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 6

ATR inhibition during EMT in vivo impedes tumor growth and metastasis.

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ATR inhibition during EMT in vivo impedes tumor growth and metastasis. (...
(A) Schematic of in vivo experiment in B. (B) Line graph plotting tumor bioluminescence in indicated treatment groups from A. Log fold change in tumor bioluminescence was normalized to day 0 (day of tail vein injection). Data are presented as mean ± SEM for 4 mice in each treatment group. Representative images are shown on the right. (C) Schematic of the in vivo experiments in D–G. (D) Immunofluorescence of nuclear S9.6 intensity in BT549 subcutaneous tumors. Slides were incubated overnight with RNaseH as control and then subject to immunofluorescence with the S9.6 antibody. Representative images are shown on the left. Quantification of nuclear S9.6 intensity is shown on the right. Original magnification, ×10. (E) Line graph plotting primary tumor bioluminescence in the indicated cohorts from the experiment in C. Each value represents fold change in respective cohorts compared with measurements from week 1. Data are presented as mean ± SEM for 3–4 mice in each group. The difference in fold change at each time point is plotted below. (F) Line graph plotting primary tumor volume in indicated cohorts from the orthotopic injection experiment in C. Each value represents fold change in respective cohorts compared with measurements from day 1 of treatment. Data are presented as mean ± SEM for 3–4 mice in each group. The difference in fold change at each time point is plotted below. Statistical significance was determined using repeated measures 2-way ANOVA with Geisser-Greenhouse correction for B, E, and F and 1-way ANOVA followed by Tukey’s multiple-comparison test for D, and each replicate is indicated by a different symbol. P values are indicated in the figure.