Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice
- The University of Queensland, Australia
- Lowy Cancer Research Centre, The University of New South Wales, Australia
- QIMR Berghofer Medical Research Institute, Australia
- The University of Oxford, United Kingdom
- Cancer Research UK, The University of Cambridge, Li Ka Shing Centre, United Kingdom
- Cardiovascular Research Institute, The University of California, San Francisco, United States
- Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, China
- Guangzhou Medical University, China
- School of Medicine, The University of Queensland, Australia
Figures
Figure 1 with 2 supplements
Mapping of SOX18 interactome and disruption of interactions by Sm4.
(A) Schematic of the experimental strategy to deconvolute SOX18-dependent protein-protein interactions (PPIs) combining Chromatin immunoprecipitation-mass spectrometry (ChIP-MS) and Amplified …
Figure 1—figure supplement 1
QC of SOX18 PPIs and effect of Sm4.
(A) Mass spectrometry spectrum for a representative double charged DDX17 peptide with the sequence KAPILIATDVASRG (Muscat ion score 51.6), identified from immunoprecipitation of cMyc-SOX18 with …
Figure 1—figure supplement 2
Differential disruption of SOXF PPI by Sm4.
The left panel shows a matrix of protein-protein interactions between SOXF, MEF2C and RBPJ and OCT4 as measured by ALPHAScreen. The right panel shows the effects of 50 µM Sm4 on PPIs (blue = no …
Figure 2 with 3 supplements
Sm4 selectively affects SOX18 transcriptional output in vitro.
(A) Schematic representation of the correlation analysis between genome-wide TF ChIP-seq data and Sm4 affected genes from transcriptomics data. The chromatin around the transcription start sites …
Figure 2—figure supplement 1
Transcriptome-wide analysis of Sm4 selectivity in vitro.
(A) Top motif identified from SOX18 ChIP-seq peaks (MEME software) performed in HUVECs. (B) UCSC browser view of representative ChIP-seq peaks (arrowheads) for known SOX18 target genes VCAM and PROX1…
Figure 2—figure supplement 2
c-JUN motifs are enriched in SOX18 binding sites.
(A) HOMER motif analysis on SOX18 ChIP-seq peaks revealed an enrichment of the c-JUN motif 5’-TGAC/GTCA-3’. (B) ALPHA-Screen binding curve for SOX18-c-JUN and SOX18-SOX18 (positive control), …
Figure 2—figure supplement 3
Sm4 does not interfere with SOX9 or SOX17 activity in vitro.
(A) Cell based reporter assay for SOX9 homodimer activity. COS-7 cell were transfected with Sox9 and Col2a1:luc reporter construct. Sox9 overexpression caused a >8 fold induction of Col2a1 …
Figure 3 with 2 supplements
Sm4 blocks SoxF transcriptional activity in vivo.
(A) Lateral brightfield (top) and fluorescent (bottom) images of 60 hpf zebrafish larvae carrying the tg(−6.5kdrl:eGFP) SoxF reporter. Treatment was initiated at late stage (20 hpf) with either DMSO …
Figure 3—figure supplement 1
Sox9 activity is not perturbed by treatment in vivo.
(A) Timeline of treatment: Zebrafish larvae were treated continuously for four days during chondrogenesis. Medium was refreshed daily throughout the experiment to maintain Sm4 levels. (B) tg(col2a1:Y…
Figure 3—figure supplement 2
Sm4 interferes with SoxF activity in vivo.
(A) Timeline of Sm4 treatment in zebrafish larvae. Treatment for SOXF reporter gene studies was initiated at 20 hpf, while for the phenotypic studies treatment was initiated at precedes that for, to …
Figure 4 with 4 supplements
Metastasis and tumor vascularization is suppressed by Sm4 treatment.
(A) Timeline of mouse model for breast cancer metastasis. 4T1.2 tumor was inoculated at day 0, and resected at day 12. Sm4 (25 mg/kg/day), Aspirin (25 mg/kg/day) or vehicle control (PBS), was …
Figure 4—figure supplement 1
Sm4 efficacy is not a result of surgery-induced inflammation.
4T1.2 tumor was inoculated at day 0, and surgery was performed at day 12, without resecting the tumor (n = 6). (A) Survival (n = 6) was monitored in PBS vehicle control mice and Sm4-treated mice (25 …
Figure 4—figure supplement 2
Penetrance of blood vessels into 4T1.2 tumors is impaired by Sm4.
Brightfield images of serial vibratome sections (300 μm) from a whole 4T1.2 mammary tumor for mice treated with PBS vehicle or Sm4. Main blood vessels and haemorrhagic areas are distinctive in red.
Figure 4—figure supplement 3
Sm4-treated mice have decreased tumor vascular density.
Immunofluorescent staining for ERG and Endomucin (EMCN) on tumor sections. Two representative regions for both vehicle PBS and Sm4 are shown. Detailed blow-up shows distinct nuclear staining for …
Figure 4—figure supplement 4
Sm4 treatment disrupts tumour-induced lymphangiogenesis.
Lymphatic vessels images of serial vibratome sections (200 μm) from a whole 4T1.2 mammary tumor for mice treated with PBS vehicle or Sm4 (25 mg/kg/day). Immunofluorescence for lymphatic specific …
Author response image 1
Snapshot of FANTOM5 database, showing (absence of) OCT4 transcript levels in arterial, venous and lymphatic endothelial cell types.
https://doi.org/10.7554/eLife.21221.018
Author response image 2
Sm4-treatment causes mild malformations to the lateral dorsal aorta (LDA), reminiscent of partial interference with Sox7 function.
Head circulation is unaffected by Sm4.
Author response image 3
Effect of Sm4 on endogenous dll4 transcript in 27 hpf zebrafish larvae.
Both the dorsal aorta and intersomitic vessels (ISV) were labeled by dll4 ish probe. In presence of Sm4(1 μM) ISV show a mild decrease of signal intensity.
Additional files
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Supplementary file 1
(A) GO term analysis (PANTHER) on top 5K SOX18 ChIP-seq peaks, reveals over-representation of biological processes, which are in agreement with known roles for SOX18 (e.g. blood vessel morphogenesis, angiogenesis, blood vessel development).
(B) Summary of sequencing statistics, listing the sample with the number of the replicate (#n). Percentage of mapped reads is consistently high across all samples (>87%). Mapping was performed with STAR aligner (Dobin et al., 2013). (C) Summary of endothelial specific TF expression levels and summary of distance from peak to TSS analysis on DE SOX18oe vs. Sm4 genes. A subtraction of SOX18, or cJUN peaks from all TF peaks was performed to reduce overlap bias (column #2 and #3). Sm4 down regulated genes are significantly closer to SOX18 and c-JUN ChIP-seq peaks.
- https://doi.org/10.7554/eLife.21221.017
