ORCID ID

0009-0005-8397-7590

Graduation Date

Summer 8-9-2024

Document Type

Thesis

Degree Name

Master of Science (MS)

Programs

Biochemistry & Molecular Biology

First Advisor

Parthasarathy Seshacharyulu

Abstract

Prostate cancer (PCa) stands as the top frequently diagnosed malignancy and the second-most leading cause of death in males in the United States in 2024. Androgen-targeted therapies are effective for early-stage PCa. However, it recurs with castration resistant PCa, leading to survival for less than 2 years. Further advancement of PCa causes it to metastasize and develop “de novo” and treatment related neuroendocrine PCa cancer (NEPC), a lethal form of PCa with a poor survival of 7 months. Hence, to understand the biology beyond PCa metastasis, we developed a composite mouse model with loss of Pten and p53 function and gain of MYC amplification. Our composite mouse triple transgenic model exhibited poor survival due to high metastasis to visceral organs. To understand the mechanism behind this aggressive phenotype of the mouse we performed global transcriptomic analysis of less aggressive and highly aggressive mice (which differs by gains of p53 function). We found ASPORIN (ASPN) to be the top differentially regulated ECM gene in the aggressive mouse model of PCa. ECM and associated proteins are known to be significant contributors to PCa development, growth, proliferation, and EMT, but their role in metastasis is not explored completely in PCa. ASPN displayed significantly high protein expression in human and mouse PCa tissues and cells compared to their normal counterparts. To understand the functional significance of ASPN, we generated stable knockdown of ASPN in 22Rv1 cells and simultaneously overexpressed (OE) ASPN in normal immortalized RWPE1 cells and PC-3 PCa cells. We performed in vitro functional studies such as sphere formation, growth kinetics, proliferation, colony formation, wound-healing, transwell-migration assays, and biochemical assays on ASPN knockdown and ASPN overexpressing cells. Our results revealed a reduction in tumor-forming-, colony-forming-, wound healing-, migratory abilities, growth kinetics, and associated signaling (P-ERK, -AKT), and cell cycle (Cyclin D1 and D3) proteins upon ASPN knockdown while the opposite effects were observed in ASPN OE cells. RNA sequencing analysis of ASPN KD revealed the downregulation of top significant genes related to EMT- and neuroendocrine-like phenotype. Interestingly, ASPN KD and OE are associated with EMT switching by downregulating E-cadherin and ZO-1 with subsequent increased protein expression of N-cadherin and Zeb1. Upon RNA sequencing validation, CRABP2 emerged to be a neuroendocrine-associated protein significantly regulated by ASPN. Further, ASPN KD and OE regulate neuroendocrine markers such as Chromogranin A, NeuroD1, and INSM1 along with loss of RB function.

In conclusion, our studies revealed the significance of the ASPN/CRABP2 axis in the PCa initiation, progression, and cell-type phenotypic switch to neuroendocrine PCa (NEPC). In the future, this study will help to develop drugs targeting the ASPN/CRABP2 axis to control the specific mechanisms driving PCa towards NEPC, which would be crucial in overcoming t-NEPC.

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