Everolimus downregulates estrogen receptor and induces autophagy in aromatase inhibitor-resistant breast cancer cells

Abstract

BackgroundmTOR inhibition of aromatase inhibitor (AI)-resistant breast cancer is currently under evaluation in the clinic. Everolimus/RAD001 (Afinitor®) has had limited efficacy as a solo agent but is projected to become part of combination therapy for AI-resistant breast cancer. This study was conducted to investigate the anti-proliferative and resistance mechanisms of everolimus in AI-resistant breast cancer cells.MethodsIn this study we utilized two AI-resistant breast cancer cell lines, MCF-7:5C and MCF-7:2A, which were clonally derived from estrogen receptor positive (ER+) MCF-7 breast cancer cells following long-term estrogen deprivation. Cell viability assay, colony formation assay, cell cycle analysis and soft agar anchorage-independent growth assay were used to determine the efficacy of everolimus in inhibiting the proliferation and tumor forming potential of MCF-7, MCF-7:5C, MCF-7:2A and MCF10A cells. Confocal microscopy and transmission electron microscopy were used to evaluate LC3-II production and autophagosome formation, while ERE-luciferase reporter, Western blot, and RT-PCR analyses were used to assess ER expression and transcriptional activity.ResultsEverolimus inhibited the proliferation of MCF-7:5C and MCF-7:2A cells with relatively equal efficiency to parental MCF-7 breast cancer cells. The inhibitory effect of everolimus was due to G1 arrest as a result of downregulation of cyclin D1 and p21. Everolimus also dramatically reduced estrogen receptor (ER) expression (mRNA and protein) and transcriptional activity in addition to the ER chaperone, heat shock protein 90 protein (HSP90). Everolimus restored 4-hydroxy-tamoxifen (4OHT) sensitivity in MCF-7:5C cells and enhanced 4OHT sensitivity in MCF-7 and MCF-7:2A cells. Notably, we found that autophagy is one method of everolimus insensitivity in MCF-7 breast cancer cell lines.ConclusionThis study provides additional insight into the mechanism(s) of action of everolimus that can be used to enhance the utility of mTOR inhibitors as part of combination therapy for AI-resistant breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2490-z) contains supplementary material, which is available to authorized users.

IL‐1 induces p62/SQSTM1 and autophagy in ERα+/PR+ BCa cell lines concomitant with ERα and PR repression, conferring an ERα−/PR− BCa‐like phenotype

“…We show that IL‐1 signaling mediates HS‐5 BMSC repression of ERα and PR levels concomitant with p62 upregulation and autophagy induction in ERα + /PR + BCa cell lines (Figures ); and we contend that IL‐1‐induced p62 upregulation and autophagy induction also contribute to BCa cell survival, growth, and endocrine resistance when hormone receptors are repressed. In support of this notion, knockout or silencing of p62 or autophagy have been shown to prevent BCa initiation, progression, or metastasis in mouse models and in vitro studies demonstrate that autophagy is cytoprotective against endocrine therapy …”

Mechanisms of resistance in estrogen receptor positive breast cancer: overcoming resistance to tamoxifen/aromatase inhibitors

“…Genomic alterations in PIK3CA , the gene encoding the catalytic subunit of PI3K, are common in ER+ tumors [28 ● ,29,30]. It is thought that suppression of the PI3K/AKT/mTOR pathway can lead to increased activity of ER, facilitating resistance, and therefore many groups have used preclinical models to elucidate the mechanisms of PI3K/AKT/mTOR pathway inhibitors and the influence these compounds have on estrogen-mediated signaling [19 ● ,21 ●● ,25,31–33]. …”

“…Evidence suggests that, while inhibition of the PI3K/AKT/mTOR pathway at all levels results in reduced cell proliferation and survival, the complexity of this signaling pathway ensures that compensatory mechanisms are activated that confer resistance to single inhibitors [32,33]. For example, inhibition of mTOR results in activation of AKT as well as extracellular signal-regulated kinase (ERK), leading to increased signaling through alternative branches of these pathways [19 ● ,21 ●● ].…”

PHENSIM: Phenotype Simulator

“…phosphorylates ULK1 (unc-51 like autophagy activating kinase 1) and DAP (death-associated protein) inhibiting autophagy. 15 All these functions of mTORC1 are reversed by everolimus and other mTORC1 inhibitors 16,17 (fig. 2).…”