Cisplatin­resistant germ cell tumor models: An exploration of the epithelial­mesenchymal transition regulator SLUG.

Germ cell tumors (GCTs) constitute diverse neoplasms arising in the gonads or extragonadal locations. Testicular GCTs (TGCTs) are the predominant solid tumors in adolescents and young men. Despite cisplatin serving as the primary therapeutic intervention for TGCTs, 10­20% of patients with advanced disease demonstrate resistance to cisplatin­based chemotherapy, and epithelial­mesenchymal transition (EMT) is a potential contributor to this resistance. EMT is regulated by various factors, including the snail family transcriptional repressor 2 (SLUG) transcriptional factor, and, to the best of our knowledge, remains unexplored within TGCTs. Therefore, the present study investigated the EMT transcription factor SLUG in TGCTs. In silico analyses were performed to investigate the expression of EMT markers in TGCTs. In addition, a cisplatin­resistant model for TGCTs was developed using the NTERA­2 cell line, and a mouse model was also established. Subsequently, EMT was assessed both in vitro and in vivo within the cisplatin­resistant models using quantitative PCR and western blot analyses. The results of the in silico analysis showed that the different histologies exhibited distinct expression profiles for EMT markers. Seminomas exhibited a lower expression of EMT markers, whereas embryonal carcinomas and mixed GCT demonstrated high expression. Notably, patients with lower SLUG expression had longer median progression­free survival (46.4 months vs. 28.0 months, P=0.022). In the in vitro analysis, EMT­associated genes [fibronectin; vimentin (VIM); actin, α2, smooth muscle; collagen type I α1; transforming growth factor­ß1; and SLUG] were upregulated in the cisplatin­resistant NTERA­2 (NTERA­2R) cell line after 72 h of cisplatin treatment. Consistent with this finding, the NTERA­2R mouse model demonstrated a significant upregulation in the expression levels of VIM and SLUG. In conclusion, the present findings suggested that SLUG may serve a crucial role in connecting EMT with the development of cisplatin resistance, and targeting SLUG may be a putative therapeutic strategy to mitigate cisplatin resistance.

Is epithelial-mesenchymal transition related to the biological behavior of salivary gland neoplasms?

OBJECTIVE: To evaluate and compare the expression of E-cadherin, Snail1 and Twist1 in pleomorphic adenomas (PAs), adenoid cystic carcinomas (AdCCa) and carcinoma ex-pleomorphic adenomas (CaexPA) of salivary glands, as well as investigate possible associations with clinicopathological parameters. STUDY DESIGN: E-cadherin, Snail1 and Twist1 antibody immunostaining were analyzed semiquantitatively in 20 PAs, 20 AdCCas and 10 CaexPAs. Cases were classified as low and high expression for analysis of the association with clinicopathological parameters. RESULTS: Compared to PAs, AdCCas and CaexPAs exhibited higher nuclear expression of Snail1 (p = 0.021 and p = 0.028, respectively) and Twist1 (p = 0.009 and p = 0.001). Membranous and cytoplasmic expression of E-cadherin were positively correlated in PAs, AdCCas and CaexPAs (r = 0.645, p = 0.002; r = 0.824, p < 0.001; r = 0.677, p = 0.031). In PAs, positive correlation was found between nuclear expression of Snail1 and membrane expression of E-cadherin (r = 0.634; p = 0.003), as well as between nuclear expression of Snail1 and Twist1 (r = 0.580; p = 0.007). Negative correlations were detected between membrane expression of E-cadherin and cytoplasmic expression of Snail1 in AdCCas (r = - 0.489; p = 0.029). CONCLUSIONS: E-cadherin, Twist1, and Snail1 may participate in modulating events related to cell differentiation and adhesion in PAs and to biological behavior in AdCCas and CaexPAs, which indicates the involvement of EMT in these processes. Furthermore, the expression of these proteins in these carcinomas may reflect the plasticity feature of EMT.

Low-dose cisplatin exposure and SNAIL, Vimentin, E-cadherin expression in HEPG2 cell line / Exposición a dosis bajas de cisplatino y expresión de SNAIL, Vimentina, E-cadherina en línea celular HEPG2

Cisplatin, the first platinum compound approved for cancer treatment, is widely used in the treatment of various cancers including hepatocellular carcinoma (HCC). HCC incidence rates rise globally. Epithelial mesenchymal transition (EMT) is implicated in cancer invasion and metastasis, which are associated with increased mortality. Cisplatin dose might influence cancer invasion and metastatic behavior of the cells. The aim of the study was to investigate the effect of low-dose cisplatin treatment on EMT- related changes in HepG2 cells. Following treatment with 4 µM cisplatin, HepG2 cells were evaluated morphologically. Gene expression of E-cadherin, Vimentin, Snail1 was assessed by quantitative PCR. Immunofluorescence analyses of NA-K ATPase were performed. Although the low-dose cisplatin treated cells exhibited a more stretched morphology, no statistical difference was detected in gene expression of E-cadherin, Vimentin, Snail1 and immunofluorescence of NA-K ATPase. Findings on low-dose cisplatin effects in HepG2 might contribute to the knowledge of antineoplastic inefficacy by further understanding the molecular mechanisms of drug action.

