β1 Integrin Adhesion Enhances IL-6–Mediated STAT3 Signaling in Myeloma Cells: Implications for Microenvironment Influence on Tumor Survival and Proliferation

Multiple myeloma (MM) is a B-cell malignancy characterized by the monoclonal expansion of plasma cells. Although numerous genetic alterations have been implicated in MM pathogenesis, it is hypothesized that the bone marrow (BM) microenvironment also contributes to MM cell pathogenesis (1–6). The BM microenvironment consists of a complex array of factors that promote cell growth and survival, and may contribute to minimal residual disease and the development of acquired drug resistance (7–14). These BM factors can be divided into two categories: (a) soluble factors including cytokines, chemokines, and growth factors, and (b) physical factors that include extracellular matrix glycoproteins and BM stromal cells.

Interleukin (IL)-6, regarded as one of the most important cytokines in MM disease progression, promotes the proliferation and survival of MM cells through the activation of Janus kinase (Jak)/signal transducers and activators of transcription 3 (STAT3), Ras/extracellular signal-regulated kinase (ERK) 1/2, and PI3 kinase/Akt/PKB signaling pathways (15–19). Signal transduction follows binding of IL-6 to gp80/IL-6Rα and recruitment of gp130/CD130 (20). The subsequent formation of a hexameric signaling complex, containing two molecules each of IL-6, gp80, and gp130, induces dimerization of gp130 and receptor phosphorylation by constitutively bound Jak family tyrosine kinases (Jak1, Jak2, and Tyk2; refs. 21, 22). In regard to STAT3 signaling, phosphorylation of four c-terminal tyrosine residues in the gp130 subunit has been proposed to facilitate the recruitment and phosphorylation of STAT proteins. STAT phosphorylation promotes its dimerization, nuclear translocation, and DNA-binding, followed by the transactivation of genes involved in a number of cellular functions including proliferation, differentiation, and survival (15, 18, 20, 23).

Of the physical factors of the BM microenvironment, the glycoprotein fibronectin (FN) has also been shown to control the growth, survival, and drug resistance of MM cells (1, 4, 9–12). FN adhesion activates α4 or α5 and β1 (α4β1 & α5β1) integrin heterodimers via unique molecular aggregates using associated nonreceptor protein tyrosine kinases (PTK) and, in some cases, recruitment of receptor PTKs (RPTK) for intracellular signaling (24, 25). Signaling via these integrin complexes confers resistance to a multitude of proapoptotic effectors including serum deprivation, chemotherapeutic drugs, and death receptor ligation (CD95/Fas; refs. 13, 26).

Because of the influence of the BM microenvironment in MM progression, it is important to define not only the role of individual components of the BM but also the manner by which both soluble and physical factors of the microenvironment collaborate in MM tumorigenesis. To date, the soluble factor IL-6 and the physical factor FN have been examined independently. However, in vivo, it is most likely that these factors collaborate to influence myeloma cell survival. Therefore, elucidation of biochemical mechanisms associated with these factors alone, and in combination, is important in defining the effects of the microenvironment on cell survival and proliferation.

In this report, using a simple cellular model, we examined intracellular signaling and the biological sequelae after stimulation with IL-6 alone, FN adhesion alone, or their combination in MM cell lines. Importantly, we show that IL-6 and FN adhesion collaborate to activate STAT3 but not ERK1/2 or Akt. Furthermore, this collaboration parallels an increase in gp130 complex phosphorylation and a novel IL-6–independent preassociation of STAT3 with gp130 when cells are adhered to FN. This work is significant because it shows that IL-6 stimulation does not reverse CAM-DR but does reverse FN-mediated cell cycle arrest. Taken together, these results suggest a mechanism by which collaborative signaling by β1 integrin and gp130 confer a survival advantage to MM cells.

Cells and materials. Four MM cell lines used were as follows: RPMI 8226 [RPMI supplemented with 5% heat inactivated fetal bovine serum (FBS)], MM1.S (kindly provided by Dr. Steve Rosen, Northwestern University, Chicago, Illinois; 10% FBS, RPMI), H929 (10% FBS, RPMI supplemented with 0.0004% β-mercaptoethanol), and U266 (10% FBS, RPMI). Cells were maintained in culture in the respective concentration of FBS in RPMI supplemented with 1.0% penicillin/streptomycin and l-Glutamine (Life Technologies). Experiments (up to 6 h) were carried out under serum-free conditions. Antibodies used were as follows: STAT3 (1:2,000) and phosphor-STAT3 (1:1,000) in 5% bovine serum albumin (BSA)/TBS 0.1% Tween 20 (Cell Signaling), gp130 (1:250; BD Pharmingen), β1 integrin (1:250; Chemicon), β-actin (1:10,000; Sigma-Aldrich). Recombinant human IL-6 was purchased from Sigma-Aldrich.

