Bone Marrow Interaction in Multiple Myeloma Pathogenesis: Phenotypical Analysis, Kinetics and Novel Therapy Approaches Based On CXCR4 Inhibition.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2450-2450
Author(s):  
Johannes Waldschmidt ◽  
Dagmar Wider ◽  
Marie Follo ◽  
Josefina Udi ◽  
Martina Kleber ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Adhesion Molecules ◽  
Cell Lines ◽  
Cxcr4 Expression ◽  
Cell Trafficking ◽  
Newly Diagnosed ◽  
Infiltration Rates ◽  
Dependent Cell ◽  
And Migration

Abstract Abstract 2450 Introduction: The interaction between malignant plasma cells and their microenvironment is central in multiple myeloma (MM) pathogenesis. Binding of MM cells to bone marrow (BM) stroma cells triggers the expression of adhesion molecules and secretion of chemo- and cytokines, promoting MM cell growth, drug resistance and migration. Stromal-derived factor-1 (SDF-1) and its receptor CXCR4 are essential for normal hematopoietic progenitor cell movement and adherence within the BM microenvironment. In leukemia and lymphoma, oncoproteins may inhibit SDF-1-dependent cell trafficking within the BM through a mechanism that is not fully understood. For that reason, understanding SDF-1-dependent cell trafficking within the BM and targeting MM-cell - host-BM interactions display a promising approach for the development of novel therapeutic strategies. Methods: BM samples of MM patients (n=59) were analysed using flow cytometry and compared to MGUS patients (n=3) and healthy volunteers (n=7). We compared patient samples with low BM infiltration (≤5%; n=13) intermediate (5–30%; n=29) and high infiltration rates (≥30%; n=17). We also assessed expression of adhesion molecules in MM patients with long-term disease control (n=20) vs. both newly diagnosed (n=16) and symptomatic MM patients (n=23) as previously grouped by San Miguel et al. (Haematologica July 6,2012). We also sought to elucidate in vitro, whether specific anti-MM agents (bortezomib, vorinostat, pomalidomide, EGCG), with and without M210B4 stroma support, and with and without the CXCR4 inhibitor AMD3100, target the interaction of MM cells. Experiments were performed using MM cell lines (U266, RPMI8226, L363, NCI-H929), the control T-cell line MOLT-4 and MM-patient BM samples. Cell viability was assessed via Trypan Blue- and AnnexinV/PI-staining. CD138, CXCR4 (SDF1-receptor), CD49d (VLA-4), CD11a (LFA-1) and CD44 (HERMES antigen) expression was evaluated by flow cytometry and ScanR microscopy. Results: In BM samples of MM patients as compared to MGUS and healthy volunteers, the CXCR4/CD138- (p=.036), CD49d/CD138- (p=.0013) and CD44/CD138-expression (p=.0072) was significantly amplified and correlated with increasing BM infiltration rates (p=.001). Both newly diagnosed and symptomatic MM patients confirmed significantly increased CXCR4/CD138-, CD49d/CD138- (p=.0013) and CD44/CD138-expression as compared to patients with long-term disease control. Of note, in newly diagnosed patients, the expression of adhesion molecules was even more enhanced than in symptomatic myeloma patients, underlining their critical and future potential role as targets for novel therapeutics. Comparison of MM cell lines' adhesion and migration markers with that of MM-patient BM specimens revealed U266 as the cell line most closely resembling human specimens. Cytotoxic effects with use of MM cell lines and bortezomib, vorinostat and pomalidomide confirmed prior cytotoxic concentrations. Cocultivation with stroma substantially reduced apoptosis and induced tumor protective effects. Additional AMD3100 treatment restored sensitivity to bortezomib, vorinostat and pomalidomide. CXCR4 expression was substantially reduced after AMD3100 treatment, while that of CD49d, CD44 and CD11a remained widely unchanged. Toxic or therapeutic effects of AMD3100 monotherapy were excluded for used doses of 50μM. Additional use of ScanR microscopy visualized co-localisation of CXCR4 expression both on the cell surface and within the cytoplasm of MM cells. ScanR microscopy results correlated with flow cytometry-determined CXCR4 expression. Ongoing analyses of both ScanR microscopy and flow cytometry will allow the detailed assessment of treatment studies with and without anti-MM agents and AMD3100. Conclusions: Our findings underline the critical role of adhesion and migration molecules in MM and may pave the way for novel therapeutic approaches targeting these microenvironmental mediators. Disclosures: No relevant conflicts of interest to declare.

