Overcome Cell Adhesion-mediated Drug Resistance Research Articles

157 - Overcoming cell adhesion-mediated drug resistance (CAM-DR) in multiple myeloma by targeting possible crosstalk between the thioredoxin system and mucins

Multiple myeloma (MM) is a clonal plasma B-cell neoplasm that resides in the bone marrow (BM) cavity. While in the BM, the cells interact with BM stromal cells resulting in the secretion of chemokines and growth factors as well as expression of cell adhesion molecules that allow the myeloma cells to migrate to secondary BM sites where they eventually invade and proliferate. These events allow the cells to not only disseminate but also to become chemo-resistant to MM drugs through cell-adhesion mediated drug resistance (CAM-DR). The thioredoxin (Trx) system is one of the major cellular antioxidant systems involved in maintaining redox homeostasis in the cell. Studies have shown that the Trx system system redox-regulates a large number of transcription factors including NF-κβ, p53 and PTEN and is responsible for increased cancer cell proliferation, survival and chemo-resistance. Although inhibition of the Trx system has been shown to re-sensitise bortezomib-resistant MM cells, the effects of its redox activity to overcome CAM-DR remains to be fully elucidated. Mucins are a group of glycoproteins and their functions range from establishing molecular barriers for epithelial surfaces to signal transduction. In addition the mucins are able to mediate cell adhesion properties in cancer cells. Mucins are aberrantly expressed in MM and are also involved in the regulation of ROS. These findings suggest a possibility of crosstalk between the redox systems and mucins that may be involved in the pathogenesis of MM. This project aims to discover the possible crosstalk between the Trx system and mucins with respect to CAM-DR in MM.

Y-box-binding protein-1 (YB-1) promotes cell proliferation, adhesion and drug resistance in diffuse large B-cell lymphoma

YB-1 is a multifunctional protein, which has been shown to correlate with resistance to treatment of various tumor types. This study investigated the expression and biologic function of YB-1 in diffuse large B-cell lymphoma (DLBCL). Immunohistochemical analysis showed that the expression statuses of YB-1 and pYB-1S102 were reversely correlated with the clinical outcomes of DLBCL patients. In addition, we found that YB-1 could promote the proliferation of DLBCL cells by accelerating the G1/S transition. Ectopic expression of YB-1 could markedly increase the expression of cell cycle regulators cyclin D1 and cyclin E. Furthermore, we found that adhesion of DLBCL cells to fibronectin (FN) could increase YB-1 phosphorylation at Ser102 and pYB-1S102 nuclear translocation. In addition, overexpression of YB-1 could increase the adhesion of DLBCL cells to FN. Intriguingly, we found that YB-1 overexpression could confer drug resistance through cell-adhesion dependent and independent mechanisms in DLBCL. Silencing of YB-1 could sensitize DLBCL cells to mitoxantrone and overcome cell adhesion-mediated drug resistance (CAM-DR) phenotype in an AKT-dependent manner.

Romidepsin Overcomes Cell Adhesion-Mediated Drug Resistance in Multiple Myeloma Cells

Romidepsin Overcomes Cell Adhesion-Mediated Drug Resistance in Multiple Myeloma Cells

Abstract 4524: Designer nanoparticles for selective targeting of multiple myeloma and overcoming CAM-DR.

