Novel monoclonal antibodies detect Smad-interacting protein 1 (SIP1) in the cytoplasm of human cells from multiple tumor tissue arrays

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Abstract

Smad-interacting protein 1 (SIP1, also known as ZEB2) represses the transcription of E-cadherin and mediates epithelial–mesenchymal transition in development and tumor metastasis. Due to the lack of human SIP1-specific antibodies, its expression in human tumor tissues has not been studied in detail by immunohistochemistry. Hence, we generated two anti-SIP1 monoclonal antibodies, clones 1C6 and 6E5, with IgG1 and IgG2a isotypes, respectively. The specificity of these antibodies was shown by Western blotting studies using siRNA mediated downregulation of SIP1 and ZEB1 in a human osteosarcoma cell line. In the same context, we also compared them with 5 commercially available SIP1 antibodies. Antibody specificity was further verified in an inducible cell line system by immunofluorescence. By using both antibodies, we evaluated the tissue expression of SIP1 in paraffin-embedded tissue microarrays consisting of 22 normal and 101 tumoral tissues of kidney, colon, stomach, lung, esophagus, uterus, rectum, breast and liver. Interestingly, SIP1 predominantly displayed a cytoplasmic expression, while the nuclear localization of SIP1 was observed in only 6 cases. Strong expression of SIP1 was found in distal tubules of kidney, glandular epithelial cells of stomach and hepatocytes, implicating a co-expression of SIP1 and E-cadherin. Squamous epithelium of the esophagus and surface epithelium of colon and rectum were stained with moderate to weak intensity. Normal uterus, breast and lung tissues remained completely negative. By comparison with their normal tissues, we observed SIP1 overexpression in cancers of the kidney, breast, lung and uterus. However, SIP1 expression was found to be downregulated in tumors from colon, rectum, esophagus, liver and stomach tissues. Finally we did nuclear/cytoplasmic fractionation in 3 carcinoma cell lines and detected SIP1 in both fractions, nucleus being the dominant one. To our best knowledge, this is the first comprehensive immunohistochemical study of the expression of SIP1 in a series of human cancers. Our finding that SIP1 is not exclusively localized to nucleus suggests that the subcellular localization of SIP1 is regulated in normal and tumor tissues. These novel monoclonal antibodies may help elucidate the role of SIP1 in tumor development.

Introduction

Smad-interacting protein 1 (SIP1, also known as ZEB2) encoded by ZFHX1B is a member of ZEB family of transcription factors. The protein contains a central homeodomain, CtBP-binding and Smad-interacting domains and two zinc finger clusters each at either end (Remacle et al., 1999, Verschueren et al., 1999). SIP1 directly binds to bipartite E-boxes on the promoters of different targets by means of its zinc finger domains and mediates transcriptional repression (Verschueren et al., 1999). One of these targets is CDH1, the gene encoding for the epithelial adherens junction protein E-cadherin, whose transcriptional downregulation induces epithelial-to-mesenchymal transition (EMT) in developmental processes and during tumor cell invasion and metastasis (Comijn et al., 2001). Transcriptional repression is mediated through the association of SIP1 with the corepressor CtBP, however this interaction is dispensable at least for the attenuation of CDH1 transcription (Postigo et al., 2003, van Grunsven et al., 2003). Overexpression of SIP1 in epithelial cells has also been shown to downregulate constituents of cell–cell junctions other than E-cadherin (Vandewalle et al., 2005). Although binding of SIP1 to p300 or pCAF was proposed as a mechanism for transactivation and other transcriptional activators associated to SIP1 are yet to be determined, SIP1-mediated upregulation of EMT and invasion related genes, such as vimentin and matrix metalloproteases, have been reported (Bindels et al., 2006, Miyoshi et al., 2004, Postigo et al., 2003).

