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

in Endocrine-Related Cancer
Authors:
Yuanzhong Wang Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California, USA

Search for other papers by Yuanzhong Wang in
Current site
Google Scholar
PubMed Close
,
Yen-Dun Tony Tzeng Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California, USA
Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung City, Taiwan, Republic of China

Search for other papers by Yen-Dun Tony Tzeng in
Current site
Google Scholar
PubMed Close
,
Gregory Chang Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California, USA

Search for other papers by Gregory Chang in
Current site
Google Scholar
PubMed Close
,
Xiaoqiang Wang Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California, USA

Search for other papers by Xiaoqiang Wang in
Current site
Google Scholar
PubMed Close
https://orcid.org/0000-0002-1892-5613
, and
Shiuan Chen Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California, USA

Search for other papers by Shiuan Chen in
Current site
Google Scholar
PubMed Close

Correspondence should be addressed to S Chen: schen@coh.org
DOI:
https://doi.org/10.1530/ERC-20-0258
Volume/Issue:
Volume 27: Issue 12
Article Type:
Research Article
Page Range:
671–683
Online Publication Date:
Dec 2020
Copyright:
© 2020 Society for Endocrinology 2020
Restricted access
Rent on DeepDyve

Sign up for journal news

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.

Endocrine-Related Cancer is committed to supporting researchers in demonstrating the impact of their articles published in the journal.

The two types of article metrics we measure are (i) more traditional full-text views and pdf downloads, and (ii) Altmetric data, which shows the wider impact of articles in a range of non-traditional sources, such as social media.

More information is on the Reasons to publish page.

Sept 2018 onwards Past Year Past 30 Days
Full Text Views 770 37 0
PDF Downloads 637 26 0

 

  • Collapse
  • Expand
Print ISSN:
1351-0088
Online ISSN:
1479-6821
Copyright:
© 2020 Society for Endocrinology 2020
Page(s):
13
Received Date:
26 Aug 2020
Accepted Date:
29 Sep 2020
Print Publication Date:
01 Dec 2020
Online Publication Date:
Dec 2020
Keywords:
endocrine resistance; estrogen receptor; mTOR; FOXM1; SGK3

  • Aggelis V & Johnston SRD 2019 Advances in endocrine-based therapies for estrogen receptor-positive metastatic breast cancer. Drugs 79 1849–1866. (https://doi.org/10.1007/s40265-019-01208-8)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Alayev A, Salamon RS, Berger SM, Schwartz NS, Cuesta R, Snyder RB & Holz MK 2016 mTORC1 directly phosphorylates and activates ERalpha upon estrogen stimulation. Oncogene 35 3535–3543. (https://doi.org/10.1038/onc.2015.414)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Araki K & Miyoshi Y 2018 Mechanism of resistance to endocrine therapy in breast cancer: the important role of PI3K/Akt/mTOR in estrogen receptor-positive, HER2-negative breast cancer. Breast Cancer 25 392–401. (https://doi.org/10.1007/s12282-017-0812-x)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Baselga J, Campone M, Piccart M, Burris HA 3rd, Rugo HS, Sahmoud T, Noguchi S, Gnant M, Pritchard KI & Lebrun F et al.2012 Everolimus in postmenopausal hormone-receptor-positive advanced breast cancer. New England Journal of Medicine 366 520–529. (https://doi.org/10.1056/NEJMoa1109653)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Berasain C & Avila MA 2014 Amphiregulin. Seminars in Cell and Developmental Biology 28 31–41. (https://doi.org/10.1016/j.semcdb.2014.01.005)

