RESUMEN

BACKGROUND: ZEB1, a core transcription factor involved in epithelial-mesenchymal transition (EMT), is associated with aggressive cancer cell behavior, treatment resistance, and poor prognosis across various tumor types. Similarly, the expression and activity of CD73, an ectonucleotidase implicated in adenosine generation, is an important marker of tumor malignancy. Growing evidence suggests that EMT and the adenosinergic pathway are intricately linked and play a pivotal role in cancer development. Therefore, this study focuses on exploring the correlations between CD73 and ZEB1, considering their impact on tumor progression. METHODS: We employed CRISPR/Cas9 technology to silence CD73 expression in cell lines derived from papillary thyroid carcinoma. These same cells underwent lentiviral transduction of a reporter of ZEB1 non-coding RNA regulation. We conducted studies on cell migration using scratch assays and analyses of cellular speed and polarity. Additionally, we examined ZEB1 reporter expression through flow cytometry and immunocytochemistry, complemented by Western blot analysis for protein quantification. For further insights, we applied gene signatures representing different EMT states in an RNA-seq expression analysis of papillary thyroid carcinoma samples from The Cancer Genome Atlas. RESULTS: Silencing CD73 expression led to a reduction in ZEB1 non-coding RNA regulation reporter expression in a papillary thyroid carcinoma-derived cell line. Additionally, it also mitigated ZEB1 protein expression. Moreover, the expression of CD73 and ZEB1 was correlated with alterations in cell morphology characteristics crucial for cell migration, promoting an increase in cell polarity index and cell migration speed. RNA-seq analysis revealed higher expression of NT5E (CD73) in samples with BRAF mutations, accompanied by a prevalence of partial-EMT/hybrid state signature expression. CONCLUSIONS: Collectively, our findings suggest an association between CD73 expression and/or activity and the post-transcriptional regulation of ZEB1 by non-coding RNA, indicating a reduction in its absence. Further investigations are warranted to elucidate the relationship between CD73 and ZEB1, with the potential for targeting them as therapeutic alternatives for cancer treatment in the near future.

Asunto(s)

Neoplasias de la Tiroides , Factores de Transcripción , Humanos , Cáncer Papilar Tiroideo , Línea Celular Tumoral , Factores de Transcripción/genética , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , ARN no Traducido , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética

RESUMEN

OBJECTIVES: Nasopharyngeal carcinoma (NPC) is an aggressive epithelial cancer. The expression of miR-186 is decreased in a variety of malignancies and can promote the invasion and metastasis of cancer cells. This study aimed to explore the role and possible mechanism of miR-186 in the metastasis and epithelial-mesenchymal transformation (EMT) of NPC. METHODS: The expression of miR-186 in NPC tissues and cells was detected by RT-PCR. Then, miR-186 mimic was used to transfect NPC cell lines C666-1 and CNE-2, and cell activity, invasion and migration were detected by CCK8, transwell and scratch assay, respectively. The expression of EMT-related proteins was analyzed by western blotting analysis. The binding relationship between miR-186 and target gene Zinc Finger E-Box Binding Homeobox 1 (ZEB1) was confirmed by double luciferase assay. RESULTS: The expression of miR-186 in NPC was significantly decreased, and transfection of miR-186 mimic could significantly inhibit the cell activity, invasion, and migration, and regulate the protein expressions of E-cadherin, N-cadherin and vimentin in C666-1 and CNE-2 cells. Further experiments confirmed that miR-186 could directly target ZEB1 and negatively regulate its expression. In addition, ZEB1 has been confirmed to be highly expressed in NPC, and inhibition of ZEB1 could inhibit the activity, invasion, metastasis and EMT of NPC cells. And co-transfection of miR-186 mimic and si-ZEB1 could further inhibit the proliferation and metastasis of NPC. CONCLUSION: miR-186 may inhibit the proliferation, metastasis and EMT of NPC by targeting ZEB1, and the miR-186/ZEB1 axis plays an important role in NPC.

