Effect of C1q null mutation on mammary gland development and breast cancer susceptibility

Abstract

The complement protein C1q promotes rapid macrophage-mediated clearance of dying cells and tolerance to self antigens. mRNA encoding C1q is a key gene upregulated during mammary gland regression, and we hypothesise that C1q complement protein promotes mammary gland tumourigenesis through induction of tolerance to tumour antigens. We have investigated the role of C1q in normal mammary gland development, hormone-mediated regression, and in tumour development using two different mammary tumour mouse models together with C1q ⁻ʹ⁻ mice. In the absence of C1q, mammary gland development proceeds normally during puberty, with a similar abundance of terminal end buds and rate of epithelial cell proliferation at 6 weeks of age compared to wildtype C1q ⁺ʹ⁺ mice. However, deficiency in C1q perturbed mammary gland regression, with 45% increased number of ductal branch points 24 hours following progesterone receptor antagonist RU486 (also known as mifepristone)-induced epithelial cell apoptosis in C1q ⁻ʹ⁻ mice compared to C1q ⁺ʹ⁺ mice (p=0.027, n=7-11). There was a reduction of macrophage abundance (p=0.002, n=6-7) and a 3.5-fold increase of TUNEL positive apoptotic cells (p=0.011, n=5-6) in the ductal epithelium of C1q ⁻ʹ⁻ mice compared to C1q ⁺ʹ⁺ mice 24 hours following RU486 administration. To investigate the role of C1q in mammary tumour development, MMTV-PyMT transgenic mice were crossed with C1q ⁻ʹ⁻ mice and monitored weekly from 6 weeks by palpation to determine tumour latency, and mammary tumours dissected to assess total tumour burden. Lack of C1q did not affect development of mammary hyperplasia at 10 weeks, however development of palpable tumours was increased by 1 week in PyMT⁺/C1q ⁻ʹ⁻ mice compared to PyMT⁺/C1q ⁺ʹ⁺ mice (p=0.012, n=43-45). The number of tumours was significantly reduced in 15-week old PyMT⁺/C1q ⁻ʹ⁻ mice compared to PyMT⁺/C1q ⁺ʹ⁺ mice (p=0.028, n=15-16). There was also a significant reduction in the total tumour burden in PyMT⁺/C1q ⁻ʹ⁻ mice compared to PyMT⁺/C1q ⁺ʹ⁺ mice at 15 weeks of age (p=0.036, n=15-16). The frequency of tumours that had progressed to carcinoma stage was also reduced in PyMT⁺/C1q ⁻ʹ⁻ mice at 15 weeks of age (p=0.05, n=11-15) and 18 weeks of age (p=0.003, n=28-29). Carcinogen-induced mammary tumourigenesis was also investigated in mice administered 7,12-dimethylbenz[α]anthracene (DMBA) by oral gavage for 6 weeks. DMBA-treated C1q ⁻ʹ⁻ mice were highly resistant to mammary tumourigenesis, with tumours detected in only 2 of 20 mice over the 30 week monitoring period, compared to 10 of 20 DMBA-treated C1q ⁺ʹ⁺ mice (p=0.02). T lymphocytes were skewed to the CD4⁺ subset, with a significant increase in IFNG-producing CD4⁺ T cells in the mammary gland draining lymph nodes in DMBA-treated C1q ⁻ʹ⁻ mice (p=0.014, n=6-10). These findings suggest that C1q promotes tissue remodeling and clearance of dying cells during mammary gland regression, and increases mammary cancer susceptibility and tumour progression.