The Role of B Cell-Activating Factor (BAFF) in the Biology of Multiple Myeloma (MM).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3380-3380 ◽  
Author(s):  
Noopur Raje ◽  
Shaji Kumar ◽  
Teru Hideshima ◽  
Kenji Ishitsuka ◽  
Hiroshi Yasui ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Cell Development ◽  
Newly Diagnosed ◽  
Growth And Survival ◽  
Affymetrix U133a ◽  
Apoptotic Proteins

Abstract BAFF is a member of the tumor necrosis factor (TNF) family and is critical for the maintenance and homeostasis of normal B-cell development. Importantly, BAFF promotes the generation of rapidly dividing immunoglobulin secreting plasmablasts from activated memory B cells by enhancing their survival. Given that MM is a cancer of plasma cells and that the signaling cascades implicated in receptor ligand interactions of BAFF are crucial in MM cell biology, we hypothesized that this cytokine may play a critical role in MM cell development, survival, and proliferation. We performed gene expression profiling (GEP) on CD 138+ plasma cells isolated from 90 MM patients (45 newly diagnosed and 45 relapsed) and 11 healthy controls using the Affymetrix U133A arrays. Our data demonstrates increased expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA), 2 receptors used by BAFF to exert its effects. Our data also shows an increased expression of a proliferation-inducing ligand (APRIL), another member of the TNF family with homology to BAFF. Expression levels of BAFF and BAFF-R could not be determined because of lack of these probe sets on the Affymetrix U133A arrays. GEP analysis shows increased BCMA expression (p<0.0001, student T test) on newly diagnosed and relapsed MM versus normal plasma cells. Flow cytometry on MM cell lines demonstrated a differential expression of the three receptors of BAFF, with BCMA present on most cell lines but BAFF-R expressed at low levels only on LR5 cells and DOX40 MM cells. In contrast, flow cytometry performed on MM patient cells demonstrated the presence of all 3 receptors on CD 138+ cells. ELISA assays performed on 30 MM sera demonstrated a mean BAFF level of 618 pg/ml (range: 128–2126pg/ml) versus 235pg/ml (range: 158–326pg/ml) in 7 normal donor sera. Fifty six% (17/30) of MM patients had BAFF levels in excess of the highest value noted in normals. To understand the role BAFF might play in the biology of MM, we studied the effects of recombinant BAFF (rh-BAFF) on MM cells directly and in the context of its bone marrow microenvironment. (abstract # 554746) rh-BAFF conferred a survival advantage to MM cells and protected them against dexamethasone-induced cytotoxicity. Importantly, anti-apoptotic proteins Bcl2 and Mcl-1 were upregulated, as were growth and survival signals belonging to the JAK/STAT and MAPKinase pathways. Conversely, neutralizing antibody to BAFF blocked, at least in part, blocked the upregulation of anti-apoptotic proteins with associated growth and survival, confirming that these effects were due to BAFF. Importantly, all of these signals were downregulated even in the presence of bone marrow stromal cells (BMSCs). These data therefore show a role for BAFF mediating MM cell survival and provide the framework for inhibiting BAFF, either alone or in combination with dexamethasone, to improve patient outcome in MM.

