Multiple myeloma BM-MSCs increase the tumorigenicity of MM cells via transfer of VLA4-enriched microvesicles.

Multiple myeloma (MM) cells accumulate in the bone marrow (BM) where their interactions impede disease therapy. We have shown that microvesicles (MVs) derived from BM mesenchymal stem cells (MSCs) of MM patients promote the malignant traits via modulation of translation initiation (TI), whereas MVs from normal donors (ND) do not. Here, we observed that this phenomenon is contingent on a MVs' protein constituent, and determined correlations between the MVs from the tumor microenvironment, for example, MM BM-MSCs and patients' clinical characteristics. BM-MSCs' MVs (ND/MM) proteomes were assayed (mass spectrometry) and compared. Elevated integrin CD49d (X80) and CD29 (X2) was determined in MM-MSCs' MVs and correlated with patients' staging and treatment response (free light chain, BM plasma cells count, stage, response to treatment). BM-MSCs' MVs uptake into MM cell lines was assayed (flow cytometry) with/without integrin inhibitors (RGD, natalizumab, and anti-CD29 monoclonal antibody) and recipient cells were analyzed for cell count, migration, MAPKs, TI, and drug response (doxorubicin, Velcade). Their inhibition, particularly together, attenuated the uptake of MM-MSCs MVs (but not ND-MSCs MVs) into MM cells and reduced MM cells' signaling, phenotype, and increased drug response. This study exposed a critical novel role for CD49d/CD29 on MM-MSCs MVs and presented a discriminate method to inhibit cancer promoting action of MM-MSCs MVs while retaining the anticancer function of ND-MSCs-MVs. Moreover, these findings demonstrate yet again the intricacy of the microenvironment involvement in the malignant process and highlight new therapeutic avenues to be explored.

Niche origin of mesenchymal stem cells derived microvesicles determines opposing effects on NSCLC: Primary versus metastatic.

Novel therapeutic approaches that address the malignant cells in their stroma microenvironment are urgently needed in lung cancer. The stroma resident mesenchymal stem cells (MSCs) interact with cancer cells in diverse ways including microvesicles (MVs) that transfer proteins and RNA species thereby modulating recipient cells' phenotype. Previously, we have demonstrated that MSCs' secretome from the primary non-small cell lung cancer (NSCLC) niche (lung) and metastatic niche (bone marrow (BM)) demonstrate opposite effects on NSCLC cells in a translation initiation (TI) dependent manner. Here, we examined the effect of MVs secreted from BM-MSCs' or lung-MSCs (healthy, NSCLC) to NSCLC phenotype. Briefly, NSCLC cell lines treated with Lung or BM-MSCs' MVs were assayed for viability (WST-1), cell count/death (trypan), migration (scratch), TI status and MAPKs activation (immunoblotting). Corresponding to previous published trends, Lung-MSCs' MVs promoted NSCLC cells' assayed traits whereas, BM-MSCs' MVs suppressed them. Activation of MAPKs and autophagy was registered in lung-MSCs MVs treated NSCLC cell lines only. Furthermore, lung-MSCs' MVs' treated NSCLC cells demonstrated an early (5min) activation of MAPKs and TI factors (peIF4E/peIF4GI) not evident in BM-MSCs MVs treated cells. These observations depict a role for MSCs'-MVs in NSCLC phenotype design and display distinct differences between the primary and metastatic niches that correspond to disease progression. In conclusion, the systemic nature of MVs marks them as attractive therapeutic markers/targets and we propose that identification of specific cargoes/signals that differentiate between MSCs MVs of primary and metastatic niches may introduce fresh therapeutic approaches.

BM-MSCs-derived ECM modifies multiple myeloma phenotype and drug response in a source-dependent manner.

