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. 2015 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 2015 American Association for Cancer Research. tumor invasion and metastasis.

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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 (6to290) 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 2 104 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, 1 106 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 2 106 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 within 6 months. The last time of visual examination and manual counting of formalin- xed lungs authentication was November 2015. All cell lines were routinely and livers. maintained in DMEM (Invitrogen) supplemented with 10% FBS (Biowest), penicillin (107 U/L), and streptomycin (10 mg/L) at Statistical analysis 37 C in a humidified chamber under 5% CO2. Statistical analyses and graphics were undertaken using Prism version 6.00 (GraphPad Software) and R software, version 3.1.2 Patients and follow-up (R Core Team). A one-way ANOVA and Pearson c2 test were used This study enrolled two independent cohorts of colorectal for the univariate analyses where appropriate. The survival anal- cancer patients at the Department of Gastrointestinal Surgery, ysis was performed as previously reported (16). The Kaplan– West China Hospital (Chengdu, P.R. China). Ethics approval for Meier method was used to calculate the survival rates. Each single these studies was obtained from the Biomedical Ethics Committee variable was analyzed using the Cox proportional hazards model. of West China Hospital. A total of 127 tumor tissues and paired A P value of <0.05 was considered statistically significant. adjacent normal tissues (Cohort 1) were collected from patients undergoing resection for colorectal cancer between 2008 and Results 2009. Cohort 2 was collected from patients who underwent resection for colorectal cancer liver metastasis with matched PDLIM1 is downregulated in human colorectal cancers and primary tumor tissue available from 2010 to 2013. Data, including correlates with liver metastasis clinical information, pathologic characteristics, and AJCC stages, To explore the role of PDLIM1 in the progression of colorectal were collected (14). The follow-up data were acquired at 3-month cancer, the expression of PDLIM1 was evaluated in tissue micro- intervals through outpatient visits, telephone calls, or office visits. arrays containing 127 samples of colorectal cancers with patient- The follow-up data were censored in December 2014. Detailed matched normal mucosal tissues. Immunohistochemical analy- clinicopathologic features are listed in Supplementary Table S1. ses demonstrated decreased expression of PDLIM1 in colorectal cancer samples compared with adjacent normal mucosal tissues Bisulfite genomic DNA sequencing (Fig. 1A and B). In addition, PDLIM1 expression in six colorectal Genomic DNA was prepared using the TIANamp Genomic cancer tissues and paired normal mucosa was detected by Western DNA Kit (TIANGEN), followed by the treatment of sodium blot analysis. Despite interindividual variations, PDLIM1 was

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Figure 1. PDLIM1 is downregulated in human colorectal cancers. A, immunohistochemical staining of PDLIM1 in parafﬁn-embedded human colorectal cancer tissues; scale bar, 200 mm (left), 50 mm (right). B, the relative protein level of PDLIM1 is signiﬁcantly lower in colorectal cancer tissues than in adjacent normal tissue. Data represent the mean SD. C, Western blot analysis of PDLIM1 expression in colorectal cancer tissues and patient-matched adjacent normal tissues. D, the relative mRNA expression of PDLIM1 in three Oncomine datasets, GDS2947, GDS3756, and GDS2609. Data represent the mean SD. E, relative PDLIM1 protein levels in six colorectal cancer (CRC) cell lines as determined by Western blot analysis. F, immunohistochemical staining of PDLIM1 in colorectal cancer liver metastasis (mCRC) and patient- matched primary colorectal cancer tissues; scale bar, 50 mm. G, the relative protein level of PDLIM1 is lower in colorectal cancer liver metastases than patient-matched primary colorectal cancer tissues; , P < 0.001.

