Transcriptional Silencing of the Wnt-Antagonist DKK1 by Promoter Methylation Is Associated with Enhanced Wnt Signaling in Advanced Multiple Myeloma

The Wnt/β-catenin pathway plays a crucial role in the pathogenesis of various human cancers. In multiple myeloma (MM), aberrant auto-and/or paracrine activation of canonical Wnt signaling promotes proliferation and dissemination, while overexpression of the Wnt inhibitor Dickkopf1 (DKK1) by MM cells contributes to osteolytic bone disease by inhibiting osteoblast differentiation. Since DKK1 itself is a target of TCF/β-catenin mediated transcription, these findings suggest that DKK1 is part of a negative feedback loop in MM and may act as a tumor suppressor. In line with this hypothesis, we show here that DKK1 expression is low or undetectable in a subset of patients with advanced MM as well as in MM cell lines. This absence of DKK1 is correlated with enhanced Wnt pathway activation, evidenced by nuclear accumulation of β-catenin, which in turn can be antagonized by restoring DKK1 expression. Analysis of the DKK1 promoter revealed CpG island methylation in several MM cell lines as well as in MM cells from patients with advanced MM. Moreover, demethylation of the DKK1 promoter restores DKK1 expression, which results in inhibition of β-catenin/TCF-mediated gene transcription in MM lines. Taken together, our data identify aberrant methylation of the DKK1 promoter as a cause of DKK1 silencing in advanced stage MM, which may play an important role in the progression of MM by unleashing Wnt signaling

Attention-dependent modulation of cortical taste circuits revealed by granger causality with signal-dependent noise

We show, for the first time, that in cortical areas, for example the insular, orbitofrontal, and lateral prefrontal cortex, there is signal-dependent noise in the fMRI blood-oxygen level dependent (BOLD) time series, with the variance of the noise increasing approximately linearly with the square of the signal. Classical Granger causal models are based on autoregressive models with time invariant covariance structure, and thus do not take this signal-dependent noise into account. To address this limitation, here we describe a Granger causal model with signal-dependent noise, and a novel, likelihood ratio test for causal inferences. We apply this approach to the data from an fMRI study to investigate the source of the top-down attentional control of taste intensity and taste pleasantness processing. The Granger causality with signal-dependent noise analysis reveals effects not identified by classical Granger causal analysis. In particular, there is a top-down effect from the posterior lateral prefrontal cortex to the insular taste cortex during attention to intensity but not to pleasantness, and there is a top-down effect from the anterior and posterior lateral prefrontal cortex to the orbitofrontal cortex during attention to pleasantness but not to intensity. In addition, there is stronger forward effective connectivity from the insular taste cortex to the orbitofrontal cortex during attention to pleasantness than during attention to intensity. These findings indicate the importance of explicitly modeling signal-dependent noise in functional neuroimaging, and reveal some of the processes involved in a biased activation theory of selective attention

The Role of the BMP Signaling Antagonist Noggin in the Development of Prostate Cancer Osteolytic Bone Metastasis

Members of the BMP and Wnt protein families play a relevant role in physiologic and pathologic bone turnover. Extracellular antagonists are crucial for the modulation of their activity. Lack of expression of the BMP antagonist noggin by osteoinductive, carcinoma-derived cell lines is a determinant of the osteoblast response induced by their bone metastases. In contrast, osteolytic, carcinoma-derived cell lines express noggin constitutively. We hypothesized that cancer cell-derived noggin may contribute to the pathogenesis of osteolytic bone metastasis of solid cancers by repressing bone formation. Intra-osseous xenografts of PC-3 prostate cancer cells induced osteolytic lesions characterized not only by enhanced osteoclast-mediated bone resorption, but also by decreased osteoblast-mediated bone formation. Therefore, in this model, uncoupling of the bone remodeling process contributes to osteolysis. Bone formation was preserved in the osteolytic lesions induced by noggin-silenced PC-3 cells, suggesting that cancer cell-derived noggin interferes with physiologic bone coupling. Furthermore, intra-osseous tumor growth of noggin-silenced PC-3 cells was limited, most probably as a result of the persisting osteoblast activity. This investigation provides new evidence for a model of osteolytic bone metastasis where constitutive secretion of noggin by cancer cells mediates inhibition of bone formation, thereby preventing repair of osteolytic lesions generated by an excess of osteoclast-mediated bone resorption. Therefore, noggin suppression may be a novel strategy for the treatment of osteolytic bone metastases

Expression of a protein involved in bone resorption, Dkk1, is activated by HTLV-1 bZIP factor through its activation domain

