ROLE OF STAT1 AND CXCL10 IN THE TUMOUR IMMUNE MICROENVIRONMENT OF HIGH-GRADE SEROUS OVARIAN CANCER

High-grade serous ovarian cancer (HGSC) is the most prevalent epithelial ovarian cancer characterized by late detection, metastasis and resistance to chemotherapy. Previous studies on the tumour immune microenvironment in HGSC identified STAT1 and CXCL10 as the most differentially expressed genes between treatment naïve chemotherapy resistant and sensitive tumours. Interferon-induced STAT1 is a transcription factor, which induces many genes including tumour suppressor genes and those involved in recruitment of immune cells to the tumour immune microenvironment (TME), including CXCL10. CXCL10 is a chemokine that recruits tumour infiltrating lymphocytes (TILs) and exhibits angiostatic function. The current study was performed to determine the effects of differential STAT1 and CXCL10 expression on HGSC disease progression and TME. STAT1 expression and intratumoural CD8+ T cells were evaluated as prognostic and predictive biomarkers via immunohistochemistry on 734 HGSC tumours accrued from the Terry Fox Research Institute-Canadian Ovarian Experimental Unified Resource. The combined effect of STAT1 expression and CD8+ TIL density was confirmed as prognostic and predictive companion biomarkers in the second independent biomarker validation study. Significant positive correlation between STAT1 expression and intratumoral CD8+ TIL density was observed. The effects of enforced CXCL10 expression on HGSC tumour growth, vasculature and immune tumour microenvironment were studied in the ID8 mouse ovarian cancer cell engraftment in immunocompetent C57BL/6 mice. Significant decrease in tumour progression in mice injected with ID8 CXCL10 overexpressing cells compared to mice injected with ID8 vector control cells was observed. Multiplexed cytokine analysis of ascites showed differential expression of IL-6, VEGF and CXCL9 between the two groups. Endothelial cell marker staining showed differences in tumour vasculature between the two groups. Immune transcriptomic profiling identified distinct expression profiles in genes associated with cytokines, chemokines, interferons, T cell function and apoptosis between the two groups. These findings provide evidence that STAT1 is an independent biomarker and in combination with CD8+ TIL density could be applied as novel immune-based biomarkers in HGSC. These results provide the basis for future studies aimed at understanding mechanisms underlying differential tumour STAT1 and CXCL10 expression and its role in pre-existing tumour immunologic diversity, thus potentially contributing to biomarker guided immune modulatory therapies.

FES KINASE SIGNALING PROMOTES MAST CELL RECRUITMENT TO TUMOURS

FES protein-tyrosine kinase (PTK) activation downstream of the KIT receptor in mast cells (MC) promotes cell polarization and migration towards the KIT ligand Stem cell factor (SCF). A variety of tumours secrete SCF to promote MC recruitment and release of mediators that enhance tumour vascularization and growth. This study investigates whether FES promotes MC migration via regulation of microtubules (MTs), and if FES is required for MC recruitment to the tumour microenvironment. MT binding assays showed that FES has at least two MT binding sites, which likely contribute to the partial co-localization of FES with MTs in polarized bone marrow-derived mast cells (BMMCs). Live cell imaging revealed a significant defect in chemotaxis of FES-deficient BMMCs towards SCF embedded within an agarose drop, which correlated with less MT organization compared to control cells. To extend these results to a tumour model, mouse mammary carcinoma AC2M2 cells were engrafted under the skin and into the mammary fat pads of immune compromised control (nu/nu) or FES-deficient (nu/nu:fes-/-) mice. A drastic reduction in tumour-associated MCs was observed in FES-deficient mice compared to control in both mammary and skin tissue sections. This correlated with a trend towards reduced tumour volumes in FES-deficient mice. These results implicate FES signaling downstream of KIT, in promoting MT reorganization during cell polarization and for chemotaxis of MCs towards tumour-derived SCF. Thus, FES is a potential therapeutic target to limit recruitment of stromal mast cells or macrophages to solid tumours that enhance tumour progression.