CXCR7 Regulates SDF-1 Induced Adhesion and Homing in Multiple Myeloma.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1674-1674 ◽  
Author(s):  
Nicholas Burwick ◽  
Anne-Sophie Moreau ◽  
Xiaoying Jia ◽  
Xavier Leleu ◽  
Judith Runnels ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Adhesion ◽  
Cell Lines ◽  
Cxcr4 Expression ◽  
Rt Pcr ◽  
Growth And Survival ◽  
Tumor Types

Abstract BACKGROUND: Multiple myeloma (MM) is a plasma cell malignancy that depends on interactions with the bone marrow (BM) microenvironment for growth and survival. In turn, adhesion of MM cells to the BM stroma provides a mechanism of resistance from standard chemotherapeutic agents. Recently, our lab has shown that by disrupting this adhesion using a selective CXCR4 inhibitor named AMD3100, MM cells are more sensitive to the proteasome inhibitor Bortezomib (Ghobrial lab, unpublished data). CXCR4 has been a particularly attractive target because its ligand SDF-1 is known to induce p42/44 MAPK, AKT, and the down-stream anti-apoptotic protein bad in MM cells, leading to increased MM growth and survival. Until recently, CXCR4 was thought to be a canonical receptor for the SDF-1 ligand. However, a second chemokine receptor for SDF-1 was subsequently discovered and named CXCR7. CXCR7 is a novel chemokine receptor that is important in cell adhesion, growth and survival in several tumor types. However, the role of CXCR7 in multiple myeloma (MM) has yet to be explored. Furthermore, the ability of SDF-1 ligand to regulate MM function via CXCR7 has not been studied. METHODS: The MM cell lines (U266, MM1.S, RPMI, OPM2, OPM1) were used. After informed consent was obtained, primary bone marrow samples from MM patients were collected. CD138 positive mononuclear cells were isolated by microbead selection. The expression of CXCR7 on MM cell lines and patient samples was confirmed using flow cytometry and RT-PCR analysis. For functional in vitro and ex-vivo assays, the CXCR7 selective antagonist 733 was used (ChemoCentryx Inc., Mountain View, CA). RESULTS: Here we show that CXCR7 was expressed on all tested MM cell lines and primary patient samples as demonstrated by flow cytometry and RT-PCR. Furthermore, CXCR7 was found to regulate SDF-1 induced MM cell adhesion, as demonstrated by in vitro assays using a small molecule compound specific for CXCR7 (733). The CXCR7 antagonist showed significant inhibition of adhesion of MM cell lines and patient samples to fibronectin, endothelial cells and stromal cells, with 50% reduction of adhesion at 5nM of the CXCR7 inhibitor, and with similar activity compared to 20uM of AMD3100 (CXCR4 inhibitor). However, unlike CXCR4, CXCR7 did not effect trans-well migration to SDF-1 chemokine. Interestingly, both receptors were found to be important for trans-endothelial migration of MM cells. Moreover, pre-treatment with 733 reduced homing of MM cells to the BM niche in vivo. Previous studies have failed to show signaling in response to CXCR7 in many tumor types. Here, we demonstrate that treatment with 733 inhibited SDF-1 induced pERK and pAKT, ribosomal pS6Kinase, pGSK3, pSTAT3, pFAK and pPAK signaling pathways, confirming a role for CXCR7 in facilitating SDF-1 signaling. This effect was further confirmed using immunofluorescence. To investigate whether CXCR7 and CXCR4 interact directly, we examined the effect of 733 and AMD3100 on CXCR4 expression and found that AMD3100 significantly inhibited CXCR4 expression, while 733 had no effect on CXCR4 expression, even in the presence of SDF-1. The CXCR7 inhibitor had no effect on the survival of MM cells using MTT and flow cytometry analysis, while high doses of 733 (1uM) had modest inhibition of proliferation. Interestingly, 733 prevented the growth advantage induced by 30nM SDF-1 at 24 hrs. CONCLUSION: Together, these results demonstrate the importance of CXCR7 in regulating MM adhesion and homing, and highlight the differential effects of CXCR4 and CXCR7 in regulating SDF-1 signaling in MM, thus providing a rationale for targeting the SDF-1/CXCR7 axis in MM.

