ATF3 downmodulates its new targets IFI6 and IFI27 to suppress the growth and migration of tongue squamous cell carcinoma cells.

Activating transcription factor 3 (ATF3) is a key transcription factor involved in regulating cellular stress responses, with different expression levels and functions in different tissues. ATF3 has also been shown to play crucial roles in regulating tumor development and progression, however its potential role in oral squamous cell carcinomas has not been fully explored. In this study, we examined biopsies of tongue squamous cell carcinomas (TSCCs) and found that the nuclear expression level of ATF3 correlated negatively with the differentiation status of TSCCs, which was validated by analysis of the ATGC database. By using gain- or loss- of function analyses of ATF3 in four different TSCC cell lines, we demonstrated that ATF3 negatively regulates the growth and migration of human TSCC cells in vitro. RNA-seq analysis identified two new downstream targets of ATF3, interferon alpha inducible proteins 6 (IFI6) and 27 (IFI27), which were upregulated in ATF3-deleted cells and were downregulated in ATF3-overexpressing cells. Chromatin immunoprecipitation assays showed that ATF3 binds the promoter regions of the IFI6 and IFI27 genes. Both IFI6 and IFI27 were highly expressed in TSCC biopsies and knockdown of either IFI6 or IFI27 in TSCC cells blocked the cell growth and migration induced by the deletion of ATF3. Conversely, overexpression of either IFI6 or IFI27 counteracted the inhibition of TSCC cell growth and migration induced by the overexpression of ATF3. Finally, an in vivo study in mice confirmed those in vitro findings. Our study suggests that ATF3 plays an anti-tumor function in TSCCs through the negative regulation of its downstream targets, IFI6 and IFI27

The ARE-binding protein Tristetraprolin (TTP) is a novel target and mediator of calcineurin tumor suppressing function in the skin

<div><p>An increased incidence of skin inflammatory diseases is frequently observed in organtransplanted patients being treated with calcineurin inhibitor-based immunosuppressive agents. The mechanism of increased skin inflammation in this context has however not yet been clarified. Here we report an increased inflammation following inhibition of calcineurin signaling seen in both chemically induced mouse skin tumors and in tumors grafted from <i>H-ras</i>^<i>V12</i> expressing primary human keratinocytes (HKCs). Following UVB or TPA treatment, we specifically found that deletion of the calcineurin gene in mouse keratinocytes (MKCs) resulted in increased inflammation, and this was accompanied by the enhanced production of pro-inflammatory cytokines, such as TNFα, IL-8 and CXCL1. Furthermore, expression of the RNA-binding protein, tristetraprolin (TTP) was down-regulated in response to calcineurin inhibition, wherein TTP was shown to negatively regulate the production of pro-inflammatory cytokines in keratinocytes. The induction of TTP following TPA or UVB treatment was attenuated by calcineurin inhibition in keratinocytes, and correspondingly, disruption of calcineurin signaling down-regulated the amounts of TTP in both clinical and <i>H-ras</i>^<i>V12</i>-transformed keratinocyte tumor models. Our results further demonstrated that calcineurin positively controls the stabilization of TTP in keratinocytes through a proteasome-dependent mechanism. Reducing the expression of TTP functionally promoted tumor growth of <i>H-ras</i>^<i>V12</i> expressing HKCs, while stabilizing TTP expression counteracted the tumor-promoting effects of calcineurin inhibition. Collectively these results suggest that calcineurin signaling, acting through TTP protein level stabilization, suppresses keratinocyte tumors by downregulating skin inflammation.</p></div

CnB1 regulates TTP stability through proteasomal mediation.

