Graduation Date

Summer 8-12-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Kaustubh Datta

Abstract

Despite the development of new therapies, clinical management of advanced metastatic castrationresistant prostate cancer (mCRPC) still remains challenging owing to the emergence of therapy resistance. Resistance to the therapies is driven by both AR and non-AR pathways. Non-AR pathway is characterized by a lineage switch that leads to the development of neuroendocrine-like (NE-like) prostate cancer (PCa), which has a fulminant clinical course due to the lack of effective treatment strategy. Therefore, novel molecular targets need to be identified to develop effective therapeutic regimen to improve the outcomes in advanced PCa patients including NE-like PCa. Recent investigation on advanced PCa patients revealed that a set of genes regulating important neuronal functions and implicated in the poor cancer prognosis are upregulated in patients with NE-like phenotype. This opened an avenue to identify a potential molecular target in NE-like advanced CRPC. Neuropilin-2 (NRP2), a transmembrane, non-kinase protein, is involved in vascular and nervous system development by regulating angiogenesis and axon guidance cues. Several published reports have established NRP2’s role in tumorigenesis. In addition to the cancer cells, NRP2 is expressed in endothelial and immune cells in the tumor microenvironment and promotes functions such as lymphangiogenesis and immune suppression important for cancer progression. The expression of NRP2 is increased with the prostate cancer (PCa) progression and is associated with poor cancer specific survival. In addition, published reports showed that NRP2 depletion sensitizes mCRPC PCa cells to chemotherapy. In view of the above, the current study was carried out to evaluate NRP2’s function in NE-like PCa and devise a strategy to target NRP2 expressing advanced PCa. We have identified significant NRP2 expression in NE-like PCa patients by using bioinformatics and immunohistochemical analysis of patient tissues. To further understand NRP2’s role in advanced therapy resistant PCa, we have generated NE-like PCa cell lines from established adenocarcinoma CRPC lines. NRP2 expression has been detected to be significantly more in NE-like cells than the adenocarcinoma cells. In vitro and in vivo studies suggested the importance of the NRP2 axis in promoting growth and survival of NE-like PCa upon treatment with chemotherapeutic drugs. As an underlying mechanism, NRP2 was found to be required to regulate the secretory function of NE-like PCa cells and thus is important for establishing paracrine communication with neighboring NE-like and adenocarcinoma cells. By inhibiting the NRP2- dependent secretory function of NE-like PCa cells, we could sensitize both the neighboring adenocarcinoma and NE-like cells to the front line of chemotherapies. Therefore, this study identifies a novel NE-like PCa specific mechanism of NRP2 in conferring therapy resistance to cancer cells. However, there is no effective inhibitor of NRP2. Therefore, in this current project, we have performed computer aided drug discovery followed by biophysical characterization, in vitro and in vivo experiments to discover lead small molecule inhibitor of NRP2. We have also performed lead optimization and fragment-based drug discovery to develop novel small molecule inhibitors of NRP2 with higher efficacy, superior binding affinity and better specificity. Furthermore, we have performed molecular dynamics simulation study to gain structural insight of NRP2 functional inhibition by the novel small molecule inhibitors. In addition, we have developed LC-MS/MS based bioanalytical method to evaluate pharmacokinetic properties of the novel inhibitors including absorption, distribution, metabolism and excretion, and reported the novel inhibitor’s preliminary bio-distribution. Thus, this project raises an opportunity to develop future NRP2-axis targeting therapeutic strategy for enhanced efficacy against aggressive and therapy-resistant PCa.

Comments

2022 Copyright, the authors

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