Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer
Cunxi Li, … , Lila Solnica-Krezel, Robert J. Coffey
Cunxi Li, … , Lila Solnica-Krezel, Robert J. Coffey
Published April 1, 2014
Citation Information: J Clin Invest. 2014;124(5):2172-2187. https://doi.org/10.1172/JCI71103.
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Research Article Oncology

Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer

Abstract

The epithelial-to-mesenchymal transition (EMT) transcriptional program is characterized by repression of E-cadherin (CDH1) and induction of N-cadherin (CDH2), and mesenchymal genes like vimentin (VIM). Placenta-specific 8 (PLAC8) has been implicated in colon cancer; however, how PLAC8 contributes to disease is unknown, and endogenous PLAC8 protein has not been studied. We analyzed zebrafish and human tissues and found that endogenous PLAC8 localizes to the apical domain of differentiated intestinal epithelium. Colon cancer cells with elevated PLAC8 levels exhibited EMT features, including increased expression of VIM and zinc finger E-box binding homeobox 1 (ZEB1), aberrant cell motility, and increased invasiveness. In contrast to classical EMT, PLAC8 overexpression reduced cell surface CDH1 and upregulated P-cadherin (CDH3) without affecting CDH2 expression. PLAC8-induced EMT was linked to increased phosphorylated ERK2 (p-ERK2), and ERK2 knockdown restored cell surface CDH1 and suppressed CDH3, VIM, and ZEB1 upregulation. In vitro, PLAC8 directly bound and inactivated the ERK2 phosphatase DUSP6, thereby increasing p-ERK2. In a murine xenograft model, knockdown of endogenous PLAC8 in colon cancer cells resulted in smaller tumors, reduced local invasion, and decreased p-ERK2. Using MultiOmyx, a multiplex immunofluorescence-based methodology, we observed coexpression of cytosolic PLAC8, CDH3, and VIM at the leading edge of a human colorectal tumor, supporting a role for PLAC8 in cancer invasion in vivo.

Authors

Cunxi Li, Haiting Ma, Yang Wang, Zheng Cao, Ramona Graves-Deal, Anne E. Powell, Alina Starchenko, Gregory D. Ayers, Mary Kay Washington, Vidya Kamath, Keyur Desai, Michael J. Gerdes, Lila Solnica-Krezel, Robert J. Coffey

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Figure 3

plac8.1 RNA expression and Plac8.1 protein localization in zebrafish embryos.

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plac8.1 RNA expression and Plac8.1 protein localization in zebrafish em...
(A) Whole-mount ISH using an antisense probe against full-length plac8.1 in zebrafish embryos at indicated hours post-fertilization (hpf). Asterisks denote position of animal poles. The arrow and arrowhead denote future dorsal and ventral side, respectively. Scale bar: 200 μm. (B) Confocal immunofluorescent images of whole-mount zebrafish embryos stained with anti-Plac8.1 antibody (green) and Texas red–conjugated phalloidin (red, F-actin). Cartoons on the left of each panel illustrate corresponding stages. The dashed lines correspond to their right section planes. Scale bar: 10 μm. (C) Whole-mount ISH using plac8.1 antisense probe in zebrafish embryos at 4 days post-fertilization (dpf). Dashed line indicates approximate position for transverse section shown in inset with strong signal in gut. Scale bars: 250 μm. (D) Cryosections through the gut of embryos at 4 dpf were stained with anti-Plac8.1 antibody (green), Texas red–conjugated phalloidin (red, F-actin), and TO-PRO-3 (blue, DNA). Scale bar: 10 μm.