The role of hypoxic signalling in the primary tumour microenvironment and metastasis in breast cancer

© 2013 Dr. Jaclyn Elise SceneayPublications included in thesis:Sceneay, J., Liu, M. C. P., Chen, A., Wong, C. S. F., Bowtel, D. D. L., & Möller, A. (2013). The antioxidant N-acetylcysteine prevents HIF-1 stabilization under hypoxia in vitro but does not affect tumorigenesis in multiple breast cancer models in vivo. PLOS ONE, 8(6), e66388. DOI: 10.1371/journal.pone.0066388Sceneay, J., Chow, M. T., Chen, A., Halse, H. M., Wong, C. S., Andrews, D. M., et al. ( 2012). Primary tumor hypoxia recruits CD11b+/Ly6Cmed/Ly6G+ immune suppressor cells and compromises NK cell cytotoxicity in the premetastatic niche. Cancer Research, 72, 3906-3911. DOI: 10.1158/0008-5472.CAN-11-3873Intratumoural hypoxia is a poor prognostic factor associated with reduced disease-free survival in many cancer types, including breast cancer. Under hypoxic conditions, tumour cells signal via the hypoxic response pathway and stabilisation of the HIF-1 transcription factor to increase the expression of genes that promote survival, proliferation, angiogenesis, invasion and metastatic spread. The factors secreted from hypoxic tumour cells help to recruit various stromal and bone marrow-derived cell (BMDC) lineages that together create a growth-supporting primary tumour microenvironment. The aims of this thesis are twofold; to investigate novel treatments targeting HIF-1 in the primary tumour microenvironment, and to investigate the effects of hypoxic signalling on pre-metastatic niche formation in breast cancer. Antioxidants have been identified as potential anti-cancer drugs that reduce tumour growth and metastasis in a HIF-1-dependent manner. N-acetylcysteine (NAC) is one such antioxidant that has a promising but somewhat controversial role in cancer therapy. This study utilised three orthotopic, syngeneic murine models of breast cancer, the PyMT, EO771 and 4T1.2 models, to investigate the ability of NAC to regulate the hypoxic response in breast tumour cells, and prevent tumour growth and metastasis. NAC demonstrated an ability to prevent HIF-1 stabilisation and signalling via the hypoxic response pathway in breast tumour cells in vitro. When administered in vivo however, NAC treatment did not inhibit primary tumour growth or metastasis. Furthermore, NAC treatment promoted metastases formation in an experimental metastasis model. This work brings into question the validity of NAC as an anti-tumourigenic agent in breast cancer, and highlights the need to further investigate its properties in vivo in different cancer models. Recently, it has been demonstrated that a primary tumour promotes metastasis through the formation of supportive microenvironments, termed pre-metastatic niches, in secondary organs predisposed to metastases. Cytokines, chemokines and growth factors secreted from tumour cells promote the mobilisation and recruitment of BMDCs to pre-metastatic organs, where they interact with the extracellular matrix and other stromal components to create tumour-promoting niches. While hypoxic tumour cells themselves have an increased ability to metastasize, they also secrete a variety of factors independently identified in pre-metastatic niches. This study investigated the combinatorial effect of factors secreted from hypoxic breast tumour cells on pre-metastatic niche formation in distant organs. Using immune competent eGFP bone marrow chimeric mice and different syngeneic mammary cancer models, hypoxic factors were shown to create an immunosuppressed environment in pre-metastatic organs. A subset of granulocytic CD11b+/Ly6Cmed/Ly6G+ myeloid-derived suppressor cells with increased CCR2 expression and STAT3 signalling were identified in the pre-metastatic niche. As were CD3-/NK1.1+ Natural Killer cells with reduced maturity, cytotoxicity, and expression of the activating receptor NKG2D. Together, factors secreted from hypoxic breast tumour cells promote metastasis by creating immunosuppressed pre-metastatic niches in distant organs. This work identifies novel components of the pre-metastatic niche that provide potential avenues for therapeutic intervention in metastatic disease