The Role Of Transcriptional Regulator HNF4α And FoxM1 In Gastric Carcinogenesis And The Relative Mechanism

BackgroundGastric cancer (GC) is a life-threatening malignancy and the fourth most common cancer worldwide. The incidence and mortality of gastric cancer in China ranked first in the world. Because the symptom of gastric cancer is often mild in early stage, many patients with overt clinical symptoms have been late for the treatment. Many patients with gastric cancer have missed the best timing for surgery and the conventional chemotherapies have many side effects. Therefore, it is very important to reveal the mechanism of gastric cancer, find the key tumor markers and develop new stategy for early prevention and treatment of gastric cancer.The process of gastric cancer is caused by the cumulative effect of multiple factors. Helicobacter pylori infection is one of the major causes in the development of gastric cancer. Infection of H. pylori can cause gastric mucosal injury, inflammatory cell infiltration and lead to chronic gastritis. Persistent inflammation can cause gastric atrophy, intestinal metaplasia, and ultimately result in gastric cancer. Meanwhile, gastric cancer is a highly heterogeneous disease, and ultimately only a small number of patients with H. pylori infection will develop into irreversible malignant stage. The host gene change during H. pylori infection plays a key role in the development of gastric cancer. Therefore, exploration of the differentially expressed molecules during transition from gastritis to gastric cancer is critical for early diagnosis and prevention of gastric cancer.Transcriptional regulators play key roles in gene expression and cellular biological functions. Nuclear receptor superfamily is one of the most abundant elements in charge of transcriptional regulation of human cells, which plays an important role in cell proliferation, differentiation and development of the body’s metabolism. Nuclear receptor binds to a corresponding ligand and regulates transcription of target genes. They can also combine with other transcription factors forming a transcription complex. Less is known about the function of nuclear receptor superfamily in gastric cancer. By microdissection and DNA microarray analysis, we found that, one of the nuclear receptor superfamily member-hepatic nuclear factor 4a (HNF4α) tended to increase during the progression of gastritis to gastric cancer. Homodimer of HNF4α can bind to DNA repeat sequence (AGGTCA)and regulate the transcription of target genes, including molecules involved in cellular metabolism, differentiation and other biological processes. This study reveals the biological function of HNF4α in gastric cancer cells and relative mechanism during evolution of gastric carcinoma.Fox family is another type of transcriptional regulators. FoxM1, one member of Fox family, is increased in many human solid tumors. Inhibition of its expression can significantly delay tumor progression. It can bind to a DNA sequence (5’-A (C/T) AAA (C/T) AA-3’) and regulate target gene expression. Our previous study has showed that FoxM1 is up-regulated in gastric cancer and H. pylori can induce FoxM1 expression. Nowadays, associated drugs targeting FoxM1 has become a hotspot for research. Small molecule siomycin A can reduced FoxM1 expression levels effectively and inhibit the binding of FoxM1 to downstream target gene promoter region. Our study further confirmed the mechanism of FoxM1 in gastric cancer as a drug target.Aims1. Reveal the biological function of HNF4α in gastric cancer cells and relative mechanism during evolution of gastric carcinoma.2. Confirm the mechanism of FoxM1 in gastric cancer and its value as a drug target in treatment of gastric cancer.Methods and Results1. H. pylori can activate HNF4a expression by NF-κB signaling pathway. HNF4a and its target IL-1R1 may form a positive feedback loop involved in malignant transformation of gastric cells.(1)Real-time quantitative PCR (qRT-PCR) and immunohistochemistry found that HNF4a expression level in atrophic gastritis tissues was significantly lower than that of gastric cancer tissues. HNF4a expression levels in gastric cancer cell line were significantly higher than that in the immortalized gastric epithelial cell line GES-1. Clony formation assay and tumor formation assay in nude mice found that HNF4a knockdown reduced proliferation ability of gastric cancer cells.(2)Real-time PCR and western blot experiments confirmed that different strains of H. pylori can induce HNF4a expression in gastric cancer cell lines. HNF4a expression was up-regulated in mice fed with low doses of chemical carcinogens MNU and H. pylori.(3)Ensemble database suggests the key transcription factor p65 in NF-κB signaling pathway can bind to both P1 and P2 promoter region of HNF4a. HNF4a up-regulation induced by H. pylori infection can receded by NF-κB signaling pathway inhibitor or p65 siRNA. Dual luciferase assay and CHIP experiments confirmed that p65 can bind to two promoter region of HNF4a. Therefore, we conclude that H. pylori can activate HNF4a expression by NF-κB signaling pathway.(4)H. pylori infection can promote the secretion of inflammatory cytokines. Epithelial cell surface receptors bind to corresponding inflammatory cytokines and act as communicators with outside environment. DNA microarray analysis indicated that IL-1R1 shows the most significant rising trend in the conversion process from gastritis to gastric cancer. Real-time PCR of clinical samples showed that the expression of IL-1R1 and HNF4a is positively correlated. Western blot and immunofluorescence confirmed the expression of IL-1R1 is down-regulated when HNF4a was knockdown, and vice versa. Dual luciferase experiments identified that HNF4α can bind to promoter region of IL-1R1. Up-regulation of IL-1R1 by HNF4α can sensitize the response of gastric epithelial cells to exogenous inflammatory cytokine IL-1β. Binding of IL-1β and IL-1R1 may further activate NF-κB signaling pathway, thereby induce HNF4α expression and form a positive inflammatory feedback loop promoting the malignant transformation of gastric cells.(5)Immunohistochemistry confirmed HNF4α and IL-1R1 is gradually increased with the severity of atrophic gastritis and their expression reached higher level in gastric cancer. Both high expression of HNF4α and IL-1R1 indicated poor prognosis.2. FoxM1 can regulate TERT expression directly and promote gastric cell proliferation and siomycin A can inhibit gastric cancer proliferation by targeting FoxM1-TERT axis.(1)Real-time PCR and immunohistochemistry of clinical specimens found a positive correlation between FoxM1 and telomerase reverse transcriptase (TERT).(2)Real-time PCR and western blot experiments demonstrated that TERT expression level decreased when FoxM1 was knockdown in gastric cancer cells. Colony formation assay confirmed that knockdown of FoxM1 expression can significantly inhibit the proliferation of gastric cancer cells. Further experiments showed that TERT over-expression retrieved the proliferation ability of gastric cancer cells when FoxM1 was knockdown.(3)We found a highly conserved FoxMl binding motif 1285 bp upstream of the TERT translation initiation site by using Ensemble database. Dual luciferase experiments revealed that cells transfected with luciferase plasmids containing TERT promoter showed decreased luciferase activity when FoxM1 was knockdown. CHIP assay proved that FoxM1 can bind to TERT promoter region in a direct way.(4)Clony formation assay and tumor formation assay in nude mice found that siomycin A can reduce the rate of proliferation of gastric cancer cells and inhibit expression of FoxM1 and TERT.Conclusions1. HNF4a can promote proliferation of gastric cancer. Hp infection induces expression of HNF4a via the NF-κB pathway. HNF4a subsequently activates the expression of IL-1R1 and amplifies gastric cell inflammatory responses to IL-1β, which further stimulates the already over-active NF-κB pathway, completing a positive feedback loop that drives continuous inflammation to gastric cancer.2. FoxMl and TERT expression shows a positive correlation in gastric cancer. FoxMl is a direct upsteam transcription factor regulating TERT expression.Siomycin A can inhibit gastric cancer cell proliferation both in vitro and in vivo, which partially relies on its inhibition of FoxM1-TERT pathway.