El cisplatino, el primer compuesto de platino aprobado para el tratamiento del cáncer, es ampliamente utilizado en el tratamiento de varios tipos de cáncer, incluido el carcinoma hepatocelular (CHC). Las tasas de incidencia de CHC aumentan a nivel mundial. La transición mesenquimal epitelial (EMT) está implicada en la invasión del cáncer y la metástasis, que se asocian con un aumento de la mortalidad. La dosis de cisplatino podría influir en la invasión del cáncer y el comportamiento metastásico de las células. El objetivo del estudio fue investigar el efecto del tratamiento con dosis bajas de cisplatino en los cambios relacionados con la EMT en las células HepG2. Tras el tratamiento con cisplatino de 4 µM, se evaluaron morfológicamente las células HepG2. La expresión génica de E-cadherina, vimentina, caracol1 se evaluó mediante PCR cuantitativa. Se realizaron análisis de inmunofluorescencia de NA-K ATPasa . Aunque las células tratadas con cisplatino en dosis bajas exhibieron una morfología más estirada, no se detectaron diferencias estadísticas en la expresión génica de E-cadherina, vimentina, Snail1 e inmunofluorescencia de NA-K ATPasa. Los hallazgos sobre los efectos del cisplatino en dosis bajas en HepG2 podrían contribuir al conocimiento de la ineficacia antineoplásica al comprender mejor los mecanismos moleculares de la acción del fármaco.

[Identification of epithelial-mesenchymal transition markers (EMT) by immunohistochemistry in pediatric osteosarcoma and association with clinical outcomes]. / Identificación de marcadores de transición epitelio-mesénquima (TEM) por inmunohistoquímica en osteosarcoma pediátrico y asociación con desenlaces clínicos.

The epithelial-mesenchymal transition (EMT) is the ability of epithelial and mesenchymal cells to exchange phenotypes transiently. Its identification in carcinomatous cells has been associated with aggressive clinical phenotypes. In sarcomas, this ability is under study. OBJECTIVE: to evaluate the expression of two transcription factors involved in EMT by immunohistochemistry in pediatric osteosarcoma and its association with clinical outcomes. PATIENTS AND METHOD: A retrospective cohort study in children under 18 years of age with osteosarcoma diagnosis. Immunohistochemistry was performed for Snail and Twist-1 expressions from samples collected at the time of diagnosis. Correlations between immunohistochemistry and the clinical outcomes and overall survival were performed. RESULTS: 53 patients were included. There were 26 positive cytoplasmic cases (49.1%) in Snail expression and were correlated with the presence of multiple metastases (p = 0.02) and distant bone metastases (p = 0.01). On the other hand, 45 cases (84.9%) were positive in Twist-1 expression in the nuclear location, showing no association with the analyzed clinical variables. CONCLUSIONS: Snail and Twist-1 were frequently expressed in pediatric cases of osteosarcoma. Cytoplasmic Snail was correlated with the presence of multiple metastatic disease and distant bone metastases. The positivity of both markers suggests the activation of these proteins as regulators of EMT events in this tumor, suggesting a role in the phenomena related to the clinical presentation of the disease.

The Transcription Factors Zeb1 and Snail Induce Cell Malignancy and Cancer Stem Cell Phenotype in Prostate Cells, Increasing Androgen Synthesis Capacity and Therapy Resistance.

Prostate cancer (PCa) incidence has increased during the last decades, becoming one of the leading causes of death by cancer in men worldwide. During an extended period of prostate cancer, malignant cells are androgen-sensitive being testosterone the main responsible for tumor growth. Accordingly, treatments blocking production and action of testosterone are mostly used. However, during disease progression, PCa cells become androgen insensitive producing a castration-resistant stage with a worse prognosis. Overcoming castration-resistant prostate cancer (CRPC) has become a great challenge in the management of this disease. In the search for molecular pathways leading to therapy resistance, the epithelial-mesenchymal transition (EMT), and particularly the transcription factors zinc finger E-box-binding homeobox 1 (Zeb1) and zinc finger protein SNAI1 (Snail), master genes of the EMT, have shown to have pivotal roles. Also, the discovery that cancer stem cells (CSCs) can be generated de novo from their non-CSCs counterpart has led to the question whereas these EMT transcription factors could be implicated in this dynamic conversion between non-CSC and CSC. In this review, we analyze evidence supporting the idea that Zeb1 and Snail induce cell malignancy and cancer stem cell phenotype in prostate cells, increasing androgen synthesis capacity and therapy resistance.

Silencing IKBKE inhibits the migration and invasion of glioblastoma by promoting Snail1 degradation.