Protein isolation and Western blot analysis. Cells, after incubation under the indicated conditions, were washed twice with ice cold PBS, and incubated for 10 min at 4°C in Triton X-100 lysis buffer [30 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 25 mmol/L NaF, 1% Triton X-100, 10% glycerol, 2 mmol/L Na-orthovanadate], with 1 Complete, Mini protease inhibitor cocktail tablet (Roche) per 10 mL Triton X-100 buffer. Protein lysates were quantitated with Bio-Rad reagent (Bio-Rad) and 10 to 50 μg of cellular lysates were separated on 12.5% polyacrylamide gels and transferred to polyvinylidene difluoride membrane. Protein levels were examined with specific antisera and visualized with SuperSignal-Pico or SuperSignal-Dura Light (Pierce).

EMSA analysis. STAT3 DNA-binding assays were performed on nuclear extracts prepared in hypertonic buffer [20 mmol/L HEPES (pH 7.9), 420 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 20% glycerol, 20 mmol/L NaF, 1 mmol/L Na₃VO₄, 1 mmol/L Na₄P₂O₇, 1 mmol/L DTT, and 0.5 mmol/L phenylmethylsulfonyl fluoride]. Five micrograms of total protein were incubated with a double-stranded ³²P-radiolabeled hSIE oligonucleotide probe and visualized as previously described (15).

Immunoprecipitation. Cells were maintained in suspension or adhered to FN for 60 to 90 min in the presence or absence of 1.0 ng/mL rhIL-6. Cells were washed twice in ice cold PBS and harvested as previously described (1). Cells were incubated in TBS [30 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl] containing 1% NP40, 0.1% sodium azide, 20 mmol/L NaF, 1 mmol/L Na₃VO₄, 1 mmol/L Na₄P₂O₇, and 1 Complete, Mini protease inhibitor cocktail tablet for 10 min on ice. Cellular debris was removed by centrifugation at 10,000 × g for 10 min at 4°C. Lysates were quantified via Bio-Rad reagent and between 2 and 4 mg of protein were used per sample. Samples were precleared with 10 μL of Protein Plus A/G agarose beads (Santa Cruz Biotech) for 60 min rotating at 4°C. Slurries were centrifuged (800 × g for 1 min at 4°C) and supernatant was transferred to fresh tubes containing 1 μg anti-gp130 antibodies or isotype control antibodies (BD Pharmingen) and incubated overnight rotating at 4°C. Protein Plus A/G agarose beads (10 μL) were added and lysates were incubated an additional 2 h. Immunoprecipitates were subsequently washed twice in TBS containing 1% NP40 and 0.1% sodium azide, and 3 additional times with TBS containing 0.1% sodium azide and 0.1% BSA (Roche). Immunoprecipitates were mixed with 4× SDS loading dye and 100 mmol/L DTT and incubated for 5 min at 95°C. After centrifugation (800 × g for 2 min) samples were separated on 4% to 12% NuPAGE SDS-PAGE gels (Invitrogen).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. All assays were performed on cells in logarithmic growth. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described (1). Briefly, cells were incubated with serial dilutions of mitoxantrone or doxorubicin (Sigma) under the various conditions for 96 h. Cells were then incubated with 50 μL MTT dye (Sigma) for 4 h. Plates were centrifuged, aspirated, and formazan was solubilized with DMSO. Absorbance was determined with a Dynex II ELISA plate reader at 540 nm (Dynex Technologies).

BrdUrd analysis. The effects of FN adhesion ±IL-6 on cell cycle progression were measured by two-color flow cytometry using FITC-conjugated anti-bromodeoxyuridine (BrdUrd) antibody and PI staining (BD Biosciences) of DNA content. Cells were incubated on FN-coated 60-mm dishes or maintained in suspension on noncoated plates for 48 h. After the 48-h incubation, samples were prepared as previously described (4). After preparation, samples were analyzed for BrdUrd incorporation and PI staining by flow cytometry.

