4) to isolate highly enriched populations of human breast CSCs, and we identified the gene regulatory network operating in ITGB4⁺ CSCs. Specifically, we identified ΔNp63 and p73, the latter of which transactivates ΔNp63, as centrally important transcriptional regulators of quasi-mesenchymal CSCs that reside in an intermediate EMT state. We found that the transcriptional program controlled by ΔNp63 in CSCs is largely distinct from the one that it orchestrates in normal basal mammary stem cells and, instead, it more closely resembles a regenerative epithelial stem cell response to wounding. Moreover, quasi-mesenchymal CSCs repurpose this program to drive metastatic colonization via autocrine EGFR signaling.

Keywords: EMT; breast cancer; cancer stem cells; epigenetics; metastasis.

Figure 1.. p63 and p73 are transcriptional drivers of quasi-mesenchymal metastatic CSCs.

(A) Experimental approach used in this study. (B) Motif enrichment scores across 547 open chromatin regions specific to ITGB4^hi cells. (C) Enrichment of TP63 and TP73 motifs in the indicated H3K27ac regions. (D) Clustergram heatmap showing ITGB4^hi- and ITGB4^lo-specific H3K27ac ChIP-seq peaks (left) and p63/p73 binding at these sites in ITGB4^hi cells (right). (E) Venn diagram comparing p73 and p63 binding sites defined by ChIP-seq. (F) Average gene expression of H3K27ac-associated genes bound or unbound by p63 (left) or p73 (right) in ITGB4^hi cells. (G) Differential super-enhancers (SEs) in the ITGB4^hi vs. ITGB4^lo cells. Statistically significant SEs are shown in blue. (H) ALDH activity in ITGB4^lo and ITGB4^hi cells. Representative flow cytometry plots (above) and quantification (below). Data are ± SEM (n = 3), ** p ≤ 0.01. (I) Metastatic colonization of ITGB4^lo and ITGB4^hi cells. Representative lung images (top) and quantification (below). Data are ± SEM (n = 3), * p ≤ 0.05. Scale bars, 1 mm. (J) Immunohistochemistry (IHC) staining for p63 and p73 in representative ITGB4^hi lung metastases. Scale bars, 100 μm. See also Figure S1.

Figure 2.. Loss of ΔNp63 and p73 impairs the post-extravasation proliferation of CSCs.

(A) Representative flow cytometry plots in the indicated ITGB4^hi CRISPR-KO cells. (B) Western blot analysis of the indicated proteins in control or KO cells that were successively sorted for loss of ITGB4 expression. (C) Quantification of ALDH-positive cells in ITGB4^hi control (NT) or KO cells. Data are ± SEM (n = 3), ** p ≤ 0.01, *** p ≤ 0.001. (D) Metastatic colonization of ITGB4^hi control or KO cells. Representative lung images (left) and quantification (right). Data are ± SEM (n = 10), *** p ≤ 0.001, **** p ≤ 0.0001. Scale bars, 1 mm. (E) IHC staining for ΔNp63 and p73 in metastatic lung lesions. Scale bars, 100 μm. (F) Quantification of luminescence after injection of ITGB4^hi NT or dKO cells (n=8, days 0–2, n=4, days 3–10) and representative IVIS images (right). (G) Representative IF staining for tdTom and CD31 in lung cryosections and quantification of extravasated ITGB4^hi NT (n=29 cells from 2 mice) and dKO cells (n=28 cells from 2 mice) 48 hours after tail-vein injection (right). Scale bars, 20 μm. (H) Representative IF staining for tdTom and Ki67 in lung cryosections and quantification of Ki67-positive cells 48 hours or 10 days after injection of ITGB4^hi NT or dKO cells. 48hr: ITGB4^hi NT (n=41 cells from 2 mice), dKO (n=79 cells from 2 mice). 10 days: ITGB4^hi NT (n=166 cells from 2 mice), dKO (n=26 cells from 2 mice). Scale bars, 20 μm. (I) Representative IF staining for tdTom and CD31 in lung cryosections 10 days after tail-vein injection of ITGB4^hi dKO cells. Scale bar, 20 μm. (J) GSEA of single-cell RNA-seq data comparing ITGB4^hi NT and dKO cells isolated from the lungs of mice 3 days after tail-vein injection. Positive enrichment indicates higher expression in ITGB4^hi dKO cells. (K) t-SNE plot of tdTom-positive ITGB4^hi NT and dKO cells isolated from the lungs. Corresponding plots show expression of genes of interest. See also Figure S2 and S3.

Figure 3.. ΔNp63 regulates a distinct transcriptional program in CSCs and normal basal cells.

