Tumor and Stem Cell Biology LIN 28 Expression in Malignant Germ Cell Tumors Downregulates let-7 and Increases Oncogene Levels

Despite their clinicopathologic heterogeneity, malignant germ cell tumors (GCT) share molecular abnormalities that are likely to be functionally important. In this study, we investigated the potential significance of downregulation of the let-7 family of tumor suppressor microRNAs in malignant GCTs. Microarray results from pediatric and adult samples (n1⁄4 45) showed that LIN28, the negative regulator of let-7 biogenesis, was abundant in malignant GCTs, regardless of patient age, tumor site, or histologic subtype. Indeed, a strong negative correlation existed between LIN28 and let-7 levels in specimens with matched datasets. Low let-7 levels were biologically significant, as the sequence complementary to the 2 to 7 nt common let-7 seed "GAGGUA" was enriched in the 30 untranslated regions of mRNAs upregulated in pediatric and adult malignant GCTs, compared with normal gonads (a mixture of germ cells and somatic cells). We identified 27 mRNA targets of let-7 that were upregulated in malignant GCT cells, confirming significant negative correlations with let-7 levels. Among 16 mRNAs examined in a largely independent set of specimens by quantitative reverse transcription PCR, we defined negative-associations with let-7e levels for six oncogenes, includingMYCN, AURKB, CCNF, RRM2,MKI67, and C12orf5 (when including normal control tissues). Importantly, LIN28 depletion in malignant GCT cells restored let-7 levels and repressed all of these oncogenic let-7 mRNA targets, with LIN28 levels correlating with cell proliferation andMYCN levels. Conversely, ectopic expression of let-7e was sufficient to reduce proliferation and downregulate MYCN, AURKB, and LIN28, the latter via a double-negative feedback loop. We conclude that the LIN28/let-7 pathway has a critical pathobiologic role in malignant GCTs and therefore offers a promising target for therapeutic intervention. Cancer Res; 73(15); 4872–84. 2013 AACR. Introduction Germ cell tumors (GCT) are clinically and histopathologically complex. They present from early infancy through to late adulthood, occur at both gonadal and extragonadal sites, and comprise diverse histologic subtypes (1). Benign forms show somatic differentiation and are termed teratomas, whereas malignant GCTs are classified into germinomas (a collective term for testicular seminoma, ovarian dysgerminoma, and extragonadal germinoma) and non-germinomatous tumors, the main types of which are yolk sac tumors (YST) and embryonal carcinoma (1). Although most patients with malignant GCTs have a good prognosis, some patients still have inferior outcomes, and testicular germ cell malignancy remains a leading cause of death in young men (2). Improved understanding of the molecular pathogenesis of malignant GCTs would represent an important step toward developing novel therapeutic agents with favorable toxicity profiles, whichmay improve survival for patients with high-risk disease and reduce toxicity for low-risk patients. It is particularly important to identify abnormalities that are shared across the diverse spectrumofmalignantGCTs, as these are likely to be of fundamental significance in disease pathogenesis. Usingmicroarray profiling, we previously identified that all 9 members of the lethal-7 (let-7) microRNA family were significantly underexpressed in pediatric malignant GCTs, when compared with nonmalignant control tissues (3). MicroRNAs regulate gene expression via their 50 seed region (nucleotides at positions 1–8; 1–8 nt), which binds the corresponding seed complementary region (SCR) located predominantly in the 30untranslated region (30UTR) of mRNA targets (4). Within the seed, 2 to 7 nt are most critical for binding specificity (5). Importantly, all 9 let-7microRNAs share the same 2 to 7 nt seed sequence (GAGGUA) and therefore share mRNA targets containing the 30UTR SCR "TACCTC. " Authors' Affiliations: Department of Pathology, Cambridge University; Departments of Paediatric Oncology and Histopathology, Addenbrooke's Hospital; EMBL European Bioinformatics Institute, Hinxton, Cambridge; and Department of Pathology, Royal Group of Hospitals Trust, Belfast, United Kingdom Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Matthew J. Murray, Department of Pathology, Cambridge University, Cambridge, United Kingdom. Phone: 44-1223765066; Fax: 44-1223-333346; E-mail: mjm16@cam.ac. uk doi: 10.1158/0008-5472.CAN-12-2085 2013 American Association for Cancer Research. Cancer Research Cancer Res; 73(15) August 1, 2013 4872 on July 19, 2017. © 2013 American Association for Cancer Research. cancerres.aacrjournals. org Downloaded from Published OnlineFirst June 17, 2013; DOI: 10.1158/0008-5472.CAN-12-2085