ALBERT

Description: Enhanced angiogenesis is a hallmark of cancer. Pleiotrophin (PTN) is an angiogenic factor that is produced by many different human cancers and stimulates tumor blood vessel formation when it is expressed in malignant cancer cells. Recent studies show that monocytes may give rise to vascular endothelium. In these studies, we show that PTN combined with macrophage colony-stimulating factor (M-CSF) induces expression of vascular endothelial cell (VEC) genes and proteins in human monocyte cell lines and monocytes from human peripheral blood (PB). Monocytes induce VEC gene expression and develop tube-like structures when they are exposed to serum or cultured with bone marrow (BM) from patients with multiple myeloma (MM) that express PTN, effects specifically blocked with antiPTN antibodies. When coinjected with human MM cells into severe combined immunodeficient (SCID) mice, green fluorescent protein (GFP)–marked human monocytes were found incorporated into tumor blood vessels and expressed human VEC protein markers and genes that were blocked by anti-PTN antibody. Our results suggest that vasculogenesis in human MM may develop from tumoral production of PTN, which orchestrates the transdifferentiation of monocytes into VECs.

Description: Recent studies suggest that zoledronic acid (ZOL) and other nitrogen-containing bisphosphonates (BPs) inhibit angiogenesis by reducing angiogenic factor production and signaling by these factors. However, few studies have addressed the potential role of BPs in blocking the formation of new vasculature or so-called vasculogenesis. Thus, we determined whether ZOL could impact this process using a chorioallantoic membrane (CAM) vasculogenesis model. First, fertilized chick eggs were incubated horizontally at 37.5°C in a humidified incubator and windowed on day 8. Another set of E8 chicken embryonic skins were cultured in insert dishes with different concentrations of ZOL (1.0 or 10 uM) or without drug treatment for 24 hours. The embryonic skins were transferred to CAM. Endothelial cells of CAM normally started to proliferate and migrate into feather buds after two days. Blood vessel formation was determined after four days of culturing under the microscope. We found that endothelial cell proliferation and migration of CAM was completely inhibited and development of embryonic skin buds was blocked by ZOL at 10 uM. At a lower concentration of ZOL (1.0 uM), the vasculogenesis was also decreased and embryonic skin buds were also reduced in size (but to a lesser extent than at the higher drug concentration) compared with a control group without ZOL. We also examined whether zoledronic acid affected development of embryonic feather buds (epithelial cell) directly. The results showed that embryonic feather bud growth was unaffected when buds were cultured on insert dishes with 10uM ZOL compared to buds without this drug. This suggests that ZOL blocks endothelial cell proliferation in CAM and migration but does not effect epithelial cell development. We further examined endothelial cell gene expression of the bud cells on CAM treated with and without zoledronic acid. We measured vascular endothelial growth factor receptor-2 (Flk-1) expression with Western blot analysis. The results showed that Flk-1 is markedly reduced after buds were treated with ZOL at 10 uM and there was also some reduction in the expression of Flk-1 at the lower concentration (1.0 um) of ZOL. To determine which signal transduction pathway(s) may be involved in blocking endothelial cell proliferation and migration by ZOL, we determined gene expression of β-catenin, Runx2, and smad7 by RT-PCR in the embryonic buds. Runx2 is a target of β-catenin /TCF1 for the stimulation of bone formation, and Smad7 gene expression is increased in human hematopoietic stem cells (HSCs) and is required for TGF-β-induced expression of β-catenin. The results showed that expression of β-catenin was down-regulated by ZOL whereas expression of Runx2 and smad7 was up-regulated by this bisphosphonate. These results suggest that ZOL blocks the β-catenin pathway, and also add to other studies suggesting the important role of this pathway in blood vessel development. We have also shown that zoledronic acid profoundly suppresses vasculogenesis, and this effect adds another potential mechanism by which this bisphosphonate may possess anti-tumor effects.

Description: Pleiotrophin (PTN, Ptn) is an 18 kD cytokine that is expressed in many human breast cancers and its gene is inappropriately expressed in cell lines derived from these breast cancers. To study the siginificance of inappropriate expression of Ptn in human breast cancer cells on surrounding stromal cells, we first compared nude mouse xenografts of MCF-7 and MCF-7-Ptn cells. MCF-7-Ptn cells lack the Receptor Protein Tyrosine Phosphatase (RPTP)b/z, the PTN receptor, and thus are not responsive to PTN through autocrine or paracrine stimulation. The MCF-7-Ptn cell xenografts grew rapidly whereas MCF-7 cells xenografts were barely detectable 6 weeks after injection. MCF-7-Ptn cells that were co-injected with equal numbers of NIH3T3 cells grew even more rapidly in the flanks of the nude mice. Surprisingly, the MCF-7-Ptn cell explants developed a morphological phenotype remarkably similar to that of the human invasive ductal carcinoma. We then co-cultured MCF-7 cells that express Ptn (MCF-7-Ptn cells) with NIH 3T3 cells. Secretion of PTN from MCF-7-Ptn cells induced formation of sharply defined clusters of MCF-7-Ptn cells, termed “epithelial islands”, that were surrounded by dense fibrous bands interspersed with NIH 3T3 cells that morphologically closely resemble carcinoma associated fibroblasts (CAFs). A striking increase in tropoelastin and expression of type IV procollagen mRNA was identified in NIH3T3 cells co-cultured with MCF-7-Ptn cells. Furthermore, different markers often resulting from stromal cell-carcinoma cell interactions in breast cancer, including protein kinase C (PKC)-d, and both human and murine matrix metalloproteinase (MMP) 9 were identified either in cells or in the culture media taken from MCF-7-Ptn/NIH3T3 cell co-cultures. The induction of these biochemical and morphological features in the co-cultures of MCF-7-Ptn and NIH3T3 cells was demonstrated to be Ptn expression dependent, PTN-secretion dependent, and NIH3T3 cell dependent. The data suggest that PTN secretion alone from human breast cancer cells with inappropriate expression of Ptn is sufficient to markedly remodel the microenvironment of the breast cancer cell and induce a morphological transition of the MCF-7-Ptn cells and NIH3T3 cells to patterns resembling breast carcinomas through activation of the PTN/RPTPb/z signaling pathway in NIH3T3 cells and reciprocal signaling between the carcinoma stromal cells and the PTN secreting breast cancer cells.

