LOXL2-induced PEAR1 Ser891 phosphorylation suppresses CD44 degradation and promotes triple-negative breast cancer metastasis
Yingzhi Shen, … , Junling Liu, Xuemei Fan
Yingzhi Shen, … , Junling Liu, Xuemei Fan
Published August 15, 2024
Citation Information: J Clin Invest. 2024;134(16):e177357. https://doi.org/10.1172/JCI177357.
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Research Article Cell biology Oncology

LOXL2-induced PEAR1 Ser891 phosphorylation suppresses CD44 degradation and promotes triple-negative breast cancer metastasis

Abstract

CD44 is associated with a high risk of metastasis, recurrence, and drug resistance in various cancers. Here we report that platelet endothelial aggregation receptor 1 (PEAR1) is a CD44 chaperone protein that protected CD44 from endocytosis-mediated degradation and enhances cleavage of the CD44 intracellular domain (CD44-ICD). Furthermore, we found that lysyl oxidase–like protein 2 (LOXL2), an endogenous ligand of PEAR1, bound to the PEAR1-EMI domain and facilitated the interaction between PEAR1 and CD44 by inducing PEAR1 Ser891 phosphorylation in a manner that was independent of its enzyme activity. Levels of PEAR1 protein and PEAR1 phosphorylation at Ser891 were increased in patients with triple-negative breast cancer (TNBC), were positively correlated with expression of LOXL2 and CD44, and were negatively correlated with overall survival. The level of PEAR1 Ser891 phosphorylation was identified as the best independent prognostic factor in TNBC patients. The prognostic efficacy of the combination of PEAR1 phosphorylation at Ser891 and CD44 expression was superior to that of PEAR1 phosphorylation at Ser891 alone. Blocking the interaction between LOXL2 and PEAR1 with monoclonal antibodies significantly inhibited TNBC metastasis, representing a promising therapeutic strategy for TNBC.

Authors

Yingzhi Shen, Jie Yan, Lin Li, Huiyan Sun, Lin Zhang, Guoming Li, Xinxia Wang, Ruoyan Liu, Xuefeng Wu, Baosan Han, Xueqing Sun, Junling Liu, Xuemei Fan

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Figure 3

PEAR1 protects CD44 from endocytosis-mediated degradation.

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PEAR1 protects CD44 from endocytosis-mediated degradation. (A) Co-IP of ...
(A) Co-IP of whole-cell lysates of HEK293T cells transiently transfected with PEAR1-ICD–Flag and CD44-ICD–HA plasmids with anti-Flag and anti-HA agarose beads. Cells transfected with the corresponding empty vector plasmids were used as negative controls. (B) The interaction between PEAR1-ECD and CD44-ECD was detected using pulldown with anti-His Dynabeads in a solution containing purified PEAR1-ECD–His and CD44-ECD–Fc protein. Fc was used as a negative control. (C) Quantification of the mammosphere diameters and quantities of shPEAR1 and oe-PEAR1 MDA-MB-231 cells (n = 3; mean ± SEM). (D) Expression levels of CD44, OCT4, SOX2, NANOG, E-cadherin, and vimentin in total lysates and of CD44-ICD in nuclear proteins of shPEAR1 and oe-PEAR1 MDA-MB-231 cells as determined by Western blotting analysis. Quantitative analysis of the relative gray values of full-length CD44 with GAPDH and nuclear CD44-ICD with histone H3 (n = 3; mean ± SD). (E) CD44 mRNA expression levels in shPEAR1 and oe-PEAR1 MDA-MB-231 cells were determined by RT-qPCR. The diagram shows the relative expression of mRNAs normalized to that of 18S rRNA (n = 3; mean ± SD). (F and G) Western blotting of CD44 (F) and IF staining of CD44 (red), LAMP1 (green), and nuclei (blue) (G) in shPEAR1 MDA-MB-231 cells treated with or without dynasore, SGC-AAK-1, pitstop 2, or MG132 for 1 hour. β-Actin served as an internal control (F). Number of endocytotic CD44 foci per cell and mean intensity of CD44 (G). Scale bars: 20 μm. One-way ANOVA followed by Dunnett’s test was used for C–E and G; unpaired 2-tailed t tests were used for C–E. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The Western blotting results are representative of 3 independent experiments.