Copy number amplification of ENSA promotes the progression of triple-negative breast cancer via cholesterol biosynthesis

Copy number alterations (CNAs) are pivotal genetic events in triple-negative breast cancer (TNBC). Here, our integrated copy number and transcriptome analysis of 302 TNBC patients reveals that gene alpha-endosulfine (ENSA) exhibits recurrent amplification at the 1q21.3 region and is highly expressed in TNBC. ENSA promotes tumor growth and indicates poor patient survival in TNBC. Mechanistically, we identify ENSA as an essential regulator of cholesterol biosynthesis in TNBC that upregulates the expression of sterol regulatory element-binding transcription factor 2 (SREBP2), a pivotal transcription factor in cholesterol biosynthesis. We confirm that ENSA can increase the level of p-STAT3 (Tyr705) and activated STAT3 binds to the promoter of SREBP2 to promote its transcription. Furthermore, we reveal the efficacy of STAT3 inhibitor Stattic in TNBC with high ENSA expression. In conclusion, the amplification of ENSA at the 1q21.3 region promotes TNBC progression and indicates sensitivity to STAT3 inhibitors.

1. The FUSCC TNBC used for integrated analysis included 302 women with TNBC. This cohort was from Fudan University Shanghai Cancer Center (FUSCC) TNBC cohort (Sequence Read Archive (SRA) dataset: SRP157974; Gene Expression Omnibus (GEO) dataset: GSE118527) .Among these 465 patients, 302 patients who had both RNA-seq data and copy number data were included in our study for screening candidate genes. 2. Other sample sizes determined for experiments are included in the relevant figure legends.
no data were excluded The reproducibility for each analysis was confirmed by at least three independent experiments.
For animal experiments, Female 6-week-old NOD.CB17-Prkdc scid/JSlac mice underwent randomization before cell injection. For the treatment groups, when the tumor volumes reached 50-100 mm3, mice underwent the second randomization before beding assigned to vehicle or Stattic (10 mg/kg) treatment.
Data acquisition in the studies, was conducted in a blinded manner. For animal experiments, tumor volumes were measured without knowing the cell type injected (by assigning numerical IDs). For the other experiments, samples were also assigned numerical IDs to process in a blind fashion.
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Female 6-week-old NOD.CB17-Prkdc scid/JSlac mice were used for the in vivo mouse xenograft models. Female 5-week-old nu/nu mice were used for the establishment of mini-PDX models. Mice were exposed to 12h light, 12h darkness cycle at temperature of 21 ±3 ‫خ‬ and an average of 55% humidity.

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For cell cycle analysis, a total of 1×106 cells were fixed with precooled 70% ethanol overnight and then processed using the Cell Cycle and Apoptosis Analysis Kit (Yeasen, #40301ES50) according to the manufacturer's instructions. For the cell apoptosis assay, 5×105 cells were collected and incubated with annexin V-fluorescin isothiocyanate (FITC) and propidium iodide (PI) staining solution from the Annexin V-FITC/PI Apoptosis Detection Kit (Yeasen, #40302ES50).