Lysophosphatidic Acid–Induced EGFR Transactivation Promotes Gastric Cancer Cell DNA Replication by Stabilizing Geminin in the S Phase

Geminin, an inhibitor of the DNA replication licensing factor, chromatin licensing and DNA replication factor (Cdt) 1, is essential for the maintenance of genomic integrity. As a multifunctional protein, geminin is also involved in tumor progression, but the molecular details are largely unknown. Here, we found that lysophosphatidic acid (LPA)–induced upregulation of geminin was specific to gastric cancer cells. LPA acted via LPA receptor (LPAR) 3 and matrix metalloproteinases (MMPs) signaling to transactivate epidermal growth factor receptor (EGFR) (Y1173) and thereby stabilize geminin expression level during the S phase. LPA also induced the expression of deubiquitinating protein (DUB) 3, which prevented geminin degradation. These results reveal a novel mechanism underlying gastric cancer progression that involves the regulation of geminin stability by LPA-induced EGFR transactivation and provide potential targets for the signaling pathway and tumor cell–specific inhibitors.

Targeting PAR2 Overcomes Gefitinib Resistance in Non-Small-Cell Lung Cancer Cells Through Inhibition of EGFR Transactivation

Drug resistance can notably restrict clinical applications of gefitinib that is a commonly used EGFR-tyrosine kinase inhibitors (EGFR-TKIs) for non-small cell lung cancer (NSCLC). The attempts in exploring novel drug targets and reversal strategies are still needed, since gefitinib resistance has not been fully addressed. Protease-activated receptor 2 (PAR2), a G protein-coupled receptor, possesses a transactivation with EGFR to initiate a variety of intracellular signal transductions, but there is a lack of investigations on the role of PAR2 in gefitinib resistance. This study established that protease-activated receptor 2 (PAR2), actively participated in NSCLC resistant to gefitinib. PAR2 expression was significantly up-regulated when NSCLC cells or tumor tissues became gefitinib resistance. PAR2 inhibition notably enhanced gefitinib to modulate EGFR transactivation, cell viability, migration and apoptosis in gefitinib-sensitive and-resistant NSCLC cells, suggesting its reversal effects in gefitinib resistance. Meanwhile, the combination of a PAR2 inhibitor (P2pal-18S) and gefitinib largely blocked ERK phosphorylation and epithelial-mesenchymal transition (EMT) compared to gefitinib alone. Importantly, we probed its underlying mechanism and uncovered that PAR2 blockade sensitized gefitinib and reversed its resistance mainly via β-arrestin-EGFR-ERK signaling axis. These effects of PAR2 inhibition were further confirmed by the in vivo study which showed that P2pal-18S reactivated gefitinib to inhibit tumor growth via restricting ERK activation. Taken together, this study could not only reveal a new mechanism of receptor-mediated transactivation to modulate drug resistance, but also provide a novel drug target and direction for overcoming gefitinib resistance in NSCLC.

Lysophosphatidic Acid-induced EGFR Transactivation Promotes Gastric Cancer DNA Replication Through Up-Regulation of Geminin

BackgroundLysophosphatidic acid (LPA) is one of the simplest active phospholipid molecules. Binding to its receptors on the cell surface, LPA initiates various intracellular signal cascades, involving in numerous biological processes, such as cell proliferation, migration, and apoptosis. If abnormalities occur in the processes of LPA production, receptor expression or signal transduction, it may induce certain diseases and even contribute to the occurrence, development and metastasis of cancer. However, whether the initiation of DNA replication is regulated by LPA has not yet been investigated.MethodsFirst, diverse public databases were analyzed to explore the genetic abnormalities affecting geminin. Next, an LPA gradient treatment was performed on gastric cancer cells, followed by detecting geminin expression variation using western blot analysis. Finally, RNAi technology or inhibitors were used to block the biological activity of related factors in the GPCR induced EGFR transactivation signaling pathway to verify whether the effect of LPA evoked gastric cancer DNA replication is dependent on geminin upregulation.ResultsWe found that LPA specifically up-regulated expression of an essential replication negative regulator geminin in early S phase in gastric cancer cell lines, and that the deletion of geminin selectively induced DNA re-replication. Neither of these phenomena has been observed in normal gastric epithelial cells, indicating LPA-induced geminin up-regulation is restricted to tumor cells. Using RNAi or specific inhibitors to block the activity of related factors in the signaling pathway, we found that LPA acts through LPAR3 and downstream coupled MMPs signaling to trans-activate EGFR, increasing the expression level of geminin in S phase. On the other hand, LPA stimulation induced the up-regulation of de-ubiquitinating enzyme 3 (DUB3) in a short time and inhibited the ubiquitination degradation of geminin to enhance geminin stability and positively regulate the DNA replication initiation in gastric cancer cells. Taken together, our results suggested that LPA mediated DNA replication and S-phase cell-cycle progression through a LPAR3/MMPs/EGFR/PI3K/mTOR signaling axis in gastric cancer. ConclusionsOur research is for the first time to study the regulatory effect of LPA-induced EGFR transactivation in DNA replication of tumor cells, and to uncover a novel mechanism for regulating the stability of geminin through LPA and related downstream signaling pathways. All of which will provide potential targets for the development of signaling pathways and tumor cell-specific EGFR transactivation inhibitor for the treatment of gastric cancer.