Asunto(s)

Carcinoma , MicroARNs , Neoplasias Nasofaríngeas , Humanos , Carcinoma Nasofaríngeo/patología , MicroARNs/genética , MicroARNs/metabolismo , Línea Celular Tumoral , Carcinoma/genética , Carcinoma/patología , Transición Epitelial-Mesenquimal/genética , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patología , Regulación Neoplásica de la Expresión Génica/genética , Proliferación Celular , Invasividad Neoplásica/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo

RESUMEN

Cancer biologists have focused on studying cancer stem cells (CSCs) because of their ability to self-renew and recapitulate tumor heterogeneity, which increases their resistance to chemotherapy and is associated with cancer relapse. Here, we used two approaches to isolate CSCs: the first involved the metabolic enzyme aldehyde dehydrogenase ALDH, and the second involved the three cell surface markers CD44, CD117, and CD133. ALDH cells showed a higher zinc finger E-box binding homeobox 1 (ZEB1) microRNA (miRNA) expression than CD44/CD117/133 triple-positive cells, which overexpressed miRNA 200c-3p: a well-known microRNA ZEB1 inhibitor. We found that ZEB1 inhibition was driven by miR-101-3p, miR-139-5p, miR-144-3p, miR-199b-5p, and miR-200c-3p and that the FaDu Cell Line inhibition occurred at the mRNA level, whereas HN13 did not affect mRNA expression but decreased protein levels. Furthermore, we demonstrated the ability of the ZEB1 inhibitor miRNAs to modulate CSC-related genes, such as TrkB, ALDH, NANOG, and HIF1A, using transfection technology. We showed that ALDH was upregulated upon ZEB1-suppressed miRNA transfection (Mann-Whitney ** p101 = 0.009, t-test ** p139 = 0.009, t-test ** p144 = 0.002, and t-test *** p199 = 0.0006). Overall, our study enabled an improved understanding of the role of ZEB1-suppressed miRNAs in CSC biology.

Asunto(s)

Neoplasias de Cabeza y Cuello , MicroARNs , Humanos , MicroARNs/metabolismo , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Recurrencia Local de Neoplasia/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Neoplasias de Cabeza y Cuello/genética , Células Madre Neoplásicas/metabolismo , ARN Mensajero/genética , Movimiento Celular/genética , Proliferación Celular

RESUMEN

BACKGROUND: Lung squamous cell carcinoma (LUSC) is recognized as the major subtypes of non-small cell lung cancer (NSCLC). Circulating tumor cells (CTCs) are critical players in tumor metastasis. A molecular profiling of CTCs has previously identified notch receptor 1 (Notch1) as an important mediator in NSCLC. Therefore, we investigate Notch1 roles in LUSC and its related mechanisms. METHODS: The serum levels of Notch1 were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The CTCs isolated from blood samples were characterized via an immunofluorescence method. Cell motion was determined using Transwell chambers. The regulatory relationship between Notch1 and zinc finger E-box-binding homeobox 1 (ZEB1) was verified by chromatin immunoprecipitation (ChIP) and luciferase reporter assays. The protein levels were detected by western blotting. RESULTS: Higher Notch1 expression in patients with LUSC than that in normal controls was observed. Notch1 knockdown inhibited cell motion and epithelial-mesenchymal transition (EMT). ZEB1 transcriptionally activated Notch1. ZEB1 upregulation exacerbated the malignant phenotypes of CTCs. CONCLUSION: ZEB1-activated Notch1 promotes malignant phenotypes of CTCs in LUSC and indicates poor prognosis.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , MicroARNs , Células Neoplásicas Circulantes , Humanos , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Carcinoma de Células Escamosas/patología , Pulmón , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Invasividad Neoplásica/genética , MicroARNs/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Receptor Notch1

RESUMEN

Medulloblastoma (MB) is a malignant brain tumor that afflicts mostly children and adolescents and presents four distinct molecular subgroups, known as WNT, SHH, Group 3, and Group 4. ZEB1 is a transcription factor that promotes the expression of mesenchymal markers while restraining expression of epithelial and polarity genes. Because of ZEB1 involvement in cerebellum development, here we investigated the role of ZEB1 in MB. We found increased expression of ZEB1 in MB tumor samples compared to normal cerebellar tissue. Expression was higher in the SHH subgroup when compared to all other MB molecular subgroups. High ZEB1 expression was associated with poor prognosis in Group 3 and Group 4, whereas in patients with WNT tumors poorer prognosis were related to lower ZEB1 expression. There was a moderate correlation between ZEB1 and MYC expression in Group 3 and Group 4 MB. Treatment with the immunomodulator and histone deacetylase (HDAC) inhibitor fingolimod (FTY720) reduced ZEB1 expression specifically in D283 cells, which are representative of Group 3 and Group 4 MB. These findings reveal novel subgroup-specific associations of ZEB1 expression with survival in patients with MB and suggest that ZEB1 expression can be reduced by pharmacological agents that target HDAC activity.

