ABSTRACT
Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial-mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. Although intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of calcium-signaling pathways controlling EMT induction in cancer cells may therefore be an important therapeutic strategy for preventing metastases.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Calcium Signaling , Calcium/metabolism , Epithelial-Mesenchymal Transition/physiology , Cell Hypoxia , Cell Line, Tumor , Epidermal Growth Factor/metabolism , Epidermal Growth Factor/pharmacology , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Female , Humans , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , Protein Serine-Threonine Kinases , RNA, Small Interfering/genetics , STAT3 Transcription Factor/metabolism , TRPM Cation Channels/antagonists & inhibitors , TRPM Cation Channels/genetics , TRPM Cation Channels/metabolism , Vimentin/biosynthesisABSTRACT
Spontaneously rhythmic pacemaker activity produced by interstitial cells of Cajal (ICC) is the result of the entrainment of unitary potential depolarizations generated at intracellular sites termed pacemaker units. In this study, we present a mathematical modeling framework that quantitatively represents the transmembrane ion flows and intracellular Ca2+ dynamics from a single ICC operating over the physiological membrane potential range. The mathematical model presented here extends our recently developed biophysically based pacemaker unit modeling framework by including mechanisms necessary for coordinating unitary potential events, such as a T-Type Ca2+ current, Vm-dependent K+ currents, and global Ca2+ diffusion. Model simulations produce spontaneously rhythmic slow wave depolarizations with an amplitude of 65 mV at a frequency of 17.4 cpm. Our model predicts that activity at the spatial scale of the pacemaker unit is fundamental for ICC slow wave generation, and Ca2+ influx from activation of the T-Type Ca2+ current is required for unitary potential entrainment. These results suggest that intracellular Ca2+ levels, particularly in the region local to the mitochondria and endoplasmic reticulum, significantly influence pacing frequency and synchronization of pacemaker unit discharge. Moreover, numerical investigations show that our ICC model is capable of qualitatively replicating a wide range of experimental observations.
Subject(s)
Biophysical Phenomena , Membrane Potentials , Animals , Biological Transport , Calcium/metabolism , Cell Membrane , Electrophysiological Phenomena , Ion Channel Gating , Models, Biological , Patch-Clamp Techniques , Pyloric Antrum/cytologyABSTRACT
Unitary potential (UP) depolarizations are the basic intracellular events responsible for pacemaker activity in interstitial cells of Cajal (ICCs), and are generated at intracellular sites termed "pacemaker units". In this study, we present a mathematical model of the transmembrane ion flows and intracellular Ca(2+) dynamics from a single ICC pacemaker unit acting at near-resting membrane potential. This model quantitatively formalizes the framework of a novel ICC pacemaking mechanism that has recently been proposed. Model simulations produce spontaneously rhythmic UP depolarizations with an amplitude of approximately 3 mV at a frequency of 0.05 Hz. The model predicts that the main inward currents, carried by a Ca(2+)-inhibited nonselective cation conductance, are activated by depletion of sub-plasma-membrane [Ca(2+)] caused by sarcoendoplasmic reticulum calcium ATPase Ca(2+) sequestration. Furthermore, pacemaker activity predicted by our model persists under simulated voltage clamp and is independent of [IP(3)] oscillations. The model presented here provides a basis to quantitatively analyze UP depolarizations and the biophysical mechanisms underlying their production.
