Neratinib induces ErbB2 ubiquitylation and endocytic degradation via HSP90 dissociation in breast cancer cells

doi:10.1016/j.canlet.2016.08.026

Cancer Letters

Volume 382, Issue 2, 28 November 2016, Pages 176-185

https://doi.org/10.1016/j.canlet.2016.08.026 Get rights and content

Highlights

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Tyrosine kinase inhibitors lapatinib and neratinib show opposite effects on ErbB2 levels.
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Both lapatinib and neratinib treatments elevate ErbB2 mRNA.
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Neratinib induces more potent endocytic degradation via endolysosome system than lapatinib.
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Neratinib triggers ErbB2 ubiquitylation through HSP90 dissociation.

Abstract

Receptor tyrosine kinase ErbB2/HER2 is frequently observed to be overexpressed in human cancers, leading to over activation of downstream signaling modules. HER2 positive is a major type of breast cancer for which ErbB2 targeting is already proving to be an effective therapeutic strategy. Apart from antibodies against ErbB2, the small molecule tyrosine kinase inhibitor lapatinib has had successful clinical outcomes, and other inhibitors such as neratinib are currently undergoing clinical investigations. In this study we report the effects of lapatinib and neratinib on the mRNA and protein levels of the ErbB2 receptor. We provide evidence that neratinib-induced down regulation of ErbB2 occurs through ubiquitin-mediated endocytic sorting and lysosomal degradation. At the mechanistic level, neratinib treatment leads to HSP90 release from ErbB2 and its subsequent ubiquitylation and endocytic degradation. Our findings provide novel insights into the mechanism of ErbB2 inhibition by neratinib.

Introduction

ErbB2/HER2 belongs to the ErbB family of receptor tyrosine kinases, with amplification frequently observed in a number of malignancies [1]. ErbB2/HER2 amplification identifies a major subtype of breast cancer called HER2-positive breast cancer that accounts for approximately 25–30% of all breast cancer cases [2]. Targeting ErbB2 has proven to be an effective strategy in the clinical treatment of HER2-positive breast malignancies. Two approaches have been approved by the US Food and Drug Administration (FDA) for clinical use: humanized monoclonal antibodies targeting the extracellular domain of ErbB2 and small molecule tyrosine kinase inhibitors (TKI) that block the kinase activity of ErbB2 [3]. Antibody based therapies include trastuzumab, pertuzumab, and the antibody-drug conjugate ado-trastuzumab emtansine (T-DM1). Lapatinib is a tyrosine kinase inhibitor treatment and is reversible, targeting both ErbB2 and EGFR.

Neratinib (HKI-272) is another small molecule inhibitor that blocks the kinase activities of EGFR and ErbB2 [4]. Unlike lapatinib, neratinib irreversibly binds to the kinase domain of EGFR and ErbB2 via covalent interaction with conserved cysteine residues: C773 of EGFR and C805 of ErbB2 [5], [6]. Multiple clinical investigations of neratinib treatment of HER2-positive breast cancer and other solid tumors are ongoing, with promising results for future treatment of HER2-positive malignancies [2].

The cell surface levels of many receptor tyrosine kinases are tightly regulated by endocytosis, which internalizes membrane receptors into the cells and subsequently sorts them into lysosomes for degradation via the endosome and multivesicular body systems [7]. ErbB2 is normally endocytosis impaired and requires the chaperone HSP90 for stability on the cell surface [8], [9]. HSP90 inhibitors have been reported to induce ErbB2 degradation through either proteasomal or lysosomal pathways, which are regulated by CHIP-mediated post-translational modifications of ErbB2 by ubiquitin (ubiquitylation) [10], [11], [12].

