Snail is essential for constitutive and TPA-induced gene expression of FN and LEF in HepG2 and HCC340 cells
Initially, by RT-PCR, we found TPA elevated mRNA of FN and LEF as well as SNA by 2.0~2.7-fold within 1-6 h in HepG2, one of the conventional HCC cell lines (Fig. 1a). This was also quantitatively confirmed by Q-RT-PCR (supplemental Fig. 1a). Also, Western blot confirmed FN and LEF proteins were elevated by TPA at 6 h by 1.6-1.8-fold, and declined at 12 h whereas TPA induced dramatic elevation of SNA earlier at 2h and 4h and gradually declined at 6 and 12 h (supplemental Fig. 1b) implicating SNA is an upstream regulator of FN and LEF. In both conventional RT-PCR (supplemental Fig. 1c) and quantitative (Q) RT-PCR (Fig. 1b) analysis, TPA-induced elevation of FN and LEF mRNA were found to be attenuated by three shRNAs of SNA, shSN18, shSN19 and shSN20 by 50-80% at 4h in HepG2 and HCC340, a patient-derived HCC cell lines used for studying the SNA-upregulated MMP9 and ZEB1 transcription [17]. On the contrary, overexpression of SNA for 6-16 h elevated FN and LEF mRNAs by 3-10-fold in both HepG2 and HCC340 (Fig. 1c and 1d). Collectively, SNA is essential for gene expression of FN and LEF in HCC.
SNA is essential for constitutive and TPA-induced transcriptional activation of FN and LEF in HCC
We further examined whether TPA can induce promoter activation of FN and LEF in a SNA-dependent manner. To this end, full-length promoter plasmid of FN (FNpro1938) and LEF (LEFpro1897) were constructed by inserting the promoter fragment containing 1938 bp and 1897 bp, respectively, upstream of the translation start site of FN and LEF, into PGL3 vector. As shown in Fig. 2a, TPA elevated promoter activity of FNpro1938 (left panel) and LEFpro1897 (right panel) by 20- and 10-fold, respectively, in HepG2. Transfection of SNA shRNAs, including shSN18, shSN19 or shSN20 suppressed the TPA-induced promoter activity of FNpro1938 and LEF pro1897 by 38-60% compared with that of the control (Luciferase, Luc) shRNA group. Similarly, TPA induced promoter activity of FNpro1938 and LEFpro1891 by 25 and 12-fold, respectively, in HCC340, which can be suppressed by aforementioned SNA shRNA by 60-70% (Fig. 2b). Moreover, overexpression of SNA increased constitutive promoter activity of FNpro1938 and LEFpro1897 by 2-2.5-fold in HCC340 (Fig. 2c) and HepG2 (data not shown). Collectively, SNA is essential for constitutive and TPA-induced transcriptional activation of FN and LEF in HCC.
