The following day Ad-null, Ad-shor Ad-ATF/were infected at a MOI of 250 for EBC2 cells at a MOI of 1500 for TE1and at a MOI of 50 for TE4 cells for 24 hours. plays an important role in squamous cell differentiation [10, 11]. Amplification of chromosome 3q26 is the most common of the genetic alterations found in lung SCC . is a candidate oncogene present in this locus and amplification of has been reported in lung and esophageal SCC . Bass et al. showed that inhibition of suppresses cell growth. There is a data that supports a role for this gene as a lineage survival oncogene . In D8-MMAE a previous study, we demonstrated that silencing SOX2 by siRNA induced G1 cell cycle arrest mediated by upregulation of CDKN1A expression resulting in an anti tumor effect in SOX2-expressing lung SCC cells both and . As shown in Figure ?Figure1A,1A, SOX2 expression was detected in 3 of 6 kinds of lung SCC cells, in 2 of 4 kinds of pulmonary adenocarcinoma cells and in all kinds of esophageal SCC cells, while expression of SOX2 was not detected in normal human foreskin fibroblast HFF1 and normal human lung fibroblast NHLF and human umbilical vein endothelial cells (HUVEC). In human lung SCC and esophageal SCC sections, SOX2 expression was detected in more than 87.5% of sections (Figure ?(Figure1B1B and ?and1C)1C) suggesting that molecular targeting of SOX2 might be useful for treating SCC. Open in a separate window Figure 1 SOX2 expression in lung, esophageal SCC and pulmonary adenocarcinoma(A) Immunoblot analysis of SOX2 in indicated cells. The expression level of -actin is shown as a control. (B) The intensity of SOX2 staining was assigned the following scores: none = -, weak = +, moderate = ++, and strong = +++ expression. Examples of representative immunohistochemistry results are shown. Bars, 50 m. (C) SOX2 expression in primary pulmonary adenocarcinoma samples of 40 patients, lung SCC samples of 40 patients and esophageal SCC samples of 40 patients who underwent surgical tumor resection at the Kawasaki Hospital Attached to Kawasaki Medical School between 2007 and 2012. Percentage values are given in parentheses. As we reviewed previously, zinc finger D8-MMAE -based artificial transcription factors (ATFs)  can be designed to regulate the expression of target genes and can provide powerful biotechnological tools for the investigation and treatment of disease [17, 18]. We demonstrated that a simple mode of DNA recognition by zinc Rabbit polyclonal to Complement C3 beta chain finger domains makes it possible to design ATFs with novel sequence specificities . The designed ATFs can perform DNA-binding activity and conduct natural transcription and they have been used to either activate or repress miscellaneous endogenous target genes [20C22]. We have also demonstrated that designed regulatory proteins (DRPs), in which artificial transcription factors (ATFs) are fused to cell-penetrating peptides (CPPs) effectively activate or repress target genes . In a previous study, Stolzenburg et al. developed zinc finger-based artificial transcription factors (ATFs) to target SOX2 and showed their possible therapeutic use against breast cancer . In order to target SOX2 as a candidate oncogene of lung SCC and esophageal SCC, we have developed a zinc finger-based artificial transcription factor (ATF) to selectively suppress SOX2 expression in D8-MMAE cancer cells. We termed this system ATF/(Figure ?(Figure2A2A and ?and2B).2B). Ad-ATF/(Figure ?(Figure2C)2C) up-regulated mRNA and protein expression more significantly than did Ad-shin SOX2-expressing lung and esophageal SCC cells induced an antitumor effect in SOX2-expressing SCC more effectively than did Ad-shboth and activated CDKN1A and showed a greater antitumor effect more strongly than post-transcriptional SOX2 inhibition by shRNA. Here we report a novel SOX2-targeting therapy using an ATF for the treatment of lung and esophageal SCC. Open in a separate window Figure 2 Schematic representation of ATF/distal promoter region. TSS; transcription start site. (B) The Artificial Transcription Factor (ATF) contains the KOX suppressor domain, a nuclear localization signal (NLS), the Artificial zinc finger protein (AZP) and a FLAG tag. We termed this ATF as ATF/and Ad-ATF/The PCR-generated expression cassette of ATF/from pcDNA3.1 ATF/or shfrom pBAsi-mU6 sh(described in Materials and Methods section) were subcloned into the linearized E1 deleted adenovirus type 5 genome. E1; Adenovirus early region 1, E3; Adenovirus early region 3, LITR; Left Inverted Terminal Repeat, RITR; Right Inverted Terminal Repeat. RESULTS ATF/suppressed transcriptional activity and protein expression in lung and esophageal SCC cells In order to suppress SOX2 expression in lung SCC, we have generated a zinc finger-based artificial repressor for SOX2 termed ATF/using six zinc finger arrays designed to bind a 19 bp site in the D8-MMAE distal.