  • Bergamaschi A, Madak-Erdogan Z, Kim YJ, Choi YL, Lu H & Katzenellenbogen BS 2014 The forkhead transcription factor FOXM1 promotes endocrine resistance and invasiveness in estrogen receptor-positive breast cancer by expansion of stem-like cancer cells. Breast Cancer Research 16 436. (https://doi.org/10.1186/s13058-014-0436-4)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Campbell RA, Bhat-Nakshatri P, Patel NM, Constantinidou D, Ali S & Nakshatri H 2001 Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance. Journal of Biological Chemistry 276 9817–9824. (https://doi.org/10.1074/jbc.M010840200)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Ciarloni L, Mallepell S & Brisken C 2007 Amphiregulin is an essential mediator of estrogen receptor alpha function in mammary gland development. PNAS 104 5455–5460. (https://doi.org/10.1073/pnas.0611647104)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • DeRose YS, Gligorich KM, Wang G, Georgelas A, Bowman P, Courdy SJ, Welm AL & Welm BE 2013 Patient-derived models of human breast cancer: protocols for in vitro and in vivo applications in tumor biology and translational medicine. Current Protocols in Pharmacology Chapter 14 Unit14.23. (https://doi.org/10.1002/0471141755.ph1423s60)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Eeckhoute J, Keeton EK, Lupien M, Krum SA, Carroll JS & Brown M 2007 Positive cross-regulatory loop ties GATA-3 to estrogen receptor alpha expression in breast cancer. Cancer Research 67 6477–6483. (https://doi.org/10.1158/0008-5472.CAN-07-0746)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Hsu PY, Wu VS, Kanaya N, Petrossian K, Hsu HK, Nguyen D, Schmolze D, Kai M, Liu CY & Lu H et al.2018 Dual mTOR kinase inhibitor MLN0128 sensitizes HR(+)/HER2(+) breast cancer patient-derived xenografts to trastuzumab or fulvestrant. Clinical Cancer Research 24 395–406. (https://doi.org/10.1158/1078-0432.CCR-17-1983)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Jeong Y, Bae SY, You D, Jung SP, Choi HJ, Kim I, Lee SK, Yu J, Kim SW & Lee JE et al.2019 EGFR is a therapeutic target in hormone receptor-positive breast cancer. Cellular Physiology and Biochemistry 53 805–819. (https://doi.org/10.33594/000000174)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Kornblum N, Zhao F, Manola J, Klein P, Ramaswamy B, Brufsky A, Stella PJ, Burnette B, Telli M & Makower DF et al.2018 Randomized Phase II trial of fulvestrant plus everolimus or placebo in postmenopausal women with hormone receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer resistant to aromatase inhibitor therapy: results of PrE0102. Journal of Clinical Oncology 36 1556–1563. (https://doi.org/10.1200/JCO.2017.76.9331)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Lin NU & Winer EP 2008 Advances in adjuvant endocrine therapy for postmenopausal women. Journal of Clinical Oncology 26 798–805. (https://doi.org/10.1200/JCO.2007.15.0946)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Lui K, Tamura T, Mori T, Zhou D & Chen S 2008 MCF-7aro/ERE, a novel cell line for rapid screening of aromatase inhibitors, ERalpha ligands and ERRalpha ligands. Biochemical Pharmacology 76 208–215. (https://doi.org/10.1016/j.bcp.2008.04.011)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Ma CX, Reinert T, Chmielewska I & Ellis MJ 2015 Mechanisms of aromatase inhibitor resistance. Nature Reviews: Cancer 15 261–275. (https://doi.org/10.1038/nrc3920)

  • Madureira PA, Varshochi R, Constantinidou D, Francis RE, Coombes RC, Yao KM & Lam EW 2006 The Forkhead box M1 protein regulates the transcription of the estrogen receptor alpha in breast cancer cells. Journal of Biological Chemistry 281 25167–25176. (https://doi.org/10.1074/jbc.M603906200)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Manning BD & Toker A 2017 AKT/PKB signaling: navigating the network. Cell 169 381–405. (https://doi.org/10.1016/j.cell.2017.04.001)