Deletion mapping identified SNA motif coupled with the adjacent EGR1/SP1 overlapping regions within TPA-response element on both FN and LEF promoters
Further, we sought to identify the TPA-responsive element in promoters of FN and LEF and examined whether it contained the proposed SNA binding region “TCACA” and putative EGR1/SP1 overlapping motif as described previously [17]. According to the Genomatix software, there are three and two proposed SNA binding regions, respectively, locating on promoter of FN (-1187 to -547 bp upstream of the translation start site) and LEF (-1621 to -1211 bp upstream of the translation start site) (supplemental Fig. 3a, b). To examine whether any of these SNA binding motifs were required for promoter activation of both FN and LEF, deletion mapping analysis using 5’ end truncated mutants including FNpro1150, FNpro750, FNpro700 and FNpro568 (excluding 0, 1, 2 and 3 SNA region, respectively, from 5’ end on full-length FN promoter) (Fig. 3a, b upper left panel) and LEFpro1595, LEFpro1300 and LEFpro1231 (excluding 0, 1, and 2 SNA region, respectively, from 5’ end on full-length LEF promoter) (Fig. 3 a, b lower left panel) were performed. Surprisingly, we found a significantly higher TPA-induced promoter activity of FNpro1150 (deleted with 5’ end region upstream of the 1st SNA motif), compared with full-length FNpro1938 in both HepG2 (Fig. 3a upper right panel) and HCC340 (Fig. 3b upper right panel). Similarly, LEF pro1595 (with small deletion in the 5’ end region upstream of the 1st SNA motif) has significantly higher TPA-induced promoter activity compared with that of full-length LEFpro1897 in both HepG2 (Fig. 3a lower right panel) and HCC340 (Fig. 3b lower right panel). These implicated that a negative regulatory region located upstream of SNA region in full-length promoter of both FN and LEF, as will be described in below section. Further, the TPA-induced promoter activity of FNpro750 and FNpro700, the FN mutants lacking one and two distal SNA motifs, respectively, decreased only 10-30% compared with FNpro1150. Strikingly, FNpro568, the mutant lacking all 3 SNA regions, exhibited a dramatic reduction (by 68%) of TPA-induced promoter activity compared with FNpro1150 in HepG2 (Fig. 3a upper right panel) and HCC340 (Fig. 3b upper right panel). Similarly, the TPA-induced promoter activity of LEFpro1300, the LEF mutants lacking one SNA motif, exhibited minor difference (by 10-20%) compared with LEF pro1595, whereas LEFpro1231, the mutant lacking both SNA motifs exhibited a greater reduction (by 70-80%) of TPA-induced promoter activity than those of LEFpro1595 and LEFpro1300 in HepG2 (Fig. 3a lower right panel) and HCC340 (Fig. 3b lower right panel). Together, these implied that the most proximal (3’) SNA motifs are required for TPA-induced promoter activity of both FN and LEF. Interestingly, the TPA-responsive SNA motifs on both promoters are close to a EGR1/SP1 (E/S) overlapping region which was known to be required for TPA-induced, SNA-mediated MMP9 and ZEB1 promoter activation [17]. Thus, it is very probable that the E/S overlapping regions close to the candidate SNA site on both promoters are also involved in SNA-dependent transcriptional upregulation in FN and LEF. Indeed, FNpro330 and LEFpro1100 which lack E/S overlapping and all the SNA regions, showed a further decrease (by 60-80%) of TPA-induced promoter activity compared with those of the FNpro568 and LEFpro1231 that still contained E/S (Fig. 3a, b). Collectively, these strongly suggest that the most proximal (3’) SNA region coupled with the adjacent E/S overlapping regions are essential for TPA-induced promoter activations of both FN and LEF.
Mutagenesis on promoter validated SNA binding motif and E/S overlapping region required for transcription of FN and LEF
To prove whether the candidate SNA binding motifs are required for transcription of LEF and FN, site directed mutagenesis were performed, producing promoter mutants with altered sequence (TCACA®TTGTA) at indicated SNA motifs (PM 1-3). As shown in Fig. 3 (c, d), FN promoter mutant with altered sequence at the proximal (3’) SNA binding motif (PM3) exhibited a 60% decrease of TPA-induced promoter activity compared with that of the wild type FNpro1150 in both HepG2 (Fig. 3c, upper panel) and HCC340 (Fig. 3d, upper panel) at 12 h, whereas mutation of FN promoter at the middle SNA (PM2) and the distal (5’) SNA region (PM1) didn’t decrease the TPA-induced promoter activity. Similarly, LEF promoter mutant with the altered sequence at the proximal (3’) SNA (PM2) but not the distal (5’) SNA (PM1) exhibited a significantly decrease (by 50-70%) of TPA-induced promoter activity compared with that of the wild type LEFpro1595 in both HepG2 (Fig. 3c, lower panel) and HCC340 (Fig. 3d, lower panel). Collectively, these validated that the most proximal (3’) SNA binding regions on promoters of both FN and LEF are essential for TPA-induced promoter activation. In addition, mutation (CCCCGCCT ®CCCTATCT) on the E/S region (-1195 to -1179 bp) of LEFpro1595 (LEFpro1595 E/S-PM1) close to the active SNA motif, but not the next (proximal) E/S region (-1134 to -1119 bp) (LEFpro1595 E/S-PM2) also attenuated the TPA-enhanced promoter activity of LEFpro1595 by 33% in HepG2 (Fig. 3c, lower panel) and HCC340 (Fig. 3d, lower panel). As a negative control, mutagenesis on the FOXA2 region close to the active SNA region has no effect on TPA-induced promoter activity of LEF (Fig. 3c, d, lower panel). Taken together, the proximal (3’) SNA region coupled with the adjacent E/S overlapping region are responsible for TPA-induced promoter activation of FN and LEF.