Extended Exposure To The CXCR4 Inhibitor Plerixafor May Lead To Enhanced Microenvironment Interactions in Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1295-1295
Author(s):  
Edward Allan R. Sison ◽  
Daniel Magoon ◽  
Patrick Brown
Keyword(s):  
Bone Marrow ◽  
Adhesion Molecules ◽  
Cell Lines ◽  
Prolonged Exposure ◽  
Surface Expression ◽  
Cxcr4 Expression ◽  
Antibody Binding ◽  
Drug Binding ◽  
Dose Dependent ◽  
Extended Exposure

Abstract Background We have previously demonstrated that inhibition of CXCR4 in ALL decreases CXCR4 antibody binding, inhibits SDF-1α-(CXCL12)-induced chemotaxis, and overcomes chemotherapy resistance conferred by the bone marrow microenvironment. Specifically, we found that treatment with plerixafor and araC significantly decreased leukemic burden in a xenograft model of infant ALL, compared to treatment with araC alone. In those experiments, we treated mice on 3 consecutive days per week for 2 weeks with plerixafor and araC. However, the combination did not eradicate the leukemia in our model. We hypothesized that extended exposure to plerixafor may have led to increased interactions between surviving leukemic blasts and the bone marrow microenvironment. In our current experiments, we sought to characterize the effects of prolonged exposure to plerixafor in ALL. Methods/Results We treated pre-B (HB-1119, Nalm-6) and T (CCRF-CEM-1301, Jurkat) ALL cell lines with a dose range of plerixafor and harvested cells for FACS over an extended time course. We measured surface CXCR4 (s-CXCR4) expression using 3 antibodies: 12G5, which attaches to the SDF-1α and drug-binding site of CXCR4, and 1D9 and 2B11, which do not compete with SDF-1α or drug binding. 12G5 binding was decreased by plerixafor even at 1 hour and this effect was concentration-dependent. Interestingly, we found a time and dose-dependent increase in 1D9 and 2B11 antibody binding, suggesting that plerixafor caused an actual increase in s-CXCR4 over time. Increases in 1D9 and 2B11 binding were inversely proportional to decreases in 12G5 binding. We also measured surface expression of CD49d (VLA-4), which binds to fibronectin and VCAM-1; CXCR7, which binds to SDF-1α and CXCL11; and CXCR3, which binds to CXCL9, 10, and 11. We hypothesized that CXCR4 inhibition would lead to upregulation of parallel pathways of leukemia-stroma interactions. CD49d was highly expressed at baseline, while CXCR7 and CXCR3 were expressed to a lesser degree. Treatment with plerixafor led to dose-dependent increases in CXCR7 and variable changes in CD49d and CXCR3 surface expression, suggesting that plerixafor can modulate surface expression of adhesion molecules other than CXCR4. Next, we treated ALL cell lines with plerixafor (0, 10, 100 nM) for 72 hours, washed with PBS, and resuspended the cells in fresh medium to determine the effects of extended exposure to plerixafor and subsequent withdrawal. First, we measured surface expression of s-CXCR4 after 72 hours of treatment with plerixafor and found that 12G5 binding was decreased, while 1D9/2B11 binding was increased in an inversely proportional manner. After withdrawal, 12G5 binding increased to untreated levels between 4 and 24 hours, while 1D9/2B11 binding decreased to untreated levels between 4 and 72 hours. We also measured surface expression of CD49d, CXCR7, and CXCR3 and found that the effects of plerixafor treatment and withdrawal were variable by cell line. For example, after plerixafor treatment, surface expression of CD49d and CXCR7 was increased in Nalm-6 and surface expression of CXCR7 and CXCR3 was increased in CCRF-CEM-1301. Interestingly, 4 hours after plerixafor withdrawal, CD49d expression was increased in Jurkat and Nalm-6, and CXCR7 expression was increased in CCRF-CEM-1301, HB-1119, and Jurkat. Finally, we measured migration of washed cells from each treatment condition through a permeable membrane toward medium containing SDF-1α or medium alone. Despite CXCR4 inhibition for 72 hours, all plerixafor-treated cells migrated in response to SDF-1α. In addition, some plerixafor-treated cells exhibited significantly increased SDF-1α-induced chemotaxis compared to control-treated cells. These findings imply that increases in s-CXCR4 induced by 72 hours of treatment with plerixafor are functional. Conclusions Treatment of ALL cell lines with plerixafor led to a dose-dependent decrease in 12G5 antibody binding with a simultaneous overall increase in s-CXCR4 expression. Prolonged exposure to plerixafor led to increased s-CXCR4 expression that persisted for up to 72 hours after drug withdrawal, modulated surface expression of additional adhesion molecules, and enhanced SDF-1α-induced chemotaxis. Therefore, additional careful studies of CXCR4 inhibitors and other microenvironment-targeted agents must be performed in order to determine their optimal use in ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals B7-H6, an immunoligand for the natural killer cell activating receptor NKp30, reveals inhibitory effects on cell proliferation and migration, but not apoptosis, in cervical cancer derived-cell lines

