title: Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models.
abstract: Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center; Department of Hematology, VU University Medical Center; Department of Medical Oncology, VU University Medical Center.
authors.forename: Rocco
authors.initials: R
authors.lastname: Sciarrillo
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center.
authors.forename: Anna
authors.initials: A
authors.lastname: Wojtuszkiewicz
authors.affiliation: Department of Clinical Genetics, VU University Medical Center.
authors.forename: Irsan E
authors.initials: IE
authors.lastname: Kooi
authors.affiliation: Department of Medical Oncology, VU University Medical Center.
authors.forename: Valentina E
authors.initials: VE
authors.lastname: Gómez
authors.affiliation: Division of General and Transplant Surgery, Azienda Ospedaliera Universitaria Pisana, Universita' di Pisa.
authors.forename: Ugo
authors.initials: U
authors.lastname: Boggi
authors.affiliation: Amsterdam Immunology and Rheumatology Center, VU University Medical Center.
authors.forename: Gerrit
authors.initials: G
authors.lastname: Jansen
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center; Princess Máxima Center for Pediatric Oncology.
authors.forename: Gert-Jan
authors.initials: GJ
authors.lastname: Kaspers
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Cloos
authors.affiliation: Department of Medical Oncology, VU University Medical Center; Cancer Pharmacology Lab, AIRC Start-Up Unit, University of Pisa; Institute of Nanoscience and Nanotechnology, CNR-Nano; e.giovannetti@vumc.nl.
authors.forename: Elisa
authors.initials: E
authors.lastname: Giovannetti
mesh_terms: D002352:Carrier Proteins / Q000235:genetics; D053487:DEAD-box RNA Helicases / Q000235:genetics; D019175:DNA Methylation; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D009369:Neoplasms / Q000235:genetics*; D020033:Protein Isoforms / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D012313:RNA; D017423:Sequence Analysis, RNA; D013963:Thyroid Hormones / Q000235:genetics; D000097810:Thyroid Hormone-Binding Proteins
chemical_list: D002352:Carrier Proteins; D053487:DEAD-box RNA Helicases; D008565:Membrane Proteins; D020033:Protein Isoforms; D012313:RNA; D013963:Thyroid Hormones; D000097810:Thyroid Hormone-Binding Proteins; C506749:Ddx5 protein, human