Stat3 Regulates Genes Common to Both Wound Healing and Cancer

Oncogene (2005) 24, 3397–3408 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc Stat3 regulates genes common to both wound healing and cancer Daniel J Dauer1,3, Bernadette Ferraro1,3, Lanxi Song1,3, Bin Yu1,3, Linda Mora2,3, Ralf Buettner2,3, Steve Enkemann2,3, Richard Jove2,3 and Eric B Haura*,1,3 1Thoracic Oncology/Experimental Therapeutics, H Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA; 2Molecular Oncology Programs, H Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA; 3Department of Interdisciplinary Oncology, University of South Florida College of Medicine, Tampa, FL 33612, USA Wound healing and cancer are both characterized by cell hallmarks of cancer consist of an analogous program proliferation, remodeling of extracellular matrix, cell that allows for deregulated cell growth, tumor cell invasion and migration, new blood vessel formation, and invasion, angiogenesis, and metastasis (Hanahan and modulation of blood coagulation. The mechanisms that Weinberg, 2000). These observations have been the link wound healing and cancer are poorly understood. We backdrop for the concept that tumors are ‘wounds that report here that Stat3, a common signaling mechanism do not heal’ (Dvorak, 1986). While the relationship involved in oncogenesis and tissue injury, regulates a between inflammation/wound healing and cancer is common set of genes involved in wound healing and widely accepted, the molecular mechanisms that link cancer. Using oligonucleotide gene arrays and quantita- inflammation and cancer remain unclear (Coussens and tive real-time PCR, we evaluated changes in global gene Werb, 2002). Here, we evaluated the ability of Stat3 to expression resulting from expression of Stat3 in lung coordinate pathways common to both wound healing epithelial cells. We report here previously uncharacterized and cancer. We postulated that signaling pathways genes induced by Stat3 implicated in signaling pathways regulated by bothoncogenic signals and tissue injury common to both wound healing and cancer including cell signals coordinate gene expression changes common to invasion and migration, angiogenesis, modulation of bothevents. coagulation, and repression of interferon-inducible genes. Originally identified as a key component linking Consistent with these results, we found increased Stat3 normal cytokine signals to transcriptional events in activity associated with wound healing in chronically cells, Stat3 is now thought to play a major role in tumor inflamed mouse lungs and increased Stat3 activity was formation (Levy and Darnell, 2002; Yu and Jove, 2004). identified at the leading edge of lung tumors invading Stat3 is activated by oncogenic signals suchas Src and adjacent nontumor stroma. These findings provide a EGFR signaling as well as by cytokines and mitogens molecular basis for understanding cancer as a deregula- involved in wound healing such as interleukin-6 and tion of normal wound healing processes. hepatocyte-growth factor (HGF) (Song et al., 2003). A Oncogene (2005) 24, 3397–3408. doi:10.1038/sj.onc.1208469 constitutively activated mutant of Stat3 can promote Published online 28 February 2005 cellular proliferation and survival as well as lead to cellular transformation, providing genetic evidence for Keywords: STAT proteins; cancer; wound healing; the intrinsic oncogenic potential of Stat3 (Bromberg Stat3; lung cancer et al., 1999). Stat3 regulates a number of pathways important in tumorigenesis including cell cycle progres- sion, apoptosis, tumor angiogenesis, invasion and metastasis, and tumor cell evasion of the immune Introduction system (Bromberg et al., 1999; Grandis et al., 2000; Yu and Jove, 2004). In addition to processes involved in In response to tissue injury, local cytokines and growth tumor formation in epithelial cells, Stat3 plays a crucial factors act on epithelial, mesenchymal, and immune cells role in normal wound healing and response to injury. to orchestrate healing of the wound. Wound healing is Stat3 is activated in lung tissues following either generally characterized by processes involved in cell lipopolysaccharide (LPS) exposure or intrapulmonary proliferation, remodeling of extracellular matrix, cell deposition of IgG immune complexes (Gao et al., 2004; invasion and migration, new blood vessel formation, Severgnini et al., 2004). Selective Stat3 deletion in lung and modulation of blood coagulation. Similarly, the epithelial cells does not compromise postnatal lung function but mice exposed to hyperoxia develop rapid *Correspondence: EB Haura, Thoracic Oncology and Experimental lung injury characterized by alveolar capillary leak and Therapeutics Programs, H Lee Moffitt Cancer Center and Research acute respiratory distress (Hokuto et al., 2004). Similary, Institute, MRC3 East, Room 3056, 12902 Magnolia Drive, Tampa, FL 33612-9497, USA; E-mail: hauraeb@moffitt.usf.edu selective loss of Stat3 in keratinocytes results in impaired Received 10 November 2004; revised 8 December 2004; accepted 20 wound healing and defects in keratinocyte migration December 2004; published online 28 February 2005 (Sano et al., 1999). Stat3, wound healing and cancer DJ Dauer et al 3398 Results repressed by Stat3C. The top 50 genes in each of these two groups are shown in Tables 1 and 2, respectively. Identification of Stat3-regulated genes using Complete lists of the significant upregulated and down- oligonucleotide microarrays regulated genes are included in the Supplementary Data (Tables A–C). Importantly, we identified targets that To investigate whether Stat3 regulates genes involved in have been previously reported as Stat3 targets in the bothwound healing and cancer, we investigated literature. These include upregulated genes such as genome-wide changes in gene expression in lung fibrinogen, lipopolysaccharide-binding protein (LBP), epithelial cells. We developed an adenoviral vector that SOCS3, c/EBPd, BCL6, JunB, and Myc (Fujitani et al., expresses a mutant of Stat3, designated Ad-Stat3C, 1994; Schumann et al., 1996; Hutt et al., 2000; Reljic which is constitutively active in the absence of tyrosine et al., 2000; Bowman et al., 2001; Duan and Simpson- phosphorylation and can transform rodent fibroblasts Haidaris, 2003; He et al., 2003). These putative Stat3- (Bromberg et al., 1999). Quiescent A549 lung cells were regulated genes were then grouped into distinct known infected witheither Ad-Stat3C or a control adenoviral biological functions of the encoded proteins, including vector expressing green fluorescent protein (Ad-GFP). apoptosis, cell cycle, cell signaling, invasion, and These cells were established from a human bronchio- inflammation. We used Onto-Express to aid in under- loalveolar cell carcinoma and have features resembling standing functional roles for genes identified as differ- et al type II alveolar epithelial cells of the lung (Lieber ., entially expressed (Khatri et al., 2002; Khatri et al., 1976). Preliminary results had identified an optimal 2004). Onto-Express is a tool designed to mine available multiplicity of infection (MOI) of 50 resulting in functional annotation data and identify relevant and equivalent levels of Stat3 DNA binding compared with significant functional biological processes. In addition, IL-6 stimulation and resulting in nearly 90% of the cells eachgene was researched by manual examination of expressing GFP (data not shown). Figure 1 demon- published literature using PubMed. This analysis is strates that Stat3–DNA-binding activity is detected after shown in Table 3. as early as 8 hfollowing infection and increases One of the more interesting findings was the throughout the infection. identification of a large number of genes induced by To control for biological and experimental variation, Stat3 that are involved in cell invasion/migration and five 10 cm dishes of cells were infected with either Ad- remodeling of extracellular matrix. These included genes GFP or Ad-Stat3C (10 individual plates). At 24 hafter in the chemoattractants family such as CCL2 and infection, total RNA was collected and used as the RNA CXCL2, proteases in the cathepsin and SERPIN source for microarray analysis. Eachdishof cells was families suchas uPA and its receptor uPAR, and genes used for one Affymetrix U133A Human GeneChip. involved in bothcell invasion and blood coagulation Differentially expressed genes between Ad-Stat3C and pathways (such as PAI-1). Several genes encoding Ad-GFP were identified using significance analysis of coagulation proteins were similarly upregulated by Stat3 microarrays (SAM) (Tusher et al., 2001). This is a including fibrinogen, PAI-1 (SERPINE1), and throm- method of identifying statistically significant changes in bomodulin. We also identified genes involved in gene expression taking into account multiple testing and angiogenesis including EPAS1, adrenomedullin, and a family-wise error rate. We accepted all genes identified angiopoietin-like protein 4 (ANGPTL4; also known as by SAM as differentially regulated by at least 1.5-fold. PPARg-angiopoietin-related protein), as being upregu- While this choice was somewhat arbitrary, it was largely lated by Stat3. VEGF, previously identified as a Stat3 based on a report demonstrating that a 50% change in target gene, was identified by SAM as being upregulated gene expression can result in tumorigenesis (Yan et al., by Stat3 but with a fold change of slightly less than 1.5 2002). We identified 200 genes induced by Stat3C that in our analysis (Niu et al., 2002). were considered

Load more