Asunto(s)

Neoplasias Encefálicas , Neoplasias Cerebelosas , Meduloblastoma , Niño , Adolescente , Humanos , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/genética , Cerebelo , Inhibidores de Histona Desacetilasas/uso terapéutico , Clorhidrato de Fingolimod/uso terapéutico , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo

RESUMEN

OBJECTIVE: This study aimed to investigate the effect of Zinc Finger E-box Binding Homeobox 1 (ZEB1) regulation by Micro Ribonucleic acid (miR)-448 on Breast Cancer (BC) cells and their sensitivity to chemotherapy. METHODS: miR-448 and ZEB1 mRNA levels in BC and normal tissues were detected by qPCR, and ZEB1 protein was detected by Western Blotting (WB). The correlation between miR-448 and tumor metastasis, clinical staging, and ZEB1 expression was analyzed. MCF-7 cells were transfected or co-transfected with the miR-448 mimic, oe-ZEB1, or their negative controls. Changes in miR-448 and ZEB1 expression were detected by qPCR and WB. Cell proliferation was determined by CCK-8 assays, invasion changes were analyzed by Transwell assays, and apoptosis was detected by flow cytometry. RESULTS: miR-448 expression in BC tissues was lower than that in normal tissues, while ZEB1 expression was increased in the former. ZEB1 expression was lower in BC patients with lymph node metastasis than in those without. In patients with clinical stage I-III BC, miR-448 expression decreased with an increase in tumor stage, which was negatively correlated with ZEB1 expression. Upregulation of miR-448 expression can suppress MCF-7 cell proliferation and invasion and promote apoptosis. Upregulation of ZEB1 expression in cells overexpressing miR-448 can partially reverse the inhibition of BC cell growth induced by miR-448. miR-448 can enhance the sensitivity of cells toward paclitaxel and 5-fluorouracil. CONCLUSIONS: miR-448 suppresses cell proliferation and invasion and promotes apoptosis by targeting ZEB1. Moreover, it can increase the sensitivity of cells toward paclitaxel and 5-fluorouracil.

Asunto(s)

Neoplasias de la Mama , MicroARNs , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Fluorouracilo , Regulación Neoplásica de la Expresión Génica , Genes Homeobox , Humanos , Invasividad Neoplásica , Paclitaxel , Homeobox 1 de Unión a la E-Box con Dedos de Zinc , Dedos de Zinc

RESUMEN

Secreted protein acidic and rich in cysteine (SPARC), or osteonectin, is a matricellular protein that modulates interactions between cells and their microenvironment. SPARC is expressed during extracellular matrix remodeling and is abundant in bone marrow and high-grade prostate cancer (PCa). In PCa, SPARC induces changes associated with epithelial-mesenchymal transition (EMT), enhancing migration and invasion and increasing the expression of EMT transcriptional factor Zinc finger E-box-binding homeobox 1 (ZEB1), but not Zinc finger protein SNAI1 (Snail) or Zinc finger protein SNAI2 (Slug). It is unknown whether the SPARC-induced downregulation of E-cadherin in PCa cells depends on ZEB1. Several integrins are mediators of SPARC effects in cancer cells. Because integrin signaling can induce EMT programs, we hypothesize that SPARC induces E-cadherin repression through the activation of integrins and ZEB1. Through stable knockdown and the overexpression of SPARC in PCa cells, we demonstrate that SPARC downregulates E-cadherin and increases vimentin, ZEB1, and integrin ß3 expression. Knocking down SPARC in PCa cells decreases the tyrosine-925 phosphorylation of FAK and impairs focal adhesion formation. Blocking integrin αvß3 and silencing ZEB1 revert both the SPARC-induced downregulation of E-cadherin and cell migration enhancement. We conclude that SPARC induces E-cadherin repression and enhances PCa cell migration through the integrin αvß3/ZEB1 signaling pathway.

Asunto(s)

Neoplasias de la Próstata , Factores de Transcripción , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Transición Epitelial-Mesenquimal/genética , Humanos , Integrina alfaVbeta3/metabolismo , Masculino , Invasividad Neoplásica , Osteonectina/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Microambiente Tumoral , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo

RESUMEN

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.

