When breast cancer progresses to a metastatic stage, survival rates decline rapidly and it is considered incurable. Thus, deciphering the critical mechanisms of metastasis is of vital importance to develop new treatment options. We hypothesize that studying the proteins that are newly synthesized during the metastatic processes of migration and invasion will greatly enhance our understanding of breast cancer progression. We conducted a mass spectrometry screen following bioorthogonal noncanonical amino acid tagging to elucidate changes in the nascent proteome that occur during epidermal growth factor stimulation in migrating and invading cells. Annexin A2 was identified in this screen and subsequent examination of breast cancer cell lines revealed that Annexin A2 is specifically upregulated in estrogen receptor negative (ER-) cell lines. Furthermore, siRNA knockdown showed that Annexin A2 expression promotes the proliferation, wound healing and directional migration of breast cancer cells. In patients, Annexin A2 expression is increased in ER- breast cancer subtypes. Additionally, high Annexin A2 expression confers a higher probability of distant metastasis specifically for ER- patients. This work establishes a pivotal role of Annexin A2 in breast cancer progression and identifies Annexin A2 as a potential therapeutic target for the more aggressive and harder to treat ER- subtype.

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Breast cancer is the most prevalent form of cancer worldwide, with surgery remaining a standard treatment. Although a treatable disease, with the survival rate improving due to enhancements in screening and treatment, cancer recurrence remains a dominant contributor to breast cancer related deaths. It has been suggested that systemic factors during the postoperative period, such as surgical site infection (SSI), may increase the risk of recurrence, although the exact role that these infections play in breast cancer recurrence has yet to be elucidated. To investigate the influence of these SSIs in breast cancer recurrence following primary breast cancer surgery, we conducted a systematic literature review1 to examine both the incidence and risk factors related to SSI after primary breast cancer surgery and the contribution of SSIs to breast cancer recurrence. Data were extracted from 99 studies for the SSI-focused searches, and 53 studies for recurrence-focused searches and we found that there was a 13.07% mean incidence of SSIs. 638 Gram-positive and 442 Gram-negative isolates were identified, with Staphylococcus aureus and Escherichia coli appearing as the most abundant bacteria in SSIs of breast cancer patients. 11.8% cases of cancer recurrence were noted, however confounding factors of retrospective study design, surgery type and SSI definition make results challenging to compare and interpret. Only five studies investigated the association between SSI and breast cancer recurrence, three of which highlighting a positive correlation between SSI and breast cancer recurrence. To further explore this link, we are using in vitro models to investigate the molecular mechanisms by which both Gram-positive and Gram-negative bacteria affect the phenotype of breast cancer. Using LTA (a component of the cell wall of Staphylococcus aureus) to model Gram-positive bacteria and LPS (a component of the Escherichia coli cell wall) to model Gram-negative bacteria, we examined the effects of LTA and LPS on the behavior of MCF-7, MDA-MB-231 and ZR-75-1 cells. Using qRT-PCR, we found that treatment of cells with LPS and LTA dysregulates the expression of pro-tumorigenic inflammatory markers TNF-a and IL-6 in MDA-MB-231 cells. Separately, using metabolomic profiling on LPS and LTA treated MDA-MB-231 cells, we observe significant metabolic reprogramming of the cells when stimulated with the bacterial mimics. We believe that these findings may lead to a better understanding of how SSI promotes breast cancer recurrence and may help design surgical procedures and target therapies. 1O’Connor, R. Í., Kiely, P. A., & Dunne, C. P. (2020). The relationship between post-surgery infection and breast cancer recurrence. A systematic review. Journal of Hospital Infection. 106, (522-535) https://doi.org/10.1016/j.jhin.2020.08.004. Citation Format: Ruth Í. O'Connor, Amira F. Mahdi, Joanne Nolan, Catríona M. Dowling, Colum P. Dunne, Patrick A. Kiely. How surgical site infection influences breast cancer cell recurrence and cancer cell reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6345.

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Breast cancer is the most frequently invasive cancer type among women worldwide, accounting for over 2 million new cases annually and 25% of all cancers diagnosed in women. Considering significant advances in the diagnostics and treatment of breast cancer it remains a leading cause of cancer mortality worldwide. The extracellular matrix (ECM) is a meshwork of fibrous proteins and proteoglycans that is essential for providing a physical framework and facilitating biochemical cues to maintain tissue homeostasis. However, the ECM also has a well-established role in tumour development and progression. Increasing evidence suggests that the ECM may impede targeted therapies and novel approaches that target the composition of the ECM may improve drug delivery and efficacy. Using the xCELLigence real time analysis platform, we monitored the growth of several breast cancer cell lines on the major fibrous proteins of the ECM to elucidate the contribution of individual ECM components on cell behaviour. Coupling this with endpoint assays, we have identified diverse adhesion, proliferation and migration profiles for each cell line on each substrate. RNA was extracted from cells in 2-Dimensional culture on 1) collagen, 2) fibronectin, 3) laminin and 4) stimulated by various growth factors and from cells in 3-Dimensional culture. Using qRT-PCR, we examined the expression of 40 genes that code for proteins known to directly or indirectly regulate the composition of the ECM. We determined that there is significant differential gene expression amongst each cell line in response to individual ECM components. Using a hierarchical clustering analysis of our gene panel, we have identified a strong relationship amongst cell lines maintained on collagen and fibronectin. We used STRING[1] analysis to help us refine a gene signature and are currently examining our gene set in patient tissue samples. Our hypothesis is that the composition of the ECM regulates gene expression and this correlates with advanced cancer stage. This work has the potential to create a clinically relevant gene signature to be used in the prediction of invasive cancers. Understanding how the ECM influences these proteins may provide information on the design of novel ECM targeted therapies in breast cancer. 1. Szklarczyk, D., et al., The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic acids research, 2017. 45(D1):p. D362-D368. Citation Format: Joanne Nolan, Amira F. Mahdi, Aoife J. Lowery, Colum P. Dunne, Patrick A. Kiely. Using hierarchical clustering of differential gene expression patterns to study the contribution of the extracellular matrix in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1908.