Hematoxylin and eosin staining was performed by routine methods. components. Pharmacologic inhibition of the mTOR pathway reduces BT183 cell viability. == Conclusions == BT183 retains key genetic and histologic features of ETMR. In ETMR, Lin28 is not only a diagnostic marker but also a regulator of genes involved in growth and metabolism. Our findings indicate that inhibitors of the IGF/PI3K/mTOR pathway may be promising novel therapies for these fatal embryonal tumors. As the first patient-derived cell line of these rare tumors, BT183 is an important, unique reagent for investigating ETMR biology and therapeutics. Keywords:ETMR, ETANTR, C19MC, Lin28, mTOR Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are a class of malignant childhood brain tumors featuring poorly differentiated cells of neural origin. Among these, tumors that fall under the histologic category of embryonal tumor with multilayered rosettes (ETMR) (first described as embryonal tumor with abundant neuropil and true rosettes or ETANTR) occur primarily in infants and young children and are particularly aggressive, with common survival of <1 year after diagnosis despite intensive therapy.1,2Recently, ETMRs have been found to harbor recurrent focal amplifications of chr19q13.41-42, encoding the large polycistronic microRNA cluster C19MC, a genetically defining lesion that is likely critical to the biology of the disease.3,4 Amplification of C19MC is associated with marked overexpression of miRNAs in the cluster that can drive proliferation, promote survival, and increase tumorigenicity of cells.3The oncogenic properties of the overexpressed miRNAs reflect some of the known functions of C19MC. In normal development, the C19MC locus is usually posited to control general stemness programs, as many C19MC miRNAs show expression restricted to undifferentiated tissues or germinal tissues, and their expression decreases with human embryonic stem cell differentiation.57Further supporting the association of C19MC with stemness, the pluripotency-associated transcription factors Oct4 and Nanog may regulate C19MC miRNAs in human embryonic stem cells.5 Interestingly, C19MC-amplified CNS-PNETs also show striking upregulation of other distinctive primitive markers including the RNA-binding proteins Lin28A and LIN28B, 8a finding that is diagnostically useful as a marker of ETMRs.9Beyond its diagnostic utility, however, Lin28 overexpression may be ENTPD1 functionally important as well. Through binding and repression of let-7 microRNAs, Lin28 has been found to regulate lineage, stemness, cellular metabolism,and the cell cycle in normal and disease says.1012Lin28 also directly binds other mRNAs to increase their translation, with the cumulative effect that Lin28 may coordinately control both proliferative growth and metabolism.1319 Despite our increasing knowledge of C19MC and Lin28 functions, the biology of ETMR has remained poorly understood. Furthermore, the ability to translate the molecular findings to potential therapeutic targets for these tumors has been hampered Lesopitron dihydrochloride by a lack of model systems for the disease. Here, we have established a patient-derived ETMR cell line that maintains the Lesopitron dihydrochloride characteristic genetic and histologic features of the disease including C19MC-amplification and Lin28 expression. We found that Lin28 expression Lesopitron dihydrochloride was inversely correlated with expression of let-7 microRNAs and positively associated with mTOR pathway activation. Lin28 knockdown also increased let-7 expression and decreased expression of mTOR signaling pathway components, supporting the notion that Lin28 dysregulation is usually functionally important in ETMR. This novel cell line will be an important tool for further investigating ETMR biology and for identifying future therapies for this and other C19MC-overexpressing cancers. == Materials and Methods == == Tumor and Nucleic Acid Samples == Formalin-fixed paraffin embedded (FFPE) tumor specimens and fresh frozen tumor specimens were collected with consent per protocols approved by the Hospital for Sick Children Research Ethics Board in Toronto and the Conjoint Health Research Ethics Board of the University of Calgary. Clinical and genetic information on these tumors was reported previously.3Only tumors classified histologically as CNS-PNET according to 2007 WHO CNS tumor classification criteria20were included. DNA and RNA were extracted from frozen tumor material using Qiagen DNeasy (Qiagen) and Trizol (Invitrogen), respectively, per manufacturer’s protocol. In this paper, we use a genetic definition of ETMR to refer to histologic.
