Immune modulation of mammographic density and breast cancer risk

Mammographic density (MD) is a strong risk factor for breast cancer that can increase breast cancer risk by 4-6 fold independently of age and BMI. High MD is characterised by breast tissue containing high proportions of stroma, containing fibroblasts, collagen and immune cells, indicative of an inflammatory pro-tumour environment. Currently, the biological mechanisms that drive MD and the associated breast cancer risk are not yet understood. Immune signalling factors such as monocyte chemotactic protein 1 (CCL2), peroxidase enzymes, transforming growth factor beta (TGFB) and tumour necrosis factor alpha (TNFA) have been implicated in breast cancer risk, and are also known to influence functions of stromal fibroblasts. The work in this thesis aims to investigate how these immune signalling factors and the immune microenvironment may act as drivers of MD through their interactions with mammary fibroblasts in vitro, and this effect mammary tumorigenesis in a transgenic tumour mouse model. Firstly, primary mammary fibroblasts from women with high and low MD were treated in vitro with immune factors myeloperoxidase (MPO), eosinophil peroxidase (EPO), TGFB, TNFA and CCL2 for 72 hours. The abundance of mRNA encoding cancer associated fibroblast (CAF) markers and genes involved in extracellular matrix (ECM) regulation were investigated. Production of soluble collagen 1 and insoluble collagen fibres were also measured. No significant differences were observed in gene expression and collagen production between fibroblasts from women with low or high mammographic density. MPO and EPO significantly increased production of collagen 1 in mammary fibroblasts. TGFB and TNFA induced variable changes in CAF and ECM gene expression. TGFB and CCL2 increased deposition of insoluble collagen fibres. Further investigation of the crosstalk between mammary fibroblasts and macrophages utilised an indirect co-culture of primary mammary fibroblasts with THP-1 derived macrophages in the presence or absence of CCL2 for 72 hours. THP-1 derived macrophages underwent differentiation to an M2 phenotype, and had high expression of genes involved in tissue remodelling including MMP2, TIMP1 and VEGF. Co-culture with THP-1 macrophages induced high expression of ECM remodelling genes by mammary fibroblasts such as MMP1, MMP3, MMP9 and TIMP1, as well as inflammatory genes COX2, IL6 and IL8. Production of insoluble collagen fibres was increased in mammary fibroblasts in co-culture with THP-1 macrophages and CCL2. Thirdly, the effect of CCL2 on mammary tumourigenesis and global gene expression was investigated. Mmtv-CCL2 mice overexpressing CCL2 in the mammary gland were crossbred with Mmtv-PyMT tumour model mice. Tumour development was monitored until 9 and 12 weeks of age. CCL2 overexpressing mice were found to have increased macrophage infiltration to primary tumours and increased number of areas of early tumorigenesis in the mammary gland at 9 weeks age. No differences were observed in tumour latency, tumour burden, tumour grade, or pulmonary metastasis between CCL2 overexpressing mice and controls. Global gene expression was analysed by RNAseq in mammary glands from 12 week old Mmtv-CCL2 mice and FVB controls. Mmtv-CCL2 mice exhibited increased expression of genes involved in cancer, DNA damage and extracellular matrix deposition. Expression of genes involved in fatty acid metabolism and adaptive immunity were reduced in Mmtv-CCL2 mice. These results suggest that fibroblasts from women with high mammographic density are not inherently different to those from women with low mammographic density. The role of fibroblasts in MD and breast cancer risk may be a result of immune signals from surrounding cells in the breast microenvironment, which may be through interactions with macrophages and inflammation driven by CCL2.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 202

A Phylogeny and Timescale for the Evolution of Pseudocheiridae (Marsupialia: Diprotodontia) in Australia and New Guinea

Pseudocheiridae (Marsupialia: Diprotodontia) is a family of endemic Australasian arboreal folivores, more commonly known as ringtail possums. Seventeen extant species are grouped into six genera (Pseudocheirus, Pseudochirulus, Hemibelideus, Petauroides, Pseudochirops, Petropseudes). Pseudochirops and Pseudochirulus are the only genera with representatives on New Guinea and surrounding western islands. Here, we examine phylogenetic relationships among 13 of the 17 extant pseudocheirid species based on protein-coding portions of the ApoB, BRCA1, ENAM, IRBP, Rag1, and vWF genes. Maximum parsimony, maximum likelihood, and Bayesian methods were used to estimate phylogenetic relationships. Two different relaxed molecular clock methods were used to estimate divergence times. Bayesian and maximum parsimony methods were used to reconstruct ancestral character states for geographic provenance and maximum elevation occupied. We find robust support for the monophyly of Pseudocheirinae (Pseudochirulus + Pseudocheirus), Hemibelidinae (Hemibelideus + Petauroides), and Pseudochiropsinae (Pseudochirops + Petropseudes), respectively, and for an association of Pseudocheirinae and Hemibelidinae to the exclusion of Pseudochiropsinae. Within Pseudochiropsinae, Petropseudes grouped more closely with the New Guinean Pseudochirops spp. than with the Australian Pseudochirops archeri, rendering Pseudochirops paraphyletic. New Guinean species belonging to Pseudochirops are monophyletic, as are New Guinean species belonging to Pseudochirulus. Molecular dates and ancestral reconstructions of geographic provenance combine to suggest that the ancestors of extant New Guinean Pseudochirops spp. and Pseudochirulus spp. dispersed from Australia to New Guinea ∼12.1–6.5 Ma (Pseudochirops) and ∼6.0–2.4 Ma (Pseudochirulus). Ancestral state reconstructions support the hypothesis that occupation of high elevations (>3000 m) is a derived feature that evolved on the terminal branch leading to Pseudochirops cupreus, and either evolved in the ancestor of Pseudochirulus forbesi, Pseudochirulus mayeri, and Pseudochirulus caroli, with subsequent loss in P. caroli, or evolved independently in P. mayeri and P. forbesi. Divergence times within the New Guinean Pseudochirops clade are generally coincident with the uplift of the central cordillera and other highlands. Diversification within New Guinean Pseudochirulus occurred in the Plio-Pleistocene after the establishment of the Central Range and other highlands

