Role of adipose renin-angiotensin system inhibition and omega-3-fatty acids in obesity and breast cancer crosstalk

Both obesity and breast cancer (BC) are complex diseases of diverse etiologies. Obesity significantly increases the risk for BC; especially in postmenopausal women. Moreover, inflammation is an important underlying basis for both diseases. Thus, it is critical to understand the mechanisms of obesity-related BC and the role of inflammation in linking these diseases. In obesity, several adipocyte-derived inflammatory substances can potentially drive the effects of adipocytes on BC cells. Among these, angiotensin II (Ang II) produced by adipocytes increases local and systemic inflammation in adipose tissue. Ang II is classically known as a hormone that regulates blood pressure and fluid balance. Its unusual production in adipose tissue suggests that it plays an important role in obesity-related hypertension. Various components of the renin angiotensin system (RAS, the system that generates Ang II) are expressed in adipose tissue as well as in BC cells. Interestingly, in obesity, RAS is overexpressed in adipose tissue, leading to increased secretion of Ang II, which is the primary factor behind most RAS-mediated active inflammation. Similarly, overexpression of RAS components drives BC progression via increased cancer cell proliferation, invasion and favorable tumor microenvironment formation. However, limited work has been done to address mechanistic interactions between adipocyte-derived Ang II and obesity-related BC, especially with a focus on modulation of inflammatory pathways by Ang II. On the other hand, given the importance of nutrition in both obesity and BC, it is equally critical to identify nutrients or bioactive food components that can reduce effects of obesity on BC through their anti-inflammatory properties, including reducing RAS-related inflammation in obesity and BC. Omega-3 polyunsaturated fatty acids are among potent anti-inflammatory bioactive compounds known for their anti-obesity and anti-cancer effects. Previous research from our laboratory has shown that part of the anti-inflammatory effects of these fatty acids include reduced secretion of angiotensinogen (Agt, which is Ang II precursor protein in RAS) and other inflammatory cytokines from adipocytes. Accordingly, this dissertation research focused on dissecting the interactions between adipocytes and BC cells via modulation of inflammatory RAS pathway. More specifically, we proposed to (1) determine the role of the adipocyte RAS in BC cell inflammation, proliferation, and invasiveness; (2) determine the role of omega-3 fatty acids alone or combined with RAS inhibitors in reducing effects of adipocytes on BC cells; and (3) identifying potential associations between obesity, antihypertensive medication use (including RAS inhibitors) and BC aggressiveness in BC patients. To accomplish these objectives, we have designed a series of in vitro experiments involving both cultured human or murine mature adipocytes and two types of human BC cells (receptor positive, MCF7 and receptor triple negative MDA-MB-231) to investigate effects of anti-inflammatory RAS inhibitors and omega-3 fatty acids (specifically eicosapentaenoic acid, EPA) on adipocyte-BC cell interactions. In our studies, we attempted to mimic the breast environment in vitro by testing effects of adipocyte-conditioned medium on BC cells. We performed gene and protein analyses along with cell viability and migration assays to determine effects of Ang II, RAS inhibitors and/or EPA either directly on breast cancer cells or using pretreated adipose-conditioned medium (CM). In addition, we also conducted a retrospective pilot study by collecting clinical, pathological and histological data from a convenient sample of female breast cancer patients in collaboration with the Southwest Cancer Center at the Texas Tech Health Science Center to determine associations between body mass index (BMI), antihypertensive medication use (including RAS inhibitors), cancer aggressiveness, and expression of RAS markers in the BC tissues. We demonstrated that direct treatments of BC cells with Ang II, or RAS inhibitors did not alter inflammatory cytokines in either BC cell line. CM from Ang II-pretreated adipocytes significantly increased secretion of pro-inflammatory markers at protein level while RAS inhibitors reduced their secretion in triple negative BC cell line. Additionally, CM from adipocytes treated with RAS inhibitors significantly reduced mRNA levels for markers of inflammation, fat synthesis, and angiogenesis in both BC cell lines. Furthermore, CM from Ang II inhibitor pretreated adipocytes reduced cell migration in both BC cell lines. Similarly, we found CM from human adipocytes pretreated with Ang II inhibitor ± EPA significantly reduced pro-inflammatory cytokines expression and secretion in both BC cell lines, while direct treatments did not alter pro-inflammatory cytokine profile in either BC cells. Moreover, we observed reduced cell migration by triple negative BC cells in response to both direct and CM mediated Ang II inhibitor ± EPA treatments. However, these changes were not significant between individual and combined treatments of Ang II inhibitor and EPA. In the human retrospective pilot study, we demonstrated a positive association between higher BMI with higher cancer aggressiveness, as measured by percent Ki-67 index (marker of cancer cell proliferation). In addition, RAS associated markers including Ang II and its type 1 receptor were expressed in histologic sections of breast cancer tissues. Surprisingly, antihypertensive RAS inhibitor use was positively associated with higher histological grade of BC, which merits further confirmation in a larger cohort of BC patients. In summary, the proposed research is highly significant given the high prevalence of both obesity and BC in women, especially after menopause. One acknowledged limitation is that we did not use adipocytes from healthy control versus breast cancer patients affected with obesity; rather we used mesenchymal stem cells differentiated into adipocytes. This needs to be kept in consideration while interpreting findings from our in vitro studies. We also need to test the mechanistic effect of adipose-Ang II inhibition in multiple breast cancer and control epithelium cell lines for future translational studies. However, our research is novel since it fills an important gap in both pharmacological and nutrition knowledge by repurposing currently used antihypertensive therapies (RAS inhibitors) and anti-inflammatory dietary components (EPA) to attenuate obesity-associated BC. Overall, our findings will aid in identifying new mechanisms to help understand the link between obesity and BC as well as will assist in designing future powered clinical studies with larger patient cohorts.