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 4

PEAR1 phosphorylation at Ser891 is crucial for CD44 function.

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PEAR1 phosphorylation at Ser891 is crucial for CD44 function. (A) PEAR1 ...
(A) PEAR1 was immunoprecipitated from MDA-MB-231 cell lysates with anti-PEAR1 antibody, after which the levels of serine/threonine phosphorylation or tyrosine phosphorylation were measured with Western blotting. IgG served as an isotype control. (B) Serine/threonine phosphorylation levels of PEAR1 in HEK293T cells transiently transfected with oe-PEAR1–WT-Flag, oe-PEAR1–All SA-Flag, oe-PEAR1–S795A-Flag, oe-PEAR1–S891A-Flag, oe-PEAR1–S953A-Flag, oe-PEAR1–S976A-Flag, or oe-PEAR1–S1029A-Flag plasmids. “All SA” indicates that all serine was mutated to alanine at all 5 residues. (C) PEAR1 serine/threonine phosphorylation levels in MDA-MB-231 cells stably expressing PEAR1-WT-Flag, SA-Flag, or S891A-Flag. (D) The interaction between PEAR1 and CD44 in MDA-MB-231 cells was detected by co-IP with anti-PEAR1 or anti-CD44 antibodies. (E) CD44, OCT4, SOX2, NANOG, E-cadherin, vimentin, and CD44-ICD protein expression in total lysates and nuclear fractions of the indicated MDA-MB-231 cells. (F and G) Western blotting of CD44 (F) and IF staining of CD44 (red), LAMP1 (green), and nuclei (blue) (G) in oe-PEAR1–WT-Flag and oe-PEAR1–S891A-Flag MDA-MB-231 cells treated with or without dynasore, SGC-AAK-1, pitstop 2, or MG132 for 1 hour. Number of endocytotic CD44 foci per cell and mean intensity of CD44. Scale bars: 20 μm. (H and I) Quantification of mammosphere diameters and quantities (H); and quantification of invasive cells in Transwell assay and relative migration area in wound healing assay (I) of the indicated MDA-MB-231 cells (n = 3; mean ± SEM). (J) Quantification of metastatic foci by H&E staining in the lungs and livers of nude mice i.v. injected with the indicated MDA-MB-231 cells (n = 5 mice per group; mean ± SEM). One-way ANOVA followed by Dunnett’s test was used for G–J. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The Western blotting results are representative of 3 independent experiments.