Abstract In the continuing search for effective treatments for cancer, here we report the targeting and efficacy studies pursued with a designer multifunctional nanoparticle that combines traditional chemotherapy with cell targeting and anti-cellular-adhesion functionalities to selectively target multiple myeloma (MM) cells and overcome cell adhesion-mediated drug resistance (CAM-DR). Anti-cellular-adhesion evolves as a promising target in oncology. VLA-4-mediated adhesion to the bone marrow extracellular matrix and stromal cells confers MM cells with CAM-DR. In our design, we used micellar nanoparticles as dynamic self-assembling scaffolds to present VLA-4-antagonist peptides and doxorubicin conjugates simultaneously to selectively target MM cells and to overcome CAM-DR. Doxorubicin was conjugated to the nanoparticles through an pH-sensitive hydrazone bond to prevent premature release and thus non-specific toxicity. Peptides were conjugated via a multifaceted synthetic procedure for generating highly effective targeting. Each component of the nanoparticle was purified to >98% purity prior to particle formation, and by controlling the stoichiometries of each component during particle formation, we were able to precisely control the number of targeting functionalities per nanoparticle. The nanoparticles, which exhibited a size of ∼20nm, were efficiently internalized by MM cells with an optimal peptide valency of 20 per micelle, and induced cytotoxicity to MM cells. Mechanistic studies revealed that nanoparticles induced DNA double strand breaks as evidenced by H2AX phosphorylation, and triggered apoptosis, which was associated with PARP and caspase-8 cleavage. Importantly, multifunctional nanoparticles were more efficacious than doxorubicin in the presence of fibronectin (IC50=0.15±0.04 μM and 0.42±0.09 μM, respectively), and overcame CAM-DR induced by adherence of MM cells to fibronectin. Finally, in a MM xenograft model, nanoparticles preferentially homed to MM tumors, with a ∼10 fold more drug accumulation when compared to doxorubicin, and demonstrated dramatic tumor growth inhibition with much reduced overall systemic toxicity. Taken together, we demonstrate the disease-driven engineering of a nanoparticle-based drug delivery system, enabling the model of an integrative approach in the treatment of MM. Citation Format: Tanyel Kiziltepe, Jonathan Ashley, Jared Stefanick, Basar Bilgicer. Designer nanoparticles for selective targeting of multiple myeloma and overcoming CAM-DR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4524. doi:10.1158/1538-7445.AM2013-4524

Abstract 5686: Engineering multifunctional nanoparticles to selectively target multiple myeloma cells and overcome cell adhesion-mediated drug resistance

Abstract In the continuing search for effective treatments for cancer, here we report the rational engineering of a novel multifunctional nanoparticle that combines traditional chemotherapy with cell targeting and anti-cellular-adhesion functionalities to selectively target multiple myeloma (MM) cells and overcome cell adhesion-mediated drug resistance (CAM-DR). Anti-cellular-adhesion evolves as a promising target in oncology. VLA-4-mediated adhesion to the bone marrow extracellular matrix and stromal cells confers MM cells with CAM-DR. In our design, we used micellar nanoparticles as dynamic self-assembling scaffolds to present VLA-4-antagonist peptides and doxorubicin conjugates simultaneously to selectively target MM cells and to overcome CAM-DR. Doxorubicin was conjugated to the nanoparticles through an pH-sensitive hydrazone bond to prevent premature release and thus non-specific toxicity. Peptides were conjugated via a multifaceted synthetic procedure for generating precisely controlled number of targeting functionalities per nanoparticle. The nanoparticles, which exhibited a size of ∼20nm, were efficiently internalized by MM cells with an optimal peptide valency of 20 per micelle, and induced cytotoxicity to MM cells. Mechanistic studies revealed that nanoparticles induced DNA double strand breaks as evidenced by H2AX phosphorylation, and triggered apoptosis, which was associated with PARP and caspase-8 cleavage. Importantly, multifunctional nanoparticles were more efficacious than doxorubicin in the presence of fibronectin (IC50=0.15±0.04 μM and 0.42±0.09 μM, respectively), and overcame CAM-DR induced by adherence of MM cells to fibronectin. Finally, in a MM xenograft model, nanoparticles preferentially homed to MM tumors, with a ∼10 fold more drug accumulation when compared to doxorubicin, and demonstrated dramatic tumor growth inhibition with much reduced overall systemic toxicity. Taken together, we demonstrate the disease-driven engineering of a nanoparticle-based drug delivery system, enabling the model of an integrative approach in the treatment of MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5686. doi:1538-7445.AM2012-5686

Arsenic Trioxide overcomes cell adhesion-mediated drug resistance through down-regulating the expression of β1-integrin in K562 chronic myelogenous leukemia cell line

