Heregulin selectively upregulates vascular endothelial growth factor secretion in cancer cells and stimulates angiogenesis

The interaction between the erbB tyrosine kinase receptors and their ligands plays an important role in tumor growth via the regulation of autocrine and paracrine loops. We report the effect of heregulin beta1, the ligand for erbB-3 and erbB-4 receptors, on the regulation of vascular endothelial growth factor (VEGF) expression, using a panel of breast and lung cancer cell lines with constitutive erbB-2 overexpression or engineered to stably overexpress the erbB-2 receptor. We demonstrate that heregulin beta1 induces VEGF secretion in most cancer cell lines, while no significant effect was observed in normal human mammary and bronchial primary cells. Overexpression of erbB-2 receptor results in induction of the basal level of VEGF and exposure to heregulin further enhances VEGF secretion. This is associated with increased VEGF mRNA expression. In contrast, VEGF induction is significantly decreased in a T47D cell line where erbB-2 is functionally inactivated. Conditioned media from heregulin-treated cancer cells, but not from normal cells, stimulates endothelial cell proliferation; this paracrine stimulation is inhibited by co-exposure to a specific VEGF neutralizing antibody. Furthermore, heregulin-mediated angiogenesis is observed in the in vivo CAM assay. This study reports the first evidence of VEGF regulation by heregulin in cancer cells. Oncogene (2000) 19, 3460 - 3469     

In vitro growth of hematopoietic progenitors and stromal bone marrow cells from patients with multiple myeloma

In the present study we have determined the content of hematopoietic and stromal progenitors in multiple myeloma (MM) bone marrow, and assessed their in vitro growth. Marrow cells were obtained from 17 MM patients at the time of diagnosis, and from 6 hematologically normal subjects. When mononuclear cells (MNC) from MM marrow were cultured, reduced numbers of hematopoietic progenitors were detected and their growth in long-term cultures was deficient, as compared to cultures of normal cells. When cell fractions enriched for CD34⁺ Lin⁻ cells were obtained, the levels of hematopoietic progenitors from MM marrow were within the normal range, and so was their growth kinetics in liquid suspension cultures. The levels of fibroblast progenitors in MM were not statistically different from those in normal marrow; however, their proliferation potential was significantly reduced. Conditioned media from MM-derived MNC and stroma cells contained factors that inhibited normal progenitor cell growth. Our observations suggest that hematopoietic progenitors in MM marrow are intrinsically normal; however, their growth in LTMC may be hampered by the presence of abnormal accessory and stroma cells. These results suggest that besides its role in the generation of osteolytic lesions and the expansion of the myeloma clone, the marrow microenvironment in MM may have a negative effect on hematopoiesis.     

The vascular endothelial growth factor receptor tyrosine kinase inhibitor PTK787/ZK222584 inhibits growth and migration of multiple myeloma cells in the bone marrow microenvironment

Our prior studies show that multiple myeloma (MM) cell lines and patient cells express high-affinity vascular endothelial growth factor (VEGF) receptor (VEGFR) Flt-1 but not Flk-1/KDR. Moreover, these studies have shown that VEGF induces proliferation and migration of MM cells, and we have begun to delineate the signaling cascades mediating those sequelae. In this study, we examined the activity of PTK787/ZK 222584 (PTK787), a molecule designed to bind specifically to the tyrosine kinase domain of VEGFR and inhibit angiogenesis. We show that PTK787 acts both directly on MM cells and in the bone marrow microenvironment. Specifically, PTK787 (1-5 micro M) inhibits proliferation of MM cells by 50%, as assayed by [(3)H]thymidine uptake. This effect of PTK787 is dose dependent in both MM cell lines and patient cells that are both sensitive and resistant to conventional therapy. PTK787 enhances the inhibitory effect of dexamethasone on growth of MM cells and can overcome the protective effect of interleukin 6 (IL-6) against dexamethasone-induced apoptosis. PTK787 (1 micro M) also blocks VEGF-induced migration of MM cells across an extracellular matrix. Importantly, PTK787 also inhibits the increased MM cell proliferation and increased IL-6 and VEGF secretion in cultures of MM cells adherent to bone marrow stem cells. These findings therefore demonstrate that PTK787 both acts directly on MM cells and inhibits paracrine IL-6-mediated MM cell growth in the bone marrow milieu. The demonstrated anti-MM activity of PTK787, coupled with its antiangiogenic effects, provides the framework for clinical trials of this agent to overcome drug resistance and improve outcome in MM.     

The bone marrow stromal microenvironment influences myeloma therapeutic response in vitro.

