Impact of the acidic environment on gene expression and functional parameters of tumors in vitro and in vivo

Mandy Rauschner,Anne Riemann,Alexander Nolze,Luisa Lange,Thea Hüsing,Oliver Thews,Sarah Reime,Marcel Maschek

doi:10.1186/s13046-020-01815-4

Mandy Rauschner, Anne Riemann + Show 6 more

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https://doi.org/10.1186/s13046-020-01815-4

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Abstract

BackgroundThe low extracellular pH (pHe) of tumors resulting from glycolytic metabolism is a stress factor for the cells independent from concomitant hypoxia. The aim of the study was to analyze the impact of acidic pHe on gene expression on mRNA and protein level in two experimental tumor lines in vitro and in vivo and were compared to hypoxic conditions as well as combined acidosis+hypoxia.MethodsGene expression was analyzed in AT1 prostate and Walker-256 mammary carcinoma of the rat by Next Generation Sequencing (NGS), qPCR and Western blot. In addition, the impact of acidosis on tumor cell migration, adhesion, proliferation, cell death and mitochondrial activity was analyzed.ResultsNGS analyses revealed that 147 genes were uniformly regulated in both cell lines (in vitro) and 79 genes in both experimental tumors after 24 h at low pH. A subset of 25 genes was re-evaluated by qPCR and Western blot. Low pH consistently upregulated Aox1, Gls2, Gstp1, Ikbke, Per3, Pink1, Tlr5, Txnip, Ypel3 or downregulated Acat2, Brip1, Clspn, Dnajc25, Ercc6l, Mmd, Rif1, Zmpste24 whereas hypoxia alone led to a downregulation of most of the genes. Direct incubation at low pH reduced tumor cell adhesion whereas acidic pre-incubation increased the adhesive potential. In both tumor lines acidosis induced a G1-arrest (in vivo) of the cell cycle and a strong increase in necrotic cell death (but not in apoptosis). The mitochondrial O2 consumption increased gradually with decreasing pH.ConclusionsThese data show that acidic pHe in tumors plays an important role for gene expression independently from hypoxia. In parallel, acidosis modulates functional properties of tumors relevant for their malignant potential and which might be the result of pH-dependent gene expression.

Highlights

The low extracellular pH of tumors resulting from glycolytic metabolism is a stress factor for the cells independent from concomitant hypoxia
Results mRNA and protein expression Next Generation Sequencing (NGS) analyses were performed in both tumor lines in cultured cells and in experimental tumors
Most of the genes were consistently regulated in both tumor cells lines either up or down

Summary

Introduction

The low extracellular pH (pHe) of tumors resulting from glycolytic metabolism is a stress factor for the cells independent from concomitant hypoxia. The aim of the study was to analyze the impact of acidic pHe on gene expression on mRNA and protein level in two experimental tumor lines in vitro and in vivo and were compared to hypoxic conditions as well as combined acidosis+hypoxia. In comparison to healthy tissue many tumors show pronounced extracellular acidosis with pH values even below 6.0 [1] These adverse environmental conditions result from increased glycolytic metabolism due to an insufficient oxygen supply. Low extracellular pH (pHe) has been shown to modulate tumor cell function. Different mechanisms have been discussed by which the H+-level may affect the sensitivity, for instance, by affecting the drug permeability of the cell membrane [16], by the activation of active drug efflux pumps [17] or by acidosis-dependent changes of cytokine expression of tumor-associated immune cells [16, 18]

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