PURPOSE: Glioblastoma multiforme (GBM) is one of the most common malignant brain tumors in adults and has high mortality and relapse rates. Over the past few years, great advances have been made in the diagnosis and treatment of GBM, but unfortunately, the five-year overall survival rate of GBM patients is approximately 5.1%. Inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKBKE) is a major oncogenic protein in tumors and can promote evil development of GBM. Snail1, a key inducer of the epithelial-mesenchymal transition (EMT) transcription factor, is subjected to ubiquitination and degradation, but the mechanism by which Snail1 is stabilized in tumors remains unclear. Our study aimed to investigate the mechanism of IKBKE regulating Snail1 in GBM. METHODS: First, we analyzed the correlation between the expression of IKBKE and the tumor grade and prognosis through public databases and laboratory specimen libraries. Second, immunohistochemistry (IHC) and western blot were used to detect the correlation between IKBKE and Snail expression in glioma samples and cell lines. Western blot and immunofluorescence (IF) experiments were used to detect the quality and distribution of IKBKE and Snail1 proteins. Third, In situ animal model of intracranial glioma to detect the regulatory effect of IKBKE on intracranial tumors. RESULTS: In this study, Our study reveals a new connection between IKBKE and Snail1, where IKBKE can directly bind to Snail1, translocate Snail1 into the nucleus from the cytoplasm. Downregulation of IKBKE results in Snail1 destabilization and impairs the tumor cell migration and invasion capabilities. CONCLUSION: Our studies suggest that the IKBKE-Snail1 axis may serve as a potential therapeutic target for GBM treatment.

Imunoexpressão das proteínas e-caderina, snail1 e vimentina em neoplasias de glândula salivar / Immunoexpression of e-cadherin, snail1 and vimentin proteins in salivary gland neoplasms

Os tumores de glândula salivar (TGS) representam cerca de 3% a 6% das neoplasias da região de cabeça e pescoço e são caracterizados por sua diversidade morfológica e de comportamentos biológicos. Sabe-se que algumas das características de tumores malignos, como a invasão tumoral e metástases à distância, possuem envolvimento da transição epitélio-mesênquima (TEM), evento no qual proteínas como a E-caderina, Snail1 e Vimentina estão diretamente envolvidas. Esta pesquisa se propôs a analisar e relacionar a expressão imuno-histoquímica dessas proteínas com as características clínico-patológicas em adenomas pleomórficos (APs), carcinomas adenoides císticos (CACs) e carcinomas ex-adenomas pleomórficos (CaExAPs) de glândulas salivares maiores e menores. A análise da imunoexpressão dessas proteínas foi feita de forma semiquantitativa em 20 casos de APs, 20 de CACs e 10 de CaExAPs. Na avaliação de E-caderina, considerou-se a expressão em membrana e/ou citoplasma das células do parênquima tumoral. Para a Snail1, foi considerada a expressão nos compartimentos nuclear e/ou citoplasmático dessas células. A Vimentina foi analisada no citoplasma de células fusiformes presentes no estroma dos TGS. Os dados obtidos foram comparados e correlacionados utilizando o nível de significância de 5% (p ≤ 0,05). Observou-se imunopositividade para E-caderina principalmente em citoplasma das células neoplásicas nãoluminais; a marcação membranar, perceptível em células luminais, estava mais presente nas neoplasias malignas (p = 0,041). A expressão de Snail1 foi mais frequente no compartimento nuclear, sendo mais evidenciada em células não-luminais dos TGS e apresentou maior reatividade nuclear em tumores malignos (p = 0,012). A expressão nuclear dessa proteína também foi maior para CACs e CaExAPs ao compará-los, de forma separada, com os APs (p = 0,037 e p = 0,025, respectivamente). Não foram observadas diferenças estatisticamente significativas entre a expressão de E-caderina e Snail1 e outros parâmetros clinico-patológicos e os subtipos histopatológicos das lesões (p > 0,05). A positividade para Vimentina foi vista no estroma de todos os casos de TGS, sendo mais difusa e intensa em CACs. Não foram verificadas diferenças estatisticamente significativas entre a expressão desse biomarcador e os parâmetros clínico-patológicos e subtipos histopatológicos das lesões (p > 0,05). Houve correlações positivas entre as expressões membranar e citoplasmática de E-caderina em APs, CACs e CaExAPs (r = 0,645, p = 0,002; r = 0,781, p < 0,001; r = 0,677 p = 0,031), bem como entre a expressão nuclear e citoplasmática de Snail1 e a expressão citoplasmática de E-caderina e nuclear de Snail1 em APs (r = 0,569, p = 0,009; r = 0,481, p = 0,032). Foram verificadas correlações negativas entre a expressão membranar de E-caderina e citoplasmática de Snail1, bem como entre Snail1 nuclear e Vimentina em CACs (r = -0,471, p = 0,036; r = -0,514; p = 0,021). Essa última correlação, bem como correlação positiva entre a expressão membranar e citoplasmática de E-caderina, também foi vista entre os casos de CACs e CaExAPs quando agrupados (r = -0,457; p = 0,0011; r = 0,746, p < 0,001). Os resultados do presente estudo sugerem que a participação de E-caderina e Snail1 no processo da TEM pode estar relacionada ao estágio de diferenciação celular em APs e à progressão tumoral nas neoplasias malignas bem como é possível que a expressão dessas proteínas em neoplasias malignas seja indicativa da plasticidade presente na TEM. Ressalta-se, também, a expressão de Vimentina em células fusiformes presentes no estroma tumoral, provavelmente refletindo estágios tardios da TEM (AU).

