Constitutive Activation Of Stat3 Signaling Pathway Correlates Positively With LIF Expression Status In Gastric Cancer And Its Potential Molecular Mechanisms

Background and Objective Gastric cancer (GC) is the fourth most common cancer and the second leading cause of cancer-related deaths worldwide because of its rapid growth and strong tendency of local invasiveness and distal metastasis. Although early diagnosis and treatment of gastric cancer significantly improves its dismal prognosis, the survival rate of patients with advanced disease is still low. Therefore, the primary prevention of gastric cancer is particularly important.Although the etiology of gastric cancer is not fully understood, gastric carcinogenesis has been known as a stepwise and multifactorial process. In this process, many transcription factors are involved and activated such as Stat3. Stat3 is one of the signal transducer and activator of the transcription (STAT)-family proteins that can transmit signals from cytokines and growth factors to the nucleus. Many investigations have suggested that the Stat3 is constitutively activated in hematological malignancies and various solid tumors including gastric cancer, suggesting a possible role of Stat3 in gastric carcinogenesis and development. Several studies have shown that IL-6-like cytokines such as leukemia inhibitory factor (LIF) can make cytoplasmic Stat3 activated via phorsphorylation and the phorsphorylated Stat3 can gain the chance to translocalize into the nucleus. LIF/Stat3 signaling pathway regulates the expression of a panel of genes that involve in cell differentiation, growth, apoptosis and angiogenesis. Vascular endothelial growth factor (VEGF) and Survivin are two typical downstream genes targeted by LIF/Stat3 signaling, which play critical roles in promoting angiogenesis and cell survival. Previous investigations indicated that VEGF and Survivin up-regulations were associated with Stat3 constitutively activation in many solid malignancies including gastric cancer, While few studies referred to LIF and considered them as a whole. The aims of current study are to investigate the expression status of LIF, Stat3,VEGF and Survivin in gastric cancer and to analyze the possible relationship between LIF abnormal expression and constitutive Stat3 activation of signaling pathway, so as to explore the potential molecular mechanisms of gastric carcinogenesis and development.Materials and Methods Surgical specimens were selected from the gastric tissue bank of Liaoning Key Lab of Cancer genomics, Dalian Medical University. Human gastric cancer cell lines AGS and HGC-27 were ordered from American Type Culture Collection (ATCC, USA), MGC803 and BGC823 were kindly provided by Beijing Institute for Cancer Research, Peking University. The different gastric tissues were classified histologically as premalignant (intestinal type and diffuse type) and gastric cancer tissues and indicated by the methods of paraffin tissue array-based immunohistochemical staining. Paralleled Western blot and RT-PCR analyses were conducted to check the expression statuses of LIF, Stat3, VEGF and Survivin in four human gastric cancer cell lines. Statistical significance was determined by The Mann-Whitney Test, X2 test and Spearman correlation test in this work and p<0.01 was considered statistically significant.Results1. The statuses of LIF, VEGF and Survivin expressions were increased distinctively in gastric cancer in comparison with that in premalignant group. Stat3 nuclear translocation can also be observed in most gastric cancer tissue spots (p<0.01).2. Among 65 gastric tissue spots, 45(45/65, 69.23%) showed positive nuclear translocation of Stat3 and 43(43/65, 66.15%)were positive for LIF staining,which were essentially observed in gastric cancer tissues. Importantly, of the 45 nuclear Stat3 positive cases, 40(40/45, 88.89%)were also positive for LIF staining, and there was a distinct correlation between nuclear positive staining of Stat3 and LIF(X2=33.762; p<0.01).3. VEGF overexpression and nuclear translocation of Stat3 appeared in great proportion of gastric cancer tissues (r=0.502; p<0.01) and similar phenomenon could also be observed in the examination of Survivin expression (r=0.533; p<0.01). Consequently, a significant correlation can be found between nuclear translocation of Stat3 and VEGF or Survivin expression.4. The level of mRNA was determined using RT-PCR analysis and we could detect different mRNA amount of LIF. LIF mRNA expression level was apparently higher in AGS than that in other 3 cell lines, while the levels of Stat3, VEGF and Survivin appeared similar among those four cell lines. Meanwhile, production of correspondingproteins were determined via Western blot analysis.The amounts of LIF, Stat3, VEGF and Survivin proteins remain similar among the 4 cell lines.Conclusion Our current study demonstrates a constitutive Stat3 activation and frequent expressions of its downstream genes, VEGF and Survivin in gastric tumors and cell lines. The above phenomena were closely associated with LIF expression. In accompany with LIF up-regulation, cytoplasmic Stat3 is translocalized into the nucleus, which is correlated with VEGF and Survivin expression. Our results reveal a positive relationship between LIF expression and Stat3 activation as well as up-regulated VEGF and Survivin expression in gastric cancers. Consequently, gastric cancer cell are conferred on strong ability of angiogenesis and apoptosis suppression.