Breast cancer plasticity is restricted by a LATS1-NCOR1 repressive axis

Breast cancer, the most frequent cancer in women, is generally classified into several distinct histological and molecular subtypes. However, single-cell technologies have revealed remarkable cellular and functional heterogeneity across subtypes and even within individual breast tumors. Much of this heterogeneity is attributable to dynamic alterations in the epigenetic landscape of the cancer cells, which promote phenotypic plasticity. Such plasticity, including transition from luminal to basal-like cell identity, can promote disease aggressiveness. We now report that the tumor suppressor LATS1, whose expression is often downregulated in human breast cancer, helps maintain luminal breast cancer cell identity by reducing the chromatin accessibility of genes that are characteristic of a “basal-like” state, preventing their spurious activation. This is achieved via interaction of LATS1 with the NCOR1 nuclear corepressor and recruitment of HDAC1, driving histone H3K27 deacetylation near NCOR1-repressed “basal-like” genes. Consequently, decreased expression of LATS1 elevates the expression of such genes and facilitates slippage towards a more basal-like phenotypic identity. We propose that by enforcing rigorous silencing of repressed genes, the LATS1-NCOR1 axis maintains luminal cell identity and restricts breast cancer progression.

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March 2021
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy The RNA-Seq and ATAC-Seq data generated in this study were aligned to mouse genome assembly (mm10) and have been deposited in NCBI's Gene Expression Omnibus database and are accessible through GEO Series accession number GSE195716. The CyTOF data was uploaded to the FlowRepository, FR-FCM-Z5L5 and FR-FCM-Z5L6. All other relevant data are available from the corresponding authors upon reasonable request.

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Sample size
No sample size calculations were performed. Animal sample size was determined by the literature and the number of biological replicates necessary for ensuring statistical significance. 5 WT-PyMT mouse tumors were used for in vivo studies. 3 PyMT cell lines for each genotype were independently derived. The number of biological replicates are reported in the relevant figure legends in the manuscript.
Data exclusions No data was excluded.

Replication
Experiments were performed at least 2 times independently. Similar observation was obtained for each replicate and replicated data points are presented in the figures.
Randomization Not relevant for our study. Comparisons were performed between samples with determined status.

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The experiments and analyses were performed in a non-blinded fashion since the same investigators performed group allocations during data collection and analysis. Our analyses all generate objective outcomes that are not subject to observer bias.

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We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Validation All antibodies were validated by their respective manufacturer. The LATS1 antibody (CST, #3477) was validated by CST https:// www.cellsignal.com/products/primary-antibodies/lats1-c66b5-rabbit-mab/3477?site-search-type=Products&N=4294956287&Ntt=lats1&fromPage=plp and also validated for mouse LATS1 by us by Western blot comparison of WT LATS1 vs LATS1-null lysates (see figure 4b).

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Cell line source(s) MDA-MB-468, MCF 10A and HEK293T-phoenix cells were obtained from ATCC. PyMT-derived cell lines were generated in house (as described in Methods section of manuscript).
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All cell lines tested negative for mycoplasma.
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Laboratory animals
Mice were housed in a pathogen-free facility in single-unit cages with 12-hr alternate light and dark cycles and at controlled ambient temperature (21-23 C) with humidity between 40%-60% with free access to water and irradiated food. For PyMT tumor samples, tumors were harvested from five individual 3.5 month old FBV/N-PyMT female mice (purchased from Jackson, strain #002374). Maximum tumor size (10% of body weight) permitted by the Animal Ethics Committee was not exceeded.

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This study did not involve wild animals.
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All mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Weizmann Institute (approval #06320720-2).
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Population characteristics
The breast cancer sub-study (N=16) derive from a German prospective observational trial of ER-positive, postmenopausal female patients with early-stage invasive breast cancer diagnosis, who were recruited between 2005 and 2011 (N=1286). The median age at diagnosis of the N=16 samples is 63 years (range 51-75 years). Treatment included surgical removal, followed by radiation (N=10), chemotherapy (N=4), and intended 5 years of endocrine treatment (Tamoxifen N=5, Aromatase inhibitors N=7, agent switch usually after 2.5 years N=4).

Recruitment
The study is multicentric with >25 national hospitals plus one centre from Liverpool, UK, for patients recruitment. Patients represent incidental cases from the respective hospital. There is no evidence for selection bias.

Ethics oversight
The study was carried out in accordance with the provisions of the declaration of Helsinki of 1975 and ethics approval was obtained from the Ethics Commission of the University of Tübingen, Germany, and respective local ethics committees of participating centers. The immunohistological assays of breast cancer patient samples presented in this study complied to the Weizmann Institutional Review Board (IRB) approval.
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Flow Cytometry
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For FACS analyses a Guava EasyCyte (Milipore) was used. For FACS-based sorting a FACSAria III instrument (BD Biosciences) was used.