Proteome-wide profiling of the MCF10AT breast cancer progression model

Abstract

Background: Mapping the expression changes during breast cancer development should facilitate basic and translational research that will eventually improve our understanding and clinical management of cancer. However, most studies in this area are challenged by genetic and environmental heterogeneities associated with cancer.

Methodology/principal findings: We conducted proteomics of the MCF10AT breast cancer model, which comprises of 4 isogenic xenograft-derived human cell lines that mimic different stages of breast cancer progression, using iTRAQ-based tandem mass spectrometry. Of more than 1200 proteins detected, 98 proteins representing at least 20 molecular function groups including kinases, proteases, adhesion, calcium binding and cytoskeletal proteins were found to display significant expression changes across the MCF10AT model. The number of proteins that showed different expression levels increased as disease progressed from AT1k pre-neoplastic cells to low grade CA1h cancer cells and high grade cancer cells. Bioinformatics revealed that MCF10AT model of breast cancer progression is associated with a major re-programming in metabolism, one of the first identified biochemical hallmarks of tumor cells (the "Warburg effect"). Aberrant expression of 3 novel breast cancer-associated proteins namely AK1, ATOX1 and HIST1H2BM were subsequently validated via immunoblotting of the MCF10AT model and immunohistochemistry of progressive clinical breast cancer lesions.

Conclusion/significance: The information generated by this study should serve as a useful reference for future basic and translational cancer research. Dysregulation of ATOX1, AK1 and HIST1HB2M could be detected as early as the pre-neoplastic stage. The findings have implications on early detection and stratification of patients for adjuvant therapy.

Figure 1. Overview of iTRAQ-based protein expression profiling of MCF10AT breast cancer progression model.

Biological duplicates were prepared. Lysates from A1, 1k, 1h and 1a cells were labeled with 114, 115, 116 and 117 iTRAQ isotopic labels, respectively, and analyzed with nanospray-ESI tandem mass spectrometry.

Figure 2. Functional characterization of proteins detected to display aberrant expression across the MCF10AT model of breast cancer progression.

(A) Proteins were organized via KO (KEGG Orthology) using KEGG pathway database into major processes such as Metabolism (carbohydrate, lipid, amino acid etc.); Genetic Information Processing (transcription, translation, replication, repair etc.); Environmental Information Processing (membrane transport, signal transduction etc.); and Cellular Processes (cell growth, death, motility etc.). (B) Gene list was imported and analyzed by the Core Analysis Module in Ingenuity Pathway Analysis software to statistically determine the functions/pathways most strongly associated with the gene list.

Figure 3. Expression profiles of Vimentin, LGALS3 and VCP across the MCF10AT model.

(A) Immunofluorescence of Vimentin in MCF10AT model using Cy3-conjugated anti-Vimentin antibodies. (B) Top panels - Immunoblotting of LGALS3 and VCP. Cells were processed as per iTRAQ experiments. The lysates were immunoblotted with the protein-specific antibodies to reveal the relative expression levels across the 4 MCF10AT cell lines. Actin was included as a loading control. Bottom panel -Densitometry readings for the signals corresponding to the respective protein bands were obtained and expressed as a ratio using the signal from A1 normal cells as the denominator

Figure 4. Validation of the expression of novel breast cancer-associated proteins AK1, ATOX1 and HIST1H2BM in vitro and ex-vivo.

(A) Top panels, Immunoblotting of ATOX1, AK1 and HIST1H2BM. Cells were processed as per iTRAQ experiments. The lysates were immunoblotted with the protein-specific antibodies to reveal the relative expression levels across the 4 MCF10AT cell lines. Actin was included as a loading control. Bottom panel, Densitometry readings for the signals corresponding to the respective protein bands were obtained and expressed as a ratio using the signal from A1 normal cells as the denominator. (B) Immunohistochemistry of ATOX1, AK1 and HIST1H2BM were performed on 26 matched adjacent normal and tumor tissues containing a spectrum of lesions including DCIS and IDC. Top panels-A representative case showing the predominant expression trend for each candidate is shown. Bottom panel–Summary of the expression trends of candidate proteins between normal and breast cancer lesions.