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Motor Protein–Dependent Membrane Trafficking of KCl Cotransporter-4 Is Important for Cancer Cell Invasion

¹ Institute of Basic Medical Sciences and Departments of ² Pharmacology and ³ Obstetrics and Gynecology, College of Medicine, National Cheng Kung University, Tainan, Taiwan; ⁴ Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom; and ⁵ Renal Division, Brigham and Women's Hospital and VA Boston Healthcare System, Harvard Medical School, Boston, Massachusetts

Requests for reprints: Meng-Ru Shen, Department of Obstetrics and Gynecology, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan 704, Taiwan. Phone: 886-6-2353535, ext. 5505; Fax: 886-6-2766185; E-mail: mrshen{at}mail.ncku.edu.tw.

The KCl cotransporter (KCC) is a major determinant of osmotic homeostasis and plays an emerging role in tumor biology. This study stresses the important role of KCC4 in tumor malignant behavior. Real-time reverse transcription-PCR on samples collected by laser microdissection and immunofluorescent stainings with different KCC isoform antibodies indicate that KCC4 is abundant in metastatic cervical and ovarian cancer tissues. Insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulate KCC4 recruitment from a presumably inactive cytoplasmic pool of endoplasmic reticulum and Golgi to plasma membrane along actin cytoskeleton that is significantly inhibited by LY294002 and wortmannin. Throughout the trafficking process, KCC4 is incorporated into lipid rafts that function as a platform for the association between KCC4 and myosin Va, an actin-dependent motor protein. KCC4 and ezrin, a membrane cytoskeleton linker, colocalize at lamellipodia of migratory cancer cells. Interference with KCC activity by either an inhibitor or a dominant-negative loss-of-function mutant profoundly suppressed the IGF-I–induced membrane trafficking of KCC4 and the structural interaction between KCC4 and ezrin near the cell surface. Endogenous cancer cell invasiveness was significantly attenuated by small interfering RNA targeting KCC4, and the residual invasiveness was much less sensitive to IGF-I or EGF stimulation. In the metastatic cancer tissues, KCC4 colocalizes with IGF-I or EGF, indicating a likely in vivo stimulation of KCC4 function by growth factors. Thus, blockade of KCC4 trafficking and surface expression may provide a potential target for the prevention of IGF-I– or EGF-dependent cancer spread. [Cancer Res 2009;69(22):8585–93]

Key Words: IGF-I • KCl cotransport • membrane trafficking • metastasis • invasion