<p><b>A.</b> qRT-PCR analysis of TTP expression level from the dorsal epidermis of two wild-type (<i>wt</i>) and two CnB1-/- (<i>KO</i>) mice 24 hr after treatment with UVB (+) or sham-irradiation (-) as control. <b>B.</b> Ad-GFP- or Ad-Cre-infected CnB1^flox/flox MKCs were treated with UVB (+) or sham-irradiated (-). Four hours after treatment, cells were analyzed for TTP mRNA level by qRT-PCR. <b>C,D.</b> HKCs were transfected with siRNAs of scramble RNA (siCtrl), calcineurin B1(siCnB1), NFATc1(siC1) or NFATc4 (siC4) or were treated with DMSO, CsA, Veet or Vivit. 48 hr after treatment, cells were analyzed for TTP mRNA level by qRT-PCR. <b>A-D</b>: Normalized to 36B4 gene, *p<0.05, **p<0.01, ***p<0.001; n = 3. <b>E.</b> Ad-GFP- or Ad-Cre-infected CnB1^flox/flox MKCs, UVB-irradiated as in <b>B,</b> were incubated with puromycin (<i>PURO</i>) or DMSO (<i>ctrl</i>). Cells were then collected at the indicated times and analyzed by immuoblot for TTP (tubulin as loading control). <b>F.</b> Ad-GFP- or Ad-Cre-infected CnB1-loxP MKCs were treated with UVB as in <b>B,</b> in the presence of epoxomicin 1μM (<i>EPOX</i>) or <i>DMSO</i> at the indicated times. Cells were then analyzed by immunoblot for TTP, tubulin as a loading control. <b>G.</b> HKCs with over-expression of TTP (TTP-OE) with treated either with CsA with or without MG132 were collected at indicated times for analysis of TTP protein level. <b>H.</b> qRT-PCR analysis of TTP mRNA level at indicated time points in HKCs treated with CsA with or without MG132.</p

TTP plays a tumor-suppressing function in keratinocytes.

<p><b>A.</b> H-<i>ras</i>^V12 expressing HKCs with siRNA-mediated TTP knockdown (siTTP) versus control (siCtrl) were injected into the dermal-epidermal junction of nu/nu mouse skin. Mice were sacrificed 6 wks later for histological analysis and IF staining of Ki67 and Gr-1. Results were quantified by establishing the percentage of Ki67 (<b>B</b>) and Gr-1 (<b>C</b>) positive cells over DAPI positive cells. <b>D.</b> Similar assays were performed with H-<i>ras</i>^V12 expressing HKCs with or without stable expression of TTP, followed by treatment with CsA or DMSO. Four wks later, the mice were sacrificed for histological analysis and Ki67 (<b>F</b>) and Gr-1 (<b>G</b>) positive cells were counted. According to the histological analysis, the quantification of lesions was shown in <b>E</b>. The results were quantified by calculating the percentage of Ki67 (<b>F</b>) and Gr-1 (<b>G</b>) positive cells divided by DAPI positive cells. <b>B, D, F, G</b>: *p<0.05, ** p<0.01, n = 3.</p

TTP is down-modulated in keratinocytes and in tumor tissues when calcineurin signaling is inhibited.

<p><b>A.</b> Ad-GFP- or Ad-Cre-infected CnB1^flox/flox MKCs were irradiated with UVB (+) or were sham-irradiated (-). Four hr after irradiation, the cells were analyzed by immunoblot for TTP, TIA-1, TIAR and HuR proteins, and tubulin as a loading control. <b>B.</b> Ad-GFP (<i>CnB+</i>) and Ad-Cre infected (<i>CnB-</i>) CnB1^flox/flox MKCs were sham-irradiated (<i>untr</i>) or were UVB-treated (<i>UVB</i>); after 4 hr they were stained with an anti-TTP antibody (red) and DAPI (blue) for nuclei. <b>C.</b> HKCs were either transfected with siRNA of CnB1 (siCnB1), NFATC1 (siC1), siNFATc4 (siC4) or a control scramble siRNA, or were treated with Vivit (Veet as control) or with CsA (DMSO as a control). After 72 hr, cells were lysed and immunoblotted for TTP, with tubulin as a loading control. The quantification of band density is shown in the lower panel. ** indicates p<0.001. <b>D,E.</b> IHC analysis of TTP in tumor sections from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007366#pgen.1007366.s002" target="_blank">S2A Fig</a>; <b>F</b>. Immunoblot analysis of TTP from 10 different SCC lines and two primary HKC lines; tubulin as a loading control. The full-blot image is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007366#pgen.1007366.s005" target="_blank">S5 Fig</a>. Quantification of band densities is shown in the lower panel. *: p<0.05, **p<0.01 when compared to primary HKCs. n = 3. <b>G-H.</b> Tissue microarrays (TMAs) of cutaneous SCCs from patients with or without CsA treatment versus the normal skin (NS) from general untreated population were analysed for TTP expression by immunohistochemistry. The quantification of TTP is shown in <b>H</b>, * indicates p<0.05 when compared with each other as indicated.</p

