Ivana Catacchio

Bone marrow fibroblasts parallel multiple myeloma progression in patients and mice: in vitro and in vivo studies

The role of cancer-associated fibroblasts (CAFs) has not been previously studied in multiple myeloma (MM). Here, cytofluorimetric analysis revealed higher proportions of bone marrow (BM) CAFs in patients with active MM (both at diagnosis and relapse) compared with patients in remission or those with monoclonal gammopathy of undetermined significance or deficiency anemia (controls). CAFs from MM patients produced increased levels of transforming growth factor-β, interleukin-6, stromal cell-derived factor-1α, insulin-like growth factor-1, vascular endothelial growth factor and fibroblast growth factor-2 and displayed an activated and heterogeneous phenotype, which supported their origin from resident fibroblasts, endothelial cells and hematopoietic stem and progenitor cells via the endothelial–mesenchymal transition as well as mesenchymal stem cells via the mesenchymal transition, as both of these processes are induced by MM cells and CAFs. Active MM CAFs fostered chemotaxis, adhesion, proliferation and apoptosis resistance in MM cells through cytokine signals and cell-to-cell contact, which were inhibited by blocking CXCR4, several integrins and fibronectin. MM cells also induced the CAFs proliferation. In syngeneic 5T33MM and xenograft mouse models, MM cells induced the expansion of CAFs, which, in turn, promoted MM initiation and progression as well as angiogenesis. In BM biopsies from patients and mice, nests of CAFs were found in close contact with MM cells, suggesting a supportive niche. Therefore, the targeting of CAFs in MM patients may be envisaged as a novel therapeutic strategy.

Evidence for Bone Marrow Adult Stem Cell Plasticity: Properties, Molecular Mechanisms, Negative Aspects, and Clinical Applications of Hematopoietic and Mesenchymal Stem Cells Transdifferentiation

In contrast to the pluripotent embryonic stem cells (ESCs) which are able to give rise to all cell types of the body, mammalian adult stem cells (ASCs) appeared to be more limited in their differentiation potential and to be committed to their tissue of origin. Recently, surprising new findings have contradicted central dogmas of commitment of ASCs by showing their plasticity to differentiate across tissue lineage boundaries, irrespective of classical germ layer designations. The present paper supports the plasticity of the bone marrow stem cells (BMSCs), bringing the most striking and the latest evidences of the transdifferentiation properties of the bone marrow hematopoietic and mesenchymal stem cells (BMHSCs, and BMMSCs), the two BM populations of ASCs better characterized. In addition, we report the possible mechanisms that may explain these events, outlining the clinical importance of these phenomena and the relative problems.

Four proteins governing overangiogenic endothelial cell phenotype in patients with multiple myeloma are plausible therapeutic targets

Bone marrow (BM) angiogenesis has an important role in the initiation and progression of multiple myeloma (MM). We looked at novel mechanisms of vessel formation in patients with MM through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with active MM (MMECs) and ECs of patients with monoclonal gammopathy of undetermined significance (MGECs) and of subjects with benign anemia (normal ECs). Four proteins were found overexpressed in MMECs: filamin A, vimentin, α-crystallin B and 14-3-3ζ/δ protein, not yet linked to overangiogenic phenotype. These proteins gave a typical distribution in the BM of MM patients and in MMECs versus MGECs, plausibly according to a different functional state. Their expression was enhanced by vascular endothelial growth factor, fibroblast growth factor 2, hepatocyte growth factor and MM plasma cell conditioned medium in step with enhancement of MMEC angiogenesis. Their silencing RNA knockdown affected critical MMEC angiogenesis-related functions, such as spreading, migration and tubular morphogenesis. A gradual stabilization of 14-3-3ζ/δ protein was observed, with transition from normal ECs to MGECs and MMECs that may be a critical step for the angiogenic switch in MMECs and maintenance of the cell overangiogenic phenotype. These proteins were substantially impacted by anti-MM drugs, such as bortezomib, lenalidomide and panobinostat. Results suggest that these four proteins could be new targets for the antiangiogenic management of MM patients.

HIF-1α of bone marrow endothelial cells implies relapse and drug resistance in patients with multiple myeloma and may act as a therapeutic target

