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PDLIM1 Stabilizes the E-Cadherin/B-Catenin Complex To Published OnlineFirst December 23, 2015; DOI: 10.1158/0008-5472.CAN-15-1962 Cancer Molecular and Cellular Pathobiology Research PDLIM1 Stabilizes the E-Cadherin/b-Catenin Complex to Prevent Epithelial–Mesenchymal Transition and Metastatic Potential of Colorectal Cancer Cells Hai-Ning Chen1, Kefei Yuan1, Na Xie1, Kui Wang1, Zhao Huang1, Yan Chen1, Qianhui Dou1, Min Wu2, Edouard C. Nice3,4, Zong-Guang Zhou1, and Canhua Huang1 Abstract Metastasis is a major cause of death in patients with colorectal and promoted distant metastatic colonization in an experimental cancer, and increasing evidence supports the contribution of the lung metastasis model. Mechanistic investigations revealed that epithelial–mesenchymal transition (EMT) to cancer progression. PDLIM1 interacted with and stabilized the E-cadherin/b-catenin The dissociation of the E-cadherin/b-catenin adhesion complex complex, thereby inhibiting the transcriptional activity of b-cate- represents a key step in EMT and promotes cancer invasion and nin and preventing EMT. Accordingly, PDLIM1 overexpression metastasis, but the upstream signaling pathways regulating this attenuated EMT of colorectal cancer cells. Moreover, the down- interaction are poorly understood. Here, we show that PDLIM1, a regulation of PDLIM1 in colorectal cancer samples correlated member of the PDZ and LIM protein family, was downregulated with reduced E-cadherin and membrane b-catenin levels, and in highly metastatic colorectal cancer cells and liver metastases was associated with shorter overall survival. In conclusion, our from colorectal cancer patients. We found that loss of PDLIM1 study demonstrates that PDLIM1 suppresses EMT and meta- promoted the expression of EMT markers and increased the static potential of colorectal cancer cells by stabilizing b-catenin invasive and migratory properties of multiple colorectal cancer at cell–cell junctions, and its loss in metastatic tissues may cell lines. Furthermore, PDLIM1 knockdown increased colon- represent a potential prognostic marker of aggressive disease. derived liver metastasis in an orthotopic colorectal cancer model Cancer Res; 76(5); 1122–34. Ó2015 AACR. Introduction Although several therapeutic strategies, including targeted drugs, have been developed, the management of metastatic colorectal Colorectal cancer is the third most common cancer in men and cancer remains problematic due to distinct molecular alterations the second most common cancer in women worldwide, with an in the tumor (3). Thus, a better understanding of the molecular estimated incidence of more than 1.3 million in 2012, and mechanism underlying colorectal cancer metastasis may lead to mortality of 700,000 (1). Despite various advances in the diag- new therapeutic strategies. nosis and treatment of colorectal cancer over the past decades, the The epithelial–mesenchymal transition (EMT) is a biologic overall prognosis for patients with colorectal cancer remains poor process involving multiple molecular events by which the (2). Tumor metastasis is a significant factor in the management of polarized epithelial cells acquire mesenchymal cell pheno- colorectal cancer and is a major obstacle to successful treatment. types (4). In colorectal cancers, loss of E-cadherin has been characterized as a unique trait of EMT cells at the invasive front 1Department of Gastrointestinal Surgery, State Key Laboratory of and is usually concomitant with deregulation of the Wnt Biotherapy and Cancer Center, West China Hospital, Sichuan Univer- signaling pathway (4). b-Catenin, a key component of the sity, Collaborative Innovation Center for Biotherapy, Chengdu, P.R. canonical Wnt signaling pathway, plays a crucial role in the China. 2Department of Biochemistry and Molecular Biology, University of North Dakota, Grand Forks, North Dakota. 3Department of Bio- negative regulation of E-cadherin and the induction of EMT chemistry and Molecular Biology, Monash University,Clayton,Victoria, (5). Under normal circumstances, most b-catenin and E-cad- Australia. 4The State Key Laboratory of Biotherapy/Collaborative herin is present as an E-cadherin/catenin adhesion complex Innovation Center of Biotherapy, West China Hospital, Sichuan Uni- located in cell–cell adherent junctions at the membrane. versity, Chengdu, P.R. China. Signaling pathways like TGFb and EGF may activate EMT by Note: Supplementary data for this article are available at Cancer Research causing disassociation of this complex to release b-catenin, Online (http://cancerres.aacrjournals.org/). which is a prerequisite for its nuclear translocalization and H.