found to be significantly downregulated in colorectal cancer resection of the affected liver tissues. The PDLIM1 protein levels tissues (Fig. 1C). Furthermore, the downregulated expression of were found to be significantly reduced in colorectal cancer PDLIM1 in colorectal cancer was verified by gene expression liver metastasis compared with primary colorectal cancer tissues analysis based on three Oncomine datasets (Fig. 1D). To explore (Fig. 1F and G). Together, these results indicate that PDLIM1 is the role of PDLIM1 in colorectal cancer progression, six human frequently downregulated in human colorectal cancers, particu- colorectal cancer cell lines, KM12C, SW480, HT29, DLD1, SW620 larly in metastatic lesions, suggesting that loss of PDLIM1 may and Lovo, were employed to evaluate PDLIM1 expression. The play a role in colorectal cancer metastasis. protein expression of PDLIM1 was significantly reduced in the two highly metastatic cell lines (SW620 and Lovo) compared with the Downregulation of PDLIM1 in colorectal cancer cells involves weakly metastatic colorectal cancer cell lines (KM12C, SW480, DNA methylation HT29, DLD1; Fig. 1E). To further investigate the correlation To investigate the mechanism of PDLIM1 repression in colo- between PDLIM1 and colorectal cancer metastasis, colorectal rectal cancer cells, we examined the mRNA level of six colorectal cancer liver metastases with the corresponding patient-matched cancer cell lines. Consistently, a higher mRNA level of PDLIM1 primary tumors were obtained from 32 patients who underwent was observed in highly metastatic colorectal cancer cell lines

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Loss of PDLIM1 Induces EMT and Promotes CRC Metastasis

Figure 2. Downregulation of PDLIM1 in colorectal cancer cells involves DNA methylation. A, relative mRNA levels of PDLIM1 in indicated cells were determined by real-time PCR; bars, SD (n ¼ 4). B, cells were treated with 5-aza-dC (5 mmol/L) for 48 hours. Methylation status of PDLIM1 promoter was determined by bisulfite genomic DNA sequencing as described in Materials and Methods. Each dot represents a CpG site; white dots represent unmethylated CpG dinucleotides, whereas each black dot represents a methylated cytosine residue within the CpG islands. C, methylation statuses of PDLIM1 promoter in five pairs of colorectal cancer tissues and patient-matched normal tissues were determined by bisulfite genomic DNA sequencing. D, methylation statuses of PDLIM1 promoter in five pairs of colorectal cancer tissues and patient-matched liver metastatic tissues were determined by bisulfite genomic DNA sequencing. E, mRNA levels of PDLIM1 were determined by real-time PCR. Changes in PDLIM1 mRNA levels following 5-aza-dC treatment are represented as fold induction relative to control cells; bars, SD (n ¼ 4). F, Western blot analysis of PDLIM1 expression in indicated cells as treated in B.

(SW620, Lovo) compared with primary colorectal cancer cell lines was observed in the CpG islands of the PDLIM1 promoter of (KM12C, SW480, HT29, and DLD1; Fig. 2A). To date, PDLIM2, highly metastatic colorectal cancer cells (SW620, Lovo; Fig. 2B). PDLIM3, and PDLIM4, which belong to the same family as Consistently, similar results were observed in the methylation PDLIM1, have been demonstrated to be downregulated status of PDLIM1 promoter in clinical samples. On the basis of by promoter hypermethylation. In line with these ﬁndings, we colorectal cancer tissues with patient-matched normal tissues, we evaluated the potential role of DNA methylation in PDLIM1 found that the PDLIM1 promoter was methylated in colorectal repression. A total of 44 CpG sites located between nucleotides cancer tissues (Fig. 2C). This tendency was further validated in 6 and 290 in the PDLIM1 promoter were examined using hypermethylated colorectal cancer liver metastatic tissues com- bisulﬁte genomic DNA sequencing. Dense cytosine methylation pared with primary foci (Fig. 2D). These results suggest that the

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Loss of PDLIM1 Induces EMT and Promotes CRC Metastasis

