Adaptation to acidic extracellular pH induces epithelial-mesenchymal transition as stable phenotype in Lewis lung carcinoma model
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1
Ohu University School of Dentistry, Department of Oral Function & Molecular Biology, Japan
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2
Ohu University School of Dentistry, Department of General Clinical Medicine, Japan
Background and aim. Acidic extracellular pH (pHe) is a hallmark phenotype of solid tumor. We have shown that acidic pHe induces morphological changes to fibroblastic and matrix metalloproteinase-9 (MMP-9) and other several gene expressions that are associated with tumor invasion and metastasis in mouse B16 melanoma cells (1). Aim of this study was to determine whether acute and chronic extracellular acidification contributes epithelial-mesenchymal transition (EMT).
Methods. LLC variants with different metastatic ability from parental LLC cells were established by repeating injection of tumor cells into tail vein of C57Bl/6 mouse. Steady state levels of mRNA expression were quantified by RT-qPCR. Gelatinolytic activities were determined by gelatin zymography. Motility activity was measured by wound-healing assay.
Results. When low metastatic LLCm1 cells showing cobble-stone like morphology were cultured at acidic pHe, those cell shape became fibroblastic which was similar to high metastatic LLCm4. RT-qPCR analysis showed that acidic pHe induced vimentin expression but reduced E-cadherin expression, whose changes were typical features of EMT. Among E-cadherin suppressor genes, zeb2, twist1, and twist2 expressions were induced 24 h after acidification and those expression patterns were inversely correlated with the E-cadherin expression level at acidic pHe. MMP-9 and MMP-3 expression and migration activity were induced by acidic pHe. These results suggested that acute acidic pHe transiently affects EMT. As the next experiment, to determine chronic effect of acidic pHe on EMT, we established the acidic pHe-adapted LLCm1 variant (LLCm1A) by culturing at pH 6.2 for more than two months (more than 16 passage generation) and compared the EMT phenotype with LLCm1 and LLCm4 cells. LLCm1A cells showed fibroblastic at neutral pHe even in several passage numbers. E-cadherin mRNA expression was constitutively lower, with high levels of zeb1, zeb2, twist1, and twist2 mRNA expression, regardless the pHe, than that of LLCm1 and LLCm4 cells. N-cadherin and MMP-9 was highly expressed at neutral pHe and those expressions were further stimulated by acidic pHe. Unexpectedly, vimentin expression was low level regardless the pHe. Our data suggested that acute and chronic acidic pHe stimulation induces EMT with slightly different mechanism and that especially chronic effect of acidic pHe provides constitutively “active” for EMT-like changes.
Conclusions. Acidic pHe is thought to be a microenvironmental niche to induce EMT in some kinds of cancer.
References
1. Kato Y, et al, Cancer Cell Int. 13(1):89, 2013.
Keywords:
microenvironment,
invasion and metastasis,
EMT,
MMP,
Gene Expression Regulation
Conference:
4th Annual Meeting of the International Society of Proton Dynamics in Cancer, Garching, Germany, 10 Oct - 12 Oct, 2013.
Presentation Type:
Abstract
Topic:
6. pH control of immune functions and tumor cell plasticity
Citation:
Kato
Y,
Suzuki
A,
Maeda
T and
Baba
Y
(2014). Adaptation to acidic extracellular pH induces epithelial-mesenchymal transition as stable phenotype in Lewis lung carcinoma model.
Front. Pharmacol.
Conference Abstract:
4th Annual Meeting of the International Society of Proton Dynamics in Cancer.
doi: 10.3389/conf.fphar.2014.61.00024
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Received:
15 Jan 2014;
Published Online:
07 Feb 2014.
*
Correspondence:
Prof. Yasumasa Kato, Ohu University School of Dentistry, Department of Oral Function & Molecular Biology, Koriyama, 963-8611, Japan, yasumasa_kato@live.jp