Crosstalk between VEGF and novel angiogenic protein regulates tumor angiogenesis and contributes to aggressiveness of breast carcinoma.

We have identified and characterized a novel proangiogenic glycoprotein (NAP) with molecular weight of 67 kDa from synovial fluid of rheumatoid arthritis patients. Proteomic analysis of the protein revealed 29% sequence coverage with maximum identity for human retinoblastoma binding protein 2. N-terminal amino acid sequence showed no identity to recently discovered protein sequences. NAP was also identified in both normal and tumor cell lines by Western blotting. NAP is a permeability factor as verified by miles permeability assay. The proangiogenic potential of NAP was identified using shell less CAM, rat cornea and tumor on CAM assays. NAP induces expression of VEGF and Flt-1 gene as verified by promoter reporter gene analysis. Further NAP induces proliferation of endothelial cells and formation of tube like structures. NAP is also involved in migration and invasion of tumor cells. Clinical data revealed the presence of NAP in breast cancer biopsies. We have developed monoclonal antibody (mAb), and specific ELISA, which confirmed the presence of NAP in the cytosol of tumor cells. The mAb effect was evaluated with established angiogenic assays. Further, we investigated the detailed mechanism by which NAP induces angiogenesis. NAP is phosphorylated by VEGF induced activation of MAPK and JNK pathways through VEGFR2 phosphorylation. NAP involves JNK pathway predominantly with further activation of NFκB in downstream processing of VEGF activation. Together these findings establish that NAP displays angiogenic properties and promotes efficient neovascularization both in vitro and in vivo models. These observations suggest that anti-NAP-mAb can be targeted for antiangiogenic therapy of cancer.

Belugali Nataraj N ，Salimath BP 《-》

Crosstalk between VEGF and MTA1 signaling pathways contribute to aggressiveness of breast carcinoma.

The expression of metastasis associated protein (MTA1) correlates well with tumor metastasis; however its role as a proangiogenic protein and the molecular mechanisms underlying the same are not fully understood. In this study the MTA1 protein was expressed and purified to evaluate its angiogenic potential. In both MCF-7 and MDA-MB-231 cells, endogenous MTA1 protein was localized in the nucleus; while added recombinant MTA1 protein was bound to cell membrane as per immunofluorescence data. MTA1 was detected both in conditioned media and in human serum samples. Recombinant MTA1 regulated cellular functions of HUVEC's such as, proliferation, tube formation, and migration. MTA1 was more potent than VEGF in inducing invasion of breast cancer cells. Analogous to VEGF, MTA1 could induce angiogenesis in both non-tumor and tumor context, as verified by rat cornea, shell less CAM and xenograft models respectively. However MTA-1 was more potent an inducer of angiogenesis. VEGF or Flt-1 gene promoter, luciferase gene reporter analysis revealed that MTA1 up regulates the expression of VEGF and its receptor Flt-1 genes. Kinetics of VEGF-induced expression of MTA1 and qPCR studies showed that there is an increased expression of MTA1 in tumor cells. VEGF induced phosphorylation of endogenous MTA1 on tyrosine residues; phosphorylation was mediated through VEGFR2 and p38-MAP kinase. Recombinant MTA1 activated signaling, in MCF-7 and MDA-MB-231 cells, involved ERK and JNK pathways. In conclusion, MTA1 is a potent angiogenic molecule and cross talk between VEGF and MTA1 protein regulates tumor angiogenesis and metastasis.

Nagaraj SR ，Shilpa P ，Rachaiah K ，Salimath BP ... -  《-》

Antiangiogenic mechanisms of PJ-8, a novel inhibitor of vascular endothelial growth factor receptor signaling.

Angiogenesis occurs not only during tissue growth and development but also during wound healing and tumor progression. Angiogenesis is a balanced process controlled by proangiogenic and antiangiogenic molecules. As a critical factor in the induction of angiogenesis, vascular endothelial growth factor (VEGF) has become an attractive target for antiangiogenic and cancer therapeutic agents. In an effort to develop novel inhibitors to block VEGF signaling, we selected Pj-8, a benzimidazole derivative, and investigated its inhibitory mechanisms in human umbilical vascular endothelial cells (HUVECs). Pj-8 concentration-dependently inhibited VEGF-induced proliferation, migration and tube formation of HUVECs. Pj-8 also suppressed VEGF-induced microvessel sprouting from aortic rings ex vivo and suppressed neovascularization of implanted matrigel plugs in vivo. Pj-8 inhibited VEGF-induced phosphorylation of VEGF receptor (VEGFR) 2 and the downstream protein kinases, including Akt, focal adhesion kinase, extracellular signal-regulated kinases and Src. Results from in vitro kinase assay further demonstrated that Pj-8 suppressed the kinase activity of 3-phosphoinositide-dependent kinase 1 (PDK1). Using xenograft tumor angiogenesis model, Pj-8 markedly eliminated tumor-associated angiogenesis. Taken together, our findings suggest that Pj-8 inhibits VEGF and tumor cells MDA-MB-231-induced angiogenesis, and it may be a potential drug candidate in anticancer therapy. Downregulation of VEGFR2-mediated signaling may contribute to its antiangiogenic actions.

Huang SW ，Lien JC ，Kuo SC ，Huang TF ... -  《-》

GABARBP down-regulates HIF-1α expression through the VEGFR-2 and PI3K/mTOR/4E-BP1 pathways.

Human γ-aminobutyrate type A (GABAA) receptor-binding protein (GABARBP), a tumor suppressor protein with apoptotic function, can be inhibited in response to angiogenesis through the PI3K/Akt signaling cascades. Here, we investigated whether GABARBP over-expression could regulate vascular endothelial growth factor (VEGF)/hypoxia-inducible factor-1α (HIF-1α) expression and angiogenic activity in a carcinoma model system. GABARBP dramatically inhibited VEGF-induced endothelial cell proliferation, migration, and tube formation, as well as VEGFR-2 phosphorylation in vitro. At the same time, GABARBP exposed potent anti-angiogenic activity and remarkably down-regulated the levels of VEGF and HIF-1α protein expression, key components for angiogenesis. In addressing its biological molecular mechanism, GABARBP was found to effectively inhibit the phosphorylation of down-stream PI3K components, such as PDK1, Akt, mTOR, TSC-2, p70S6K, and 4E-BP1 by directly binding with VEGFR-2. In contrast, p38/JNK phosphorylation was not suppressed by GABARBP. These findings disclose a novel function of GABARBP in suppressing VEGF and HIF-1α protein expression, which is important for tumor angiogenesis and tumor growth. Thus, our data strongly provides novel biological mechanistic insights into the regulatory function of GABARBP in ovarian tumor progression, and the important of pre-clinical certification of GABARBP as a potential angiogenesis agent targeting ovarian tumorigenesis.

Park SH ，Kim BR ，Lee JH ，Park ST ，Lee SH ，Dong SM ，Rho SB ... -  《-》

Stemness marker ALDH1A1 promotes tumor angiogenesis via retinoic acid/HIF-1α/VEGF signalling in MCF-7 breast cancer cells.

Ciccone V ，Terzuoli E ，Donnini S ，Giachetti A ，Morbidelli L ，Ziche M ... -  《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》