Deletion of CnB1 in keratinocytes enhances the skin acute inflammatory response to TPA treatment and to UVB exposure.

<p><b>A</b>. Ear thickness was measured at different time points as indicated. ## p<0.01 when comparing the TPA-treated group with the untreated group, ** p<0.01 when comparing the CnB1-/- (KO) group (KO+TPA) with the control CnB1+/+ group (Ctrl+TPA); <b>B.</b> Histological analysis of ears of Ctrl and KO mice at 48 hr after TPA treatment; <b>C.</b> Thickness of dorsal skin collected at the indicated time points was measured using a microscope. ## p<0.01 when comparing the UVB-exposed group with the untreated group, ** p<0.01 when comparing the KO group (KO+UVB) with the control group (Ctrl+UVB); <b>D.</b> Histological analysis of dorsal skins of Ctrl and KO mice at 48 hr after exposure to UVB; <b>E-H.</b> Sections from <b>C</b> and <b>D</b> were analyzed for infiltration of inflammatory cells by IF analysis of Gr-1 (<b>E,</b> green) and F4/80 (<b>G</b>, green), DAPI is used as a counter-stain for nuclei. The corresponding quantification analysis of <b>E</b> and <b>G</b> is shown in <b>F</b> (Gr-1) and <b>H</b> (F4/80). * p<0.05 when comparing the data indicated with the brackets. Bars = 500 μm for <b>B, D</b>; 100 μm for <b>E, G</b>. Student’s t test analysis was used for all quantification data to compare two groups as indicated, n = 3, Standard error bars are provided in <b>A, C, F</b> and <b>H</b>.</p

Inhibition of calcineurin signaling induces the expression of pro-inflammatory cytokines in keratinocytes.

<p><b>A-D</b>: qRT-PCR analysis of TNFα, IL-8 and CXCL1 expression; results were normalized to levels of 36B4 mRNA. <b>A.</b> Total mRNA isolated from mouse ears from the experiments shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007366#pgen.1007366.g001" target="_blank">Fig 1A</a>; <b>B.</b> Total mRNA extracted from primary MKCs from CnB1f^flox/flox mice infected with either Ad-Cre or Ad-GFP viruses, with or without UVB treatment; <b>C.</b> Total mRNA isolated from TPA-treated HKCs transfected with siRNA of CnB1 (siCnB1) or a control scramble siRNA (siCtrl) at 24 hr; <b>D.</b> Total mRNA from HKCs treated with CsA or DSMO (Ctrl) for 24 hr, followed by treatment with TPA for another 24 hr. <b>E-G:</b> ELISA analysis of indicated cytokine protein levels in the medium. <b>E.</b> Conditioned medium collected from MKC cultures shown in <b>B</b>; <b>F,G</b>. Conditioned medium collected from HKC cultures shown in <b>C</b>. Student’s t-test was used for statistical analysis in <b>A-G</b>, standard error bars provided, * p<0.05, **p<0.01, n = 3.</p