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

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.

Overexpression of ZEB2 at the Invasion Front of Colorectal Cancer Is an Independent Prognostic Marker and Regulates Tumor Invasion In Vitro

“…ZEB2 was predominantly expressed in the cytoplasm, whereas nuclear staining was observed only in a few cells. These findings are consistent with a recent report, comparing the expression of ZEB2 by 2 monoclonal antibodies in a small cohort of patients with colorectal cancer (31). Due to the overexpression of ZEB2 at the invasion front, we postulated an invasion-promoting role of this transcription factor.…”

ZEB2/SIP1 as novel prognostic indicator in eyelid sebaceous gland carcinoma

“…Immunohistochemical staining of ZEB2 predominantly displayed a cytoplasmic staining, whereas nuclear staining was observed only in a few cells. These results are consistent with a previous study, in which Oztas et al [21] generated 2 clones of anti-ZEB2 antibodies and assessed their immunoreactivity in an immunohistochemical study of cell lines and multiple tumor tissue arrays, yielding predominantly cytoplasmic staining. Similar expression patterns were also earlier reported in pancreatic cancer [12], colorectal cancer [13], non-small cell lung cancer [22], and hepatocellular carcinoma [23].There was a significant association of ZEB2 overexpression and orbital invasion with poor disease-free survival.…”

PTPRDis Homozygously Deleted and Epigenetically Downregulated in Human Hepatocellular Carcinomas

“…The intensity of PTPRD staining for each sample was scored as previously described (Chen et al, 2006;Oztas et al, 2010). Statistical analysis were performed by Student's t-test.…”