Abstract
A growing body of evidence supports that the epithelial-to-mesenchymal transition (EMT), which occurs during cancer development and progression, has a crucial role in metastasis by enhancing the motility of tumor cells. Transforming growth factor-β (TGF-β) is known to induce EMT in a number of cancer cell types; however, the mechanism underlying this transition process is not fully understood. In this study we have demonstrated that TGF-β upregulates the expression of tumor suppressor protein Par-4 (prostate apoptosis response-4) concomitant with the induction of EMT. Mechanistic investigations revealed that exogenous treatment with each TGF-β isoform upregulates Par-4 mRNA and protein levels in parallel levels of phosphorylated Smad2 and IκB-α increase. Disruption of TGF-β signaling by using ALK5 inhibitor, neutralizing TGF-β antibody or phosphoinositide 3-kinase inhibitor reduces endogenous Par-4 levels, suggesting that both Smad and NF-κB pathways are involved in TGF-β-mediated Par-4 upregulation. NF-κB-binding sites in Par-4 promoter have previously been reported; however, using chromatin immunoprecipitation assay we showed that Par-4 promoter region also contains Smad4-binding site. Furthermore, TGF-β promotes nuclear localization of Par-4. Prolonged TGF-β3 treatment disrupts epithelial cell morphology, promotes cell motility and induces upregulation of Snail, vimentin, zinc-finger E-box binding homeobox 1 and N-Cadherin and downregulation of Claudin-1 and E-Cadherin. Forced expression of Par-4, results in the upregulation of vimentin and Snail expression together with increase in cell migration. In contrast, small interfering RNA-mediated silencing of Par-4 expression results in decrease of vimentin and Snail expression and prevents TGF-β-induced EMT. We have also uncovered a role of X-linked inhibitor of apoptosis protein in the regulation of endogenous Par-4 levels through inhibition of caspase-mediated cleavage. In conclusion, our findings suggest that Par-4 is a novel and essential downstream target of TGF-β signaling and acts as an important factor during TGF-β-induced EMT.
Highlights
Matrix adhesion, transcriptional regulation and migration capacity.[2,3] Members of the transforming growth factor-b (TGF-b) family have been identified as important inducers of epithelial-to-mesenchymal transition (EMT) during development as well as carcinogenesis.[4,5]
As the cells used in the present study constitutively produce the precursor protein of TGF-b isoforms and express abundant levels of TGF-b receptors[27,28,29] (Supplementary Figure S1C), they are a valuable model for studying autocrine and paracrine TGF-b signaling
It has been demonstrated that TGF-b acts as a tumor suppressor during the early stages of tumor; the mechanisms involved in these pro-apoptotic responses are diverse and cell type dependent
Summary
Matrix adhesion, transcriptional regulation and migration capacity.[2,3] Members of the transforming growth factor-b (TGF-b) family have been identified as important inducers of EMT during development as well as carcinogenesis.[4,5]. Several studies have shown that during early stages of carcinogenesis, TGF-b acts as a tumor suppressor principally through its ability to promote cell cycle arrest or apoptosis.[12] when the tumor progresses, TGF-b shifts its role from tumor suppressor to tumor promoter, inducing neoplastic cell invasiveness and metastasis through EMT and via its reprogramming of cell microenvironments.[13,14] EMT is characterized by the downregulation of the expression of epithelial markers such as. E-cadherin, which is critical in mediating epithelial cell integrity and cell–cell adhesion[15] and the upregulation of mesenchymal markers N-cadherin, which has been linked to elevated cell motility and invasive phenotype.[1,3] TGF-b stimulation of EMT is mostly achieved through its ability to induce the expression of the Snail/ZEB family of basic helix–loop–helix transcription factors, including that of Snail[1], zinc-finger. We have found prostate apoptosis response-4 (Par-4) as a novel target of TGF-b signaling
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