Weida Gong

Drastic Down-regulation of Krüppel-Like Factor 4 Expression Is Critical in Human Gastric Cancer Development and Progression

Krüppel-like factor 4 (KLF4) is highly expressed in epithelial tissues such as the gut and skin. However, the role of KLF4 in human gastric cancer development and progression is unknown. Here we show that KLF4 protein expression was decreased or lost in primary tumors and, in particular, lymph node metastases when compared with that in normal gastric mucosa. Moreover, loss of KLF4 expression in the primary tumors was significantly associated with poor survival, and also an independent prognostic marker in a multivariate analysis. Consistently, most human gastric cancer cell lines exhibited loss of or a substantial decrease in KLF4 expression at both RNA and protein levels. Enforced restoration of KLF4 expression resulted in marked cell growth inhibition in vitro and significantly attenuated tumor growth and total abrogation of metastasis in an orthotopic animal model of gastric cancer. Mechanism studies indicated that promoter hypermethylation and hemizygous deletion contributed to the down-regulation of KLF4 expression and the induction of apoptosis contributed to the antitumor activity of KLF4. Collectively, our data provide first clinical and casual evidence and potential mechanism that the alteration of KLF4 expression plays a critical role in gastric cancer development and progression.

Association between Expression of Transcription Factor Sp1 and Increased Vascular Endothelial Growth Factor Expression, Advanced Stage, and Poor Survival in Patients with Resected Gastric Cancer

The biological and clinical behaviors of cancer are affected by multiple molecular pathways that are under the control of transcription factors. Improved understanding of how transcription factors affect cancer biology may lead to improved ability to predict clinical outcome and discovery of novel therapeutic strategies. We evaluated the relationship between Sp1 and vascular endothelial growth factor (VEGF) expression, as well as their effect on survival in 86 cases of resected human gastric cancer. The degree of VEGF expression correlated highly with Sp1 expression (P < 0.01). Patients with high Sp1 expression were 98 times more likely to have high VEGF expression compared with those with negative Sp1 expression. Clinically, negative or weak Sp1 expression was associated with early stage (IA) in gastric cancer. Strong Sp1 expression was more frequently observed among patients with stage IB–IV disease (P = 0.035). Similarly, whereas strong Sp1 expression was uncommonly observed among patients with N0 or N1 disease (19 and 16%), N2/N3 gastric cancer was associated with strong Sp1 expression (48%; P = 0.034). Strong Sp1 expression was also associated with inferior survival. The median survival duration in patients who had a tumor with a negative, weak, and strong Sp1 expression was 44, 38, and 8 months (P = 0.0075), respectively, whereas patients with strong VEGF expression had a shorter survival duration; the difference was not statistically significant. When Sp1 and VEGF expression, stage, completeness of resection, histology, and patient age were entered in a Cox proportional hazards model, strong Sp1 expression (P = 0.021) and an advanced disease stage (P < 0.001) were independently prognostic of poor survival. Given the importance of Sp1 in the expression of VEGF, our data suggest that dysregulated Sp1 expression and activation play important roles in VEGF overexpression and, thus, gastric cancer development and progression.

Aberrant FoxM1B expression increases matrix metalloproteinase-2 transcription and enhances the invasion of glioma cells

We recently showed that FoxM1 is overexpressed in human glioblastomas and that forced FoxM1B expression in anaplastic astrocytoma cells leads to the formation of highly invasive glioblastoma multiforme (GBM) in nude mice. However, the molecular mechanisms by which FoxM1 enhances glioma invasion are unknown. In this study, we found that FoxM1 overexpression increased matrix metalloproteinase (MMP)-2 expression in glioma cells, whereas blockade of FoxM1 expression suppressed MMP-2 expression. Transfection of FoxM1 into glioma cells directly activated the MMP-2 promoter, whereas inhibition of FoxM1 expression by FoxM1-siRNA suppressed its activation. We identified a FoxM1-binding site in the MMP-2 promoter and demonstrated that FoxM1 protein bound directly to it. Mutation of this FoxM1-binding site significantly attenuated MMP-2 promoter activity. Furthermore, FoxM1 overexpression increased the invasiveness of glioma cells, whereas inhibition of FoxM1 expression suppressed the invasiveness of GBM cells. Inhibition of MMP-2 by a specific MMP-2 inhibitor reversed the invasive phenotype of glioma cells overexpressing FoxM1. Finally, immunohistochemical analysis of 45 human GBM specimens showed a significant correlation between FoxM1 overexpression and elevated MMP-2 expression. Collectively, these findings provide evidence that FoxM1 contributes to glioma progression by enhancing MMP-2 gene transcription and thus tumor-cell invasion.

