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Molecular characterization of the breast cancer associated antigen NY-BR-1

Bitzer, Julia

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Breast cancer is one of the most common malignancies with increasing incidence every year and a leading cause of death among women. Although early stage breast cancer can be effectively treated, there are limited numbers of treatment options available for patients with advanced and metastatic disease. The breast cancer associated antigen NY-BR-1 was identified by a serological screening strategy (SEREX). NY-BR-1 is expressed in the majority of breast tumours (>70 %) as well as in metastases, in normal breast tissue, in testis, and occasionally in prostate tissue. Due to its restricted expression pattern, its immunogenicity and its subcellular localization to the cytoplasm and plasma-membrane, NY-BR-1 represents a promising target for immunotherapeutic approaches in breast cancer patients. The aim of this thesis was the molecular and functional characterization of NY-BR-1 to reveal its biological function in normal and tumorigenic breast tissues. To examine the effect of NY-BR-1 over-expression on cellular processes, such as proliferation, apoptosis and the cell cycle, HEK293, HEK293T, MCF-10A, and MCF-7 were transiently transfected with NY-BR-1. The results showed that NY-BR-1+ cells do not proliferate, do not have an increased apoptosis rate and accumulate in the G1 phase compared to the control cells. This finding is supported by IHC stainings of normal breast tissue sections for NY-BR-1 and Ki-67 (proliferation marker) displaying NY-BR-1+/Ki-67- cells. Another approach to clarify the biological function of NY-BR-1 was the identification of specific protein interaction partners by co-immunoprecipitations with lysates of NY-BR-1 transfected cells (HEK293, MCF-10A, MCF-7) followed by mass spectrometry. The protein identified in all three analysed cell lines is the tubulin beta-4B chain, which amongst other is involved in mitosis, underlining a crucial role of NY-BR-1 during the cell cycle. To investigate the transcriptional regulation of the NY-BR-1 gene, an in silico prediction for potential transcription factor binding sites in the NY-BR-1 promoter region was performed with the Alibaba 2.0 algorithm. Tissue pieces as well as isolated epithelial cells from healthy breast tissue and tumour cells from pleural effusions were treated with different hormones, a demethylation agent and histone deacetylase inhibitors. The NY-BR-1 expression was analysed via qPCR. Several estrogen receptor binding sites were mapped in the NY-BR-1 promoter region and within the gene (up to Intron 21) by ChIP-Seq. The NY-BR-1 protein and the estrogen receptor (ER) are mainly co-expressed in normal breast tissues as assessed by IHC stainings and in silico analyses of RNA-seq data showed that NY-BR-1 is predominantly expressed in ER positive luminal A and B breast cancers. However, the stimulation of normal breast cells as well as tumour cells from pleural effusions with estrogen, tamoxifen or progesterone did not systematically show an up- or down-regulatory effect. On the other hand, short term stimulations of tumour cells from pleural effusions with progesterone, estrogen, vitamin D3 and retinoic acid lead to an up-regulation of NY-BR-1 expression (up to 8 fold) compared to the untreated controls. VPA treatment also induced NY-BR-1 expression. Of note, the combination of VPA with the above mentioned hormones only showed a weak up-regulation of NY-BR-1 expression (up to 3 fold) compared to the controls. These results suggest that NY-BR-1 expression is further regulated on histone level and/or non-histone proteins such as transcription factors modified by acetylation. Treatment of the cells with the demethylation agent 5´Aza-deoxycytidine did not show a distinct induction or up-regulation of NY-BR-1 expression. Moreover, the methylation status of selected promoter regions containing the predicted CpG islands was assessed in a selection of treated samples, but no association between methylation patterns and NY-BR-1 expression was observed. This finding could be confirmed by an in silico analysis of the TCGA database. Strikingly, a decreased NY-BR-1 expression in cultured normal breast tissue pieces and isolated epithelial cells versus snap-frozen cells was seen, which was not observed in breast tumour cells from pleural effusions cultured in conditioned medium. An in depth analysis of the conditioned medium containing the cell free effusion supernatant as well as cell culture experiments with normal breast epithelial cells in conditioned medium will reveal, which factors, e.g. cytokines, chemokines, hormones or growth factors, lead to a sustained NY-BR-1 expression. Considering the complex regulation of NY-BR-1 gene expression, the mosaic-like protein expression in normal breast tissue and the inhibitory effect of NY-BR-1 over-expression on proliferation and the G1 phase cell cycle arrest in normal cells it was hypothesized, that NY-BR-1 may be expressed by a specific breast progenitor cell population. Thus, mammospheres from isolated normal breast epithelial cells were generated and analysed by immunofluorescent staining and qPCR for NY-BR-1 expression and the presence of progenitor cell markers. In four analysed patients, NY-BR-1 was expressed in primary spheres and co-expression with integrin-α 6, HER2, GATA-3 and FOXA1 could be observed whereas no ER or progesterone receptor (PR) mRNA was detected. IHC staining with ER and NY-BR-1 in normal breast tissue showed that as well ER+/NY-BR-1+ as ER-/NY-BR-1+ cells in the mammary gland are existing. These results suggest that NY-BR-1 is expressed in the ER+/ER- luminal progenitor cells of the mammary gland. The NY-BR-1 gene was investigated regarding genetical variations to receive a better understanding of splice variants. Thereby, 69 damaging nsSNPs within the coding region of NY-BR-1 gene and 39 potential splicing SNPs could be identified by using in silico anlaysis. Taken together, NY-BR-1 is expressed mainly in well differentiated hormone sensitive estrogen receptor positive breast cancer subtypes and it is likely that NY-BR-1 expression is influenced by ERα and/or PR expression, but the association of NY- BR-1 expression and ER signaling still needs to be elucidated. Furthermore, transiently NY-BR-1 expressing cells show an inhibited proliferation rate and accumulate in the G1 phase. In vivo, endogenous NY-BR-1 is expressed in non proliferating (Ki-67 negative) cells in normal breast tissue. The protein potentially interacts with the tubulin beta-4B chain suggesting a crucial role during mitosis. Moreover, NY-BR-1 was shown to be expressed in progenitor cells of the mammary gland. The phenotypic characterization of these progenitor cells and the question whether the protein is also expressed in cancer stem cells is part of ongoing studies. In summary, the presented work could show for the first time that NY-BR-1 is a progenitor cell marker in the mammary gland and is influences the mitotic process.

Item Type: Dissertation
Supervisor: Gruss, Prof. Dr. Oliver
Date of thesis defense: 23 June 2015
Date Deposited: 21 Jul 2015 12:26
Date: 2016
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
570 Life sciences
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