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NEOPLASIA
From the Laboratory of Cellular and Molecular Biology,
National Cancer Institute, National Institutes of Health, Bethesda, MD.
Multiple myeloma (MM) is an invariably fatal disease that accounts
for approximately 1% to 2% of all human cancers. Surprisingly little
is known about the cellular pathways contributing to growth of these
tumors. Although the cytokine interleukin-6 has been suggested to be
the major stimulus for myeloma cell growth, the role of a second
potential growth factor, insulin-like growth factor I (IGF-I), has been
less clearly defined. The IGF-I signaling cascade in 8 MM cell lines
was examined. In 7 of these, the IGF-I receptor (IGF-IR) was expressed
and autophosphorylated in response to ligand. Downstream of
IGF-IR, insulin receptor substrate 1 was phosphorylated, leading to the
activation of phosphatidylinositol-3'-kinase (PI-3K). PI-3K, in turn,
regulated 2 distinct pathways. The first included Akt and Bad, leading
to an inhibition of apoptosis; the second included the
mitogen-activated protein kinase (MAPK), resulting in proliferation.
Biologic relevance of this pathway was demonstrated because in vitro
IGF-I induced both an antiapoptotic and a proliferative effect.
Importantly, in vivo administration of IGF-I in SCID mice inoculated
with the OPM-2 line led to approximately twice the growth rate of tumor
cells as in controls. These results suggest that IGF-I activates at
least 2 pathways effecting myeloma cell growth and contributes
significantly to expansion of these cells in vivo.
(Blood. 2000;96:2856-2861) Multiple myeloma (MM) is a progressive B-lineage
neoplasia characterized by the accumulation of malignant plasma cells
in the bone marrow and the occurrence of extensive osteolytic bone destruction.1,2 The disease is incurable and accounts for approximately 1% to 2% of total human cancers.3 In spite
of extensive study, little is known about the molecular lesions or biochemical abnormalities contributing to the initiation or development of this disease. To date, the interleukin-6 (IL-6) pathway is the
single signaling cascade most consistently implicated in MM. The role
of IL-6 in this disease has been clearly demonstrated in model systems
wherein IL-6 null mice normally fail to develop plasma cell
tumors,4,5 though these tumors can be induced in an
IL-6-independent manner by selected agents.6 In human myeloma a critical role for IL-6 is suggested based largely on associative observations. Some MM cell lines are IL-6
dependent,7-9 and IL-6 is a proliferative signal for
primary MM explants.10 Furthermore, patient IL-6 levels
have been suggested in some studies11,12 to be a
prognosticator of disease severity, and attempts to interfere with IL-6
signaling in clinical trials have led to transient effects on tumor
growth.13
It should be noted, however, that it is unclear whether all MM passes
through an IL-6-dependent phase or whether a subset of tumors arises
as IL-6 independent. It is also unknown whether IL-6-dependent tumors
routinely become IL-6 independent during disease progression. Given
these possibilities, it is critical to identify other pathways that may
coordinately enhance MM growth or substitute as a growth stimulus in
the absence or loss of IL-6 dependence. Herein, we describe studies of
the insulin-like growth factor I (IGF-I) signaling cascade, which
suggest that this pathway may play such a role in the development of MM.