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Nehla Banu ◽  
Annie Riera-Leal ◽  
Jesse Haramati ◽  
Pablo Cesar Ortiz-Lazareno ◽  
Sandeep Surendra Panikar ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cellular Localization ◽  
Cell Proliferation And Migration ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background Although great progress has been made in treatment regimens, cervical cancer remains as one of the most common cancer in women worldwide. Studies focusing on molecules that regulate carcinogenesis may provide potential therapeutic strategies for cervical cancer. B7-H6, an activating immunoligand expressed by several tumor cells, is known to activate NK cell-mediated cytotoxicity once engaged with its natural receptor NKp30. However, the opposite, that is, the effects in the tumor cell triggered by B7-H6 after interacting with NKp30 has not yet been well explored. Methods In this study, we evaluated the surface expression of B7-H6 by flow cytometry. Later, we stimulated B7-H6 positive cervical cancer derived-cell lines (HeLa and SiHa) with recombinant soluble NKp30 (sNKp30) protein and evaluated biological effects using the impedance RTCA system for cell proliferation, the scratch method for cell migration, and flow cytometry for apoptosis. Cellular localization of B7-H6 was determined using confocal microscopy. Results Notably, we observed that the addition of sNKp30 to the cervical cancer cell lines decreased tumor cell proliferation and migration rate, but had no effect on apoptosis. We also found that B7-H6 is selectively maintained in tumor cell lines, and that efforts to sort and purify B7-H6 negative or positive cells were futile, as negative cells, when cultured, regained the expression of B7-H6 and B7-H6 positive cells, when sorted and cultivated, lost a percentage of B7-H6 expression. Conclusions Our results suggest that B7-H6 has an important, as of yet undescribed, role in the biology of the cervical tumor cells themselves, suggesting that this protein might be a promising target for anti-tumor therapy in the future.

scholarly journals Pim1 Serine/Threonine Kinase Regulates the Number and Functions of Murine Hematopoietic Stem Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2303-2303
Author(s):  
Ningfei An ◽  
Yingwei Lin ◽  
Sandeep Mahajan ◽  
Yong Wang ◽  
Andrew Kraft ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cxcr4 Expression ◽  
Cancer Center ◽  
Serine Threonine Kinase ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Threonine Kinase

Abstract Abstract 2303 Background: The genes and pathways that govern the functions and expansion of hematopoietic stem cells (HSC) remain to be elucidated. Pim (proviral insertion in murine lymphomas) protein kinases are a small family of constitutively active, highly conserved oncogenic serine/threonine kinases and have 3 members: Pim1, Pim2, and Pim3. Currently, little is known about the contribution of each Pim kinase in hematopoiesis. Pim1 was recently found to be important in the regulation of CXCR4 expression in HSCs (Grundler R et al, J. Exp Med 2009, 206:1957). However, the roles of Pim1 in HSC proliferation, self-renewal, and long-term repopulation are unclear. In the current study, we performed detailed hematological and bone marrow transplant studies to address these questions. Methods: We generated Pim1 transgenic (Tx) mice bearing human PIM1 under the control of vav hematopoietic promoter (vav-hPIM1 Tx). Pim1−/−, Pim2−/−, Pim3−/− single knockout (KO) mice were also utilized. We quantified the number of hematopoietic stem/progenitor cells (HSPCs) in these mice using flow cytometry, colony forming units (CFUs) and cobblestone area forming cell colonies (CAFCs) assays. In vivo BrdU labeling was performed to determine the proliferation status of long-term HSCs in these mice. CXCR4 expression and the homing of HSCs to bone marrow and spleen were also analyzed. Additionally, we performed noncompetitive, competitive, and serial transplantation assays to examine the role of Pim1, Pim2 and Pim3 in hematological reconstitution in lethally irradiated recipient mice. Finally, a limiting dilution competitive bone marrow transplantation assay using purified long-term HSC cells (Lin− Sca-1+c-kit+ CD34−) was performed to calculate the frequency of active HSCs in vav-hPIM1 Tx and Pim1−/−mice. Results: Conclusion: Our current studies demonstrate a novel role of Pim1 serine/threonine kinase in the regulation of self-renewal, proliferation, and long-term repopulation of HSCs. The functions of Pim1 in hematopoiesis do not overlap with Pim2 or Pim3. Acknowledgment: We thank Richard Peppler at the Hollings Cancer Center Flow Cytometry Core for performing flow cytometry analysis. This work is supported by MUSC Hollings Cancer Center Startup Fund, Hollings Cancer Center ACS IRG, ASCO Conquer Cancer Foundation Career Development Award, NIH 1K08HL 103780–01A1, and NIH 3P30CA138313–01S3. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Characterizing the Tumor Suppressor Role of CEACAM1 in Multiple Myeloma