PDLIM1 promoter was hypermethylated in colorectal cancers knockdown in SW480 significantly increased the number of with high metastatic potential. To further determine whether metastatic nodules in the liver, whereas overexpression of PDLIM1 promoter hypermethylation was responsible for PDLIM1 decreased the metastatic capacity of SW620 (Fig. 3G PDLIM1 repression, the effect of 5-aza-dC (a DNA methyltrans- and I). Collectively, these data from both in vitro and in vivo ferase inhibitor) on PDLIM1 expression was evaluated in colo- experiments demonstrate that loss of PDLIM1 promotes both rectal cancer cells. Treatment with 5-aza-dC increased the mRNA invasion and metastasis in colorectal cancer. level of PDLIM1 and restored the protein expression of PDLIM1 in colorectal cancer cells (Fig. 2E and F). Consistently, cytosine Loss of PDLIM1 induces EMT in colorectal cancer methylation of the PDLIM1 promoter is largely reversed in Given the essential role of EMT in tumor metastasis, we response to 5-aza-dC treatment (Fig. 2B). Together, these data evaluated the protein expression levels of EMT markers, suggest that PDLIM1 repression in colorectal cancer is attributed E-cadherin, vimentin, and a panelofEMT-associatedtranscrip- to promoter methylation. tion factors, including Snail, Slug, ZEB1, ZEB2, and TWIST in SW480, KM12C, SW620, and Lovo cells. Knockdown of Loss of PDLIM1 promotes colorectal cancer cell invasion and PDLIM1 in SW480 and KM12C cells led to significantly lower metastasis in vitro and in vivo levels of E-cadherin and higher levels of vimentin and Snail To identify the role of PDLIM1 in colorectal cancer metastasis, (Fig. 4A). In contrast, the enhanced expression of PDLIM1 in we evaluated the invasive and metastatic potential in SW480 SW620 and Lovo cells resulted in a concomitant reduction of cells transfected with PDLIM1 shRNA transfected, and SW620 vimentin and Snail, accompanied by upregulation of E-cad- transfected with PDLIM1 cDNA. Successful shRNA-mediated herin (Fig. 4A). However, PDLIM1 showed no significant knockdown of PDLIM1 in SW480 cells and cDNA-mediated impact on the expression levels of other transcription factors, overexpression of PDLIM1 in SW620 cells were confirmed using including Slug, ZEB1, ZEB2, and TWIST (Fig. 4A). Changes in Western blot analysis (Fig. 3A). In the Transwell migration and EMT markers following stable downregulation or upregulation Matrigel invasion assays, PDLIM1 shRNA promoted the migra- of PDLIM1 were further verified by immunofluorescence anal- tion and invasion of SW480 cells, whereas the exogenous expres- ysis (Fig. 4B). Thus, these data suggest that loss of PDLIM1 may sion of PDLIM1 led to a reduction in the migratory and invasive be an inducer of EMT in colorectal cancer cells. property of SW620 (Fig. 3B). In wound-healing migration assays, On the basis of these findings, we next assessed a potential microscopic examination after wounding revealed a significantly clinical relationship between PDLIM1 and EMT in human colo- increased rate of wound closure in SW480-PDLIM1 shRNA-trea- rectal cancer tissues. On the basis of the immunohistochemical ted (hereafter referred to as SW480-shPDLIM1) cells compared staining on tissue microarrays composed of primary tumors and with SW480 cells with control shRNA transfected (hereafter matched peritumoral tissues from 127 colorectal cancer patients, referred to as SW480-NC; Supplementary Fig. S1). In addition, we found a similar pattern of distribution for PDLIM1, E-cad- PDLIM1 cDNA–transfected SW620 cells (hereafter referred to as herin, and membrane b-catenin at the cell–cell junctions in SW620-PDLIM1) had a significant delay in the wound closure rate normal mucosal tissues (Fig. 4D). Moreover, we found that compared with SW620 cells transfected with vector (hereafter colorectal cancer patients with low PDLIM1 expression tended referred to as SW620-Vector; Supplementary Fig. S1). to have lower E-cadherin and membrane b-catenin levels as well To determine whether loss of PDLIM1 is involved in colo- as higher vimentin and Snail levels, suggesting that loss of rectal cancer metastasis in vivo, a lung metastasis model was PDLIM1 promoted the EMT process (Fig. 4E and Supplementary generated by tail injection of two paired cell lines, SW480- Fig. S2). Indeed, as shown in Fig. 4F, the protein expression of shPDLIM1/SW480-NC and SW620-PDLIM1/SW620-Vector. PDLIM1 is closely associated with E-cadherin (R2 ¼ 0.1761, P < Each group was assigned with 6 mice. Representative MRI slides 0.001) and membrane b-catenin (R2 ¼ 0.3274, P < 0.001), taken4weeksafterinjectionareshowninFig.3C,confirming respectively. Representative images from four cases are shown to the occurrence of lung metastases. Six weeks after injection, a illustrate the correlation between these proteins in colorectal higher number of lung metastatic nodules was observed in the cancer tissues (Fig. 4G). Taken together, this specific expression SW480-shPDLIM1 group (n ¼ 6) compared with the SW480- pattern suggests a potential role of PDLIM1 in the EMT of NC group (n ¼ 6; Fig. 3D and E). In contrast, SW620-PDLIM1 colorectal cancer. cells formed fewer lung metastatic nodules compared with SW620-Vector cells (Fig. 3D and E). We next investigated the Loss of PDLIM1 activates b-catenin by disassociating the impact of PDLIM1 on colorectal cancer metastasis using an E-cadherin/b-catenin complex orthotopic mouse model of colorectal cancer (6 mice for each To explore the mechanism by which PDLIM1 regulates EMT in group). Representative MRI slides taken 4 weeks after injection colorectal cancer, we evaluated whether the E-cadherin/b-catenin are shown in Fig. 3F. Eight weeks after injection, the PDLIM1 complex was affected by the manipulation of PDLIM1 expression.

