IL-22 has a pathogenetic role in psoriasis, where it is responsible for the altered proliferation and differentiation of keratinocytes and induces inflammatory molecules. The IL-22-induced effects are mediated by STAT3, whose activity is proportional to acetylation in lysine (Lys)685 and phosphorylation in tyrosine (Tyr)705. Lys 685 acetylation of STAT3 is inhibited by sirtuin (SIRT)1, a class III deacetylase promoting keratinocyte differentiation. Due to the opposite effects of IL-22 and SIRT1, we investigated whether IL-22-induced effects in keratinocytes could be regulated by SIRT1 through control of STAT3. We found that SIRT1 opposes the IL-22-induced STAT3 activity by deacetylating STAT3 and reducing STAT3 Tyr705 phosphorylation. By controlling STAT3, SIRT1 also influences the IL-22-induced expression of molecules involved in proliferation and inflammation as well as proliferation and migration processes in cultured keratinocytes. Although SIRT1 levels were similar in keratinocytes of healthy individuals and patients with psoriasis, they were reduced in psoriatic skin lesions, with the lymphokine IFN-γ inhibiting SIRT1 expression. Concomitantly, IFN-γ enhanced basal acetylation of STAT3 and its phosphorylation induced by IL-22. In conclusion, STAT3-dependent IL-22 signaling and effects in keratinocytes are negatively regulated by SIRT1. In skin affected by psoriasis, SIRT1 is down-regulated by IFN-γ, which thus renders psoriatic keratinocytes more prone to respond to IL-22.

The endothelin-1 (ET-1)/ET A receptor (ETAR) signalling pathway is a well-established driver of epithelial ovarian cancer (EOC) progression. One key process promoted by ET-1 is tumor cell invasion, which requires the scaffolding functions of β-arrestin-1 (β-arr1) downstream of the receptor; however, the potential role of ET-1 in inducing invadopodia, which are crucial for cellular invasion and tumor metastasis, is completely unknown. We describe here that ET-1/ETAR, through β-arr1, activates RhoA and RhoC GTPase and downstream ROCK (Rho-associated coiled coil-forming kinase) kinase activity, promoting actin-based dynamic remodelling and enhanced cell invasion. This is accomplished by the direct interaction of β-arr1 with PDZ-RhoGEF (postsynaptic density protein 95/disc-large/zonula occludens-RhoGEF). Interestingly, ETAR-mediated invasive properties are related to the regulation of invadopodia, as evaluated by colocalization of actin with cortactin, as well as with TKS5 and MT1-MMP (membrane type 1-matrix metalloproteinase) with areas of matrix degradation, and activation of cofilin pathway, which is crucial for regulating invadopodia activity. Depletion of PDZ-RhoGEF, or β-arr1, or RhoC, as well as the treatment with the dual ET-1 receptor antagonist macitentan, significantly impairs invadopodia function, MMP activity and invasion, demonstrating that β-arr1/PDZ-RhoGEF interaction mediates ETAR-driven ROCK-LIMK-cofilin pathway through the control of RhoC activity. In vivo, macitentan is able to inhibit metastatic dissemination and cofilin phosphorylation. Collectively, our data unveil a noncanonical activation of the RhoC/ROCK pathway through the β-arr1/PDZ-RhoGEF complex as a regulator of ETAR-induced motility and metastasis, establishing ET-1 axis as a novel regulator of invadopodia protrusions through the RhoC/ROCK/LIMK/cofilin pathway during the initial steps of EOC invasion.

Telomeres consist of DNA tandem repeats that recruit the multiprotein complex shelterin to build a chromatin structure that protects chromosome ends. Although cancer formation is linked to alterations in telomere homeostasis, there is little understanding of how shelterin function is limited in cancer cells. Using a small-scale screening approach, we identified miR-155 as a key regulator in breast cancer cell expression of the shelterin component TERF1 (TRF1). miR-155 targeted a conserved sequence motif in the 3 0 UTR of TRF1, resulting in its translational repression. miR-155 was upregulated commonly in breast cancer specimens, as associated with reduced TRF1 protein expression, metastasis-free survival, and relapse-free survival in estrogen receptor-positive cases. Modulating miR-155 expression in cells altered TRF1 levels and TRF1 abundance at telomeres. Compromising TRF1 expression by elevating miR-155 increased telomere fragility and altered the structure of metaphase chromosomes. In contrast, reducing miR-155 levels improved telomere function and genomic stability. These results implied that miR-155 upregulation antagonizes telomere integrity in breast cancer cells, increasing genomic instability linked to poor clinical outcome in estrogen receptor-positive disease. Our work argued that miRNAdependent regulation of shelterin function has a clinically significant impact on telomere function, suggesting the existence of "telo-miRNAs" that have an impact on cancer and aging. Cancer Res; 74(15); 4145-56. Ó2014 AACR.

