An acidic extracellular pH induces Src kinase-dependent loss of β-catenin from the adherens junction

doi:10.1016/j.canlet.2008.03.005

Cancer Letters

Volume 267, Issue 1, 18 August 2008, Pages 37-48

https://doi.org/10.1016/j.canlet.2008.03.005 Get rights and content

Abstract

Little attention has been paid to the role of adherens junctions (AJs) in acidic extracellular pH (pHe)-induced cell invasion. Incubation of HepG2 cells in acidic medium (pH 6.6) induced cell dispersion from tight cell clusters, and this change was accompanied by downregulation of β-catenin at cell junctions and a rapid activation of c-Src. Pretreatment with PP2 prevented the acidic pH-induced downregulation of β-catenin at AJ and in the membrane fractions. The acidic pHe-induced c-Src activation increased tyrosine phosphorylation of β-catenin and decreased the amount of β-catenin-associated E-cadherin. The depletion of membrane-bound β-catenin coincided with enhanced cell migration and invasion, and this acidic pHe-increased cell migration and invasion was prevented by PP2. In conclusion, this study characterizes a novel signaling pathway responsible for acidic microenvironment-promoted migration and invasive behaviors of cancer cells.

Introduction

Interstitial acidification occurs in inflammatory sites, in peritoneal fluids during laparatomy, and in the tumor microenvironment [1], [2]. The pHe in a variety of solid tumors ranges from 5.8 to 7.4 [3], [4], [5]. Mechanisms reported to be responsible for this acidity include the production of lactic acid and ATP hydrolysis in hypoxic tumors [3]. In addition, effects of CO₂ pneumoperitoneum on tumor growth have been reported. CO₂ pneumoperitoneum stimulates the growth of malignant colonic cells and subcutaneous tumors [6]. In addition to a decrease in partial oxygen pressure in the abdominal wall, dramatic decreases in extra- and intra-cellular pH values are observed during CO₂ insufflation in vitro [7]. Severe peritoneal acidosis down to a pH of 6.59–6.74 has been observed after prolonged CO₂ pneumoperitoneum and may help tumor metastasis to port sites [8]. Thus, alterations in the peritoneal pH (6.59–6.74) may be responsible for providing an environment favorable to tumor cell implantation during laparoscopy [8]. Extracellular acidification is often associated with tumor progression [9], [10], [11], but the mechanism is unknown.

The AJ is composed of transmembrane cadherin molecules and the associated proteins, β-catenin and α-catenin [12]. At the plasma membrane adjacent to the AJ, β-catenin binds directly to the cytoplasmic region of E-cadherin and α-catenin, the latter of which is linked to the actin-based cytoskeleton [13], [14]. Disruption of AJs precedes mesenchymal transition and enhances invasion and metastasis in several types of cancer cells [15]. In many invasive carcinomas, expression of E-cadherin and β-catenin is either reduced or absent [16], [17], [18]. Moreover, in epithelial cells, β-catenin plays a role in both cell adhesion at the AJ and in Wnt signal transduction in the cytoplasm/nucleus [19]. Retention of β-catenin in the E-cadherin/catenin complex at the AJ is regulated by tyrosine phosphorylation of β-catenin [20]. Three Src family kinases, Fer, Fyn, and c-Src, are involved in phosphorylation of β-catenin at different tyrosine residues [21]. For example, transformation of MDCK cells with a temperature-sensitive v-Src mutant results in increased tyrosine phosphorylation of E-cadherin and β-catenin, a rapid loosening of cell–cell contact, and promotion of invasiveness [22]. Treatment with the protein tyrosine phosphatase inhibitor, pervanadate, increases tyrosine phosphorylation of β-catenin and leads to its redistribution from the cell–cell junction to the cytoplasm or the nucleus [23], [24].