Adhesion of the RMPI 8226 MM cell line to FN enhances IL-6 signaling. Signaling by the soluble BM effector IL-6 elicits biological effects in MM cells via Jak/STAT3, ERK1/2, and PI3-K/Akt signaling pathways (15, 16, 19). Cell adhesion to FN via β1 integrins also initiates several signaling pathways involving ERK1/2, Akt, src-family tyrosine kinases, and RelB (25, 27). To date, the intracellular signaling of these two determinants has been examined independently. Here, we investigated the combination of both IL-6– and FN-mediated cell adhesion. As shown in Fig. 1A, adhesion of RPMI 8226 cells to FN enhanced their response to IL-6 stimulation. Treatment of FN-adhered RPMI 8226 cells with 0 to 10 ng/mL recombinant IL-6 results in a dose-dependent increase in the phosphorylation of STAT3 and ERK1/2, with no consistent stimulation of Akt, at 2 hours poststimulation with IL-6 (ERK and Akt data not shown; Fig. 1A). Further comparison of IL-6–dependent STAT3 phosphorylation in suspension and adhered cells revealed a 10-, 6.25-, and 3-fold increase in STAT3 phosphorylation in adhered cells over suspension cells at 0.1, 1.0, and 10 ng/mL of recombinant IL-6, respectively. A parallel 3-fold increase in STAT3/DNA complexes was seen in cells adhered to FN relative to cells in suspension, as measured by electrophoretic mobility shift assay (Fig. 1B). Super-shift assays showed primarily STAT3 homodimers, with a paucity of STAT3:STAT1 heterodimers being observed (data not shown). These results show that adhesion of MM cells to FN alters STAT3 signaling via the IL-6 receptor. One explanation for the increased capacity of IL-6 signaling in RPMI 8226 cells adhered to FN may involve an up-regulation of the IL-6 receptor α (gp80) or gp130. Flow cytometric analysis showed that surface expression of both gp80 and gp130 remained unchanged after adhesion of RPMI 8226 cells to FN (data not shown), indicating that the observed changes in STAT3 phosphorylation are not mediated by changes in the levels of IL-6 receptor components.

To examine the possibility that FN-adhesion stimulates IL-6 production in an autocrine fashion, we measured IL-6 production by ELISA in supernatants from RPMI 8226 myeloma cells cultured in suspension versus those adhered to FN. Under both conditions, soluble IL-6 remained undetectable from 0.5 to 4.0 hours (data not shown). Based on these findings, we conclude that an FN-adhesion–mediated production of IL-6 does not contribute to STAT3 signaling in costimulated cells.

FN adhesion results in a sustained activation of STAT3 phosphorylation and DNA binding. Signaling via cytokines and associated receptors occurs via a transient early activation of specific signaling determinants on the order of minutes. As shown in Fig. 2, costimulation of RPMI 8226 cells with IL-6 and FN augments STAT3 phosphorylation (30 minutes), and this increase in STAT3 phosphorylation is maintained for at least 6 hours (Fig. 2A). Mobility shift assays similarly showed amplified and sustained STAT3 DNA-binding in cells adhered to FN after incubation with IL-6 compared with cells maintained in suspension (Fig. 2B). These results show that the augmentation of IL-6–mediated STAT3 signaling is maintained for up to 6 hours in RPMI 8226 cells adhered to FN relative to cells not engaged with FN.

The collaboration between β1 integrin–mediated adhesion and IL-6 is observed in all four MM cell lines examined. In this report, we have shown that cellular adhesion to FN potentiated IL-6 stimulated STAT3 activity in the RPMI 8226 cell line. Similar results were observed in three additional MM cell lines. As shown in Fig. 3, STAT3 phosphorylation in MM1.S, U266, and H929 MM cells was increased 1.9 (P < 0.05), 4.1 (P < 0.05), and 5.5-fold (P < 0.05), respectively, in cells costimulated with IL-6 and adhesion to FN when compared with cells treated with IL-6 in suspension. Importantly, the increased ERK1/2 phosphorylation observed in the RPMI 8226 cell line after costimulation was not consistent across the cell lines (data not shown). Furthermore, although phosphorylation of Akt was observed in the MM1.S and H929 MM cell lines after IL-6 stimulation, this response was unchanged by FN-mediated cell adhesion (data not shown). Together, these results suggested that the effects of FN adhesion on IL-6 signaling may be relatively specific for STAT3.

