Changes in uPA, PAI-1, and TGF-β Production during Breast Cancer Cell Interaction with Human Mesenchymal Stroma/Stem-Like Cells (MSC)

Catharina Melzer,Hendrik Ungefroren,Ralf Hass,Hannah Otterbein,Juliane Von Der Ohe

doi:10.3390/ijms20112630

Abstract

The interactions of cancer cells with neighboring non-malignant cells in the microenvironment play an important role for progressive neoplastic development and metastasis. Long-term direct co-culture of human MDA-MB-231cherry breast cancer cells with benign human mesenchymal stroma/stem-like cells (MSC) MSC544GFP stably expressing mCherry and eGFP fluorescence proteins, respectively, was associated with the formation of three-dimensional (3D) tumor spheroids in vitro. The quantification of the breast tumor marker urokinase plasminogen activator (uPA) in mono-cultured MDA-MB-231 cells revealed an approximately 14-fold enhanced expression when compared to five different normal human MSC mono-cultures. Moreover, uPA levels in 3D tumor spheroids remained elevated 9.4-fold above the average of five different human MSC cultures. In contrast, the expression of the corresponding plasminogen activator inhibitor type-1 (PAI-1) declined by 2.6-fold in the breast cancer cells and was even further reduced by 3.2-fold in the MDA-MB-231cherry/MSC544GFP 3D co-culture spheroids when compared to the various MSC populations. The supportive data were obtained for the production of TGF-β1, which is an important growth factor in the regulation of tumor growth and metastasis formation. Whereas, TGF-β1 release in MDA-MB-231cherry/MSC544GFP co-cultures was elevated by 1.56-fold as compared to MSC544 mono-cultures after 24 h; this ratio further increased to 2.19-fold after 72 h. Quantitative PCR analyses in MSC544 and MDA-MB-231 cells revealed that MSC, rather than the breast cancer cells, are responsible for TGF-β1 synthesis and that TGF-β1 contributes to its own synthesis in these cells. These findings suggested potential synergistic effects in the expression/secretion of uPA, PAI-1, and TGF-β during the co-culture of breast cancer cells with MSC.

Highlights

In the course of tumor development and formation of metastases, cancer cells change cell fate, adhesive properties, cell movements, and motility during interactions with the microenvironment [1]
We demonstrate the alterations in the production of urokinase plasminogen activator (uPA), plasminogen activator inhibitor type-1 (PAI-1), and transforming growth factor-β (TGF-β) during co-culture of mesenchymal stroma/stem-like cells (MSC) with highly malignant breast cancer cells that may support continued tumor growth
In contrast to our normal primary human MSC that maintain proliferative capacity, cell fate, and marker expression for up to 10 cell passages [11,12,13], the neoplastic tissue-derived MSC544 continued to grow and it maintained the expression of typical MSC markers beyond passage 10 (P10)

Summary

Introduction

In the course of tumor development and formation of metastases, cancer cells change cell fate, adhesive properties, cell movements, and motility during interactions with the microenvironment [1]. Reciprocal release and uptake of extracellular vesicles and factors, including proteinases and growth factors, contribute to cancer cell interactions. Among those are the serine proteinase urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type-1 (PAI-1), and the regulatory cytokine transforming growth factor-β (TGF-β). Physiological uPA can exist in different forms, including a high molecular weight (HMW)-uPA with two amino acid chains that were connected by a disulfide bridge and a low molecular weight (LMW)-uPA form exhibiting proteolytic activity. The substrates for uPA are several components of the extracellular matrix and a predominant target of uPA is represented by plasminogen with the subsequent conversion to plasmin upon uPA-mediated cleavage of a specific Arg-Val amino acid bond. The activation of uPA and subsequent degradation of target proteins can further relay proteolytic cascades, which play an important role during neoplastic development [5]

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Results

Conclusion