Pdcd4, a colon cancer prognostic that is regulated by a microRNA

ED10-02

Department of Experimental Surgery and Molecular Oncology of Solid Tumors, Medical Faculty Mannheim, University Heidelberg, and DKFZ Heidelberg, Germany
 The novel tumor suppressor Pdcd4 inhibits neoplastic transformation, tumor progression, and translation. Furthermore, we recently showed that Pdcd4 suppresses invasion and intravasation, this at least in part by suppressing expression of the invasion-related urokinase receptor (u-PAR) gene via transcription factors Sp1/Sp3. However, relatively little is known about mechanisms regulating Pdcd4 expression in cancer. Recently, microRNAs (miRNAs) have been discovered as naturally occurring non-coding RNAs, controlling gene expression via specific sites at the 3’-UTR of target mRNAs. We conducted a first study to investigate the regulation of Pdcd4, and invasion/intravasation, by miRNAs.
 A bioinformatic search revealed a conserved target site for miR-21 within the Pdcd4-3’-UTR at 228-249 nucleotides. In 10 colorectal cell lines, an inverse correlation of miR-21 and Pdcd4-protein was observed. Transfection of Colo206f-cells with miR-21 significantly suppressed a luciferase reporter containing the Pdcd4-3’-UTR, whereas transfection of RKO with anti-miR-21 increased activity of this construct. This was abolished when a construct mutated at the miR-21/nt228-249 target site was used instead. Anti-miR-21-transfected RKO-cells showed an increase of Pdcd4-protein and reduced invasion. Moreover, these cells showed reduced intravasation and lung metastasis in a chicken-embryo-metastasis assay. In contrast, overexpression of miR-21 in Colo206f significantly reduced Pdcd4-protein amounts and increased invasion, while Pdcd4-mRNA was unaltered. Resected normal and tumor tissues of 22 colorectal cancer patients demonstrated an inverse correlation between miR-21 and Pdcd4-protein. This is the first study to show that Pdcd4 is negatively regulated by miR-21. Furthermore, it is the first report to demonstrate that miR-21 induces invasion/intravasation/metastasis.
 This study adds to the increasing knowledge about diverse important roles played by Pdcd4 in suppressing different phenoma associated with cancer. However, only few translational or clinical studies so far have investigated or supported the potential use of Pdcd4 as a prognostic factor. Also, the role of Pdcd4 in the progression from benign to malignant tissue has rarely been supported by in vivo studies at resected patient tissues. Therefore, we conducted a second study to 1. determine Pdcd4 as a diagnostic marker in the adenoma- carcinoma sequence of patients with colorectal cancer, 2. obtain the first prognostic evidence of Pdcd4 in colorectal cancer. Since recent studies also suggested that Pdcd4 is regulated by Akt, one of the most important antiapoptotic regulators within the PI3-kinase pathway, and that Akt is able to regulate the nuclear localization of Pdcd4, this study was also conducted, to 3. support phosphorylated Akt (pAkt) - mediated Pdcd4 regulation in vivo.
 Tumor samples and normal tissues from 71 patients with colorectal cancer who were followed prospectively (median follow-up, 36 months) and 42 adenomas were analyzed for Pdcd4, Akt, and pAkt in immunohistochemical and Western blot analyses.
 A significant reduction in Pdcd4 was observed between normal mucosa and adenomas and between adenomas and tumor samples (P < .01 and P < .01, respectively). Normal mucosa demonstrated strong nuclear Pdcd4, which was reduced significantly in adenomas (P <.01) and almost was lost in tumors (P <.01). pAkt was correlated inversely with Pdcd4 and with the transition of Pdcd4 from nucleus to cytoplasm (P <.01). Kaplan-Meier analysis (using the Mantel-Cox log-rank test) indicated a significant correlation between the loss of total and nuclear Pdcd4 in tumors and overall survival (P <.05 and P <.02, respectively), and with disease-specific survival (P <.01 and P <.01, respectively). In multivariate analysis, loss of total or nuclear Pdcd4 was an independent predictor of disease-specific or overall survival.
 This is the first study to demonstrate an independent prognostic impact of Pdcd4 and its expression pattern in colorectal cancer. Data from this study support the regulation of Pdcd4 localization by pAkt in vivo. Pdcd4 immunohistochemistry may be useful as a supportive diagnostic tool for the transition between normal, adenoma, and tumor tissues.
 Taken together, tumor suppressor Pdcd4 marks adenoma/carcinoma transition, is an independent prognostic factor in resected colorectal cancer, and is negatively regulated by miR-21 in colorectal cancer, this being associated with miR-21 inducing invasion and intravasation.