The bone marrow microenvironment protects leukemia cells from drug-induced damage, and cell adhesion-mediated drug resistance (CAM-DR) is the main obstacle for the eradication of bone marrow minimal residual disease. In this study, we evaluated the survival response of K562 cells co-cultured with bone marrow stromal cells (BMSCs) derived from patients with chronic myelogenous leukemia (CML). Co-cultured K562 cells had a survival advantage after exposure to cytotoxic drugs compared to suspended K562 cells, but there was no survival difference between the two groups following exposure to As2O3. The cytotoxicity of As2O3 combined with cytotoxic drugs had an additive effect in the suspension group and a synergistic effect in the co-cultured group. Similar apoptotic responses were found using flow cytometry assays. As2O3 treatment caused a dose dependent reduction of the adhesion ability of K562 cells to BMSCs, and Western blot analysis revealed that the treatment of K562 cells with As2O3 led to down-regulation of β1-integrin.

Bortezomib overcomes cell adhesion-mediated drug resistance through downregulation of VLA-4 expression in multiple myeloma

Multiple myeloma (MM) is incurable, mainly because of cell adhesion-mediated drug resistance (CAM-DR). In this study, we performed functional screening using short hairpin RNA (shRNA) to define the molecule(s) responsible for CAM-DR of MM. Using four bona fide myeloma cell lines (KHM-1B, KMS12-BM, RPMI8226 and U266) and primary myeloma cells, we identified CD29 (beta1-integrin), CD44, CD49d (alpha4-integrin, a subunit of VLA-4), CD54 (intercellular adhesion molecule-1 (ICAM-1)), CD138 (syndecan-1) and CD184 (CXC chemokine receptor-4 (CXCR4)) as major adhesion molecules expressed on MM. shRNA-mediated knockdown of CD49d but not CD44, CD54, CD138 and CD184 significantly reversed CAM-DR of myeloma cells to bortezomib, vincristine, doxorubicin and dexamethasone. Experiments using blocking antibodies yielded almost identical results. Bortezomib was relatively resistant to CAM-DR because of its ability to specifically downregulate CD49d expression. This property was unique to bortezomib and was not observed in other anti-myeloma drugs. Pretreatment with bortezomib was able to ameliorate CAM-DR of myeloma cells to vincristine and dexamethasone. These results suggest that VLA-4 plays a critical role in CAM-DR of MM cells. The combination of bortezomib with conventional anti-myeloma drugs may be effective in overcoming CAM-DR of MM.

Combination therapy of an anticancer drug with the FNIII14 peptide of fibronectin effectively overcomes cell adhesion-mediated drug resistance of acute myelogenous leukemia

We investigated whether FNIII14, a 22-mer peptide derived from fibronectin (FN) that potently impairs interaction of FN with beta1-integrin, could overcome cell adhesion-mediated drug resistance (CAM-DR) induced by very late antigen (VLA)-4-to-FN interaction in acute myelogenous leukemia (AML). Two AML cell lines, U937 cells and HL-60 cells, and fresh leukemic cells from six AML patients with high alpha4-integrin expression exhibited CAM-DR to cytosine arabinoside (Ara C) through VLA-4-to-FN interaction, while fresh leukemic cells from two AML patients with low alpha4-integrin expression did not display CAM-DR to Ara C. FNIII14 impaired VLA-4-to-FN interaction and restored sensitivity to Ara C in the CAM-DR leukemic cells. In these CAM-DR leukemic cells, upregulation of Bcl-2, which was induced through the focal adhesion kinase/Akt signal pathway upon VLA-4-to-FN interaction, was inhibited by FNIII14 treatment. In a mouse model of minimal residual disease (MRD) in bone marrow, 100% survival was achieved by combining FNIII14 with Ara C, whereas Ara C alone prolonged survival only slightly. The myelosuppression induced by Ara C was not augmented by the combination of FNIII14 in mouse experiments. Thus, the combination of anticancer drugs and FNIII14 holds promise to eradicate MRD in bone marrow after chemotherapy.

