NDRG4 promoter hypermethylation is a mechanistic biomarker associated with metastatic progression in breast cancer patients

Luiz Felipe Campesato,Ana Paula M Da Silva,Elisa Helena Farias Jandrey ,Raphael B Parmigiani ,Fernando Augusto Soares ,Helena Brentani ,Paula Fontes Asprino ,André Carvalho ,Alfredo Carlos Simões Dornellas De Barros ,Anamaria A Camargo ,Dirce Maria Carraro ,Vladmir Cláudio Cordeiro De Lima ,Luciana Nogueira De Sousa Andrade ,Ricardo P Moura ,Kátia R M Leite ,Lilian T Inoue ,Camila Longo Machado ,Isabela Werneck Da Cunha ,Érico T Costa ,Roger Chammas

doi:10.1038/s41523-019-0106-x

Abstract

The risk of developing metastatic disease in breast cancer patients is traditionally predictable based on the number of positive axillary lymph nodes, complemented with additional clinicopathological factors. However, since lymph node-negative patients have a 20–30% probability of developing metastatic disease, lymph node information alone is insufficient to accurately assess individual risk. Molecular approaches, such as multigene expression panels, analyze a set of cancer-related genes that more accurately predict the early risk of metastasis and the treatment response. Here, we present N-Myc downstream-regulated gene 4 (NDRG4) epigenetic silencing as a mechanistic biomarker of metastasis in ductal invasive breast tumors. While aberrant NDRG4 DNA hypermethylation is significantly associated with the development of metastatic disease, downregulation of NDRG4 transcription and protein expression is functionally associated with enhanced lymph node adhesion and cell mobility. Here, we show that epigenetic silencing of NDRG4 modulates integrin signaling by assembling β1-integrins into large punctate clusters at the leading edge of tumor cells to promote an “adhesive switch,” decreasing cell adhesion to fibronectin and increasing cell adhesion and migration towards vitronectin, an important component of human lymph nodes. Taken together, our functional and clinical observations suggest that NDRG4 is a potential mechanistic biomarker in breast cancer that is functionally associated with metastatic disease.

Highlights

Breast cancer patients with localized disease present a nearly 100% 5-year survival rate, but this number falls to 85% and 25% for patients with regional and distant metastasis, respectively
By interrogating this list of 11 genes using the Molecular Signatures Database (MSigDB),[18,19] we observed significant enrichment of genes associated with integrin signaling pathways and extracellular matrix (ECM) remodeling (Supplementary Data 3).[17]
Integrins are ECM receptors that are expressed on the surface of cells that activate essential programs associated with survival, proliferation and mobility, among other processes.20 5Aza-dC treatment induced the expression of integrin subunit α5 (ITGA5) and the secreted proteins plasminogen activator, urokinase (PLAU), cysteine rich angiogenic inducer 61 (CYR61) and collagen type IX α3 chain (COL9A3), which play critical roles in ECM formation and remodeling

Summary

Introduction

Breast cancer patients with localized disease present a nearly 100% 5-year survival rate, but this number falls to 85% and 25% for patients with regional and distant metastasis, respectively. We demonstrated that NDRG4 is expressed in normal breast tissue and is epigenetically silenced by DNA promoter hypermethylation in breast primary tumors and tumor cell lines.

Results

Conclusion