Cancer Biology and Signal Transduction PseudogenePTENP1FunctionsasaCompetingEndogenous RNA to Suppress Clear-Cell Renal Cell Carcinoma Progression

PTENP1 is a pseudogene of the PTEN tumor suppression gene (TSG). The functions of PTENP1 in clearcell renal cell carcinoma (ccRCC) have not yet been studied. We found that PTENP1 is downregulated in ccRCC tissues and cells due to methylation. PTENP1 and PTEN are direct targets of miRNA miR21 and their expression is suppressed by miR21 in ccRCC cell lines. miR21 expression promotes ccRCC cell proliferation, migration, invasion in vitro, and tumor growth and metastasis in vivo. Overexpression of PTENP1 in cells expressing miR21 reduces cell proliferation, invasion, tumor growth, and metastasis, recapitulating the phenotypes induced by PTEN expression. Overexpression of PTENP1 in ccRCC cells sensitizes these cells to cisplatin and gemcitabine treatments in vitro and in vivo. In clinical samples, the expression of PTENP1 and PTEN is correlated, and both expressions are inversely correlated with miR21 expression. Patients with ccRCC with no PTENP1 expression have a lower survival rate. These results suggest that PTENP1 functions as a competing endogenous RNA (ceRNA) in ccRCC to suppress cancer progression. Mol Cancer Ther; 13(12); 3086–97. 2014 AACR. Introduction Renal cell carcinoma (RCC) is the third most common urological cancer after prostate and bladder cancer, accounting for about 3% of all human malignancies in adults (1, 2). With more than 200,000 individuals diagnosedwith renal caner andmore than 100,000 individuals who died from this type of cancer every year, RCC is the seventh most common cancer in women and the fifth in men (3). Statistics from the previous research on the incidence of RCC in most areas of the world are available (4, 5). The majority of RCC cases are of the clear-cell subtype (ccRCC), but approximately 10% of tumors are of the papillary subtype and approximately 5% the chromophobe subtype. The other histologic subtypes like collecting duct, transitional cell (urothelial cell) carcinoma, and medullary together account for approximately 5% to 10% of cases (6–8). Besides surgical intervention, RCC is not sensitive to chemotherapy or radiotherapy. Theabsence of biomarkers is responsible for latediagnosis and subsequent poor prognosis. Identification and characterization of genetic andbiologic changes is necessary in understanding the pathogenesis of RCC and identifying new biomarkers. Recent studies have shown that genetic events and histopathologically heterogeneous disorder play a major role in the development of ccRCC (9). Currently, there are at least 12 different genes associated with the development of kidney cancer: VHL, MET, FLCN, TSC1, TSC2, TFE3, TFEB, MITF, fumarate hydratase (FH), succinate dehydrogenase B (SDHB), succinate dehydrogenase D (SDHD), and PTEN (10, 11). Genetic analysis has shown that each subtype represents distinct tumor biology. Comprehensive genome analysis and studies involving several autosomal dominant syndromes characterized by the development of RCC have greatly contributed to the identification of the genes involved in the development of RCC (12). Noncoding RNAs (ncRNA), including miRNAs, long noncoding RNAs (LncRNA), pseudogenes, etc., do not code for proteins and have recently been found to be pervasively transcribed in the genome. The noncoding transcripts range in length from 100 nt to approximately 100 kilobases (kb) and lack significant Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. The Wistar Institute, Philadelphia, Pennsylvania. Department of Urology and Helen-Diller Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). G. Yu and W. Yao contributed equally to this article. Corresponding Authors:Hua Xu, Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China. Phone: 862-7836-63454; Fax: 862-783663454; E-mail: xuhua@mail.hust.edu.cn; and Qihong Huang, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. Phone: 1-215-4956835; Fax: 1-215-898-7952; E-mail: qhuang@wistar.org doi: 10.1158/1535-7163.MCT-14-0245 2014 American Association for Cancer Research. Molecular Cancer Therapeutics Mol Cancer Ther; 13(12) December 2014 3086 open reading frames. The great majority of ncRNAs are transcribed by RNA polymerase II (RNA pol II) and are polyadenylated (13, 14). It has been suggested that pseudogenes arise from protein-coding genes that lost the protein production function mostly due to mutation or aberrant duplication. These noncoding transcripts, including pseudogenes, were once considered useless transcription products with no functions. However, recent evidence increasingly links mutations and dysregulations of ncRNAs to diverse human diseases, including human cancers (15). NcRNAs have been validated to have important functions, including tumor