 2c shows that siRNAWWOX increased TwistLuc activity in respect to untransfected (c) and siRNAcontrol-transfected cells. In contrast, WWOX expression vector (e.v.) reduced Twist-luciferase activity in untransfected, siRNAcontrol- and siRNAWWOX-transfected cells. The data of Western blots gave an explanation, because siRNAWWOX transfection largely reduced Wwox-protein levels in cytosol and nuclei, while WWOX e.v. co-transfection caused Wwox-protein accumulation in the cytosol, much more than in nuclei. siRNAWWOX reduced (−70 %) the protein level of Wwox under expression vector co-transfection (Fig. 2d).
As shown in Fig. 2e, the separate transfection of siRNAWWOX and WWOX e.v. oppositely affected TwistLuc, and overexpression of Wwox almost completely prevented HGF-dependent luciferase activation.
Altogether, the high-cytosolic Wwox seemed related to Twist-transactivation decrease, opposite to Wwox depletion being stimulatory for TwistLuc. The nuclear depletion of Wwox was, indeed, correlated with Twist1 access to the nucleus in the phosphorylated form, while Wwox overexpression augmented cytosolic-unphosphorylated Twist. Therefore, Wwox levels might participate in the nuclear phosphoTwist1 translocation and function.
Figure 3a reports that HGF between 4 and 16 h strongly enhanced nuclear Twist and that, thereafter, the signal diffused to all the cell, as shown at 24 h. Under 4-h HGF, WWOX e.v. and siRNAWWOX caused Twist accumulation in the cytosol and in the nuclei, respectively (Fig. 3a, left panels). The transfection of siRNA control did not affect Twist distribution due to HGF (data not shown). Additional file 1: Figure S1 reports cellular Wwox distribution under the above reported experimental conditions. Figure 3a also shows that the cellular Snail slightly and progressively augmented under HGF, but it was strongly enhanced by TGFβ1 starting from 4 h until the end of the observation period. Cellular Twist slightly increased at later times after TGFβ1. After 4-h HGF, nuclear phosphoTwist1 signal was found (Fig. 3b).
Thus, in response to HGF and TGFβ1, protein levels and intracellular localization of Twist and Snail were consistent with the distribution of their signals observed by immunofluorescence. Of note, the 1833 cells exposed to HGF for 24 h had a morphology more elongated (mesenchymal)- with preponderant Snail- than 1833 cells at early times after HGF, that seemed epithelial and clustered showing nuclear Twist. This might be index of Twist program activation. Also, the data led to hypothesize that HGF regulated TGFβ signalling, since Snail is a TGFβ-target gene [29], and suggested to study the gene patterns typical of EMT and MET under the two microenvironment stimuli. These studies were performed in invasive parental MDA-MB231 breast carcinoma cells and the derived 1833-bone metastatic clone. The comparative study of transcriptomic profile of the two cell lines identifies a gene set whose expression is associated with, and promotes the formation of metastasis to bone [20]. MDA-MB231 cells are invasive, and when metastasize they have tropism for various organs, and give metastasis to bone less efficiently and more slowly than 1833 cells [2].
First, we studied the role of AMPK under HGF on the luciferase activity of 3TPLux, a construct containing a strong TGFβ-responsive element that is routinely used to assay TGFβ signalling [30]. In other cell systems, AMPK is involved in HGF-TGFβ reciprocal regulation [31]. In 1833 cells exposed to HGF, AMPK phosphorylation progressively increased starting from 0.5 h, reaching the maximal values between 16 and 24 h (Fig. 3c). Moreover, we found that HGF increased 3TPLux activity (about 6-fold), and that the specific AMPK blockade with Compound C almost completely prevented the HGF-dependent luciferase activation (Fig. 3d). Additional file 2: Figure S2 shows 3TPLux activity in MDA-MB231 cells, also under AMPK blockade with Compound C.
Next, experiments were performed dealing with HGF and TGFβ1 involvement in MET versus EMT, by examining the patterns of the principal genes responsible for these phenotypes (Fig. 4).
