[Show abstract][Hide abstract] ABSTRACT: Multiple myeloma (MM) is characterized by the clonal expansion of malignant plasma cells within the bone marrow. There is a growing literature that tumor cells release biologically active microvesicles (MVs) that modify both local and distant microenvironments. In this study, our goals were to determine if MM cells release MVs, and if so, begin to characterize their biologic activity. Herein we present clear evidence that not only do both patient MM cells and human MM cell lines (HMCLs) release MVs, but that these MVs stimulate MM cell growth. Of interest, MM-derived MVs were enriched with the biologically active form of CD147, a transmembrane molecule previously shown by us to be crucial for MM cell proliferation. Using MVs isolated from HMCLs stably transfected with a CD147-GFP fusion construct (CD147GFP), we observed binding and internalization of MV-derived CD147 with HMCLs. Cells with greater CD147GFP internalization proliferated at a higher rate than did cells with less CD147GFP association. Lastly, MVs obtained from CD147 downregulated HMCLs were attenuated in their ability to stimulate HMCL proliferation. In summary, this study demonstrates the significance of MV shedding and MV-mediated intercellular communication on malignant plasma cell proliferation, and identifies the role of MV-enriched CD147 in this process.
[Show abstract][Hide abstract] ABSTRACT: Increased use of the glycolytic pathway, even in the presence of oxygen, has recently been recognized as a key characteristic of malignant cells. However, the glycolytic phenotype results in increased lactic acid production and, in order to prevent cellular acidosis tumor cells, must increase proton efflux via upregulation of pH regulators such as proton-pumps, sodium-proton exchangers, and/or monocarboxylate transporters (MCT) (e.g., MCT1, MCT4). Interestingly, expression of MCT1 and MCT4 has been previously shown to be dependent upon expression of the transmembrane glycoprotein CD147. Recently, we demonstrated that primary patient multiple myeloma (MM) cells and human MM cell lines (HMCLs) overexpress CD147. Therefore, the goal of the current study was to specifically determine if MCT1 and MCT4 were also overexpressed in MM cells. RT-PCR analysis demonstrated both primary patient MM cells and HMCLs overexpress MCT1 and MCT4 mRNA. Notably, primary MM cells or HMCLs were found to express variable levels of MCT1 and/or MCT4 at the protein level despite CD147 expression. In those HMCLs positive for MCT1 and/or MCT4 protein expression, MCT1 and/or MCT4 were found to be associated with CD147. Specific siRNA-mediated downregulation of MCT1 but not MCT4 resulted in decreased HMCL proliferation, decreased lactate export, and increased cellular media pH. However, western blot analysis revealed that downregulation of MCT1 also downregulated CD147 and vice versa despite no effect on mRNA levels. Taken together, these data demonstrate the association between MCT1 and CD147 proteins in MM cells and importance of their association for lactate export and proliferation in MM cells.
[Show abstract][Hide abstract] ABSTRACT: The introduction of novel immunomodulatory drugs (IMiDs) has dramatically improved the survival of patients with multiple myeloma (MM). While it has been shown that patients with specific cytogenetic subtypes, namely t(4;14), have the best outcomes when treated with bortezomib-based regimens, the relationship between cytogenetic subtypes and response to IMiDs remains unclear. Using DNA synthesis assays, we investigated the relationship between cytogenetic subtype and lenalidomide response in a representative panel of human myeloma cell lines (HMCLs). We examined HMCL protein expression levels of the lenalidomide target cereblon (CRBN) and its downstream target interferon regulatory factor 4 (IRF4), which have previously been shown to be predictive of lenalidomide response in HMCLs. Our results reveal that lenalidomide response did not correlate with specific cytogenetic translocations. There were distinct groups of lenalidomide-responsive and non-responsive HMCLs, as defined by inhibition of cellular proliferation; notably, all of the hyperdiploid HMCLs fell into the latter category. Repeated dosing of lenalidomide significantly lowered the IC50 of the responsive HMCL ALMC-1 (IC50 = 2.6 μM versus 0.005 μM, p<0.0001), but did not have an effect on the IC50 of the non-responsive DP-6 HMCL (p>0.05). Moreover, no association was found between lenalidomide responsiveness and CRBN and IRF4 expression. Our data indicate that lenalidomide sensitivity is independent of cytogenetic subtype in HMCLs. While CRBN and IRF4 have been shown to be associated with response to lenalidomide in patients, these findings do not translate back to HMCLs, which could be attributable to factors present in the bone marrow microenvironment. This article is protected by copyright. All rights reserved.
