Daoyan Wei

Stat3 activation regulates the expression of vascular endothelial growth factor and human pancreatic cancer angiogenesis and metastasis

Expression of vascular endothelial growth factor (VEGF), a key angiogenic protein, has been linked with pancreatic cancer progression. However, the molecular basis for VEGF overexpression remains unclear. Immunohistochemical studies have indicated that VEGF overexpression coincides with elevated Stat3 activation in human pancreatic cancer specimens. In our study, more than 80% of the human pancreatic cancer cell lines used exhibited constitutively activated Stat3, with Stat3 activation correlated with the VEGF expression level. Blockade of activated Stat3 via ectopic expression of dominant-negative Stat3 significantly suppressed VEGF expression, angiogenesis, tumor growth, and metastasis in vivo. Furthermore, constitutively activated Stat3 directly activated the VEGF promoter, whereas dominant-negative Stat3 inhibited the VEGF promoter. A putative Stat3-responsive element on the VEGF promoter was identified using a protein–DNA binding assay and confirmed using a promoter mutagenesis assay. These results indicate that Stat3 directly regulates VEGF expression and hence angiogenesis, growth, and metastasis of human pancreatic cancer, suggesting that Stat3 signaling may be targeted for treatment of pancreatic cancer.

Stat3 activation regulates the expression of matrix metalloproteinase-2 and tumor invasion and metastasis

The expression of matrix metalloproteinase-2 (MMP-2) has been linked with tumor invasion, angiogenesis, and metastasis. However, the molecular basis for MMP-2 overexpression in tumor cells remains unclear. In this study, by using K-1735 melanoma system, we demonstrated that highly metastatic C4, M2, and X21 tumor cells express elevated MMP-2 mRNA and enzymatic activity, whereas poorly metastatic C10, C19, and C23 tumor cells express much lower levels. Moreover, a concomitant elevated Stat3 activity has been detected in these metastatic tumor cells that overexpress MMP-2. Transfection of constitutively activated Stat3 into poorly metastatic C23 tumor cells directly activated the MMP-2 promoter, whereas the expression of a dominant-negative Stat3 in highly metastatic C4 tumor cells inhibited the MMP-2 promoter. A high-affinity Stat3-binding element was identified in the MMP-2 promoter and Stat3 protein bound directly to the MMP-2 promoter. Blockade of activated Stat3 through expression of a dominant-negative Stat3 significantly suppressed MMP-2 expression in the metastatic tumor cells. Therefore, overexpression of MMP-2 in the metastatic melanoma cells can be attributed to elevated Stat3 activity, and Stat3 upregulates the transcription of MMP-2 through direct interaction with the MMP-2 promoter. Furthermore, blockade of activated Stat3 in highly metastatic C4 cells significantly suppressed the invasiveness of the tumor cells, inhibited tumor growth, and prevented metastasis in nude mice. Collectively, these studies suggest that Stat3 signaling directly regulates MMP-2 expression, tumor invasion, and metastasis, and that Stat3 activation might be a crucial event in the development of metastasis.

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.

Celecoxib Inhibits Vascular Endothelial Growth Factor Expression in and Reduces Angiogenesis and Metastasis of Human Pancreatic Cancer via Suppression of Sp1 Transcription Factor Activity

The aggressive biology of human pancreatic adenocarcinoma has been linked with overexpression of vascular endothelial growth factor (VEGF). Constitutive activation of the transcription factor Sp1 plays a critical role in VEGF overexpression. Recent studies indicated that celecoxib, a selective cyclooxygenase-2 inhibitor, exhibits potent antitumor activity. However, the underlying molecular mechanisms of this activity remain unclear. In the present study, we used a pancreatic cancer model to determine the role of Sp1 in the antitumor activity of celecoxib. Treatment of various pancreatic cancer cells with celecoxib suppressed VEGF expression at both the mRNA and protein level in a dose-dependent manner. VEGF promoter deletion and point mutation analyses indicated that a region between nucleotide −109 and −61 and its intact Sp1-binding sites were required for the inhibition of VEGF promoter activity by celecoxib. Also, celecoxib treatment reduced both Sp1 DNA binding activity and transactivating activity. This decreased activity correlated with reduced Sp1 protein and its phosphorylation as determined using Western blot analysis. Furthermore, in an orthotopic pancreatic cancer animal model, celecoxib treatment inhibited tumor growth and metastasis. The antitumor activity was consistent with inhibition of angiogenesis as determined by evaluating tumor microvessel formation, which correlated with decreased Sp1 activity and VEGF expression. Collectively, our data provide a novel molecular mechanism for the antitumor activity of celecoxib and may help further improve its effectiveness in controlling pancreatic cancer growth and metastasis.