Figure 3. Loss of PDLIM1 enhances invasive and metastatic potential of colorectal cancer cells. A, PDLIM1 knockdown and overexpression as detected by Western blot analysis. B, representative data for Transwell Matrigel invasion and migration assay for indicated colorectal cancer cells. Magniﬁcation, 200. Data represent the mean SD and are representative of three independent experiments. C, representative slides of lung tumor metastasis monitored by MRI 4 weeks after injection. D, the number of lung metastasis nodules from experimental metastases generated by indicated cells 6 weeks after tail vein injection. Data represent the mean SD. E, representative images of lung metastasis from mice as in D. F, representative slides of liver tumor metastasis monitored by MRI 4 weeks after injection. G, the number of liver metastasis nodules from the orthotopic colorectal cancer models generated by indicated cells 8 weeks after injection. Data represent the mean SD. H, representative images of liver metastasis from mice as in G. I, Kaplan–Meier curves of mice from G; , P < 0.01; , P < 0.001.

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Loss of PDLIM1 Induces EMT and Promotes CRC Metastasis

The cell lysate was immunoprecipitated by b-catenin, and domains of PDLIM1 in 293T cells transfected with PDLIM1 then immunoblotted with anti–E-cadherin antibodies to analyze truncation mutants. Only N-terminal fragments containing PDZ the E-cadherin/b-catenin complex formation. As shown in Fig. 5A, domain, but not other regions of PDLIM1, interacted with the the abundance of b-catenin–associated E-cadherin was remark- b-catenin/E-cadherin complex (Fig. 6C). Moreover, PDZ-contain- ably decreased following PDLIM1 knockdown in SW480 and ing fragments were able to inhibit the EMT process, as evidenced KM12C cells, indicating that the E-cadherin/b-catenin complex by induction of E-cadherin and repression of Snail (Fig. 6C), formation was reduced by knockdown of PDLIM1. Next, we tested indicating that the PDZ domain of PDLIM1 forms the molecular whether disassociation of the E-cadherin/b-catenin complex basis of the interaction between PDLIM1 and the E-cadherin/ might contribute to the release of b-catenin. The immunoblot b-catenin complex. analysis showed that PDLIM1 knockdown in SW480 and KM12C Given the essential role of the E-cadherin/b-catenin complex in cells resulted in downregulation of the epithelial marker E-cad- EMT induction, TGFb1 was used as an EMT inducer to investigate herin (Fig. 4A). However, the level of b-catenin was not signifi- the role of the interaction between PDLIM1 and this complex. cantly altered (Fig. 5B). We separated cytosolic and nuclear Treating SW480 and KM12C cells with TGFb1 for 48 hours fractions of cell lysates and found that PDLIM1 knockdown resulted in EMT as evidenced by the nuclear translocation of resulted in a decreased level of cytosolic b-catenin and an b-catenin, decreased E-cadherin expression, and increased vimen- increased level of nuclear b-catenin, suggesting that loss of mem- tin and Snail levels (Fig. 6D and E). Furthermore, the interaction brane-associated forms of b-catenin was due to redistribution to between PDLIM1 and E-cadherin/b-catenin complex was the nucleus (Fig. 5B). Subcellular location of b-catenin was then decreased in response to TGFb1 treatment (Fig. 6A and B). evaluated using immunofluorescence analysis. Consistently, we However, enforced PDLIM1 expression in SW480 and KM12C found that b-catenin was located primarily in the plasma cells significantly attenuated TGFb1-induced EMT (Fig. 6D). membrane in SW480-NC