2018 ◽  
Vol 45 (4) ◽  
pp. 1631-1640 ◽  
Author(s):  
Jinge Xu ◽  
Bin Liu ◽  
Shoubao Ma ◽  
Jubin Zhang ◽  
Yuhan Ji ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Tumor Suppressor ◽  
Carcinoembryonic Antigen ◽  
Cell Lines ◽  
Western Blotting ◽  
Cell Invasion ◽  
Caspase 3 ◽  
Invasion And Migration ◽  
And Migration

Background/Aims: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), also known as CD66a, is a member of the immunoglobulin (Ig) superfamily that belongs to the carcinoembryonic antigen (CEA) family which plays a dual role in cancer. Previous studies showed high expression of CEACAM1 in multiple myeloma (MM). The aim of this study was to investigate the biological consequences of CEACAM1 overexpression in MM. Methods: pEGFP-N1-CEACAM1 and pcDNA3.1-CEACAM1 expression plasmids were transfected into U-266 and RPMI8266 cell lines . Effect of CEACAM1 overexpression on the proliferation of two cell lines were tested by the CCK8 assay. Cell cycle and Apoptotic changes after CEACAM1 transfection were examined with AnnexinV–FITC/PI by flow cytometry. Hochest staining assay was used to confirm the apoptotic changes. Caspase-3 activity was examined by Western blotting. The cell invasion and migration activity change after CEACAM1 transfection were performed by well chamber assays and a wound healing, respectively. MMP-2 and MMP-9 proteins expression were detected by Western blotting. Flow cytometry immunophenotyping was be evaluated on myeloma cells from bone marrow taken from 50 patients with symptomatic MM newly diagnosed. The correlations between CEACAM1 expression levels and the clinical features across all groups were investigated. Results: CEACAM1 overexpression significantly suppressed MM cell proliferation, induced cell apoptosis, and inhibited cell invasion and migration possibly through activation of caspase-3 and downregulation of MMP-2 and MMP-9. CEACAM1 expression in patients with DS stage I was more frequent (61.5%) than those with DS stage II (21.1%) or III (22.2%). Furthermore, patients with β2-microglobulin levels equal to or less than 3.5 mg/L had higher CEACAM1 expression than those with β2-microglobulin levels greater than 3.5 mg/L. Conclusion: Our findings suggest that CEACAM1 may act as a tumor suppressor in MM.

Class I PI3K Isoforms Exert a Differential Role On Survival and Cell Trafficking In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3159-3159
Author(s):  
Ilyas Sahin ◽  
Feda Azab ◽  
Michele Moschetta ◽  
Yuji Mishima ◽  
Brian Tsang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tumor Growth ◽  
Tumor Progression ◽  
Tumor Cells ◽  
Research Funding ◽  
Class I ◽  
Cell Trafficking ◽  
And Migration

