Proteomic identification of tumor- and metastasis-associated galectin-1 in claudin-low breast cancer

https://doi.org/10.1016/j.bbagen.2020.129784Get rights and content

Highlights

  • Proteomics identify differentially-expressed claudin-low breast cancer biomarkers.

  • Murine primary and metastatic claudin-low breast cancers express high galectin-1.

  • Biochemical and immunohistochemical methods validate Galectin-1 proteomics results.

  • Imaging mass spectrometry maps galectin-1 spatial distribution to lung metastases.

  • Genomics analysis reveals high galectin-1 in murine and human claudin-low cancer.

Abstract

Background

Metastasis and mortality remain high among breast cancer patients with the claudin-low subtype because these tumors are aggressive, chemoresistant, and lack targeted therapies. Our objective was to utilize discovery-based proteomics to identify proteins associated with claudin-low primary and metastatic tumors to gain insight into pathways and mechanisms of tumor progression.

Methods

We used nano-LC-MS/MS proteomics to analyze orthotopic and metastatic tumors from the syngeneic murine T11 tumor model, which displays gene expression profiles mirroring human claudin-low tumors. Galectin-1 identity, expression and spatial distribution were investigated by biochemical and immunochemical methods and MALDI/IMS. RNA seq data from mouse and human tumors in our study and publicly available microarray data were analyzed for differential galectin-1 expression across breast cancer subtypes.

Results

Galectin-1, an N-acetyllactosamine-binding protein, exhibited the highest sequence coverage and high abundance rank order among nano-LC-MS/MS-identified proteins shared by T11 claudin-low tumors but not normal tissue. Label-free quantitation, Western immunoblot and ELISA confirmed galectin-1 identity and significant differential expression. MALDI/IMS spatial mapping and immunohistochemistry detected galectin-1 in T11 metastatic lung foci. Immunohistochemistry of human claudin-low tumors demonstrated intermediate-to-high intensity galectin-1 staining of tumor and stroma. Gene expression analysis of mouse and human tumors found the highest galectin-1 levels in the claudin-low breast cancer subtype.

Conclusions

Proteomics and genomics reveal high expression of galectin-1 protein and RNA in primary and metastatic claudin-low breast cancer.

General significance

This work endorses proteomic approaches in cancer research and supports further investigations of the function and significance of galectin-1 overexpression in claudin-low tumor progression.

Introduction

A hallmark of triple negative breast cancer (TNBC) is the absence or reduced expression of the commonly targeted estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), which limits therapeutic options. The primary cause of death in TNBC patients is metastasis. Compared to non-TNBC subtypes, TNBC is 2–3 times more likely to metastasize to the lung and brain [1,2]. Although grouped into a single subtype when classified solely by expression of ER, PR and HER2, upon molecular subtyping TNBC clusters into additional biologically heterogeneous subtypes, including basal-like and claudin-low tumors [3].

Claudin-low tumors have reduced expression of E-cadherin and the tight junction proteins, claudin-3, −4 and −7 [4]. Exhibiting mesenchymal, stem cell-like properties, claudin-low tumors are clinically aggressive and exhibit high rates of metastasis, recurrence and chemoresistance; this results in a particularly poor prognosis [2,5,6]. Disease progression can be affected by soluble mediators and infiltrating immune cells that comprise the tissue microenvironment. Thus, it is imperative to study both primary and metastatic tumors in order to identify clinical targets to effectively interrupt cancer progression.

Proteomic research approaches, such as nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) and matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI/IMS), have enabled the identification of promising diagnostic and prognostic biomarkers in various diseases, including cancer [7,8]. Nano-LC-MS/MS utilizes low flow chromatography separation combined with nano-electrospray ionization to improve detection limits and provide accurate peptide masses to enhance protein identification [[9], [10], [11]]. MALDI/IMS integrates spatial distribution maps of mass spectra with histopathological methods, which can be used to identify the distribution of proteins and peptides within tissues [12], and is particularly useful to capture heterogeneity both within primary tumors and within distant tissues with varying metastatic burdens. Several studies have employed electrospray ionization and MALDI techniques to measure treatment responses and to identify potential biomarkers in primary tumors and sera from TNBC patients [[13], [14], [15]]. To our knowledge, this is the first study to exploit these proteomic approaches in the claudin-low subtype to identify and subsequently validate tumor-associated proteins. To our knowledge, the data are also the first to show overexpression of galectin-1 (Gal-1) RNA and protein in primary and metastatic claudin-low tumors.

The objective of the present study was to identify proteins associated with claudin-low primary and metastatic tumors to gain insight into pathways and mechanisms that could uncover biomarkers or targets for intervention. We employed a discovery-based, exploratory proteomic approach, using both nano-LC-MS/MS and MALDI/IMS, to examine primary orthotopic and metastatic claudin-low breast tumors using the T11 syngeneic, transplantable tumor in an immunocompetent mouse. The T11 murine tumor mirrors the gene expression profile of human claudin-low breast cancer and exhibits similar immune profile, drug responsiveness (or lack thereof), and metastatic behavior, making it a faithful model for these studies [16,17]. We extended these findings to translational studies investigating galectin-1 expression in human breast tumors.

Section snippets

Animal use

Experimental research protocols as designed per the National Institutes of Health's Guide for the Care and Use of Laboratory Animals were approved by the East Carolina University Institutional Animal Care and Use Committee. All procedures performed in studies involving animals were in accordance with the ethical standards of East Carolina University. Female (6-8-week-old) Balb/c mice were purchased from Charles River. All mice were housed in ventilated cages within a pathogen-free facility at

Proteomic profile of T11 claudin-low breast cancer

The overall study approach and workflow including proteomics, validation studies and data analysis, are depicted in Fig. 1. Proteomic profiling of murine claudin-low tumors and control tissues was first conducted by nano-LC-MS/MS methodology (Fig. 1a). The local and global Protein Pilot False Discovery Report (FDR) of the number of proteins and peptides identified in T11 cultures, primary and metastatic tumor samples and control tissues are summarized in Supplement 1. Accession numbers for all

Discussion

In this study, a discovery-based exploratory proteomic approach revealed that Gal-1, an N-acetyllactosamine binding protein, is highly expressed in a mouse model of claudin-low breast cancer. When compared to normal tissue from healthy control mice, Gal-1 was documented by mass spectrometry as overexpressed in claudin-low primary tumors and in lung metastases, with the highest protein coverage of all identified T11 metastasis-associated proteins and a high abundance rank order. LFQ, Western

Conclusions

In summary, our findings endorse multiple mass spectrometry approaches for cancer research and support further investigations focusing on the function, role and significance of Gal-1 overexpression in claudin-low tumor progression. Our documentation of Gal-1 overexpression in a murine claudin-low tumor model offers the tantalizing possibility that Gal-1 inhibitors, which have been tested in clinical trials for other cancers, may be a promising therapeutic strategy for this very aggressive

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by awards from the Leo W. Jenkins Cancer Center Oncology Research and Education Fund (KV and NV). We acknowledge the support of the Brody School of Medicine Mass Spectrometry Core at East Carolina University and the instrument that was sponsored by the Golden Leaf Foundation. This project used the North Carolina Tissue Consortium (NCTC) shared resource which is supported in part by the University Cancer Research Fund (UCRF) to the UNC Lineberger Cancer Center. We thank

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