Author ORCID Identifier

0000-0002-1100-1768

Date of Graduation

5-2017

Document Type

Dissertation (PhD)

Program Affiliation

Cancer Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Raghu Kalluri, M.D., Ph.D.

Committee Member

Giulio Draetta, M.D., Ph.D.

Committee Member

Michael Andreeff, M.D., Ph.D.

Committee Member

Frederick Lang, M.D., F.A.C.S., F.A.A.N.S.

Committee Member

Anirban Maitra, M.B.B.S.

Abstract

Despite the current standard of care, the prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) remains dismal. Genetic analyses of PDAC show that mutations in the GTPase KRAS are encountered in the majority of patients that present with the disease, and are key drivers of cancer initiation, progression and metastasis. However, while it is recognized that the Ras mediated signaling pathway is a key mediator of PDAC progression, a direct and specific targeting of Ras has been elusive. Therefore, due to the indisputable evidence for the causal functional role of RAS in the biology of PDAC, new approaches to specifically and efficiently target it must be explored.

Currently, many different strategies to target oncogenic Ras, including RNA interference, are being explored. However, a major hurdle in employing specific RNAi constructs to inhibit Ras is the lack of an optimal delivery system. Therefore, we probed whether exosomes could function as an efficient carrier of RNAi molecules. Exosomes are nano-sized extracellular vesicles (40-150nm) with a membrane lipid bilayer that are released by all cells of the body and efficiently enter other cells. Therefore exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry RNA interference molecules to specifically target oncogenic KRASG12D, a mutation identified in about 50% of patients with pancreatic cancer. Compared to liposomes, exosomes containing siRNA or shRNA specific to KrasG12D mRNA (iExosomes) targeted oncogenic Kras with an enhanced efficacy. iExosomes treatment suppressed tumor growth in multiple pre-established orthotopic human pancreatic cancer models, genetically engineered mouse models (GEMMs), as well as in a patient derived xenograft (PDX) model of pancreatic cancer, and significantly increased their overall survival. In addition we demonstrate that the enhanced efficacy iExosomes is in part due to enhanced survival of exosomes in the circulation, as compared to liposomes, which is mediated by CD47, a widely expressed integrin associated transmembrane protein found on exosomes. We also reveal the role of macropinocytosis in aiding the entry of exosomes into oncogenic Ras mutant pancreatic cancer cells. Overall, our results inform on a novel approach of direct and specific targeting of oncogenic Kras in tumors using iExosomes.

Keywords

Pancreatic cancer, Exosomes, Kras, Drug delivery, CD47, RNAi

Available for download on Saturday, May 05, 2018

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