Targeted Proteomics Enables Simultaneous Quantification of Folate Receptor Isoforms and Potential Isoform-based Diagnosis in Breast Cancer.

Ting Yang,Feifei Xu,Danjun Fang,Yun Chen

doi:10.1038/srep16733

Abstract

The distinct roles of protein isoforms in cancer are becoming increasingly evident. FRα and FRβ, two major isoforms of the folate receptor family, generally have different cellular distribution and tissue specificity. However, the presence of FRβ in breast tumors, where FRα is normally expressed, complicates this situation. Prior to applying any FR isoform-based diagnosis and therapeutics, it is essential to monitor the expression profile of FR isoforms in a more accurate manner. An LC-MS/MS-based targeted proteomics assay was developed and validated in this study because of the lack of suitable methodology for the simultaneous and specific measurement of highly homologous isoforms occurring at low concentrations. FRα and FRβ monitoring was achieved by measuring their surrogate isoform-specific peptides. Five human breast cell lines, isolated macrophages and 60 matched pairs of breast tissue samples were subjected to the analysis. The results indicated that FRβ was overexpressed in tumor-associated macrophages (TAMs) but not epithelial cells, in addition to an enhanced level of FRα in breast cancer cells and tissue samples. Moreover, the levels of the FR isoforms were evaluated according to the histology, histopathological features and molecular subtypes of breast cancer. Several positive associations with PR/ER and HER2 status and metastasis were revealed.

Highlights

The clinical relevance of specific protein isoforms is becoming increasingly clear, in cancer diagnosis and treatment
The appearance of FRβ in tumor-associated macrophages (TAMs) suggested that FRβ may be a useful breast cancer marker and a potential target for anti-cancer therapy, in addition to FRα, because breast cancer development and progression are highly dependent on specialized tumor-associated stroma[7], as tumors rarely develop in the absence of this microenvironment
This assay was applied to the quantitative analysis of folate receptor (FR) isoforms in the MCF-10 A normal breast cell line; the MCF-7/ WT, T47D, MDA-MB-231 and MCF-7/ADR breast cancer cell lines; macrophages isolated from fresh breast tissue; and 60 pairs of primary human breast tumors and adjacent normal tissue samples

Summary

Introduction

The clinical relevance of specific protein isoforms is becoming increasingly clear, in cancer diagnosis and treatment. Antibody-based techniques, including Western blotting[8] and immunohistochemistry (IHC)[9], have been traditionally employed to measure protein levels Using these techniques, the detection of protein isoforms is mainly based on a panel of antibodies that recognize distinct epitopes and different electrophoresis motilities[10]. This study first developed and validated an LC-MS/MS-based targeted proteomics assay for the simultaneous quantification of FRα and FRβ in human breast cells and tissue samples. This assay was applied to the quantitative analysis of FR isoforms in the MCF-10 A normal breast cell line; the MCF-7/ WT, T47D, MDA-MB-231 and MCF-7/ADR breast cancer cell lines; macrophages isolated from fresh breast tissue; and 60 pairs of primary human breast tumors and adjacent normal tissue samples. The biological distribution of the FRs and their differential levels based on breast cancer histology, clinical histopathological features and molecular subtypes were discussed

Methods

Results

Conclusion