cells; however, PDLIM1 inhibition Moreover, the overexpression of PDLIM1 was sufficient to abolish led to the nuclear accumulation of b-catenin (Fig. 5C). On the the increased cell invasion and migration induced by TGFb1 (Fig. other hand, the expression of membrane b-catenin in SW620 6F). Similar results were also obtained when EGF was used as an cells significantly increased after enhanced expression of EMT inducer (Supplementary Fig. S3). Taken together, these PDLIM1, whereas nuclear b-catenin was almost absent (Fig. results suggest that PDLIM1 binds b-catenin and attenuates 5C). Next, we evaluated the transcriptional activity of b-cate- TGFb1-induced EMT. nin–TCF by using TOP-Flash and FOP-Flash luciferase reporter. PDLIM1 knockdown increased the transcriptional activity of Loss of PDLIM1 predicts poor survival in patients with b-catenin–TCF in SW480, whereas enhanced expression of colorectal cancer PDLIM1 decreased the transcriptional activity of b-catenin–TCF Detailed clinicopathologic features are listed in Supplementary in SW620 (Fig. 5D). To investigate whether PDLIM1-regulated Table S1. Pearson c2 tests indicated that PDLIM1 was significantly EMT involves b-catenin activation, we used siRNA to inhibit related to microvascular invasion (P ¼ 0.002), tumor invasiveness b-catenin expression. As shown in Fig. 5E, the knockdown of (P ¼ 0.044), and lymph node involvement (P < 0.001; Supple- b-catenin results in significant reduction of both Snail and mentary Table S2). Using a median cutoff for PDLIM1 expression, vimentin levels. Moreover, the EMT induced by PDLIM1 down- we noted a trend toward better overall survival (OS) for patients regulation was abolished on b-catenin knockdown (Fig. 5E), with a high level of PDLIM1 expression (Fig. 7A). Supplementary leading to the impairment of cell invasion and migration (Fig. Table S3 shows the results of univariate analyses of 5-year OS and 5F). Taken together, these results demonstrate that the loss of HRs, according to subgroups based on several prognostic variables. PDLIM1 leads to nuclear translocation of b-catenin from the E- OS was significantly higher for colorectal cancer patients who had cadherin/b-catenin complex, thereby resulting in the activation high PDLIM1 expression (72% vs. 36%, P < 0.001). Interestingly, of Wnt signaling and induction of EMT. patients with no detectable lymph node metastasis had better survival when PDLIM1 was highly expressed (76% in the PDLIM1 PDLIM1 binds the E-cadherin/b-catenin complex and high group vs. 33% in the PDLIM1 low group, P < 0.001; Fig. 7B), as attenuates EMT did those who had no distant metastasis (74% in the PDLIM1 high To explore the mechanism by which PDLIM1 regulates the E- group vs. 39% in the PDLIM1 low group,P < 0.001). These findings, cadherin/b-catenin complex, we investigated whether PDLIM1 in conjunction with our previous findings that PDLIM1 correlates can interact with the E-cadherin/b-catenin complex. During with tumor invasiveness and metastasis, implicate PDLIM1 as a growth in normal media when EMT was not observed, PDLIM1 potential predictor of tumor micrometastasis in patients with no interacts with b-catenin and E-cadherin, respectively, in SW480 metastatic focus detectable in pathologic specimens. and KM12C cells, indicating an interaction between PDLIM1 and In addition, we also observed significant OS differences for E-cadherin/b-catenin complex in the absence of EMT in colorectal patients based on E-cadherin and membrane b-catenin expres- cancer cells (Fig. 6A and B). Next, we mapped the binding sion (Fig. 7C and D). When PDLIM1/E-cadherin and PDLIM1/

