Brachyury-YAP Regulatory Axis Drives Stemness and Growth in Cancer

Abstract

Molecular factors that define stem cell identity have recently emerged as oncogenic drivers. For instance, brachyury, a key developmental transcriptional factor, is also implicated in carcinogenesis, most notably of chordoma, through mechanisms that remain elusive. Here, we show that brachyury is a crucial regulator of stemness in chordoma and in more common aggressive cancers. Furthermore, this effect of brachyury is mediated by control of synthesis and stability of Yes-associated protein (YAP), a key regulator of tissue growth and homeostasis, providing an unexpected mechanism of control of YAP expression. We further demonstrate that the brachyury-YAP regulatory pathway is associated with tumor aggressiveness. These results elucidate a mechanism of controlling both tumor stemness and aggressiveness through regulatory coupling of two developmental factors.

Keywords: YAP; brachyury; chordoma; glioblastoma; growth; lung carcinoma; stremness.

Figure 1. Brachyury regulates cancer stemness and tumor-initiating capacity in chordoma

a, Distribution of biological functions associated to brachyury (T) direct positive targets, identified by GO terms. b, mRNA expression of cell cycle regulator genes in shCtrl or shT JHC7 cells. c, Average percentage of cells in each cell cycle phase in shCtrl or shT JHC7 cells. d, Gene set enrichment analysis (GSEA) of T signature for signatures specific for embryonic stem cells (ES expressed), Nanog, Oct4, and Sox2 (NOS) targets, and polycomb targets. e, Representative in vitro extreme limiting dilution assay plating decreasing numbers of shCtrl or shT JHC7 cells. Solid lines: mean; dotted lines: 95% confidence interval; circles: values obtained in each cell dilution. f, In vivo limiting dilution assay showing relationship between number of JHC7 cells injected subcutaneously in mice and tumor formation capacity after a year (as assessed by tumor frequency in %). g, Left: Table showing tumor frequency in mice injected subcutaneously with the 1×10⁶ shCtrl or shT JHC7 cells after 1 year. Right: Images of isolated JHC7 tumor mass from mice injected subcutaneously with 1×10⁶ shCtrl cells after 1 year. All error bars are s.e.m. * = P<0.05. See also Figures S1, S2, and S3.

Figure 2. Brachyury regulates proliferation and stemness in glioblastoma

a, Immunoblots of brachyury expression in non-cancer cortex and primary glioblastoma patient tissues. b, Densitometric quantification of brachyury expression (normalized to β-actin) from the immunoblots shown in a. a.u. = arbitrary units. c, Percentage of T-positive or T-negative expression in patient-derived glioblastoma tissues from the RNA-seq dataset of TCGA. d, Percentage of T-positive or T-negative GBM patients in each TCGA subtype. Fisher’s exact test. e, Representative immunoblot of brachyury expression in shCtrl or shT GBM1A and GBM1049 cells. f, g, Representative long-term MTT proliferation assay of shCtrl or shT GBM1A and GBM1049 cells. h, i, Representative in vitro extreme limiting dilution assay plating decreasing numbers of shCtrl or shT GBM1A and GBM1049 cells. Solid lines: mean; dotted lines: 95% confidence interval; circles: values obtained in each cell dilution. All error bars are s.e.m. * = P<0.05. See also Figure S4.

Figure 3. Brachyury regulates YAP in CNS-derived cancers

a, Statistical enrichment of the T signature genes among other pathway-specific signatures (Fisher’s exact test). b, Representative immunoblot of brachyury and YAP expression in shCtrl or shT JHC7, UCH1, and UCH2 cells. c, YAP mRNA expression in shCtrl vs. shT JHC7, UCH1, and UCH2 cells. d, Mcl-1 mRNA expression in shCtrl vs. shT JHC7, UCH1, and UCH2 cells. e, Representative images of immunohistochemical staining of Brachyury, YAP, and H&E in patient-derived chordoma tissues. Scale bar = 200μm. f, Immunoblots of brachyury and YAP expression in primary and recurrent sacral, clival, or mobile spine chordoma patient tissues. g, Correlation plot of relative brachyury and YAP protein expression (normalized to β-actin) based on densitometric quantification of immunoblots in e; a.u. = arbitrary units. h, Correlation plot of relative brachyury and YAP mRNA expression (normalized to β-actin) in a subset of patient-derived chordoma tissues (n=10). All error bars are s.e.m. * = P<0.05. See also Figure S4, S5, S6, and S7.

Figure 4. Brachyury directly activates transcription of YAP

a, Brachyury binding to the proximal promoter region of YAP as observed in a publicly available ChIP-sequencing dataset using UCH1 cells. b, ChIP-PCR querying across the −1.5 kb region upstream of the transcriptional start site (TSS) of the YAP promoter in JHC7 cells using two independent antibodies specific for epitopes on the C- (Ab1) and N-terminus (Ab2) of brachyury. c, Representative immunoblot of brachyury and YAP expression in siCtrl or siT JHC7 cells. d, YAP gene promoter luciferase assay using the −105bp and −608bp regions upstream of the TSS in siCtrl or siT JHC7 cells. e, Schematic of transcriptional regulation of YAP by brachyury. All error bars are s.e.m. * = P<0.05. See also Figure S5.

Figure 5. Brachyury regulates stemness and proliferation through YAP

a, Average percentage of cells in each cell cycle phase in shCtrl or shYAP JHC7 cells. b, Representative in vitro extreme limiting dilution assay plating decreasing numbers of shCtrl or shYAP JHC7 cells. c, Representative long-term MTT proliferation assay of shCtrl or shYAP JHC7 cells. d, Representative immunoblot of YAP, Cyclin-D1, and Survivin expression in shCtrl or shT JHC7 cells transfected with empty vector (CONT) or YAP-overexpression (YAP-OE) plasmids. e, mRNA expression of cell cycle regulator genes in shCtrl or shT JHC7 cells with CONT or YAP-OE overexpression. f, Average percentage of cells in G1 phase in shCtrl or shYAP JHC7 cells with CONT or YAP-OE overexpression. g, Representative in vitro extreme limiting dilution assay plating decreasing numbers of shCtrl or shYAP JHC7 cells with CONT or YAP-OE overexpression. Solid lines: mean; dotted lines: 95% confidence interval; circles: values obtained in each cell dilution. h, Representative long-term MTT proliferation assay of shCtrl or shYAP JHC7 cells with CONT or YAP-OE overexpression. In h, * = significant versus Day 0, # = significant versus shCtrl CONT group for the same day. All error bars are s.e.m. *, # = P<0.05. See also Figure S6.