TET2 directs mammary luminal cell differentiation and endocrine response

Epigenetic regulation plays an important role in governing stem cell fate and tumorigenesis. Lost expression of a key DNA demethylation enzyme TET2 is associated with human cancers and has been linked to stem cell traits in vitro; however, whether and how TET2 regulates mammary stem cell fate and mammary tumorigenesis in vivo remains to be determined. Here, using our recently established mammary specific Tet2 deletion mouse model, the data reveals that TET2 plays a pivotal role in mammary gland development and luminal lineage commitment. We show that TET2 and FOXP1 form a chromatin complex that mediates demethylation of ESR1, GATA3, and FOXA1, three key genes that are known to coordinately orchestrate mammary luminal lineage specification and endocrine response, and also are often silenced by DNA methylation in aggressive breast cancers. Furthermore, Tet2 deletion-PyMT breast cancer mouse model exhibits enhanced mammary tumor development with deficient ERα expression that confers tamoxifen resistance in vivo. As a result, this study elucidates a role for TET2 in governing luminal cell differentiation and endocrine response that underlies breast cancer resistance to anti-estrogen treatments.

. n=4 data points analyzed from four independent tissue section staining images of two animals for each group in (e), (h), (i), (j). n=8 data points analyzed from eight independent tissue section staining images of two animals for each group in (f). n=6 data points analyzed from six independent tissue section staining images of two animals for each group in (g). Quantification of TEB size was determined by measuring the relative TEB area using ImageJ. Quantification of fibrosis was determined by measuring the relative tricrhome blue staining intensity over an equal vision field area using ImageJ Fiji. (k) In vitro limiting dilution analysis showing calculated frequency of sphere forming WT and KO MaSCs (n=5 independent experiments). Representative confocal immunofluorescence images of (l) acini stained for basal polarity marker (integrin-a6, green), epithelial cell marker (E-cadherin, red) and DAPI (nucleus, blue; scale bar: 50mm). The percentage of normal polarized acini was analyzed from 10-20 acini each field for each group (n=3 independent experiments).

Statistics
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Data
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Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
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Life sciences study design
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Sample size
The sample sizes in each experimental group was determined from our preliminary experiments and based on 80% power and two-sided tests for 5% level of significance.
Data exclusions All samples that met adequate experimental conditions were included in the analysis; no methods were used to determine whether the data met assumptions of the statistical approach; no inclusion/exclusion criteria/cases were applied.

Replication
Experiments were successfully performed for at least 3 times and/or with sufficient animals per group to demonstrate statistical significance as specified in the figure legend.
Randomization Only female animals were used for the purpose of studying mammary gland development and breast cancer implication. The female animals were age matched litter mates housed in the same cage with synchronized estrous cycle. Cell dishes for different treatments was allocated by randomization.

nature research | reporting summary
October 2018

Field-collected samples
No Field-collected samples were used in the study.

Ethics oversight
Experiments were conducted with approval of the Animal Care and Use Committees at Purdue University and Roswell Park Comprehensive Cancer Center.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
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Methodology Sample preparation
Mammary epithelial cells were isolated from the mouse mammary gland and prepared and stained as described in Method.

Instrument BD FACSAria Fusion, BD FACSCantoII
Software FCS express 6 (Denovo Software) Cell population abundance 10,000 cells to 100,000 cells were acquired per sample, the total population was analyzed.

Gating strategy
The gating boundaries were determined by using fsc/ssc and mouse linage marker cocktail as shown in Supplementary Fig S4. Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.