Cell lines
Immunoprecipitation and Western blot analysis
Akt immunoprecipitation kinase assay
MAPK assay Cells (1 × 106) were serum starved for 48 hours and either untreated or treated with IGF-I for 10 minutes before lysis. Immunoprecipitations were performed by incubating lysates with immobilized anti-p-p44/42 MAPK (New England BioLabs). Immunoprecipitates were incubated with Elk-2 fusion protein in the presence of adenosine triphosphate and phosphorylated product detected with anti-p-Elk-2 antibody. Phosphorylated Elk-2 protein was quantitated by densitometry tracing.3H thymidine incorporation assay Exponentially growing cells were washed with PBS, resuspended in serum-free media, and cultured in 24-well plates with or without 100 ng/mL of IGF-I at a density of 1 × 105/well for 72 hours. To measure DNA synthesis, 1 µCi 3H-thymidine (Amersham International, Arlington Heights, IL) was added during the final 4 hours of culture. Uptake of 3H-thymidine was determined in triplicate samples by liquid scintillation counting. Mitogenic activity was represented by a fold increase of 3H-thymidine uptake calculated in the same manner as described above for Akt kinase activity.In vivo tumorigenesis studies The OPM-2 line was used for in vivo inoculation into SCID mice. Cells were washed with PBS 3 times before inoculation. Ten mice were injected with 1 × 107 OPM-2 cells intraperitoneally, of which 4 mice (IGF-I group) received 20 µg IGF-I in 0.5 mL PBS 3 times per week for 4 weeks. The remaining 6 mice (control, no IGF-I group) received only PBS. Animals were monitored 2 times per week for tumor growth after inoculation. The control group was killed at day 30 because of the presence of large, palpable tumors. Animals in the IGF-I group were killed at day 60. Tumor volume was determined by measurement of the largest diameter and the diameter at a right angle.
Expression and autophosphorylation of IGF-I receptor in response to IGF-I stimulation To evaluate a possible role for IGF-I in MM, levels of IGF-IR were first analyzed in a series of MM and other lymphoid cell lines. Receptor was immunoprecipitated from the indicated lines and blotted with either antibody to the receptor or phosphotyrosine. As can be seen (Figure 1), 7 IL-6-independent lines Brown, Delta-47, OPM-2, 8226, H929, KMM1, and MM144expressed readily detectable amounts of IGF-IR,
whereas the IL-6-dependent line ANBL-6 evidenced a lower level of
receptor. The functional status of IGF-IR in terms of
autophosphorylation was examined in response to IGF-I stimulation. As
seen, autophosphorylation was observed in 7 of 8 lines with no
detectable phosphorylation observed in ANBL-6, consistent with the low
level of receptor protein. Two T-cell leukemia lines expressed IGF-IR,
which was autophosphorylated in an IGF-I-dependent manner, whereas the
NK line YT exhibited only minimal expression of IGF-IR. Among B-cell
lymphomas, BL-41 and ST486 expressed levels of IGF-IR comparable to
that seen in the IL-6-dependent MM lines, and somewhat lower levels
were observed in Daudi. IGF-I-dependent autophosphorylation was
observed in each of these 3. The downstream effect of IGF-IR activation
in terms of subsequent 3H-thymidine incorporation was
clearly distinct among these differing cell types (see below).
IGF-I-induced phosphorylation of insulin receptor substrate proteins and their association with phosphatidylinositol-3'-kinase To establish whether a functional signaling pathway was activated downstream of the IGF-IR, we next examined IRS proteins previously shown to be phosphorylated by both the insulin and the IGF-I receptors.19,20 IRS-2 (Figure 2A) and IRS-1 (Figure 2B) expression and tyrosine phosphorylation were examined in nonstimulated and stimulated cells. IRS-2 expression was observed only in Brown but not in the other 3 or 4 additional lines examined (not shown). No detectable level of tyrosine phosphorylation of IRS-2 was observed in Brown on IGF-I stimulation. In contrast, IRS-I was expressed in all 4 lines with the highest expression level in ANBL-6, in which IRS-1 was constitutively phosphorylated. In the other 3 lines, tyrosine phosphorylation of IRS-1 was strongly IGF-I-dependent in Delta-47 and OPM-2 and weakly so in Brown. Similar results, as seen in the IRS-1-expressing lines, were also obtained in 3 of 4 additional lines (8226, H929, and MM144) analyzed (Figure 1), in which IRS-1 phosphorylation was, again, IGF-I dependent (not shown).One of the mechanisms by which phosphorylated IRS proteins transduce downstream signals is interaction with the SH2 homology domains of other proteins.21 PI-3K has been identified as one such target that associates with IRS-1 or IRS-2 through the SH2 domain of its p85 regulatory subunit.22 Coimmunoprecipitation