The role of complement anaphylatoxin receptors in mammary tumour growth: identification of new therapeutic targets

Md Akhir, Fazrena Nadia (2014). The role of complement anaphylatoxin receptors in mammary tumour growth: identification of new therapeutic targets PhD Thesis, School of Biomedical Sciences, The University of Queensland. doi:10.14264/uql.2014.315

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Author Md Akhir, Fazrena Nadia
Thesis Title The role of complement anaphylatoxin receptors in mammary tumour growth: identification of new therapeutic targets
School, Centre or Institute School of Biomedical Sciences
Institution The University of Queensland
DOI 10.14264/uql.2014.315
Publication date 2014
Thesis type PhD Thesis
Supervisor Trent Woodruff
Barbara Rolfe
Wenyi Gu
Christine Wells
Total pages 111
Total colour pages 12
Total black and white pages 99
Language eng
Subjects 110707 Innate Immunity
110709 Tumour Immunology
111502 Clinical Pharmacology and Therapeutics
Formatted abstract
Breast cancer is the most common cancer diagnosed in women worldwide, accounting for 12% of all cancers. It is also the most common cause of cancer mortality in women. The idea that inflammation is a cause of cancer was out of favour for more than a century. However, there has been a resurgence in interest in the role of chronic inflammation in carcinogenesis, with recent data supporting the concept that inflammation contributes to development and progression of tumours, including breast cancers. In light of new evidence implicating the complement system in promoting tumour growth, the aim of this thesis was to elucidate the role(s) that key inflammatory mediators, the complement anaphylatoxins C3a and C5a, play in a murine model of breast cancer.

Immunofluorescence staining of cultured cells revealed that the mouse mammary tumour cell lines, EMT6 and 4T1, express all three anaphylatoxin receptors (C3aR, C5aR and C5L2), although at lower levels than the murine macrophage cell line J774, which is known to express high levels of these receptors. Given the expression of anaphylatoxin receptors by EMT6 and 4T1 cells, we next assessed whether or not these cells are responsive to stimulation by a dual C3aR/C5aR peptide agonist (YSFKPMPLaR; EP54, 0.5 nM to 5000 nM). Although treatment with EP54 did not induce a classical GPCR-mediated calcium response in either cell line, Western analysis showed that EMT6 cells are capable of calcium-independent complement receptor activation by EP54 (10 μM), a selective C3aR agonist (WWGKKYRASKLGLAR; EP141; 1 μM) or recombinant mouse C5a (10 nM), with maximal levels of phosphorylated ERK1/2 detectable after 5 minutes exposure. EP54 treatment of EMT6 cells also activated the AKT pathway. However, despite these signalling events, C3aR/C5aR agonism had no significant effect on proliferation of tumour cells in vitro.

Given that C3aR and C5aR expressed by mammary tumour cells in vitro were functionally coupled to signalling pathways, we next sought to explore the role of C3aR and C5aR receptor activation in tumour growth in vivo, by using the dual C3aR/C5aR peptide agonist, EP54. Mammary tumours were induced by injecting EMT6 tumour cell suspensions into the mammary fat pads of BALB/c mice (n=8/group; 5x105 cells/mouse). Mice received daily s.c. injections of EP54 (1 or 3 mg/kg/day) or vehicle (saline) from the time of tumour cell injection (day 0). Tumour areas were measured daily with digital callipers and all mice were terminated 14 days post-tumour induction, at which time tumours were excised and weighed. The results revealed that tumour growth was significantly reduced by both EP54 doses compared with control (vehicle-treated) mice, suggesting that stimulation of C3aR/C5aR impairs tumour growth, and that the efficacy of EP54 is similar at both doses tested.

Having established in the pilot test that an EP54 dose of 1mg/kg/day is effective, all further studies used this lower dose. Mice received daily s.c. injections of EP54 (1 mg/kg/day) or vehicle (saline), commencing either from the time of tumour cell injection (day 0) or from the time tumours became palpable (day 7). The results demonstrated that mammary tumour area and weight were significantly retarded in mice treated with EP54 from day 0 compared to vehicle-treated mice. Additionally, treatment of mice with EP54 from day 7 significantly reduced tumour area and weight compared to controls, demonstrating that C3aR/C5aR agonism can not only prevent tumour development, but also inhibit the growth of established tumours.

Given that EP54 did not affect tumour cell proliferation in vitro, we postulated that the protective effects seen with C3aR/C5aR stimulation by EP54 could be due to immune cell activation in vivo. To investigate this, we first determined whether circulating leukocyte populations are altered in tumour-bearing mice. Differential blood counts revealed that there was a significant increase in neutrophil numbers in mice with EMT6 tumours compared with naïve mice (without tumours), but there were no significance differences in monocyte or lymphocyte numbers between groups. Treatment with either dose of EP54 (1 or 3 mg/kg/day) resulted in significant increases in circulating leukocytes compared with saline (vehicle)-treated mice. Flow cytometric analysis of leukocyte sub-populations in tumour tissue showed that administration of EP54 from day 0 had no significant effect on total CD45+ leukocyte numbers. Although there were slight reductions in all leukocyte sub-populations (F4/80+ macrophages, CD11b+Gr-1+ MDSCs, Ly6G+ neutrophils, CD3+ T lymphocytes and Tie-2+ angiogenic macrophages) in EP54-treated mice compared with the control (vehicle treated) group, these were also not significant. Flow cytometric analysis of infiltrating leukocyte sub-populations in tumours from mice in which EP54 treatment commenced 7 days after tumour initiation showed a significant increase in CD3+ T lymphocytes compared with the control group; all other leukocyte populations were similar in both groups. These results suggest that delayed EP54 administration is acting to promote a T cell-mediated response against the tumour.

Taken together, the results presented in this thesis support the idea that complement anaphylatoxin receptor activation plays a role in regulating mammary tumour growth. Specifically, we show protective outcomes for EP54 in the murine EMT6 model of mammary carcinoma. Although mouse mammary tumour cells express receptors for both C3a and C5a, the results suggest that the protective effect of EP54 is indirect, via up-regulation of T cell-mediated anti-tumour immune responses. These findings have important implications for our understanding the role of the complement system in breast cancer development and for the design of future preventive and therapeutic strategies, such as promoting complement activation. The results achieved in this study warrant further investigations into the mechanisms by which EP54 exerts its effect, and the therapeutic effects of this drug in other cancer models.
Keyword Complement component C5a
Complement component C3a
Complement component C5a receptor (C5aR)
Complement component C3a receptor (C3aR)
EMT6 mouse mammary tumour
Dual C3aR/C5aR agonist (EP54)
Innate immunity

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Created: Mon, 08 Sep 2014, 13:51:44 EST by Fazrena Md Akhir on behalf of Scholarly Communication and Digitisation Service