In HGF-treated 1833 cells (Fig. 4a), E-cadherin increased early, peaking at 1 h and remaining elevated thereafter, with a return to the starvation value at 24 h, while protein levels of vimentin and MMP2 suddenly dropped almost disappearing from 6 h until the end of the observation period: the patterns of the three markers indicated prevalent MET transition. In contrast, TGFβ1 induced N-cadherin, vimentin and MMP2-active form, with MMP2-precursor (pro72) degradation contributing to the increase in the active form, while E-cadherin progressively decreased after TGFβ1 (Fig. 4a). As shown in Fig. 4b, the EMT markers Snail and vimentin were expressed in MDA-MB231 cells and were further augmented by TGFβ1, while HGF was ineffective; Twist1 phosphorylation was scarce, undergoing slight down-regulation under the two stimuli. Wwox-protein level, extremely low in MDA-MB231 compared to 1833 cells in agreement with previous data [19], was unaffected by HGF and TGFβ1. As shown in Additional file 2: Figure S2, HGF effect was studied on Twist transactivating activity in MDA-MB231 cells under HGF. Since Wwox did not seem present in this cell line, we did not study siRNAWWOX effect.
Taking into consideration all the data in 1833 cells, the return of E-cadherin to basal value 24 h after HGF might contribute to their reversion to a mesenchymal phenotype, consistent with immunofluorescence data; since HGF and TGFβ1 oppositely affected MET and EMT markers including E-cadherin, as reported for Twist, the latter might be differently involved in E-cadherin expression under the two biological stimuli. The regulation of E-cadherin deserves further attention.
Figure 4c shows that E-cadherin was expressed in 1833 clone but was practically absent in MDA-MB231 cells, as previously reported [19]. In 1833 cells, E-cadherin localized at plasma-membrane level, and increased under HGF; the merge showed a slight signal also in nuclei (Fig. 4d).
Experiments on E-cadherin transactivation in 1833 cells were performed under the two environmental stimuli to evaluate the role of Twist and Wwox, by transfecting the construct containing the entire E-cadherin promoter (Fig. 4e). The putative consensus sequences, present in the entire E-cadherin promoter and in the ΔPPRE-promoter fragment are shown. E-cadherinLuc was activated 4.5-fold by HGF, but was unaffected by TGFβ1. Twist seemed involved in the luciferase activation, because ΔTwist prevented of 40 and 60 % basal and HGF-stimulated E-cadherinLuc activity, respectively. WWOX e.v. reduced basal and HGF-induced E-cadherinLuc activity, while siRNAWWOX enhanced basal luciferase activity. All these data supported the role of Twist and Wwox in E-cadherin transactivation.
Under HGF and TGFβ1, we evaluated the activities of the transcription factors presenting numerous-consensus sequences in the E-cadherin promoter, considering the function played by Twist. As shown in Fig. 5a and b, we started to clarify the involvement of PPARγ transcription factor in E-cadherin transactivation using ECad-LucΔPPRE gene reporter, i.e., the fragment lacking PPARγ-consensus site (PPRE) and the upstream sequence, and the construct containing the PPRE multimer (PPARγLuc). Then, the activities of the gene reporters driven by Ets1 or NF-kB multimers were evaluated (Fig. 5b). HGF enhanced E-cadherinLuc ΔPPRE activity, even if at a lesser extent in respect to the entire promoter, and ΔTwist partially prevented (30 %) this stimulatory effect (Fig. 5a). HGF activated PPARγLuc, Ets1Luc and NFkBLuc, while TGFβ1 activated only PPARγLuc. Twist was involved in all the luciferases activated by HGF, especially in PPARγLuc and Ets1Luc, as demonstrated with ΔTwist (Fig. 5b).
Other regulatory mechanisms for E-cadherin transactivation were investigated, because the E-cadherin promoter also contains 3 E-boxes (see Scheme Fig. 5c), and 8 HIF-1 consensus sites (HRE). Since the candidate target genes of Twist are analyzed for containing conserved E-box sequences [26], the activation of the wild-type short promoter fragment of E-cadherin under HGF, if any occurs, might indicate Twist functionality through E-boxes. In 1833 cells, HGF did not affect E-cadherin-luciferase activity of both wild-type and mutated short promoters (Fig. 5c).
Considering all the data, Twist seemed implicated in E-cadherin induction through the activation of PPARγ/Ets1/NF-kB transcription-factor network.
Notwithstanding HGF diminished (−50 %) HRELuc activity (Fig. 5d), we examined the effect of the dominant negative of HIF-1β subunit (ΔARNT) on E-cadherin transactivation by HGF (Fig. 5e). ΔARNT prevented the stimulation of the E-cadherin luciferase activity by HGF, suggesting that HIF-1 activity was implicated in HGF-dependent E-cadherin induction only within a