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 5

Expression of PLAC8 enhances HCA-7 cell invasion and alters CDH1 subcellular localization.

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Expression of PLAC8 enhances HCA-7 cell invasion and alters CDH1 subcell...
(A) 2D invasive capacity was assessed by a magnetically attachable stencil invasion assay. After removal of a magnetically attachable stencil, movement of parental HCA-7 and PLAC8-overexpressing HCA-7 (HCA-7P8) cells was monitored over 24 hours by time-lapse microscopy. HCA-7 cells moved as a common front, whereas HCA-7P8 cell movement was uneven and cells detached. Scale bars: 100 μm. (B) At 8 and 24 hours, static images were taken and quantified by 2 parameters (deviation ratio and number of detached cells per field), based on 3 independent experiments performed in triplicate. Both parameters were significantly greater in PLAC8-overexpressing cells. Data are presented as mean ± SD. *P < 0.05; **P < 0.01. (C) After 15 days in 3D collagen culture, PLAC8 and CDH1 immunofluorescence were largely cytosolic in HCA-7 cells overexpressing PLAC8 (HCA-7P8). The boxed regions in the upper panels are magnified in the lower panels. Scale bars: 50 μm.