Amphiregulin retains ERα expression in acquired aromatase inhibitor resistant breast cancer cells

in Endocrine-Related Cancer
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  • 1 Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California, USA
  • 2 Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung City, Taiwan, Republic of China

Correspondence should be addressed to S Chen: schen@coh.org
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Acquired resistance to aromatase inhibitors (AIs) is a significant clinical issue in endocrine therapy for estrogen receptor (ER) positive breast cancer which accounts for the majority of breast cancer. Despite estrogen production being suppressed, ERα signaling remains active and plays a key role in most AI-resistant breast tumors. Here, we found that amphiregulin (AREG), an ERα transcriptional target and EGF receptor (EGFR) ligand, is crucial for maintaining ERα expression and signaling in acquired AI-resistant breast cancer cells. AREG was deregulated and critical for cell viability in ER+ AI-resistant breast cancer cells, and ectopic expression of AREG in hormone responsive breast cancer cells promoted endocrine resistance. RNA-sequencing and reverse phase protein array analyses revealed that AREG maintains ERα expression and signaling by activation of PI3K/Akt/mTOR signaling and upregulation of forkhead box M1 (FOXM1) and serum- and glucocorticoid-inducible kinase 3 (SGK3) expression. Our study uncovers a previously unappreciated role of AREG in maintaining ERα expression and signaling, and establishes the AREG-ERα crosstalk as a driver of acquired AI resistance in breast cancer.

Supplementary Materials

    • upplementary data Amphiregulin retains ERα signaling in acquired aromatase inhibitor resistant breast cancer cells
    • Figure 1. AREG is deregulated in acquired AI-resistant cells. (A) Western blotting analysis of AREG expression. MCF7aro cells were cultured in the normal growth medium (regular), or hormone-deprived for 2 days (HD) and then treated with 1nM T for 48h. LET-R cells were cultured in the normal growth medium. (B) RT-qPCR analysis of AREG expression in EXE-R cells treated with DMSO, 100nM ICI or 0.2µM Wortmannin for 45h. *, p<0.05 vs. DMSO.
    • Figure 2. AREG is critical for viability of acquired AI-resistant cells. (A) Western blotting analysis of EXE-R cells transfected with siRNA negative control or 3 individual AREG siRNAs (which target different regions of AREG mRNA) for 72 h, respectively. (B) MTT assay on EXE-R transfected with siRNA negative control or 3 individual AREG siRNAs, respectively. *, p<0.05. (C) MTT assay on LET-R transfected with siRNA negative control or 3 individual AREG siRNAs, respectively. *, p<0.05. (D, E) HCC1428aro and HCC1428aro/LET-R cells were transfected with siRNA negative control or AREG siRNA. Cell were harvested for Western blotting analysis (D) after 4 days transfection or measured cell proliferation using MTT assay (E) at the time indicated. *, p<0.05.
    • Figure 3. EGFR signaling is deregulated in acquired AI-resistant breast cancer cells. (A) The effect of EGFR inhibitors on cell viability of MCF7aro and AI-resistant cells. Cell viability was measured using MTT at the indicated time after treatment. *, p<0.05 vs. DMSO control, n=3. (B, C) HCC1428aro and HCC1428aro/LET-R cells were transfected with siRNA negative control or EGFR siRNA. Cell were harvested for Western blotting analysis (B) after 4 days transfection or measured cell proliferation using MTT assay (C) at the time indicated. *, p<0.05.
    • Figure 4. AREG maintains EnR homeostasis in AI-resistant cells. (A) Western blotting analysis of MCF7aro transfected with siRNA negative control or AREG siRNA for 72 h. (B) Western blotting analysis of HCC1428aro/LET-R transfected with siRNA negative control or AREG siRNA for 4 days.
    • Figure 5. Ectopic expression of AREG in hormone responsive breast cancer cells promotes endocrine therapy resistance. (A) MTT assay on MCF7aro/pMG and MCF7aro/AREG cells grown in normal growth medium as described in Material and Methods. *, p<0.05, n=3. (B-D) T47D/tetON/AREG cells were cultured in normal growth medium added with or without 100ng/ml DOX, and treated with DMSO, 1µM 4-hydroxyltamoxifen (4OHT) or100nM ICI182,780. Cell viability was measured by MTT assay at the indicated time (B, C). At 96h posttreatment, cells were fixed and stained with crystal violet (D).
    • Figure 6. AREG is important for ERα expression and signaling. (A) Western blotting analysis of MCF7aro and ANA-R cells after transfection with siRNA negative control or AREG siRNA for 72 h. (B) Western blotting analysis of HCC1428aro cells after transfection with siRNA negative control or AREG siRNA for 72 h. (C) Western blotting analysis of primary culture cells from COH-SC31 PDX after transfection with siRNA negative control or three individual AREG siRNAs for 4 days. (D) Western blotting analysis of primary culture cells from COH-SC1 PDX after transfection with siRNA negative control or AREG siRNAs for 4 days.
    • Figure 7. AREG regulation of ERα expression and signaling involves multiple mechanisms. (A) Heatmap of RPPA analysis of LET-R transfected with siRNA negative control or AREG siRNA. Red rectangle highlights most of downregulated proteins and is shown in main Figure 7A. (B) Western blotting analysis of HCC1428aro/LET-R cells treated with DMSO, 0.5 μM Rapamycin, or 0.5 μM INK128 for 48 h. (C) Western blotting analysis of HCC1428aro/LET-R cells transfected with siRNA negative control or AREG siRNA for 72 h. (D) Western blotting of T47D/tetON/AREG cells treated with DMSO, 50nM ICI142,480, or 200nM ICI142,480 for 48h in the presence or absence of 100ng/ml doxycycline.

 

Society for Endocrinology

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