PPAR-g is the negative feedback regulator in SNA-mediated transcription of FN and LEF
In the deletion mapping analysis, we found a negative regulatory region upstream of the distal (5’) SNA binding motif on promoters of both genes as described above (Fig. 3a, b). Notably, a putative binding motif of peroxisome proliferator-activated receptor γ (PPAR-g) transcriptional factor according to the JASPAR database, was identified within these regions (Fig. 3 and supplemental Fig. 3). Interestingly, both SNA and PPAR-g may antagonize gene expressions of each other. PPAR-g is well known to be a repressor of SNA for blocking EMT [22]. On the other hand, SNA can inhibit the expression of PPAR-g for adipocyte differentiation [23]. In a Q-RT-PCR analysis, PPAR-g mRNA was increased by treatment of TPA at 6 h by 2.8-fold, further increased to 3.3-fold at 12 h and declined to 2.0-fold at 14 h (supplemental Fig. 2a, left panel). As expected, it can be suppressed by GW9662, one of the PPAR-g antagonists, by 50% at 12 h time point (supplemental Fig. 2a, left panel). Moreover, TPA-induced PPAR-γ mRNA at 12 h was enhanced by SNA shRNA, shSN18, and on the contrary, it was diminished by overexpression of SNA approximately by 1.5-2-fold at 12 h (supplemental Fig. 2b, right panel). Thus, SNA can be a negative regulator of PPAR-γ in TPA-treated HCC. On the other hand, we examined whether PPAR-γ plays a negative regulatory role in the expression of SNA and the SNA-upregulated mesenchymal genes. In Q-RT-PCR of SNA, FN and LEF (supplemental Fig. 1a), GW9662 reversed the down regulation of mRNA of SNA, FN and LEF by 50~60 % at 12 h of TPA treatment. Similarly, in Western blot of SNA (supplemental Fig. 1b, upper panel), FN and LEF (supplemental Fig. 1b, lower panel), GW9662 dramatically reversed the down regulation of protein of SNA, FN and LEF at 12 h to a level close to those at 6 h of TPA treatment. On the transcriptional level, GW9662 (2.5-5.0mM) dose dependently elevated the TPA-induced promoter activation of both full-length promoters, FNpro1938 and LEFpro1897 (containing PPAR-g region) at 12 h to a level close to FNpro1150 and LEFpro1595 (without PPAR-g region) in both HepG2 (Fig. 4a) and HCC340 (Fig. 4b). To validate the negative regulatory role of PPAR-g, we investigated whether it is also involved in the SNA-mediated MMP9 and ZEB1 upregulation as reported previously [17]. Interestingly, TPA-induced promoter activation of full-length promoter of MMP9 and ZEB1 (MMP9pro1920 and ZEB1pro2052, containing PPAR-g) were also lower than those of shorter promoter fragments lacking PPAR-g region but contained the TPA-responsive SNA motif (MMP9pro950 and ZEB1pro1079) [17] in both HepG2 (supplemental Fig. 4a) and HCC340 (supplemental Fig. 4b). As expected, cotreatment of GW9662 (2.5-5.0 mM) elevated TPA-induced promoter activation of full-length promoter of MMP9pro1920 and ZEB1pro2052 (containing PPAR-g region) close to that of MMP9pro950 and ZEB1pro1079 (without PPAR-g region) in a dose dependent manner (supplemental Fig. 4a, b). However, cotreatment of GW9662 has no rescue effect on TPA-induced promoter activation of the