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Investigating the role of G-protein coupled receptor (GPCR) signaling as a potential target for anti-angiogenic therapy

  • Author / Creator
    Azad, Abul
  • Angiogenesis is an essential process for normal growth and development whereby new blood vessels are formed from pre-existing ones. The balance between pro-angiogenic and anti-angiogenic growth factors is critical to maintain normal physiological condition. Prolonged activation of angiogenesis leads to different pathological conditions including diabetic retinopathy, ischemic heart disease and cancer progression. In 1971, Folkman proposed that tumor could not grow beyond 2mm3 without the supply of nutrients and oxygen via neovascularization, suggesting that inhibition of angiogenesis would keep the tumor in a small size and dormant state. In 1990s, Ferrara’s group showed that the loss of single allele of vascular endothelial growth factor (VEGF) resulted in embryonic lethality due to vessels abnormalities and inhibition of VEGF suppressed tumor angiogenesis and tumor growth in vivo. These works led the foundation for the development of several anti-angiogenic drugs targeting VEGF-pathway. To this end, several anti-angiogenic inhibitor (AI) drugs have been approved by FDA for the treatment of many types of cancers. While benefits of AI therapies are clinically evident, the initial response to therapy is not durable, and in many cases, patients develop drug resistance over time due to the activation of multiple other mechanisms compensating blockade of VEGF-signaling. One of the key mechanisms that is over activated due to vessels pruning by AI therapy is intra-tumoral hypoxia, which in turn contributes to the production of plethora of alternative pro-angiogenic growth factors, thereby enabling the tumors with the continuous supply of oxygen and nutrients. Besides, patients with high level of tumor hypoxia have higher risk of tumor progression and metastasis and reduce the tumor cells sensitivity to radiation, chemo- and immunotherapies as it becomes harder for the delivery of drugs in chronically hypoxic microenvironment. Thus, it is of utmost importance to identify broad-spectrum anti-angiogenic target(s) that can block multiple pro-angiogenic pathways, and at the same will alleviate hypoxia, for the development of better anti-angiogenic therapy. This thesis investigates the role of alternative pro-angiogenic signaling, especially with regard to the G-protein coupled receptor (GPCR)-mediated phosphoinositide 3-kinases (PI3K) signaling activation in tumor angiogenesis. First, we investigated the role of endothelial cell (EC) specific p110 (PI3K) isoform of PI3K signaling in regulating angiogenesis. We showed that p110 functions as a downstream target of GPCR signaling and controls angiogenesis in ex-vivo and in vivo models. We found that selective inactivation of PI3Kβ reduced patient-derived renal cell carcinoma (PD-RCC)-stimulated EC spheroid sprouting. In mice, loss of EC PI3K in combination with receptor tyrosine kinase inhibitor (TKI) sunitinib treatment decreased primary tumor growth and tumor metastasis, which was accompanied by decreased tumor vessel density and angiogenesis. Second, we studied the role of apelin, a GPCR ligand, in tumor angiogenesis. We found that loss of apelin in mice delayed the primary tumor growth in combination with sunitinib treatment, accompanied by a marked reduction in tumor vessels density, angiogenic sprouting and tip cell marker gene expression in comparison to the control group. Interestingly, in our single cell RNA (scRNA) sequencing, we observed that the loss of apelin prevented EC tip cell differentiation in comparison to the control group, suggesting that blockade of apelin captures most of the effect of PI3K inhibition in EC to limit the tumor angiogenesis and the subsequent tumor growth. Third, we investigated the role of endothelial cell specific Facio-genital dysplasia 5 (FGD5) in regulating C-X-C motif chemokine receptor 4 (CXCR4)/PI3K signaling pathways. We observed that CXCL12 augmented EC sprouting angiogenesis and tip cell marker gene expression under conditions where VEGF was limiting, and that CXCL12-stimulated CXCR4/PI3K signaling activation depends on PI3K isoform. Knockdown of FGD5 decreases CXCL12-stimulated CXCR4/PI3K signaling. FGD5 modulates the CXCR4/PI3K signaling by acting as a Rho guanine exchange factor (RhoGEF) for Rac1 to control p110 activity. In summary, we show that GPCR/PI3K signaling participates in tumor angiogenesis along with the RTK/PI3K signaling. In endothelium, PI3K is the dominant isoform of PI3K to regulate GPCR-mediated tumor angiogenesis. Inhibition of apelin, a pro-angiogenic GPCR ligand, mostly arrests the contribution of GPCR/PI3K signaling in tumor angiogenesis. Finally, we show that EC-specific FGD5 could be a potential target for the development of an effective anti-angiogenic therapy as FGD5 regulates parallel pro-angiogenic signaling coming from both RTK/PI3K and GPCR/PI3K signaling pathways.

  • Subjects / Keywords
  • Graduation date
    Fall 2021
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.