Golgi scaffold protein PAQR3 as a candidate suppressor of gastric cardia adenocarcinoma via regulating TGF-ß/Smad pathway.

OBJECTIVES: To investigate the function of PAQR3 in gastric cardia adenocarcinoma (GCA) and understand the possible mechanism of PAQR3 in regulating epithelial-mesenchymal transition (EMT). METHODS: We detected PAQR3 protein in 146 GCA tissues and paired normal adjacent tissues (PNTs) specimens using immunohistochemical analysis, and explored its clinical significance. The expression levels of PAQR3 protein in 20 GCA tissues, their paired PNTs, HGC27, SGC7901, and GES-1 cells were analyzed by Western blot. Wild-type PAQR3 was overexpressed in HGC27 cells. The effects of PAQR3 overexpression on the function of HGC27 cells and its underlying mechanisms were then analyzed through a series of cell and molecular biology experiments. RESULTS: PAQR3 was significantly down-regulated in GCA tissues when compared with paired PNTs (p < 0.0001). The expression level of PAQR3 in GCA tissues was significantly negatively correlated with Helicobacter pylori infection (p = 0.000), venous invasion (p = 0.000), invasion depth (p = 0.000), lymph node metastasis (p = 0.022), tumor stage (p = 0.000), and patient survival (p = 0.009). Downregulation of PAQR3 was highly correlated with increased EMT signature and activated TGF-ß/Smad pathway in GCA tissues. Overexpression of PAQR3 in HGC27 cells negatively regulates its cellular functions, such as cell proliferation and migration, and suppresses EMT. Mechanistically, overexpression of PAQR3 significantly down-regulates the protein expression levels of TGF-1, p-Smad2, and p-Smad3 in HGC27 cells. CONCLUSION: PAQR3 was significantly down-regulated in GCA tissues, HGC27, and SGC7901 cells. PAQR3 significantly inhibits the proliferation, migration, and invasion of HGC27 cells. Mechanistically, PAQR3 can inhibit the EMT process in HGC27 cells by regulating TGF-ß/Smad signaling pathway.

Circulating methylated THBS1 DNAs as a novel marker for predicting peritoneal dissemination in gastric cancer.

OBJECTIVES: Thrombospondin 1 (THBS1) is known to play a key role in tumor metastasis, and aberrant DNA methylation is one of the mechanisms regulating THBS1. The present study investigated whether methylated THBS1 in circulating cell-free DNA from preoperative peritoneal lavage fluid (PPLF) and peripheral blood could be used as a potential biomarker for predicting peritoneal dissemination in gastric cancer (GC) patients. METHODS: The status of THBS1 methylation was detected by quantitative methylation-specific PCR (MSP) in tumor tissues, paired PPLF, and serum from 92 GC patients. The correlation between methylated THBS1 levels and peritoneal dissemination of GC was studied, and its diagnostic value for predicting peritoneal dissemination was clarified by the receiver operating characteristic (ROC) curve. RESULTS: Aberrant THBS1 methylation in tumor tissues was significantly higher than that in paracancerous normal tissues (p < 0.0001). No THBS1 methylation was found in 40 healthy controls, and partial methylation was detected in 3 of 48 patients with chronic non-atrophic gastritis. The frequency of THBS1 methylation in pairing PPLF and serum from 92 GC patients was 52.2% (48/92) and 58.7% (54/92), respectively. The results of methylated THBS1 in pairing PPLF and serum were similar to those of tumor tissues. Aberrant THBS1 methylation in tumor tissues and pairing PPLF or serum was closely related to peritoneal dissemination, tumor progression, and poor prognosis (all p < 0.0001). CONCLUSION: Circulating methylated THBS1 DNAs in PPLF/serum may predict peritoneal dissemination, a potential poor prognostic factor for GC patients.

A Potential Oncogenic Role for PFKFB3 Overexpression in Gastric Cancer Progression.

OBJECTIVES: PFKFB3 regulates glycolysis in tumor cells, might function as an oncogene, and is associated with cancer metastasis. However, its role in gastric cancer (GC) remains largely unknown. METHODS: PFKFB3 expression was assessed by immunohistochemistry (IHC) in GC tissues and paired paracancerous histological normal tissues (PCHNTs). The associations of PFKFB3 expression with clinical features and HIF-1α, Ki-67, E-cadherin, Snail, and Vimentin expression levels were assessed. A series of in vivo and in vitro experiments were performed to investigate the effects of PFKFB3 on the growth, migration, and invasion of GC cells. RESULTS: We found that PFKFB3 expression was significantly higher in GC tissues compared with PCHNTs (P = 0.000). PFKFB3 expression was positively correlated with tumor size (P = 0.000), differentiation (P = 0.025), venous invasion (P = 0.084), nerve invasion (P = 0.014), lymphatic invasion (P = 0.000), local invasion (P = 0.000), invasive depth (P = 0.000), nodal metastasis (P = 0.000), tumor-node-metastasis stage (P = 0.000), and patient survival (P = 0.000). Notably, PFKFB3 upregulation was highly correlated with increased epithelial-mesenchymal transition (EMT) in GC samples. PFKFB3 overexpression positively modulated cell proliferation, migration, and EMT in GC cells in vitro, with concomitant activation of NF-κB signaling. Administration of an NF-κB inhibitor attenuated PFKFB3-induced EMT in GC cells. PFKFB3 overexpression promoted tumor development and EMT in nude mice, which were attenuated by PFK-15, a PFKFB3 inhibitor. DISCUSSION: PFKFB3 could potentiate malignancy in GC cells through NF-κB pathway-mediated EMT, suggesting PFKFB3 represents a potential target for GC therapy.

Oxidative stress-mediated AMPK inactivation determines the high susceptibility of LKB1-mutant NSCLC cells to glucose starvation.

The liver kinase B1 (LKB1) is an important tumor suppressor and its loss-of-function mutations are observed in around 16% of non-small cell lung cancer (NSCLC) cases. One of the main functions of LKB1 is to activate AMP-activated protein kinase (AMPK) via direct phosphorylation. Under metabolic or energy stress conditions, the LKB1-AMPK axis inhibits the anabolic pathways and activates the catabolic pathways to maintain metabolic homeostasis for cell survival. In this study, we found that LKB1-mutant NSCLC cells are particularly susceptible to cell death induced by glucose starvation, but not by other forms of starvation such as amino acid starvation or serum starvation. Reconstitution of LKB1 in LKB1-mutant cells or LKB1 knockout in LKB1-wild type cells highlighted the importance of the LKB1-AMPK axis for cell survival under glucose starvation. Mechanistically, in LKB1-mutant cells, glucose starvation elicits oxidative stress, which causes AMPK protein oxidation and inactivation, and eventually cell death. Importantly, this process could be effectively reversed and rescued by 2DG (a glucose analog capable of producing NADPH, a key antioxidant), A769662 (an allosteric AMPK activator), and N-acetyl cysteine (NAC) (a ROS scavenger), indicating the presence of a vicious circle between AMPK inactivation and ROS in LKB1-mutant NSCLC cells under glucose starvation. Our study thus elucidates the critical role of redox balance in determining the susceptibility to cell death under glucose starvation in LKB1-mutant NSCLC cells. The findings from this study reveal important clues in search of novel therapeutic strategies for LKB1-mutant NSCLC by targeting glucose metabolism and redox balance.