Microenvironmental Snail1-induced immunosuppression promotes melanoma growth.

Melanoma is an aggressive form of skin cancer due to its high metastatic abilities and resistance to therapies. Melanoma cells reside in a heterogeneous tumour microenvironment that acts as a crucial regulator of its progression. Snail1 is an epithelial-to-mesenchymal transition transcription factor expressed during development and reactivated in pathological situations including fibrosis and cancer. In this work, we show that Snail1 is activated in the melanoma microenvironment, particularly in fibroblasts. Analysis of mouse models that allow stromal Snail1 depletion and therapeutic Snail1 blockade indicate that targeting Snail1 in the tumour microenvironment decreases melanoma growth and lung metastatic burden, extending mice survival. Transcriptomic analysis of melanoma-associated fibroblasts and analysis of the tumours indicate that stromal Snail1 induces melanoma growth by promoting an immunosuppressive microenvironment and a decrease in anti-tumour immunity. This study unveils a novel role of Snail1 in melanoma biology and supports its potential as a therapeutic target.

SNAIL1: Linking Tumor Metastasis to Immune Evasion.

The transcription factor Snail1, a key inducer of epithelial-mesenchymal transition (EMT), plays a critical role in tumor metastasis. Its stability is strictly controlled by multiple intracellular signal transduction pathways and the ubiquitin-proteasome system (UPS). Increasing evidence indicates that methylation and acetylation of Snail1 also affects tumor metastasis. More importantly, Snail1 is involved in tumor immunosuppression by inducing chemokines and immunosuppressive cells into the tumor microenvironment (TME). In addition, some immune checkpoints potentiate Snail1 expression, such as programmed death ligand 1 (PD-L1) and T cell immunoglobulin 3 (TIM-3). This mini review highlights the pathways and molecules involved in maintenance of Snail1 level and the significance of Snail1 in tumor immune evasion. Due to the crucial role of EMT in tumor metastasis and tumor immunosuppression, comprehensive understanding of Snail1 function may contribute to the development of novel therapeutics for cancer.

Tumor-associated macrophages promote pancreatic ductal adenocarcinoma progression by inducing epithelial-to-mesenchymal transition.

In this study, we investigated the role of tumor-associated macrophages (TAMs) in the progression of pancreatic ductal adenocarcinoma (PDAC). PDAC patients with higher levels of CD68+ TAMs exhibited shorter overall survival. In Transwell assays, PDAC cells incubated with TAMs or conditioned media from TAM cells (TAM-CM) showed higher migration and invasion rates than controls. PET/CT scan analysis of orthotopic PDAC model mice revealed greater primary tumor growth and liver metastasis in the TAM-CM treatment group than the controls. H&E staining of liver tissues showed significantly higher numbers of metastatic nodules in the TAM-CM treatment group. Heat inactivation of TAM-CM significantly reduced Transwell migration by PDAC cells, suggesting the involvement of one or more secreted proteins in PDAC progression. Transcriptome sequencing analysis of PDAC cells treated with TAM-CM revealed significant enrichment of transforming growth factor-ß (TGF-ß) signaling pathway genes. Western blot and qRT-PCR analysis showed that TAM-CM enhanced PDAC migration cells by inducing epithelial-to-mesenchymal transition through the TGF-ß-Smad2/3/4-Snail signaling axis. The pro-tumorigenic effects of TAMs or TAM-CM were abolished by TGF-ß signaling pathway inhibitors and neutralizing TGF-ß antibody. These results demonstrate that TAMs promote PDAC progression through the TGF-ß signaling pathway.

Transactivation of Herpes Simplex Virus 1 (HSV-1) Infected Cell Protein 4 Enhancer by Glucocorticoid Receptor and Stress-Induced Transcription Factors Requires Overlapping Krüppel-Like Transcription Factor 4/Sp1 Binding Sites.

