Twist1 induces distinct cell states depending on TGFBR1-activation

Basic helix-loop-helix transcription factor Twist1 is a master regulator of Epithelial-Mesenchymal Transition (EMT), a cellular program implicated in different stages of development as well as metastatic dissemination of carcinomas. Here, we show that Twist1 requires TGF-beta type-I receptor (TGFBR1)-activation to bind an enhancer region of downstream effector ZEB1, thereby inducing ZEB1 transcription and EMT. When TGFBR1-phosphorylation is inhibited, Twist1 generates a distinct cell state characterized by collective invasion, simultaneous proliferation and expression of endothelial markers. By contrast, TGFBR1-activation directs Twist1 to induce stable mesenchymal transdifferentiation through EMT, thereby generating cells that display single-cell invasion, but lose their proliferative capacity. In conclusion, preventing Twist1-induced EMT by inhibiting TGFβ-signaling does not generally block acquisition of invasion, but switches mode from single-cell/non-proliferative to collective/proliferative. Together, these data reveal that transient Twist1-activation induces distinct cell states depending on signaling context and caution against the use of TGFβ-inhibitors as a therapeutic strategy to target invasiveness.


Fluorescence-Activated Cell Sorting and Flow Cytometry
Single cell suspensions were suspended in 0.1% BSA/PBS on ice prior to FACS. Cells were stained with the following antibodies: APC Mouse Anti-Human CD44 (clone G44-26; BD Biosciences: #559942) and FITC Mouse Anti-Human CD24 (clone ML5; BD Biosciences: #555427). Dead cells were excluded by 7-AAD (BD Biosciences). Cells were sorted and analyzed on a BD FACS AriaI, followed by data processing with FlowJoV10 software.

Migration Assay
2.5x10 4 cells were plated in 24-well culture inserts with 8 μm pores (BD Falcon). After 24 hours, non-migrated cells were removed from the upper side of the insert. For visualization, migrated cells were stained with the Hemacolor Rapid staining Set (Merck) according to the manufacturer's instructions.

Proliferation Assay
3000 cells per well were seeded in white polystyrene 96-well plates. After 24h, drugs were added every 24h for a total duration of 3 days. Viability of cells was measured every 24h using Cell Titer Glo (Promega) according to the manufacturer's instructions.

Live cell imaging
1x10 4 cells per well were seeded in 24-well plates. After 24h, cells were imaged every 5min at 10x magnification with an Axio Observer.Z1 microscope using in-house software over a period of 3 days.

Cell tracking and data analysis
Individual cells were tracked manually using Timm's Tracking Tool (TTT) [2,3]. Samples were run on a QuantStudio 12K Flex qPCR system (Life Technologies).

Chromatin Immunoprecipitation (ChIP)
ChIP analysis was performed as described [5] with minor modifications. Briefly, cells were cross-linked with 1% formaldehyde for 20 min at room temperature and quenched with glycine. Following nuclei isolation with nuclear preparation buffer, these were sonicated in equal volumes of sonication buffers I and II using Bioruptor Plus (Diagenode SA). Next, chromatin extracts were cleared by centrifugation, diluted, precleared with 50% sepharose 4B (Sigma-Aldrich, USA) slurry, and used for immuneprecipitation with 1 µg of anti-ERα antibody (HC20) (Santa Cruz: # sc-543) or IgG control antibody (Abcam: # ab37415). The immune complexes were pulled down using blocked protein A sepharose beads and the ChIP immune complexes were washed twice with IP buffer, wash buffer and with 1X TE buffer. The washed ChIP immune complexes and inputs (10%) were treated with RNase A (0.2 µg/µl) at 37°C and reverse cross-linked with Proteinase K (20 μg/μl, Invitrogen) overnight at 65°C. The phenolchloroform-isoamyl alcohol extraction method was used to purify DNA, followed by precipitation with LiCl and linear polyacrylamide. The precipitated DNA was dissolved in nuclease free water and amplified by PCR. PCR-reaction was performed as previously described [6]. Briefly, the reaction was carried out in 14 µl of PCR mix, 1.5 µl of 5 µM primers, 1 µl of purified DNA and 8.5 µl of dH 2 O. Input DNA was used for standard curves and normalization.

Gene
Forward Reverse

Proteomics analysis
Glycosyl residues on intact cells were labelled with aminooxybiotin under mild oxidative conditions as described before [7,8] and after cell lysis, glycosylated cell surface proteins were enriched with streptavidin beads (IBA). After stringent washing steps, proteins were on-bead proteolysed with trypsin, followed by deglycosylation with PNGase F (NEB). Eluted peptides were combined, acidified and directly used for analysis on a LTQ-OrbitrapXL connected with an Ultimate3000 nano HPLC system (Thermo Fisher Scientific) as described [9]. The full-scan MS spectra were acquired in the Orbitrap with a resolution of 60,000 and up to 10 most abundant peptide ions were selected for fragmentation in the linear ion trap. Peptides were identified and quantified using the Progenesis QI software (Nonlinear, Waters) and the Mascot search algorithm with the Ensembl Human public database as described [7][8][9]. Table S1: Cell surface proteomics analysis (related to Figure 5).