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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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 5

PI4K2A drives anterograde vesicular trafficking and promotes RAB6A+ vesicle fission.

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PI4K2A drives anterograde vesicular trafficking and promotes RAB6A+ vesi...
(A) Single-channel and merged confocal micrographs of total and surface VSV-G in H1299 cells cotransfected with siRNAs and EGFP–VSV-G and imaged 30 minutes after transfer to the permissive temperature. Plot shows the ratio of surface VSV-G to total VSV-G in each cell (dot) 30 or 60 minutes after transfer to 32°C. Scale bar: 20 μm. (B) BRET measurement of PI4P in RAB6A+ vesicles in siRNA-transfected H1299 cells. Results are expressed as a ratio of the values from GSK-A1–treated and vehicle-treated (DMSO) cells at each time point (n = 5 replicates per group). (C) Confocal micrographs of RAB6A+ vesicles (blue arrows) and unfissioned RAB6A+ tubules (red arrows) emerging from the Golgi. Scale bar: 20 μm. Dotted lines indicate the cell boundaries. Results were quantified per cell (dot plots). (D) Venn diagram of PI42KA-interacting proteins identified by TurboID and IP approaches. Overlapping proteins are listed on the right. Reported PI4K2A-interacting proteins (69, 70) are shown in bold. (E) Schematic illustration of full-length and truncated PI4K2A constructs. WB assays on whole-cell lysates (WCLs) (input) or IP proteins isolated from H1299 cells transfected with MYC-tagged PI4K2A constructs (gel). Full-length (1–479) and truncated constructs are indicated under the gels. IgG was used as the control IP. (F) WB analysis of WCLs (WCL) and Golgi-enriched fractions (Golgi) from siRNA-transfected H1299 cells. (G) Confocal micrographs of SPP1+ vesicles (arrows) in siRNA-transfected H1299 cells costained with anti-SPP1 and anti–Golgin 97 antibodies. Scale bars: 50 μm. Dot plot shows the vesicle numbers per cell. (H) WB analysis of WCLs or enriched subcellular fractions from siRNA-transfected H1299 cells. Densitometric values were normalized to siCTL (graph). 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 < 0.001, by 2-tailed Student’s t test for 2-group comparisons (B); 1-way ANOVA test for multiple comparisons (A, C, G, and H).