(A) Clustergram heatmap showing ITGB4^hi- and ITGB4^lo-specific H3K27ac ChIP-seq peaks (left) in the indicated ITGB4^hi CRISPR KO cells (right). (B) Metagene plot showing H3K27ac ChIP-seq reads in parental ITGB4^lo and ITGB4^hi cells (left) and ITGB4^hi cells with the indicated CRSIPR KOs (right). (C) Heatmap of the top differentially expressed genes (log₂ fold-change ±1, p ≤ 0.01) in control (NT) ITGB4^hi cells compared to the indicated KO lines (left). Venn diagram of genes downregulated across the three KO cell lines (right). (D) GSEA based on RNA-seq data comparing ITGB4^hi NT and dKO cells. Positive enrichment indicates higher expression in ITGB4^hi dKO cells. (E) Venn diagram showing ΔNp63 target genes in ITGB4^hi cells and MCF10A cells . Top gene ontology (GO) terms of the ITGB4^hi-specific ΔNp63 targets are shown below. (F) Venn diagram showing ΔNp63 target genes in ITGB4^hi cells and two subpopulations of CD10⁺ myoepithelial/basal . (G) GSEA of RNA-seq data comparing ITGB4^hi NT and dKO cells using the indicated epithelial wounding signatures. See also Figure S4.

Figure 4.. ΔNp63 is sufficient to activate the CSC transcriptional circuit.

(A) Western blot analysis of ΔNp63 and p73 in ITGB4^hi cells ectopically expressing the indicated vectors. (B) Clustergram heatmap showing ITGB4^hi- and ITGB4^lo-specific H3K27ac ChIP-seq peaks in the indicated ITGB4^hi dKO overexpression cells. (C) Metagene plot showing H3K27ac ChIP-seq peaks in the indicated ITGB4^hi dKO overexpression cells. (D) Flow cytometry analysis of the indicated ITGB4^hi dKO overexpression cells. (E) Volcano plots showing genes differentially expressed between ITGB4^hi dKO cells expressing the indicated overexpression vectors compared to cells expressing an empty vector (EV). Genes with significant expression changes are highlighted in blue (down) or red (up). (F) Venn diagram showing the genes downregulated in all ITGB4^hi KO lines and those upregulated upon re-expression of ΔNp63 in dKO cells. (log₂ fold-change ±1, p ≤ 0.01). (G) Western blot analysis of ΔNp63 and p73 in ITGB4^lo cells ectopically expressing the indicated vectors. (H) Clustergram heatmap showing ITGB4^hi- and ITGB4^lo-specific H3K27ac ChIP-seq peaks in control or ΔNp63-expressing ITGB4^lo cells. (I) Metagene plot showing H3K27ac ChIP-seq peaks in control or ΔNp63-expressing ITGB4^lo cells. (J) Volcano plot showing genes differentially expressed between ITGB4^lo ΔNp63 and control EV cells. (K) Flow cytometry analysis of control or ΔNp63-expressing ITGB4^lo cells. (L) ALDH activity in ITGB4^lo cells expressing a doxycycline-inducible ΔNp63 expression vector. (M) Representative lung images after injection of ITGB4^lo cells expressing a doxycycline-inducible ΔNp63 expression vector (left) and quantification of macrometastatic lesions (right). Data are ± SEM (n = 3 in −dox group and n=4 in +dox group). Scale bars, 1 mm.

Figure 5.. ITGB4^hi CSCs are dependent on EGFR signaling for metastatic colonization.

(A) GSEA of RNA-seq data comparing ITGB4^hi NT and the indicated KO lines using an EGF response signature. (B) Western blot analysis of EGFR and ERK activation in the indicated parental or KO cell lines. (C) Representative qRT-PCR analysis of the indicated EGFR ligands in the ITGB4^lo and ITGB4^hi cells. Data are ± SEM, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (D) Relative expression of the indicated EGFR ligands (from RNA-seq data) in control ITGB4^hi cells (NT) or KO cell lines. Data are ± SEM (n=3), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, (E) Western blot analysis of EGFR and ERK activation in serum-starved ITGB4^lo cells treated with media conditioned from ITGB4^hi cells. Negative control (−)cells were maintained in serum-free media. (F) ITGB4^lo or ITGB4^hi tumorsphere growth after treatment with DMSO or erlotinib (1 μM). Representative images (left) and quantification (right). Data are ± SEM (n = 3), ** p ≤ 0.01. Scale bars, 100 μm. (G) Correlation of TP63 expression with a 4 gene EGFR ligand score (TGFA, EREG, AREG, EPGN) in breast cancer specimens from the TCGA database. (H) Representative IF staining for tdTom and pEGFR in lung cryosections prepared 48 hours after tail-vein injection of the indicated cell lines. Scale bars, 20 μm. (I) Metastatic colonization of ITGB4^hi control or EGFR KO cells. Representative lung images (left) and quantification (right). Data are ± SEM (n = 5), ** p ≤ 0.01. Scale bars, 1 mm. (J) Metastatic colonization of ITGB4^lo cells expressing a doxycycline-inducible TGFA expression vector. Representative lung images (left) and quantification (right). Data are ± SEM (n = 4 in −dox group and n=3 in +dox group), * p ≤ 0.05. Scale bars, 1 mm. See also Figure S5.