Figure 4. Loss of PDLIM1 induces EMT in colorectal cancer. A, EMT marker expression following stable downregulation of PDLIM1 expression as detected by Western blot analysis. B, EMT marker expression following stable upregulation of PDLIM1 expression as detected by Western blot analysis. C, PDLIM1, E-cadherin, vimentin, and Snail expression in indicated cells as detected by immunoﬂuorescence assay; scale bar, 20 mm. D, representative images of normal colon epithelial cells with intact PDLIM1, E-cadherin, and b-catenin membrane expression; scale bar, 20 mm. E, the association between expression of PDLIM1, E-cadherin, membrane b-catenin, Snail, and vimentin in colorectal cancer tissues; P < 0.001. F, the correlation test of immunostaining intensity between PDLIM1 and membrane b-catenin or E-cadherin, respectively. G, immunohistochemical analyses of PDLIM1, E-cadherin, b-catenin, Snail, and vimentin expression in colorectal cancer tissues (scale bar, 50 mm).

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Figure 5. Loss of PDLIM1 activates the WNT/b-catenin signal pathway by disassociating the E-cadherin/b-catenin complex. A, immunoprecipitation of b-catenin with E-cadherin in indicated colorectal cancer cells as detected by Western blot analysis. B, b-Catenin expression in whole-cell lysates, cytoplasm, and nuclear fractions as detected by Western blot analysis. C, immunoﬂuorescence analysis of b-catenin (green) in indicated colorectal cancer cells. Merged images represent overlays of b-catenin (green) and nuclear staining by DAPI (blue). D, TOP-Flash/FOP-Flash assay depicting Wnt activity in indicated colorectal cancer cells. E, protein expression of PDLIM1, E-cadherin, b-catenin, vimentin, and Snail in SW480 cells with indicated treatments as detected by Western blot analysis. F, Matrigel invasion and Transwell migration assay for SW480 cells as treated in E. Data, mean SD and are representative of three independent experiments; , P < 0.001.

b-catenin were analyzed using immunohistochemistry, the PDLIM1, E-cadherin, and membrane b-catenin had the best median density was determined and used as a cutoff in sub- survival outcomes (Fig. 7E). These clinical data provide evi- sequent analyses. Colorectal cancer patients with low levels of dence that PDLIM1 in combination with E-cadherin and mem- PDLIM1, E-cadherin, and membrane b-catenin showed worse brane b-catenin could be useful biomarkers in colorectal cancer prognoses compared with other groups (Fig. 7E and F). Con- to assess the invasive tumor biology and predict the prognosis versely, colorectal cancer patients who expressed high levels of of colorectal cancer patients.

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Loss of PDLIM1 Induces EMT and Promotes CRC Metastasis

Figure 6. PDLIM1 binds the E-cadherin/b-catenin complex and attenuates EMT. A, immunoprecipitation of PDLIM1 with E-cadherin and b-catenin in SW480 and KM12C cells as detected by Western blot analysis. Cells were treated with 10 ng/mL TGFb1 for 24 hours. B, immunoprecipitation of b-catenin with E-cadherin and PDLIM1 in SW480 and KM12C cells as detected by Western blot analysis. Cells were treated with 10 ng/mL TGFb1 for 24 hours. C, 293T cells transfected with myc-tagged full-length or PDLIM1 fragments were immunoprecipitated with an anti-myc antibody. b-Catenin, E-cadherin, Snail, and myc-PDLIM1 (full-length or fragments) levels were shown. D, protein expression of PDLIM1, E-cadherin, vimentin, and Snail in SW480 and KM12C cells with indicated treatment. E, immunoﬂuorescence analysis of b-catenin (green) in SW480 cells as treated in C. merged images represent overlays of b-catenin (green) and nuclear staining by DAPI (blue). F, data of Matrigel invasion and Transwell migration assay for SW480 cells as treated in D. Data, mean SD and are representative of three independent experiments; , P < 0.001.

