Dkk1-induced inhibition of Wnt signaling in osteoblast differentiation is an underlying mechanism of bone loss in multiple myeloma.

Expression of the Wnt signaling inhibitor, DKK1 by multiple myeloma cells is correlated with lytic bone disease in multiple myeloma. However, the mechanism(s) by which DKK1 contributes to this process is not clear. Herein, we analyzed the functional role of canonical Wnt signaling and Dkk1 inhibition of this pathway in bone morphogenic protein (BMP)-2-induced osteoblast differentiation. Osteoblast differentiation was measured by alkaline phosphatase (ALP) activity in murine (C2C12) and human pre-osteoblast (hFOB1.19) and osteoblast-like (Saos-2 and MG63) cell lines. Cytoplasmic beta-catenin protein was separated by E-cadherin-GST pull-down assay and analyzed by Western blotting. A dominant negative form of beta-catenin, Dkk1 and TCF reporter constructs were transfected into C2C12 cells. C2C12 cells were also transfected with siRNA specific to LRP5/6 to knockdown receptor expression. Canonical Wnt signaling was activated in these cell lines in response to Wnt3a as assessed by increased cytoplasmic, non-phosphorylated beta-catenin and TCF/LEF transcription activity. Recombinant Dkk1 and plasma from MM patients containing high levels of Dkk1 blocked Wnt3a-induced beta-catenin accumulation. Importantly, Dkk1 abrogated BMP-2 mediated osteoblast differentiation. The requirement for Wnt signaling in osteoblast differentiation was confirmed by the following observations: 1) overexpression of Dkk1 decreased endogenous beta-catenin and ALP activity; 2) silencing of Wnt receptor mRNAs blocked ALP activity; and 3) a dominant negative form of beta-catenin eliminated BMP-2-induced ALP activity. Furthermore, Wnt3a did not increase ALP activity nor did BMP-2 treatment result in beta-catenin stabilization indicating that cooperation between these two pathways is required, but they are not co-regulated by either ligand. These studies have revealed that autocrine Wnt signaling in osteoblasts is necessary to promote BMP-2-mediated differentiation of pre-osteoblast cells, while Wnt signaling alone is not capable of inducing such differentiation. Dkk1 inhibits this process and may be a key factor regulating pre-osteoblast differentiation and myeloma bone disease.

IL-6 mediated activation of STAT3 bypasses Janus kinases in terminally differentiated B lineage cells.

Cytokine signaling generally occurs through receptors lacking tyrosine kinase activity. Aggregation of receptors leads to activation of receptor associated Janus kinases (Jaks) which in turn phosphorylate members of a family of transcription factors (STATs) that translocate to the nucleus and regulate gene expression. In the case of Interleukin-6 (IL-6), the consensus for signaling in B lineage cells has been that Jak1, Jak2 and Tyk2 are all phosphorylated upon ligand binding and participate in activation of downstream elements, in particular STAT3. In other cell types, Jak1 has been demonstrated to be absolutely required for IL-6 mediated activation of STAT3. In the present studies, we have identified a series of end stage B cell (plasma cell) lines that fail to express Jak1, but phosphorylate STAT3 in response to IL-6. No evidence was found for a requirement of other Jak family members in the activation of STAT3. STAT3 tyrosine phosphorylation was inhibited in a dose dependent manner by the MEK inhibitor U0126, but not by inhibitors of PI-3K or Src kinases. Moreover, STAT3 phosphorylation was similarly inhibited in lines expressing Jak1 wherein Jak1 was phosphorylated upon IL-6 stimulation and Jak1 phosphorylation was not inhibited by U0126. These results indicate that the MAPK pathway plays a critical role in IL-6 mediated tyrosine phosphorylation of STAT3 and suggests that Jak kinases may not be required in this cascade. Thus, it may be important to re-evaluate the role of Jak kinases in other cytokine signaling pathways as well.

Wnt signaling in B-cell neoplasia.

