BAG-1 expression and function in HER2-positive breast cancer

Abstract

Background: In the United Kingdom every year 50,000 women are diagnosed with breast cancer, and 12,000 deaths are attributed to the disease. Human epidermal growth factor receptor 2 (HER2/ neu) is overexpressed in 15- 25% of breast cancer and is a marker of poor prognosis. Treatment with the monoclonal antibody trastuzumab (Herceptin™) can reduce the risk of death by a third and halve the risk of recurrence, but many of those treated do not benefit. The potential for combination treatments is seen in neoadjuvant studies where combination anti-HER2 therapies show greater efficacy than single anti-HER2 therapy.

Methods: BAG-1 is a multifunctional protein frequently overexpressed in breast cancer. BAG-1 interacts with Hsc70/ Hsp70 and the serine/threonine kinase RAF-1 to promote cell survival. This study investigated the potential of targeting BAG-1 in HER2-positive breast cancer. The functional significance of BAG-1 expression in HER2-positive breast cancer cells was investigated using BAG-1 overexpression, and siRNA knockdown. The effect of HER2 inhibition in combination with BAG-1 protein-protein inhibition using Thioflavin S was investigated with cell viability and cell yield assays. Western blots of upstream and downstream proteins were used to explore proposed signalling mechanisms involved. Previous evidence for BAG-1 as a biomarker in breast cancer was assessed against published reporting criteria (REMARK). An immunohistochemical study of BAG-1 expression was performed in a test cohort of ERpositive breast cancers, using the pan-isoform antibody, 3.10G3E2, and a novel BAG1L isoform-specific antibody.

Results: An increase in endogenous BAG-1L expression occurred following trastuzumab treatment of SKBR3 cells. Additionally overexpression of BAG-1 isoforms by transfection in SKBR3 cells exposed to trastuzumab resulted in an increase in cell number and relative cell viability compared to vector controls. Conversely, the combination of siRNA targeting of BAG-1 and exposure to trastuzumab enhanced reduction of cell viability. The decrease in relative cell viability following trastuzumab treatment was also enhanced following combination with Thioflavin S, a BAG-1:HSC70 interaction inhibitor, in BT-474 cells. This was accompanied by reduced expression of phospho-Akt, suggesting a possible mechanistic link between BAG-1, which interacts directly with RAF-1, and the HER2 signalling pathway. With a view to determining whether the expression of BAG-1L is elevated in HER2 positive cancers, a pan-isoform anti-BAG-1 antibody (3.10G3E2) and the novel BAG1L-specific antibody were optimised for immunohistochemical study using tissue microarrays. Further work is required prior to use on tissue from a large cohort of patients diagnosed with HER2-positive breast cancer, however in a second cancer type in which HER2 is known to be overexpressed, oesophageal adenocarcinoma, immunohistochemistry demonstrated an association between advanced tumour regression grade and nuclear BAG-1 expression. In summary, this work has demonstrated potentiating effects of BAG-1 expression on HER2 signalling pathways, possibly mediated through Akt phosphorylation. The work provides evidence that BAG-1 has the potential to play a role in HER2-positive breast cancer and affect the response to trastuzumab; therefore inhibition of the BAG-1/ RAF1/Akt pathway may be a potential therapeutic target when used in combination with anti-HER2 therapies.

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ORCID for Graham Packham: orcid.org/0000-0002-9232-5691

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