Abstract
IntroductionEndocrine resistance in breast cancer is associated with enhanced metastatic potential and poor clinical outcome, presenting a significant therapeutic challenge. We have established several endocrine insensitive breast cancer lines by shRNA induced depletion of estrogen receptor (ER) by transfection of MCF-7 cells which all exhibit enhanced expression profile of mesenchymal markers with reduction of epithelial markers, indicating an epithelial to mesenchymal transition. In this study we describe their behaviour in response to change in extracellular pH, an important factor controlling cell motility and metastasis.MethodsMorphological changes associated with cell exposure to extracellular alkaline pH were assessed by live cell microscopy and the effect of various ion pumps on this behavior was investigated by pretreatment with chemical inhibitors. The activity and expression profile of key signaling molecules was assessed by western blotting. Cell motility and invasion were examined by scratch and under-agarose assays respectively. Total matrix metalloproteinase (MMP) activity and specifically of MMP2/9 was assessed in conditioned medium in response to brief alkaline pH exposure.ResultsExposure of ER –ve but not ER +ve breast cancer cells to extracellular alkaline pH resulted in cell shrinkage and spherical appearance (termed contractolation); this was reversed by returning the pH back to 7.4. Contractolation was blocked by targeting the Na+/K+ and Na+/H+ pumps with specific chemical inhibitors. The activity and expression profile of key signaling molecules critical for cell adhesion were modulated by the exposure to alkaline pH. Brief exposure to alkaline pH enhanced MMP2/9 activity and the invasive potential of ER –ve cells in response to serum components and epithelial growth factor stimulation without affecting unhindered motility.ConclusionsEndocrine resistant breast cancer cells behave very differently to estrogen responsive cells in alkaline pH, with enhanced invasive potential; these studies emphasise the crucial influence of extracellular pH and caution against indiscriminate application of alkalinising drug therapy.
Highlights
Endocrine resistance in breast cancer is associated with enhanced metastatic potential and poor clinical outcome, presenting a significant therapeutic challenge
This is indicative of an epithelial to mesenchymal transition (EMT) [8,10], a process that is being increasingly implicated in facilitation of breast cancer metastasis
For routine culture all cell lines were maintained as monolayers at 37°C in an incubator gassed with an atmosphere of 5% CO2 at 95% humidity, in advanced dulbecco’s minimum essential medium (DMEM) containing phenol red as a pH indicator and supplemented with 5% fetal bovine serum (FBS), 600 μg/ml Lglutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 6 ml/500 ml 100 x non-essential amino acids
Summary
Endocrine resistance in breast cancer is associated with enhanced metastatic potential and poor clinical outcome, presenting a significant therapeutic challenge. We have previously described several endocrine insensitive cell lines generated by shRNA induced depletion of estrogen receptor (ER) by transfection of MCF-7 cells [8,9] These lines exhibit distinct changes in morphology, reduced expression profile of epithelial markers such as E-cadherin, catenin, occludins, and claudins, enhanced expression of mesenchymal-associated markers such as N-cadherin, vimentin, integrin β4 and α5 and various metalloproteinase (MMPs), and enhanced motility and invasive potential compared to the parental cells. This is indicative of an epithelial to mesenchymal transition (EMT) [8,10], a process that is being increasingly implicated in facilitation of breast cancer metastasis. Other key downstream modifiers of intracellular activity such as Snail, Slug and Sip-1, and the TGF β mediated Smad-dependent pathways all contribute to mesenchymal-like behaviour and have been extensively described [1,14]
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