Abstract
Activation of Epithelial-to-Mesenchymal Transition (EMT) is important for tumor metastasis. Although growth factors such as TGFβ and EGF have been shown to induce EMT in breast epithelial cells, the mechanism resulting in migration is not well understood. Herein, we provide evidence that Ca2+ entry into the cell, especially upon store-depletion, plays an important role in TGFβ-induced EMT by promoting cellular migration and potentially leading to metastasis. The increased migration by TGFβ in non-cancerous cells was due to the loss of E-cadherin along with a subsequent increase in N-cadherin levels. Importantly, TGFβ-treatment increases store-mediated Ca2+ entry, which was essential for the activation of calpain leading to the loss of E-cadherin and MMP activation. Inhibition of Ca2+ entry by using Ca2+ channel blocker SKF-96365, significantly decreased Ca2+ entry, decreased TGFβ-induced calpain activation, and suppressed the loss of E-cadherin along with inhibiting cell migration. Furthermore, TRPC1 function as an endogenous Ca2+ entry channel and silencing of either TRPC1 or its activator, STIM1, significantly decreased TGFβ induced Ca2+ entry, inhibited TGFβ-mediated calpain activation and cell migration. In contrast, overexpression of TRPC1 showed increased Ca2+ entry and promoted TGFβ-mediated cell migration. Moreover, increased TRPC1 expression was observed in ductal carcinoma cells. Together these results suggest that disrupting Ca2+ influx via TRPC1/STIM1 mechanism reduces calpain activity, which could restore intercellular junction proteins thereby inhibiting EMT induced motility.
Highlights
Tumor metastasis is the principal cause of cancerassociated deaths and accounts for over 90% of all cancer deaths [1, 2]
Similar to NMuMG cells, the MCF-10A, a normal breast epithelial cell line, undergoes Epithelial-to-Mesenchymal Transition (EMT) when treated with TGFβ and increased motility is observed [35]
The human breast cancer cell line MDA-MB-231 was used as a positive control as it naturally expresses high levels of EMT markers, is innately metastatic, and increases migration when treated with TGFβ due to degradation of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) [24, 35, 36]
Summary
Tumor metastasis is the principal cause of cancerassociated deaths and accounts for over 90% of all cancer deaths [1, 2]. Metastasis occurs when a malignant cell migrates from a primary organ through the blood stream or lymphatic system to a secondary site causing new tumor growth [3,4,5]. The steps of metastasis include: a) invasion of cancer cells followed by entry into systemic circulation (intravasation), b) movement from the circulatory system into a new host tissue (extravasation), and c) initiating proliferation and growth of the secondary tumor. A known inducer of EMT is the cytokine Transforming Growth Factor Beta, TGFβ, which has been shown to induce EMT via the non-conical pathway in epithelial cells along with increasing cancer cell invasiveness [9,10,11]. It could be anticipated that a common signaling molecule could be essential in regulating EMT
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