NANOGP8 is the key regulator of stemness, EMT, Wnt pathway, chemoresistance, and other malignant phenotypes in gastric cancer cells

Abstract

BackgroundAccumulating evidence demonstrated that NANOG1, the key transcription factor for embryonic stem cells, is associated with human cancers. NANOGP8, one of the pseudogenes in NANOG gene family, contains an intact open reading frame and also said to be expressed in cancer tissues. Therefore, a systematic study is greatly needed to address the following questions: among NANOG1 and NANOGP8, which gene is the main contributor for NANOG expression in cancer cells and which one is the key regulator responsible for stemness, epithelial-mesenchymal transition (EMT), metastasis, chemoresistance and other malignant phenotypes. Here we try to explore these issues with gastric adenocarcinoma cell lines in vitro using variety of molecular and cellular techniques.MethodsSpecial primers were designed to distinguish PCR products from NANOG1 and NANOGP8. Sphere-forming cells were cultured with serum-free and selective medium. A stable cell line was established with infection of lentivirus containing NANOGP8. qPCR was performed to measure NANOGP8 expression and its association with stemness, EMT and CSC markers in adherent cells and sphere-forming cells. Western blot analysis was deployed to confirm results of the transcript analysis. Experiments of cell proliferation, migration, invasion, clonogenic assay, sphere cell growth assays, cell cycle analysis, β-catenin accumulation and translocation in nucleus, and drug resistance were conducted to measure the impact of NANOGP8 on malignant statuses of gastric cancer cells. Immunofluorescence staining was used to analyze cell subpopulations with different markers.ResultsNANOGP8 is mainly responsible for NANOG expression in sphere-forming (stem cell-like) cells derived from gastric cancer cell lines regardless their differentiation status. Ectopic expression of NANOGP8 significantly up-regulates stemness transcription factors, EMT inducers, and cancer stem cell markers (CSC) including Lgr5. NANOGP8 also promotes expression of the signature genes vimentin and N-caderin for mesenchymal cells and down-regulates the signature gene E-caderin for epithelial cells whereby confer the cells with mesenchymal cell phenotype. In NANOGP8 over-expressed adherent and sphere-forming cells, Lgr5+ cells are significantly increased. Ectopic expression of NANOGP8 endows gastric cells with enhanced proliferation, migration, invasion, sphere-forming and clonogenic capacity, and chemoresistance. NANOGP8 expression also enhances β-catenin accumulation in nucleus and strengthens Wnt signal transduction.ConclusionNANOGP8 is the main regulator of gastric cancer stem cells. It is closely associated with EMT, stemness, and CSC marker as well as Wnt signal pathway. NANOGP8 is correlated with cell proliferation, migration, invasion, clonogenic capacity, β-catenin accumulation in nucleus, and chemoresistance in gastric cancer. NANOGP8 is a promising molecular target for clinical intervention of gastric cancer.

Pseudogene-Derived lncRNAs and Their miRNA Sponging Mechanism in Human Cancer

“…NANOGP8 expression was significantly elevated in gastric cancer and knockdown of NANOGP8 resulted in decreased proliferation and promoted apoptosis of gastric cancer cells . Ma X. et al (2018) also suggested that NANOGP8 was a positive regulator of gastric cancer stem cells, associated with epithelial-mesenchymal transition, stemness, cancer stem cell markers as well as Wnt signaling pathway. DUXAP8, a 2107 nt RNA derived from pseudogene, was obviously upregulated in gastric cancer and significantly associated with greater tumor size, advanced clinical stage and lymphatic metastasis .…”

The importance of epithelial-mesenchymal transition and autophagy in cancer drug resistance

“…In gastric cancer, NANOGP8 overexpression leads to anti-oxaliplatin (L-OHP) resistance. It upregulates EMT markers and increases β-catenin accumulation in the nucleus and strengthens Wnt signalling [68] . Activation of the Hh pathway has also been linked to drug resistance in both non-small cell lung cancer with resistance to EGFR-TKIs [61] and in CRC with resistance to cetuximab [69] .…”

“…Several other EMT-inducing pathways have also been directly linked to drug resistance in cancer. Wnt has been demonstrated to cause drug resistance in HER2-overexpressing breast cancer, Type-1 epithelial ovarian cancer (EOC) and gastric cancer [60,67,68] . In HER2-overexpressing breast cancer cells, it is considered that Wnt3 overexpression may activate Wnt/β-catenin transactivating EGFR, which can lead to a partial-EMT that could be important in understanding trastuzumab resistance in these cells [60] .…”

Mechanisms of the Epithelial–Mesenchymal Transition and Tumor Microenvironment in Helicobacter pylori-Induced Gastric Cancer

“…MSCs promote EMT, tumor growth, and metastasis [295,296]. Nanog Homeobox Retrogene P8 (NANOGP8) makes up the main regulator of gastric CSCs [297].…”