Activation of β1 integrins further augments IL-6–mediated STAT3 activity. We have shown that MM cell adhesion to FN augments IL-6–induced STAT3 signaling. However, the specific contribution of β1 integrins has yet to be addressed. Our lab has previously shown that pretreatment of RPMI 8226 cells with antibodies that block β1-integrins inhibits adhesion to FN (1). In the following experiments, we used antibodies to β1 integrins that instead promote an active conformation, thereby increasing the binding potential of β1 integrins. To this end, pretreatment of cells with β1 integrin activating antibody should enhance the amplification of IL-6–dependent STAT3 activation caused by FN adhesion, if β1 receptors are involved. The U266 MM cell line only modestly adhered to FN and pretreatment of cells with β1 integrin–activating antibodies increased U266 cellular FN adhesion (data not shown). In Fig. 4, we showed that adhesion of U266 cells to FN enhances IL-6 mediated STAT3 protein phosphorylation and DNA binding. Furthermore, preincubation of U266 MM cells with activating β1 integrin antibodies further enhanced the effects of adhesion on STAT3 phosphorylation (Fig. 4A), nuclear translocation, and STAT3 DNA binding (Fig. 4B) in costimulated cells. These data show a central role for β1 integrin–mediated adhesion in the heightened response observed with the combination of IL-6 and FN adhesion on STAT3 signaling. Additionally, we show that pretreatment with IL-6 blocking antibodies attenuates the signaling effects mediated by the collaboration between IL-6 and β1 integrin mediated adhesion. IL-6 receptor blocking antibodies also completely blocked the modest increase in STAT3 phosphorylation and DNA binding observed in U266 cells adhered to FN with β1 activating antibodies in the absence of exogenous IL-6 (Fig. 4A, and B versus 6 versus 8). These results indicate that the observed slight increase in STAT3 phosphorylation in the absence of recombinant IL-6 may result from the potentiation of relatively low levels of cytokine produced in the IL-6 autocrine loop known to be present in the U266 MM cell line (15). This slight increase, however, is very minor compared with the dramatic increase observed when these cells are costimulated with both IL-6 and β1 integrin–mediated adhesion (Fig. 4), suggesting that any autocrine IL-6 produced by U266 cells does not contribute substantially to the observed amplification of IL-6–dependent STAT3 activation mediated by adhesion to FN.

Adhesion of MM cells to FN facilitates an IL-6–independent recruitment of STAT3 to gp130. The signaling events initiated after IL-6 ligation are relatively well-characterized. IL-6 binding to its cognate receptor facilitates gp130 homodimerization, activation of Jak family kinases, phosphorylation of specific tyrosine residues within the cytoplasmic domain of gp130, and activation of STAT3 (20). To further characterize the receptor proximal effects of FN adhesion on IL-6 signaling, we immunoprecipitated the IL-6R complex with antisera to gp130. Immunoprecipitation of gp130 revealed enhanced tyrosine phosphorylation of the gp130/Jak family complexes after stimulation of FN-adhered RPMI 8226 cells with IL-6 (phosphotyrosine at 125–130 kDa; Fig. 5). Consistent with increased phosphorylation of the receptor complex, increased levels of phospho-STAT3 were found to be associated with gp130 under costimulatory conditions relative to IL-6 or FN adhesion alone. Interestingly, immunoprecipitation with gp130 antibodies also revealed an association between STAT3 (nonphosphorylated) and gp130 in the absence of IL-6 stimulation in cells adhered to FN (Fig. 5, lane 1 versus 3). To our knowledge, this is the first example of β1 integrin–mediated preloading of STAT3 to the receptor complex. Moreover, these data suggest that FN adhesion facilitates an altered localization of STAT3, priming cells for signaling.

CAM-DR is maintained despite a reversal of FN-mediated cell cycle arrest after incubation with IL-6. In MM, IL-6 and FN adhesion have been shown to protect cells from a host of cytotoxic stimuli (1, 2, 4, 7, 23). These reports suggest that the increased levels of STAT3 observed after IL-6 stimulation of FN-adhered cells may confer a greater protection against chemotherapeutics than FN adhesion alone. However, MTT cytotoxicity assays show that although adhesion to FN provides significant protection against treatment with either mitoxantrone or doxorubicin (P < 0.0002 and P < 0.0001, respectively), the addition of IL-6 provides no further protection (Fig. 6A and B). Further analysis of drug-mediated apoptosis using FCM by Annexin-V/7-AAD corroborated these findings (data not shown).

