Investigation of the localization pattern and the role of anti apoptotic BAG-1 in healthy and tumor tissues from patients with breast carcinoma

Abstract

Breast cancer is the second most common cancer in world, and by far the most frequent cancer among women. In less developed region, it is the most frequent reason of death in women, and second cause of cancer death in more developed regions after lung cancer. Besides, in Turkey the most common cancer in women is breast cancer. Immunohistochemical biomarkers oestrogen receptor, progesterone receptor, human epidermal growth factor receptor and Ki-67 are considered with the combination of tumor grade, size, and nodal status for the disease status. Presently, there are 20 major types of breast cancer. However, mainly four subgroups that has been defined clearly based on gene expression profiles and molecular classification were chosen for this study: Luminal A, luminal B, HER2 enriched and triple negative breast cancers. Estrogen and progesterone receptors are steroid hormone receptors that have roles in cell growth and proliferation. PR is ligand activated transcription factor and activate signal transduction pathway in pro-proliferative signalling. It induces the activation of MAPK signalling in cell. ER induces PI3K/AKT signalling and stimulates cell growth in breast cancer cells. Besides, activated HER2 receptor phosphorylates RAS and PI3K pathways also. In nucleus, steroid hormone receptors recognise and bind to hormone responsive enhancer sequences. Bcl-2 associated anthanogene BAG-1 is commonly increased in pre-invasive breast disease and breast cancer compared to normal breast epithelium. In addition, Bag family proteins express differentially in a range of human cancers and tumour cell lines, particularly leukaemia, breast, prostate, and colon cancers. Bag family, a multifunctional group of protein that interacts with a wide range of target molecules and regulate apoptosis, proliferation, transcription, metastasis, motility and autophagy. BAG-1 also has anti-apoptotic effects, which are independent of Bcl-2; it binds to numerous receptor tyrosine kinases and improves their ability to inhibit apoptosis. Studies show that BAG-1 can be accepted as a biomarker for clinical purposes in early breast cancer prognosis. In case of breast cancer, Bag-1 is a co-chaperone for the heat-shock protein HSP70 that interacts AKT, BCL-2, B-RAF and C-RAF, steroid hormone receptors and other proteins. In the other hand, it increases estrogen dependent transcription by regulating ER function. Reported associations between BAG-1 and PR, ER are variable. To address these issues we tried to assess the expression of BAG-1 and BAG-1 related survival factors with cell lines with breast cancer that differ from the receptor type in our laboratory.Our early studies encourage us to enlighten to find out exact role of BAG-1 in survival pathway. Three isoforms of BAG-1 that are generated by alternative translation mechanism are localized various location in the cell. Bag-1 isoforms, which can be differentially localized in the cell, are functionally distinct. Alternative translation initiation from a single mRNA transcript produces 3 major and 1 minor isoform. All isoforms have BAG domain, but nuclear localization signalling is found in BAG-1L and a part of NLS in BAG-1M. Thus, BAG-1L localizes on nucleus and BAG-1M can also be translocated to nucleus. The isoforms of BAG-1 are specialized according to the tissue and cell lines they localized. Because of the involvement in number of cellular pathways, BAG-1 is regarded as an important marker for diseases, regulating cell survival. Apoptotic protein BAD is known that reside in mitochondrial membrane with dimerized with BCL-2 and BCL-XL. This dimerization promotes BAD mediated apoptosis in cell. If BAD is phosphorylated on Ser112 and Ser136 that residues are considered to be in BCL-binding site, phosphorylated BAD unable to bind mitochondrial membrane via BCL and exist cytoplasm. Cytoplasmic BAD promotes cell survival. The previous studies in our lab demonstrated that Bag-1 and its interaction partners have impprtant roles in cell survival and apoptosis pathways. The studies were managed by using tumorigenic MCF-7 and triple-negative MDA-MB-231 and non-tumorigenic MCF-10A epithelial cell lines. In our laboratory, BAG-1 interactions with p-AKT, BRAF, CRAF, BCL-2 was verified. In further, the member of this interaction complex AKT, phosphorylates BAD at Ser 136 residue and Ser 112 residue of BAD is phosphorylated via p-CRAF. The phosphorylated BAD on these two residues promotes cell survival. This work exited us to find these pathways in breast carcinoma patients' tissue. To begin with, we think about that "Are these pathways also present in tissues from breast carcinoma patients?'' and "How is the situation in breast cancer linked to hormone receptors?'' In this study, it was intended to enlighten hormone receptor and HER2 dependent expression patterns of BAG-1 and survival factors affecting BAD in breast cancer tissues. To achieve this aim, BAG-1, BAD, AKT, BRAF, CRAF and phosphorylated forms were investigated in normal and tumor breast tissues across ER, PR, HER2 status. Simultaneously, proteomic effects if the mutation in common tumor related genes were searched to identify tumor progression for same patients' sample. This study consist of two workflow, one is conducted with tissue sample from patients. Tumor tissue samples were supplied from Univesity of Ministry of Health - Umraniye Training and Research Hospital of Istanbul. Tissues were taken from the lumpectomy, mastectomy surgeons. The fresh tissues were taken into RNA Later solution. Protein isolation of both normal and tumor tissue samples were done immediately. Immunobloting studies were done further. Besides, immunohistochemistry studies were conducted. To make this experiments, tumorous sections of tissues were embedded in paraffin blocks. Formaline fixed parafine embedded tissues then stained with interested antibodies. In the other hand, we conducted NGS study to detect major common tumor suppressor and oncogenic genes variations via the genetic material isolated from the blood sample from the same patients. We conducted to library preparation step via PCR based detection kit for our breast cancer panel. Sequencing was conducted further. Aligned fragments were then analysed to searched the potential variation by using genetic tools. Many small cohort studies in literature have measured expression of BAG-1 with different outcomes in breast cancer. Researchers and clinicians found that many different results such as association between nuclear BAG-1 expression and decreased survival or association between high cytoplasmic Bag-1 and improved survival. However the estrogen, progesterone, human epidermal growth factor receptor and Bag-1 interactions came up as an important issue. Also, described associations between Bag-1, ER and PR are different. Some studies have shown no association between Bag-1 expression and ER or PR expression while other studies have shown that Bag-1 and ER tend to co-express, as do BAG-1 and PR. There are many contradictions about it. Total forty eight tissue samples from different patients were used to make protein experiments in this study. The main finding in this study is that in tumors, BAG-1 L overexpression accompanied by BAG-1 localization in nucleus. Besides, ER positivity is parallel for this relationship. In lumainal A type, BAG-1L, p-AKT and p-BAD Ser 136 resided in nucleus. Depends on the finding, we suggested that possible BAG-1, p-AKT, BAD/p-BAD Ser 136 triple complex might be co-localized in nucleus in the condition of ER presence and BAG-1L could be the main target for the ER-related breast tumors.