Epidermal keratinocytes can counteract the detrimental effects of IFN-γ by inducing the expression of suppressor of cytokine signaling (SOCS)1, which plays an important anti-inflammatory and self-protective role. To date, limited information exists on its expression and regulation in human diseased keratinocytes. In this study, we compared the expression levels of SOCS1 in keratinocytes isolated from skin affected by psoriasis with cells obtained from healthy donors, unveiling that keratinocytes are more prone than healthy cells to upregulate SOCS1 mRNA expression in response to IFN-γ. We explored the regulatory mechanisms involved in socs1 gene transcription, and found that Sp1 and IFN regulatory factor-1 transcription factors are, respectively, responsible for the basal and IFN-γ–induced activity of human socs1 promoter. In parallel, we demonstrated that socs1 promoter is negatively regulated by two transcriptional repressors, namely, growth factor independence-1b and Krüppel-like factor 4, which tightly control SOCS1 transcription on IFN-γ stimulation. Interestingly, although the expression of Sp1 and IFN regulatory factor-1 activators of socs1 promoter is unaltered, growth factor independence-1b and Krüppel-like factor 4 are significantly reduced in psoriatic compared with healthy keratinocytes. This reduction and the consequent unbalanced binding of transcriptional activators and repressors to socs1 promoter after IFN-γ stimulation might be responsible for the enhanced expression of SOCS1 in psoriatic cells. We suggest that SOCS1 exaggerated upregulation in psoriatic keratinocytes could represent a mechanism through which these cells attempt to protect themselves from IFN-γ effects. However, the SOCS1 increased levels in psoriatic keratinocytes are not sufficient to completely inhibit the expression of proinflammatory genes.

Drug resistance remains the major clinical barrier to successful treatment in epithelial ovarian carcinoma (EOC) patients, and the evidence of microRNA involvement in drug resistance has been recently emerging. Endothelin-1 (ET-1)/ETA receptor (ETAR) axis is aberrantly activated in chemoresistant EOC cells and elicits pleiotropic effects promoting epithelial-to-mesenchymal transition (EMT) and the acquisition of chemoresistance. However, the relationship between ETAR and miRNA is still unknown. Hence, in this study we evaluated whether dysregulation of miRNA might enhance ETAR expression in sensitive and resistant EOC cells. Based on bioinformatic tools, we selected putative miRNA able to recognize the 3′UTR of ETAR. An inverse correlation was observed between the expression levels of miR-30a and ETAR in both EOC cell lines and tumor samples. miR-30a was found to specifically bind to the 3′UTR of ETAR mRNA, indicating that ETAR is a direct target of miR-30a. Overexpression of miR-30a decreased Akt and mitogen activated protein kinase signaling pathway activation, cell proliferation, invasion, plasticity, EMT marker levels, and vascular endothelial growth factor release. Interestingly, ectopic expression of miR-30a re-sensitized platinum-resistant EOC cells to cisplatinum-induced apoptosis. Consistently, resistant EOC xenografts overexpressing miR-30a resulted in significantly less tumor growth than controls. Together our study provides a new perspective on the regulatory mechanism of ETAR gene. Interestingly, our findings highlight that blockade of ETAR regulatory axis is the mechanism underlying the tumor suppressor function of miR-30a in chemoresistant EOC cells.

