Glioblastoma (GBM) is a devastating brain tumour characterized by local invasion, microvascular proliferation, and therapeutic resistance. The highly infiltrative nature of glioma cells makes complete surgical resection unlikely, and 90% of tumors recur. High-grade gliomas are among the most angiogenic of all tumors. Adrenomedullin (AM) is a 52 aminoacid C-terminal amidated peptide widely expressed in a variety of tumor types and was shown to be mitogenic for many human cancer cell lines in vitro. AM binds to and mediates its activity through the G protein-coupled receptor calcitonin receptor-like receptor (CLR), with specificity for AM being conferred by the receptor activity modifying protein -2 (RAMP2) and -3 (RAMP3). The genetic ablation of AdM, calcrl, Ramp2 or the enzyme responsible for functional AM amidation, peptidylglycine α-amidating monooxygenase (PAM) all result in midgestational lethality associated with severe interstitial edema and cardiovascular defects. In vivo studies highlight the significance of AM as an important factor to promote tumor growth and to affect the tumor microenvironment by inducing pathologic angiogenesis and lymphangiogenesis. Accumulating studies suggest a new role for AM as a cross-talk molecule that integrates tumor and microenvironment stroma cells underlying promotion mechanisms to facilitate angiogenesis and tumor growth. Several in vivo studies have shown a regression of tumor neovessels and growth upon the treatment with neutralizing AM antibodies, AM receptor antagonist, or AM receptor interference. These findings highlight the implication of AM in the progression of tumor growth and angiogenesis, suggesting that targeting the AM system may be a useful therapeutic strategy in brain cancer. Therefore, understanding the molecular mechanisms by which AM can determine the integrity of tumor neovessels will be the main focus of our research with the use of GBM as model to pursue our projects.
Our general objective is to study the specific role of adrenomedullin in glioblastoma growth and vasculature. Our working hypothesis is that AM is involved in a cascade of molecular and cellular events that ultimately lead to the stabilization of functional vessels to promote glioblastoma growth. Accordingly, we intend to develop the following aims: Aim 1: will determine the role of AM in the recruitment and incorporation of provascular and pro-angiogenic bone marrow-derived cells (BMDCs) in tumor neovascularization (endothelial and mural progenitor cells); Aim 2: will decipher the molecular mechanisms activated by AM to stabilize the tumor blood neovessels; Aim 3: will determine the role of AM in the neoangiogenesis and/or vasculogenesis during glioma recurrence after radiotherapy; Aim 4: will investigate the expression and role of the AM system in glioblastoma-associated vascular cells; Aim 5: will investigate a combined treatment by targeting the AM system using classical or innovative drugs in the GBM field.
1. Role of AM in the recruitment of pro-vascular and pro-angiogenic bone marrow-derived cells in tumor neovascularization
We will assess whether the AM receptors are expressed in different BMDCs types, whether these cells can be mobilized by AM in vitro as well as in vivo, whether AM can induce recruitment of provascular cells (endothelial progenitor cells EPCs and pericytes cells), whether proangiogenic marrow-derived cells are recruited to the neo-angiogenic site in GBMs. We will also address the role of metalloproteases (MMP) activities such MMP2, MMP9, MMP14 as well as uPA expressed by the recruited BMDCs. We will determine whether AM induces the expression of MMPs and uPA, what are the roles of these MMPs and uPA in the remodeling of the microenvironment. Finally, we will assess how blockade of the AM system by aAM and aAMR as well as by antagonists interfere in this recruitment.
2. Role and mechanisms of action of AM in the tumor blood vessels stabilization in GBM
Mature blood vessels are characterized histologically by associated pericytes/smooth muscle cells (SMC) and a mature basement membrane. Vascular basement membrane is a self-assembled, supra-molecular complex of proteins, glycoproteins and proteoglycans that tightly envelops endothelial cells and pericytes of blood vessels. Type IV collagen, laminin, fibronectin and heparan sulfate proteoglycan are among the main components. Whereas a number of the molecular players that mediate vessel assembly have been identified, less is known about the cellular and molecular control of vessel remodeling and stabilization. We will thus focus on the role of AM in the endothelial cell-matrix adhesive interactions and on the role of the AM system in the tight interactions between endothelial cells and mural cells (pericytes).
3. Role of AM in the neoangiogenesis and/or vasculogenesis during glioma recurrence after radiotherapy
We will focus on several experimental paradigms with orthotopic injection of U87 MG cells in nude mice to demonstrate and validate our working hypothesis. Large screening of expression of AM as well as other growth and angiogenic factors will be performed in the U87 orthotopic models xenografts and in cohorts of pair samples of GBM taken in pre- and post-radiotherapy.
4. Expression of the AM system in glioblastoma-associated vascular cells
Our working hypothesis is that GBM-associated vascular cells (GVCs) could express and secrete high levels of AM to support, promote and stabilize a vascular network with an independent manner of GBM tumor cells. We will isolate GBM-associated endothelial cells (GECs), GBM-associated pericyte cells (GPCs) and GBM cells from primary GBM tumors to assess the expression of AM mRNA and AM receptors mRNAs (CLR, RAMP2, and RAMP3). We will maintain these cells in culture to assay the immunoreactive AM secretion and maintain GBM cells in coculture with GECs or GPCs to determine whether the communication between GBM cells and vascular cells (GECs, GPCs) can modulate the levels of expression of the AM system in GECs and GPCs. We will determine whether AM is able to induce GECs differentiation into cord-like structures on Matrigel, determine the role of AM system in the tight interaction between GECs and GPCs and determine whether AM is one of the GECs-derived factors responsible for endothelial cell-like and GPCs recruitment to build a neovascularization, angiogenesis assay in vivo with Matrigel admixed to medium secreted by GECs and GECs transfected with shRNA targeting AM mRNA will be used. Different groups of animals will be treated with aAM and aAMR to block the AM system, or IgG-control for control group will be used in this experimental paradigm.
5. Therapeutic issues: inhibition of glioblastoma tumor growth by targeting only AM system using monoclonal antibodies or in combination with classical or innovative drugs
We developed neutralizing monoclonal antibodies generated against AM peptide. Accordingly, we plan to study the effects of AM-mAb and chemAMR-mAb on proliferation, migration and invasion of GBM cells (U87, U138, U251, T98G) in vitro. We will evaluate the effects of AM-mAb on angiogenesis in vitro, analyse the effect of AM by AM mAb in in vivo models of angiogenesis and on tumor growth in GBM xenograft tumor models. We will investigate the cellular and molecular mechanisms by which AM mAb inhibit tumor growth. Finally, treatment with mAbs against the AM system combined to either temozolomide, Avastin (anti-VEGF mAb), DC101 (anti-VEGFR2 mAb), OTX015 (MK-8628) and inhibitors to CXCL12-CXCR4 pathway (AMD 3100) will be evaluated in heterotopic and orthotopic models.