Multiple myeloma (MM) malignant plasma cells accumulate in the bone marrow (BM) where their interaction with the microenvironment promotes disease progression and drug resistance. Previously, we have shown that MM cells cocultured with BM-mesenchymal stem cells (MSCs) comodulated cells' phenotype in a MAPKs/translation initiation (TI)-dependent manner. Dissection of the coculture model showed that BM-MSCs secretomes and microvesicles (MVs) participate in this crosstalk. Here, we addressed the role of the BM-MSCs extracellular matrix (ECM). MM cell lines cultured on decellularized ECM of normal donors' (ND) or MM patients' BM-MSCs were assayed for phenotype (viability, cell count, death, proliferation, migration, and invasion), microRNAs (MIR125a-3p, MIR199a-3p) and targets, MAPKs, TI epithelial-to-mesenchymal transition (EMT), CXCR4, and autophagy. Drug (doxorubicin, velcade) response of MM cells cultured on ND/MM-MSCs' ECM with/without adhered MVs was also evaluated. ECM evoked opposite responses according to its origin: MM cells cultured on ND-MSCs' ECM demonstrated a rapid and continued decrease in MAPK/TI activation (↓10%-25%, P < 0.05) (15-24 hours) followed by diminished viability, cell count, proliferation, migration, and invasion (16-72 hours) (↓10%-50%, P < 0.05). In contrast, MM cells cultured on MM-MSCs' ECM displayed activated MAPK/TI, proliferation, EMT, and CXCR4 (↑15%-250%, P < 0.05). Corresponding changes in microRNAs relevant to the MM cells' altered phenotype were also determined. The hierarchy and interdependence of MAPKs/TI/autophagy/phenotype cascade were demonstrated. Finally, we showed that the ECM cooperates with MVs to modulate MM cells drug response. These data demonstrate the contribution of BM-MSCs' ECM to MM niche design and underscore the clinical potential of identifying targetable signals.

The effect of mesenchymal stem cells' secretome on lung cancer progression is contingent on their origin: primary or metastatic niche.

The fatality of non-small-cell lung cancer (NSCLC) and the role of the cancer microenvironment in its resistance to therapy are long recognized. Accumulating data allocate a significant role for mesenchymal stem cells (MSCs) in the malignant environment. Previously, we have demonstrated that MSCs from NSCLC metastatic bone marrow (BM) niche deleteriously affected NSCLC cells. Here, we have decided to examine the effect of MSCs from the primary niche of the lung (healthy or adjacent to tumor) on NSCLC phenotype. We cultured NSCLC cell lines with healthy/NSCLC lung-MSCs conditioned media (secretome) and showed elevation in cells' MAPKs and translation initiation signals, proliferation, viability, death, and migration. We also established enhanced autophagy and epithelial to mesenchymal transition processes. Moreover, we observed that MSCs from tumor adjacent sites (pathological niche) exhibited a more profound effect than MSCs from healthy lung tissue. Our findings underscore the capacity of the lung-MSCs to modulate NSCLC phenotype. Interestingly, both tumor adjacent (pathological) and distant lung-MSCs (healthy) promoted the NSCLC's TI, proliferation, migration, and epithelial to mesenchymal transition, yet the pathological MSCs displayed a greater affect. In conclusion, by comparing the effects of normal lung-MSCs, NSCLC adjacent MSCs, and BM-MSCs, we have established that the primary and metastatic niches display opposite and critical effects that promote the cancerous systemic state. Specifically, the primary site MSCs promote the expansion of the malignant clone and its dispersion, whereas the metastatic site MSCs facilitates the cells re-seeding. We suggest that sabotaging the cross-talk between MSCs and NSCLC affords effective means to inhibit lung cancer progression and will require different targeting strategies in accordance with niche/disease stage.

Microvesicles derived from normal and multiple myeloma bone marrow mesenchymal stem cells differentially modulate myeloma cells' phenotype and translation initiation.

