Microvesicle particles (MVP) secreted by a variety of cell types in response to reactive oxygen species (ROS)-generating pro-oxidative stressors have been implicated in modifying the cellular responses including the sensitivity to therapeutic agents. Our previous studies have shown that expression of a G-protein coupled, platelet-activating factor-receptor (PAFR) pathway plays critical roles in pro-oxidative stressors-mediated cancer growth and MVP release. As most therapeutic agents act as pro-oxidative stressors, the current studies were designed to determine the role of the PAFR signaling in targeted therapies (i.e., gefitinib and erlotinib)-mediated MVP release and underlying mechanisms using PAFR-expressing human A549 and H1299 non-small cell lung cancer (NSCLC) cell lines. Our studies demonstrate that both gefitinib and erlotinib generate ROS in a dose-dependent manner in a process blocked by antioxidant and PAFR antagonist, verifying their pro-oxidative stressor’s ability, and the role of the PAFR in this effect. We observed that these targeted therapies induce MVP release in a dose- and time-dependent manner, similar to a PAFR-agonist, carbamoyl-PAF (CPAF), and PAFR-independent agonist, phorbol myristate acetate (PMA), used as positive controls. To confirm the PAFR dependency, we demonstrate that siRNA-mediated PAFR knockdown or PAFR antagonist significantly blocked only targeted therapies- and CPAF-mediated but not PMA-induced MVP release. The use of pharmacologic inhibitor strategy suggested the involvement of the lipid ceramide-generating enzyme, acid sphingomyelinase (aSMase) in MVP biogenesis, and observed that regardless of the stimuli used, aSMase inhibition significantly blocked MVP release. As mitogen-activated protein kinase (MAPK; ERK1/2 and p38) pathways crosstalk with PAFR, their inhibition also significantly attenuated targeted therapies-mediated MVP release. These findings indicate that PAFR signaling could be targeted to modify cellular responses of targeted therapies in lung cancer cells.

Accumulating evidence indicates that microRNAs (miRs) play critical roles in essentially all biological processes and their altered expression has been documented in various disease conditions, including human malignancies. Although several cellular mechanisms have been identified in mediating the effects of miRs, the involvement of G-protein-coupled, platelet-activating factor-receptor (PAFR) signaling in miR-149-5p-induced effects on lung cancer growth and therapeutic potential has not been studied. To that end, we first evaluated the functional significance of PAFR and miR-149-5p in A549 and H1299 human non-small cell lung cancer (NSCLC) cell lines. We observed that these tumor lines express endogenous PAFR and miR-149-5p and that PAFR activation by PAF agonist (CPAF) significantly increased, whereas miR-149-5p mimic transfection inhibited cell proliferation in a dose-dependent manner. Interestingly, miR-149-5p mimic significantly attenuated CPAF-mediated increased proliferation of NSCLC cells, as confirmed by miR-149-5p, cyclin D1, and forkhead box protein M1 (FOXM1) expression analysis via qPCR. Our next studies examined PAFR- and miR-149-5p-mediated effects on targeted therapy (i.e., erlotinib and gefitinib) responses. We observed that erlotinib and gefitinib inhibited A549 and H1299 cell survival in a dose- and time-dependent manner, and CPAF significantly blocked this effect. These findings indicate that miR-149-5p blocks PAFR-mediated increased cell proliferation, and PAFR activation attenuates the cytotoxic effects of targeted therapy.

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) signaling in augmenting tumor growth or limiting therapy effectiveness in various experimental cancer models. While several mechanisms of the PAF-R pathway have been proposed, its effect with microRNAs (miRs) has 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. The current study aimed to determine the effects of PAF-R and miR-149 in lung cancer growth and therapy effectiveness. We first evaluated the functional significance of PAF-R and miR-149 using A549 and H1299 human non-small cell lung cancer (NSCLC) cell lines as tools. These tumor lines express endogenous PAF-R and miR-149, 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 CPAF-mediated 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 inhibited the survival of A549 and H1299 cell lines in a dose-and time-dependent manner. While CPAF significantly blocked erlotinib and gefitinib (at wIC 50 dose)-mediated decreased cell proliferation, PAF-R antagonist and miR-149 mimic did not exert any effects. While additional studies are needed, these findings indicate that miR-149 overcomes PAF-Rmediated increased cell proliferation effect, and PAF-R activation attenuates cytotoxic response of targeted therapy.

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) signaling in augmenting tumor growth or limiting therapy effectiveness in various experimental cancer models. While several mechanisms of the PAF-R pathway have been proposed, its effect with microRNAs (miRs) has 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. The current study aimed to determine the effects of PAF-R and miR-149 in lung cancer growth and therapy effectiveness. We first evaluated the functional significance of PAF-R and miR-149 using A549 and H1299 human non-small cell lung cancer (NSCLC) cell lines as tools. These tumor lines express endogenous PAF-R and miR-149, 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 CPAF-mediated 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 inhibited the survival of A549 and H1299 cell lines in a dose-and time-dependent manner. While CPAF significantly blocked erlotinib and gefitinib (at wIC 50 dose)-mediated decreased cell proliferation, PAF-R antagonist and miR-149 mimic did not exert any effects. While additional studies are needed, these findings indicate that miR-149 overcomes PAF-Rmediated increased cell proliferation effect, and PAF-R activation attenuates cytotoxic response of targeted therapy.