STAT3 Knockdown Reduces Pancreatic Cancer Cell Invasiveness and Matrix Metalloproteinase-7 Expression in Nude Mice

et al. (2011) STAT3 Knockdown Reduces Pancreatic Cancer Cell Invasiveness and Matrix Metalloproteinase-7 Expression in Nude Mice. PLoS ONE 6(10): e25941. doi:10.1371/journal.pone.0025941 STAT3 Knockdown Reduces Pancreatic Cancer Cell Invasiveness and Matrix Metalloproteinase-7 Expression in Nude Mice Hai dong Li 0 Chen Huang 0 Ke jian Huang 0 Wei dong Wu 0 Tao Jiang 0 Jun Cao 0 Zhen zhong Feng 0 Zheng jun Qiu 0 Marcelo G. Bonini, University of Illinois at Chicago, United States of America 0 1 Department of General Surgery, Affiliated First People's Hospital, Shanghai Jiao Tong University , Shanghai , China , 2 Shanghai Key Laboratory of Pancreas Disease , Shanghai , China , 3 Pancreatic Cancer Center of Shanghai Jiao Tong University , Shanghai , China , 4 Department of Pathology, Shanghai Jiao Tong University-Affiliated First People's Hospital , Shanghai , China Aims: Transducer and activator of transcription-3 (STAT3) plays an important role in tumor cell invasion and metastasis. The aim of the present study was to investigate the effects of STAT3 knockdown in nude mouse xenografts of pancreatic cancer cells and underlying gene expression. Methods: A STAT3 shRNA lentiviral vector was constructed and infected into SW1990 cells. qRT-PCR and western immunoblot were performed to detect gene expression. Nude mouse xenograft assays were used to assess changes in phenotypes of these stable cells in vivo. HE staining was utilized to evaluate tumor cell invasion and immunohistochemistry was performed to analyze gene expression. Results: STAT3 shRNA successfully silenced expression of STAT3 mRNA and protein in SW1990 cells compared to control cells. Growth rate of the STAT3-silenced tumor cells in nude mice was significantly reduced compared to in the control vector tumors and parental cells-generated tumors. Tumor invasion into the vessel and muscle were also suppressed in the STAT3-silenced tumors compared to controls. Collagen IV expression was complete and continuous surrounding the tumors of STAT3-silenced SW1990 cells, whereas collagen IV expression was incomplete and discontinuous surrounding the control tumors. Moreover, microvessel density was significantly lower in STAT3-silenced tumors than parental or control tumors of SW1990 cells. In addition, MMP-7 expression was reduced in STAT3-silenced tumors compared to parental SW1990 xenografts and controls. In contrast, expression of IL-1b and IgT7a was not altered. Conclusion: These data clearly demonstrate that STAT3 plays an important role in regulation of tumor growth, invasion, and angiogenesis, which could be act by reducing MMP-7 expression in pancreatic cancer cells. - . These authors contributed equally to this work. Pancreatic cancer is a lethal disease and is the fourth most common cause of cancer-related death in Western countries [1]. Most recent data estimated that 43,140 new cases were diagnosed in 2010, with approximately 36,800 associated deaths in the United States [2]. Although surgical resection provides a potential cure, pancreatic cancer is often diagnosed at the advanced stages due to lack of symptoms or nonspecific symptoms, making surgery impossible. Palliative chemotherapy could improve the quality of life and potentially affect survival. These have led to a very dismal prognosis for pancreatic cancer patients, i.e., the median survival is approximately 3 to 6 months and 5-year survival is ,5%. Risk factors for pancreatic cancer include smoking, alcohol consumption, diets high in red meat but low in vegetables and fruits, chronic pancreatitis, and family history. However, the molecular mechanisms responsible for pancreatic cancer development have not been elucidated and there are no established guidelines for pancreatic cancer prevention. Therefore, novel approaches to diagnose pancreatic cancer early to ensure effective therapy are drastically needed. The signal transducer and activator of transcription (STAT3) gene is a member of the STAT protein family of transcription factors. In response to cytokines and growth factors, STAT family members are phosphorylated by receptor-associated kinases, forming homo- or heterodimers and translocating in the nucleus of cells to bind to DNA and regulate expression of the target genes. STAT3 is activated via phosphorylation of tyrosine 705 and serine Figure 1. Suppression of STAT3 mRNA and protein expression using STAT3 shRNA in pancreatic cancer SW1990 cells. A, qRT-PCR. Stable STAT3 shRNA and control vector-transfected SW1990 cells and parental SW1990 cells were grown and RNA was isolated for qRT-PCR analysis of STAT3 mRNA expression *P,0.01 compared to SW1990 or control vector-transfected cells. B, Western immunoblot. These cells were grown for total cellular protein extraction and western immunoblot analyses of STAT3 protein expression. doi:10.1371/journal.pone.0025941.g001 727 in response to various cytokines and growth factors (such as interferon, EGF, and interleukins). STAT3 plays an important role in mediating various cellular processes (e.g., cell growth, apoptosis, and differentiation) [3]. Previous studies have shown that the oncogenic STAT-3 protein was constitutively activated in many human cancers [46]. In contrast, inhibition of the STAT-3 signaling pathway suppressed cancer cell invasion in various cancers [79]. In pancreatic cancer, activation of STAT-3 promoted tumor cell growth, invasion, and metastasis, leading to poor patient survival. Molecularly, STAT3 can regulate expression of VEGF and MMP-2 genes [1011]. These two genes, together with MMP-7, MMP-9, bFGF, IL-1b and IgT7aa are closely related to tumor progression (angiogenesis, invasion and metastasis) [1214]. In the present study, we utilized the STAT3 shRNA lentivirus to silence STAT3 expression. Previous data from our group suggests that knockdown of STAT3 inhibited invasion of pancreatic cancer SW1990 cells in vitro and markedly decreased VEGF and MMP-2 expression [7]. In the present study, we investigated whether silencing of STAT3 in pancreatic cancer cells modulates tumor cell growth and invasiveness in nude mouse xenografts and determined the underlying signaling mechanisms involved using a cDNA microarray. Materials and Methods Cell culture and lentivirus infection The human pancreatic cancer cell line SW1990 was obtained from American Type Culture Collection (Manassas, VA, USA) and cultured with Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/ streptomycin at 37uC in a 5% CO2 and 95% air incubator. To silence STAT3 expression, we constructed a lentiviral vector carrying STAT3 shRNA as described previously [7]. SW1990 cells (16105) were seeded into each well and grown in 6-well plates and then infected with a negative control lentiviral vector (Genechem, Shanghai, China) or the lentiviral vector carrying STAT3 shRNA at a multiplicity of infection (MOI) of 40. After three days, the cells were subjected to vivo i (...truncated)