CLIC1 recruits PIP5K1A/C to induce cell-matrix adhesions for tumor metastasis
Jei-Ming Peng, … , Ming-Chin Yu, Sen-Yung Hsieh
Jei-Ming Peng, … , Ming-Chin Yu, Sen-Yung Hsieh
Published October 20, 2020
Citation Information: J Clin Invest. 2021;131(1):e133525. https://doi.org/10.1172/JCI133525.
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Research Article Cell biology Hepatology

CLIC1 recruits PIP5K1A/C to induce cell-matrix adhesions for tumor metastasis

Abstract

Membrane protrusion and adhesion to the extracellular matrix, which involves the extension of actin filaments and formation of adhesion complexes, are the fundamental processes for cell migration, tumor invasion, and metastasis. How cancer cells efficiently coordinate these processes remains unclear. Here, we showed that membrane-targeted chloride intracellular channel 1 (CLIC1) spatiotemporally regulates the formation of cell-matrix adhesions and membrane protrusions through the recruitment of PIP5Ks to the plasma membrane. Comparative proteomics identified CLIC1 upregulated in human hepatocellular carcinoma (HCC) and associated with tumor invasiveness, metastasis, and poor prognosis. In response to migration-related stimuli, CLIC1 recruited PIP5K1A and PIP5K1C from the cytoplasm to the leading edge of the plasma membrane, where PIP5Ks generate a phosphatidylinositol 4,5-bisphosphate–rich (PIP2-rich) microdomain to induce the formation of integrin-mediated cell-matrix adhesions and the signaling for cytoskeleon extension. CLIC1 silencing inhibited the attachment of tumor cells to culture plates and the adherence and extravasation in the lung alveoli, resulting in suppressed lung metastasis in mice. This study reveals what we believe is an unrecognized mechanism that spatiotemporally coordinates the formation of both lamellipodium/invadopodia and nascent cell-matrix adhesions for directional migration and tumor invasion/metastasis. The unique traits of upregulation and membrane targeting of CLIC1 in cancer cells make it an excellent therapeutic target for tumor metastasis.

Authors

Jei-Ming Peng, Sheng-Hsuan Lin, Ming-Chin Yu, Sen-Yung Hsieh

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Figure 3

CLIC1 facilitates adherence and extravasation of tumor cells for metastasis in mice.

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CLIC1 facilitates adherence and extravasation of tumor cells for metasta...
Luciferase-transduced SK-Hep1 and Huh7 cells were used for in vivo metastasis in nude mice. Cells transfected with siRNAs containing scrambled sequences (siNC) or targeting CLIC1 (siCLIC1), or constitutively transduced with shRNAs targeting CLIC1 (shCLCI1) or an empty vector (shEV). (A) Lung metastasis assays by injecting SK-Hep1 cells (1 × 106 cells in 200 μL PBS/mouse, n = 6) through tail veins. (B) The statistical results of the luminescence signals for lung metastasis. Statistical analysis was performed by using Mann-Whitney U test between 2 groups (n = 6/group; *P < 0.05; ***P < 0.001). (C) Representative lung tissues 63 days after injection. Arrowheads, metastatic tumors. Lower panel: H&E staining for the lung sections. A, lung alveoli; T, metastatic tumors. Scale bar: 120 μm. (D) Dot blot of metastatic tumor foci per lung. Statistical analysis was performed by using Mann-Whitney U test between 2 groups (n = 6/group; ***P < 0.001). (E) Western blots for CLIC1 silencing efficiency in HCC cells. (F and G) Cumulative survival curves for the mice bearing xenograft tumors. P values were determined by the log-rank test. (H) Liver metastasis in nude mice by injection of luciferase-transduced Huh7 cells through the spleen. (I) Liver metastasis was inspected by IVIS imaging at weeks 1 and 6 after injection. Lower panel: quantification of luciferase signals in the liver 6 weeks after injection. Statistical analysis was performed by using Mann-Whitney U test (n = 6/group; ***P < 0.001). (J) H&E staining for liver sections. Scale bar: 120 μm. (K) Western blots for CLIC1 silencing efficiency. (L) GFP-transduced SK-Hep1 cells with or without CLIC1 depletion were injected through tail veins. Lungs were assayed at 4, 8, 12, 16, 20, and 24 hours after injection. Representative lung section images at 8, 16, and 24 hours after injection are shown. GFP-labeled tumor cells were detected and quantified. Statistical analysis was performed by using Mann-Whitney U test between 2 groups (n = 8; NS, no statistical significance; ***P < 0.001). (M) In vitro cell adhesion assays. The experiments were conducted twice independently. Statistical analysis was performed by using 2-tailed Student’s t test between 2 groups (n = 8/group; ***P < 0.001).