Wnts comprise a family of secreted proteins that interact with receptors consisting of a Frizzled (Fz) family member alone or complexed with LDL receptor-related proteins (LRP5/6). Wnt signaling plays a crucial role in both development and differentiation, and activation of a 'canonical' Wnt pathway resulting in beta-catenin stabilization is associated with several types of human cancers. To date, little is known about potential Wnt signaling in mature lymphocytes or lymphoid neoplasia. Herein, we have analysed Wnt signaling in mature B cells (lymphomas) and plasma cells (multiple myeloma). Both Fz and LRP5/6 mRNAs were expressed in myeloma lines, but LRP5/6 were not observed in lymphomas. In myelomas, a canonical Wnt signaling pathway was activated following treatment with Wnt-3a as assessed by accumulation of beta-catenin, but beta-catenin levels actually decreased in lymphoma cells. Wnt-3a treatment further led to striking morphological changes in myeloma cells accompanied by rearrangement of the actin cytoskeleton. Morphological changes were associated with a second Wnt pathway dependent on Rho activation. These results suggest that Wnt responsiveness is a stage-specific phenomenon in B-cell development and that the morphological changes associated with Wnt signaling may play a role in the motility and metastatic potential of myeloma cells.

An unusual H-Ras mutant isolated from a human multiple myeloma line leads to transformation and factor-independent cell growth.

Multiple myeloma (MM) is an incurable plasma cell malignancy. To investigate biochemical lesions associated with MM, we constructed an expression cDNA library from the OPM-2 human myeloma line. A highly transforming H-Ras mutant was identified by transfection analysis using NIH 3T3 cells. DNA sequencing demonstrated a single-point mutation at position 117 located in the guanine nucleotide-binding site resulting in a lysine-to-glutamic acid substitution. This mutant, H-Ras (K117E), was found to be constitutively activated in terms of GTP binding. We compared the biological effects of H-Ras (K117E) and H-Ras (G12V) in 32D murine hematopoietic progenitor cells. Whereas both Ras proteins are constitutively activated, 32D cells expressing H-Ras (G12V) are still dependent on IL-3 for survival and proliferation while cells carrying H-Ras (K117E) become IL-3 independent. Similar experiments conducted with the B9 line, an IL-6-dependent hybridoma, also demonstrated that B9/H-Ras (K117E) became IL-6-independent. Expression of H-Ras (K117E) in the human IL-6-dependent ANBL-6 myeloma line resulted in enhanced proliferation at suboptimal concentrations of IL-6. These observations suggest that H-Ras mutations at the binding site for the GTP nucleotide ring structure may also represent activating lesions and have additional biological effects when compared to previously described Ras mutants.

Wnt signaling in B and T lymphocytes.

Wnt signaling has been shown to be critical for proper embryonic development as well as growth regulation of certain adult tissues. Defects in Wnt pathways have additionally been associated with a number of human cancers. However, it is only recently that a role for Wnts in the immune system has come to be appreciated. Wnts have now been shown to play significant roles in early stage development of both B and T lineage cells. Current studies suggest that proliferation and/or survival of these cells is associated with activation of the 'canonical' Wnt/beta-catenin pathway. Functional Wnt signaling appears to also occur in end stage B (plasma) cells where both the 'canonical' and the Wnt/RhoA pathways are activated. Herein, we review the current understanding of Wnt signaling in B and T cell development and the potential involvement of Wnt cascades in lymphoid neoplasia.

PML mediates IFN-alpha-induced apoptosis in myeloma by regulating TRAIL induction.

Interferon (IFN) induces expression of proapoptotic genes and has been used in the clinical treatment of multiple myeloma. The promyelocytic leukemia (PML) gene is an IFN-induced target that encodes a tumor suppressor protein. PML protein is typically localized within discrete speckled nuclear structures termed PML nuclear bodies (NBs). Multiple myeloma cells demonstrate differential responses to IFN treatment, the mechanism of which is largely unknown. Herein, we show that growth inhibition effects of IFN-alpha in myeloma cells correlate with PML NBs and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induction, whereas known IFN targets including signal transducer and activator of transcription-1 (STAT1), STAT3, p38, and Daxx cannot account for these differential responses. RNAi silencing of PML blocks IFN-alpha-induced apoptosis in myeloma cells and correspondingly down-regulates TRAIL expression. Similarly, stable expression of a dominant negative TRAIL receptor DR5 partially blocks IFN-induced cell death. These results demonstrate that PML and TRAIL play important roles in IFN-induced apoptosis and identify TRAIL as a novel downstream transcriptional target of PML. Identification of PML and PML NBs as effectors of IFN responses provides insights into mechanisms by which tumor cells exhibit resistance to this class of agents and may prove useful in assessing treatment regimens.