Salivary gland tumors (SGTs) comprise about 2% to 10% of head and neck tumors and are known for their morphologic diversity and biologic behavior. Some features of malignant neoplasms, such as tumor invasion and distant metastasis, might have the participation of epithelial-mesenchymal transition (EMT), event in which proteins like E-cadherin, Vimentin and Snail1 are directly involved. This study aimed to analyze, by means of immunohistochemistry, the expression of these proteins, as well as to relate their expressions to clinical-pathological features of pleomorphic adenomas (PAs), adenoid cystic carcinomas (ACCs) and carcinomas ex-pleomorphic adenomas (CXPAs) located in minor and major salivary glands. This was a semi-quantitatively analysis which comprised 20 PAs, 20 ACCs and 10 CXPAs. Analysis of E-cadherin was made considering membranar and/or cytoplasmatic expression in parenchymal cells. For Snail1, it was considered the positivity in nucleus and/or cytoplasm of parenchymal cells. Vimentin was evaluated in the cytoplasm of fusiform stromal cells. Data were compared and correlated adopting a level of significance of 5% (p ≤ 0,05). Marked immunoexpression for E-cadherin was mostly found in the cytoplasm of non-luminal neoplastic cells in SGTs; membrane reaction for the protein, seen in luminal cells, was higher in malignant tumors (p = 0,041). Snail1 was more expressed in the nucleus, mostly of nonluminal cells of SGTs, with this reactivity being higher in malignant tumors(p = 0,012). Nuclear positivity for this marker was also higher for ACCs and CXPAs when compared with PAs separately (p = 0,037 e p = 0,025, respectively). No significant differences between E-cadherin and Snail1 and other clinical-pathological parameters were found (p > 0,05). Vimentin was seen in the stroma of all cases, being more diffuse and intense in ACCs. No significant differences between this marker and clinical-pathological parameters were found (p > 0,05). Positive correlations between membranar and cytoplasmatic E-cadherin in PAs, ACCs and CXPAs were observed (r = 0,645, p = 0,002; r = 0,781, p < 0,001; r = 0,677 p = 0,031), as well as between nuclear and cytoplasmatic Snail1 and between cytoplasmatic E-cadherin and nuclear Snail1 in PAs APs (r = 0,569, p = 0,009; r = 0,481, p = 0,032). Negative correlations between membranar E-cadherin and cytoplasmatic Snail1 were observed, as well as between nuclear Snail1 and Vimentin in ACCs (r = -0,471, p = 0,036; r = -0,514; p = 0,021). This last correlation and a positive correlation between membranar and cytoplasmatic E-cadherin were also seen when ACCs and CXPAs were grouped (r = -0,457; p = 0,0011; r = 0,746, p < 0,001). These results suggest that the participation of these proteins in EMT might be related to cellular differentiation in PAs and to tumoral progression in malignant tumors. In addition, it can be infered that the expression of E-cadherin and Snail1 in malignant tumors might reflect the plasticity seen in EMT process. Furthermore, the expression of Vimentin in fusiform stromal cells, probably in later stages of EMT, in the stroma of SGTs, is highlighted (AU).

The Nek2 centrosome-mitotic kinase contributes to the mesenchymal state, cell invasion, and migration of triple-negative breast cancer cells.

Nek2 (NIMA-related kinase 2) is a serine/threonine-protein kinase that localizes to centrosomes and kinetochores, controlling centrosome separation, chromosome attachments to kinetochores, and the spindle assembly checkpoint. These processes prevent centrosome amplification (CA), mitotic dysfunction, and chromosome instability (CIN). Our group and others have suggested that Nek2 maintains high levels of CA/CIN, tumor growth, and drug resistance. We identified that Nek2 overexpression correlates with poor survival of breast cancer. However, the mechanisms driving these phenotypes are unknown. We now report that overexpression of Nek2 in MCF10A cells drives CA/CIN and aneuploidy. Besides, enhanced levels of Nek2 results in larger 3D acinar structures, but could not initiate tumors in a p53+/+ or a p53-/- xenograft model. Nek2 overexpression induced the epithelial-to-mesenchymal transition (EMT) while its downregulation reduced the expression of the mesenchymal marker vimentin. Furthermore, either siRNA-mediated downregulation or INH6's chemical inhibition of Nek2 in MDA-MB-231 and Hs578t cells showed important EMT changes and decreased invasion and migration. We also showed that Slug and Zeb1 are involved in Nek2 mediated EMT, invasion, and migration. Besides its role in CA/CIN, Nek2 contributes to breast cancer progression through a novel EMT mediated mechanism.

