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Phosphatase PRL-3 Is a Direct Regulatory Target of Tgfb in Colon Cancer Metastasis

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Phosphatase PRL-3 Is a Direct Regulatory Target of Tgfb in Colon Cancer Metastasis Published OnlineFirst November 16, 2010; DOI: 10.1158/0008-5472.CAN-10-1487 Cancer Tumor and Stem Cell Biology Research Phosphatase PRL-3 Is a Direct Regulatory Target of TGFb in Colon Cancer Metastasis Yanjun Jiang1, Xiao-Qiong Liu1, Ashwani Rajput3, Liying Geng1, Melanie Ongchin4, Qi Zeng5, Gregory S. Taylor2, and Jing Wang1 Abstract Metastasis causes most deaths from cancer yet mechanistic understanding and therapeutic options remain limited. Overexpression of the phosphatase PRL-3 (phosphatase of regenerating liver) is associated with metastasis of colon cancer. Here, we show that PRL-3 is a direct target of signaling by TGFb, which is broadly implicated in progression and metastasis. We found that suppression of PRL-3 expression by TGFb was mediated by Smad-dependent inhibition of PRL-3 transcription at the level of promoter activity. PRL-3 activation stimulated PI3K/AKT signaling that caused resistance to stress-induced apoptosis. PRL-3 over- expression promoted metastatic colonization in an orthotopic mouse model of colon cancer, whereas PRL-3 knockdown reduced metastatic potential. Altered metastatic phenotypes were not derivative of primary tumor development or local invasion but could be attributed to PRL-3–mediated cell survival. Our findings suggest that inhibiting PRL-3 expression might be an important mechanism through which TGFb suppresses metastasis in colon cancer. In addition, our findings suggest that loss of TGFb signaling, which occurs commonly during colon cancer progression, is sufficient to activate a PRL-3–mediated cell survival pathway that can selectively promote metastasis. Therefore, a major implication of our findings is that PRL-3 antagonists may offer significant value for antimetastatic therapy in patients with colon cancer. Cancer Res; 71(1); 234–44. Ó2010 AACR. Introduction promotes metastasis (6), others have demonstrated that TGFb suppressesmetastasis(7,8).Recently,studiesof TGFb plays an important role in tumorigenesis and human tumor samples indicate that loss or reduction of metastasis. Upon ligand binding, TGFb type II receptor TGFb signaling in human colorectal tumors is associated (RII) recruits and activates TGFb type I receptor (RI), which with the development of metastasis (9, 10). then activates Smad2 and Smad3. Activated Smad2 and Our previous studies indicate that abrogation of TGFb signal- Smad3 form complexes with Smad4 and translocate to ing enables increased survival under stress in colon cancer cells the nucleus, where they regulate gene expression (1). We (11). In addition, we have shown that loss of TGFb signaling is and others have demonstrated that TGFb suppresses tumor associated with increased metastasis, whereas enhanced TGFb initiation in a variety of cancers, including colon cancer, and signaling suppresses metastasis in an orthotopic model of colon that loss of TGFb signaling leads to malignancy (2–5). cancer (Simms et al., unpublished data). These results suggest However, the role of TGFb signaling in metastasis has been that endogenous TGFb increases stress-induced apoptosis to controversial. Although many studies have shown that TGFb prevent metastatic progression. In contrast, abrogation of TGFb signaling leads to activation of oncogenic signals that promote survival and protect tumor cells from stress-induced apoptosis, thereby increasing their metastatic potential. Identification of 1 Authors' Affiliations: University of Nebraska Medical Center, Eppley oncogenes that are suppressed by TGFb signaling would shed Institute for Research in Cancer and Allied Diseases, and 2University of Nebraska Medical Center, Department of Biochemistry and Molecular light on mechanisms of TGFb tumor suppressor function and Biology, Omaha, Nebraska; 3Division of Surgical Oncology, Department provide new opportunities of molecular targeting for novel of Surgery, The University of New Mexico, Albuquerque, New Mexico; cancer treatment. 4Department of Surgery, Roswell Park Cancer Institute, Buffalo, New York; and 5Institute of Molecular and Cell Biology, Proteos, Singapore PRL-3 (phosphatase of regenerating liver), a metastasis- Note: Supplementary data for this article are available at Cancer Research associated protein, belongs to the PRL family of protein Online (http://cancerres.aacrjournals.org/). tyrosine phosphatases, which includes 2 other members Y. Jiang, X.