Targeting MARCKS Inhibits Growth of Bortezomib-Resistant Multiple Myeloma Cells through E2F1 and SKP2-Mediated Signal Pathway
Yijun Yang, Manujendra N Saha, Hong Chang. University Health Network, Toronto, ON, Canada; University of Toronto, Toronto, ON, Canada
Background: Multiple myeloma (MM) remains an incurable disease because of its inevitable resistance to antimyeloma drug. Using proteomic profiling, we recently identified that MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) is highly differentially expressed in bortezomib-resistant MM cells but not in sensitive cells. Here we sought to investigate the molecular mechanisms of MARCKS associated with drug resistance in MM in vitro and in vivo.
Design: Transfection with MARCKS RNAi and treatment with enzastaurin, a small molecular inhibitor for phospho-MARCKS (pMARCKS), were used to block MARCKS in bortezomib-resistant MM cells (8226R5 and MM1R) and to determine the cell viability, cell-cycle distribution and apoptosis. Regulation of the activation among MARCKS, SKP2, and E2F 1 were assessed by immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) assays. To evaluate the impact of pMARCKS on tumor growth in vivo, a xenograft mouse model was established by subcutaneous injection of SCID mice with 8226R5 cells. When tumors were palpable, mice were treated once with an interval of 2 days with shMARCKS lentiviurs (25 mmol/kg) and/or bortezomib (0.5 mg/kg) or vehicle (PBS).
Results: Reduction of pMARCKS through knockdown of MARCKS or inhibition by enzastaurin led to a decreased SKP2 and an increase in p27 and p21, as well as a reduction of Cyclin E and CDK2 resulting in G1/S cell cycle arrest and apoptosis in both MM cell lines. In addition, pMARCKS interacted with E2F1, and subsequently activated SKP2 by binding with SKP2 promoter affecting cell-cycle distribution and increase cell survival. Moreover, in vivo studies with shMARCKS showed that mice treated with shMARCKS lentivirus or bortezomib alone had modest effects, however, cotreatment with both agents significantly suppressed tumor growth compared with either agent alone. shMARCKS lentivirus plus bortezomib treated mice showed significantly increased survival compared with the vehicle treated group (p=0.0268), or with either agent alone (p=0.05 and p=0.034 for shMARCKS and bortezomib, respectively).
Conclusions: These findings suggest that pMARCKS is required for the survival of bortezomib-resistant human MM cells through E2F1 activation on SKP2/p27 mediated cell cycle signaling pathway. Targeting pMARCKS sensitizes MM cells to bortezomib treatment and enhances the inhibition of tumor growth, thus provides a potential novel therapeutic strategy in MM patiens with bortezomib resistance.
Wednesday, March 6, 2013 1:00 PM
Poster Session VI # 232, Wednesday Afternoon