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Figure 7. Loss of PDLIM1 predicts poor prognosis in patients with colorectal cancer. Kaplan–Meier analyses of OS based on PDLIM1, E-cadherin, and membrane b-catenin expression. A, OS was signiﬁcantly higher in the PDLIM1 high group compared with the PDLIM1 low group. B, for patients with no lymph node metastasis detected, OS was signiﬁcantly higher in the PDLIM1 high group compared with the PDLIM1 low group. C, patients in the E-cadherin high group have higher OS when compared with the E-cadherin low group. D, patients in the membrane b-catenin high group have higher OS when compared with the membrane b-catenin low group. E, prognostic values of PDLIM1 combined with E-cadherin expression. F, prognostic values of PDLIM1 combined with membrane b-catenin expression.

Discussion two independent in vivo models to elucidate the effect of Tumor metastasis is a multistage process in which malignant PDLIM1 on the multistages of colorectal cancer metastasis. In cells disseminate from the site of origin and colonize distant line with the in vitro results, PDLIM1 knockdown increases organs or nodes. Although the general steps of this process may colon-derived experimental liver metastasis in the orthotopic be the same, metastasis of different cancer types shows hetero- colorectal cancer model as well as colonization in the distant geneous biology and might be dependent on distinct sets of metastatic site using the experimental lung metastasis model. factors according to the tissue of origins and region of metas- Together, the ﬁndings from our study indicate a pivotal and tasis (17). Thus, the need to understand the biology of metas- negative role of PDLIM1 in colorectal cancer metastasis. tasis in different types of cancer becomes increasingly impor- Experimental and clinical studies have shown that EMT plays a tant. PDLIM1 is a cytoskeletal protein that is diffusely expressed gatekeeper role in the regulation of tumor invasion and metastasis in heart, muscle, and gastrointestinal tract tissues, whereas (4). Recent microarray studies have investigated the molecular normal breast tissues show nondetectable or weak expression alterations associated with EMT in colorectal cancers and found of PDLIM1 (18, 19). Previous studies implicate PDLIM1 as a that PDLIM1 is downregulated in cells with an EMT phenotype prometastasis factor in glioma and breast cancer (20, 21). (22, 23). Here, we report that colorectal cancer cells with low However, given the tissue speciﬁcity of PDLIM1, the role of levels of PDLIM1 expressed high levels of vimentin, Snail, and PDLIM1 in different tumor types might vary accordingly. On low levels of E-cadherin, indicating a potential correlation the basis of the results derived from two cohorts of clinical between PDLIM1 and EMT, which might account for the invasive samples, our study demonstrated that the expression of and metastatic potential of these cells. Moreover, knockdown of PDLIM1 was downregulated in colorectal cancer tissues com- PDLIM1 in colorectal cancer cells reduced E-cadherin expression, pared with peritumoral tissues. More importantly, the samples whereas enhanced expression of PDLIM1 induced E-cadherin from colorectal cancer liver metastases exhibited lower levels of expression. As downregulation or loss of E-cadherin marks the PDLIM1 than patient-matched primary tumors. On the basis of initiation of EMT and the gain of invasive and metastatic potential a number of depletion and overexpression experiments in vitro, of cancer cells, we postulated that PDLIM1-dependent regulation we revealed a negative role for PDLIM1 in regulating colorectal of E-cadherin expression was a potential mechanism that results cancer cell invasion and migration. Moreover, we employed in colorectal cancer metastasis.