Multiple myeloma (MM) cells' interaction with the bone marrow (BM) microenvironment critically hinders disease therapy. Previously, we showed that MM co-culture with BM-mesenchymal stem cells (MSCs) caused co-modulation of translation initiation (TI) and cell phenotype and implicated secreted components, specifically microvesicles (MVs). Here, we studied the role of the BM-MSCs [normal donors (ND) and MM] secreted MVs in design of MM cells' phenotype, TI and signaling. BM-MSCs' MVs collected from BM-MSCs (MM/ND) cultures were applied to MM cell lines. After MVs uptake confirmation, the MM cells were assayed for viability, cell count and death, proliferation, migration, invasion, autophagy, TI status (factors, regulators, targets) and MAPKs activation. The interdependence of MAPKs, TI and autophagy was determined (inhibitors). ND-MSCs MVs' treated MM cells demonstrated a rapid (5 min) activation of MAPKs followed by a persistent decrease (1-24 h), while MM-MSCs MVs' treated cells demonstrated a rapid and continued (5 min-24 h) activation of MAPKs and TI (↑25-200%, P < 0.05). Within 24 h, BM-MSCs MVs were internalized by MM cells evoking opposite responses according to MVs origin. ND-MSCs' MVs decreased viability, proliferation, migration and TI (↓15-80%; P < 0.05), whereas MM-MSCs' MVs increased them (↑10-250%, P < 0.05). Inhibition of MAPKs in MM-MSCs MVs treated MM cells decreased TI and inhibition of autophagy elevated cell death. These data demonstrate that BM-MSCs MVs have a fundamental effect on MM cells phenotype in accordance with normal or pathological source implemented via TI modulation. Future studies will aim to elucidate the involvement of MVs-MM receptor ligand interactions and cargo transfer in our model.

Migration and epithelial-to-mesenchymal transition of lung cancer can be targeted via translation initiation factors eIF4E and eIF4GI.

Metastasis underlies cancer morbidity and accounts for disease progression and significant death rates generally and in non-small cell lung cancer (NSCLC) particularly. Therefore, it is critically important to understand the molecular events that regulate metastasis. Accumulating data portray a central role for protein synthesis, particularly translation initiation (TI) factors eIF4E and eIF4G in tumorigenesis and patients' survival. We have published that eIF4E/eIF4GI activities and consequently NSCLC cell migration are modulated by bone-marrow mesenchymal stem cell secretomes, suggesting a role for TI in metastasis. Here, we aimed to expand our understanding of the TI factors significance to NSCLC characteristics, particularly epithelial-to-mesenchymal transition (EMT) and migration, supportive of metastasis. In a model of NSCLC cell lines (H1299, H460), we inhibited eIF4E/eIF4GI's expressions (siRNA, ribavirin) and assessed NSCLC cell lines' migration (scratch), differentiation (EMT, immunoblotting), and expression of select microRNAs (qPCR). Initially, we determined an overexpression of several TI factors (eIF4E, eIF4GI, eIF4B, and DHX29) and their respective targets in NSCLC compared with normal lung samples (70-350%↑, P<0.05). Knockdown (KD) of eIF4E/eIF4GI in NSCLC cell lines (70%↓, P<0.05) also manifested in decreased target levels (ERα, SMAD5, NFkB, CyclinD1, c-MYC, and HIF1α) (20-50%↓, P<0.05). eIF4E/eIF4GI KD also attenuated cell migration (60-75%↓, P<0.05), EMT promoters (15-90%↓, P<0.05), and enhanced EMT suppressors (30-380%↑, P<0.05). The importance of eIF4E KD to NSCLC phenotype was further corroborated with its inhibitor, ribavirin. Changes in expression of essential microRNAs implicated in NSCLC cell migration concluded the study (20-100%, P<0.05). In summary, targeting eIF4E/eIF4GI reduces migration and EMT, both essential for metastasis, thereby underscoring the potential of TI targeting in NSCLC therapy, especially the already clinically employed agents (ribavirin/4EGI). Comparison of these findings with previously reported effects of eIF4E/eIF4GI KD in multiple myeloma suggests a collective role for these TI factors in cancer progression.

Multiple myeloma cells promote migration of bone marrow mesenchymal stem cells by altering their translation initiation.