Exosome-transmitted lncRNA PCGEM1 promotes invasive and metastasis in gastric cancer by maintaining the stability of SNAI1.

PURPOSE: Clinically, hypoxia is associated with increased distant metastasis and poor survival in gastric cancer (GC). In this study, we set out from the cellular interaction to further explain the molecular mechanism of invasion in GC cells under hypoxic conditions. METHODS: Gastric cancer cells were cultured under 1% O2 (hypoxia-cultured gastric cancer cells, HGC) and 20% O2 condition (normoxic-cultured gastric cancer cells, NGC). NGC was co-cultured with HGC-medium. Scrape and Transwell were used to evaluate invasion and migration. Exosomes from GC were extracted by ultracentrifugation. Electron microscopy images, western-blot used to analyze the size distributions and the number of exosomes. RESULTS: HGC-medium induced NGC dissociated. Long non-coding RNA (lncRNA) prostate cancer gene expression marker 1 (PCGEM1) was specifically expressed in HGC exosomes. HGC-derived PCGEM1-riched exosomes could promote the invasion and migration of NGC. On the mechanism, PCGEM1 maintained stability and reduced the degradation of SNAI1, which could induce the epithelial-mesenchymal transition of GC. CONCLUSION: LncRNA PCGEM1 was overexpressed in GC cells. And part of the PCGEM1 can be encapsulated into exosomes. These exosomes promoted invasion and migration of other GC cells. We considered PCGEM1 might act as a "scaffold" combined with SNAI1 and prompt the invasion and migration of GC.

miR-143-3p inhibits proliferation and invasion of hepatocellular carcinoma cells by regulating its target gene FGF1.

PURPOSE: To explore FGF1 and miR-143-3p expression in hepatocellular carcinoma (HCC) cells and its related mechanisms. METHODS: Eighty-two HCC patients treated at our hospital from January 2018 to January 2019 were enrolled as Group A, while further 80 healthy people undergoing physical examinations during the same time period were enrolled as Group B. HCC cells and normal human liver cells were purchased, with HepG2 and SMMC-7721 cells transfected with pcDNA3.1-FGF1, si-FGF1, NC, miR-143-3p-inhibitor and miR-143-3p-mimics. FGF1 and miR-143-3p expression was detected by qRT-PCR. The expression of N-cadherin, vimentin, Snail, Slug, E-cadherin and γ-catenin was detected by Western Blotting (WB). Cell proliferation was detected by MTT assay. Cell invasion was detected by Transwell. Cell apoptosis was detected by flow cytometry (FCM). RESULTS: FGF1 was highly expressed but miR-143-3p was poorly expressed in HCC cells. Areas under the curves (AUCs) of the two indicators were > 0.8. The indicators were correlated with the age, gender, tumor invasion, degree of differentiation, tumor location and TNM staging of the patients. Silencing FGF1 and overexpressing miR-143-3p could promote cell apoptosis, inhibit cell growth, cell epithelial-mesenchymal transition (EMT) and the expression of N-cadherin, vimentin, Snail and Slug, and increase the expression of E-cadherin and γ-catenin. Dual luciferase reporter gene assay (DLRGA) confirmed that FGF1 and miR-143-3p had a targeted relationship. The rescue experiment showed that the proliferation, invasion and apoptosis of HepG2 and SMMC-7721 cells in the miR-143-3p-mimics+pcDNA3.1-FGF1 and miR-143-3p-inhibitor+Si-FGF1 groups were not different from those in the miR-NC group. CONCLUSION: Inhibiting FGF1 can upregulate miR-143-3p-mediated Hedgehog signaling pathway, and affect cells' EMT, proliferation and invasion, so FGF1 is expected to become a potential therapeutic target for HCC.

Turnera diffusa extract attenuates profibrotic, extracellular matrix and mitochondrial markers in activated human hepatic stellate cells (HSC).

INTRODUCTION AND OBJECTIVES: Hepatic fibrosis is characterized by the accumulation of extracellular matrix which includes the accumulation of α-smooth muscle actin (α-SMA), collagen type I (COL1α1), as well as remodeling induced by metalloproteinases and tissue inhibitor of metalloproteinase (TIMPs), where hepatic stellate cells (HSCs) play a central role. In addition, the transcription factor SNAI1 (which participates in epithelial-mesenchymal transition, EMT) and mitofusin 2 (MFN2, a mitochondrial marker) plays an important role in chronic liver disease. Turnera diffusa (TD), a Mexican endemic plant, has been shown to possess antioxidant and hepatoprotective activity in vitro. We treated human HSC (LX2 cells) with a methanolic extract of Turnera diffusa (METD) to evaluate the mechanism involved in its hepatoprotective effect measured as fibrosis modulation, EMT, and mitochondrial markers. MATERIALS AND METHODS: HSC LX-2 cells were treated with METD (100 and 200ng/mL) alone or combined with TGF-ß (10ng/mL) at different time points (24, 48, and 72h). α-SMA, COL1α1, MMP2, TIMP1, SNAI1, and MFN2 mRNAs and protein levels were determined by real-time quantitative PCR and Western Blot analysis. RESULTS: We found that METD decreases COL1α1-mRNA, α-SMA, and TIMP1 protein expression in LX2 cells treated with and TGF-ß. This treatment also decreases MFN2 and TIMP1 protein expression and induces overexpression of MMP2-mRNA. CONCLUSIONS: Our results suggest that a methanolic extract of Turnera diffusa is associated with an antifibrotic effect by decreasing profibrotic and mitochondrial markers together with the possible induction of apoptosis through SNAI1 expression in activated HSC cells.