<p>Abstract</p> <p>Background</p> <p>Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia, a malignancy characterized by uncontrolled proliferation of virally-infected CD4+ T-cells. Hypercalcemia and bone lesions due to osteoclast-mediated bone resorption are frequently associated with more aggressive forms of the disease. The HTLV-1 provirus contains a unique antisense gene that expresses HTLV-1 basic leucine zipper (bZIP) factor (HBZ). HBZ is localized to the nucleus where it regulates levels of transcription by binding to certain cellular transcriptional regulators. Among its protein targets, HBZ forms a stable complex with the homologous cellular coactivators, p300 and CBP, which is modulated through two N-terminal LXXLL motifs in the viral protein and the conserved KIX domain in the coactivators.</p> <p>Results</p> <p>To determine the effects of these interactions on transcription, we performed a preliminary microarray analysis, comparing levels of gene expression in cells with wild-type HBZ versus cells with HBZ mutated in its LXXLL motifs. <it>DKK1</it>, which encodes the secreted Wnt signaling inhibitor, Dickkopf-1 (Dkk1), was confirmed to be transcriptionally activated by HBZ, but not its mutant. Dkk1 plays a major role in the development of bone lesions caused by multiple myeloma. In parallel with the initial findings, activation of Dkk1 expression by HBZ was abrogated by siRNA-mediated knockdown of p300/CBP or by a truncated form of p300 containing the KIX domain. Among HTLV-1-infected T-cell lines tested, the detection of Dkk1 mRNA partially correlated with a threshold level of HBZ mRNA. In addition, an uninfected and an HTLV-1-infected T-cell line transfected with an HBZ expression vector exhibited <it>de novo </it>and increased DKK1 transcription, respectively. In contrast to HBZ, The HTLV-1 Tax protein repressed Dkk1 expression.</p> <p>Conclusions</p> <p>These data indicate that HBZ activates Dkk1 expression through its interaction with p300/CBP. However, this effect is limited in HTLV-1-infected T-cell lines, which in part, may be due to suppression of Dkk1 expression by Tax. Consequently, the ability of HBZ to regulate expression of Dkk1 and possibly other cellular genes may only be significant during late stages of ATL, when Tax expression is repressed.</p

Citrullination of histone H3 drives IL-6 production by bone marrow mesenchymal stem cells in MGUS and multiple myeloma

Multiple myeloma (MM), an incurable plasma cell malignancy, requires localisation within the bone marrow. This microenvironment facilitates crucial interactions between the cancer cells and stromal cell types that permit the tumour to survival and proliferate. There is increasing evidence that the bone marrow mesenchymal stem cell (BMMSC) is stably altered in patients with MM – a phenotype also postulated to exist in patients with monoclonal gammopathy of undetermined significance (MGUS) a benign condition that precedes MM. In this study, we describe a mechanism by which increased expression of peptidyl arginine deiminase 2 (PADI2) by BMMSCs in patients with MGUS and MM directly alters malignant plasma cell phenotype. We identify PADI2 as one of the most highly upregulated transcripts in BMMSCs from both MGUS and MM patients, and that through its enzymatic deimination of histone H3 arginine 26, PADI2 activity directly induces the upregulation of interleukin-6 (IL-6) expression. This leads to the acquisition of resistance to the chemotherapeutic agent, bortezomib, by malignant plasma cells. We therefore describe a novel mechanism by which BMMSC dysfunction in patients with MGUS and MM directly leads to pro-malignancy signalling through the citrullination of histone H3R26

Growth factors in multiple myeloma: a comprehensive analysis of their expression in tumor cells and bone marrow environment using Affymetrix microarrays

<p>Abstract</p> <p>Background</p> <p>Multiple myeloma (MM) is characterized by a strong dependence of the tumor cells on their microenvironment, which produces growth factors supporting survival and proliferation of myeloma cells (MMC). In the past few years, many myeloma growth factors (MGF) have been described in the literature. However, their relative importance and the nature of the cells producing MGF remain unidentified for many of them.</p> <p>Methods</p> <p>We have analysed the expression of 51 MGF and 36 MGF receptors (MGFR) using Affymetrix microarrays throughout normal plasma cell differentiation, in MMC and in cells from the bone marrow (BM) microenvironment (CD14, CD3, polymorphonuclear neutrophils, stromal cells and osteoclasts).</p> <p>Results</p> <p>4/51 MGF and 9/36 MGF-receptors genes were significantly overexpressed in plasmablasts (PPC) and BM plasma cell (BMPC) compared to B cells whereas 11 MGF and 11 MGFR genes were overexpressed in BMPC compared to PPC. 3 MGF genes (AREG, NRG3, Wnt5A) and none of the receptors were significantly overexpressed in MMC versus BMPC. Furthermore, 3/51 MGF genes were overexpressed in MMC compared to the the BM microenvironment whereas 22/51 MGF genes were overexpressed in one environment subpopulation compared to MMC.</p> <p>Conclusions</p> <p>Two major messages arise from this analysis 1) The majority of MGF genes is expressed by the bone marrow environment. 2) Several MGF and their receptors are overexpressed throughout normal plasma cell differentiation. This study provides an extensive and comparative analysis of MGF expression in plasma cell differentiation and in MM and gives new insights in the understanding of intercellular communication signals in MM.</p

Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

BACKGROUND: Glycogen Synthase Kinase-3 (GSK-3) \u3b1 and \u3b2 are two serine-threonine kinases controlling insulin, Wnt/\u3b2-catenin, NF-\u3baB signaling and other cancer-associated transduction pathways. Recent evidence suggests that GSK-3 could function as growth-promoting kinases, especially in malignant cells. In this study, we have investigated GSK-3\u3b1 and GSK-3\u3b2 function in multiple myeloma (MM). METHODS: GSK-3 \u3b1 and \u3b2 expression and cellular localization were investigated by Western blot (WB) and immunofluorescence analysis in a panel of MM cell lines and in freshly isolated plasma cells from patients. MM cell growth, viability and sensitivity to bortezomib was assessed upon treatment with GSK-3 specific inhibitors or transfection with siRNAs against GSK-3 \u3b1 and \u3b2 isoforms. Survival signaling pathways were studied with WB analysis. RESULTS: GSK-3\u3b1 and GSK-3\u3b2 were differently expressed and phosphorylated in MM cells. Inhibition of GSK-3 with the ATP-competitive, small chemical compounds SB216763 and SB415286 caused MM cell growth arrest and apoptosis through the activation of the intrinsic pathway. Importantly, the two inhibitors augmented the bortezomib-induced MM cell cytotoxicity. RNA interference experiments showed that the two GSK-3 isoforms have distinct roles: GSK-3\u3b2 knock down decreased MM cell viability, while GSK-3\u3b1 knock down was associated with a higher rate of bortezomib-induced cytotoxicity. GSK-3 inhibition caused accumulation of \u3b2-catenin and nuclear phospho-ERK1, 2. Moreover, GSK-3 inhibition and GSK-3\u3b1 knockdown enhanced bortezomib-induced AKT and MCL-1 protein degradation. Interestingly, bortezomib caused a reduction of GSK-3 serine phosphorylation and its nuclear accumulation with a mechanism that resulted partly dependent on GSK-3 itself. CONCLUSIONS: These data suggest that in MM cells GSK-3\u3b1 and \u3b2 i) play distinct roles in cell survival and ii) modulate the sensitivity to proteasome inhibitors

Human Umbilical Cord Blood-Derived CD34+ Cells Reverse Osteoporosis in NOD/SCID Mice by Altering Osteoblastic and Osteoclastic Activities

Osteoporosis is a bone disorder associated with loss of bone mineral density and micro architecture. A balance of osteoblasts and osteoclasts activities maintains bone homeostasis. Increased bone loss due to increased osteoclast and decreased osteoblast activities is considered as an underlying cause of osteoporosis.The cures for osteoporosis are limited, consequently the potential of CD34+ cell therapies is currently being considered. We developed a nanofiber-based expansion technology to obtain adequate numbers of CD34(+) cells isolated from human umbilical cord blood, for therapeutic applications. Herein, we show that CD34(+) cells could be differentiated into osteoblastic lineage, in vitro. Systemically delivered CD34(+) cells home to the bone marrow and significantly improve bone deposition, bone mineral density and bone micro-architecture in osteoporotic mice. The elevated levels of osteocalcin, IL-10, GM-CSF, and decreased levels of MCP-1 in serum parallel the improvements in bone micro-architecture. Furthermore, CD34(+) cells improved osteoblast activity and concurrently impaired osteoclast differentiation, maturation and functionality.These findings demonstrate a novel approach utilizing nanofiber-expanded CD34(+) cells as a therapeutic application for the treatment of osteoporosis

Integration of the β-Catenin-Dependent Wnt Pathway with Integrin Signaling through the Adaptor Molecule Grb2

THE COMPLEXITY OF WNT SIGNALING LIKELY STEMS FROM TWO SOURCES: multiple pathways emanating from frizzled receptors in response to wnt binding, and modulation of those pathways and target gene responsiveness by context-dependent signals downstream of growth factor and matrix receptors. Both rac1 and c-jun have recently been implicated in wnt signaling, however their upstream activators have not been identified.Here we identify the adapter protein Grb2, which is itself an integrator of multiple signaling pathways, as a modifier of beta-catenin-dependent wnt signaling. Grb2 synergizes with wnt3A, constitutively active (CA) LRP6, Dvl2 or CA-beta-catenin to drive a LEF/TCF-responsive reporter, and dominant negative (DN) Grb2 or siRNA to Grb2 block wnt3A-mediated reporter activity. MMP9 is a target of beta-catenin-dependent wnt signaling, and an MMP9 promoter reporter is also responsive to signals downstream of Grb2. Both a jnk inhibitor and DN-c-jun block transcriptional activation downstream of Dvl2 and Grb2, as does DN-rac1. Integrin ligation by collagen also synergizes with wnt signaling as does overexpression of Focal Adhesion Kinase (FAK), and this is blocked by DN-Grb2.These data suggest that integrin ligation and FAK activation synergize with wnt signaling through a Grb2-rac-jnk-c-jun pathway, providing a context-dependent mechanism for modulation of wnt signaling