Theses and Dissertations

Date of Award

7-2022

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry and Molecular Biology

First Advisor

Dr. Sheema Khan

Second Advisor

Dr. Subhash Chauhan

Third Advisor

Dr. Murali Yallapu

Abstract

The poor patient survival rate in pancreatic ductal adenocarcinoma (PDAC) remains a challenge. KRAS activating point mutation on codon-12 is found in 70–95 percent of PDAC patients, and no progress in inhibiting KRAS has been obtained thus far. KRASG12D is a transcription factor that controls cell proliferation, differentiation, and apoptosis. Recent preliminary and published research indicates that Galectin-1 (Gal-1) levels are high in both PDAC and stromal cells, which modulates tumor microenvironment and metastasis. As a result, we created a new combination treatment for PDAC that targets both proliferation and metastasis in PDAC by targeting mutant KRASG12D and Gal-1. This includes the delivery of KRASG12D inhibiting siRNA (siKRASG12D) using a superparamagnetic iron oxide nanoparticle (SPION) and a gal-1 inhibitor.

Our patented SPION nano-formulation was used to deliver siKRASG12D and investigated in conjunction with Gal-1 inhibitor for its anticancer efficacy. Particles were investigated for size, physico-chemical characterization (Dynamic light scattering), hemocompatibility (hemolysis assay) and the complexation of siKRAS (gel retardation assay). Cellular internalization and uptake of the particles were investigated. Anti-cancer efficacy was determined using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), clonogenicity, wound healing, tumor spheroid formation, and in a mouse model.

Our findings show that the SP-siKRAS formulation has an excellent particle size/zeta potential. SP-siKRAS internalized effectively in PDAC cells, suppressing KRASG12D as well as its downstream targets, YAP and PDL-1. Cell growth was decreased when siKRAS and Gal-1 were both targeted. It decreased PDAC cell growth, clonogenicity, migration, and invasion. This resulted in the activation of death-related pathways in KRASG12D cells, such as Bax, bcl-2, and PARP cleavage. Surprisingly, the formulation was particularly efficient in reducing KRASG12D and tumor spheroid formation in 3D cell models, resembling the heterogeneity and pathogenesis of PDAC. This adds to the clinical confirmation of SP-siKRAS particles' ability to effectively suppress KRAS expression. SP-siKRAS also demonstrated hemocompatibility and stability, suggesting that it has the potential to silence KRAS without being hazardous to the body. In KPC mouse model C57BL/6J mice, the formulation effectively silenced KRasG12D and reduced tumor development and metastasis.

This gene therapy targeting KRASG12D mutation with Gal-1 inhibition has the ability to modify the oncogenic network and tumor microenvironment, resulting the repression of growth, metastasis, chemoresistance, and improvement in patient survival. This project will create a fresh, long-term treatment strategy to target PDAC development and patient survival.

Comments

Copyright 2022 Ana Izavelle Martinez Bulnes. All Rights Reserved.

https://go.openathens.net/redirector/utrgv.edu?url=https://www.proquest.com/dissertations-theses/targeting-mutated-kras-galectin-1-pancreatic/docview/2742637797/se-2?accountid=7119

Download

Share

COinS