PLAG Exerts Anti-Metastatic Effects by Interfering with Neutrophil Elastase/PAR2/EGFR Signaling in A549 Lung Cancer Orthotopic Model

The effectiveness of chemotherapy and radiotherapy to treat lung cancer is limited because of highly metastatic nature. Novel strategies and drugs to attenuate metastatic activity are urgently required. In this study, red fluorescence proteins (RFP)-labeled A549 human lung cancer cells were orthotopically implantation, where they developed primary tumors. Metastasis in brain and intestines were reduced by up to 80% by treatment with 100 mpk 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) compared with that in control mice. PLAG treatment also reduced the migration of the primary tumors. Interestingly, substantial neutrophil infiltration was observed in the tumors in control mice. The neutrophil contribution to A549 cell metastatic activity was examined in in vitro co-culture system. Metastatic activity could be achieved in the A549 cells through epidermal growth factor receptor (EGFR) transactivation mediated by protease activating receptor 2 (PAR2) receptor. Neutrophil elastase secreted from tumor-infiltrating neutrophils stimulated PAR2 and induced EGFR transactivation. However, this transactivation was inhibited by inducing PAR2 degradation following PLAG treatment and metastatic activity was effectively inhibited. PLAG attenuated cancer metastatic activity via modulated PAR2/EGFR transactivation by accelerating PAR2 degradation. These results suggest PLAG as potential therapeutic agent to combat tumor metastasis via regulating the activation signal pathway of PAR2 by tumor infiltrate-neutrophils.

Secreted Phospholipase A2-IIA Modulates Transdifferentiation of Cardiac Fibroblast through EGFR Transactivation: An Inflammation–Fibrosis Link

Secreted phospholipase A2-IIA (sPLA2-IIA) is a pro-inflammatory protein associated with cardiovascular disorders, whose functions and underlying mechanisms in cardiac remodelling are still under investigation. We herein study the role of sPLA2-IIA in cardiac fibroblast (CFs)-to-myofibroblast differentiation and fibrosis, two major features involved in cardiac remodelling, and also explore potential mechanisms involved. In a mice model of dilated cardiomyopathy (DCM) after autoimmune myocarditis, serum and cardiac sPLA2-IIA protein expression were found to be increased, together with elevated cardiac levels of the cross-linking enzyme lysyl oxidase (LOX) and reactive oxygen species (ROS) accumulation. Exogenous sPLA2-IIA treatment induced proliferation and differentiation of adult rat CFs. Molecular studies demonstrated that sPLA2-IIA promoted Src phosphorylation, shedding of the membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) ectodomain and EGFR phosphorylation, which triggered phosphorylation of ERK, P70S6K and rS6. This was also accompanied by an up-regulated expression of the bone morphogenic protein (BMP)-1, LOX and collagen I. ROS accumulation were also found to be increased in sPLA2-IIA-treated CFs. The presence of inhibitors of the Src/ADAMs-dependent HB-EGF shedding/EGFR pathway abolished the CF phenotype induced by sPLA2-IIA. In conclusion, sPLA2-IIA may promote myofibroblast differentiation through its ability to modulate EGFR transactivation and signalling as key mechanisms that underlie its biological and pro-fibrotic effects.