Abstract
Multiple myeloma (MM) is incurable in virtually all patients due to the presence of innate and emergent drug-resistance. To identify potential drug resistance mechanisms in MM we used iTRAQ (isobaric tags for relative and absolute quantitation) mass spectrometry to compare protein expression profiles of drug-resistant (RPMI 8226-R5) and sensitive (RPMI 8226-S) isogenic cell lines. We identified selective overexpression of myristoylated alanine-rich C-kinase substrate (MARCKS) in drug-resistant R5 cells. MARCKS overexpression was also observed in several drug-resistant human myeloma cell lines (HMCLs) and in drug-resistant primary MM samples. Functionally, inhibition of MARCKS phosphorylation by enzastaurin or knockdown of the gene by RNAi significantly enhanced the sensitivity of resistant HMCLs and primary MM samples to bortezomib and to other anti-myeloma drugs, providing evidence that MARCKS can modulate drug response. Mechanistically, pMARCKS (phosphorylated form of MARCKS) was found to function as an E2F-1 cofactor to regulate SKP2 transcription. pMARCKS promoted cell-cycle progression by facilitating SKP2 expression, suppressing p27Kip1 and potentially counteracting drug-induced cell-cycle arrest by promoting Cyclin E/CDK2 activity. Importantly, MARCKS knockdown in combination with bortezomib treatment overcame bortezomib resistance, significantly inhibited tumor growth and prolonged host survival in a MM xenograft model. These data provide a rationale for therapeutic targeting of pMARCKS to improve the outcome of patients with refractory/relapsed MM.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Anderson KC . Oncogenomics to target myeloma in the bone marrow microenvironment. Clin Cancer Res 2011; 17: 1225–1233.
Dimopoulos MA, San-Miguel JF, Anderson KC . Emerging therapies for the treatment of relapsed or refractory multiple myeloma. Eur J Haematol 2011; 86: 1–15.
Feinman R, Koury J, Thames M, Barlogie B, Epstein J, Siegel DS . Role of NF-kappaB in the rescue of multiple myeloma cells from glucocorticoid-induced apoptosis by bcl-2. Blood 1999; 93: 3044–3052.
Ni H, Ergin M, Huang Q, Qin JZ, Amin HM, Martinez RL et alAnalysis of expression of nuclear factor kappa B (NF-kappa B) in multiple myeloma: downregulation of NF-kappa B induces apoptosis. Br J Haematol 2001; 115: 279–286.
Keats JJ, Fonseca R, Chesi M, Schop R, Baker A, Chng WJ et alPromiscuous mutations activate the noncanonical NF-kappaB pathway in multiple myeloma. Cancer Cell 2007; 12: 131–144.
Bhardwaj A, Sethi G, Vadhan-Raj S, Bueso-Ramos C, Takada Y, Gaur U et alResveratrol inhibits proliferation, induces apoptosis, and overcomes chemoresistance through down-regulation of STAT3 and nuclear factor-kappaB-regulated antiapoptotic and cell survival gene products in human multiple myeloma cells. Blood 2007; 109: 2293–2302.
Ma MH, Yang HH, Parker K, Manyak S, Friedman JM, Altamirano C et alThe proteasome inhibitor PS-341 markedly enhances sensitivity of multiple myeloma tumor cells to chemotherapeutic agents. Clin Cancer Res 2003; 9: 1136–1144.
Markovina S, Callander NS, O'Connor SL, Kim J, Werndli JE, Raschko M et alBortezomib-resistant nuclear factor-kappaB activity in multiple myeloma cells. Mol Cancer Res 2008; 6: 1356–1364.
Kuhn DJ, Berkova Z, Jones RJ, Woessner R, Bjorklund CC, Ma W et alTargeting the insulin-like growth factor-1 receptor to overcome bortezomib resistance in preclinical models of multiple myeloma. Blood 2012; 120: 3260–3270.
Moschetta M, Basile A, Ferrucci A, Frassanito MA, Rao L, Ria R et alNovel targeting of phospho-cMET overcomes drug resistance and induces antitumor activity in multiple myeloma. Clin Cancer Res 2013; 19: 4371–4382.