Following acute infection, herpes simplex virus 1 (HSV-1) lytic cycle viral gene expression is silenced; consequently, lifelong latency in neurons is established. Certain external stimuli that trigger reactivation from latency also activate the glucocorticoid receptor (GR). The synthetic corticosteroid dexamethasone, but not a GR-specific antagonist, increases the frequency of explant-induced reactivation from latency and stimulates productive infection. Furthermore, dexamethasone increases expression of cellular transcription factors in trigeminal ganglionic neurons: for example, SLUG and three Krüppel-like transcription factor (KLF) family members, KLF4, KLF15, and promyelocytic leukemia zinc finger protein (PLZF). Consequently, we hypothesized that stress-induced transcription factors stimulate expression of ICP4, a viral transcriptional regulator required for productive infection. New studies demonstrated that GR and KLF4, PLZF, or SLUG cooperatively transactivate the ICP4 enhancer upstream of a minimal promoter in monkey kidney cells (Vero) and mouse neuroblastoma cells (Neuro-2A). Strikingly, mutagenesis of two KLF4/Sp1 binding sites reduced GR- plus KLF4-, PLZF-, or SLUG-mediated transactivation to basal levels. A consensus enhancer (E)-Box adjacent to a KLF4/Sp1 binding site was also required for GR- and SLUG-, but not KLF family member-, mediated transactivation of the ICP4 promoter. Chromatin immunoprecipitation studies (ChIP) revealed GR and stress-induced transcription factors occupy ICP4 enhancer sequences. Conversely, specific binding was generally reduced in the KLF4/Sp1 mutant. Furthermore, GR and SLUG occupancy of ICP4 enhancer sequences was reduced in the E-Box mutant. Based on these studies, we suggest stressful stimuli can trigger productive infection because GR and specific stress-induced transcription factors activate ICP4 expression.IMPORTANCE Certain stressful stimuli activate the glucocorticoid receptor (GR) and increase the incidence of herpes simplex virus 1 (HSV-1) reactivation from latency. For example, a corticosteroid antagonist impairs productive infection and virus shedding following explant of trigeminal ganglia from latently infected mice. Infected cell protein 4 (ICP4) is the only immediate early viral transcriptional regulator required for productive infection, suggesting stressful stimuli stimulate ICP4 expression. New studies revealed GR and stress-induced transcription factors identified during reactivation from latency, SLUG and three Krüppel-like transcription factor family members (KLF4, KLF15, and promyelocytic leukemia zinc finger protein), cooperatively transactivate the ICP4 enhancer. Two KLF4 consensus binding sites were crucial for cooperative transactivation of the ICP4 enhancer. A consensus enhancer-box also mediated cooperative transactivation of the ICP4 enhancer by GR and SLUG. The ability of GR and stress-induced transcription factors to transactivate ICP4 enhancer activity is predicted to trigger productive infection following stressful stimuli.

E-cadherin, fibronectin and Slug immunoexpression in non-melanoma skin cancers.

Epithelial-mesenchymal transition (EMT) is an essential biological process involved in the initiation and progression of cancer by which epithelial tumor cells lose their differentiated characteristics, such as cell-cell adhesion and apical-basal polarity and acquire a more invasive and∕or metastatic mesenchymal phenotype. The present study investigated the expression of immunomarkers with a role in EMT of non-melanoma skin cancers (NMSCs), such as E-cadherin, fibronectin and Slug, for a number of 50 NMSCs, represented by 30 cases of basal cell carcinomas (BCCs) and 20 cases of squamous cell carcinomas (SCCs). For BCC, the statistical analysis of the investigated immunomarkers indicated significantly differences in relation to the depth of invasion, and for E-cadherin and fibronectin with the degree of risk. In the case of SCC, the statistical analysis indicated significant differences of E-cadherin and Slug with the degree of tumor differentiation, and for fibronectin and Slug with the depth of invasion. The analysis of the distribution for the percentage values of the investigated immunomarkers in the case of BCC indicated a significant negative linear relation between E-cadherin/fibronectin and E-cadherin/Slug, and in SCC a significant negative linear relation between E-cadherin∕fibronectin, E-cadherin∕Slug and a positive linear one in the case of fibronectin∕Slug. The study indicates through the statistically significant relation between E-cadherin∕fibronectin and E-cadherin∕Slug, the EMT intervention in carcinogenesis of NMSC.

Immunoexpression of Snail, Twist1 and Slug in clear cell renal cell carcinoma.

Clear cell renal cell carcinomas (ccRCC) represent about 80% of the malignant neoplasia with this localization. Snail, Twist1 and Slug are transcription factors and play a central role in the epithelial-mesenchymal transition (EMT), which is involved in the progression of renal cell carcinoma (RCC). In this study, we analyzed the immunoexpression of these transcription factors in 50 cases of ccRCC, in relation to histopathological aggressiveness parameters of the lesions. The results indicated the association of Snail and Twist1 expression with high Fuhrman grade, as well as the association of Slug expression with low Fuhrman grade. The immunoexpression of Snail and Twist1 was significantly superior for advanced stages and Slug was overexpressed in early stages of ccRCC. Our study supports the usefulness of the Snail, Twist1 and Slug expression for the appreciation of aggressiveness in ccRCC, the panel being attractive for targeted therapy.

Immunoexpression of E-cadherin, Snail and Twist in colonic adenocarcinomas.