Despite the overwhelming evidence that SIP1 induces EMT phenotype, its role in tumorigenesis was ill-defined. In fact, SIP1 was originally identified as a binding partner of R-Smads, and shown to be part of the TGF-β pathway, which is frequently involved in carcinogenesis (Verschueren et al., 1999). hTERT repression in breast cancer cells was partly mediated by SIP1 in a TGF-β dependent manner (Lin and Elledge, 2003). Also, analysis of senescence arrest of clonal hepatocellular carcinoma cells revealed SIP1 as a mediator of hTERT repression (Ozturk et al., 2006). Impaired G1/S progression was observed upon repression of cyclin D1 by SIP1 (Mejlvang et al., 2007). SIP1 was also shown to contribute to tumorigenesis in a transgenic mouse model of lymphoma by retroviral tagging (Mikkers et al., 2002). The differential expression of SIP1 has been described, mostly by RT-PCR, in several human tumors due to the lack of human SIP1-specific antibodies. E-cadherin downregulation was associated to increased SIP1 expression in intestinal type gastric carcinoma but not in diffuse type gastric carcinoma (Rosivatz et al., 2002). Elevated SIP1 expression correlated inversely with E-cadherin in advanced stages of pancreatic tumors (Imamichi et al., 2007). Surprisingly, SIP1 and E-cadherin expression were positively correlated in malignant mesothelioma (Sivertsen et al., 2006). In the esophagus, differential expression of SIP1 was observed during keratinocyte differentiation. Only stem cell containing basal cells, but not parabasal cells and keratinocytes expressed SIP1. Consistent with this, SIP1 transcripts were present in all studied esophageal carcinoma cases (Isohata et al., 2009). High SIP1/E-cadherin ratio correlated with metastatic disease and poor patient survival in breast and ovarian carcinomas (Elloul et al., 2005). Elevated SIP1 transcripts were observed in von Hippel–Lindau-null renal cell carcinomas in a hypoxia-inducible factor 1 alpha (HIF1α)-dependent manner (Krishnamachary et al., 2006). Immunohistochemical analysis of ovarian tumors revealed a stepwise increase of SIP1 from benign to borderline to malignant tumors (Yoshida et al., 2009). In oral squamous cell carcinoma, SIP1 was immunohistochemically detected in a relatively low proportion of tumors and its expression correlated with poor prognosis (Maeda et al., 2005). In a previous study, we have found that SIP1 was overexpressed in a series of bladder cancers. Its expression was found to be an independent prognostic factor in bladder cancers and positively stained cases correlated with poor therapeutical outcome (Sayan et al., 2009). With the exception of a few and as described above, most of the expression studies of SIP1 were done using RT-PCR technique, but SIP1protein levels have been shown to be tightly regulated by post-transcriptional mechanisms. For instance, Pc2-mediated sumoylation of SIP1 affects the transcriptional regulation of E-cadherin (Long et al., 2005). SIP1 has been identified as a direct target of miR-200 family and miR-205 (Gregory et al., 2008, Park et al., 2008).

In this study, we generated 2 new monoclonal antibodies (MAb) against the N-terminus of SIP1 protein and validated their specificity by specifically downregulating SIP1 protein, and the other ZFHX1 family member protein ZEB1, by siRNA in endogenous levels. Then, by using these antibodies we explored the expression pattern of SIP1 in human tumor cell lines and in a variety of tissues. We detected predominantly cytoplasmic but also nuclear SIP1 staining. Finally, subcellular fractionation of cell lines showed that SIP1 protein can be present in the cytoplasm and nucleus of multiple carcinoma cell lines. To our knowledge, this study is the first description of SIP1 protein expression in a multiple tumor tissue arrays.

Section snippets

Cell lines, tissues and siRNA transfections

Wild-type mouse SIP1 expressing squamous epidermoid carcinoma cell line A431/WTSIP1 with Tet-on doxycycline-inducible SIP1 expression was previously described (Mejlvang et al., 2007). Osteosarcoma cell line HOS2, hepatocellular carcinoma cell line SK-HEP-1 and colorectal carcinoma cell lines SW480 and SW620 were maintained in DMEM supplemented with 10% fetal bovine serum, 100 IU penicillin, 100 μg streptomycin and nonessential amino acids. Multiple Tumor Tissue arrays were purchased from BioChain

Monoclonal antibodies 1C6 and 6E5 detect both endogenous and overexpressed SIP1

Two MAbs, clones 1C6 and 6E5 were obtained by immunizing BALB/c mice with a partial human SIP1 recombinant protein (aa 1-360). The isotypes of antibodies were IgG2a and IgG1, respectively (data not shown). ZEB1 and SIP1 (ZEB2) are members of the zinc finger Homeobox 1 gene family. Among these 2 proteins, there is more than 45% overall protein homology which is much higher (up to 93% identity at C-terminal zinc finger cluster) in the functional domains (Vandewalle et al., 2009). Thus, as a first

Discussion

SIP1 has been reportedly shown to mediate EMT and disease aggressiveness in human tumors (Comijn et al., 2001, Elloul et al., 2005). Several studies indicated increased levels of SIP1 transcripts in association with invasion and metastasis in cancers with advanced stages (Imamichi et al., 2007, Miyoshi et al., 2004). However, a comprehensive study on SIP1 protein expression in human normal and tumors tissues has not been performed. We produced two MAbs using the N-terminal 360 amino acids of

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the Scientific and Technological Research Council of Turkey Grant 104S243 (to T.Y.) and the Cancer Research United Kingdom Grant C8851/A10844 (to E.T.).

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