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"MicroRNA-9 is central to Glioblastoma Temozolomide resistance: Potential for RNAi delivery by Mesenchymal Stem Cells"

by
Jessian L. Munoz
MD/Ph.D. Program
B.S. 2009, University of Puerto Rico



Thesis Advisor: Pranela Rameshwar, Ph.D.
Professor
Department of Medicine- Division of Hematology/Oncology

Friday, April 26, 2013
11:00 A.M., MSB B610


Abstract

Glioblastoma Multiforme (GBM) is the most common and lethal adult primary tumor of the central nervous system. Despite therapeutic interventions, surgery, chemotherapy and radiation, patient outcome remains dismal. The molecular mechanisms underlying chemoresistance of GBM are poorly understood. The Sonic Hedgehog (SHH) pathway is dysregulated in chemoresistant GBM cells. Improved chemosensitivity occurred when the SHH pathway was inhibited, e.g., cyclopamine treatment. SHH is activated when its repressor receptor, PTCH1, is decreased. This thesis studied the mechanisms by which PTCH1 was decreased since this could be key to reversing temozolomide (TMZ) resistance. The resistant GBM cells showed an increase in PTCH1 mRNA and a decrease in its protein. Dicer knockdown GBM cells indicated a role for miRNA in reduced PTCH1 protein. Computational studies, real-time PCR, reporter gene studies, western blots, target protector oligos and, ectopic and knockdown of miR-9 identified miR-9 in PTCH1 decrease and TMZ resistance. MiR-9 increased the drug efflux transporters, MDR1 and ABCG2. TMZ regulated MDR1 expression in a bimodal manner, first by inducing the translocation and activation of MDR1 protein (P-gp) to the cell membrane. The later phase required new MDR1 transcript, through EGF-medated activation of ERK1/2-JNK-AP-1, which interacted with the 5′ regulatory region of MDR1 to increase its transcription. EGF also induced the expression of connexin 43 (Cx43), which established gap junctional intercellular communication (GJIC) between GBM cells. Cx43 knockdown resulted in enhanced chemosensitivity of the GBM cells. The findings were translated to identify methods for improved chemosensitivity. This was done by exploring GJIC to target miR-9 with anti-miR and to block SHH signaling pathways and the drug transporters with pharmacological inhibitors. Anti-miR was delivered with human bone marrow-derived mensenchymal stem cells (MSCs), which formed GJIC with the GBM cells. Although the GJIC was functional, the transfer of anti-miR-9 occurred mostly through exosomes. The inhibitor, GDC-0449, enhanced the sensitivity of GBM cells to TMZ. The findings with GBM cell lines were consistent with the information in the array database with >500 GBM patients and with early passaged GBM cell lines from a na´ve and recurrent GBM patients. Taken together, the data showed a role for miR-9 in the chemoresistance of GBMs, identified potential treatment targets and, showed a role for MSCs in the delivery of anti-miR-9.


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