Pro-survival pathways in triple negative breast cancer: Focus on the roles of RIP2 and HSPB1

Abstract

Triple negative breast cancers (TNBC) lack estrogen and progesterone receptors, and HER-2 markers rendering them resistant to current targeted therapies. Most TNBCs show basal-like characteristics and the terms TNBC and basal-like breast cancer (BC) are used interchangeably. Both have high histological grade, characterized by exceptionally high mitotic indices, and the presence of central necrotic or fibrotic zones. Also, they have been reported to overexpress anti-apoptotic proteins, such as inhibitors of apoptosis proteins (IAPs), and release growth factors and cytokines that are important for pro-survival signaling and enhance carcinogenesis. Understanding the mechanism and role of these proteins in breast cancer would help develop effective and targeted therapy. In Chapter 1 of this thesis, proteins essential for the induction of survival pathways were identified and their relative messenger ribonucleic acid (mRNA) expression was analyzed among different (BC) patient samples obtained from online GEO NCBI. This is the first study that comprises expression analyses of different isoforms of mRNA expression of genes important in pro-survival pathways regulation, to understand basal-like BC tumourigenesis, and determine main proteins involved in BC prognosis and identify potential therapy targets. mRNA expression of genes involved in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and the heat shock response (HSR) pathways were found to be most associated with TNBC subtype, and the mRNA expression of genes involved in these pathways were found to be correlated with tumour size, tumour grade and worse prognosis. In addition, the genes involved in these pathways were highly expressed in patient samples of basal-like or TNBC subtypes and positively correlated with mRNA gene expression of TP53 and the proliferation marker MKI-67. Interestingly, the NF-κB pathway related genes’ mRNA expression was highly correlated with TP53 and MKI-67 mRNA expression in the basal-like BC subtype. Therefore, MDA-MB-231 cell line was used in order to understand the involvement of the NF-κB pathway and HSR in TNBC. In Chapter 2 of this thesis, the role of receptor interacting serine/threonine protein kinase 2 (RIP2) in enhancing BC development was studied. RIP2 protein has been reported to increase NF-κB activation and have a role in increasing cell proliferation and survival. It has been reported to associate with TNBC enhancing metastasis. Therefore, the role of RIP2 protein in TNBC was studied. An anti-apoptotic role of RIP2 in TNBC via activation of NF-κB was demonstrated. It is well known that NF-κB signaling mediates cancer cell proliferation and protects breast cancer cells from apoptosis. Therefore, targeting RIP2 could potentially sensitize cells to therapeutic agents. In this study, inactive mutant forms of RIP2 or shRNA against RIP2 sensitized TNBC cells to apoptosis. Anti-apoptotic proteins 13 expression levels, such as, B-cell lymphoma 2 (Bcl-2), B-cell lymphoma 2-extra large (Bcl- xL), and IAPs, increased upon RIP2 overexpression in MDA-MB-231 cells. This overexpression of anti-apoptitic proteins might explain the resistance of TNBC to drug induced cell death. Upon inhibition of NF-κB, the expression of anti-apoptotic proteins enhanced by RIP2 was reduced, and the cells were sensitized to drug treatment. RIP2 also mediated the release of cytokines and growth factors from these cells. Released factors include interleukin (IL) IL-6, IL-8, vascular endothelial growth factor (VEGF) and (C-X-C motif) ligand 1 (CXCL-1). These results were confirmed by data analysis of patients’ breast cancer databases, where high RIP2 expression was associated with poor prognosis, and decreased survival of patients. RIP2 expression was also associated with TNBC subtype; in addition, it was correlated with high mRNA expression of Bcl-xL, X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein (cIAP) anti-apoptotic proteins and IL-6, tumour growth factor (TGF) and VEGF released factors. The last section of the thesis focused on the role of intracellular chaperone heat shock proteins (HSPs), HSPB1 and HSPA1in TNBC pro-survival signaling. HSPB1 belongs to the family of small HSPs and is a potent regulator of apoptosis. However, the role of HSPB1 in endoplasmic reticulum (ER) stress induced apoptosis has not been defined. Previous work in the lab showed that heat shock could protect cells from ER stress induced cell death. Treating cells with ER stress inducing drugs did not affect HSPB1 or HSPA1 expression, and neither phosphorylation level of HSPB1 in different TNBC cell lines. Therefore, a crosstalk between ER stress and HSR pathways in TNBC was not observed. An interesting finding was the release of non-phosphorylated form on HSPB1 into culture media of MDA-MB-231 and MDA-MB-453 cells. Neutralizing HSPB1 by treating culture media with anti-HSPB1 antibody did not show any effect on cell survival, resistance to drug treatments or cell migration.