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.

Critical Role and Regulation of Transcription Factor FoxM1 in Human Gastric Cancer Angiogenesis and Progression

The mammalian forkhead box (Fox) transcription factor FoxM1b is implicated in tumorigenesis. However, the presence of expression and role of FoxM1b in gastric cancer remain unknown. Therefore, we investigated FoxM1b expression in 86 cases of primary gastric cancer and 57 normal gastric tissue specimens. We further investigated the underlying mechanisms of altered FoxM1b expression in and the effect of this altered expression on gastric cancer growth and metastasis using in vitro and animal models of gastric cancer. We found weak expression of FoxM1b protein in the mucous neck region of gastric mucosa, whereas we observed strong staining for FoxM1b in tumor cell nuclei in various gastric tumors and lymph node metastases. A Cox proportional hazards model revealed that FoxM1b expression was an independent prognostic factor in multivariate analysis (P < 0.001). Experimentally, overexpression of FoxM1b by gene transfer significantly promoted the growth and metastasis of gastric cancer cells in orthotopic mouse models, whereas knockdown of FoxM1b expression by small interfering RNA did the opposite. Promotion of gastric tumorigenesis by FoxM1b directly and significantly correlated with transactivation of vascular endothelial growth factor expression and elevation of angiogenesis. Given the importance of FoxM1b to regulation of the expression of genes key to cancer biology overall, dysregulated expression and activation of FoxM1b may play important roles in gastric cancer development and progression.

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.

Krüppel-like Factor 4 Induces p27Kip1 Expression in and Suppresses the Growth and Metastasis of Human Pancreatic Cancer Cells

The zinc finger transcription factor Krüppel-like factor 4 (KLF4) has been implicated in both tumor suppression and progression. However, its function in pancreatic cancer has not been well characterized. Here, we show that pancreatic cancer cell lines expressed various levels of KLF4 RNA and protein. Ectopic expression of KLF4 by FG and BxPC-3 pancreatic cancer cells resulted in cell cycle arrest and marked inhibition of cell growth in vitro and attenuation of tumor growth and metastasis in an orthotopic mouse model. Overexpression of KLF4 also led to significant induction of p27(Kip1) expression, at both the RNA and protein levels, in a dose- and time-dependent manner, indicating that KLF4 transcriptionally regulates the expression of p27(Kip1). Chromatin immunoprecipitation assays consistently showed that KLF4 protein physically interacts with the p27(Kip1) promoter. Promoter deletion and point mutation analyses indicated that a region between nucleotides -435 and -60 of the p27(Kip1) promoter and intact of the three KLF4-binding sites within that region were required for the full induction of p27(Kip1) promoter activity by KLF4. Our findings suggest that KLF4 transactivates p27(Kip1) expression and inhibits the growth and metastasis of human pancreatic cancer.

Concise Review: Emerging Role of CD44 in Cancer Stem Cells: A Promising Biomarker and Therapeutic Target

The reception and integration of the plethora of signals a cell receives from its microenvironment determines the cells fate. CD44 functions as a receptor for hyaluronan and many other extracellular matrix components, as well as a cofactor for growth factors and cytokines, and thus, CD44 is a signaling platform that integrates cellular microenvironmental cues with growth factor and cytokine signals and transduces signals to membrane‐associated cytoskeletal proteins or to the nucleus to regulate a variety of gene expression levels related to cell‐matrix adhesion, cell migration, proliferation, differentiation, and survival. Accumulating evidence indicates that CD44, especially CD44v isoforms, are cancer stem cell (CSC) markers and critical players in regulating the properties of CSCs, including self‐renewal, tumor initiation, metastasis, and chemoradioresistance. Furthermore, there is ample evidence that CD44, especially CD44v isoforms, are valuable prognostic markers in various types of tumors. Therefore, therapies that target CD44 may destroy the CSC population, and this holds great promise for the cure of life‐threatening cancers. However, many challenges remain to determining how best to use CD44 as a biomarker and therapeutic target. Here we summarize the current findings concerning the critical role of CD44/CD44v in the regulation of cancer stemness and the research status of CD44/CD44v as biomarkers and therapeutic targets in cancer. We also discuss the current challenges and future directions that may lead to the best use of CD44/CD44v for clinical applications.