First clinical experience with simvastatin to overcome drug resistance in refractory multiple myeloma

In vitro statins overcome cell adhesion-mediated drug resistance at non-toxic concentrations that are achievable in humans by standard dose simvastatin. A pilot phase-II trial was initiated to determine feasibility and antimyeloma efficacy. In six myeloma patients refractory to two cycles of bortezomib or bendamustine simvastatin was concomitantly administered during further two cycles. The therapy was well tolerated without grade 3/4 toxicity. Intrapatient (cycles I/II vs. III/IV) and interpatient comparison (vs. ten patients without simvastatin) showed reduction of drug resistance by inhibition of HMG-CoA-reductase. In summary, this is the first phase II experience to study antimyeloma activity of statins in humans.

Combination Therapy of Anti-Cancer Drug with FNIII14 Peptide of Fibronectin Effectively Overcome Cell Adhesion Mediated-Drug Resistance of Acute Myelogenous Leukemia.

We have previously reported that in acute myelogenous leukemia (AML), drug resistance is induced by the attachment of α4β1-integrin (very late antigen (VLA)-4) on leukemic cells (AML cells) to fibronectin (FN) on bone marrow (BM) stromal cells, and this interaction is crucial in BM minimal residual disease (MRD) and prognosis after chemotherapy (Matsunaga T et al, Nature Med 2003, 9, 1158–1165). We also reported a successful overcome of drug resistance in human AML cells transplanted mice with the use of anti-VLA-4 antibody. However, a drawback of progressive multifocal leukoencephalopathy by anti-VLA-4 antibody administration was reported in multiple sclerosis and Chron's disease patients. Therefore, in this study, we examined whether FNIII14, a 22-mer peptide derived from FN which was reportedly potent to block the interaction between FN and VLA-4, VLA-5 or αvβ3-integrin (Fukai F et al. Clin Cancer Res 2002, 8, 2455–2462), can overcome cell adhesion mediated drug resistance (CAM-DR) induced by VLA-4/FN interaction in AML cells. By in vitro culturing system, FNIII14 dose dependently inhibited adhesion of two AML cell lines (U937 cells, HL-60 cells) and fresh AML cells from patients to FN, and significantly circumvented CAM-DR of these AML cells to cytosine arabinoside (Ara C). The mechanism for this circumvention of CAM-DR involved conformational inactivation of β1-integrin as revealed by FACS through inside out inhibitory signal from complex FNIII14/its 50kDa receptor resulting in down regulation of FAK/Akt/Bcl-2 signal which is responsible for VLA-4/FN induced CAM-DR. In a mouse model of MRD, a 100% survival rate was achieved by combining FNIII14 and Ara C, in contrast to a slight prolongation of survival by Ara C alone. These results indicate that combination treatment of anticancer drugs and FNIII14 is the potent modality for eradication of MRD in BM, and should improve the prognosis of AML.

Tipifarnib and Bortezomib Are Synergistic and Overcome Cell Adhesion–Mediated Drug Resistance in Multiple Myeloma and Acute Myeloid Leukemia

It has been established in preclinical models of multiple myeloma and acute myeloid leukemia (AML) that the bone marrow microenvironment provides protection from chemotherapy- and death receptor-mediated apoptosis. This form of resistance, termed de novo drug resistance, occurs independent of chronic exposure to cancer-related therapies and likely promotes the development of multidrug resistance. Consequently, it is of major interest to identify compounds or drug combinations that can overcome environment-mediated resistance. In this study, we investigated the activity of tipifarnib (Zarnestra, formerly R115777) combined with bortezomib (Velcade, formerly PS-341) in microenvironment models of multiple myeloma and AML. The combination proved to be synergistic in multiple myeloma and AML cell lines treated in suspension culture. Even in tumor cells relatively resistant to tipifarnib, combined activity was maintained. Tipifarnib and bortezomib were also effective when multiple myeloma and AML cells were adhered to fibronectin, providing evidence that the combination overcomes cell adhesion-mediated drug resistance (CAM-DR). Of importance, activation of the endoplasmic reticulum stress response was enhanced and correlated with apoptosis and reversal of CAM-DR. Multiple myeloma and AML cells cocultured with bone marrow stromal cells also remained sensitive, although stromal-adhered tumor cells were partially protected (relative to cells in suspension or fibronectin adhered). Evaluation of the combination using a transwell apparatus revealed that stromal cells produce a protective soluble factor. Investigations are under way to identify the cytokines and/or growth factors involved. In summary, our study provides the preclinical rationale for trials testing the tipifarnib and bortezomib combination in patients with multiple myeloma and AML.

