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

Zhang, Yingqiu; Zhang, Jinrui; Liu, Congcong; Du, Sha; Lü, Feng; Luan, Xuelin; Zhang, Yayun; Shi, Yulin; Wang, Taishu; Wu, Yue; Cheng, Wei; Shao, Meng; Li, Man; Liu, Han

doi:10.1016/j.canlet.2016.08.026

Cited by 68 publications

(59 citation statements)

References 19 publications

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“…Our work demonstrates that covalent EGFR/HER2 inhibitors such as afatinib possess TRIB2 degrading-activity in human cells at micromolar concentrations, in a similar range to those reported for other destabilizing kinase inhibitors, including the covalent EGFR/HER2 drug neratinib [85].…”

Section: Discussionsupporting

confidence: 70%

Repurposing covalent EGFR/HER2 inhibitors for on-target degradation of human Tribbles 2 (TRIB2) pseudokinase

Foulkes

Byrne

Bailey

et al. 2018

Preprint

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ONE SENTENCE SUMMARY:A Tribbles 2 pseudokinase small molecule screen led to the identification of known EGFR/HER2 inhibitors that alter the stability of TRIB2 in vitro and lead to rapid on-target degradation of TRIB2 in human cancer cells. SHORT ABSTRACT: (150 words)Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the AKT signaling module. Substantial evidence demonstrates that TRIB2 dysregulation is important in multiple human tumors. The non-canonical TRIB2 pseudokinase domain contains a unique cysteine rich region and interacts with a peptide motif in its own C-terminal tail. We demonstrate that TRIB2 is a target for previously described small molecule protein kinase 'inhibitors', which were originally designed to inhibit the catalytic domain of EGFR/HER2 tyrosine kinases. Using thermal-shift assays and drug repurposing, we classify ligands that stabilize or destabilize the TRIB2 pseudokinase domain. TRIB2 destabilizing agents, including the clinical inhibitor afatinib, lead to rapid and ontarget TRIB2 protein degradation in tumor cells, eliciting tractable effects on cell signaling and survival. Our data identifies leads for further development of TRIB2-degrading drugs and highlights 2 compound-induced TRIB2 downregulation, which might be mechanistically relevant for other catalytically-deficient (pseudo)kinases targeted by small molecules. FULL ABSTRACT: (232 words)A major challenge associated with biochemical and cellular analysis of pseudokinases is the lack of target-validated small molecule ligands with which to probe molecular function. Human Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, which includes the canonical AKT signaling module. There is substantial evidence that human TRIB2 is a therapeutic target in both solid tumors and blood cancers. The non-canonical TRIB2 pseudokinase domain contains a unique cysteine-rich region and interacts with a peptide motif in its own C-terminal tail, which was previously shown to drive interaction with cellular E3 ubiquitin ligases. In this study we demonstrate that TRIB2 is a target for previously described small molecule protein kinase inhibitors, which were originally designed to inhibit the canonical catalytic domain of the tyrosine kinases EGFR/HER2.Using a thermal-shift assay, we discovered TRIB2 ligands within the Published Kinase Inhibitor Set (PKIS), and employed a drug repurposing approach to classify compounds that either stabilize or destabilize TRIB2 in vitro. Remarkably, TRIB2 destabilizing agents, including the clinical covalent drug afatinib, lead to rapid and on-target TRIB2 degradation in human cells, eliciting tractable effects on signaling and survival. Our data reveal the first drug-leads for development of TRIB2-degrading ligands, which will also be invaluable for unravelling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule-induced protein downregulation through drug 'off-targets' might be relevant for other inhibitors th...