aforementioned full-length promoters at 4h when PPAR-g was not yet induced (supplemental Fig. 4c). Consistently, the downregulation of MMP9 and ZEB1 protein in TPA-treated HCC340 at 12h can be rescued by co-treatment of GW9926 to a level close to those at 6 h (supplemental Fig. 1b, lower panel). Moreover, overexpression of PPAR-g (using a PPAR-g expression plasmid, p-PPAR-g) suppressed the TPA-induced activations of full-length FN and LEF promoters, FNpro1938 and LEFpro1897, but not 5’-truncated mutants FNpro568 and LEFpro1231 (without PPAR-g region) by 60-70% (Fig. 4c). Prior transfection of p-PPAR-g also prevented TPA-induced activation of full-length promoter of FN, LEF, MMP9 and ZEB1 (FNpro1938, LEFpro1897, MMP9pro1920 and ZEB1pro2052, respectively) at 12 h in HCC340 by 40-50% (Fig. 4d). The same phenomenon was also observed in HepG2 (data not shown). On the contrary, full-length promoters of FN, LEF, MMP9 and ZEB1 with alteration (AAAGG or CCTTT®AGTCT) in PPAR-g regions (FNpro1938*PPAR-g, LEFpro1897*PPAR-g, MMP9pro1920*PPAR-g and ZEB1pro2052*PPAR-g) exhibited higher TPA-induced promoter activity at 12 h by 2.0-6.0-fold, compared with each of wild type full-length promoters in both HepG2 (Fig. 4e) and HCC340 (Fig. 4f).
In summary, PPAR-g is responsible for the negative feedback against the SNA-upregulated transcription for FN, LEF, MMP9 and ZEB1 at late stage of TPA treatment.
ChIP and EMSA assay validated the binding of key transcription factors on putative regions
Thus far, it appears that SNA coupled with EGR1/SP1 activate transcription of the aforementioned mesenchymal genes, and PPAR-g plays as a negative feedback role, whether the indicated putative regions can be bound by the relevant transcriptional factors in these processes was examined, using ChIP assay. As shown in Fig. 5a (upper panel), TPA can induce binding of SNA on a FN promoter fragment (FN280), containing sequences of the critical SNA coupled with EGR1/SP1 region, at 4 h, further increased at 6h and returned to basal level at 12 h in HCC340. Also, TPA can induce binding of EGR1 on the same promoter fragment at 2-4 h, further increased at 6 h and declined at 12 h. In addition, TPA can induce sustained binding of SP1 on the same fragment during 4-12 h (Fig. 5a, upper panel). Similarly, TPA-induced binding of SNA, EGR1 and SP1 on the LEF promoter fragment (LEF280) containing the critical SNA coupled with EGR1/SP1 region begin at 2 h, further increased at 4 h and gradually declined during 6-12 h (Fig. 5a, lower panel). Moreover, GW9662 recovered the binding of SNA on FN280 and LEF280 at 12h (Fig. 5a, last lane), consistent with the rescue effect of this PPAR-g inhibitor on TPA-induced promoter activation (Fig. 4b) and expression (supplemental Fig. 1) of both genes at 12 h time point. Moreover, double ChIP assay validated the association of SNA with EGR1 (by 1st ChIP EGR1, 2nd ChIP SNA), and SNA with SP1 (by 1st ChIP SP1, 2nd ChIP SNA) on FN280 at 4 h, further increased at 6 h and declined at 12 h (Fig. 5b, upper panel). Similarly, TPA induced association of SNA with EGR1, and SNA with SP1 at 2 h on LEF280, further increased at 4-6 