Association of elevated GRP78 expression with increased lymph node metastasis and poor prognosis in patients with gastric cancer

Glucose-regulated protein 78 (GRP78) has been implicated in the protection of tumor cells from cytotoxic damage and apoptosis and thus assists cells in survival under oxygen-deprivation and nutrient-stress conditions. However, its expression and potential role in gastric cancer development and progression have not been reported. In the present study, we determined the level of GRP78 expression in the primary tumor in 86 cases of resected gastric cancer by using immunohistochemistry and analyzed the relationships between GRP78 and clinicopathological characteristics. We found that GRP78 was overexpressed in the tumor specimens when compared with the expression in adjacent tumor-free gastric mucosa. Furthermore, the level of GRP78 expression in both primary tumors and metastatic lymph nodes was inversely correlated with patient survival. Overexpression of GRP78 was directly correlated with Sp1 expression and increased lymph node metastasis. Knocking down GRP78 expression inhibited tumor cell invasion in vitro and growth and metastasis in a xenograft nude mouse model. Therefore, our data imply that dysregulated expression of GRP78 may contribute to the development and progression of gastric cancer.

Expression of activated signal transducer and activator of transcription 3 predicts expression of vascular endothelial growth factor in and angiogenic phenotype of human gastric cancer

Purpose: Angiogenic behavior is a critical aspect of cancer biology and subject to regulation by multiple molecular pathways. Because the signal transducer and activator of transcription 3 (Stat3) transcription factor regulates multiple genes important to angiogenesis, we sought to determine whether Stat3 expression is related to vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) in gastric cancer and whether these factors predict survival in gastric cancer patients. Experimental Design: The expression of Stat3 and VEGF was determined by immunohistochemistry using archival tissues from 86 cases of resected human gastric cancer and confirmed by Western blot analysis. Angiogenic phenotype was determined by CD34 staining and microvessel counting. Results: Stat3 expression correlated with VEGF expression and MVD. In univariate survival analyses, Stat3 expression (P = 0.013) and MVD (P = 0.036) were associated with inferior survival. However, when Stat3 expression, VEGF expression, MVD, stage, completeness of resection, Laurens histologic classification, and age were entered into a Cox proportional hazards model, only strong Stat3 expression (P = 0.049) and advanced stage (P < 0.01) were independently prognostic of poor survival. Furthermore, genetically enforced alterations of activated Stat3 expression led to altered VEGF expression and angiogenic potential in human gastric cancer cells. Conclusion: Dysregulated Stat3 activation may play an important role in VEGF overexpression and elevated angiogenic phenotype in gastric cancer and contribute to gastric cancer development and progression.

Loss of RUNX3 expression significantly affects the clinical outcome of gastric cancer patients and its restoration causes drastic suppression of tumor growth and metastasis

Identification of precise prognostic marker and effective therapeutic target is pivotal in the treatment of gastric cancer. In the present study, we determined the level of RUNX3 expression in gastric cancer cells and gastric cancer specimens and the impact of its alteration on cancer biology and clinical outcome. There was a loss or substantial decrease of RUNX3 protein expression in 86 cases of gastric tumors as compared with that in normal gastric mucosa (P < 0.0001), which was significantly associated with inferior survival duration (P = 0.0005). In a Cox proportional hazards model, RUNX3 expression independently predicted better survival (P = 0.036). Moreover, various human gastric cancer cell lines also exhibited loss or drastic decrease of RUNX3 expression. Enforced restoration of RUNX3 expression led to down-regulation of cyclin D1 but to up-regulation of p27, caspase 3, 7, and 8 expression, cell cycle arrest, and apoptosis in vitro, and dramatic attenuation of tumor growth and abrogation of metastasis in animal models. Therefore, we offered both clinical and mechanistic evidence that RUNX3 was an independent prognostic factor and a potential therapeutic target for gastric cancer.

A high expression level of insulin-like growth factor I receptor is associated with increased expression of transcription factor Sp1 and regional lymph node metastasis of human gastric cancer.

Insulin-like growth factor I receptor (IGF-IR) is critical to cell survival and growth and altered IGF-IR expression is found in many human cancers. However, its expression and potential role in gastric cancer development and progression has not been explored. The IGF-IR expression level was determined via immunohistochemistry in primary tumor and lymph node metastasis of 86 cases of resected gastric cancer. Relationships of IGF-IR expression with transcription factor Sp1 expression and clinicopathological features were analyzed. The impact of altered Sp1 expression on IGF-IR expression and gastric cancer biology was further determined using small inhibitory RNA for Sp1 mRNA. We found that IGF-IR was overexpressed in 62% of the tumor samples when compared with adjacent tumor-free gastric mucosa. Patients with lymph node metastases had strong expression of IGF-IR in both primary and metastatic tumor cells. IGF-IR overexpression in the primary tumor correlated with increased lymph node metastasis. Furthermore, the level of IGF-IR expression directly correlated with that of Sp1, an important transcription factor for IGF-IR regulation. Knocking-down of Sp1 expression by small inhibitory RNA led to decreased IGF-IR expression and attenuated growth and metastasis of gastric cancer cells. Therefore, dysregulated expression of IGF-IR and/or Sp1 may contribute to the growth and metastasis of gastric cancer and potentially can be a target of therapeutic intervention.