The bone marrow microenvironment supports growth and differentiation of normal hematopoietic cells and can contribute to malignant growth. Since myeloma cells localize and accumulate in bone marrow, it is important to understand the influence of the bone marrow microenvironment not only on the growth of the malignant cells, but also on the therapeutic response of myeloma cells. Growth factors such as interleukin-6 (IL-6) produced by bone marrow stromal cells can protect myeloma cells from glucocorticoid-induced apoptosis. We examined the effect of myeloma cells-bone marrow stromal cells interaction in vitro on several therapeutic treatments. An interleukin-6-dependent myeloma cell line ANBL6 was used and treated with dexamethasone, doxorubicin, and melphalan in the presence of bone marrow stromal cells. Stromal cells were able to protect ANBL6 from dexamethasone, but significantly enhanced the effect of doxorubicin and melphalan. IL-6-induced bcl-XL and cyclin D2 expression in ANBL6 cells, but dexamethasone was able to suppress both bcl-XL and cyclin D2 expression in ANBL6. Doxorubicin and melphalan were able to suppress bcl-XL expression only in the presence of IL-6. We also looked at the effect of activating mutations of N-ras in myeloma cells interacting with stromal cells on therapeutic responses. Surprisingly, ANBL6 N-ras shows significant resistance to all drugs used. Notably, the presence of stromal cells did not alter ANBL6 Nras cells' drug resistance. These results suggest both the bone marrow microenvironment and genetic alterations of myeloma cells can independently impact on therapeutic responses.     

血管内皮生长因子与多发性骨髓瘤

多发性骨髓瘤(MM)是目前仍缺乏根治手段的浆细胞肿瘤,与骨髓微环境异常密切相关.研究表明,血管内皮生长因子(VEGF)是肿瘤微环境中血管形成最重要的生长因子,直接参与MM的发病和进展.目前正在MM患者中开展VEGF相关途径的靶向治疗.     

Targeting the vascular endothelial growth factor pathway in the treatment of multiple myeloma

Multiple myeloma is a clonal plasma cell malignancy within the bone marrow associated with bone loss, renal disease and immunodeficiency. Despite new insights into the pathogenesis of multiple myeloma and novel targeted therapies, the median survival remains 3-5 years. It is now well established that the intimate relation between the tumor cells and components of the microenvironment plays a key role in multiple myeloma pathogenesis. Specifically, tumor cells impact the bone marrow and thereby cause immune suppression and lytic bone lesions; conversely, components of the bone marrow provide signals that influence the behavior of multiple myeloma cells, including tumor cell growth, survival, migration and drug resistance. Important contributing effectors are tumor cell-stroma cell and cell-extracellular matrix contacts, the bone marrow vasculature, and a variety of cytokines and growth factors in the bone marrow milieu.     

Targeting the vascular endothelial growth factor pathway in the treatment of multiple myeloma

Multiple myeloma is a clonal plasma cell malignancy within the bone marrow associated with bone loss, renal disease and immunodeficiency. Despite new insights into the pathogenesis of multiple myeloma and novel targeted therapies, the median survival remains 3–5 years. It is now well established that the intimate relation between the tumor cells and components of the microenvironment plays a key role in multiple myeloma pathogenesis. Specifically, tumor cells impact the bone marrow and thereby cause immune suppression and lytic bone lesions; conversely, components of the bone marrow provide signals that influence the behavior of multiple myeloma cells, including tumor cell growth, survival, migration and drug resistance. Important contributing effectors are tumor cell–stroma cell and cell–extracellular matrix contacts, the bone marrow vasculature, and a variety of cytokines and growth factors in the bone marrow milieu.     

Thalidomide downregulates angiogenic genes in bone marrow endothelial cells of patients with active multiple myeloma.

PURPOSE: To study the antiangiogenic effect of thalidomide. PATIENTS AND METHODS: The expression of key angiogenic genes was studied in bone marrow endothelial cells (ECs) of patients with active and nonactive multiple myeloma (MM), monoclonal gammopathies unattributed/unassociated (MG[u]), diffuse large B-cell non-Hodgkin's lymphoma, in a Kaposi's sarcoma (KS) cell line, and in healthy human umbilical vein ECs (HUVECs) following exposure to therapeutic doses of thalidomide. RESULTS: Thalidomide markedly downregulates the genes in a dose-dependent fashion in active MMECs and KS cell line, but upregulates them or is ineffective in nonactive MMECs, MG(u)ECs, NHL-ECs, and in HUVECs. Secretion of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor also diminishes according to the dose in culture conditioned media (CM) of active MMECs and KS, whereas it does not change in the other CM. CONCLUSION: Inhibition by thalidomide is probably confined to the genes of active MMECs and KS. This would account for its higher efficacy in these diseases.     

