“Translational Genomics and Precision Cancer Medicine” Nikhil Wagle, MD Instructor in Medicine Harvard Medical School Dana-Farber...
Nader Rahimi, Ph.D.
Department of Pathology and Laboratory of Medicine
M.Sc., Ph.D. Queen’s University, Kingston, Ontario, Canada
Elucidation of angiogenic signaling of VEGFR-2: Our systematic analysis of VEGFR-2 during the past 10 years has identified key signaling pathways activated by VEGFR-2. Activation of VEGFR-2 stimulates a number of key signal transduction pathways in endothelial cells. Activation of PI3 kinase (phosphoinositide 3-OH kinase), requires tyrosine phosphorylation of Y799 and Y1173 on mouse VEGFR-2 (Y801 and Y1175 on human VEGFR-2) which stimulates endothelial cell survival and proliferation. Phosphorylation of Y1173 on VEGFR-2 also is responsible for activation of PLCg1 (phospholipase Cg1), which stimulates endothelial cells tubulogenesis and cell growth. Src kinases are also activated by VEGFR-2 and contribute to VEGFR-2 mediated cellular events. In particular, c-Src kinase directly through its Src homology 2 (SH2) domain and indirectly via c-Cbl binds to phospho-Y1057 (Y1059 in human VEGFR-2) of VEGFR-2. In turn, c-Src kinase phosphorylates VEGFR-2 at multi-docking site, Y1173 and also catalyzes tyrosine phosphorylation of IQGAP1 and acts as an adaptor to bridge IQGAP1 to VEGFR-2. IQGAP1 activates b-Raf and mediates proliferation of endothelial cells. c-Cbl, an ubiquitin E3 ligase also is activated by VEGFR-2 and mediates ubiquitination of PLCg1 resulting in the inhibition of its activity and with it angiogenesis (Figure 1). The current projects in our laboratory are focused in the identification of additional signaling molecules involved in VEGFR-2 signaling, and to determine molecular mechanisms of their activation by VEGFR-2 and their biological importance in pathological angiogenesis.
Role of ubiquitination pathway in angiogenesis: Attachment of ubiquitin to proteins regulates a broad range of key cellular events such as proteosomal degradation, tumor suppression, inflammation, cell cycle progression, and modulation of signaling pathways. Together with ubiquitin activating enzyme E1 and ubiquitin-conjugating enzyme E2, E3 ubiquitin ligases catalyze the ubiquitination of a variety of biologically significant protein substrates for targeted degradation through the 26S proteasome, as well as for non-proteolytic regulation of their functions or subcellular localizations. Our recent work has identified Casitas B-lineage lymphoma (c-Cbl) E3 ubiquitin ligase as a negative regulator of angiogenesis. Upon stimulation by VEGF, VEGFR-2 recruits and activates c-Cbl. As a result of activation by VEGFR-2, c-Cbl ubiquitinates PLCg1 and inhibits VEGFR-2/ PLCg1 driven angiogenesis. We are currently investigating role of c-Cbl ubiquitin E3 ligase and other related proteins in angiogenesis and mechanisms involved in this process. In particular, we are studying role of ubiquitin E3 ligases in ubiquitination of VEGFR-2, and its major substrate, PLCg1. Various in vivo and in vitro models of angiogenesis including genetically engineered mouse models are used to determine role of ubiquitin pathway in VEGFR-2 signaling and angiogenesis.