Expression of endothelial nitric oxide synthase correlates with the angiogenic phenotype of and predicts poor prognosis in human gastric cancer.

BackgroundAngiogenesis is a critical aspect of cancer biology and is subject to regulation by multiple molecular pathways. We evaluated the expression of nitric oxide synthase (NOS) III and its relationship with the angiogenic phenotype and expression of the transcription factor Sp1, as well as their effect on survival in patients with gastric cancer.MethodsThe NOS III expression level and tumor microvessel density (MVD) status were determined via immunohistochemistry, using archived tissue specimens from 86 resected gastric cancer cases; these findings were then correlated with Sp1 expression and clinicopathological features.ResultsNOS III protein expression was significantly higher in both primary tumors and lymph node metastases than in normal gastric mucosa. In primary tumors, NOS III expression correlated highly with Sp1 expression (P = 0.001) and MVD status (P = 0.001). Patients with strong Sp1 expression were more likely to have strong NOS III expression (15 times) and a high MVD (7 times) than were those with negative Sp1 expression. In univariate survival analyses, strong NOS III expression (P = 0.042), strong Sp1 expression (P = 0.007), and a high MVD (P = 0.036) were associated with worse survival. However, when patients’ NOS III and Sp1 expression levels, MVD status, disease stage, completeness of resection, Lauren’s classification, and age were entered in a Cox proportional hazards model, only strong NOS III (P = 0.019) and Sp1 expression (P = 0.029) and advanced disease stage (P = 0.006) were independently prognostic of poor survival.ConclusionOur results indicated that the expression of NOS III, a potential downstream effector of Sp1, may play an important role in tumor angiogenesis and aggressiveness in gastric cancer.

Expression of autocrine motility factor correlates with the angiogenic phenotype of and poor prognosis for human gastric cancer

Autocrine motility factor (AMF) is a cytokine known to regulate tumor cell motility. Recent studies have extended its role to many other aspects of cancer biology. In the present study, we examined the level of AMF expression and its relationship with vascular endothelial growth factor (VEGF) expression and the angiogenic phenotype in human gastric cancer and their effect on survival. The AMF and VEGF expression level and tumor microvessel density (MVD) status in archived tissue specimens from 86 resected gastric cancer cases were determined. AMF expression was significantly higher in both primary tumors and lymph node metastases than in adjacent normal gastric mucosa and normal gastric mucosa from individuals without gastric cancer. In univariate survival analyses, strong AMF expression was associated with inferior survival (P = 0.028). In a Cox proportional hazards model, strong AMF expression (P = 0.019) was independently prognostic of poor survival. Strong AMF expression in the lymph node metastases was associated with poor survival (P = 0.011). Furthermore, AMF expression in the primary tumors was directly correlated with VEGF expression and MVD status. We found the first clinical evidence that AMF expression is directly correlated with VEGF expression and MVD status and predicts clinical outcome in patients with gastric cancer, supporting the hypothesis that the AMF/AMF receptor pathway plays an important role in multiple aspects of cancer biology.

10 – Immunohistochemical Detection of Activated Stat3 Protein in Pancreatic Cancer

In this chapter, the expression of activated Stat3 (transcription factor) in human pancreatic cancers using a phosphotyrosine-specific Stat3 antibody (p-Stat3 [Tyr-705]) is examined. The tyrosine phosphorylation of Stat3 constitutes an early event in the activation of this transcription factor and is required for its dimerization and DNA-binding activity. Of particular importance, the constitutive tyrosine phosphorylation of Stat3 is necessary for this transcription factor to exert its oncogenic effect. Activated Stat3 is overexpressed in human pancreatic cancers as compared with normal pancreatic tissues. Stat3 may contribute to human pancreatic cancer development and progression by collectively affecting the expression of genes related to cell survival, cell cycle control, and angiogenesis, such as the genes that encode Bcl-x L , p21 wAF1 , and vascular endothelial growth factor (VEGF). The activated Stat3 might serve as both a molecular marker for the early detection of human pancreatic cancer and a prognostic indicator for use in determining the aggressiveness of the disease.