-N. Chen and K. Yuan contributed equally to this article. subsequent posttranscriptional regulations (6, 7). Thus, the E- Corresponding Authors: Canhua Huang, Sichuan University, No. 1 Keyuan 4 cadherin/b-catenin complex provides a major strength for cell– Road, Gaopeng St. High-Tech Zone, Chengdu, Sichuan 610041, China. Phone: cell association. However, the molecular mechanisms that act 135-4065-1810; Fax: 86-28-8516-4060; E-mail: [email protected]; and Zong- upstream of these factors are still not well understood. Thus, Guang Zhou, E-mail: [email protected] establishing the signaling networks associated with these fac- doi: 10.1158/0008-5472.CAN-15-1962 tors may elucidate the molecular mechanisms underlying Ó2015 American Association for Cancer Research. tumor invasion and metastasis. 1122 Cancer Res; 76(5) March 1, 2016 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst December 23, 2015; DOI: 10.1158/0008-5472.CAN-15-1962 Loss of PDLIM1 Induces EMT and Promotes CRC Metastasis PDLIM1 (also known as CLP36, Elfin, or CLIM1) is a cytoskeletal bisulfite using the EZ DNA Methylation-Gold Kit (Zymo protein that associates with actin filaments and stress fibers (8). The Research). The products were amplified by PCR. Primer pairs PDZ and LIM protein family comprises proteins containing a C- used to recognize the bisulfite-modified regions (À6toÀ290) of terminal PDZ and one or more N-terminal LIM domains (9). These the PDLIM1 promoter were: forward 50-GTAGAGTTGTTGA- proteins are capable of recruiting signal proteins to actin and GAGTTTTGG-30, reverse 50-AATCTATCTACTAAATAATCATAA- organizing signaling complexes at the cytoplasmic or cellular CAC-30. membranes (10–12). To date, PDLIM2 andPDLIM4, which belong to the same family as PDLIM1, have been implicated as candidate TOP/FOP Flash assay tumor suppressors in several tumors (12, 13). As an actin-binding The TOP/FOP Flash assay was performed in accordance with protein, PDLIM1 is diffusely expressed in heart, muscle, and the protocol provided by the manufacturer (Upstate Biotechnol- gastrointestinal tract tissues and acts as a regulator of cell–cell ogy). To measure the transcriptional activity of b-catenin, SW480 adhesion and focal adhesion (10). However, the functional sig- cells were seeded in 24-well plates at 2 Â 104 cells per well. The nificance of the PDLIM1 in cancers remains to be determined. next day, cells were transfected with Top-flash plasmid (Upstate) In this study, we show that PDLIM1 is downregulated in plus pRL-CMV plasmid (Promega) or Fop-flash plasmid colorectal cancer tissues, particularly in colorectal cancer liver (Upstate) plus pRL-CMV plasmid. Two days later, luciferase metastasis. Furthermore, we show that loss of PDLIM1 results in activity was measured in cell extracts using a Dual Luciferase Kit disassociation of the E-cadherin/b-catenin complex, leading to (Promega). The transfection efficiency was normalized to the the nuclear translocation of b-catenin and the repression of E- activity of pRL-CMV (Renilla luciferase). cadherin, resulting in enhanced invasive and metastatic potential of colorectal cancer. The enhanced expression of PDLIM1 attenu- In vivo models ates EMT induction in colorectal cancer cells. In addition, we find a Male BALB/c nu/nu mice (7-week-old) were raised in a specific positive correlation between PDLIM1, E-cadherin, and mem- pathogen-free condition. Animal care and experimental protocols brane b-catenin expression in colorectal cancer samples and their were in accordance with guidelines provided by the Institutional associations with prognosis. Taken together, our data indicate Animal Care and Use Committee of Sichuan University that loss of PDLIM1 in colorectal cancer is an inducer of EMT (Chengdu, P.R. China). For the experimental mouse lung metas- and predicts poor outcomes in patients with colorectal cancer, tasis model, 1 Â 106 cells were injected into nude mice through implicating PDLIM1 as both a potential therapeutic target and a the tail vein. The orthotopic mouse model of colorectal cancer predictor of survival in colorectal cancer. was generated as described previously (15). Briefly, mice were anesthetized and 2 Â 106 cells were injected into the wall of Materials and Methods the cecum. Mice were inspected daily, and the date of death for Cell lines each mouse was documented. Tumor metastasis was monitored Human colorectal cancer cell lines SW480, HT29, DLD1, by MRI at week 4 after injection. Mice were killed 6 and 8 weeks SW620, Lovo were purchased from the ATCC. The KM12C cell after injection to examine the lung and liver metastasis of tumor fi line was a kind gift from Prof. Min Wu (University of North cells, respectively. The tumor metastases were con rmed by fi Dakota, Grand Forks, ND). All cell lines were tested by short hematoxylin and eosin staining and quanti ed on the basis of fi tandem repeat analysis and used
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