Role of VEGFR-1 in angiogenesis and human cancers: VEGFR-1 plays a dichotomous role in angiogenesis and tumor growth. It negatively regulates angiogenesis by acting as a “decoy receptor” and its expression and activation in cancer cells plays a positive role which may contribute to the development of human cancers. VEGFR-1 is expressed as a membrane bound receptor tyrosine kinase and as an alternatively spliced soluble protein, sVEGFR-1. sVEGFR-1 is known as a naturally occurring inhibitor of angiogenesis, as a surrogate marker for cancer progression, and its is linked to pregnancy-induced hypertension called preeclampsia and avascularity of normal cornea. Our recent study has demonstrated that in leukemic cancer cells PlGF and VEGF-A both induces tyrosine phosphorylation of VEGFR-1 and renders it susceptible to ectodomain shedding resulting in the generation of sVEGFR-1 and an intracellular cytoplasmic fragment (Figure 2). Activation of protein kinase C (PKC) and TACE family metalloproteases are critically required for occurrence of sVEGFR-1. Following the removal of ectodomain, the remnant of VEGFR-1 remains attached to membrane and activity of g-secretase/Presenilin is required for its release from cell membrane. We propose sVEGFR-1 produced via ectodomain shedding plays a prominent role in VEGF receptor system by antagonizing VEGF receptors signaling by acting as a dominant negative and or forming a non-signaling dimerizing complex with VEGF receptors. Studies are underway to further establish molecular basis of ectodomain shedding of VEGFR-1 and its ramification for VEGFR-1 activation, tumor growth and angiogenesis.
Identification of novel genes involved in the tumor growth and angiogenesis: Completion of the human genome project has created a great opportunity to translate this unprecedented scientific accomplishment into tangible improvements in the diagnosis and the treatment of human diseases. A recent work in our laboratory has identified a number of novel and uncharacterized transmembrane proteins through a unique strategy of bioinformatics coupled with cell culture-based analysis of human genome. Preliminary results have demonstrated unique expression profiles of several of these uncharacterized transmembrane proteins throughout a variety of human organs and tumor cell lines. The overall goal of this project is to establish role of these novel gene products in angiogenesis and tumor growth and metastasis. Various biological assays including, in vivo mouse model, conditional knockout strategy and in vitro cell culture system are used to elucidate function of these gene products in angiogenesis and tumor growth. These studies will provide insight into aspects of tumor development that have been poorly explored because the expression and function of these transmembrane proteins has remained uncharacterized.
Amrik J. Singh, Rosana D. Meyer, Hamid Band and Nader Rahimi. The carboxyl terminus of VEGFR-2 is required for protein kinase C-mediated downregulation. Molecular Biology of the Cell, 2005, 16(4):2106-18., 2005.
Meyer RD, Mossa Mohammadi, and Rahimi N. A single amino acid substitution in the activation loop defines the decoy characteristic of VEGFR-1. Journal of Biological Chemistry, 2006, 281(2):867-75.
Rosana D. Meyer, and Nader Rahimi. Leucine motif-dependent tyrosine autophosphorylation of type III receptor tyrosine kinases. Journal of Biological Chemistry, 2006, 281(13):8620-7.
N. Rahimi. VEGFR-1 and VEGFR-2: Two non-identical twins with a unique physiognomy. Frontiers in Bioscience, 2006, 11:818-29.
Singh AJ, Meyer RD, Navruzbekov G, Shelke R, Duan L, Band H, Leeman SE, Rahimi N. A critical role for the E3-ligase activity of c-Cbl in VEGFR-2-mediated PLCgamma1 activation and angiogenesis. Proceeding National Academy of Science USA, 2007, 104: 5413-8.
Rosana D. Meyer, David B. Sacks, and Nader Rahimi. IQGAP1-Dependent Signaling Pathway Regulates Endothelial Cell Proliferation and Angiogenesis. PLoS ONE, 2008, 3(12):e3848.
Rahimi N, Golde TE and Meyer RD Identification of ligand-induced proteolytic cleavage and ectodomain shedding of VEGFR-1/FLT-1 in leukemic cancer Cells Cancer Research, 2009; 69:2607–14.
Rahimi Lab Members
|Rosana Meyer, M.D.|
|3rd Year Pathology PhD|
| Contact Info:
670 Albany Street – 4th Floor
|Jed Mahoney||2nd Year Pathology PhD|
670 Albany Street – 4th Floor
|Srimathi Srinivasan||3rd Year Pathology PhD|
670 Albany Street – 4th Floor