Our laboratory has previously showed that adhesion of the RPMI 8226 cell line to FN-mediated a p27^Kip1-dependent G₀-G₁ cell cycle arrest (4). As shown in Fig. 6C, by BrdUrd/PI analysis, adhesion of 8226 cells to FN for 24 h results in an increased number of cells in G₀-G₁ relative to cells maintained in suspension (P = 0.0028), corroborating our previously published data. In contrast, when cells were adhered to FN in the presence of IL-6 no accumulation of cells in G₀-G₁ was observed, with levels similar to that observed in cells maintained in suspension with or without stimulation by IL-6 (Fig. 6C). These results suggest that the addition of IL-6 bypasses or reverses the cell cycle arrest mediated by cellular adhesion to FN. Examination of p27^Kip1 expression showed increased levels of p27^Kip1 protein after adhesion to FN relative to cells maintained in suspension, consistent with our original findings. Interestingly, although the combination of FN adhesion and IL-6 reversed/bypassed the cell cycle arrest mediated by adhesion alone, p27^Kip1 protein expression remained elevated under costimulatory conditions (data not shown). These data indicate that communication between integrins and IL-6 alters proliferative response mediated by either effector alone independent of p27^Kip1. Importantly, although the combination of IL-6 and FN adhesion does not further increase drug resistance compared with FN adhesion alone, the cell cycle arrest mediated by adhesion to FN is “reversed” by costimulation with IL-6. IL-6–induced cell proliferation may give adhered tumor cells an added survival advantage over the long term in vivo.

Research that focuses on the biology/pathogenesis of MM has long included the effectors of the BM microenvironment. The soluble factor IL-6 and FN-mediated cell adhesion are two factors that independently control MM cell cycle progression and sensitivity to proapoptotic agents (1, 4, 9, 10, 12, 14, 23). However, the BM microenvironment likely affords MM cells a network of proliferative and survival factors; therefore, in vivo a MM cell receives stimulation from soluble and physical BM determinants simultaneously. In this report, using a simple model, consisting of only MM cell lines, IL-6 and FN, we begin to recapitulate this network of extracellular factors to investigate the molecular and biological consequences of costimulation of MM cells. We showed that the combination of IL-6 and cellular adhesion to FN results in a dramatic amplification of STAT3 phosphorylation, nuclear translocation and DNA-binding via a novel preloading of gp130 with STAT3. These observations show collaboration between IL-6 receptor complexes and β1 integrins and underscore the complexity of interactions between MM cells and the BM microenvironment. Although interactions between integrins and cell surface receptors have been shown to enhance STAT activation (22, 28–31), to our knowledge, these results are the first illustrating specific STAT3 crosstalk between gp130 and integrins. Most previous reports demonstrating this phenomenon involved RPTKs (30, 31); however, DeFilippi and colleagues (29) showed a β1 integrin–mediated enhancement of STAT5 activation by the common IL-3 β receptor. This receptor, like gp130, is a Type I cytokine receptor without intrinsic kinase activity (29). Here, we identify a second Type I cytokine receptor involved in crosstalk between soluble and adherent factors, suggesting that this signaling mechanism involves a common mechanism across diverse receptor families. Furthermore, we describe a unique molecular mechanism by which cooperative signaling between gp130 and β1 integrins provides a survival advantage to MM cells treated with chemotherapeutic agents.

Stimulation of cells with IL-6 results in receptor multimerization and the activation of three major signaling pathways, the Jak/STAT3 pathway, the Ras/ERK1/2 pathway and PI3 kinase/Akt/PKB pathway (20). In this report, enhanced STAT3 activation was observed in all four MM cell lines examined. In contrast, enhanced ERK1/2 activation was observed in only one of four cell lines, and, although Akt activation was observed in three of four cell lines, in all three cases, it occurred independently of FN adhesion. These results indicate that the mechanism of collaboration is relatively specific for STAT3. Consistent with these observations, we showed that the enhanced activation of STAT3 correlated with a FN adhesion–mediated binding of STAT3 to gp130.

