In this exploratory study, RPPM analysis allowed us to investigate a wide array of proteins that play a role in cell survival, growth, differentiation, and death. The preliminary results presented herein is a first report suggesting that mammary epithelial cells obtained from high-risk obese women may differ in vimentin expression compared to non-obese women. Vimentin is often used as a general marker of mesenchymal cells and has been shown to play a critical role in epithelial-to-mesenchymal transition (EMT) necessary for cancer cell invasion and metastasis (17
). Additionally, vimentin is an integral structural component in fat droplet organization in adipocytes, as well as key for lipid metabolism and adipogenesis (18
). Treatment of MCF10A cells with arachidonic acid, a metabolite obtained from a high-fat diet, induced EMT and increased vimentin expression with concomitant decrease in E-cadherin expression (21
). Considering the established associations between adiposity, adipokines, and vimentin, further investigation into how adipocyte microenvironment influences mammary epithelial cell protein expression in high-risk obese women is warranted.
Few members of the apoptotic pathway have been implicated in adipocyte apoptosis induced by leptin, a hormone that regulates food intake and energy metabolism (22
). Animal model studies have shown no difference in protein expression of pro-apoptotic Bax in mammary tumors of obese and non-obese mice that were fed with high-fat diet and low-fat diet, respectively (23
). In the present study, Bax expression is not significantly different (p=0.055) in obese and non-obese women (), while pro-survival p-Bad S136 expression is higher in non-obese women (). In the absence of dietary information at the time of recruitment and RPFNA collection, we cannot attribute the differential expression of these proteins to a recent change in caloric intake. But, it is interesting to speculate that diet or exercise may impact apoptotic signaling proteins independently of BMI.
An important limitation of this study is small sample size that does not allow for statistical significance of associations between protein expression and risk factors for breast cancer, such as age, menopausal status and cytologic atypia. Thus, it is not possible to draw definitive conclusion on causal relationship between differential protein expression and breast cancer risk in obese versus non-obese women.
In this study, we did not archive adequate RPFNA cytologic samples for a statistically meaningful comparison of RPPM and IHC data. However, our limited data demonstrates that it is possible to prospectively compare RPPM and IHC analysis of specific proteins. The direct comparison of RPPM data with western blot analysis and/or IHC is clearly important for the future use of RPPM as a tool for multiplexed cell signaling analysis of human samples. Recent studies by Accordi et al. demonstrate concordance between RPPM results and western blot analysis of proteins, such as Bcl-2 S70, that are activated in primary leukemia samples with MML translocations (24
). In the present study, we lack statistical power to draw definitive conclusion about the correlation between RPPM and IHC data, given the insufficient or small number of samples. Moreover, the IHC analysis that we employed has obvious limitations, such as variable cellularity, heterogeneity of staining, and subjective nature of scoring the cytologic samples. In the future, we will address these limitations by using comparable number of cells for IHC and employing image analysis of stained cells to quantify protein expression.
Our future proteomic studies will include a larger cohort with equal number of obese and non-obese women of Caucasian and African American descent and analysis of adipokines to better understand the underlying pathology of breast cancer initiation and progression in obese women. African American women have higher rates of obesity and more aggressive forms of breast cancer with greater likelihood of dying from breast cancer in pre- and post-menopausal women (25
). Future proteomic studies will better delineate whether these two factors are causally related as well as identify potential biomarkers of response to preventative interventions, such as dietary changes and exercise.