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

Abstract

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.

Lysophosphatidic acid suppresses apoptosis of high-grade serous ovarian cancer cells by inducing autophagy activity and promotes cell-cycle progression via EGFR-PI3K/Aurora-AThr288-geminin dual signaling pathways

“…The pellet was resuspended with 20 μL 5× SDS sample buffer. Proteins were then detected by immunoblotting assay (Zhao et al, 2021).…”

“…A HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme Biotech) was used to generate the cDNA template. qRT-PCR was performed with TransStart Tip Green qPCR SuperMix (TransGen Biotech) on a LightCycler 480 II system (Roche, Basel, Switzerland) (Zhao et al, 2021). Oligonucleotide sequences (Sangon Biotech) are described in Table S2.…”

“…DNA was visualized by staining with DAPI (1:5,000 in water). The immunofluorescence analysis was visualized with a laser scanning confocal microscope (LSM 710, CarlZeiss, Wetzlar, Germany) (Zhao et al, 2021).…”