Reparameterization gradient message passing

In this paper we consider efficient message passing based inference in a factor graph representation of a probabilistic model. Current message passing methods, such as belief propagation, variational message passing or expectation propagation, rely on analytically pre-computed message update rules. In practical models, it is often not feasible to analytically derive all update rules for all factors in the graph and as a result, efficient message passing-based inference cannot proceed. In related research on (non-message passing-based) inference, a “reparameterization trick” has lead to a considerable extension of the class of models for which automated inference is possible. In this paper, we introduce Reparameterization Gradient Message Passing (RGMP), which is a new message passing method based on the reparameterization gradient. In most models, the large majority of messages can be analytically derived and we resort to RGMP only when necessary. We will argue that this kind of hybrid message passing leads naturally to low-variance gradients

Biological Mechanisms and Therapeutic Opportunities in Mammographic Density and Breast Cancer Risk

Mammographic density is an important risk factor for breast cancer; women with extremely dense breasts have a four to six fold increased risk of breast cancer compared to women with mostly fatty breasts, when matched with age and body mass index. High mammographic density is characterised by high proportions of stroma, containing fibroblasts, collagen and immune cells that suggest a pro-tumour inflammatory microenvironment. However, the biological mechanisms that drive increased mammographic density and the associated increased risk of breast cancer are not yet understood. Inflammatory factors such as monocyte chemotactic protein 1, peroxidase enzymes, transforming growth factor beta, and tumour necrosis factor alpha have been implicated in breast development as well as breast cancer risk, and also influence functions of stromal fibroblasts. Here, the current knowledge and understanding of the underlying biological mechanisms that lead to high mammographic density and the associated increased risk of breast cancer are reviewed, with particular consideration to potential immune factors that may contribute to this process

Circadian Rhythm Disruption as a Contributor to Racial Disparities in Prostate Cancer

In the United States, African American (AA) men have a 2.4 times higher mortality rate due to prostate cancer than White men. The multifactorial causes of the racial disparities in prostate cancer involve various social determinants of health, socioeconomic status, and access to healthcare. However, emerging evidence also suggests that circadian rhythm disruption (CRD) contributes to prostate cancer, and AA men may be more susceptible to developing CRDs. Circadian rhythms play a significant role in metabolism, hormone secretion, and sleep/wake cycles. Disruption in these circadian rhythms can be caused by airplane travel/jetlag, night shift work, exposure to light, and neighborhood noise levels, which can contribute to sleep disorders and chronic conditions such as obesity, diabetes, cardiovascular disease, and depression. The drivers of the racial disparities in CRD include night shift work, racial discrimination, elevated stress, and residing in poor neighborhoods characterized by high noise pollution. Given the increased vulnerability of AA men to CRDs, and the role that CRDs play in prostate cancer, elucidating the clock-related prostate cancer pathways and their behavior and environmental covariates may be critical to better understanding and reducing the racial disparities in prostate cancer

CCL2-Mediated Stromal Interactions Drive Macrophage Polarization to Increase Breast Tumorigenesis

CCL2 is an inflammatory cytokine that regulates macrophage activity and is implicated in increased mammographic density and early breast tumorigenesis. The role of CCL2 in mediating stromal interactions that contribute to breast tumorigenesis has yet to be fully elucidated. THP-1-derived macrophages and mammary fibroblasts were co-cultured for 72 h. Fibroblasts and macrophages were analysed for phenotype, expression of inflammatory and ECM-regulatory genes and collagen production. Mice overexpressing CCL2 in the mammary glands were analysed for global gene expression by RNAseq at 12 weeks of age. These mice were cross-bred with PyMT mammary tumour mice to examine the role of CCL2 in tumorigenesis. The co-culture of macrophages with fibroblasts resulted in macrophage polarization towards an M2 phenotype, and upregulated expression of CCL2 and other genes associated with inflammation and ECM remodelling. CCL2 increased the production of insoluble collagen by fibroblasts. A global gene expression analysis of CCL2 overexpressing mice revealed that CCL2 upregulates cancer-associated gene pathways and downregulates fatty acid metabolism gene pathways. In the PyMT mammary tumour model, CCL2 overexpressing mice exhibited increased macrophage infiltration and early tumorigenesis. Interactions between macrophages and fibroblasts regulated by CCL2 can promote an environment that may increase breast cancer risk, leading to enhanced early tumorigenesis