Molecular and functional characterization of the candidate tumor antigens placenta specific 1 and claudin 6

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Identification of tumor-specific antigens is central to the effectiveness of cancer immunotherapies. Within this study, two candidate tumor antigens, placenta-specific 1 (PLAC1) and claudin 6 (CLDN6), were characterized regarding their suitability to serve as antigens for cancer immunotherapeutic approaches. PLAC1 is a tumor-specific antigen with tumor-promoting functions in breast cancer. The estradiol (E2)-responsive PLAC1 gene is known to be regulated by estrogen receptor α (ERα), specificity protein 1 (SP1) and CCAAT/enhancer binding protein β-2 (C/EBPβ-2). However, additional regulators of PLAC1 were not identified so far. Here, it was investigated whether members of the nuclear receptor co-activator (NCOA)/p160 family are involved in ERα/E2-mediated trans-activation of PLAC1 in breast cancer. Using chromatin immunoprecipitation (ChIP), NCOA3 was identified to be selectively recruited to the PLAC1 promoter upon E2-stimulation only in ERα-positive MCF-7 breast cancer cells but not in ERα-negative SK-BR-3 cells. RNAi-mediated silencing of NCOA3 revealed diminished PLAC1 mRNA and protein levels in MCF-7 but not in SK-BR-3 cells. Stimulation of MCF-7 cells with E2 resulted in decreased up-regulation of PLAC1 compared to control transfected cells. Moreover, a significant correlation of PLAC1 and NCOA3 expression was found in ERα-positive human breast cancer tissues only. Collectively, these data show that PLAC1 is a downstream target of NCOA3 and provides a relationship between NCOA3 and PLAC1 that could be important for future immunotherapies targeting hormone-responsive breast cancer. The tight junction molecule CLDN6 is an embryonic antigen that plays a major role during epithelial differentiation. In this study, expression, function and regulation of CLDN6 in cancer was characterized. Analysis of CLDN6 expression by quantitative real-time RT-PCR showed that CLDN6 is absent in adult normal tissues but frequently overexpressed in many cancers, especially in ovarian, testicular and lung cancer. RNAi-mediated silencing of CLDN6 did not reveal any function for CLDN6 in proliferation, apoptosis, cell cycle, migration and adhesion in three different cancer cell lines. However, colony formation was decreased in CLDN6-negative compared to CLDN6-positive PA-1 teratocarcinoma cells. The capacity to form colonies at distant sites is a key feature of tumor cells with tumor-initiating potential suggesting a role for CLDN6 in cancer stem cells (CSC). This was further supported by the significant correlation of CLDN6 expression with several CSC-specific markers in ovarian cancer detected in this study. Moreover, CLDN6 was found here to be specifically detected on the surface of induced pluripotent (iPS) cells and up-regulated throughout the course of reprogramming. Thus, CLDN6 could be confirmed as a specific marker for iPS cells. Bisulfite sequencing and methylation-specific qPCR (MsqPCR) revealed that CLDN6 expression in tumors is associated with promoter hypomethylation. However, it is likely that promoter demethylation is not sufficient for up-regulation of CLDN6 in cancer. Accordingly, promoter analysis using deletion constructs, ChIP and RNAi-mediated silencing suggests that CCCTC-binding factor (CTCF) and brother of regulator of imprinted sites (BORIS) are possible transcription factors regulating CLDN6 in cancer. In summary, this work introduces CLDN6 as highly specific tumor antigen with a possible role in CSC that could be used for cancer immunotherapy in particular to target CSC.

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