The role of the bone marrow microenvironment in multiple myeloma pathogenesis and progression is well recognized. Indeed, we have shown that coculture of bone marrow mesenchymal stem cells from normal donors and multiple myeloma cells comodulated translation initiation. Here, we characterized the timeline of mesenchymal stem cells conditioning by multiple myeloma cells, the persistence of this effect, and the consequences on cell phenotype. Normal donor mesenchymal stem cells were cocultured with multiple myeloma cell lines (U266, ARP1) (multiple myeloma-conditioned mesenchymal stem cells) (1.5 h,12 h, 24 h, 48 h, and 3 d) and were assayed for translation initiation status (eukaryotic translation initiation factor 4E; eukaryotic translation initiation factor 4G; regulators: mechanistic target of rapamycin, MNK, 4EBP; targets: SMAD family 5, nuclear factor κB, cyclin D1, hypoxia inducible factor 1, c-Myc) (immunoblotting) and migration (scratch assay, inhibitors). Involvement of mitogen-activated protein kinases in mesenchymal stem cell conditioning and altered migration was also tested (immunoblotting, inhibitors). Multiple myeloma-conditioned mesenchymal stem cells were recultured alone (1-7 d) and were assayed for translation initiation (immunoblotting). Quantitative polymerase chain reaction of extracted ribonucleic acid was tested for microRNAs levels. Mitogen-activated protein kinases were activated within 1.5 h of coculture and were responsible for multiple myeloma-conditioned mesenchymal stem cell translation initiation status (an increase of >200%, P < 0.05) and elevated migration (16 h, an increase of >400%, P < 0.05). The bone marrow mesenchymal stem cells conditioned by multiple myeloma cells were reversible after only 1 d of multiple myeloma-conditioned mesenchymal stem cell culture alone. Decreased expression of microRNA-199b and microRNA-125a (an increase of <140%, P < 0.05) in multiple myeloma-conditioned mesenchymal stem cells supported elevated migration. The time- and proximity-dependent conditioning of normal donor mesenchymal stem cells in our model points to a dynamic interaction between multiple myeloma cells and the bone marrow niche, which causes profound changes in the nonmalignant bone marrow constituents. Future studies are warranted to identify clinically relevant means of blocking this crosstalk and improving multiple myeloma therapy.

Multiple myeloma and bone marrow mesenchymal stem cells' crosstalk: Effect on translation initiation.

Multiple myeloma (MM) malignant plasma cells reside in the bone marrow (BM) and convert it into a specialized pre-neoplastic niche that promotes the proliferation and survival of the cancer cells. BM resident mesenchymal stem cells (BM-MSCs) are altered in MM and in vitro studies indicate their transformation by MM proximity is within hours. The response time frame suggested that protein translation may be implicated. Thus, we assembled a co-culture model of MM cell lines with MSCs from normal donors (ND) and MM patients to test our hypothesis. The cell lines (U266, ARP-1) and BM-MSCs (ND, MM) were harvested separately after 72 h of co-culture and assayed for proliferation, death, levels of major translation initiation factors (eIF4E, eIF4GI), their targets, and regulators. Significant changes were observed: BM-MSCs (ND and MM) co-cultured with MM cell lines displayed elevated proliferation and death as well as increased expression/activity of eIF4E/eIF4GI; MM cell lines co-cultured with MM-MSCs also displayed higher proliferation and death rates coupled with augmented translation initiation factors; in contrast, MM cell lines co-cultured with ND-MSCs did not display elevated proliferation only death and had no changes in eIF4GI levels/activity. eIF4E expression was increased in one of the cell lines. Our study demonstrates that there is direct dialogue between the MM and BM-MSCs populations that includes translation initiation manipulation and critically affects cell fate. Future research should be aimed at identifying therapeutic targets that may be used to minimize the collateral damage to the cancer microenvironment and limit its recruitment into the malignant process. © 2015 Wiley Periodicals, Inc.

Secretome of human bone marrow mesenchymal stem cells: an emerging player in lung cancer progression and mechanisms of translation initiation.

Non-small cell lung cancer (NSCLC) remains the most common cause of cancer-related death worldwide. Patients presenting with advanced-stage NSCLC have poor prognosis, while metastatic spread accounts for >70 % of patient's deaths. The major advances in the treatment of lung cancer have brought only minor improvements in survival; therefore, novel strategic treatment approaches are urgently needed. Accumulating data allocate a central role for the cancer microenvironment including mesenchymal stem cells (MSCs) in acquisition of drug resistance and disease relapse. Furthermore, studies indicate that translation initiation factors are over expressed in NSCLC and negatively impact its prognosis. Importantly, translation initiation is highly modulated by microenvironmental cues. Therefore, we decided to examine the effect of bone marrow MSCs (BM-MSCs) from normal donors on NSCLC cell lines with special emphasis on translation initiation mechanism in the crosstalk. We cultured NSCLC cell lines with BM-MSC conditioned media (i.e., secretome) and showed deleterious effects on the cells' proliferation, viability, death, and migration. We also demonstrated reduced levels of translation initiation factors implicated in cancer progression [eukaryotic translation initiation factor 4E (eIF4E) and eukaryotic translation initiation factor 4GI (eIF4GI)], their targets, and regulators. Finally, we outlined a mechanism by which BM-MSCs' secretome affected NSCLC's mitogen-activated protein kinase (MAPK) signaling pathway, downregulated the cell migration, and diminished translation initiation factors' levels. Taken together, our study demonstrates that there is direct dialogue between the BM-MSCs' secretome and NSCLC cells that manipulates translation initiation and critically affects cell fate. We suggest that therapeutic approach that will sabotage this dialogue, especially in the BM microenvironment, may diminish lung cancer metastatic spread and morbidity and improve the patient's life quality.