Purpose: To investigate the role of hypoxia-inducible factor-1α (HIF-1α) in angiogenesis and drug resistance of bone marrow endothelial cells of patients with multiple myeloma. Experimental Design: HIF-1α mRNA and protein were evaluated in patients with multiple myeloma endothelial cells (MMEC) at diagnosis, at relapse after bortezomib- or lenalidomide-based therapies or on refractory phase to these drugs, at remission; in endothelial cells of patients with monoclonal gammapathies of undetermined significance (MGUS; MGECs), and of those with benign anemia (controls). The effects of HIF-1α inhibition by siRNA or panobinostat (an indirect HIF-1α inhibitor) on the expression of HIF-1α proangiogenic targets, on MMEC angiogenic activities in vitro and in vivo, and on overcoming MMEC resistance to bortezomib and lenalidomide were studied. The overall survival of the patients was also observed. Results: Compared with the other endothelial cell types, only MMECs from 45% of relapsed/refractory patients showed a normoxic HIF-1α protein stabilization and activation that were induced by reactive oxygen species (ROS). The HIF-1α protein correlated with the expression of its proangiogenic targets. The HIF-1α inhibition by either siRNA or panobinostat impaired the MMECs angiogenesis–related functions both in vitro and in vivo and restored MMEC sensitivity to bortezomib and lenalidomide. Patients with MMECs expressing the HIF-1α protein had shorter overall survival. Conclusions: The HIF-1α protein in MMECs may induce angiogenesis and resistance to bortezomib and lenalidomide and may be a plausible target for the antiangiogenic management of patients with well-defined relapsed/refractory multiple myeloma. It may also have prognostic significance. Clin Cancer Res; 20(4); 847–58. ©2013 AACR.

Multiple Myeloma Macrophages: Pivotal Players in the Tumor Microenvironment

Tumor microenvironment is essential for multiple myeloma (MM) growth, progression, and drug resistance through provision of survival signals and secretion of growth and proangiogenic factors. This paper examines the importance of macrophages within MM bone marrow (BM) microenvironment, referred to as MM-associated macrophages, as a potential niche component that supports tumor plasma cells. These macrophages are derived from peripheral blood monocytes recruited into the tumor. Upon activation by MM plasma cells and mesenchymal stromal cells, macrophages can release growth factors, proteolytic enzymes, cytokines, and inflammatory mediators that promote plasma cell growth and survival. Macrophages promote tumor progression through several mechanisms including angiogenesis, growth, and drug resistance. Indeed, these macrophages are essential for the induction of an angiogenic response through vasculogenic mimicry, and this ability proceeds in step with progression of the plasma cell tumors. Data suggest that macrophages play an important role in the biology and survival of patients with MM, and they may be a target for the MM antivascular management.

Halting pro-survival autophagy by TGFβ inhibition in bone marrow fibroblasts overcomes bortezomib resistance in multiple myeloma patients

Bortezomib (bort) has improved overall survival in patients with multiple myeloma (MM), but the majority of them develop drug resistance. In this study, we demonstrate that bone marrow (BM) fibroblasts (cancer-associated fibroblasts; CAFs) from bort-resistant patients are insensitive to bort and protect the RPMI8226 and patients’ plasma cells against bort-induced apoptosis. Bort triggers CAFs to produce high levels of interleukin (IL)-6, IL-8, insulin-like growth factor (IGF)-1 and transforming growth factor (TGF) β. Proteomic studies on CAFs demonstrate that bort resistance parallels activation of oxidative stress and pro-survival autophagy. Indeed, bort induces reactive oxygen species in bort-resistant CAFs and activates autophagy by increasing light chain 3 protein (LC3)-II and inhibiting p62 and phospho-mammalian target of rapamycin. The small-interfering RNA knockdown of Atg7, and treatment with 3-methyladenine, restores bort sensitivity in bort-resistant CAFs and produces cytotoxicity in plasma cells co-cultured with CAFs. In the syngeneic 5T33 MM model, bort-treatment induces the expansion of LC3-II+ CAFs. TGFβ mediates bort-induced autophagy, and its blockade by LY2109761, a selective TβRI/II inhibitor, reduces the expression of p-Smad2/3 and LC3-II and induces apoptosis in bort-resistant CAFs. A combination of bort and LY2109761 synergistically induces apoptosis of RPMI8226 co-cultured with bort-resistant CAFs. These data define a key role for CAFs in bort resistance of plasma cells and provide the basis for a novel targeted therapeutic approach.

Hemilipin, a novel Hemiscorpius lepturus venom heterodimeric phospholipase A2, which inhibits angiogenesis in vitro and in vivo.

Phospholipases A2 (PLA2) are enzymes which specifically hydrolyze the sn-2 acyl ester bond of phospholipids producing free fatty acids and lysophospholipids. The secreted PLA2 (sPLA2) are the most common types of PLA2 purified from the snake venom, mammalian pancreatic juice and other sources. They display a variety of toxic actions and biological activities, including antitumoral and antiangiogenic effects. In this study, we report the isolation, characterization and the antiangiogenic activity of Hemilipin, a novel sPLA2 extracted from Hemiscorpius lepturus venom, the most dangerous scorpion in Iran. Hemilipin was purified by HPLC and analyzed by MALDI TOF/MS. The primary structure was determined by EDMAN degradation method and the PLA2 activity by titration of fatty acids released from the egg yolk phospholipids. Its antiangiogenic activity was studied in vitro by evaluating effects on apoptosis, Matrigel angiogenesis, migration and adhesion of human umbilical vein endothelial cells (HUVECs) and human pulmonary artery endothelial cells (HPAECs) and in vivo by the chorioallantoic membrane (CAM) assay. Mass spectrometry profile showed that Hemilipin is heterodimeric and the PLA2 test demonstrated its strong hydrolytic activity. N-terminal aminoacid sequence highlighted a significant homology of Hemilipins small and large subunits with other sPLA2 group III. Hemilipin had no effect on apoptosis, but strongly impacted angiogenesis both in vitro and in vivo. Our results demonstrate that this novel non toxic sPLA2 could be a new tool to disrupt at different steps human angiogenesis.

