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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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 4

Plac8.1 overexpression causes cell-autonomous post-transcriptional downregulation of E-cadherin.

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Plac8.1 overexpression causes cell-autonomous post-transcriptional downr...
(A) Left: Representative micrographs of control or plac8.1 RNA–injected zebrafish embryo. Horizontal white lines mark the deep cell margin. Middle: ntl ISH. The vertical white lines denote midline tissues, and the red arrowhead indicates the cluster of dorsal forerunner (df) cells separated from the deep cell margin. Right: Micrographs of control and plac8.1 RNA–injected embryos at 3 days post-fertilization (dpf), with ventral view of eyes shown in insets. Scale bars: 200 μm (7.5 hpf); 500 μm (3 dpf); 100 μm (insets). (B) Representative micrographs of a cell cluster with 3 types of membrane junctions: those shared by 2 wild-type cells (white arrows); those shared by 2 Plac8.1-EGFP–overexpressing cells (cyan arrow); and hybrid membrane junctions (green arrows). Scale bars: 10 μm. (C) Quantification of membrane Cdh1 intensity at different membrane junctions. **P < 0.01, ANOVA and subsequent pair-wise t test. (D and E) Quantification of total speed and net speed of lateral mesodermal cells. **P < 0.01, t test. (F) Left: Representative paths of lateral mesodermal cells traveling during time lapse in control and Plac8.1-EGFP–overexpressing embryos. Right: Orientations of the long axes of lateral mesodermal cells are plotted with length/width ratio (LWR) expressed as mean ± SEM. P > 0.05, Mann-Whitney U test. (G) qRT-PCR of cdh1 expression in Plac8.1-overexpressing and control embryos. Data are presented as mean ± SEM (P > 0.05, t test). (H) Immunoblotting of Cdh1 levels in control and Plac8.1-overexpressing embryos at 50% epiboly. Normalized levels are shown as mean ± SEM (P < 0.01, t test).