Zhou W, Yang Y, Xia J, Wang H, Salama ME, Xiong W et alNEK2 induces drug resistance mainly through activation of efflux drug pumps and is associated with poor prognosis in myeloma and other cancers. Cancer Cell 2013; 23: 48–62.
Gatlin JC, Estrada-Bernal A, Sanford SD, Pfenninger KH . Myristoylated, alanine-rich C-kinase substrate phosphorylation regulates growth cone adhesion and pathfinding. Mol Biol Cell 2006; 17: 5115–5130.
Micallef J, Taccone M, Mukherjee J, Croul S, Busby J, Moran MF et alEpidermal growth factor receptor variant III-induced glioma invasion is mediated through myristoylated alanine-rich protein kinase C substrate overexpression. Cancer Res 2009; 69: 7548–7556.
Rombouts K, Lottini B, Caligiuri A, Liotta F, Mello T, Carloni V et alMARCKS is a downstream effector in platelet-derived growth factor-induced cell motility in activated human hepatic stellate cells. Exp Cell Res 2008; 314: 1444–1454.
Hartwig JH, Thelen M, Rosen A, Janmey PA, Nairn AC, Aderem A . MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin. Nature 1992; 356: 618–622.
Blackshear PJ . The MARCKS family of cellular protein kinase C substrates. J Biol Chem 1993; 268: 1501–1504.
Aderem A . The MARCKS brothers: a family of protein kinase C substrates. Cell 1992; 71: 713–716.
Estrada-Bernal A, Gatlin JC, Sunpaweravong S, Pfenninger KH . Dynamic adhesions and MARCKS in melanoma cells. J Cell Sci 2009; 122: 2300–2310.
Podar K, Raab MS, Zhang J, McMillin D, Breitkreutz I, Tai YT et alTargeting PKC in multiple myeloma: in vitro and in vivo effects of the novel, orally available small-molecule inhibitor enzastaurin (LY317615.HCl). Blood 2007; 109: 1669–1677.
Raab MS, Breitkreutz I, Tonon G, Zhang J, Hayden PJ, Nguyen T et alTargeting PKC: a novel role for beta-catenin in ER stress and apoptotic signaling. Blood 2009; 113: 1513–1521.
Broyl A, Hose D, Lokhorst H, de Knegt Y, Peeters J, Jauch A et alGene expression profiling for molecular classification of multiple myeloma in newly diagnosed patients. Blood 2010; 116: 2543–2553.
Podar K, Raab MS, Chauhan D, Anderson KC . The therapeutic role of targeting protein kinase C in solid and hematologic malignancies. Expert Opin Investig Drugs 2007; 16: 1693–1707.
Bergsagel PL, Kuehl WM, Zhan F, Sawyer J, Barlogie B, Shaughnessy J Jr . Cyclin D dysregulation: an early and unifying pathogenic event in multiple myeloma. Blood 2005; 106: 296–303.
Chen CH, Thai P, Yoneda K, Adler KB, Yang PC, Wu R . A peptide that inhibits function of myristoylated alanine-rich C kinase substrate (MARCKS) reduces lung cancer metastasis. Oncogene 2013; 33: 3696–3706.
Teicher BA . Protein kinase C as a therapeutic target. Clin Cancer Res 2006; 12: 5336–5345.
Roffey J, Rosse C, Linch M, Hibbert A, McDonald NQ, Parker PJ . Protein kinase C intervention: the state of play. Curr Opin Cell Biol 2009; 21: 268–279.
Buzzeo R, Enkemann S, Nimmanapalli R, Alsina M, Lichtenheld MG, Dalton WS et alCharacterization of a R115777-resistant human multiple myeloma cell line with cross-resistance to PS-341. Clin Cancer Res 2005; 11: 6057–6064.
Ott LE, Sung EJ, Melvin AT, Sheats MK, Haugh JM, Adler KB et alFibroblast migration is regulated by myristoylated alanine-rich C-kinase substrate (MARCKS) protein. PLoS One 2013; 8: e66512.