was, therefore, used to evaluate this interaction. Tyrosine-phosphorylated IRS-1 protein was observed to associate with the p85 subunit of PI-3K in 3 lines (Figure 2B) in an IGF-dependent manner, whereas interaction in ANBL-6 was constitutive. Although phosphorylated IRS-1 or IRS-2 was not detected in Brown, association with the p85 subunit of PI-3K was still observed, most likely because of greater sensitivity of anti-p85 antibody. Again, a similar association of PI-3K with IRS-I was observed in 3 additional lines, and weak, but detectable, interaction was observed in a fourth (not shown). IGF-I induces Akt phosphorylation and its kinase activity A search for further downstream members in this pathway led to analysis of Akt/PKB. Akt has been shown to be a target of PI-3K-generated signals23-25 and to participate in growth factor maintenance of cell survival26 after activation by phosphorylation of residues Thr 308 and Ser 473. Therefore, we investigated Akt activation using specific antibodies that detect phosphorylation at these sites. Akt was expressed in all 8 lines and, with the exception of ANBL-6, phosphorylated at both positions in response to IGF-I (Figure 3A). Time-course analysis of Akt phosphorylation in lines H929 and MM144 revealed maximal phosphorylation at 3 minutes in H929 and between 3 and 10 minutes in MM144. To assess the functional consequences of Akt phosphorylation, an in vitro kinase assay was performed in which a synthetic peptide (RPRAATF) related to the sequence surrounding the phosphorylation site of GSK-3 was used as substrate. As shown in Figure 3B, the kinase activity of IGF-I-stimulated cells was increased approximately 2- to 4-fold in Delta-47, OPM-2, H929, 8226, and MM144. Only minimal increases were found in ANBL-6, Brown, and KMM1 lines. Thus, in response to IGF-I stimulation, kinase activity correlated directly with the phosphorylation levels of Akt at Ser 473 and Thr 308.Additional downstream signaling in the IGF-I pathway Targets further downstream in this pathway are the subjects of considerable investigation. Growth factor and cytokine signaling through Akt has recently been suggested to play a role in protecting cells from apoptosis, at least in part, by phosphorylation and inactivation of the proapoptotic Bcl-2 family member, Bad.27,28 Thus, the status of Bad was examined using an antibody specific for phosphorylation on Ser 136. IGF-I-dependent Bad phosphorylation was observed in 5 of 8 lines (Figure 3A), and the level of phosphorylation correlated with Akt phosphorylation and kinase activity described in Figure 3. To confirm the antiapoptotic effect of Bad phosphorylation, a caspase activity assay was performed (not shown) that revealed a decrease in caspase 3 activity after IGF-I stimulation that, again, correlated with Bad phosphorylation levels.Activation of the MAPK pathway by IGF-I The above data indicate that IGF-I activates at least one pathway that inhibits apoptosis. To explore the possibility that additional pathway(s) might be activated that promote proliferation, we next examined the status of the MAPK cascade. For these studies, an in vitro kinase assay was used after immunoprecipitation of MAPK from 6 MM lines. As seen in Figure 4, kinase activity was increased in Delta-47, OPM-2, 8226, and MM144 by approximately 5- to 10-fold and 35-fold in Brown. An increase in H929 was lowest at 3.5-fold. Time-course analysis revealed maximal activation of MAPK by 3 minutes. Thus, an additional pathway associated with mitogenesis as opposed to antiapoptosis has also been activated in these lines.
Biologic relevance of IGF-I pathway activation The data presented above clearly demonstrate activation of the IGF-I signaling pathway in most MM cell lines. It was, therefore, important to determine whether this activation was biologically significant in terms of tumor cell growth. To address this question, we first analyzed the mitogenic effect of IGF-I on in vitro growth of MM cells. Cells were serum starved for 68 hours, then pulsed with 3H-thymidine either in the presence or absence of IGF-I. Compared to controls, IGF-I stimulated approximately 2- to 3-fold increases in 3H-thymidine incorporation in 6 of 8 lines (Figure 5). This effect was completely inhibited in the presence of antibody to the IGF-I receptor. In contrast to the MM lines, the 2 T-cell leukemias, Jurkat and Molt, that expressed IGF-IR and exhibited IGF-I-dependent autophosphorylation (Figure 1) showed no increase in 3H-thymidine incorporation on IGF-I stimulation. Among the B lymphomas expressing IGF-IR (Figure 1), BL-41, with the highest level of expression, exhibited a 2-fold increase in thymidine uptake whereas increases in Daudi and ST486 were minimal at 1.3 and 1.2 fold, respectively (not shown).