In order to gain a further insight into the effect of an acidic microenvironment on cell adhesion, we cultured HepG2 cells, a human hepatocellular carcinoma cell line, in vitro at an acidic pHe and examined whether this disrupted the integrity of the AJ. The distribution and changes in the phosphorylation levels of β-catenin were assessed by immunofluorescence microscopy, immunoprecipitation, and Western blot analyses. Possible signaling pathways accounting for the phosphorylation of β-catenin were analyzed. We provide evidence that an acidic pHe causes c-Src-dependent tyrosine phosphorylation of β-catenin and decreases the binding of E-cadherin to β-catenin, which leads to destabilization of the AJ and increased cell migration and invasion.

Section snippets

Cell culture

The human hepatocellular carcinoma cell line HepG2 (ATCC, HB8065), purchased from the American Type Culture Collection (Rockville, MD), was grown in growth medium consisting of Dulbecco’s modified Eagle medium containing non-essential amino acids, sodium pyruvate, Earle’s balanced salt solution, 100 IU/ml of penicillin, 100 μg/ml of streptomycin, and 10% fetal calf serum (all from Gibco BRL, Grand Island, NY) in a 5% CO₂ humidified atmosphere at 37 °C. The cells were plated at 2 × 10⁵ cells/ml in 35

Culturing at acidic pHe disrupts compact colony formation of HepG2 cells

We first examined the effect of an acidic culture medium on cell proliferation and cell death. Cell viability, assessed by the MTT assay, showed that incubating the cells in acidic culture media (pH 6.6) was not cytotoxic for HepG2 cells (Fig. 1A). Propidium iodide vital staining and DAPI (4′,6-diamidino-2-phenylindole dilactate) nuclear staining showed very few apoptotic or necrotic cells after 24 h incubation at pH 6.6, the percentages being comparable to those in the pH 7.4 group (data not

Discussion

In this study, we examined the effect of an acidic pHe on the AJ and the migratory capability of HepG2 cells. After incubation in pH 6.6 medium, HepG2 cells acquired mesenchymal characteristics, including a loss of β-catenin from the cell junction and spreading and migratory phenotypes. At this pH, rapid phosphorylation of c-Src was observed. Activation of c-Src kinase was shown to be responsible for the tyrosine phosphorylation of β-catenin, and this reduced the binding to E-cadherin, as

Acknowledgements

Grant sponsors: Far Eastern Memorial Hospital, Taiwan and the National Science Council, Republic of China (grant NSC 95-2314-B-002-192). We thank Dr. Thomas Barkas for his critical reading and correction of this manuscript.

References (50)

P.A. Schornack et al.
Contributions of cell metabolism and H+ diffusion to the acidic pH of tumors
Neoplasia
(2003)
F.C. Wong et al.
Treatment results of endometrial carcinoma with positive peritoneal washing, adnexal involvement and serosal involvement
Clin. Oncol. (R. Coll. Radiol.)
(2004)
Y. Kato et al.
Induction of 103-kDa gelatinase/type IV collagenase by acidic culture conditions in mouse metastatic melanoma cell lines
J. Biol. Chem.
(1992)
S. Tsukita et al.
Molecular linkage between cadherins and actin filaments in cell–cell adherens junctions
Curr. Opin. Cell Biol.
(1992)
K.C. Ban et al.
GSK-3beta phosphorylation and alteration of beta-catenin in hepatocellular carcinoma
Cancer Lett.
(2003)
M. Cervello et al.
Downregulation of wild-type beta-catenin expression by interleukin 6 in human hepatocarcinoma HepG2 cells: a possible role in the growth-regulatory effects of the cytokine?
Eur. J. Cancer
(2001)
K. Kim et al.
Tyrosine phosphorylation translocates beta-catenin from cell–cell interface to the cytoplasm, but does not significantly enhance the LEF-1-dependent transactivating function
Cell Biol. Int.
(2001)
T.J. Harris et al.
Decisions, decisions: beta-catenin chooses between adhesion and transcription
Trends Cell Biol.
(2005)
J.D. Fritz et al.
Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins
Anal. Biochem.
(1989)
T.M. Leber et al.
Zymography: a single-step staining method for quantitation of proteolytic activity on substrate gels
Anal. Biochem.
(1997)

E. Avizienyte et al.

Src and FAK signalling controls adhesion fate and the epithelial-to-mesenchymal transition

Curr. Opin. Cell Biol.

