Oncogene (2014) 33, 3256–3266 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE STAT3 expression, activity and functional consequences of STAT3 inhibition in esophageal squamous cell carcinomas and Barrett’s adenocarcinomas S Timme1,11, S Ihde1,11, CD Fichter1,2,11, V Waehle1, L Bogatyreva3, K Atanasov1, I Kohler1, A Scho¨ pflin1, H Geddert4, G Faller4, D Klimstra5, L Tang5, T Reinheckel6,7,8, D Hauschke3, H Busch6,9,10, M Boerries6,9,10, M Werner1,8,9,10 and S Lassmann1,7,8,9,10 Signal transducer and activator of transcription 3 (STAT3) is altered in several epithelial cancers and represents a potential therapeutic target. Here, STAT3 expression, activity and cellular functions were examined in two main histotypes of esophageal carcinomas. In situ, immunohistochemistry for STAT3 and STAT3-Tyr705 phosphorylation (P-STAT3) in esophageal squamous cell carcinomas (ESCC, n ¼ 49) and Barrett’s adenocarcinomas (BAC, n ¼ 61) revealed similar STAT3 expression in ESCCs and BACs (P ¼ 0.109), but preferentially activated P-STAT3 in ESCCs (P ¼ 0.013). In vitro, strong STAT3 activation was seen by epidermal growth factor (EGF) stimulation in OE21 (ESCC) cells, whereas OE33 (BAC) cells showed constitutive weak STAT3 activation. STAT3 knockdown significantly reduced cell proliferation of OE21 (P ¼ 0.0148) and OE33 (P ¼ 0.0243) cells. Importantly, STAT3 knockdown reduced cell migration of OE33 cells by 2.5-fold in two types of migration assays (P ¼ 0.073, P ¼ 0.015), but not in OE21 cells (P ¼ 0.1079, P ¼ 0.386). Investigation of transcriptome analysis of STAT3 knockdown revealed a reduced STAT3 level associated with significant downregulation of cell cycle genes in both OE21 (Po0.0001) and OE33 (P ¼ 0.01) cells. In contrast, genes promoting cell migration (CTHRC1) were markedly upregulated in OE21 cells, whereas a gene linked to tight-junction stabilization and restricted cell motility (SHROOM2) was downregulated in OE21 but upregulated in OE33 cells. This study shows frequent, but distinct, patterns of STAT3 expression and activation in ESCCs and BACs. STAT3 knockdown reduces cell proliferation in ESCC and BAC cells, inhibits migration of BAC cells and may support cell migration of ESCC cells. Thereby, novel STAT3-regulated genes involved in ESCC and BAC cell proliferation and cell migration were identified. Thus, STAT3 may be further exploited as a potential novel therapeutic target, however, by careful distinction between the two histotypes of esophageal cancers. Oncogene (2014) 33, 3256–3266; doi:10.1038/onc.2013.298; published online 5 August 2013 Keywords: STAT3; esophageal cancer; inhibition; cell proliferation; cell migration INTRODUCTION Irrespective of its cause, deregulation of STAT3 was reported 7,8 9 Signal transducer and activator of transcription 3 (STAT3) is a 92- for several epithelial cancers, such as lung, head and neck, 10,11 12,13 kDa large transcription factor, whose gene is located on stomach and colorectal carcinomas. In fact, inhibition of chromosome 17q21. STAT3 becomes activated upon phosphor- STAT(3) signaling is currently discussed for therapeutic ylation on critical tyrosine residues upon cytokine and/or growth intervention, alone or in combination with inhibitors to factor receptor stimulation. Phosphorylation of STAT3 tyrosine 705 upstream signaling molecules, such as receptor tyrosine 6 results in STAT(3) hetero- or homodimerization, nuclear transloca- kinases or janus kinases Jak1 or Jak2. Indeed, direct STAT3 tion of STAT(3) dimers and binding of STAT(3) dimers to their inhibition was successful in vitro by interference with STAT3 respective target gene promoters.1–3 phosphorylation and/or dimerization using peptidomimetics or 14 In normal cells, STAT3 expression and activity is under tight small molecules targeting the SH2 domain, or by interference control to ensure physiological cell proliferation, survival, differ- with nuclear translocation and/or DNA binding using 15,16 entiation and motility. In contrast, malignant transformed cells, decoy oligonucleotides. In addition, synergistic effects of respective human cancers, frequently exhibit STAT3 ‘over’expres- direct STAT3 inhibitors with the epidermal growth factor sion and constitutive activation, which contribute to increased receptor (EGFR) drug cetuximab were demonstrated in cell lines 17,18 cancer cell proliferation and cell migration.4–6 This may also be derived from squamous cell carcinomas of the head and neck triggered by alterations in upstream cytokines and/or (receptor) or in cell lines and xenograft models of pancreatic 19,20 tyrosine kinases. adenocarcinomas. 