-Q. Liu, and A. Rajput contributed equally to the work. PRL-1 and PRL-2 (12). Whereas PRL-1 and PRL-2 are Corresponding Author: Jing Wang, University of Nebraska Medical expressed in most tissues, PRL-3 expression is restricted to Center, 985950 Nebraska Medical Center, Omaha, NE 68198. Phone: heart and skeletal muscle (13). Upregulation of PRL-3 expres- 402-559-5558; Fax: 402-559-4651; E-mail: [email protected] sion correlates with colon cancer progression: undetectable in doi: 10.1158/0008-5472.CAN-10-1487 normal colon epithelial, intermediate levels in 25% to 45% Ó2010 American Association for Cancer Research. advanced primary tumors and significantly elevated levels in 234 Cancer Res; 71(1) January 1, 2011 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst November 16, 2010; DOI: 10.1158/0008-5472.CAN-10-1487 TGFb/PRL-3 Signaling in Colon Cancer Metastasis greater than 90% metastases regardless of metastatic sites (14, GTCTGCCC to CATAGCCC) to generate Prl-3-F5-mutE1 and 15). Importantly, PRL-3 expression in primary colorectal Prl-3-F5-mutE4, respectively. Prl-3-F5-mut E1/4 was gener- tumors has prognostic significance in predicting the devel- ated by mutating both SBE-like binding sites simultaneously. opment of liver and/or lung metastases as well as shortened Luciferase activity was measured with Luciferase Assay Sys- patient survival (16–19). Therefore, PRL-3 shows promise as a tem (Promega) and values were normalized by b-galactosidase biomarker for advanced malignancy and as a prognostic activity. P values were calculated using Student's t test. indicator for poor survival in colorectal cancer. Experimental manipulation of PRL-3 expression in several cell models alters Electrophoresis mobility shift assays cancer-associated phenotypes including proliferation, migra- Double-stranded oligonucleotides were labeled with tion, invasion, tumorigenesis, and metastasis (16, 20, 21). [g-32P]ATP using T4 polynucleotide kinase (Promega). Binding These studies indicate that upregulation of PRL-3 expression reactions containing GST-Smad3 (SignalChem) or His-Smad4 has a causative role in tumor progression rather than merely (Abcam) recombinant proteins and labeled probes were per- being a consequence of these processes, which raises the formed for 30 minutes at room temperature. Protein–DNA possibility of PRL-3 being a potential therapeutic target for complexes were separated on 5% polyacrylamide gels. The colon cancer treatment. However, little is known of the sequences of the probes used are in Supplemental Materials. regulation of PRL-3 expression. Increased gene copy number is partially responsible for upregulation of PRL-3 expression in Chromatin immunoprecipitation assays colon cancer (14, 15). In this study, we demonstrate that Chromatin immunoprecipitation (ChIP) assays were per- expression of PRL-3 is suppressed by TGFb signaling in colon formed with the ChIP assay kit (Upstate Inc.) according to the cancer cells. Smad3, but not Smad2, is essential for this manufacturer's protocol. FET cells were treated with 4 ng/mL suppression. Therefore, loss of TGFb signaling, which occurs of TGFb1 for 90 minutes. Cell pellets were resuspended in lysis in 30% to 50% of colon cancer (22, 23), is likely a mechanism of buffer (1% SDS, 10 mmol/L of EDTA, 50 mmol/L of Tris-HCl, PRL-3 upregulation in colon cancer. In addition, our studies pH 8.1, with protease inhibitor cocktail from Sigma-Aldrich). show that PRL-3 activates AKT and maintains AKT activation Genomic DNA fragments were purified with a MinElute PCR under growth factor deprivation stress (GFDS). Ectopic purification kit (Qiagen). The primers used to amplify E1, E4, expression of PRL-3 increases cell survival under GFDS and and negative control are described in Supplementary Materi- promotes metastasis in vivo, whereas knockdown of PRL-3 als. expression sensitizes colon cancer cells to stress-induced apoptosis and reduces metastasis in an orthotopic model. In vivo orthotopic model and immunohistochemistry Orthotopic implantation was performed as previously Materials and Methods described (26). Briefly, exponentially growing green fluores- cent protein (GFP)-labeled cells (5 Â 106) were inoculated Cell culture and reagents subcutaneously into BALB/c nude mice. Once xenografts were The human colon carcinoma FET, GEO, and CBS cell lines established, they were excised and minced into 1-mm3 pieces. were described previously (24). They were maintained at 37C Two of these pieces were then subserosally implanted into in a humidified incubator with 5% CO2 and cultured in SM cecum of other BALB/c nude mice. Sixty days postimplanta- medium (McCoy's 5A serum-free medium; Sigma) supplemen- tion, animals were euthanized. Organs were explanted and ted with 10 ng/mL of epidermal growth factor, 20 mg/mL of imaged. Tissues were then processed and embedded in par- insulin, and 4 mg/mL of transferrin. When cells were under affin. Slides were cut for hematoxylin and eosin (H&E) and growth factor and nutrient deprivation stress, they were terminal nucleotidyl transferase–mediated nick end labeling cultured
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