(2005)

S. Roura et al.

Regulation of E-cadherin/Catenin association by tyrosine phosphorylation

J. Biol. Chem.

(1999)

P. Hu et al.

Tyrosine phosphorylation of human keratinocyte beta-catenin and plakoglobin reversibly regulates their binding to E-cadherin and alpha-catenin

J. Invest. Dermatol.

(2001)

K. Glunde et al.

Extracellular acidification alters lysosomal trafficking in human breast cancer cells

Neoplasia

(2003)

N.D. Marchenko et al.

Beta-catenin regulates the gene of MMP-26, a novel metalloproteinase expressed both in carcinomas and normal epithelial cells

Int. J. Biochem. Cell Biol.

(2004)

J. Piedra et al.

Regulation of beta-catenin structure and activity by tyrosine phosphorylation

J. Biol. Chem.

(2001)

D. Fang et al.

Phosphorylation of beta-catenin by AKT promotes beta-catenin transcriptional activity

J. Biol. Chem.

(2007)

R. Palovuori et al.

SRC-induced disintegration of adherens junctions of madin-darby canine kidney cells is dependent on endocytosis of cadherin and antagonized by Tiam-1

Lab. Invest.

(2003)

D.W. Edlow et al.

The pH of inflammatory exudates

Proc. Soc. Exp. Biol. Med.

(1971)

H.P. Simmen et al.

Analysis of pH, pO2 and pCO2 in drainage fluid allows for rapid detection of infectious complications during the follow-up period after abdominal surgery

Infection

(1994)

I.F. Tannock et al.

Acid pH in tumors and its potential for therapeutic exploitation

Cancer Res.

(1989)

P. Vaupel et al.

Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review

Cancer Res.

(1989)

C.A. Jacobi et al.

The influence of laparotomy and laparoscopy on tumor growth in a rat model

Surg. Endosc.

(1997)

P. Wildbrett et al.

Impact of laparoscopic gases on peritoneal microenvironment and essential parameters of cell function

Surg. Endosc.

(2003)

D. Fukumura et al.

Hypoxia and acidosis independently up-regulate vascular endothelial growth factor transcription in brain tumors in vivo

Cancer Res.

(2001)

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^☆: Grant sponsors: Far Eastern Memorial Hospital, Taiwan and the National Science Council (Grant NSC 96-2320-B-002-005).

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An acidic extracellular pH induces Src kinase-dependent loss of β-catenin from the adherens junction☆

Abstract

Introduction

Section snippets

Cell culture

Culturing at acidic pHe disrupts compact colony formation of HepG2 cells

Discussion

Acknowledgements

Neoplasia

Clin. Oncol. (R. Coll. Radiol.)

J. Biol. Chem.

Curr. Opin. Cell Biol.

Cancer Lett.

Eur. J. Cancer

Cell Biol. Int.

Trends Cell Biol.

Anal. Biochem.

Anal. Biochem.

Curr. Opin. Cell Biol.

J. Biol. Chem.

J. Invest. Dermatol.

Neoplasia

Int. J. Biochem. Cell Biol.

J. Biol. Chem.

J. Biol. Chem.

Lab. Invest.

The pH of inflammatory exudates

Proc. Soc. Exp. Biol. Med.

Analysis of pH, pO2 and pCO2 in drainage fluid allows for rapid detection of infectious complications during the follow-up period after abdominal surgery

Infection

Acid pH in tumors and its potential for therapeutic exploitation

Cancer Res.

Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review

Cancer Res.

The influence of laparotomy and laparoscopy on tumor growth in a rat model

Surg. Endosc.

Impact of laparoscopic gases on peritoneal microenvironment and essential parameters of cell function

Surg. Endosc.

Hypoxia and acidosis independently up-regulate vascular endothelial growth factor transcription in brain tumors in vivo

Cancer Res.