Publication Date

2020

Document Type

Thesis

Committee Members

Ravi P. Sahu, Ph.D. (Advisor); Anita Thyagarajan, Ph.D. (Committee Member); Jeffrey B. Travers, M.D., Ph.D. (Committee Member); David R. Cool, Ph.D. (Committee Member)

Degree Name

Master of Science (MS)

Abstract

Lung cancer remains the leading cause of cancer-related deaths, with low response rates to the current treatment options, indicating the need to explore potential factors, involved in lung cancer growth or impeding the efficacy of therapeutic agents. Studies, including ours, have shown the critical roles of a G-protein coupled, platelet-activating factor-receptor (PAF-R) in augmenting tumor growth or limiting therapy effectiveness in various experimental cancer models via mechanisms involving the secretion of microvesicle particles (MVPs). However, the effects of PAF-R pathway with microRNAs (miRs) have not been studied. In particular, while miR-149 has been shown to play oncogenic roles in other cancer types, it functions as a tumor suppressor in lung cancer. In this project, we tested the effects of PAF-R, and miR-149 in lung cancer growth, targeted therapy efficacy, and MVP secretion using A549 and H1299 human non-small cell lung cancer (NSCLC) cell lines as tools. Our first studies evaluated that these tumor lines express endogenous PAF-R and miR-149 expression, and PAF-R activation by PAF agonist (CPAF) significantly increased-, whereas miR-149 mimic transfection inhibited cell proliferation in a dose-dependent manner. Interestingly, miR-149 mimic significantly attenuated CPAFmediated increased proliferation of A549 cells, as also confirmed by miR-149 expression analysis via qPCR. We then examined PAF-R and miR-149 effects on currently used targeted therapy (i.e., erlotinib and gefitinib) responses. Both these agents decreased the survival of NSCLC cell lines in a dose- and time-dependent manner. While CPAF significantly blocked erlotinib and gefitinib (at ~IC₅₀ dose)-mediated decreased cell proliferation, PAF-R antagonist, and miR-149 mimic did not exert any effects. Our next studies determined the effect of PAF-R on targeted therapy-mediated MVP release using CPAF and phorbol myristate acetate (PMA), a PAF-R independent agonist as positive controls. We observed that erlotinib and gefitinib induce MVP release in a dose- and timedependent manner, and that MVP secretion peaked between 4 to 8 hours’ time points with similar effects, so our further studies used a 4-hour time point with an optimal dose of targeted therapy. As MVP biogenesis regardless of the stimuli, involves acid sphingomyelinase enzyme (aSMase), we next observed that aSMase-specific inhibitor significantly blocked not only targeted therapy-mediated, and also CPAF- and PMAinduced MVP release from A549 cells. To confirm the PAF-R dependency, we tested that PAF-R knockdown via specific siRNA as measured by qPCR or PAF-R antagonist significantly blocked only targeted therapy- and CPAF-mediated but not PMA-induced MVP release. Mechanistically, mitogen-activated protein kinase (MAPK, particularly, ERK and p38) pathway inhibitors significantly attenuated targeted therapy-mediated MVP release. Overall, our studies indicate that miR-149 overcomes PAF-R-mediated increased cell proliferation effect, and that PAF-R activation attenuates cytotoxic response of targeted therapy, which could be mediated via MVP release. These findings indicate the potential role of PAF-R signaling in modulating targeted therapy-response, which could have cellular and systemic effects in lung cancer.

Page Count

65

Department or Program

Department of Pharmacology and Toxicology

Year Degree Awarded

2020

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