EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer
Xiaochao Tan, … , Chad J. Creighton, Jonathan M. Kurie
Xiaochao Tan, … , Chad J. Creighton, Jonathan M. Kurie
Published February 9, 2023
Citation Information: J Clin Invest. 2023;133(7):e165863. https://doi.org/10.1172/JCI165863.
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EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

Abstract

Hypersecretory malignant cells underlie therapeutic resistance, metastasis, and poor clinical outcomes. However, the molecular basis for malignant hypersecretion remains obscure. Here, we showed that epithelial-mesenchymal transition (EMT) initiates exocytic and endocytic vesicular trafficking programs in lung cancer. The EMT-activating transcription factor zinc finger E-box–binding homeobox 1 (ZEB1) executed a PI4KIIIβ-to-PI4KIIα (PI4K2A) dependency switch that drove PI4P synthesis in the Golgi and endosomes. EMT enhanced the vulnerability of lung cancer cells to PI4K2A small-molecule antagonists. PI4K2A formed a MYOIIA-containing protein complex that facilitated secretory vesicle biogenesis in the Golgi, thereby establishing a hypersecretory state involving osteopontin (SPP1) and other prometastatic ligands. In the endosomal compartment, PI4K2A accelerated recycling of SPP1 receptors to complete an SPP1-dependent autocrine loop and interacted with HSP90 to prevent lysosomal degradation of AXL receptor tyrosine kinase, a driver of cell migration. These results show that EMT coordinates exocytic and endocytic vesicular trafficking to establish a therapeutically actionable hypersecretory state that drives lung cancer progression.

Authors

Xiaochao Tan, Guan-Yu Xiao, Shike Wang, Lei Shi, Yanbin Zhao, Xin Liu, Jiang Yu, William K. Russell, Chad J. Creighton, Jonathan M. Kurie

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Figure 7

PI4K2A stabilizes AXL.

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PI4K2A stabilizes AXL. (A) WB analysis of parental and PI4K2A-KO H1299 c...
(A) WB analysis of parental and PI4K2A-KO H1299 cells. Densitometric values are shown under the gels. Positive (EGFR) and negative (ITGB1) controls. (B) Correlation between AXL and PI4K2A protein levels in cell lines (dots). (C) WB analysis of parental and PI4K2A-KO H1299 cells treated with the AXL ligand Gas6. pAKT, phosphorylated AKT. Graphs show densitometric analysis of pAXL and pAKT levels normalized to t = 0 minutes. (D) Boyden chamber migration assays on parental and PI4K2A-KO H1299 cells treated with (+) or without (–) Gas6. (E) WB analysis of parental and PI4K2A-KO H1299 cells stably transfected with empty vector or AXL. (F) Boyden chamber migration assays on cells in E. (G) WB analysis of parental and PI4K2A-KO H1299 cells treated with cycloheximide (CHX). Graphs show the densitometric values. (H) Single-channel and merged confocal micrographs of parental and PI4K2A-KO cells costained with anti-AXL and anti-LAMP1 antibodies. Lysosomal AXL (inset, arrows) was quantified as the percentage of total AXL that colocalized with LAMP1 per field (n = 10 fields per condition). Scale bar: 10 μm. Original magnification, ×2.5 (enlarged insets). (I) WB analysis of PI4K2A-KO cells treated with proteasomal (MG132) or lysosomal (leupeptin or monensin) inhibitors. DMSO was used as the vehicle. l.e., long exposure duration; s.e., short exposure duration. (J) WB analysis of siRNA-transfected H1299 cells. Densitometric values are shown under the gel. (K) WB analysis of WCLs (input) and anti-HA immunoprecipitates from H1299 cells transfected with HA-tagged PI4K2A. IgG was used as the negative control IP. (L) WB analysis of WCLs (input) and anti-HSP90 immunoprecipitates from parental and PI4K2A-KO H1299 cells. IgG was used as the negative control IP. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. **P < 0.01 and ***P < 0.001, by 2-tailed Student’s t test for 2-group comparisons (C, D, G, and H); 1-way ANOVA test for multiple comparisons (F).