Description: Angiogenesis is a hallmark of a variety of malignancies including multiple myeloma (MM). We have shown that MM patients express pleiotrophin (PTN), and this protein stimulates MM growth and prevents apoptosis. We have also demonstrated that PTN when combined with mCSF stimulates angiogenesis through the transdifferentiation (TD) of monocytes into endothelial cells. Our pathway-specific microarray results have shown that the Wnt pathway was up-regulated when monocytes were induced by mCSF and PTN. This signaling pathway plays an important role in cell growth, differentiation, function, and death. To further explore the role of Wnt/β-catenin signaling in the TD of monocytes into endothelial cells by PTN and mCSF, we examined whether monocytes induced to TD with this combination involved induction of the Wnt/β-catenin signal transduction pathway using immunohistochemical (IHC) staining and RT-PCR. Normal human blood monocytes were purified using density centrifugation followed by anti-CD14+ micro-bead affinity column selection. After one week of culture in the presence of mCSF and PTN, 5-10% of purified monocytes showed TD into endothelial cells as quantified by RT-PCR whereas cells treated with mCSF or PTN alone or without these factors did not. The cells also formed tube-like structures with positive staining for the endothelial cell markers Tie2, FLK-1, and vWF. The results showed β-catenin gene expression was markedly increased when CD14+ cells were treated with mCSF and PTN as demonstrated by RT-PCR. IHC studies showed CD14+ cells induced with mCSF and PTN expressed increased amounts of β-catenin in nuclear extracts but not cells treated with mCSF or PTN alone or without treatment. The nuclear gene or protein expression of β-catenin was blocked by adding casein kinase (CK)-1 inhibitor, a Wnt pathway inhibitor. We further examined whether CK-1 inhibitor can prevent TD of monocytes into endothelial cells by mCSF and PTN. CD14+ cells were exposed to mCSF and PTN or co-cultured with cells from fresh MM bone marrow or MM cell lines (U266 or MM1s). The Wnt inhibitor markedly decreased the formation of tube-like structures and endothelial cell gene expression. We also examined Smad7 gene expression. Smad7 is highly expressed in human hematopoietic stem cells (HSCs) and required for TGF-β-induced β-catenin expression. Our RT-PCR results showed that Smad7 gene expression in CD14+ cells induced with mCSF and PTN was not changed compared with untreated cells. These results suggest that the signaling pathways involved in TD of monocytes into endothelial cells are different from pathways involved in stem cell differentiation. We also examined Runx2 gene expression and this protein is a target of β-catenin /TCF1 involved in the stimulation of bone formation. Runx2 gene expression was up-regulated when monocytes were stimulated with mCSF and PTN. Runx2 gene expression was markedly increased by adding CK-1 inhibitor. Our findings further our understanding of the mechanisms involved in the TD of monocytes in endothelial cells and suggest the importance of the Wnt/β-catenin pathway in this critical early step in tumoral angiogenesis.

Description: Pleiotrophin (PTN) is an important developmental cytokine that is highly expressed during embryogenesis but shows very limited expression in adult tissues, where it is largely restricted to the brain. High PTN serum levels are associated with a variety of solid tumors. We recently showed that patients with multiple myeloma (MM) also have elevated serum levels of this protein and the amount of PTN correlated with the patients' disease status and response to treatment. In this study, we demonstrate that MM cell lines and the malignant cells from MM patients' bone marrow produced PTN and secreted PTN protein into the supernatants during short-term culture. Moreover, Ptn gene expression correlated with the patients' disease status. Inhibition of PTN with a polyclonal anti-PTN antibody reduced growth and enhanced apoptosis of MM cell lines and freshly isolated bone marrow tumor cells from MM patients in vitro. Importantly, this antibody also markedly suppressed the growth of MM in vivo using a severe combined immunodeficiency (SCID)-hu murine model. This represents the first study showing the importance of PTN in the growth of any hematological disorder. Because the expression of this protein is very limited in normal adult tissues, PTN may represent a new target for the treatment of MM.