Role of B-cell-activating factor in adhesion and growth of human multiple myeloma cells in the bone marrow microenvironment

Recent studies have underscored the role of B-cell-activating factor (BAFF), a member of the tumor necrosis factor superfamily, in promoting the survival of malignant B cells, including human multiple myeloma. We here characterized the functional significance of BAFF in the interaction between multiple myeloma and bone marrow stromal cells (BMSC) and further defined the molecular mechanisms regulating these processes. BAFF is detected on BMSCs derived from multiple myeloma patients as evidenced by flow cytometry. BAFF secretion is 3- to 10-fold higher in BMSCs than in multiple myeloma cells, and tumor cell adhesion to BMSCs augments BAFF secretion by 2- to 5-fold, confirmed by both ELISA and immunoblotting. Adhesion of MM1S and MCCAR multiple myeloma cell lines to KM104 BMSC line transfected with a luciferase reporter vector carrying the BAFF gene promoter (BAFF-LUC) significantly enhanced luciferase activity, suggesting that nuclear factor-kappaB (NF-kappaB) activation induced by multiple myeloma adhesion to BMSCs mediates BAFF up-regulation. Moreover, BAFF (0-100 ng/mL) increases adhesion of multiple myeloma lines to BMSCs in a dose-dependent manner; conversely, transmembrane activator and calcium modulator and cyclophylin ligand interactor-Ig or B-cell maturation antigen/Fc blocked BAFF stimulation. Using adenoviruses expressing dominant-negative and constitutively expressed AKT as well as NF-kappaB inhibitors, we further showed that BAFF-induced multiple myeloma cell adhesion is primarily mediated via activation of AKT and NF-kappaB signaling. Importantly, BAFF similarly increased adhesion of CD138-expressing patient multiple myeloma cells to BMSCs. These studies establish a role for BAFF in localization and survival of multiple myeloma cells in the bone marrow microenvironment and strongly support novel therapeutics, targeting the interaction between BAFF and its receptors in human multiple myeloma.     

Vascular endothelial growth factor and its receptors in multiple myeloma.

Multiple myeloma (MM) progresses from an avascular to a vascular phase (active MM) accompanied by a significant increase in microvessel density in the bone marrow. This article summarizes the literature concerning the specific role played by vascular endothelial growth factor (VEGF) in this process. Recent applications of antiangiogenic agents that interfere with VEGF signaling and block MM progression are also described.     

Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets

Multiple myeloma is a plasma cell malignancy characterized by complex heterogeneous cytogenetic abnormalities. The bone marrow microenvironment promotes multiple myeloma cell growth and resistance to conventional therapies. Although multiple myeloma remains incurable, novel targeted agents, used alone or in combination, have shown great promise to overcome conventional drug resistance and improve patient outcome. Recent oncogenomic studies have further advanced our understanding of the molecular pathogenesis of multiple myeloma, providing the framework for new prognostic classification and identifying new therapeutic targets.     

Expression of vascular endothelial growth factor and its receptors in multiple myeloma and other hematopoietic malignancies.

Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of hematopoietic stem cell development, extracellular matrix remodeling, and inflammatory cytokine generation. The importance of angiogenic factors such as VEGF, while clearly established in solid tumors, has not been fully elucidated in human hematopoietic neoplasms. Human hematopoietic tumor cell lines, representing multiple lineages and diseases, produce and secrete VEGF and express at least one of its two receptors. Exposure of human vascular endothelial cells to VEGF increased the expression of several hematopoietic growth factors known to be involved in myeloma including interleukin-6 (IL-6). Bone marrow samples from patients diagnosed with multiple myeloma were examined for expression of VEGF and its receptors. VEGF protein production was detected in malignant plasma cells from 78% of the myeloma patients studied. While expression of the Flt-1 and KDR receptors was not observed in the malignant plasma cells, both were markedly elevated in the normal marrow myeloid and monocytic cells surrounding the tumor. In bone marrow clot sections from normal allogeneic donors, low-intensity cytoplasmic VEGF expression was detected infrequently in isolated myelocytes, macrophages, and megakaryocytes. In vitro colony-forming assays using patient-derived material revealed that antibody neutralization of VEGF resulted in an inhibition of colony growth, whereas the addition of recombinant human VEGF stimulated colony formation. Neutralization of VEGF activity also suppressed the generation of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) from bone marrow mononuclear cells. These data raise the possibility that VEGF may play a role in the growth of hematopoietic neoplasms such as multiple myeloma through paracrine and perhaps autocrine mechanisms.     

