The function of Bag-1 proteins in epidermal squamous cell carcinoma

• Main Author: Wood, Jemma Claire
• Published: University of Bristol 2011
• Subjects: 616.994
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.550326

The incidence of epidermal squamous cell carcinoma (SCC) has increased significantly over recent years, largely due to increased exposure to its major aetiological factor, UVB radiation. Despite its increasing prevalence, relatively little is known regarding the genetic changes involved in SCC development. The aim of this study was to investigate the potential involvement of the anti-apoptotic protein BcI-2-associated athanogene-l (Bag-i), which shows deregulated expression in a range of human tumours, in epidermal SCC development, in addition to investigating a potential functional role for Bag-l in the epidermal response to UVB radiation. Expression of Bag-l and its key interaction partner Hsp70, which is associated with Bag-l anti- apoptotic function, was investigated in a panel of 60 epidermal SCCs and 10 samples of normal epidermal epithelium. Bag-l expression was deregulated in a subset of tumours compared with normal epidermis, with high Bag-l protein levels associated with reduced tumour differentiation. A positive correlation was observed between cytoplasmic expression of Bag-l and Hsp70, with strong Hsp70 staining also correlating with reduced tumour differentiation. In an attempt to investigate potential functional consequences of elevated Bag-l expression in epidermal tumours, the role of Bag-l in the keratinocyte response to UVB was investigated using the non-tumorigenic, spontaneously immortalised HaCaT cell line as a model system. siRNA-mediated Bag-l knockdown sensitised HaCaT cells to UVB-induced apoptosis, implicating an anti-apoptotic role for Bag-l in this context. The anti-apoptotic function of Bag-l was found to involve the Bag-iS protein isoform, using' stablv-Bag-LS transfected clones of the HaCaT cell line, in addition to transient overexpression of Bag-iS using an adenoviral vector. Bag-l anti- apoptotic function was dependent on amino acid residues required for Hsp70 and Raf-l binding. In HaCaT cells, UVB-irradiation resulted in downregulation of anti-apoptotic McI-l 2 to 8 hours post-irradiation. Levels of the BH3-only pro-apoptotic proteins Bim and Noxa were elevated at 24 hours and 6 to 24 hours post-irradiation, respectively. Overexpression of Bag-iS attenuated UVB-induced McI-l protein reduction, highlighting a potential role for Bag-l in control of McI-l levels. Further to this, a pilot study of 8 samples of normal epidermal epithelium and 14 SCCs highlighted potential overexpression of McI-l in epidermal tumours compared with normal epidermis. The results presented in this thesis demonstrate that expression of Bag-l and Hsp70 is deregulated in epidermal SCC, implicating the Bag-l:Hsp70 axis as a potential novel therapeutic target. Elevated Bag-l expression in epidermal SCC may be associated with its role in protection against apoptosis induced by UVB irradiation in epidermal keratinocytes. Bag-l anti-apoptotic function in this context appears to be mediated, at least in part, via control of levels ofthe anti-apoptotic protein McI-1. Further to its role in protection against UVB-induced apoptosis, an anti-apoptotic role for Bag-l was identified in the response to the chemotherapeutic drug 5-fluorouracil in the SCC-13 epidermal SCC cell line. Levels of Bag-l in epidermal SCC may therefore affect the tumour response to chemotherapeutic agents.