Role of hypoxia and hypoxia-inducible factor-1 in tumour immune escape

Previous studies revealed that, upon exposure to hypoxia, tumour cells acquire resistance to the cytolytic activity of IL-2-activated lymphocytes. The MHC class I chain-related (MIC) molecules – comprised of MICA and MICB – are ligands for the activating NKG2D receptor on Natural Killer (NK) and CD8+ T cells. MIC-NKG2D interactions lead to the activation of NK and CD8+ T cells and the subsequent lysis of the tumour cells. The study also showed that the mechanism of the hypoxia-mediated immune escape involves the shedding of MIC, specifically MICA, from the tumour cell surface. The objective of the present study was to determine whether the shedding of MICA requires the expression of hypoxia inducible factor-1 (HIF-1), a transcription factor that regulates cellular adaptations to hypoxia. Exposure to hypoxia (0.5% O2 vs. 20% O2) led to the shedding of MIC from the surface of MDA-MB-231 human breast cancer cells and DU-145 human prostate cancer cells as determined by flow cytometry. Knockdown of HIF-1α mRNA using siRNA technology resulted in inhibition of HIF-1α accumulation under hypoxic conditions as determined by Western blot analysis. Parallel study revealed that knockdown of HIF-1α also blocked the shedding of MICA from the surface of MDA-MB-231 cells exposed to hypoxia. These results indicate that HIF-1 is required for the hypoxia-mediated shedding of MICA and, consequently, that HIF-1 may play an important role in tumour immune escape. Ongoing studies aim to determine the HIF-1 target genes involved in the shedding of MICA under hypoxia.

The role of vitamin D signalling in the interaction between mesenchymal mammary tumour cells and macrophages

The transition of epithelial-like tumour cells to those exhibiting mesenchymal characteristics (Epithelial-to-mesenchymal Transition; EMT) is an integral process in breast cancer metastasis. EMT can be promoted by Transforming growth factor-beta (TGF-β) which can be found at high levels in the tumour stroma. Tumour-associated macrophages (TAMs) can also induce EMT in breast cancer cells, which is one way that they promote breast cancer metastasis. Vitamin D signalling has been implicated in EMT suppression and plays a role in modulating macrophage differentiation and stimulating their anti-inflammatory functions. This project had two major aims. First, we aimed to create and verify a unique fluorescent reporter gene construct designed to evaluate the dynamics of EMT in real-time and at the single-cell level. While some components of this reporter system were successfully validated, work to complete the final reporter construct is ongoing. The second and main aspect of this project focused on exploring the ability of 1,25-dihydroxyvitamin D3 (1,25D3) to modulate the interaction between mesenchymal mammary tumour cells and TAMs. Unexpectedly, in short-term treatment (48 hours) studies of 4T1 murine mammary tumour cells, we observed that 1,25D3 and TGF-β signalling work together to increase expression of the mesenchymal markers, Snai1, Fn1, and Col1a1. 1,25D3 and TGF-β also synergistically activate transcription of the gene encoding the 1,25D3-catabolizing enzyme, Cyp24a1. The ability of 1,25D3 and TGF-β to enhance expression of these genes was diminished in a long-term treatment (14 days) of 4T1 cells, and this effect was accompanied by a decrease in cell proliferation. 1,25D3 may also cooperate with cytokines produced by normal macrophages and macrophages considered to be TAM-like. Conditioned media experiments revealed that in the presence of factors from normal macrophages, 1,25D3 enhanced expression of Fn1, and in the presence of factors from TAM-like macrophages, 1,25D3 enhanced expression of Fn1 and Cyp24a1. Rather than mitigating the interaction as hypothesized, 1,25D3 may exacerbate the tumour-promoting effects of the EMT-TAM relationship. Also, signalling pathways involved in the EMT-TAM relationship may synergize with 1,25D3 to upregulate Cyp24a1 expression. These findings are important for understanding the potential of vitamin D compounds to be used in the treatment of breast cancer.