ZEB and Snail expression indicates epithelial-mesenchymal transition in canine melanoma.

Melanoma progression is associated with the epithelial-mesenchymal transition (EMT) when tumor cells reduce E-cadherin and increase N-cadherin expression resulting in an escape from the microenvironment via loss of cellular adhesion and gain of motility. Transcription factor proteins Snail and ZEB trigger EMT by repression of epithelial markers and activation of mesenchymal properties. This study evaluated E-cadherin, N-cadherin, Snail, ZEB1 and ZEB2 expression by IHC and investigated their relationship with morphological characteristics in cutaneous and oral canine melanoma. Results from melanoma cases demonstrated E-cadherin expression in 45% (9/20) of oral and 58% (22/38) of cutaneous tumors, while N-cadherin expression was observed in 95% (18/19) of oral and 92% (34/37) of cutaneous melanoma. Cytoplasmic and nuclear N-cadherin expression was positively correlated with ZEB1 expression, while the cell membrane N-cadherin expression was positively correlated with ZEB2. In addition, an increase in nuclear N-cadherin expression was associated with reduced Snail expression in cutaneous melanoma and an increase in Snail expression in oral melanoma, indicating that the correlation between N-cadherin and Snail expression is coincident with tumor location. Our data suggest that ZEB family protein is associated with N-cadherin translocation from cell membrane to the cytoplasm and nuclei, and may act as important transcription factors of EMT regulation in canine melanoma.

LOXL3 Function Beyond Amino Oxidase and Role in Pathologies, Including Cancer.

Lysyl oxidase like 3 (LOXL3) is a copper-dependent amine oxidase responsible for the crosslinking of collagen and elastin in the extracellular matrix. LOXL3 belongs to a family including other members: LOX, LOXL1, LOXL2, and LOXL4. Autosomal recessive mutations are rare and described in patients with Stickler syndrome, early-onset myopia and non-syndromic cleft palate. Along with an essential function in embryonic development, multiple biological functions have been attributed to LOXL3 in various pathologies related to amino oxidase activity. Additionally, various novel roles have been described for LOXL3, such as the oxidation of fibronectin in myotendinous junction formation, and of deacetylation and deacetylimination activities of STAT3 to control of inflammatory response. In tumors, three distinct roles were described: (1) LOXL3 interacts with SNAIL and contributes to proliferation and metastasis by inducing epithelial-mesenchymal transition in pancreatic ductal adenocarcinoma cells; (2) LOXL3 is localized predominantly in the nucleus associated with invasion and poor gastric cancer prognosis; (3) LOXL3 interacts with proteins involved in DNA stability and mitosis completion, contributing to melanoma progression and sustained proliferation. Here we review the structure, function and activity of LOXL3 in normal and pathological conditions and discuss the potential of LOXL3 as a therapeutic target in various diseases.

GDF11 exhibits tumor suppressive properties in hepatocellular carcinoma cells by restricting clonal expansion and invasion.

Growth differentiation factor 11 (GDF11) has been characterized as a key regulator of differentiation in cells that retain stemness features, despite some controversies in age-related studies. GDF11 has been poorly investigated in cancer, particularly in those with stemness capacity, such as hepatocellular carcinoma (HCC), one of the most aggressive cancers worldwide. Here, we focused on investigating the effects of GDF11 in liver cancer cells. GDF11 treatment significantly reduced proliferation, colony and spheroid formation in HCC cell lines. Consistently, down-regulation of CDK6, cyclin D1, cyclin A, and concomitant upregulation of p27 was observed after 24 h of treatment. Interestingly, cell viability was unchanged, but cell functionality was compromised. These effects were potentially induced by the expression of E-cadherin and occludin, as well as Snail and N-cadherin repression, in a time-dependent manner. Furthermore, GDF11 treatment for 72 h induced that cells were incapable of sustaining colony and sphere capacity in the absent of GDF11, up to 5 days, indicating that the effect of GDF11 on self-renewal capacity is not transient. Finally, in vivo invasion studies revealed a significant decrease in cell migration of hepatocellular carcinoma cells treated with GDF11 associated to a decreased proliferation judged by Ki67 staining. Data show that exogenous GDF11 displays tumor suppressor properties in HCC cells.

