RESUMEN
Previously we demonstrated that Ahnak mediates transforming growth factor-ß (TGFß)-induced epithelial-mesenchymal transition (EMT) during tumor metastasis. It is well-known that circulating tumor cells (CTCs) invade the vasculature of adjacent target tissues before working to adapt to the host environments. Currently, the molecular mechanism by which infiltrated tumor cells interact with host cells to survive within target tissue environments is far from clear. Here, we show that Ahnak regulates tumor metastasis through PCSK9 expression. To validate the molecular function of Ahnak in metastasis, B16F10 melanoma cells were injected into WT and Ahnak knockout (Ahnak-/-) mice. Ahnak-/- mice were more resistant to the pulmonary metastasis of B16F10 cells compared to wild-type (WT) mice. To investigate the host function of Ahnak in recipient organs against metastasis of melanoma cells, transcriptomic analyses of primary pulmonary endothelial cells from WT or Ahnak-/- mice in the absence or presence of TGFß stimulation were performed. We found PCSK9, along with several other candidate genes, was involved in the invasion of melanoma cells into lung tissues. PCSK9 expression in the pulmonary artery was higher in WT mice than Ahnak-/- mice. To evaluate the host function of PCSK9 in lung tissues during the metastasis of melanoma cells, we established lung epithelial cell-specific tamoxifen-induced PCSK9 conditional KO mice (Scgb1a1-Cre/PCSK9fl/fl). The pulmonary metastasis of B16F10 cells in Scgb1a1-Cre/PCSK9fl/fl mice was significantly suppressed, indicating that PCSK9 plays an important role in the metastasis of melanoma cells. Taken together, our data demonstrate that Ahnak regulates metastatic colonization through the regulation of PCSK9 expression.
Asunto(s)
Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Pulmón/metabolismo , Melanoma/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas de Neoplasias/biosíntesis , Proproteína Convertasa 9/biosíntesis , Animales , Humanos , Pulmón/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Melanoma/genética , Melanoma/patología , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas de Neoplasias/genética , Proproteína Convertasa 9/genéticaRESUMEN
Previously, we reported a molecular mechanism by which Ahnak potentiates transforming growth factor-ß (TGFß) signaling during cell growth. Here, we show that Ahnak induces epithelial-mesenchymal transition (EMT) in response to TGFß. 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 TGFß 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 TGFß. Moreover, TGFß 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 TGFß-Ahnak signaling axis regulates EMT during tumor metastasis.