eIF4E and eIF4GI have distinct and differential imprints on multiple myeloma's proteome and signaling.

Accumulating data indicate translation plays a role in cancer biology, particularly its rate limiting stage of initiation. Despite this evolving recognition, the function and importance of specific translation initiation factors is unresolved. The eukaryotic translation initiation complex eIF4F consists of eIF4E and eIF4G at a 1:1 ratio. Although it is expected that they display interdependent functions, several publications suggest independent mechanisms. This study is the first to directly assess the relative contribution of eIF4F components to the expressed cellular proteome, transcription factors, microRNAs, and phenotype in a malignancy known for extensive protein synthesis-multiple myeloma (MM). Previously, we have shown that eIF4E/eIF4GI attenuation (siRNA/Avastin) deleteriously affected MM cells' fate and reduced levels of eIF4E/eIF4GI established targets. Here, we demonstrated that eIF4E/eIF4GI indeed have individual influences on cell proteome. We used an objective, high throughput assay of mRNA microarrays to examine the significance of eIF4E/eIF4GI silencing to several cellular facets such as transcription factors, microRNAs and phenotype. We showed different imprints for eIF4E and eIF4GI in all assayed aspects. These results promote our understanding of the relative contribution and importance of eIF4E and eIF4GI to the malignant phenotype and shed light on their function in eIF4F translation initiation complex.

Multiple myeloma proteostasis can be targeted via translation initiation factor eIF4E.

Intensive protein synthesis is a unique and differential trait of the multiple myeloma (MM) cells. Previously we showed that tetraspanin overexpression in MM cell lines attenuated mTOR and PI3K cascades, induced protein synthesis, activated unfolded protein response (UPR), and caused autophagic death, all suggesting breach of proteostasis. Here we assessed the role of translation initiation in the tetraspanin­induced MM cell death with emphasis on eIF4E translation initiation factor. We showed tetraspanins attenuated peIF4E and its targets [c­Myc, cyclin D1 (cycD1)]; eIF4E attenuation was Akt-dependent. eIF4E inhibition in MM cells [bone marrow (BM), lines] by siRNA and/or the anti­viral drug and competitive eIF4E inhibitor ribavirin (RBV) deleteriously affected MM cells in a similar manner to the overexpression of tetraspanins. Furthermore, combined application of RBV and velcade had a synergistic anti­MM effect. Our results demonstrate that breach of proteostasis via eIF4E inhibition is an attractive therapeutic approach that may be relatively easily achieved by employing RBV, making this strategy readily translatable into the clinic.

Targeting eIF4GI translation initiation factor affords an attractive therapeutic strategy in multiple myeloma.

