Abstract
Previously, we reported a molecular mechanism by which Ahnak potentiates transforming growth factor-β (TGFβ) signaling during cell growth. Here, we show that Ahnak induces epithelial-mesenchymal transition (EMT) in response to TGFβ. EMT phenotypes, including altered in cell morphology, and expression patterns of various EMT marker genes were detected in HaCaT keratinocytes transfected with Ahnak-specific siRNA. Knockdown of Ahnak expression in HaCaT keratinocytes resulted in attenuated cell migration and invasion. We found that Ahnak activates TGFβ signaling via Smad3 phosphorylation, leading to enhanced Smad3 transcriptional activity. To validate function of Ahnak in EMT of B16F10 cells having high metastatic and tumorigenic properties, we established B16F10 cells with stable knockdown of Ahnak. N-cadherin expression and Smad3 phosphorylation were significantly decreased in B16F10-shAhnak cells, compared to B16F10-shControl cells after treatment of TGFβ. Moreover, TGFβ failed to induce cell migration and cell invasion in B16F10-shAhnak cells. To determine whether Ahnak regulates the metastatic activity of B16F10 cells, we established a lung metastasis model in C57BL/6 mice via tail vein injection of B16F10-shAhnak cells. Lung metastasis was significantly suppressed in mice injected with B16F10-shAhnak cells, compared to those injected with B16F10-shControl cells. Taken together, we propose that TGFβ-Ahnak signaling axis regulates EMT during tumor metastasis.
Highlights
Tumor metastasis comprises multiple steps, including the generation of circulating tumor cells (CTCs) from the primary tumor, the dissemination of CTCs into target tissue to generate a secondary tumor, and metastatic colonization[1,2,3]
N-cadherin expression was up-regulated in control siRNA-transfected HaCaT cells stimulated with transforming growth factor β (TGFβ) but was unchanged in Ahnak siRNA/HaCaT cells under the same conditions (Fig. 1A)
These results indicate that Ahnak is required for epithelial-mesenchymal transition (EMT) driven by TGFβ
Summary
Tumor metastasis comprises multiple steps, including the generation of circulating tumor cells (CTCs) from the primary tumor, the dissemination of CTCs into target tissue to generate a secondary tumor, and metastatic colonization[1,2,3]. Epithelial-mesenchymal transition (EMT) seems to be one complex molecular process involved in the initial development of tumor metastasis[4,5,6]. Loss of epithelial properties, including apical-basal polarity and cell-cell adhesion, in the primary tumor leads to gain of mesenchymal cellular function, with increased cell migration and invasive activity. Various cytokines, including transforming growth factor β (TGFβ), are known to regulate the EMT process in metastasis[7,8,9]. TGFβ acts as a multifunctional cytokine in cell growth and in the regulation of EMT during tumor metastasis. It has been well established that TGFβ regulates EMT during tumor metastasis by controlling the expression of Smad3-mediated target genes. We reported that Ahnak binds and activates phospholipase C-γ1 and PKC in response to stimulation with a growth factor such as PDGF www.nature.com/scientificreports/
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