Wnts induce migration and invasion of myeloma plasma cells.

Multiple myeloma is an incurable form of lymphoid cancer characterized by accumulation of neoplastic plasma cells in the bone marrow cavity. Little is known about the mechanisms regulating myeloma cell movement within the bone marrow and metastasis to secondary sites. Herein, we identify multiple members of the wingless/int (Wnt) family as promoters of myeloma cell migration/invasion. Wnt-mediated migration was associated with the Wnt/RhoA pathway and did not necessitate signaling through beta-catenin. Activation of both RhoA and members of the protein kinase C (PKC) family, including PKCalpha, PKCbeta, and PKCmu, were required for induction of migration. Activated RhoA and PKCalpha, PKCbeta, and PKCmu appear to assemble in macromolecular signaling complexes that are associated with the cell membrane. These results suggest that Wnt responsiveness of myeloma plasma cells may be a significant factor in disease progression.

Insulinlike growth factor-I signaling in multiple myeloma: downstream elements, functional correlates, and pathway cross-talk.

In multiple myeloma cells, insulinlike growth factor-I (IGF-I) activates 2 distinct signaling pathways, mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI-3K), leading to both proliferative and antiapoptotic effects. However, it is unclear through which of these cascades IGF-I regulates these different responses. The present studies identify a series of downstream targets in the PI-3K pathway, including glycogen synthase kinase-3beta, p70S6 kinase, and the 3 members of the Forkhead family of transcription factors. The contribution of the MAPK and PI-3K pathways and, where possible, individual elements to proliferation and apoptosis was evaluated by means of a series of specific kinase inhibitors. Both processes were regulated almost exclusively by the PI-3K pathway, with only minor contributions associated with the MAPK cascade. Within the PI-3K cascade, inhibition of p70S6 kinase led to significant decreases in proliferation and protection from apoptosis. Activation of p70S6 kinase could also be prevented by MAPK inhibitors, indicating regulation by both pathways. The Forkhead transcription factor FKHRL1 was observed to provide a dual effect in that phosphorylation upon IGF-I treatment resulted in a loss of ability to inhibit proliferation and induce apoptosis. The PI-3K pathway was additionally shown to exhibit cross-talk and to regulate the MAPK cascade, as inhibition of PI-3K prevented activation of Mek1/2 and other downstream MAPK elements. These results define important elements in IGF-I regulation of myeloma cell growth and provide biological correlates critical to an understanding of growth-factor modulation of proliferation and apoptosis.

Insulin-like growth factor I induces migration and invasion of human multiple myeloma cells.

Multiple myeloma (MM) is an incurable form of cancer characterized by accumulation of malignant plasma cells in the bone marrow. During the course of this disease, tumor cells cross endothelial barriers and home to the bone marrow. In latter stages, myeloma cells extravasate through blood vessels and may seed a variety of organs. Insulin-like growth factor I (IGF-I) is one of several growth factors shown to promote the growth of MM cells. In the current study, we have assessed the ability of IGF-I to serve additionally as a chemotactic factor affecting the mobility and invasive properties of these cells. Results indicate that IGF-I promotes transmigration through vascular endothelial cells and bone marrow stromal cell lines. Analysis of endogenous signaling pathways revealed that protein kinase D/protein kinase Cmicro (PKD/PKCmicro) and RhoA were both activated in a phosphatidylinositol 3-kinase (PI-3K)-dependent manner. Inhibition of PI-3K, PKCs, or Rho-associated kinase by pharmacologic inhibitors abrogated migration, whereas mitogen-activated protein kinase (MAPK), Akt, and p70S6 kinase inhibitors had no effect. These results suggest that IGF-I promotes myeloma cell migration by activation of PI-3K/PKCmicro and PI-3K/RhoA pathways independent of Akt. The identification of IGF-I as both a proliferative and migratory factor provides a rational basis for the development of targeted therapeutic strategies directed at IGF-I in the treatment of MM.