  • Masri S, Phung S, Wang X, Wu X, Yuan YC, Wagman L & Chen S 2008 Genome-wide analysis of aromatase inhibitor-resistant, tamoxifen-resistant, and long-term estrogen-deprived cells reveals a role for estrogen receptor. Cancer Research 68 4910–4918. (https://doi.org/10.1158/0008-5472.CAN-08-0303)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Meier DR, Girtman MA, Lofgren KA & Kenny PA 2020 Amphiregulin deletion strongly attenuates the development of estrogen receptor-positive tumors in p53 mutant mice. Breast Cancer Research and Treatment 179 653–660. (https://doi.org/10.1007/s10549-019-05507-2)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Miller TW, Balko JM & Arteaga CL 2011 Phosphatidylinositol 3-kinase and antiestrogen resistance in breast cancer. Journal of Clinical Oncology 29 4452–4461. (https://doi.org/10.1200/JCO.2010.34.4879)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Millour J, Constantinidou D, Stavropoulou AV, Wilson MS, Myatt SS, Kwok JM, Sivanandan K, Coombes RC, Medema RH & Hartman J et al.2010 FOXM1 is a transcriptional target of ERalpha and has a critical role in breast cancer endocrine sensitivity and resistance. Oncogene 29 2983–2995. (https://doi.org/10.1038/onc.2010.47)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Osborne CK, Shou J, Massarweh S & Schiff R 2005 Crosstalk between estrogen receptor and growth factor receptor pathways as a cause for endocrine therapy resistance in breast cancer. Clinical Cancer Research 11 865s–870s. (https://doi.org/10.1158/1078-0432.CCR-031212)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Paridaens RJ, Dirix LY, Beex LV, Nooij M, Cameron DA, Cufer T, Piccart MJ, Bogaerts J & Therasse P 2008 Phase III study comparing exemestane with tamoxifen as first-line hormonal treatment of metastatic breast cancer in postmenopausal women: the European Organisation for Research and Treatment of Cancer Breast Cancer Cooperative Group. Journal of Clinical Oncology 26 4883–4890. (https://doi.org/10.1200/JCO.2007.14.4659)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Peterson EA, Jenkins EC, Lofgren KA, Chandiramani N, Liu H, Aranda E, Barnett M & Kenny PA 2015 Amphiregulin is a critical downstream effector of estrogen signaling in ERalpha-positive breast cancer. Cancer Research 75 4830–4838. (https://doi.org/10.1158/0008-5472.CAN-15-0709)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Quintela I, Corte MD, Allende MT, Vazquez J, Rodriguez JC, Bongera M, Lamelas M, Gonzalez LO, Vega A & Garcia-Muniz JL et al.2005 Expression and prognostic value of EGFR in invasive breast cancer. Oncology Reports 14 1655–1663. (https://doi.org/10.3892/or.14.6.1655)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Sanders DA, Ross-Innes CS, Beraldi D, Carroll JS & Balasubramanian S 2013 Genome-wide mapping of FOXM1 binding reveals co-binding with estrogen receptor alpha in breast cancer cells. Genome Biology 14 R6. (https://doi.org/10.1186/gb-2013-14-1-r6)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Shoyab M, McDonald VL, Bradley JG & Todaro GJ 1988 Amphiregulin: a bifunctional growth-modulating glycoprotein produced by the phorbol 12-myristate 13-acetate-treated human breast adenocarcinoma cell line MCF-7. PNAS 85 6528–6532. (https://doi.org/10.1073/pnas.85.17.6528)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Stoll SW, Stuart PE, Swindell WR, Tsoi LC, Li B, Gandarillas A, Lambert S, Johnston A, Nair RP & Elder JT 2016 The EGF receptor ligand amphiregulin controls cell division via FoxM1. Oncogene 35 2075–2086. (https://doi.org/10.1038/onc.2015.269)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Turnbull AK, Arthur LM, Renshaw L, Larionov AA, Kay C, Dunbier AK, Thomas JS, Dowsett M, Sims AH & Dixon JM 2015 Accurate prediction and validation of response to endocrine therapy in breast cancer. Journal of Clinical Oncology 33 2270–2278. (https://doi.org/10.1200/JCO.2014.57.8963)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Vasudevan