h and declined at 12 h on LEF promoter (Fig. 5b, lower panel). Moreover, the ChIP and double ChIP assay for binding of the aforementioned transcriptional factors on both promoters were quantitatively analyzed by Q-PCR. As shown in Fig. 5c (upper left panel), TPA can induce binding of SNA and EGR1 on FN280 by 4.2- and 8.8-fold at 4 h, further increased at 6h by 17- and 20-fold and returned to 2 and 4-fold of basal level at 12 h in HCC340 whereas binding of SP1 on FN280 can be induced at 2 h by 3.0-fold, increased to 8.0-fold at 4 h and sustained to 12 h. Similarly, TPA can induce binding of SNA, EGR1 and SP1 on LEF280 at 2h by 1.5-2.0-fold, increased to 3.5-fold at 4h, significantly decreased at 6 h and returned to basal at 12h (Fig. 5c, lower left panel). Also, double ChIP assay demonstrated 5.5- and 8.5-fold increase of association of (SNA with EGR1) and (SNA with SP1) on FN280, respectively, at 6 h of TPA treatment (Fig. 5c, upper right panel). Similarly, TPA can induce association of (SNA with EGR1) and (SNA with SP1) on LEF280 by 3.5- and 3.0-fold (Fig. 5c, lower right panel), respectively, at 6 h. Moreover, the decrease of SNA binding on both FN280 and LEF280 at 12h can be rescued by cotreatment with GW9662 to those at 6 and 4h, respectively (Fig. 5c, left panel, last bar). On the other hand, TPA can induce binding of PPAR-g on the fragments containing putative region of PPAR-g on FN, LEF, MMP9 and ZEB1 promoter, FN290, LEF230, MMP-9 280, ZEB1 200, respectively, with maximal induction during 6-12h (Fig. 5d). As expected, TPA-induced binding of PPAR-g on the aforementioned promoter fragments can be greatly suppressed by GW9926 at 12 h (Fig. 5d).
We further confirmed the binding of SNA on their putative region in vitro by EMSA. Nuclear extract of HCC340 treated with TPA at different time points were incubated with biotin labelled probe containing the SNA binding motif on FN promoter (FN-Prob). As shown in Fig. 6a, a DNA-protein complex (revealed as a band shift) was observed in the time zero group. This was increased at 4 and 6 h by 1.5- and 2.2- fold, respectively, and declined at 12 h. Addition of 200-fold of the unlabeled wild type but not mutant type of probe reduced the amount of DNA-protein complex by 80-90% at 6 h. Moreover, preincubation of the EMSA mixture with SNA antibody but not IgG control resulted in a supershift of the DNA-protein complex, validating SNA was the protein bound with the DNA probe. The same phenomenon was also observed in EMSA of the DNA probe containing the critical SN1 binding motif on LEF promoter (LEF-Prob). As demonstrated in Fig. 6b, TPA induced band shift of the DNA-protein complex maximally at 4 h, and gradually decreased at 6-12 h. As expected, the TPA-induced band shift of the DNA-protein complex at 4h can be competed by unlabeled wild type but not mutant type probe (Fig. 6b). Moreover, a supper band shift of the protein DNA complex can also be observed if EMSA mixture from 4 h-TPA sample was preincubated with SNA Ab but not IgG control (Fig. 6b).
COX2 and COL1A1 were potentially transcriptionally upregulated by SN as the aforementioned mesenchymal gene.