1132 Cancer Res; 76(5) March 1, 2016 Cancer Research

Loss of PDLIM1 Induces EMT and Promotes CRC Metastasis

The disruption of cell–cell adherens junctions by misregulation set of particular biomarkers to evaluate the risk of metastasis. of E-cadherin is a key step in EMT that contributes to colorectal Our study reveals the clinical relevance and prognostic signifi- cancer cell invasion and metastasis (4). Normally, E-cadherin and cance of PDLIM1-dependent regulation of b-catenin and E-cad- b-catenin form a complex in the cell–cell junction area, which herin, indicating that the assessment of PDLIM1, in conjunction provides the basis for cell–cell association (7). In response to with E-cadherin and b-catenin, may provide a more detailed several signaling pathways such as TGFb or EGF, the E-cadherin/ appraisal of metastasis risk for individual colorectal cancer b-catenin complex is disassociated to release b-catenin for patients, which may assist in the development of individualized subsequent posttranscriptional regulation (6). In line with therapeutic strategies. this notion, we showed that PDLIM1 inhibition induced disas- In summary, our current findings demonstrate a role for sociation of the E-cadherin/b-catenin complex and nuclear trans- PDLIM1 in the regulation of EMT via a b-catenin–dependent location of membrane b-catenin, followed by the activation of pathway. Loss of PDLIM1 in colorectal cancer is a potential b-catenin–TCF transcription, increasing Snail expression and indicator of aggressive colorectal cancer phenotypes and corre- decreasing E-cadherin levels. Thus, the frequent downregulation lates with poor clinical outcome. Thus, PDLIM1 may be used as a of PDLIM1 in colorectal cancers may account for one potential biomarker for the individualized management of patients with mechanism underlying the dysfunction of the Wnt signaling colorectal cancer. pathway in these tissues. Using either TGFb1 or EGF as the inducer of EMT (7, 24), we found that enhanced PDLIM1 expression in Disclosure of Potential Conflicts of Interest colorectal cancer cells was capable of attenuating EMT. Therefore, No potential conflicts of interest were disclosed. we conclude that PDLIM1 downregulation promotes colorectal cancer metastasis by inducing EMT through a b-catenin–depen- Authors' Contributions dent pathway. Conception and design: H.-N. Chen, E.C. Nice, Z.-G. Zhou, C. Huang Another key finding in our study is the prognostic value of Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H.-N. Chen, Z. Huang, Y. Chen, Q. Dou, M. Wu PDLIM1 protein expression in colorectal cancer patients. On the Analysis and interpretation of data (e.g., statistical analysis, biostatistics, basis of a cohort of 127 patients, we demonstrated that low computational analysis): H.-N. Chen expression of PDLIM1 in colorectal cancer was an indicator of Writing, review, and/or revision of the manuscript: H.-N. Chen, K. Yuan, poor prognosis. Given the roles of PDLIM1 in colorectal cancer N. Xie, K. Wang, E.C. Nice invasion and metastasis, these findings implicated PDLIM1 as a Administrative, technical, or material support (i.e., reporting or organizing marker for tumor aggressiveness and a predictor for colorectal data, constructing databases): N. Xie, Z.-G. Zhou, C. Huang Study supervision: Z.-G. Zhou, C. Huang cancer patients' survival. Our tissue microarray results also con- Other (providing cell lines): M. Wu firmed that low expressions of either E-cadherin or membrane b -catenin were indicators of poor prognosis for colorectal cancer Grant Support patients, which is compatible with the results of previous studies This work was supported by grants from the National 973 Basic Research (22, 25). Unlike tumors such as breast cancer where metastasis Program of China (2013CB911300 and 2012CB518900), the Chinese NSFC might take decades to acquire the function for colonization (17), (81225015 and 81430071), and Sichuan Science-Technology Innovative malignant progression and most metastatic traits in colorectal Research Team for Young Scientist (2013TD0001). cancer are acquired during progression to the invasive carcinoma The costs of publication of this article were defrayed in part by the payment of advertisement stage in the primary site (26). Under this scenario, the molecular page charges. This article must therefore be hereby marked in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. alterations of colorectal cancer cells in the primary site may carry the potential to determine whether metastatic progression may Received July 20, 2015; revised November 24, 2015; accepted November 26, proceed or not, underscoring the feasibility of using one or a 2015; published OnlineFirst December 23, 2015.

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1134 Cancer Res; 76(5) March 1, 2016 Cancer Research

PDLIM1 Stabilizes the E-Cadherin/β-Catenin Complex to Prevent Epithelial−Mesenchymal Transition and Metastatic Potential of Colorectal Cancer Cells

Hai-Ning Chen, Kefei Yuan, Na Xie, et al.

Cancer Res 2016;76:1122-1134. Published OnlineFirst December 23, 2015.

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