The small subunit of Hemilipin2, a new heterodimeric phospholipase A2 from Hemiscorpius lepturus scorpion venom, mediates the antiangiogenic effect of the whole protein

ABSTRACT In a previous study, we reported the identification of Hemilipin, the first secreted heterodimeric phospholipase A2 (sPLA2) from Hemiscorpius lepturus scorpion venom and demonstrated its effective inhibition of all angiogenesis key steps in vitro and in vivo. Here, we aimed to characterize a second sPLA2, Hemilipin2, from the same venom and to elucidate its antiangiogenic effect. The protein was purified by chromatography separation and analyzed by MALDI/TOF mass spectrometry. Its N terminal amino acid sequence was determined by Edman degradation method and the enzymatic activity by fatty acids release assay. Hemilipin2 antiangiogenic activity was investigated by studying its effect in vitro on adhesion, migration and capillary like tube formation of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Pulmonary Artery Endothelial Cells (HPAECs); and in vivo on the chick embryo chorioallantoic membrane (CAM) assay. Data to be presented show that Hemilipin2 is heterodimeric composed by two subunits: the large one has a molecular weight of 12,866 and the small one of 2461 a.m.u. It has a strong calcium‐dependent PLA2 activity and impacts angiogenesis in vitro and in vivo without showing any cytotoxic or apoptotic signs. Its chemical modification with p‐Bromophenacyl Bromide abolishes the enzymatic activity without affecting the antiangiogenic effect. Furthermore, it has been proved that Hemilipin2 small subunit was able to inhibit blood vessel formation both in vitro and in vivo. These findings may serve as a starting point for the designing of a new generation of specific inhibitor of human angiogenesis at different steps. HIGHLIGHTSHemilipin2 is heterodimeric secreted phospholipase A2 (sPLA2) purified from Hemiscorpius lepturus scorpion.Hemilipin2s large and small subunits have a molecular mass of 12866 and 2461 a.m.u. respectively.Hemilipin2’s large subunit has no homology with other sPLA2s group III. Hemilipin2 could form a subgroup of group III sPLA2.Hemilipin2 exhibits an anti‐angiogenesis activity on cells migration, adhesion and vessel length, and in vivo on CAM model.The antiangiogenic effect appears to be independent from the catalytic activity and seemingly held by the small subunit.

Multiple Myeloma: The Role of Angiogenesis in Disease Progression

Angiogenesis, the formation of new blood vessels from pre-existing ones, plays an important role in the biology of multiple myeloma and has a prognostic value in this disease. Multiple myeloma is a plasma cell malignancy that home to and expand in the bone marrow where actively interacts with stromal cells inducing neovascularization, a constant hallmark of disease progression. Myeloma-induced angiogenesis involves either the direct production of angiogenic molecules by myeloma cells and the recruitment and activation of bone marrow stromal cells. Indeed, the angiogenic factors released in the bone marrow microenvironment by multiple myeloma plasma cells stimulate stromal cells to secrete their own angiogenic factors and induce the acquisition of a phenotypic and functional adaptation by non-endothelial cells, such as macrophages, which contribute to the completion of the neovessel wall (vasculogenic mimicry). In this review we summarize recent data which give strong evidence for an increased angiogenic activity in the bone marrow microenvironment and support the hypothesis that angiogenesis is not only important for tumour growth but may also promote plasma cell growth in multiple myeloma.

Jejunal overexpression of peptide YY in celiac disease complicated with pneumatosis cystoides intestinalis

Abstract A 61-year old man with coeliac disease and chronic lack of appetite, malabsorption and weight loss, despite the gluten-free diet, was operated because of a sub-diaphragmatic free air due to a small-bowel pneumatosis cystoides intestinalis (PCI). The jejunum showed granulomatous lesions with a honeycombed appearance of air cysts in the submucosa/subserosa. We found overexpression of peptide YY (PYY) into only the jejunum with PCI, while the expression was very weak or absent in the tissue without cysts. One year after surgery, he had no abdominal pain or PCI recurrence. The above chronic symptoms were plausibly attributable to the PYY.