Epithelial-mesenchymal transition (EMT) is an important mechanism in tumor progression. Snail is a transcription factor, expressed in cells which have undergone almost complete EMT and have left the tumor, and Twist is considered important in the process of metastasis, both playing a major role in EMT by indirect inhibition of E-cadherin. The study analyzed the immunoexpression of E-cadherin, Snail and Twist in 46 cases of colonic carcinomas in comparison with some histopathological prognostic factors. The quantification of reactions was done by using a composite score (CS) resulted from multiplying the percentage of marked cells with the intensity of immunostaining. The majority of cases were moderately differentiated tumors, corresponded to stage III, with vascular and perineural invasion. All cases presented positive cytoplasmic and nuclear signals for Snail and Twist. The immunostaining for both markers was intense, with the highest values of CS in G2 and G3 advanced, invasive vascular colonic carcinomas, in comparison with G1, early stage lesions. We found positive significant linear correlation of Snail and Twist expression. The results obtained indicate the implication of Snail and Twist in colonic carcinoma aggressiveness, useful aspect in the oncological evaluation of patients and guided therapy.

Neutrophils and Snail Orchestrate the Establishment of a Pro-tumor Microenvironment in Lung Cancer.

Understanding the immune compartment of tumors facilitates the development of revolutionary new therapies. We used a Kras(G12D)-driven mouse model of lung cancer to establish an immune signature and identified a contribution of Gr1+ neutrophils to disease progression. Depletion experiments showed that Gr1+ cells (1) favor tumor growth, (2) reduce T cell homing and prevent successful anti-PD1 immunotherapy, and (3) alter angiogenesis, leading to hypoxia and sustained Snail expression in lung cancer cells. In turn, Snail accelerated disease progression and increased intratumoral Cxcl2 secretion and neutrophil infiltration. Cxcl2 was produced mainly by neutrophils themselves in response to a factor secreted by Snail-expressing tumor cells. We therefore propose a vicious cycle encompassing neutrophils and Snail to maintain a deleterious tumor microenvironment.

Identification of Antibody Against SNRPB, Small Nuclear Ribonucleoprotein-Associated Proteins B and B', as an Autoantibody Marker in Crohn's Disease using an Immunoproteomics Approach.

BACKGROUND: Current non-invasive biomarkers for Crohn's disease are limited in their utility. Progress in identifying individual autoantigens and autoantibodies in Crohn's disease has been challenging due to limitations of available immunoassays. AIMS: Our aim was to identify autoantibodies associated with Crohn's disease that may be useful in diagnosis and management using an innovative protein array technology, namely nucleic acid programmable protein arrays [NAPPA]. METHODS: Serum samples of 96 patients with established Crohn's disease and 96 healthy controls were included and evenly split into discovery and validation sets randomly. Autoantibodies of both IgG and IgA classes were profiled against ~1900 human proteins in the discovery set on NAPPA. Autoantibodies discovered to be Crohn's disease-specific were further validated in the independent validation set by enzyme-linked immunosorbent assay. RESULTS: Overall, reactivity of IgG autoantibodies was stronger than that of IgA autoantibodies; however, IgA autoantibodies showed greater differential reactivity between cases and controls. Four IgA autoantibodies against SNRPB, PRPH, PTTG1 and SNAI1 were newly identified with sensitivities above 15% at 95% specificity, among which anti-SNRPB-IgA had the highest sensitivity of 24.0%. Autoantibodies associated with specific disease subtypes were also found. CONCLUSIONS: As one of the first studies to use immunoproteomics for the identification of autoantibodies in Crohn's disease, our results support the utility of NAPPA in implementing future expanded studies with better coverage of the human proteome and microbial proteomes relevant to Crohn's disease and identifying antibody markers that may have clinical impact in diagnosis and management.

TGF-ß induces M2-like macrophage polarization via SNAIL-mediated suppression of a pro-inflammatory phenotype.

Tumor-associated macrophages (TAMs) are a major component of leukocytic infiltrate in tumors, which facilitates tumor progression and promotes inflammation. TGF-ß promotes the differentiation of non-activated macrophages into a TAM-like (M2-like) phenotype; however, the underlying mechanisms are not clear. In this study, we found that TGF-ß induces a M2-like phenotype characterized by up-regulation of the anti-inflammatory cytokine IL-10, and down-regulation of the pro-inflammatory cytokines TNF-α and IL-12. In human THP-1 macrophages, overexpression of SNAIL caused M2-like differentiation by inhibiting pro-inflammatory cytokine release and promoting the expression of M2-specific markers. By contrast, SNAIL knockdown promoted M1 polarization through up-regulation of pro-inflammatory cytokines and abolished TGF-ß-mediated M2-polarization of THP-1 macrophages. The SMAD2/3 and PI3K/AKT signaling pathways were crucial for TGF-ß-induced SNAIL overexpression in THP-1 cells. These findings suggest that TGF-ß skews macrophage polarization towards a M2-like phenotype via SNAIL up-regulation, and blockade of TGF-ß/SNAIL signaling restores the production of pro-inflammatory cytokines. This study provides new understanding of the role of SNAIL in M2 polarization of macrophages, and suggests a potential therapeutic target for antitumor immunity.