Zhang L, Wang C . F-box protein Skp2: a novel transcriptional target of E2F. Oncogene 2006; 25: 2615–2627.
Salon C, Merdzhanova G, Brambilla C, Brambilla E, Gazzeri S, Eymin B . E2F-1, Skp2 and cyclin E oncoproteins are upregulated and directly correlated in high-grade neuroendocrine lung tumors. Oncogene 2007; 26: 6927–6936.
Walker BA, Leone PE, Jenner MW, Li C, Gonzalez D, Johnson DC et alIntegration of global SNP-based mapping and expression arrays reveals key regions, mechanisms, and genes important in the pathogenesis of multiple myeloma. Blood 2006; 108: 1733–1743.
Stephens OW, Zhang Q, Qu P, Zhou Y, Chavan S, Tian E et alAn intermediate-risk multiple myeloma subgroup is defined by sIL-6r: levels synergistically increase with incidence of SNP rs2228145 and 1q21 amplification. Blood 2012; 119: 503–512.
Zhan F, Colla S, Wu X, Chen B, Stewart JP, Kuehl WM et alCKS1B, overexpressed in aggressive disease, regulates multiple myeloma growth and survival through SKP2- and p27Kip1-dependent and -independent mechanisms. Blood 2007; 109: 4995–5001.
Chen Q, Xie W, Kuhn DJ, Voorhees PM, Lopez-Girona A, Mendy D et alTargeting the p27 E3 ligase SCF(Skp2) results in p27- and Skp2-mediated cell-cycle arrest and activation of autophagy. Blood 2008; 111: 4690–4699.
Chan CH, Morrow JK, Li CF, Gao Y, Jin G, Moten A et alPharmacological inactivation of Skp2 SCF ubiquitin ligase restricts cancer stem cell traits and cancer progression. Cell 2013; 154: 556–568.
Chen HZ, Tsai SY, Leone G . Emerging roles of E2Fs in cancer: an exit from cell cycle control. Nat Rev Cancer 2009; 9: 785–797.
Jarboe JS, Anderson JC, Duarte CW, Mehta T, Nowsheen S, Hicks PH et alMARCKS regulates growth and radiation sensitivity and is a novel prognostic factor for glioma. Clin Cancer Res 2012; 18: 3030–3041.
Acknowledgements
The study was supported in part by the grants from Leukemia & Lymphoma Society of Canada, Cancer Research Society of Canada, International Collaboration Fund from National Science and Technology Committee of China (2011DFA32820) and Gan-Po 555 project, Jiangxi, China. We thank Dr E Zacksenhaus and Dr R Tiedemann for their helpful suggestions.
Author Contributions
YY, YC, NMS and JC performed the research and analyzed the data. KE, LQ and AGC contributed to vital reagents and analyzed the data. LQ and DR contributed to patients' clinical information and materials. HC designed the research and analyzed the data. YY, NMS and HC wrote the paper.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Additional information
Supplementary Information accompanies this paper on the Leukemia website
Supplementary information
Rights and permissions
About this article
Cite this article
Yang, Y., Chen, Y., Saha, M. et al. Targeting phospho-MARCKS overcomes drug-resistance and induces antitumor activity in preclinical models of multiple myeloma. Leukemia 29, 715–726 (2015). https://doi.org/10.1038/leu.2014.255
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/leu.2014.255
This article is cited by
-
The macrophage-associated prognostic gene ANXA5 promotes immunotherapy resistance in gastric cancer through angiogenesis
BMC Cancer (2024)
-
Antler-derived microRNA PC-5p-1090 inhibits HCC cell proliferation, migration, and invasion by targeting MARCKS, SMARCAD1, and SOX9
Functional & Integrative Genomics (2023)
-
SMAD1 as a biomarker and potential therapeutic target in drug-resistant multiple myeloma
Biomarker Research (2021)
-
Pathophysiological roles of myristoylated alanine-rich C-kinase substrate (MARCKS) in hematological malignancies
Biomarker Research (2021)
-
Restoration of MARCK enhances chemosensitivity in cancer
Journal of Cancer Research and Clinical Oncology (2020)