Abstract Background The phosphatidylinositol-3 kinase (PI3K) pathway is a critical regulator of tumor progression, protein translation and cytoskeletal dynamics, collectively required for cell proliferation, survival, adhesion and migration in many malignancies including multiple myeloma (MM). Despite the absence of mutations in the PI3K/Akt genes, many studies have demonstrated that this pathway is constitutively activated in MM cells. In this study, we investigated the role of inhibition of class I PI3K isoforms known as p110α, p110β, p110γ and p110δ in cell trafficking of MM cells using isoform-specific knockdown (KD). We have also evaluated the effect of pan-PI3K inhibitior, NVP-BKM120, on survival, adhesion and migration of MM cells both in vitro and in vivo. Methods The baseline expression of class I PI3K isoforms in MM cell lines (MM.1S, OPM1, OPM2, H929, RPMI, INA6, U266, and U266LR7) has been evaluated by immnunobloting. MM tumor cells (MM.1S-GFP+/luc+) were infected with lentivirus mediated shRNA targeting class I PI3K isoforms. RT-qPCR and immunoblotting were performed to show infection efficiency. In vivo tumor growth of isoform specific KDs were assessed by using in vivo bioluminescence (BLI) in SCID mice. Detection of circulating MM-GFP+ cells ex vivo was performed by flow cytometry. Analysis of circulating tumor cells for each isoform-specific KD cells against relative tumor volume was performed by lineer regression using GraphPad software. Survival, adhesion and migration of KD cells were tested by MTT, adhesion and migration in vitro assay, respectively. NVP-BKM120, a pan-PI3K-inhibitor (Novartis, MA) has been tested both in vitro and in vivo. Ex vivo detection of mobilization and tumor growth of MM cells (MM.1S-GFP+/luc+) treated with 1) vehicle; 2) NVP-BKM120 in SCID mice were assessed by using flow cytometry and in vivo BLI. Homing of MM cells to the BM of mice pre-treated with NVP-BKM120 was evaluated by in vivo confocal. Increased concentrations of NVP-BKM120 have been tested on survival, cell cycle and apoptotic pathways in MM cells, by using MTT, PI staining in flow cytometry and immunoblotting, respectively. NVP-BKM120 induced dose-dependent effect on chemotaxis and adhesion of MM.1s to BM stromal cells (BMSCs) and fibronectin were tested by migration and adhesion assays. Results PI3K-p110β was highly expressed in all cell lines; while other isoforms were expressed in some of the MM cell lines tested. Of note, MM.1S expressed all isoforms. Mice injected with PI3K isoform specific knockdown MM.1S cells presented with different tumor burdens; p110β and p110δ mice showed significantly slower tumor progression compared to scramble control cell line (P<.05), whereas tumor growth was similar in p110α and p110γ to control mice. We next compared the number of circulating tumor cells (CTCs) at the same tumor burden between groups, which showed only p110β presented with a higher number of CTCs compared to the scramble group (P=0.01). In vitro, we observed reduced adhesion and enhanced migration of KD cells compared to control with no cell survival difference. The effect of pan-inhibition of PI3K with NVP-BKM120 induced MM cell mobilization from the BM to the circulation (Vehicle: 0.002 % vs NVP-BKM120: 0.023%; P<.05). This was supported by the inhibition of homing of MM cells to the BM (84% decrease) in the mice pre-treated with NVP-BKM120 (P<.05). Furthermore, treatment of mice with 50mg/kg of NVP-BKM120 once a day by oral gavage for five weeks significantly decreased the rate of tumor progression in MM compared to the vehicle treated group, as shown by BLI (69% decrease, P<.01). NVP-BKM120 decreased the activation of adhesion-related signaling in MM cells induced by co-culture with stroma, including pFAK, pSrc, pCoffilin and pMLC, as shown by immunoblotting. Moreover, it caused cell cycle arrest, as detected by PI staining and analyzed by flow cytometry. Conclusion This study suggests that inhibition of Class I PI3K isoforms, particularly p110β and p110δ, can play an important role in the regulation of cell trafficking in MM by disrupting adhesion of MM cells to the BM and inducing mobilization. Thus, pan-PI3K inhibition by NVP-BKM120 is a promising approach, which may enhance therapeutic response and overcome resistance in the treatment of MM. Disclosures: Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

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