Asunto(s)

Neoplasias de la Próstata , Factores de Transcripción de la Familia Snail , Homeobox 1 de Unión a la E-Box con Dedos de Zinc , Humanos , Masculino , Andrógenos/metabolismo , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Células Madre Neoplásicas/patología , Fenotipo , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Testosterona/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo

RESUMEN

Connexins (Cxs) are a family of proteins that form two different types of ion channels: hemichannels and gap junction channels. These channels participate in cellular communication, enabling them to share information and act as a synchronized syncytium. This cellular communication has been considered a strong tumor suppressor, but it is now recognized that some type of Cxs can be pro-tumorigenic. For example, Cx46 expression is increased in human breast cancer samples and correlates with cancer stem cell (CSC) characteristics in human glioma. Thus, we explored whether Cx46 and glioma cells, can set up CSC and epithelial-to-mesenchymal transition (EMT) properties in a breast cancer cell line. To this end, we transfected MCF-7 cells with Cx46 attached to a green fluorescent protein (Cx46GFP), and we determined how its expression orchestrates both the gene-expression and functional changes associated with CSC and EMT. We observed that Cx46GFP increased Sox2, Nanog, and OCT4 mRNA levels associated with a high capacity to form monoclonal colonies and tumorspheres. Similarly, Cx46GFP increased the mRNA levels of n-cadherin, Vimentin, Snail and Zeb1 to a higher migratory and invasive capacity. Furthermore, Cx46GFP transfected in MCF-7 cells induced the release of higher amounts of VEGF, which promoted angiogenesis in HUVEC cells. We demonstrated for the first time that Cx46 modulates CSC and EMT properties in breast cancer cells and thus could be relevant in the design of future cancer therapies.

Asunto(s)

Neoplasias de la Mama/genética , Conexinas/genética , Transición Epitelial-Mesenquimal/genética , Células Madre Neoplásicas/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células MCF-7 , Proteína Homeótica Nanog/genética , Células Madre Neoplásicas/patología , Factores de Transcripción SOXB1/genética , Factor A de Crecimiento Endotelial Vascular/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética

RESUMEN

The transcription factor Zinc finger E-box binding 1 (ZEB1) displays a range of regulatory activities in cell function and embryonic development, including driving epithelial-mesenchymal transition. Several aspects of ZEB1 function can be regulated by its functional interactions with noncoding RNA types, namely microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). Increasing evidence indicates that ZEB1 importantly influences cancer initiation, tumor progression, metastasis, and resistance to treatment. Cancer is the main disease-related cause of death in children and adolescents. Although the role of ZEB1 in pediatric cancer is still poorly understood, emerging findings have shown that it is expressed and regulates childhood solid tumors including osteosarcoma, retinoblastoma, neuroblastoma, and central nervous system tumors. Here, we review the evidence supporting a role for ZEB1, and its interplays with miRNAs and lncRNAs, in pediatric cancers.

Asunto(s)

MicroARNs/genética , Neoplasias/genética , Neoplasias/metabolismo , ARN Largo no Codificante/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Carcinogénesis , Niño , Transición Epitelial-Mesenquimal , Humanos , Neoplasias/patología , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , Neoplasias de la Retina/genética , Neoplasias de la Retina/metabolismo , Neoplasias de la Retina/patología , Retinoblastoma/genética , Retinoblastoma/metabolismo , Retinoblastoma/patología , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética

RESUMEN

Prostate cancer (PCa) is the second most diagnosed type of cancer in men worldwide. Advanced PCa is resistant to conventional therapies and high recurrence has been associated with high rates of metastasis. Cancer stem cells (CSCs) have been proposed to be responsible for this, due to their ability of self­renewal and differentiation into other cell types. Zinc finger E­box­binding homeobox 1 (ZEB1), a transcription factor involved in the regulation of epithelial­mesenchymal transition (EMT), has been associated with the activation of several mechanisms that lead to resistance to treatment. As recent evidence has shown that CSCs may originate from non­CSCs during EMT, it was hypothesized that knocking down ZEB1 expression in PCa cell lines could revert some properties associated with CSCs. Using lentiviraltransduction, ZEB1 expression was silenced in the PCa DU145 and LNCaP cell lines. The mRNA and protein expression levels of key canonical CSC markers (Krüppel­like factor 4, SOX2, CD44 and CD133) were determined using reverse transcription­-quantitative PCR and western blot analysis, respectively. In addition, the colony forming ability of the ZEB1­knockdown cells was evaluated, and the type of colonies formed (holoclones, paraclones and meroclones) was also characterized. Finally, the ability to form prostatospheres was evaluated in vitro. It was found that in ZEB1­knockdown DU145 cells, the expression levels of CSC phenotype markers (CD44, CD133 and SOX2) were decreased compared with those in the control group. Furthermore, ZEB1­knockdown cells exhibited a lower ability to form prostatospheres and to generate colonies. In conclusion, stable silencing of ZEB1 reversed CSC properties in PCa cell lines. Since ZEB1 is associated with malignancy, therapy resistance and a CSC phenotype in PCa cell lines, targeting ZEB1 may be a key factor to eradicate CSCs and improve the prognosis of patients with advanced PCa.

