Quantitative Proteomic and Mutational Landscape of Metaplastic Breast Carcinoma and Generation of a 3D Organoid Model of Neoplastic Progression

Abstract

Triple-negative breast cancer (TNBC) is considered to be the most aggressive and has worse prognosis compared to other breast cancers and accounts for roughly 18% of all epithelial cancers of the breast, or carcinomas. TNBC exhibits complex molecular heterogeneity both inter- and intratumorally and likely consists of several distinct molecular subgroups that are currently unknown. Metaplastic breast carcinoma (MBC) is even more aggressive than triple-negative breast cancer (TNBC) but also typically presents as triple-negative histologically, and is defined by the admixture of both invasive glandular and non-glandular “metaplastic” heterologous elements of spindle, squamous or sarcomatoid subtypes. The protein profiles underpinning the phenotypic diversity and metastatic behavior of MBC are unknown. We present a quantitative multi-subtype proteomic landscape of MBC, non-metaplastic TNBC, and normal breast from small yet well-annotated cohort of 27 patients, and also present the somatic mutational landscape on the same cohort. We used multiplex isobaric tandem mass tag (TMT) labeling for proteomics and quantified 5,798 proteins, and from whole-exome sequencing for genomics analysis we found 980 total somatic mutational variants. MBCs displayed increased epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM) signaling, and reduced metabolic pathways compared to TNBC. We discovered subtype-specific profiles among MBCs including distinct upregulated profiles; translation and ribosomal events in spindle, inflammation and apical junctions in squamous, and extracellular matrix in sarcomatoid. Comparison of the proteomes of spindle MBC with MMTV-cre;Ccn6fl/fl spindle MBC mouse tumors revealed a shared spindle-specific signature of 17 upregulated proteins involved in translation (e.g. RPL4,6,18, P3H1, PYCR1). The somatic mutational landscape also revealed MBCs share common TP53 mutations, and in PLEC, MUC17, CRYBG2, and ZNF681. We identified that spindle and squamous MBC exhibit overlapping mutational profiles of genes involved in transcription, RNA metabolic processes and actin filament binding, while sarcomatoid tumors harbor distinct mutations in MAPK, WNT, protocadherin cluster genes, calcium binding and ECM organization. These data identify subtype-specific MBC protein profiles and mutational signatures that identified novel biomarkers for therapy. Three-dimensional (3D) cell culture has been widely used in recent decades, compared with monolayer (2D) culture, because they better mimic the in vivo state. 3D systems utilize different types of gels critical for their success, such as collagen or the reconstituted basement membrane, Matrigel, which has enabled recapitulation of tissue architecture and function that is more physiologic compared to 2D. However, conventional 3D models using gel-embedded platforms have large variability and slow transport of biomolecules to the matrix-encapsulated cells. Here, we developed a highly reproducible, 3D scaffold-free hanging drop method amenable for primary tissues including mouse and human tumors, and our analyses describe a one drop-one organoid format using MCF10A cells, a non-tumorigenic breast cell line. We attained high-yield production of uniform organoids that resemble normal human breast acini, express both mammary gland-specific and progenitor markers, and we developed treatment assays for EMT induction and neoplastic progression delivering rapid quantification of phenotypic and morphological changes. Integration of 3D methods with omics analyses is envisioned to enhance the study of neoplastic progression and generate novel targets of both MBC and TNBC tumors.PHDMolecular & Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/155234/1/djomehri_1.pd