European Journal Of Haematology 09/2013; 91(6). DOI:10.1111/ejh.12192 · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The biology of the malignant plasma cells (PCs) in multiple myeloma (MM) is highly influenced by the bone marrow (BM) microenvironment in which they reside. More specifically, BM stromal cells (SCs) are known to interact with MM cells to promote MM cell survival and proliferation. By contrast, it is unclear if innate immune cells within this same space also actively participate in the pathology of MM. Our study shows for the first time that eosinophils (Eos) can contribute to the biology of MM by enhancing the proliferation of some malignant PCs. We first demonstrate that PCs and Eos can be found in close proximity in the BM. In culture, Eos were found to augment MM cell proliferation that is predominantly mediated through a soluble factor(s). Fractionation of cell-free supernatants and neutralization studies demonstrated that this activity is independent of Eos-derived microparticles and a proliferation-inducing ligand (APRIL), respectively. Using a multicellular in vitro system designed to resemble the native MM niche, SCs and Eos were shown to have non-redundant roles in their support of MM cell growth. Whereas SCs induce MM cell proliferation predominantly through the secretion of IL-6, Eos stimulate growth of these malignant cells via an IL-6-independent mechanism. Taken together, our study demonstrates for the first time a role for Eos in the pathology of MM and suggests that therapeutic strategies targeting these cells may be beneficial.
PLoS ONE 07/2013; 8(7):e70554. DOI:10.1371/journal.pone.0070554 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Multiple myeloma (MM) is preceded by the asymptomatic pre-malignant state, monoclonal gammopathy of undetermined significance (MGUS). Although MGUS patients may remain stable for years, they are at increased risk of progressing to MM. A better understanding of the relevant molecular changes underlying the transition from an asymptomatic to symptomatic disease state is urgently needed. Our studies show for the first time that the CD147 molecule (extracellular matrix metalloproteinase inducer) may be having an important biological role in MM. We first demonstrate that CD147 is overexpressed in MM plasma cells (PCs) vs normal and pre-malignant PCs. Next, functional studies revealed that the natural CD147 ligand, cyclophilin B, stimulates MM cell growth. Moreover, when MM patient PCs displaying bimodal CD147 expression were separated into CD147(bright) and CD147(dim) populations and analyzed for proliferation potential, we discovered that CD147(bright) PCs displayed significantly higher levels of cell proliferation than did CD147(dim) PCs. Lastly, CD147-silencing significantly attenuated MM cell proliferation. Taken together, these data suggest that the CD147 molecule has a key role in MM cell proliferation and may serve as an attractive target for reducing the proliferative compartment of this disease.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 03/2012; 26(10):2286-96. DOI:10.1038/leu.2012.91 · 9.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA double-strand breaks (DSBs) are deleterious lesions that can lead to chromosomal anomalies, genomic instability and cancer. The histone protein H2AX has an important role in the DNA damage response (DDR) and the presence of phospho-H2AX (γH2AX) nuclear foci is the hallmark of DSBs. We hypothesize that ongoing DNA damage provides a mechanism by which chromosomal abnormalities and intratumor heterogeneity are acquired in malignant plasma cells (PCs) in patients with multiple myeloma (MM). Therefore, we assessed PCs from patients with the premalignant condition, monoclonal gammopathy of undetermined significance (MGUS) and MM, as well as human MM cell lines (HMCLs) for evidence of DSBs. γH2AX foci were detected in 2/5 MGUS samples, 37/40 MM samples and 6/6 HMCLs. Notably, the DSB response protein 53BP1 colocalized with γH2AX in both MM patient samples and HMCLs. Treatment with wortmannin decreased phosphorylation of H2AX and suggests phosphoinositide (PI) 3-kinases and/or PI3-kinase-like family members underlie the presence of γH2AX foci in MM cells. Taken together, these data imply that ongoing DNA damage intensifies across the disease spectrum of MGUS to MM and may provide a mechanism whereby clonal evolution occurs in the monoclonal gammopathies.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 05/2011; 25(8):1344-53. DOI:10.1038/leu.2011.94 · 9.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Receptor crosstalk is an emerging and recurrent theme in cytokine and growth factor signaling; however, insight into the mechanism(s) underlying these interactions remains limited. Recently, we reported that crosstalk occurs between ErbB3 and the interferon alpha (IFN-alpha) signaling complex in the myeloma cell line KAS-6/1 and that this crosstalk contributes to the regulation of cell proliferation. In this study, we examined the mechanism underlying the transactivation of ErbB3 in the IFN-alpha growth-responsive KAS-6/1 cells. The examination of IFN-alpha receptor 1 and 2 (IFNAR1 and IFNAR2) levels revealed that the KAS-6/1 cell line overexpresses IFNAR1 relative to other myeloma cell lines that are growth arrested by IFN-alpha. Subsequent investigation of Tyk2, which is constitutively associated with IFNAR1, demonstrated that Tyk2 activation is uniquely sustained in the KAS-6/1 cell line following IFN-alpha stimulation. Interestingly, silencing of Tyk2 expression via siRNA resulted in attenuation of ErbB3 transactivation. However, inhibition of Jak1 expression also decreased IFN-alpha-induced tyrosine phosphorylation of ErbB3. Finally, siRNA downregulation of Tyk2 and Jak1 was found to decrease IFN-alpha-stimulated proliferation. These findings validate our previous report of ErbB3 involvement in IFN-alpha-induced proliferation and further suggest that both Janus kinase members, Tyk2 and Jak1, play a role in the transactivation of ErbB3 in this model system.