KM, Barbie DA, Davies MA, Rabinovsky R, McNear CJ, Kim JJ, Hennessy BT, Tseng H, Pochanard P & Kim SY et al.2009 AKT-independent signaling downstream of oncogenic PIK3CA mutations in human cancer. Cancer Cell 16 21–32. (https://doi.org/10.1016/j.ccr.2009.04.012)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Vecchi M, Rudolph-Owen LA, Brown CL, Dempsey PJ & Carpenter G 1998 Tyrosine phosphorylation and proteolysis. Pervanadate-induced, metalloprotease-dependent cleavage of the ErbB-4 receptor and amphiregulin. Journal of Biological Chemistry 273 20589–20595. (https://doi.org/10.1074/jbc.273.32.20589)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Wang X, Masri S, Phung S & Chen S 2008 The role of amphiregulin in exemestane-resistant breast cancer cells: evidence of an autocrine loop. Cancer Research 68 2259–2265. (https://doi.org/10.1158/0008-5472.CAN-07-5544)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Wang Y, Zhou D, Phung S, Masri S, Smith D & Chen S 2011 SGK3 is an estrogen-inducible kinase promoting estrogen-mediated survival of breast cancer cells. Molecular Endocrinology 25 72–82. (https://doi.org/10.1210/me.2010-0294)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Wang Y, Xu W, Zhou D, Neckers L & Chen S 2014 Coordinated regulation of serum- and glucocorticoid-inducible kinase 3 by a C-terminal hydrophobic motif and Hsp90-Cdc37 chaperone complex. Journal of Biological Chemistry 289 4815–4826. (https://doi.org/10.1074/jbc.M113.518480)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Wang Y, Zhou D, Phung S, Warden C, Rashid R, Chan N & Chen S 2017 SGK3 sustains ERalpha signaling and drives acquired aromatase inhibitor resistance through maintaining endoplasmic reticulum homeostasis. PNAS 114 E1500–E1508. (https://doi.org/10.1073/pnas.1612991114)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Willmarth NE & Ethier SP 2006 Autocrine and juxtacrine effects of amphiregulin on the proliferative, invasive, and migratory properties of normal and neoplastic human mammary epithelial cells. Journal of Biological Chemistry 281 37728–37737. (https://doi.org/10.1074/jbc.M606532200)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Willmarth NE, Baillo A, Dziubinski ML, Wilson K, Riese DJ 2nd & Ethier SP 2009 Altered EGFR localization and degradation in human breast cancer cells with an amphiregulin/EGFR autocrine loop. Cellular Signalling 21 212–219. (https://doi.org/10.1016/j.cellsig.2008.10.003)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Xu J, Liao L, Qin J, Xu J, Liu D & Songyang Z 2009 Identification of flightless-I as a substrate of the cytokine-independent survival kinase CISK. Journal of Biological Chemistry 284 14377–14385. (https://doi.org/10.1074/jbc.M807770200)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Xu J, Wan M, He Q, Bassett Jr RL, Fu X, Chen AC, Shi F, Creighton CJ, Schiff R & Huo L et al.2012 SGK3 is associated with estrogen receptor expression in breast cancer. Breast Cancer Research and Treatment 134 531–541. (https://doi.org/10.1007/s10549-012-2081-x)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Xu Q, Chiao P & Sun Y 2016 Amphiregulin in cancer: new insights for translational medicine. Trends in Cancer 2 111–113. (https://doi.org/10.1016/j.trecan.2016.02.002)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Yamnik RL, Digilova A, Davis DC, Brodt ZN, Murphy CJ & Holz MK 2009 S6 kinase 1 regulates estrogen receptor alpha in control of breast cancer cell proliferation. Journal of Biological Chemistry 284 6361–6369. (https://doi.org/10.1074/jbc.M807532200)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Yang L, Yang S, Li X, Li B, Li Y, Zhang X, Ma Y, Peng X, Jin H & Fan Q et al.2019 Tumor organoids: from inception to future in cancer research. Cancer Letters 454 120–133. (https://doi.org/10.1016/j.canlet.2019.04.005)

    • PubMed
    • Search Google Scholar
    • Export Citation