Author

Saini Setua

Date of Award

5-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Track

Pharmaceutics

Research Advisor

Subhash C. Chauhan, Ph.D.

Committee

Stephen W. Behrman, MD, FACS Santosh Kumar, Ph.D. Yi Lu, Ph.D. Murali M. Yallapu, Ph.D.

Abstract

Pancreatic cancer (PanCa) is the third deadliest cancer in the USA due to the late diagnosis and development of chemo-resistance, with a 5-year survival rate of less than 10%. The prognosis of patients with pancreatic ductal adenocarcinoma is extremely poor, and current therapies such as Gemcitabine, 5-FU, Nab-paclitaxel and, FOLFIRINOX alone or in combination, have displayed improved but marginal survival rates for patients. Therefore, research efforts are underway to discover new therapeutic options to treat PanCa and overcome resistance to available therapies.

Mucin, MUC13 is transmembrane glycoprotein, which is aberrantly overexpressed in PanCa and promoting cancer growth. Structural domains of MUC13, lead to oncogenic characteristics during cancer progression. Our lab previously established the role of MUC13 in tumor progression and metastasis by alteration of signaling pathways. Recent observations suggest the role of MUC13 in drug resistance and apoptosis in several cancer types. Therefore, it is of great interest to explore the role of MUC13 in chemoresistance in PanCa. Unlike other cancer types, PanCa is highly resistant to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) that emerges as one of the most-promising cancer therapeutic drugs. It is a death ligand that can selectively induce apoptosis in cancer cells over normal cells. Our recent work has demonstrated that MUC13 expressing cells showed resistance to TRAIL induced cell death and MUC13 knockdown leads to TRAIL sensitivity in cells. We have also observed that MUC13 expression blocks the activation of caspase-8 and Bid in PanCa cells in response to TRAIL treatment. Further investigation showed that alpha and beta domains of MUC13 are indispensable for blocking caspase-8 activation and PARP cleavage, indicating that the MUC13 blocks TRAIL-induced signaling upstream to Bid by inhibiting caspase-8 activation. Current studies revealed a new role of MUC13 ininhibiting TRAIL mediated activation of extrinsic apoptotic pathway in pancreatic cancer.

MicroRNAs (miRNA) have been identified as attractive targets for therapeutic intervention. The functional significance of lost miRNAs have been reported in several human malignancies, including PanCa. Restitution of lost miRNA function can provide a potential therapeutic benefit. Prior work has identified microRNA-145 (miR-145) as a tumor suppressor miRNA in PanCa. The restoration of miR-145 downregulates a number of oncogenes including mucin MUC13 and efficiently inhibits tumor growth in mice. Inhibition of MUC13 using miR-145 restoration resulted in TRAIL mediated increase in apoptotic cell death as evidenced by sub-G0 population and inhibition of MUC13, activation of caspase 8 and, cleavage of PARP-1. MiR-145 replacement can sensitize TRAIL therapy and counteract chemoresistance mechanism in PanCa.

The main challenge for successful translation of miRNAs into clinical practice remains an effective in vivo delivery system. Hence, the focus of this study was to develop and assess the efficacy of a miR-145 based nanoparticle formulation for PanCa treatment. Magnetic nanoparticle (MNP) based nanoformulation of miR-145 (miR-145-MNPF) was developed for the intracellular delivery and sustained release of miR-145. The positively charged polyethyleneimine molecules were used to increase the loading efficiency of miR-145. Treatment of cells with miR-145-MNPF led to efficient intracellular delivery of miR-145 mimics as observed through Prussian blue staining. This led to the simultaneous upregulation of miR-145 levels in cells which resulted in significant downregulation of target oncogenes including MUC13, HER2, pAKT and p53. miR-145-MNPF efficiently restores miR-145 in PanCa cells and inhibits growth and invasion of PanCa. miR-145 restitution using miR-145-MNPF may offer a potential therapeutic strategy for pancreatic cancer.

As discussed earlier that miR-145 restoration sensitized the TRAIL therapy in PanCa cells. Herein, we demonstrate the integration of novel delivery approach to reduce the delivery challenge of TRAIL. We have engineered unique superparamagnetic nanoparticles (MNPs) for co-delivering miR-145 and plasmid TRAIL for improving TRAIL response in PanCa model. MNP-miR-145-TRAIL nanoparticles were codelivered miR-145 and TRAIL to PanCa cells, which resulted in simultaneous restoration of miR-145 and inhibition of acquired resistance to TRAIL.

The current study demonstrates that acquired resistance to TRAIL in PanCa cells can be minimized with the replenishment of miR-145 expression. Combined actions of miR-145 and TRAIL markedly improve TRAIL-induced apoptotic effects in PanCa cells through the activation of an extrinsic apoptosis pathway as indicated by activation of DR4, FLIP, FADD and enhanced expression of cleaved caspase-8. The co-delivery of miR-145 and TRAIL using MNP nanoparticles inhibited tumorigenic characteristics of PanCa cells. The results were reciprocated and were further confirmed with the inhibition of tumorsphere formation and in vivo tumorigenicity in xenograft mice. Immunohistochemical staining of excised tumor tissues demonstrates an activation of the death receptor pathway and subsequent expression of apoptotic markers.

Pancreatic tumor microenvironment is a complex dynamic space which leads to desmoplasia and involved in metastasis and impediments against intracellular drug delivery. Despite extensive research efforts, there is not considerable progress in cancer therapeutics due to genomic complexity and heterogenicity of pancreatic cancer. Modern tumor therapy must be patient specific and customized for individual patients. It should be tailored for a patient-based response to the specific treatment. Thus, novel delivery vehicles are required that are biocompatible and non-immunogenic. This is possible by utilizing an autologous biological material as delivery vehicles that can be applied as a personalized medicine. Towards this, our lab has optimized an exosome based therapeutic approach, which utilizes exosomes isolated from the cultured tumor adjacent normal (NAT) fibroblast cells. We utilized this scaffold for safe and effective delivery of therapeutic payload. Our results demonstrated that NAT derived exosomal formulation (Exo-ORM) significantly enhanced the efficacy of ormeloxifene to inhibit stroma as indicated by decreased expression of α-SMA, desmin and hyaluronic acid. Exo-ORM formulation effectively inhibit EMT/SHH signaling in PanCa cells and in vivo models. NAT derived exosomes will be a promising therapeutic carrier with preferential size for passive targeting, proficient biophysical characteristics, biocompatible and nonimmunogenic vehicle for PanCa therapy.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

http://orcid.org/0000-0002-9874-6948

DOI

10.21007/etd.cghs.2020.0503

2020-011-Setua-DOA.pdf (461 kB)
Declaration of Authorship

Available for download on Thursday, May 13, 2021

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