EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer
Xiaochao Tan, Guan-Yu Xiao, Shike Wang, Lei Shi, Yanbin Zhao, Xin Liu, Jiang Yu, William K. Russell, Chad J. Creighton, Jonathan M. Kurie
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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Research Article Cell biology Oncology

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 3

PI4K2A is a therapeutic target in mesenchymal lung cancer cells.

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PI4K2A is a therapeutic target in mesenchymal lung cancer cells. (A) WB ...
(A) WB analysis confirming target gene deletion in PI4K2A-KO H1299 cells. (B) Orthotopic lung tumor size (left plot) and mediastinal and contralateral lung metastasis numbers (right plot) generated in nude mice (dots) by the intrathoracic injection of cells described in A. (C) WB analysis confirming target gene depletion in shRNA-transfected 344SQ cells. (D) Tumor weights (left plot) and lung metastasis numbers (right plot) generated in syngeneic, immunocompetent mice (dots) by subcutaneous injection of the cells described in C. (E) Daily subcutaneous tumor volume measurements (dots) in nude mice treated with PI-273 or vehicle (DMSO). (F and G) Tumor tissues removed at necropsy in E were imaged (F) and weighed (G). (H) Orthotopic lung tumor size (left plot) and metastasis numbers (right plot) in nude mice treated with PI-273 or vehicle. (I) Kaplan-Meier survival analysis of mice bearing orthotopic lung tumors treated with PI-273 or DMSO. (J) WB analysis demonstrating reconstitution of shPI4K2A-transfected H1299 cells (shUTR) with WT or enzyme-dead mutant (D308A) PI4K2A. Empty vector (Vec). (K) Orthotopic lung tumors (arrows) generated in nude mice by the cells in J. Scale bars: 5 mm. (L) Orthotopic lung tumor size (left plot) and metastasis numbers (right plot). (M) Annexin V/propidium iodide flow cytometric analysis of the apoptotic fraction in siRNA-transfected cells. (N) Colonies formed in soft agar by siRNA-transfected cells. Values are expressed relative to siCTL. (O) Boyden chamber migration and invasion assays on siRNA-transfected cells. Values are expressed relative to siCTL. 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 values were determined by 2-tailed Student’s t test for 2-group comparisons (E–H); 1-way ANOVA test for multiple comparisons (B, D, and L–O; and log-rank test (I). shCTL, control shRNA.