VEGF及其受体在多发性骨髓瘤中的表达及意义

背景与目的:骨髓新生血管形成在多发性骨髓瘤(multiple myeloma,MM)的发生、发展和预后中起着重要的作用,血管内皮生长因子(vascular endothelialgrowth factor,VEGF)在此过程中扮演了关键角色.本研究旨在研究VEGF及其受体在MM中的表达,分析其与MM发生、发展的关系.方法:采用RT-PCR的方法检测35例MM患者、16例非肿瘤患者(下称对照组)以及KM3细胞株中VEGF及其受体Flt-1和KDR的表达,并分析阳性率以及表达相对含量在MM患者和非肿瘤患者、MM不同分期间的差异.结果:VEGF和Flt-1基因在MM组的阳性率(62.9%和80.0%)显著高于对照组(18.8%和31.3%)(P＜0.01),VEGF在两组中的表达水平为0.41±0.19 vs.0.06±0.01(P＜0.05),Flt-1为0.60±0.33 vs.0.08±0.03(P＜0.01);VEGF基因在初治组和复发/难治MM组的阳性率为66.7%vs.60.9%(P＞0.05),Flt-1基因为83.3%vs.78.3%(P＞0.05);VEGF在Ⅱ期和Ⅲ期MM的阳性率为50%vs.73.7%(P＞0.05),Flt-1为81.3%vs.78.9%(P＞0.05);复发/难治MM组的VEGF和Flt-1基因的表达水平明显高于初治组(0.49±0.20 vs.0.28±0.04,P＜0.05;0.70±0.38 vs.0.41±0.06,P＜0.05),Ⅲ期MM患者VEGF和Flt-1基因的表达水平明显高于Ⅱ期患者(0.48±0.19 vs.0.28±0.09,P＜0.05;0.75±0.35 vs.0.41±0.21,P＜0.05).KDR仅在3例MM患者中检出,对照组未检出.结论:VEGF和Flt-1在MM中高表达,并与疾病进展相关.     

Allogeneic bone marrow transplantation in multiple myeloma.

The use of high-dose chemoradiotherapy with allogeneic hemopoietic stem cell support for the treatment of MM began about a decade ago. Because this procedure has been performed increasingly and because larger numbers of patients are being followed for longer periods of time, the proper role of allogeneic BMT in this setting is becoming clearer. Data available thus far indicate that such an approach results in a complete remission rate of at least 50% to 60%, and even higher if applied as consolidation treatment in the remission phase, a transplant-related mortality reported as 40% to 50% and a long-term survival plateau at around 40%. The 40% 5-year probability of relapse-free survival is considerably higher than that observed following autologous BMT and may result from an allogeneic graft-versus-tumor effect (graft versus myeloma) similar to the well-recognized graft-versus-leukemia effect. Although follow-up is still too short to clearly identify the likelihood of cure for MM allotransplant recipients, a certain number of them are currently long-term, disease-free survivors and--we hope--cured. These promising results and the incurability of MM with conventional chemotherapy should, therefore, encourage further application of allogeneic BMT to selected patients with unfavorable prognostic features. Continued efforts to reduce the morbidity and mortality related to the procedure, as well as to design effective pretransplant regimens with lower extramedullary toxicity and to identify those patients most likely to benefit from BMT, will improve the value of allogeneic BMT in MM.     

Epidermal growth factor receptor-transfected bone marrow stromal cells exhibit enhanced migratory response and therapeutic potential against murine brain tumors

We have created a novel cellular vehicle for gene therapy of malignant gliomas by transfection of murine bone marrow stroma cells (MSCs) with a cDNA encoding epidermal growth factor receptor (EGFR). These cells (EGFR-MSCs) demonstrate enhanced migratory responses toward glioma-conditioned media in comparison to primary MSCs in vitro. Enhanced migration of EGFR-MSC was at least partially dependent on EGF-EGFR, PI3-, MAP kinase kinase, and MAP kinases, protein kinase C, and actin polymerization. Unlike primary MSCs, EGFR-MSCs were resistant to FasL-mediated cytotoxicity and were capable of stimulating allogeneic mixed lymphocyte reaction, suggesting EGFR-MSCs possess suitable characteristics as vehicles for brain tumor immuno-gene therapy. Following injection at various sites, including the contralateral hemisphere in the brain of syngeneic mice, EGFR-MSCs were able to migrate toward GL261 gliomas or B16 melanoma in vivo. Finally, intratumoral injection with EGFR-MSC adenovirally engineered to secrete interferon-alpha to intracranial GL261 resulted in significantly prolonged survival in comparison to controls. These data indicate that EGFR-MSCs may serve as attractive vehicles for infiltrating brain malignancies such as malignant gliomas.     

Autologous bone marrow transplantation for multiple myeloma.

Intensive cytotoxic chemotherapy is capable of more marked tumor cytoreduction including true complete remissions in multiple myeloma. Use of autologous marrow and blood stem cells as well as growth factors has reduced the risk of infection and severe extramedullary toxicities.