1Institute of Clinical Pathology, University Medical Center, Albert-Ludwigs-University, Freiburg, Germany; 2Faculty of Biology, Albert-Ludwigs-University, Freiburg, Germany; 3Institute of Medical Biometry and Medical Informatics, University Medical Center, Albert-Ludwigs-University, Freiburg, Germany; 4Department of Pathology, St Vincentius Kliniken, Karlsruhe, Germany; 5Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA; 6Institute of Molecular Medicine and Cell Research, University Medical Center, Albert-Ludwigs-University, Freiburg, Germany; 7BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-University, Freiburg, Germany; 8Comprehensive Cancer Center Freiburg, University Medical Center, Albert-Ludwigs-University, Freiburg, Germany; 9German Cancer Consortium (DKTK), Heidelberg, Germany and 10German Cancer Research Center (DKFZ), Heidelberg, Germany. Correspondence: Professor Dr S Lassmann, Institute of Clinical Pathology, University Medical Center, Breisacherstrasse 115A, 79106 Freiburg, Germany. E-mail: [email protected] 11These authors contributed equally to this work. Received 18 October 2012; revised 13 June 2013; accepted 13 June 2013; published online 5 August 2013 STAT3 expression and function in esophageal cancer S Timme et al 3257 The two main histotypes of esophageal cancers—esophageal As ESCCs and BACs display distinct patterns of carcinogenesis squamous cell carcinoma (ESCC) and Barrett’s adenocarcinoma and behavior, STAT3 expression and activation were assessed in (BAC)—are highly aggressive tumors with a very poor prognosis ESCCs versus BACs. These analyses showed that STAT3 protein and low 5-year survival rate, especially if diagnosed at later expression was statistically similar in ESCCs and BACs (P ¼ 0.109; stages.21–23 ESCCs and BACs evolve via distinct pathways of Table 1, Figure 1a, middle column), although ESCCs more carcinogenesis, with so far few alterations for targeted therapeutic frequently appeared to be STAT3 positive (91.2%, 41/45 of cases) intervention having been identified. To date, there is little than BACs (78.3%, 47/60 of cases). Importantly, nuclear P-STAT3- information about STAT3 expression and activation and its positive tumor cells were significantly more frequent in ESCCs potential therapeutic relevance in esophageal cancers. (71%, 35/49 of cases) as compared with BACs (47%, 29/61 of cases) In the experimental setting, Andl et al.24 reported an EGFR- (P ¼ 0.013; Table 1, Figure 1a, right column). In addition, nuclear mediated Jak-dependent activation of STAT1/STAT3 dimer activity P-STAT3 expression was significantly associated with STAT3 and cell migration in esophageal keratinocytes. In addition, several expression in ESCCs (P ¼ 0.003) or BACs (Po0.001). studies implicated bile acid or, more generally, inflammation- Finally, to exclude that potential heterogeneous STAT3 or associated STAT3 activation in esophageal cancer cell models.25,26 P-STAT3 protein expression was missed in the small biopsies, Recently, STAT3 was shown to cooperate with polo-like kinase 1 primary resected ESCCs and BACs were examined. Indeed, STAT3 (PLK1) in ESCC cell proliferation,27 an observation also of interest and P-STAT3 protein expression was homogeneously distributed in view of the availability of novel PLK1 inhibitors. In a more throughout the entire tissue sections (Figure 1b). clinicopathological situation, the data on STAT3 and phospho- Thus, STAT3 protein expression frequently occurs in esophageal STAT3 expression patterns in human tissue specimens of ESCCs cancers, and functionally active STAT3 (nuclear phospho-STAT3 at and BACs are limited. One study reported positive STAT3 protein Tyr750) is a prominent feature of ESCCs rather than BACs. Because expression in 90% of ESCC cases,28 another study detected of its homogeneous distribution within the tumors, STAT3 status phospho-STAT3 (Tyr705) positivity in 37% of ESCC cases27 and yet may equally be determined in small pre-therapeutic biopsies or another study revealed upregulation of phospho-STAT3 (Tyr705) primary resection specimens. expression along the dysplasia to carcinoma sequence in surveillance biopsies of patients diagnosed with Barrett’s esophagus.25 STAT3 expression and activity in ESCC and BAC cell lines From these studies, it remains open whether or not STAT3 To evaluate the expression, regulation and activity of STAT3 expression and activation are correlated within single cases of in vitro, two established and validated cell lines31,32 were selected esophageal carcinomas and whether or not this is different for further in vitro analyses. between ESCCs and BACs. Moreover, in view of current discussions Immunofluorescence analyses (Figure 2a) showed strong about therapeutic targeting STAT3 signaling, the functional cytoplasmic STAT3 (OE21, OE33) and negligible (OE21) or weak consequences