ATR safeguards epithelial-to-mesenchymal transition by countering R-loops and enabling transcription reprogramming
Parasvi S. Patel, Jacob P. Matson, Xiaojuan Ran, Marcello Stanzione, Ajinkya S. Kawale, Mingchao Wang, Sneha Saxena, Conrad Sander, Jacquelyn Curtis, Jessica L. Hopkins, Edmond Wong, Ryan B. Corcoran, Daniel A. Haber, Nicholas J. Dyson, Shyamala Maheswaran, Lee Zou
Parasvi S. Patel, Jacob P. Matson, Xiaojuan Ran, Marcello Stanzione, Ajinkya S. Kawale, Mingchao Wang, Sneha Saxena, Conrad Sander, Jacquelyn Curtis, Jessica L. Hopkins, Edmond Wong, Ryan B. Corcoran, Daniel A. Haber, Nicholas J. Dyson, Shyamala Maheswaran, Lee Zou
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Research Article Cell biology Oncology

ATR safeguards epithelial-to-mesenchymal transition by countering R-loops and enabling transcription reprogramming

Abstract

Transitions of cancer cells between distinct cell states, which are typically driven by transcription reprogramming, fuel tumor plasticity, metastasis, and therapeutic resistance. Whether the transitions between cell states can be therapeutically targeted remains unknown. Here, using the epithelial-to-mesenchymal transition (EMT) as a model, we show that the transcription reprogramming during a cell-state transition induces genomic instability through R-loops and transcription-replication conflicts and that the cell-state transition cannot occur without the ATR kinase, a key regulator of the replication stress response. ATR inhibition during EMT not only increased transcription- and replication-dependent genomic instability, but also disrupted transcription reprogramming. Unexpectedly, ATR inhibition elevated R-loop–associated DNA damage at the SNAI1 gene, a key driver of the transcription reprogramming during EMT, triggering ATM- and Polycomb-mediated transcription repression of SNAI1. Beyond SNAI1, ATR also suppressed R-loops and antagonized repressive chromatin at a subset of EMT genes. Importantly, inhibition of ATR in tumors undergoing EMT reduced tumor growth and metastasis, suggesting that ATR inhibition eliminates cancer cells in transition. Thus, during EMT, ATR not only protects genome integrity but also enables transcription reprogramming, revealing that ATR is a safeguard of cell-state transitions and a target to suppress tumor plasticity.

Authors

Parasvi S. Patel, Jacob P. Matson, Xiaojuan Ran, Marcello Stanzione, Ajinkya S. Kawale, Mingchao Wang, Sneha Saxena, Conrad Sander, Jacquelyn Curtis, Jessica L. Hopkins, Edmond Wong, Ryan B. Corcoran, Daniel A. Haber, Nicholas J. Dyson, Shyamala Maheswaran, Lee Zou

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Figure 1

EMT is associated with DNA replication stress and DNA damage.

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EMT is associated with DNA replication stress and DNA damage. (A) Repres...
(A) Representative immunofluorescence images (left) and quantification of S phase mean RPA32 intensity (right) in MCF10A cells treated with 5 ng/mL TGF-β for 24 h. Original magnification, ×20. (B) Western blotting of p-RPA32 (Ser33) and histone H3 in MCF10A cells treated with 5 ng/mL TGF-β for 24, 48, and 72 h. H3 was used as a loading control. (C) Quantification of γH2AX+ cells in EdU+ cell populations from the immunofluorescence experiments performed in MCF10A cells treated with 5 ng/mL TGF-β and/or 10 μM ATRi (VE821) for 24, 48, and 72 h. (D) Two-dimensional plot of the cells in A displaying total DAPI intensity and mean EdU intensity. Coloring of dots indicates γH2AX mean intensity. (E) Quantification of mean γH2AX intensity from the immunofluorescence experiment in C and D. (F) Quantification of nuclear γH2AX intensity in EdU+ population from immunofluorescence experiments performed in MCF10A cells expressing pInducer20 SNAIL1-S6A, treated with 50 ng/mL doxycycline to induce SNAIL1-S6A expression for 0, 24, 48, or 72 h and DMSO or 10 μM ATRi (VE821) for the last 24 h. (G) Quantification of γH2AX+ cells in EdU+ cell populations in indicated cells. (H) Quantification of γH2AX+ cells in EdU+ cell population from immunofluorescence experiments performed in BT549 cells treated with 5 ng/mL TGF-β for 0, 24, 48, or 72 h and at the same time DMSO control or 10 μM ATRi (VE821). Statistical significance was determined using a paired 2-tailed t test for A; 1-way ANOVA for C, G, and H; and 1-way ANOVA followed by Tukey’s multiple-comparison test for E and F. Data are presented as mean ± SEM in all graphs for 3–5 biological replicates. For scatterplots in A, E, and F, each replicate is indicated by a different symbol. P values are indicated in the figure.