Thus far, we have demonstrated four of the mesenchymal genes FN, LEF, MMP9 and ZEB1 transcriptionally upregulated by SNA in a same manner, ie, direct binding of SNA coupled with EGR1/SP1 on their putative regions, which can be negatively feedbacked by PPAR-g. To validate whether this can be a general model, we screen the mesenchymal genes that can be upregulated by SNA from PubMed and examine whether they contain the aforementioned SNA binding motif (TCACA) coupled with a downstream EGR1/SP1 overlapping region and an upstream PPAR-g binding motif on the promoter sequences, 2000 bp upstream of translational start site by using gene bank in NCBI. Among them, ten of the well-known mesenchymal genes were proposed (Table 1). Most of the candidate mesenchymal genes such as COX2, vimentin, vitronectin, COL1A1, α-SMA, N-cadherin, Twist1 have been reported to be upregulated by SNA involved in tumor progression. For example, COX2, which greatly correlates with malignancy, can be upregulated by SNA in head and neck squamous cell carcinomas [24]. To begin with, we validated whether COX2 and COL1A1 can be induced by TPA. As shown in Q RT-PCR (supplemental Fig. 5), TPA increased mRNA of both COX2 and COL1A1 at 2h by 1.5-2.0-fold, further increased at 4h and reach maximum at 6h by 8 and 10-fold, respectively, and finally declined to basal level at 12h. Moreover, GW9662 significantly rescued the downregulation of mRNA of both COX2 and COL1A1 at 12h. These suggested that while SNA is essential for gene expression of COX2 and COL1A1, PPAR-g played as a negatively feedback regulator, the same mechanisms as we observed in FN, LEF, MMP9 and ZEB1. We further investigated whether transcription regulation of COX2 and COL1A1 is also the same using deletion mapping for promoter analysis. According to the Genomatix software, there are one proposed SNA binding region and a downstream EGR1/SP1, locate on promoter of COX2 (-1007 to -502 bp upstream of the translation start site) and COL1A1 ( -1122 to -934 bp upstream of the translation start site) (supplemental Fig. 3c, d). Also, there are one putative PPAR-g binding region upstream of SNA region, locating on promoter of COX2 (-1560 to -979 bp upstream of the translation start site) and COL1A1 (-1969 to -1090 bp upstream of the translation start site) (supplemental Fig. 3c, d). Accordingly, we constructed the full-length promoter plasmid of COX2 (COX2pro1560) and COX2pro1228 containing PPAR-g coupled with one SNA motif and EGR1/SP1 overlapping binding regions. Also, COX2pro979, 5’ end truncated mutants lacking PPAR-g binding region, and COX2pro870 lacking PPAR-g and one SNA region were obtained. In addition, COX2pro362 and COX2pro175, with deletion of PPAR-g, one SNA region and the downstream EGR1/SP1 overlapping region, were also included. Strikingly, we found a 40-45% higher TPA-induced promoter activity of COX2pro979 (without PPAR-g region) compared with full-length COX2pro1560 and 1228 (with PPAR-g region) whereas the TPA-induced promoter activity of COX2pro870 and COX2pro362 decreased by 50 and 80%, respectively, compared with COX2pro979 in HCC340 (Fig. 7a, upper panel). Moreover, GW9662 (at 2.5 and 5.0 mM) dose-dependently elevated the TPA-induced promoter activity of COX2pro1560 by 33 to 45%, a level close to that of COX2pro979 (Fig. 7b, upper panel). The deletion mapping analysis for COL1A1 revealed the similar results. As demonstrated in Fig. 7a (lower panel), the TPA-induced promoter activity of full-length promoter of COL1A1 (COL1A1pro1969) containing PPAR-g and one SNA region was lower than the deleted mutant COL1A1pro1090 (lacking the 5’ PPAR-g region) by 46%, whereas the activity of COL1A1pro644 without both SNA and PPAR-g regions was lower than that of COL1A1pro1090 by 60%. Moreover, GW9662 (at 2.5 and 5.0 mM) dose-dependently elevated the TPA-induced promoter activity of COL1A1pro1969 by 39 to 46%, a level close to COL1A1 pro1090 (Fig. 7a, lower panel). Thus, we suggested SNA-mediated upregulation of promoter activation COX2 and COL1A1 is also negatively feed backed by PPAR-g, potentially in the same way as that of the aforementioned mesenchymal genes.