The mechanistic studies of TKI lapatinib and neratinib mainly focus on the inhibition of receptor kinase activity and corresponding downstream signaling pathways, most prominently the RAS–MAPK and PI3K–AKT signaling cascades, both of which can be efficiently suppressed by lapatinib and neratinib. Conversely, the effects of lapatinib and neratinib on the expression levels of EGFR and ErbB2 receptors per se remain underexplored. In the present study, we report that treatment with lapatinib and neratinib leads to increased and decreased ErbB2 expression levels respectively, although ErbB2 mRNA is increased by both inhibitors. The two TKIs also induce the endocytosis of ErbB2. Mechanistically, neratinib triggers potent ubiquitylation and endocytic degradation of ErbB2 via HSP90 dissociation from ErbB2.

Section snippets

Antibodies and other reagents

Mouse anti-ErbB2 (A-2), mouse anti-ErbB2 (9G6), mouse anti-LAMP1, rabbit anti-EEA1, and rabbit anti-GAPDH (FL-335) antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Mouse anti-GAPDH antibody was purchased from Proteintech (Wuhan, China). Goat anti-ErbB2 N-terminus antibody was purchased from R&D. Rabbit anti-HER2/ErbB2 (29D8), rabbit anti-phospho-S6RP, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-MEK1/2 (Ser217/221), mouse anti-phospho-p44/42 MAPK (Erk1/2)

Lapatinib and neratinib show opposite effects on ErbB2 levels

Given that breast cancer with ErbB2/HER2 amplification is the primary target for lapatinib and neratinib, we used three ErbB2-overexpressing breast cancer cell lines in this study: SKBR3, AU565, and HCC1954. We treated SKBR3 and AU565 cells with lapatinib and neratinib (both at 500 nM). As expected, the phosphorylation of ErbB2, mTOR, AKT, S6RP, MEK and ERK1/2 downstream of ErbB2 is potently inhibited, demonstrating the effectiveness of the inhibitors on ErbB2 kinase activity (Fig. 1A).

Discussion

Breast cancer remains the most frequently diagnosed malignancy in women, accounting for approximately 30% of estimated new female cancer cases in the US [17]. Breast cancer can be divided into four subtypes: luminal A, luminal B, basal like, and HER2 positive. ErbB2/HER2 overexpression confers results in poor prognosis for this HER2-positive subtype, while offering a druggable therapeutic target. In this regard, the FDA approved ErbB2 targeting therapies, including trastuzumab (Herceptin), a

Authors' contributions

ML and HL designed the project. YZ, JZ, CL, SD, LF, XL, YZ, YS, TW, and YW performed all experiments. YZ, JZ, WC, and SM analyzed the data. YZ, JZ, and HL wrote the manuscript. All authors reviewed the manuscript.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China [No. 81301901]; and the “Climbing Scholar” and “Excellent Talents” schemes of Liaoning Province, China.

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¹: These authors contributed equally.

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Original ArticlesNeratinib induces ErbB2 ubiquitylation and endocytic degradation via HSP90 dissociation in breast cancer cells

Highlights

Abstract

Introduction

Section snippets

Antibodies and other reagents

Lapatinib and neratinib show opposite effects on ErbB2 levels

Discussion

Authors' contributions

Conflict of interest

Acknowledgments

J. Biol. Chem

Exp. Cell Res

Dev. Cell

Gynecol. Oncol

EGF-ERBB signalling: towards the systems level

Nat. Rev. Mol. Cell Biol

Efficacy and mechanism of action of the tyrosine kinase inhibitors gefitinib, lapatinib and neratinib in the treatment of HER2-positive breast cancer: preclinical and clinical evidence

Am. J. Cancer Res

Targeting HER2 for the treatment of breast cancer

Annu. Rev. Med

Synthesis and structure-activity relationships of 6,7-disubstituted 4-anilinoquinoline-3-carbonitriles. The design of an orally active, irreversible inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) and the human epidermal growth factor receptor-2 (HER-2)

J. Med. Chem

Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase

Cancer Res

The development of HKI-272 and related compounds for the treatment of cancer

Arch. Pharm. (Weinheim)

Original Articles
Neratinib induces ErbB2 ubiquitylation and endocytic degradation via HSP90 dissociation in breast cancer cells