[Show abstract][Hide abstract] ABSTRACT: We have previously demonstrated that the responsiveness of multiple myeloma (MM) cells to interferon-alpha (IFN-alpha) stimulation is variable, with an atypical growth response displayed by some cells. Here we report the ability of IFN-alpha to induce tyrosine phosphorylation of a 180 kDa band in the KAS-6/1 MM cell line, which is growth responsive to IFN-alpha. Further characterization demonstrated that this band corresponds to ErbB3. To our knowledge, this is the first report of ErbB3 expression in a cell type of the hematopoietic lineage. Although ErbB receptors have been shown to crosscommunicate with various other receptors, our results show for the first time that the IFN-alpha receptor can crosscommunicate with ErbB3. To address the significance of these observations, we transfected ErbB3-negative DP-6 MM cells with ErbB3 and used siRNA to silence ErbB3 in the KAS-6/1 cell line. Although IFN-alpha transactivated ErbB3 in the DP-6 transfectants, it did not confer growth responsiveness to IFN-alpha. Interestingly, silencing ErbB3 expression in the KAS-6/1 cells decreased the overall growth response to IFN-alpha and to interleukin-6. These results suggest that ErbB3 expression alone does not uniquely confer IFN-alpha growth responsiveness, but instead may amplify proliferation rates in MM cells that have acquired atypical expression of this receptor.
[Show abstract][Hide abstract] ABSTRACT: Receptor transactivation, i.e., interaction between unrelated receptor systems, is a growing theme in cytokine and growth factor signaling. In this study we reveal for the first time the ability of IFN-alpha to transactivate gp130 in myeloma cells. An epidermal growth factor receptor/gp130 chimeric receptor previously shown by us to transactivate endogenous gp130, provided a complementary tool to study the underlying mechanisms of receptor cross-talk. Further analysis revealed that transactivation of gp130 by IFN-alpha did not require the extracellular or trans-membrane domain of gp130. Moreover, transactivation of gp130 was critically dependent upon Janus kinase activation by the initiating receptor and correlated with rapid and sustained Janus kinase 1 and tyrosine kinase (Tyk) 2 tyrosine phosphorylation. Finally, transactivation of gp130 may be a common theme in myeloma cells, perhaps providing a mechanism for enhanced or qualitatively distinct cellular responses to specific stimuli.
The Journal of Immunology 05/2003; 170(7):3717-23. DOI:10.4049/jimmunol.170.7.3717 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RNA interference (RNAi) is a recently described powerful experimental tool that can cause sequence-specific gene silencing, thereby facilitating functional analysis of gene function. Consequently, we became interested in using RNAi to determine the function of aberrantly expressed ErbB3 in the KAS-6/1 human myeloma cell line. Despite the wealth of information available on the use of RNAi, dsRNA target design, and the transfection of dsRNA in vitro, little information is available for transfecting dsRNA into nonadherent cells from any species. In the present study, we report that gene silencing of ErbB3 was not observed in myeloma cells when dsRNA targeting ErbB3 was introduced using conventional transfection agents and protocols that have proved successful for several adherent cell lines. Silencing of ErbB3, however, was observed in T47D cells, an adherent breast carcinoma cell line, using the same transfection methods, indicating that our target sequence was functional for gene silencing of ErbB3. Interestingly, ErbB3 was silenced in myeloma cells when the dsRNA target was introduced by electroporation. Thus, our studies illustrate the striking dependence of dsRNA-mediated gene silencing in some cells on the methods of dsRNA transfection.
Antisense and Nucleic Acid Drug Development 01/2003; 12(6):411-8. DOI:10.1089/108729002321082483