Original ArticlesNeratinib induces ErbB2 ubiquitylation and endocytic degradation via HSP90 dissociation in breast cancer cells
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.
References (20)
- et al.
All ErbB receptors other than the epidermal growth factor receptor are endocytosis impaired
J. Biol. Chem
(1996) - et al.
Endocytosis and intracellular trafficking of ErbBs
Exp. Cell Res
(2009) - et al.
Governance of endocytic trafficking and signaling by reversible ubiquitylation
Dev. Cell
(2012) - et al.
Neratinib shows efficacy in the treatment of HER2 amplified carcinosarcoma in vitro and in vivo
Gynecol. Oncol
(2015) - et al.
EGF-ERBB signalling: towards the systems level
Nat. Rev. Mol. Cell Biol
(2006) - et al.
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
(2015) - et al.
Targeting HER2 for the treatment of breast cancer
Annu. Rev. Med
(2015) - et al.
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
(2003) - et al.
Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase
Cancer Res
(2004) - et al.
The development of HKI-272 and related compounds for the treatment of cancer
Arch. Pharm. (Weinheim)
(2008)
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These authors contributed equally.