Our observations suggest that FN adhesion facilitates a preloading of gp130 with STAT3 that enhances STAT3 activation. Dogma dictates that recruitment of STAT3 follows gp130 receptor stimulation and phosphorylation. So, in this context, our results suggest that either: (a) the adhesion of MM cells to FN facilitates the phosphorylation of gp130 and recruitment of STAT3 without measurable STAT3 activation—in agreement with current dogma, or (b) FN adhesion facilitates the association between gp130 and STAT3 independent of gp130 phosphorylation—suggesting a more complex regulation of STAT3 signaling from gp130. To address the former, integrin signaling from focal adhesions has been shown to involve ligand-independent activation of RPTKs (24, 32). To this end, the collaboration between β1 integrins and gp130 may involve a direct association and activation of gp130 by integrin ligation and focal adhesion formation. To date, we have been unable to show direct association between gp130 and β1 integrins (data not shown), but that does not fully exclude a direct or indirect activation by focal adhesion–associated tyrosine kinases.

It is also possible that we have identified an additional level of regulation. Although gp130 phosphorylation has been shown to be integral in STAT3 activation, the growing list of STAT chaperone proteins suggests that regulation of gp130/Jak/STAT signaling may be more complicated. The STAT chaperone proteins, including Receptor of Activated Protein Kinase C-1 (RACK1), Glucose-Related Protein 58 (GRP58/ER-60/ERp57), and PKC-δ have been shown to enhance STAT signaling in several cellular models (33–35). One example, RACK1, a 36-kDa WD40-containing scaffold protein, has been found to participate in the enhanced activation of Src, PKC, STAT1 and most recently STAT3 (33, 36, 37). Zhang and colleagues (33) showed that RACK1 facilitates the recruitment of STAT3 to Insulin and Insulin-like Growth Factor-I receptors. Importantly, the effects of RACK1 were specific to STAT3, having no affect on Akt or ERK1/2 activation. Moreover, RACK1 has been shown to associate with β1 integrins and to facilitate formation of functional focal adhesions (38). These studies suggest that RACK1 (or other chaperones) may be a catalyst for the collaboration between β1 integrin–mediated adhesion to FN and gp130.

Beyond the biochemical changes shown in this report, the collaboration between IL-6 and FN cell adhesion also has biological consequences. Here, we show that the collaboration of these two prosurvival effectors did not afford a survival advantage beyond levels conferred by CAM-DR. This was consistent with levels of Bcl-2, Bcl-X_L, and Mcl-1, which remained unchanged in response to costimulation in the RPMI 8226 cells (data not shown). In contrast, costimulation did facilitate an altered proliferative response. The collaboration between IL-6 and β1 integrins results in a bypass of the p27^Kip1-dependent cell cycle arrest mediated by FN adhesion alone, in the face of unchanged levels of p27^Kip1 (4). Previous studies suggest that changes in cell cycle progression after enhanced STAT3 signaling involve increased levels of specific D-type cyclins (23). However, no observable increases in cyclin D expression were detected as a result of enhanced STAT3 activity in the RPMI 8226 cell line (data not shown). Therefore, it seems that β1 integrin and IL-6–mediated STAT3 signaling in RPMI 8226 cells may target alternate antiapoptotic and/or proliferative factor(s).

Lastly, an expanding number of reports have been published examining the effects of BM stromal cells on MM biology (reviewed in ref. 14), providing novel insight into the dynamics between MM cells and the BM microenvironment. In this report, we have “scaled back” the environmental cell model and used two relatively well-characterized MM BM effectors to examine the biological consequences of costimulation in MM cells. We identify STAT3 as an important signaling effector involved in the collaboration between the physical environment and the soluble environment. Our findings suggest that adhesion to FN facilitates an alteration in the cellular localization of STAT3, priming gp130 for stimulation. As such, the environmental context of the cell may have significant bearing on intracellular signaling events, modulating both myeloma cell survival and proliferation.

No potential conflicts of interest were disclosed.

Grant support: National Cancer Institute grants CA-82533 (W.S. Dalton) and 77859 (W.S. Dalton).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the Flow Cytometry Core Facility and the Biostatistics Core Facility (National Cancer Institute CA-76292) at the H. Lee Moffitt Cancer Center.