Exocytosis of polyubiquitinated proteins in bortezomib-resistant leukemia cells: a role for MARCKS in acquired resistance to proteasome inhibitors
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Niels E. Franke1, Gertjan L. Kaspers1, Yehuda G. Assaraf2, Johan van Meerloo1,3, Denise Niewerth1, Floortje L. Kessler3, Pino J. Poddighe4, Jeroen Kole9, Serge J. Smeets5, Bauke Ylstra5, Chonglei Bi6,11, Wee Joo Chng6, Terzah M. Horton7, Rene X. Menezes8, Renée J.P. Musters9, Sonja Zweegman3, Gerrit Jansen10,*, Jacqueline Cloos1,3,*
1Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands
2The Fred Wyszkowski Cancer Research Laboratory, Technion-Israel Institute of Technology, Haifa, Israel
3Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands
4Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands
5Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands
6Department of Experimental Therapeutics, Cancer Science Institute of Singapore, National University of Singapore, Singapore
7Texas Children’s Cancer Center, Baylor College of Medicine, Houston, TX, USA
8Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands
9Department of Physiology, VU University, Amsterdam, The Netherlands
10Department of Rheumatology, Amsterdam Rheumatology and immunology Center, VU University Medical Center, Amsterdam, The Netherlands
11Current address: BGI-Shenzhen, Shenzhen, China
*Joint senior authors
Jacqueline Cloos, email: [email protected]
Keywords: bortezomib, resistance, proteasome, MARCKS, leukemia
Received: October 19, 2015 Accepted: July 26, 2016 Published: August 17, 2016
PSMB5 mutations and upregulation of the β5 subunit of the proteasome represent key determinants of acquired resistance to the proteasome inhibitor bortezomib (BTZ) in leukemic cells in vitro. We here undertook a multi-modality (DNA, mRNA, miRNA) array-based analysis of human CCRF-CEM leukemia cells and BTZ-resistant subclones to determine whether or not complementary mechanisms contribute to BTZ resistance. These studies revealed signatures of markedly reduced expression of proteolytic stress related genes in drug resistant cells over a broad range of BTZ concentrations along with a high upregulation of myristoylated alanine-rich C-kinase substrate (MARCKS) gene expression. MARCKS upregulation was confirmed on protein level and also observed in other BTZ-resistant tumor cell lines as well as in leukemia cells with acquired resistance to other proteasome inhibitors. Moreover, when MARCKS protein expression was demonstrated in specimens derived from therapy-refractory pediatric leukemia patients (n = 44), higher MARCKS protein expression trended (p = 0.073) towards a dismal response to BTZ-containing chemotherapy. Mechanistically, we show a BTZ concentration-dependent association of MARCKS protein levels with the emergence of ubiquitin-containing vesicles in BTZ-resistant CEM cells. These vesicles were found to be extruded and taken up in co-cultures with proteasome-proficient acceptor cells. Consistent with these observations, MARCKS protein associated with ubiquitin-containing vesicles was also more prominent in clinical leukemic specimen with ex vivo BTZ resistance compared to BTZ-sensitive leukemia cells. Collectively, we propose a role for MARCKS in a novel mechanism of BTZ resistance via exocytosis of ubiquitinated proteins in BTZ-resistant cells leading to quenching of proteolytic stress.
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