To evaluate whether IGF-I has an effect on tumor growth in vivo, we
used OPM-2 because this line exhibited one of the highest mitogenic
responses to IGF-I and was found to grow in SCID mice. Two groups of
animals were injected with OPM-2 cells. The control group was not
treated, whereas IGF-I was given to the experimental group 3 times/wk
for 4 weeks (Table 1). As can be seen,
tumors in the IGF-I treatment group grew to approximately twice the
size in about half the time as in the control group. Thus, IGF-I
produced a marked effect on tumor growth in vivo.
Considerable attention has been focused in the past several years on the role of cytokines, particularly IL-6, in the development of MM. IL-6 has been suggested as a major growth factor in this disease based on a number of associative findings such as the IL-6 dependence of some myeloma lines,7,8 the proliferative response of primary explants to this cytokine,10 correlation of disease severity with circulating IL-6 levels,11,12 and the transient effects on tumor burden on patients treated with antibodies to IL-6 or IL-6R.13 Given that it is unknown whether all MM evolves through an IL-6-dependent phase and that the frequency with which IL-6-dependent MM progresses to IL-6 independence remains undetermined, it is surprising how little is known about other cytokines or growth factors that may either augment or replace IL-6 responsiveness. One such candidate growth factor is IGF-I. Early studies involving a limited number of IL-6-independent cell lines revealed that most responded to this growth factor with modest increases in proliferation.29,30 More recently, Jelinek et al31 demonstrated that though IGF-I could, again, induce modest proliferation in 4 IL-6-dependent lines, simultaneous treatment with both factors resulted in a markedly enhanced effect. These results, together with studies suggesting a role for IGF-I in a variety of malignancies,32 indicate that the IGF-I signaling pathway may play an important role in MM development. To date, little is certain regarding the biochemical cascade constituting this pathway in myeloma cells and regarding whether IGF-mediated proliferation observed in vitro reflects an in vivo role for this growth factor. The current studies were, therefore, performed to delineate IGF-I signaling in MM cells and to address the question of in vivo relevance. IGF-IR expression was observed in all 8 MM lines investigated. In 7 of 8, receptor autophosphorylation was ligand dependent. Among other lymphoid tumors examined, similar results were obtained with 2 of 2 T-cell leukemias and 3 of 5 B-cell lymphomas. The effect of IGF-I on MM cells is likely not to represent a promiscuous and minimal response to growth factors in general because most lines also express c-met, the receptor for hepatocyte growth factor, but do not respond to this ligand (N.-L.G. and S.R., unpublished data, March 1999). Receptor phosphorylation resulted in IRS-1 activation and subsequent association with, and activation of, PI-3K. IRS-2 was only expressed in a single line (Brown). Activation of PI-3K led to phosphorylation of Akt at Ser 473 and Thr 308, and the level of phosphorylation at these sites correlated with Akt kinase activity. Further downstream, Bad was found to be clearly phosphorylated in 5 of 8 lines and weakly phosphorylated in a sixth. The levels of Bad phosphorylation correlated with Akt activity, indicating that Akt phosphorylates Bad, as has been reported in BALB/c 3T3 and PC 12 cells.27 Bad phosphorylation is functionally important in cell death because this molecule forms heteromeric complexes with 2 antiapoptotic factors, Bcl-2 and Bcl-XL, thus inhibiting their function in cell survival.33 Phosphorylation of Bad results in dissociation of these complexes and binding to 14-3-3 protein. This interaction releases Bcl-2 and Bcl-XL, resulting in cell survival.34 The functional consequence of Bad phosphorylation was confirmed by the assessment of caspase 3 as an indicator of apoptosis. Caspase 3 activity was reduced in all lines in direct relation to the degree of Bad phosphorylation. The above results clearly define a functional IGF signaling pathway in most MM lines that can be traced to elements inhibiting apoptosis and likely represents the same pathway involved in IGF-I-mediated inhibition of dexamethasone-induced apoptosis reported in the 8226 cell line.35 In addition to inhibiting apoptosis, IGF-I