Sphingosine kinase and sphingosine-1-phosphate regulate epithelial cell architecture by the modulation of de novo sphingolipid synthesis.

Sphingolipids regulate several aspects of cell behavior and it has been demonstrated that cells adjust their sphingolipid metabolism in response to metabolic needs. Particularly, sphingosine-1-phosphate (S1P), a final product of sphingolipid metabolism, is a potent bioactive lipid involved in the regulation of various cellular processes, including cell proliferation, cell migration, actin cytoskeletal reorganization and cell adhesion. In previous work in rat renal papillae, we showed that sphingosine kinase (SK) expression and S1P levels are developmentally regulated and control de novo sphingolipid synthesis. The aim of the present study was to evaluate the participation of SK/S1P pathway in the triggering of cell differentiation by external hypertonicity. We found that hypertonicity evoked a sharp decrease in SK expression, thus activating the de novo sphingolipid synthesis pathway. Furthermore, the inhibition of SK activity evoked a relaxation of cell-cell adherens junction (AJ) with accumulation of the AJ complex (E-cadherin/ß-catenin/α-catenin) in the Golgi complex, preventing the acquisition of the differentiated cell phenotype. This phenotype alteration was a consequence of a sphingolipid misbalance with an increase in ceramide levels. Moreover, we found that SNAI1 and SNAI2 were located in the cell nucleus with impairment of cell differentiation induced by SK inhibition, a fact that is considered a biochemical marker of epithelial to mesenchymal transition. So, we suggest that the expression and activity of SK1, but not SK2, act as a control system, allowing epithelial cells to synchronize the various branches of sphingolipid metabolism for an adequate cell differentiation program.

Elevated expression of GNAS promotes breast cancer cell proliferation and migration via the PI3K/AKT/Snail1/E-cadherin axis.

PURPOSE: Although it has been well established that G protein plays pivotal roles in physiologic or pathologic conditions, including cancer formation, its role in breast cancer, especially specific subunits, remains largely unknown. Our work aimed to evaluate the correlation of the G protein alpha subunit (GNAS) with breast cancer and to investigate the underlying molecular mechanism. METHODS: The expression of GNAS was determined by breast tumor tissue microarray of 150 patients with complete follow-up information. The correlation between GNAS expression and clinical features was assessed. CCK8, EdU incorporation, flow cytometry, wound healing, transwell, western blot and tumor formation assays were carried out in nude mice to study the biological function of GNAS and the underlying molecular mechanism in breast cancer by silencing GNAS using a specific siRNA. RESULTS: High GNAS expression showed a close correlation with a reduced overall survival (p = 0.021), frequent distal metastasis (p = 0.026), advanced clinical stage (p = 0.001), stronger cell proliferation (ki67+ positive cell rate, p = 0.0351) and enhanced cancer cell migration, which was further confirmed by in vitro and in vivo assays and might be dependent on the PI3K/AKT/Snail1/E-cadherin axis. CONCLUSION: The data suggested that GNAS promoted breast cancer cell proliferation and migration (EMT) through the PI3K/AKT/Snail1/E-cadherin signaling pathway. These findings also indicate that GNAS can serve as a potential prognostic indicator and novel therapeutic target in breast cancer.

Hypoxia-induced LncRNA PCGEM1 promotes invasion and metastasis of gastric cancer through regulating SNAI1.

PURPOSE: Hypoxia is an indispensable factor in the progression of metastasis. Hypoxia inducible factor-1α (HIF-1α), the core element in generating the hypoxia response, induces invasion and metastasis by promoting epithelial-mesenchymal transition (EMT). This study explored the underlying mechanism of hypoxia associated with the invasion and metastasis of gastric cancer (GC). METHODS: Six methods were employed to assess the function of the long noncoding RNA (lncRNA) prostate cancer gene expression marker 1 (PCGEM1) including gene silencing, RT-PCR, the separation of nuclear and cytoplasmic fractions, scrape motility assay, transwell migration assay, and Western-blot. RESULTS: LncRNA PCGEM1 was overexpressed in GC cells and tissues, and was induced by hypoxia in GC cells. Additional experiments confirmed that the knockdown of PCGEM1 significantly repressed the invasion and metastasis of GC cells. SNAI1, a key transcription factor of EMT, was regulated by PCGEM1. Overexpression of SNAI1 rescued the inhibition of PCGEM1-knockdown during the invasion and metastasis of GC cells. In addition, PCGEM1 and SNAI1 jointly affected the biomarkers of EMT. CONCLUSION: Our findings indicated that PCGEM1 is a hypoxia-responsive lncRNA, and contributes to the invasion and metastasis of GC. The potential mechanism is attributed to the regulation of EMT by PCGEM1 and its influence on the expression of SNAI1.

TRPM4 channel is involved in regulating epithelial to mesenchymal transition, migration, and invasion of prostate cancer cell lines.

