Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways
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Mehdi Dehghani1,*, Reynolds K. Brobey1,*, Yue Wang1, Glauco Souza1,2, Robert J. Amato1 and Kevin P. Rosenblatt1,3,4
1Division of Oncology, Department of Internal Medicine, The University of Texas Health Science Center at Houston McGovern Medical School, Houston, Texas 77030, United States of America
2n3D Biosciences, Inc., Houston, Texas 77030, United States of America
3NX Prenatal, Inc., Bellaire, Texas 77401, United States of America
4Consultative Genomics, PLLC, Bellaire, Texas 77401, United States of America
*These authors have contributed equally to this work
Kevin P. Rosenblatt, email: [email protected]
Keywords: Klotho; clear-cell renal cell carcinoma; cell migration; wound healing assay; Caki-1
Received: March 08, 2018 Accepted: May 08, 2018 Published: June 01, 2018
Klotho is a single-pass transmembrane protein with documented anti-cancer properties. Recent reports have implicated Klotho as an inhibitor of transforming growth factor β1 induced cell migration in renal fibrosis. Overexpression of epidermal growth factor receptor (EGFR) is known to promote tumor initiation and progression in clear-cell renal cell carcinoma (cRCC). We tested our hypothesis that Klotho inhibits EGF-mediated cell migration in cRCC by interfering with the EGFR signaling complex and mitogen-activated protein kinase (MAPK) pathways. We performed cell adhesion, migration, and biochemical studies in vitro using Caki-1 cell line. In addition, we validated the cell culture studies with expression analysis of six de-identified FFPE tissues from primary and metastatic cRCC patients. Our studies show that Klotho inhibited EGF-induced Caki-1 de-adhesion and decreased spreading on collagen type 1. Klotho also inhibited EGF-induced α2β1 integrin-dependent cell migration on collagen type 1. To test the involvement of MAPK pathways in EGF-induced Caki-1 cell motility, the cells were pretreated with either SB203580, a specific p38 MAPK inhibitor, or Klotho. SB203580 blocked the EGF-induced Caki-1 cell migration. Klotho had a comparable inhibitory effect. Our FFPE clinical specimens revealed decreased Klotho mRNA expression compared to a control, non-cancer kidney tissue. The decrease in Klotho mRNA levels correlated with increased c-Src expression, while E-Cadherin was relatively reduced in metastatic FFPE specimens where Klotho was least expressed. Taken together, these results suggest that secreted Klotho inhibits EGF-induced pro-migratory cell morphological changes and migration in Caki-1 cells. Our data additionally suggest that decreased Klotho expression may be involved in cRCC metastasis.
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