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 4

PI4K2A activates a prometastatic secretory program.

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PI4K2A activates a prometastatic secretory program. (A) Relative soft ag...
(A) Relative soft agar colony numbers. siRNA-transfected H1299 cells were treated with CM samples from siRNA-transfected H1299 cells. (B) Apoptosis assays on siRNA-transfected H1299 cells treated with CM samples. WB analysis of cleaved PARP1 (C-PARP1) and PI4K2A (gel). (C) Volcano plot illustration of proteins (dots) identified by LC-MS analysis of CM samples. P value (y axis) and fold change (x axis) are shown. PI4K2A-upregulated secreted proteins (pink quadrant) of interest are labeled. (D) Gene Ontology analysis of the pink quadrant in C. (E) Subcutaneous tumors (dots) were weighed (left plot) and subjected to flow cytometry to quantify CD31+ cells (middle plot) and cleaved-caspase 3+ (CC3+) cells (right plot). (F) Heatmap illustration of the correlation between mRNAs and EMT scores (Byers or Creighton) in TCGA LUAD cohort. r values were determined by Pearson’s correlation. (G) Kaplan-Meier survival analysis of TCGA LUAD and LUSC cohorts based on 6-gene signatures of secreted proteins. Tumors were scored as being above (high) or below (low) each cohort’s median values. (H–J) Apoptosis assays and WB analysis of cleaved PARP1 (gel) (H), soft agar colony formation assays (I), and Boyden chamber migration and invasion assays (J) were carried out on siRNA-transfected H1299 cells. (K) HUVEC migration in Boyden chambers. CM samples from siRNA-transfected H1299 cells were loaded into the lower wells. Scale bars: 200 μm. (L) HUVEC spheroid invasion assay. HUVEC spheroids were seeded in 3D collagen and treated with CM samples. Scale bar: 100 μm. (M) HUVEC tube formation assay in 3D Matrigel following treatment with CM samples. Scale bars: 100 μm. (N) HUVEC migration in Boyden chambers. CM from siRNA-transfected H1299 cells was loaded into the lower chambers. 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 < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed Student’s t test for 2-group comparisons (E); 1-way ANOVA test for multiple comparisons (A, B, and H–N).