was found to activate the MAPK pathway in all MM lines tested (Figure 4). It has been suggested that this pathway accounts for the proliferative response induced by IL-6 in IL-6-dependent myeloma.36,37 It should be cautioned that this suggestion is based on limited data, including a murine IL-6-dependent hybridoma and 1 of 2 explants from patients with MM. Interestingly, Anderson et al37 subsequently demonstrated that the MAPK pathway could not be activated in MM lines that were IL-6 independent because of the failure to phosphorylate Sos 1. Consistent with this is our observation that IL-6 fails to enhance the proliferation of any of the IL-6-independent lines in the current study. The current data indicate that even though MM lines may lose the ability to activate this pathway in response to IL-6, they may still proliferate in response to IGF-I, which effectively activates the same MAPK pathway. IGF-I stimulation fails to induce phosphorylation of Stat3 in the IL-6 pathway, indicating no interaction between IGF-I and the IL-6 cascade. Thus, IGF-I may act not only to enhance IL-6-mediated proliferation in IL-6-dependent myeloma but to provide sufficient antiapoptotic and proliferative stimuli to cells that have lost the ability to respond to IL-6 to promote their survival and outgrowth. The biologic relevance of the IGF-I pathway in terms of tumor cell growth was further demonstrated in vitro and in vivo. In vitro proliferation assays revealed that 6 of 8 lines exhibited 2- to 3-fold increases in mitogenesis, as has been reported30 for other MM lines. Interestingly, the 2 T-cell leukemias that expressed IGF-IR at levels similar to those seen among the myelomas failed to demonstrate 3H-thymidine incorporation in response to IGF-I, even though receptor activation was clearly observed. This result suggests the possibility of a fundamental difference between B- and T-lineage neoplasias in response to IGF-I. Among 5 B-cell lymphomas examined, 2 expressed IGF-IR at levels comparable to those seen in the IL-6-independent MM lines, but only one evidenced 3H-thymidine incorporation in the same range as the myelomas. Thus, the IGF-I pathway may, for reasons not understood, become more capable of delivering a biologically detectable signal in the latter stages of B-lineage development than in less mature cell types.38 More important from a biologic standpoint, when the OPM-2 line was inoculated into SCID mice, tumors grew to approximately twice the size in half the time in IGF-treated animals as in controls. This finding indicates that the modest IGF-I proliferative effect, likely coupled with the inhibition of apoptosis, is reflected in a marked promotion of in vivo tumor growth. We have not detected IGF-I production by myeloma cells using an ELISA assay, and thus suggest that the IGF-I to which myeloma cells respond is produced in a paracrine fashion as it is present in virtually all body fluids. It is further suggested that IGF-I acts synergistically with IL-6 in the growth of early-stage myeloma, consistent with its effect on IL-6-dependent lines in vitro.31 Assuming that MM may become IL-6 independent at later stages, the current results with IL-6-independent lines indicate that, in the absence of IL-6 responsiveness, IGF-I may alone play a significant role in the maintenance and progression of this disease. Thus, it is possible that therapeutic approaches that modulate both the IL-6 and the IGF-I pathways may prove to be important approaches to the treatment of MM.
Submitted January 27, 2000; accepted June 14, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Stuart Rudikoff, Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; e-mail: rudikoff{at}helix.nih.gov.
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Top
Abstract
Introduction
) or
treated (+) with IGF-I. Lysates from 1 × 107 cells were
incubated with anti-IGF-IR antibody, and immunoprecipitates were
prepared and analyzed as described in "Materials and methods."
Blots were probed with antibody to the IGF-IR (A) or phosphotyrosine
(B).
-32P-ATP
and 100 µmol/L substrate for 10 minutes. The supernatant was
removed, mixed with 20 µL 40% TCA, and transferred to P81 phosphocellulose paper for washing and counting. The increase in Akt
kinase activity was calculated as a fold increase of 32P
incorporation by dividing the mean value of non-IGF-I-stimulated cells
by that of the corresponding IGF-I-stimulated cells.