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Section: Discussionsupporting

confidence: 70%

Repurposing covalent EGFR/HER2 inhibitors for on-target degradation of human Tribbles 2 (TRIB2) pseudokinase

Foulkes

Byrne

Bailey

et al. 2018

Preprint

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“…In this respect, it is interesting that inhibition of HER2 signaling with lapatinib, like HER2 knockdown, reduced the frequency of larger and more complex membrane protrusions (Fig 4E), but was less effective than genetic knockdown of HER2 at reducing the numbers of smaller tubular protrusions (Fig 1G). In addition, while some reports support our data showing that lapatinib does not reduce total cellular HER2 expression, they differ as to whether it increases the ubiquitination and/or internalization of HER2 [36, 44, 45]. One important distinction between our work and that of these other groups is that we examined internalization of HER2 after acute treatment with EGF, and, in our experiments, reductions in HER2 levels and/or activity clearly caused internalization of ubiquitinated HER2 and this correlated with the effacement of membrane protrusions.…”

Section: Discussionsupporting

confidence: 50%

HER2 signaling regulates HER2 localization and membrane retention

Jeong

Kim

et al. 2017

PLoS ONE

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ErbB2/HER2/Neu is a receptor tyrosine kinase that is overexpressed in 25–30% of human breast cancers, usually associated with amplification of the ERBB2 gene. HER2 has no recognized ligands and heterodimers between HER2 and EGFR (ErbB1/HER1) or HER2 and ErbB3/HER3 are important in breast cancer. Unlike other ErbB family members, HER2 is resistant to internalization and degradation, and remains at the cell surface to signal for prolonged periods after it is activated. Although the mechanisms underlying retention of HER2 at the cell surface are not fully understood, prior studies have shown that, in order to avoid internalization, HER2 must interact with the chaperone, HSP90, and the calcium pump, PMCA2, within specific plasma membrane domains that protrude from the cell surface. In this report, we demonstrate that HER2 signaling, itself, is important for the formation and maintenance of membrane protrusions, at least in part, by maintaining PMCA2 expression and preventing increased intracellular calcium concentrations. Partial genetic knockdown of HER2 expression or pharmacologic inhibition of HER2 signaling causes the depletion of membrane protrusions and disruption of the interactions between HER2 and HSP90. This is associated with the ubiquitination of HER2, its internalization with EGFR or HER3, and its degradation. These results suggest a model by which some threshold of HER2 signaling is required for the formation and/or maintenance of multi-protein signaling complexes that reinforce and prolong HER2/EGFR or HER2/HER3 signaling by inhibiting HER2 ubiquitination and internalization.

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“…Some of these receptors are colocalized with lysosomes. It is reported that the internalized HER2 in SK‐BR‐3 cells treated with neratinib follows the classical endosome lysosomal pathway for degradation 27. Thus, the present results suggest that both EGFR and HER2 undergo neratinib‐induced endocytosis.…”

supporting

confidence: 63%

Raman Microspectroscopic Evidence for the Metabolism of a Tyrosine Kinase Inhibitor, Neratinib, in Cancer Cells

et al. 2018

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Tyrosine kinase receptors are one of the main targets in cancer therapy. They play an essential role in the modulation of growth factor signaling and thereby inducing cell proliferation and growth. Tyrosine kinase inhibitors such as neratinib bind to EGFR and HER2 receptors and exhibit antitumor activity. However, little is known about their detailed cellular uptake and metabolism. Here, we report for the first time the intracellular spatial distribution and metabolism of neratinib in different cancer cells using label‐free Raman imaging. Two new neratinib metabolites were detected and fluorescence imaging of the same cells indicate that neratinib accumulates in lysosomes. The results also suggest that both EGFR and HER2 follow the classical endosome lysosomal pathway for degradation. A combination of Raman microscopy, DFT calculations, and LC‐MS was used to identify the chemical structure of neratinib metabolites. These results show the potential of Raman microscopy to study drug pharmacokinetics.

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

Cited by 68 publications

References 19 publications

Repurposing covalent EGFR/HER2 inhibitors for on-target degradation of human Tribbles 2 (TRIB2) pseudokinase

Repurposing covalent EGFR/HER2 inhibitors for on-target degradation of human Tribbles 2 (TRIB2) pseudokinase

HER2 signaling regulates HER2 localization and membrane retention

Raman Microspectroscopic Evidence for the Metabolism of a Tyrosine Kinase Inhibitor, Neratinib, in Cancer Cells

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