Transient Receptor Potential Melastatin 4 (TRPM4) is a Ca2+ -activated and voltage-dependent monovalent cation channel, which depolarizes the plasma cell membrane, thereby modulating Ca2+ influx across Ca2+ -permeable pathways. TRPM4 is involved in different physiological processes such as T cell activation and the migration of endothelial and certain immune cells. Overexpression of this channel has been reported in various types of tumors including prostate cancer. In this study, a significant overexpression of TRPM4 was found only in samples from cancer with a Gleason score higher than 7, which are more likely to spread. To evaluate whether TRPM4 overexpression was related to the spreading capability of tumors, TRPM4 was knockdown by using shRNAs in PC3 prostate cancer cells and the effect on cellular migration and invasion was analyzed. PC3 cells with reduced levels of TRPM4 (shTRPM4) display a decrease of the migration/invasion capability. A reduction in the expression of Snail1, a canonical epithelial to mesenchymal transition (EMT) transcription factor, was also observed. Consistently, these cells showed a significant change in the expression of key EMT markers such as MMP9, E-cadherin/N-cadherin, and vimentin, indicating a partial reversion of the EMT process. Whereas, the overexpression of TRPM4 in LnCaP cells resulted in increased levels of Snail1, reduction in the expression of E-cadherin and increase in their migration potential. This study suggests a new and indirect mechanism of regulation of migration/invasion process by TRPM4 in prostate cancer cells, by inducing the expression of Snail1 gene and consequently, increasing the EMT.

ZEB1 and ZEB2 transcription factors are potential therapeutic targets of canine mammary cancer cells.

Mammary tumours are the most frequent in female dogs as in women and half are malignant. Tumorigenicity and invasiveness are important acquired characteristics for the development and progression of cancers and could be regulated by transcription factors associated with epithelial-mesenchymal transition (EMT) as ZEB1, ZEB2, SNAI1, SLUG and STAT3. Thus, here, we evaluate the expression of EMT-associated transcription factors in canine mammary cancer (CMC) cell lines characterized for invasiveness and tumorigenicity to determine if these could be considered good targets for future development of therapies. Five CMC cell lines were characterized regarding their morphology, doubling time and expression of intermediate and actin filaments. In addition, gene expression of SLUG, STAT3, ZEB1, ZEB2 and CDH1, tumorigenicity and invasiveness were assessed. Two of these cells presented an epithelial-like morphology (E20 and E37) and three a mesenchymal-like morphology (M5, M25 and CF41.Mg). M25 and CF41.Mg presented higher invasiveness. Furthermore, only mesenchymal-like cells formed tumorspheres and CF41.Mg made more and larger tumorspheres. The mesenchymal-like cells are more malignant than the epithelial-like cells being the CF41.Mg the most malignant. This cell presented higher ZEB1 and ZEB2 and lower CDH1 gene expression. Finally, our results revealed that there is a positive correlation between ZEBs and the tumorsphere number and size. In conclusion, these findings support ZEB1 and ZEB2 as potential therapeutic targets for CMC cells, demonstrating a great potential of canine models for comparative and translational studies.

Subcellular localization and expression of E-cadherin and SNAIL are relevant since early stages of oral carcinogenesis.

The biological process of epithelial-to-mesenchymal transition (EMT) has been studied in oral squamous cell carcinoma (OSCC) metastasis, but it is rarely evaluated at several stages of oral carcinogenesis. This study aimed to analyze the presence of SNAIL and E-cadherin proteins, markers of EMT, in the development and progression of OSCC, evaluating excised specimens of potentially malignant lesions (oral leukoplakia with and without dysplasia-OL and OLD, respectively), tumor tissues (OSCC), metastatic lymph nodes (LN), and normal oral mucosa (NOM) by immunohistochemistry, considering subcellular localization. Additionally, SNAIL and E-cadherin transcripts were evaluated in vitro by qPCR, using SCC-9 cell line in comparison to human keratinocytes (HPEC). There was a significant increase in nuclear expression of SNAIL from NOM to OLD followed by a noticeable decrease in nuclear expression accompanied by increased cytoplasmic expression in OSCC (p<0.05). The E-cadherin cytoplasmic expression was remarkable and statistically significant higher in OSCC and LN, both compared to NOM (p< 0.0001), OL (p<0.01) and OLD (p< 0.0001 and p<0.001, respectively). In vitro, E-cadherin and SNAIL transcripts were lower in SCC-9 compared to HPEC cells, although only the decrease of E-cadherin was statistically significant (p<0.05). Regarding the association of E-cadherin and SNAIL expression with the clinical findings, the analysis revealed an association between the cytoplasmic expression of SNAIL and the invasion pattern (p=0.05) in OSCC. The increased nuclear SNAIL expression may be characteristic of OLD, and the presence of E-cadherin in cell cytoplasm a marker of transformation to malignancy of potentially malignant oral leukoplakias into OSCC.