Malignant tumors such as glioblastoma rely heavily on angiogenesis to endure sustain their highly proliferative and aggressive phenotype. Anti-angiogenic therapy is therefore very promising for this devastating brain cancer for which effective treatments are greatly needed. Unfortunately, the enthusiasm that greeted the advent of anti-angiogenic therapies such as the VEGF-neutralizing antibody bevacizumab, has been mitigated because, although the initial responses of cancers to this therapy are often profound, many tumors develop invasive resistance, with rapid disease progression leading to poor outcomes. Preclinical studies suggest that, unlike conventional DNA damaging chemotherapy whose resistance often requires gene mutations, anti- angiogenic therapy resistance occurs via transcriptional reprogramming or post-translational modification of proteins, allowing for tumor growth resurgence to take place despite inhibition of the antivascular target. We hypothesize that upregulated ?1 integrin and receptor tyrosine kinase c-Met drive invasive resistance to anti- angiogenic therapy through a physical interaction, resulting in a structural complex that functions not only to amplify their signaling va mutual downstream mediators, but through activation of additional pathways specific to this complex. We will investigate this hypothesis through specific systems that evoke protein-protein interaction between c-Met and ?1 integrin via both chemical and light stimulation (Aim 1). Utilizing CRISPRi technology, we will incorporate dual and near-complete knockdowns of our proteins to further optimize the biological impact of the induced complex by eliminating background complexes. The induction of this complex will be analyzed through a series of biological assays in vitro and followed up for the assessment of response to anti-angiogenic therapy in vivo. Furthermore, we will screen for the activation of pathways specific to complex formation utilizing a Human Phosphokinase Array. To investigate whether disruption of the complex abrogates resistance, we will first need to elucidate which of their domains and which specific residues within these domains allow ?1 integrin and c-Met to bind each other (Aim 2). This will be achieved by introducing targeted mutations into each receptor using PCR site-directed mutagenesis, and expressing these mutant forms in tumor cells rendered genetically deficient in these receptors through CRISPRi knockdown. Receptor binding will then be confirmed using immunoprecipitation. The response to anti-angiogenic therapy of tumor cells carrying only altered versions of ?1 integrin and c-Met which fail to bind can then be assessed in the orthotopic microenvironment in vivo. Successful completion of these aims will characterize the biological effects of the ?1 integrin and c-Met complex formation, elucidate fundamental insight into the mechanism by which this unique complex drives resistance, and determine the binding site(s) between these two proteins. The findings from this program can prove to be essential for future therapeutic targets to sustain the efficacy of anti-angiogenic therapy and delay the evolution of resistance.
Glioblastoma is one of the most invasive and devastating cancers with limited therapeutic options, and due to their highly vascular nature, therapies targeting blood vessel formation, such as anti-angiogenic therapies targeting the VEGF pathway, have generated considerable enthusiasm in the clinic. Unfortunately, the initial response to anti-angiogenic therapy is only short-lived with recurrent tumors being even more aggressive, which this proposal hypothesizes to result from an interaction between upregulated ?1 integrin and receptor tyrosine kinase c-Met, the disruption of which could avert or override therapeutic resistance. Better understanding of these intricate mechanisms is of great value, as it will allow for the development of therapies that can prevent and overcome resistance to anti-angiogenic therapy and improve survival for patients with glioblastoma.
| Jahangiri, Arman; Nguyen, Alan; Chandra, Ankush et al. (2017) Cross-activating c-Met/?1 integrin complex drives metastasis and invasive resistance in cancer. Proc Natl Acad Sci U S A 114:E8685-E8694 |
| Thompson, Eric M; Hielscher, Thomas; Bouffet, Eric et al. (2016) Prognostic value of medulloblastoma extent of resection after accounting for molecular subgroup: a retrospective integrated clinical and molecular analysis. Lancet Oncol 17:484-495 |