BACKGROUND: Deregulation of protein synthesis is integral to the malignant phenotype and translation initiation is the rate limiting stage. Therefore, eIF4F translation initiation complex components are attractive therapeutic targets. METHODS: Protein lysates of myeloma cells (cell lines/patients' bone marrow samples) untreated/treated with bevacizumab were assayed for eIF4GI expression, regulation (NQO1/proteosome dependent fragmentation) (WB, Dicumarol, qPCR) and targets (WB). eIF4GI was inhibited by knockdown and 4EGI-1. Cells were tested for viability (ELISA), death (FACS) and eIF4GI targets (WB). RESULTS: Previously, we have shown that manipulation of VEGF in myeloma cells attenuated eIF4E dependent translation initiation. Here we assessed the significance of eIF4GI to MM cells. We demonstrated increased expression of eIF4GI in myeloma cells and its attenuation upon VEGF inhibition attributed to elevated NQO1/proteasome dependent fragmentation and diminished mRNA levels. Knockdown of eIF4GI was deleterious to myeloma cells phenotype and expression of specific molecular targets (SMAD5/ERα/HIF1α/c-Myc). Finally, we showed that the small molecule 4EGI-1 inhibits eIF4GI and causes a reduction in expression of its molecular targets in myeloma. CONCLUSION: Our findings substantiate that translation initiation of particular targets in MM is contingent on the function of eIF4GI, critical to cell phenotype, and mark it as a viable target for pharmacological intervention.

Tetraspanins stimulate protein synthesis in myeloma cell lines.

Intensive protein synthesis is a unique and differential trait of multiple myeloma (MM) cells. Previously we showed that tetraspanin (CD81, CD82) overexpression in MM cell lines attenuated Akt/mTOR cascades, activated UPR, and caused autophagic death, suggesting breach of protein homeostasis. Here, we explored the role of protein synthesis in the tetraspanin-induced MM cell death. Contrary to attenuation of the major metabolic regulator, mTOR we determined elevated steady-state levels of protein in CD81N1/CD82N1 transfected MM lines (RPMI-8226, CAG). Elevated levels of immunoglobulins supported increased protein production in RPMI-8226. Changes in cell morphology consistent with elevated protein synthesis were also determined (cell, nuclei, and nucleoli sizes and ratios). Increased levels of phospho-rpS6 and decreased levels of phospho-AMPK were consistent with increased translation but independent of mTOR. Involvement of p38 and its role in tetraspanin induced translation and cell death were demonstrated. Microarray analyses of tetraspanin transfected MM cell lines revealed activation of protein synthesis signaling cascades and signals implicated in ribosome biogenesis (snoRNAs). Finally, we showed tetraspanins elevated protein synthesis was instrumental to MM cells' death. This work explores and demonstrates that excessive protein translation can be detrimental to MM cell lines and therefore may present a therapeutic target. Proteostasis is particularly important in MM because it integrates the high levels of protein production unique to myeloma cells with critically important microenvironmental cues. We suggest that increasing translation may be the path of least resistance in MM and thus may afford a novel platform for strategically designed therapy.

Bevacizumab attenuates major signaling cascades and eIF4E translation initiation factor in multiple myeloma cells.

Multiple myeloma (MM), a malignancy of plasma cells, remains fatal despite introduction of novel therapies, partially due to humoral factors, including vascular endothelial growth factor (VEGF), in their microenvironment. The aim of this study was to explore the efficacy of anti-VEGF treatment with bevacizumab directly on MM cells. Particular attention was directed to the affect of VEGF inhibition on protein translation initiation. Experiments were conducted on MM cells (lines, bone marrow (BM) samples) cultured on plastic. Inhibition of VEGF was achieved with the clinically employed anti-VEGF antibody, bevacizumab, as a platform and its consequences on viability, proliferation, and survival was assessed. VEGF downstream signals of established importance to MM cell biology were assayed as well, with particular emphasis on translation initiation factor eIF4E. We showed that blocking VEGF is deleterious to the MM cells and causes cytostasis. This was evidenced in MM cell lines, as well as in primary BM samples (BM MM). A common bevacizumab-induced attenuation of critical signaling effectors was determined: VEGFR1, mTOR, c-Myc, Akt, STAT3, (cell lines) and eIF4E translation initiation factor (lines and BM). ERK1/2 displayed a variegated response to bevacizumab (lines). Utilizing a constitutively Akt-expressing MM model, we showed that the effect of bevacizumab on viability and eIF4E status is Akt-dependent. Of note, the effect of bevacizumab was achieved with high concentrations (2 mg/ml), but was shown to be specific. These findings demonstrate that bevacizumab has a direct influence on major pathways critically activated in MM that is independent from its established effect on angiogenesis. The cytostatic effect of VEGF inhibition on MM cells underscores its potential in combined therapy, and our findings, regarding its influence on translation initiation, suggest that drugs that unbalance cellular proteostasis may be particularly effective.