suppressor-like (TSG-like) functions (16, 17), in biologic processes. Recent studies have shown that some pseudogenes contain miRNA-binding elements and serve as competitive endogenous RNAs (ceRNA; refs. 16, 17), decoying that compete for miRNAs to regulate gene expression. In human cancers, monoallelic mutation of PTEN without loss or mutation of the second allele is prevalent at presentation, whereas complete loss is observed at low frequencies with the exception of advanced cancers (18). It was reported that the PTENP1 (NM_023917), which was lost in many human cancers could increase PTEN abundance and showed tumor-suppressive activity (19, 20). Our previous research and other studies have reported that the ncRNA expression signatures of renal clear-cell carcinoma were revealed bymicroarray (21, 22) and found that the PTENP1 transcript was significantly downregulated in ccRCC. Here, we report that pseudogene PTENP1 serves as a ceRNA to modulate PTEN expression regulation by miR21. PTENP1 suppresses tumor growth, invasion, and metastasis in ccRCC. PTENP1 expression sensitizes chemotherapy treatment in human ccRCC cell lines. PTENP1 and PTEN expression is correlated with primary human ccRCC samples, and their expression is inversely correlated with miR21 expression. Lower PTENP1 and PTEN expression is correlated with worse clinical outcomes. These studies demonstrated that pseudogene PTENP1 plays critical roles in ccRCC progression and can potentially serve as a therapeutic target. Materials and Methods Patients and tumor samples Written informed consent was obtained from all patients, and the study was approved by the Institutional Review Board of Huazhong University of Science and Technology, Tongji Medical College, Tongji Hospital (Hubei, China). Ninety-four patients with clear-cell carcinoma of kidney who received nephrectomy or partial nephrectomy were included in the study. The clinical information was retrieved from the medical records. Cell culture and transfection The human renal cell carcinoma cell lines 786-O, ACHN, and SN12PM6 were maintained in DMEM containing 10% FBS, OS-RC-2, and Caki-1 were cultured in RPMI1640 supplemented with 10% FBS. The human kidney proximal tubular epithelial cell line HK-2 was maintained in DMEM containing 10% FBS streptomycin at 37 C in a humidified atmosphere of 5% CO2. The cell lines were obtained from ATCC in March 2013 and authenticated by ATCC. Cells were transiently transfected with the indicated expression plasmid using FuGENE HD Transfection Reagent (Roche) according to the manufacturer’s instructions. The pcDNA3 empty vector was used as control. miR21 and negative control mimics, anti-miR21, andnegative control inhibitors (RiboBio Co. Ltd.)were transfected into cells, respectively, with X-tremeGENE siRNA Transfection Reagent (Roche) according to the manufacturer’s instructions. Construction of expression plasmid andpackaging of lentivirus Oligonucleotides (50-UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGCAACACCAGUCGAUGGGCUGUCUGACA-30) encoding miR21 precursor was subcloned into lentiviral vector pCDH (System Biosciences, Inc.) and verified by DNA sequencing. PTENP1 and PTEN were cloned into the same vector. Dicer shRNA (50-CCGGGCCTCACTTGACCTGAAGTATCTCGAGATACTTCAGCGTCAAGTGAGGCTTTTTG-30) or control scrambled shRNA were cloned into pCDH and the lentivirus was packed as above (23). Cells were transiently transfected with the indicated expression plasmid using FuGENE HD Transfection Reagent (Roche) according to themanufacturer’s instructions. For the gain of stable cell lines, lentivirus was packed with pPACKH1 Lentivector Packaging Kit (System Biosciences, Inc.) and infected in renal cell lines, following the manufacturer’s instructions. Luciferase reporter assay PTENP1 30-UTR containing the putative binding site of miR21, and its identical sequence with a mutation of the miR21 seed sequence were inserted between the restrictive sites XhoI and NotI of hlucþ/hRluc luciferase reporter vector psiCHECK2 and validated by sequencing. The hlucþ/hRluc luciferase reporter vectors of PTEN were constructed as above. Cells were seeded in 24-well plates and transfected with wild-type or mutated reporter vectors, or miR21 mimics, negative control mimics, anti-miR21 inhibitors, negative control inhibitors or vector pCDH-PTENP1, 24 hours after transfection, firefly and Renilla luciferase activities were consecutively measured, according to the Dual-Luciferase Assay Manual (Promega). The Renilla luciferase signal was normalized to the firefly luciferase signal for each individual analysis. RNA isolation and quantitative reverse transcription-PCR TotalRNAwas isolated from the indicated cell lines and renal cancer tissues by using the RNeasy Mini Kit (Qiagen) according to themanufacturer’s protocol. Total RNA PTENP1 as ceRNA i