The limited clinical response to conventional chemotherapeutics observed in colorectal cancer (CRC) may be related to the connections between the hyperactivated β-catenin signaling and other pathways in CRC stem-like cells (CRC-SC). Here, we show the mechanistic link between the endothelin-1 (ET-1)/ET-1 receptor (ET-1R) signaling and β-catenin pathway through the specific interaction with the signal transducer β-arrestin1 (β-arr1), which initiates signaling cascades as part of the signaling complex. Using a panel of patient-derived CRC-SC, we show that these cells secrete ET-1 and express ETAR and β-arr1, and that the activation of ETAR/β-arr1 axis promotes the cross-talk with β-catenin signaling to sustain stemness, epithelial-to-mesenchymal transition (EMT) phenotype and response to chemotherapy. Upon ETAR activation, β-arr1 acts as a transcription co-activator that binds β-catenin, thereby promoting nuclear complex with β-catenin/TFC4 and p300 and histone acetylation, inducing chromatin reorganization on target genes, such as ET-1. The enhanced transcription of ET-1 increases the self-sustained ET-1/β-catenin network. All these findings provide a strong rationale for targeting ET-1R to hamper downstream β-catenin/ET-1 autocrine circuit. Interestingly, treatment with macitentan, a dual ETAR and ETBR antagonist, able to interfere with tumor and microenvironment, disrupts the ET-1R/β-arr1-β-catenin interaction impairing pathways involved in cell survival, EMT, invasion, and enhancing sensitivity to oxaliplatin (OX) and 5-fluorouracil (5-FU). In CRC-SC xenografts, the combination of macitentan and OX or 5-FU enhances the therapeutic effects of cytotoxic drugs. Together, these results provide mechanistic insight into how ET-1R coopts β-catenin signaling and offer a novel therapeutic strategy to manage CRC based on the combination of macitentan and chemotherapy that might benefit patients whose tumors show high ETAR and β-catenin expression.

Although several mechanisms have been proposed to explain the putative role of beta-carotene in cancer, no studies have investigated a possible influence of beta-carotene on caveolin-1 (cav-1) pathway, an important intracellular signaling deregulated in cancer. Here, different human colon and prostate cancer cell lines, expressing (HCT-116, PC-3 cells) or not (Caco-2, LNCaP cells) cav-1, were treated with varying concentrations of beta-carotene (0.5-30 muM) for different periods of time (3-72 h) and the effects on cell growth were investigated. The results of this study show that (i) beta-carotene acted as a growth-inhibitory agent in cav-1-positive cells, but not in cav-1-negative cells; (ii) in cav-1-positive cells, the carotenoid downregulated in a dose- and time-dependent manner the expression of cav-1 protein and messenger RNA levels and inhibited AKT phosphorylation which, in turn, stimulated apoptosis by increasing the expression of beta-catenin and c-myc and the activity of caspases-3, -7, -8 and -9; when the carotenoid was removed from culture medium, a progressive increase in cell growth was observed with respect to beta-carotene-treated cells and (iii) the transfection of cav-1 in cav-1-negative cells increased cell sensitivity to beta-carotene by inducing apoptosis. This effect was accompanied by a reduction of both cav-1 and AKT phosphorylation and by an increase of c-myc and beta-catenin expression. Silencing of c-Myc attenuated beta-carotene-induced apoptosis and beta-catenin expression. All together, these data suggest that the modulation of cav-1 pathway by beta-carotene could be a novel mechanism by which the carotenoid acts as a potent growth-inhibitory agent in cancer cells.

The imbalance of the intracellular redox state and, in particular, of the glutathione (GSH)/GSH disulfide couple homeostasis, is involved in the pathogenesis of a number of diseases. In many skin diseases, including psoriasis, oxidative stress plays an important role, as demonstrated by the observation that treatments leading to increase of the local levels of oxidant species ameliorate the disease. Recently, dehydrocostuslactone (DCE) and costunolide (CS), two terpenes naturally occurring in many plants, have been found to exert various anti-inflammatory and pro-apoptotic effects on different human cell types. These compounds decrease the level of the intracellular GSH by direct interaction with it, and, therefore, can alter cellular redox state. DCE and CS can trigger S-glutathionylation of various substrates, including the transcription factor STAT3 and JAK1/2 proteins. In the present study, we investigated on the potential role of DCE and CS in regulating inflammatory and proliferative responses of human keratinocytes to cytokines. We demonstrated that DCE and CS decreased intracellular GSH levels in human keratinocytes, as well as inhibited STAT3 and STAT1 phosphorylation and activation triggered by IL-22 or IFN-γ, respectively. Consequently, DCE and CS decreased the IL-22- and IFN-γ-induced expression of inflammatory and regulatory genes in keratinocytes, including CCL2, CXCL10, ICAM-1 and SOCS3. DCE and CS also inhibited proliferation and cell-cycle progression-related gene expression, as well as they promoted cell cycle arrest and apoptosis. In parallel, DCE and CS activated the anti-inflammatory EGFR and ERK1/2 molecules in keratinocytes, and, thus, wound healing in an in vitro injury model. In light of our findings, we can hypothesize that the employment of DCE and CS in psoriasis could efficiently counteract the pro-inflammatory effects of IFN-γ and IL-22 on keratinocytes, revert the apoptosis-resistant phenotype, as well as inhibit hyperproliferation in the psoriatic epidermis.