Abstract 3183: Proteomic characterization of aromatase inhibitor resistant mammospheres reveal the presence of a novel nuclear chaperone

Postmenopausal women with early-stage metastatic estrogen-dependent breast cancer are generally treated with aromatase inhibitors (AIs) (e.g., letrozole). However, acquired resistance remains a major clinical obstacle. Previously, our group revealed a global proteomic signature of a letrozole-resistant cell line (LTLT-Ca) associated with hormone independence, enhanced cell motility and epithelial to mesenchymal (EMT). Given recent evidence suggesting a convergence of EMT and cancer stem cells (CSC), we chose to utilize a two-dimensional (2D) vs three-dimensional (3D) culture system to compare the proteome of LTLT-Ca cells, as 3D culture not only enriches for CSC, but more accurately recapitulates the tumor microenvironment, morphology, function and response to therapy compared to conventional 2D culture. We hypothesize utilizing a novel systems biology approach may reveal previously unconsidered molecular changes that could aid in understanding complex signaling networks and be exploited as therapeutic targets. To address this hypothesis ovariectomized immunocompromised female mice were inoculated in the mammary fat pad with LTLT-Ca or letrozole sensitive cells (AC-1) and intratumoral putative CSC marker expression was assessed by immunohistochemistry. Results indicate LTLT-Ca tumors were CD44high/CD24low while AC-1 tumors were CD44-/CD24low. Mammosphere formation assays were conducted and LTLT-Ca cells formed mammospheres at a 3.4-fold higher index than AC-1 cells. A quantitative proteomic analysis of whole cell lysates from LTLT-Ca (2D adherent cells) versus LTLT-Ca (3D mammospheres) was conducted. Results identified significant protein expression changes within a panel of 1010 proteins; 173 were upregulated and 186 downregulated (p1.20). Additionally, functional enrichment analyses were performed and 19 gene ontology (GO) terms and one KEGG pathway (hsa03010:Ribosome) were over-represented (BH adjusted p-value < 0.01) by the cognate genes. Notably, there was a 35.04-fold increase in midasin (MDN1), a nuclear chaperone protein required for maturation and nuclear export of pre-60S ribosome subunit. Increased MDN1 expression was strongly correlated with highly tumorigenic breast cancer spheres. Additionally, Kaplan-Meier survival plots demonstrate that increased MDN1 levels were positively correlated with decreased relapse free survival in estrogen receptor negative breast cancer, and is constitutively expressed as breast tumors progres