The HMG-CoA reductase inhibitor simvastatin overcomes cell adhesion–mediated drug resistance in multiple myeloma by geranylgeranylation of Rho protein and activation of Rho kinase

AbstractPrimary drug resistance is a major problem in multiple myeloma, an incurable disease of the bone marrow. Cell adhesion-mediated drug resistance (CAM-DR) causes strong primary resistance. By coculturing multiple myeloma cells with bone marrow stromal cells (BMSCs), we observed a CAM-DR of about 50% to melphalan, treosulfan, doxorubicin, dexamethasone, and bortezomib, which was not reversed by secreted soluble factors. Targeting the adhesion molecules lymphocyte function–associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4) by monoclonal antibodies or by the LFA-1 inhibitor LFA703 reduced CAM-DR significantly. Only statins such as simvastatin and lovastatin, however, were able to completely restore chemosensitivity. All these effects were not mediated by deadhesion or reduced secretion of interleukin 6. Targeting geranylgeranyl transferase (GGTase) and Rho kinase by specific inhibitors (GGTI-298 and Y-27632), but not inhibition of farnesyl transferase (FTase) by FTI-277, showed similar reduction of CAM-DR. Addition of geranylgeranyl pyrophosphate (GG-PP), but not of farnesyl pyrophosphate (F-PP), was able to inhibit simvastatin-induced CAM-DR reversal. Our data suggest that the 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA)/GG-PP/Rho/Rho-kinase pathway mediates CAM-DR and that targeting this pathway may improve the efficacy of antimyeloma therapies by reduction of CAM-DR.

Molecular sequelae of histone deacetylase inhibition in human malignant B cells

AbstractHistone acetylation modulates gene expression, cellular differentiation, and survival and is regulated by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC inhibition results in accumulation of acetylated nucleosomal histones and induces differentiation and/or apoptosis in transformed cells. In this study, we characterized the effect of suberoylanilide hydroxamic acid (SAHA), the prototype of a series of hydroxamic acid–based HDAC inhibitors, in cell lines and patient cells from B-cell malignancies, including multiple myeloma (MM) and related disorders. SAHA induced apoptosis in all tumor cells tested, with increased p21 and p53 protein levels and dephosphorylation of Rb. We also detected cleavage of Bid, suggesting a role for Bcl-2 family members in regulation of SAHA-induced cell death. Transfection of Bcl-2 cDNA into MM.1S cells completely abrogated SAHA-induced apoptosis, confirming its protective role. SAHA did not induce cleavage of caspase-8, -9, or -3 in MM.1S cells during the early phase of apoptosis, and the pan-caspase inhibitor ZVAD-FMK did not protect against SAHA. Conversely, poly(ADP)ribose polymerase (PARP) was cleaved in a pattern indicative of calpain activation, and the calpain inhibitor calpeptin abrogated SAHA-induced cell death. Importantly, SAHA sensitized MM.1S cells to death receptor–mediated apoptosis and inhibited the secretion of interleukin 6 (IL-6) induced in bone marrow stromal cells (BMSCs) by binding of MM cells, suggesting that it can overcome cell adhesion–mediated drug resistance. Our studies delineate the mechanisms whereby HDAC inhibitors mediate anti-MM activity and overcome drug resistance in the BM milieu and provide the framework for clinical evaluation of SAHA, which is bioavailable, well tolerated, and bioactive after oral administration, to improve patient outcome.