Asunto(s)

Autorrenovación de las Células/genética , Regulación Neoplásica de la Expresión Génica/genética , Células Madre Neoplásicas/patología , Neoplasias de la Próstata/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Autorrenovación de las Células/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Próstata/citología , Próstata/patología , Neoplasias de la Próstata/patología , Ensayo de Tumor de Célula Madre , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/antagonistas & inhibidores , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética

RESUMEN

PURPOSE: This study set out to probe into the effect and mechanism of miR-144-3p on radiosensitivity of gastric cancer (GC) cells. METHODS: Cancer tissue and paracancerous tissue of GC patients admitted to our hospital were collected, their miR-144-3p expression was tested, GC cells were transfected, and survival and biological behavior of those cells under radiation were detected. RESULTS: After detection, miR-144-3p expression was down-regulated in GC tissue, while ZEB1 was up-regulated. There was no remarkable difference in the survival fraction of cells in each group before receiving radiation, but that of tumor cells decreased obviously (p < 0.05) after radiation exposure. Survival fraction of cells overexpressing miR-144-3p or silencing ZEB1 decreased more obviously, while the inhibition of miR-144-3p or overexpressing ZEB1 was weaker. Biological behavior of cells under 6 Gy radiation was detected. It was found that miR-144-3p overexpression or silencing ZEB1 dramatically inhibited the proliferation activity of GC cells under 6 Gy radiation, increased the levels of pro-apoptotic Bax and caspase-3 proteins (p < 0.05) and decreased the anti-apoptotic protein Bcl-2 level (p < 0.05), resulting in an increase in the apoptosis rate of cells. miR-144-3p was confirmed to be ZEB1 targeting site by dual luciferase report. Moreover, rescue experiments prove that it can increase the radiosensitivity of GC cells by regulating ZEB1 expression. CONCLUSION: miR-144-3p expression was down-regulated in GC, and it can increase the radiosensitivity of those cells by inhibiting ZEB1 expression.

Asunto(s)

MicroARNs/metabolismo , Tolerancia a Radiación , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/radioterapia , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Apoptosis , Caspasa 3/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Supervivencia Celular , Regulación hacia Abajo , Femenino , Mucosa Gástrica/metabolismo , Silenciador del Gen , Humanos , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Dosis de Radiación , Transfección/métodos , Regulación hacia Arriba , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

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.

Asunto(s)

Enfermedades de los Perros/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Melanoma/veterinaria , Neoplasias Cutáneas/veterinaria , Factores de Transcripción de la Familia Snail/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Animales , Cadherinas/metabolismo , Adhesión Celular , Movimiento Celular , Enfermedades de los Perros/genética , Perros , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/metabolismo , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Neoplasias de la Boca/veterinaria , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Factores de Transcripción de la Familia Snail/genética , Factores de Transcripción/metabolismo , Microambiente Tumoral , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética

RESUMEN

Diabetic nephropathy (DN) is one of the leading causes of mortality in diabetic patients. Long non-coding RNA zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) plays a crucial role in the development of various diseases, including DN. However, the molecular mechanism of ZEB1-AS1 in DN pathogenesis remains elusive. An in vitro DN model was established by treating HK-2 cells with high glucose (HG). Quantitative polymerase chain reaction (qRT-PCR) was utilized to detect the expression levels of ZEB1-AS1, microRNA-216a-5p (miR-216a-5p), and bone morphogenetic protein 7 (BMP7). Western blot assay was used to evaluate the protein levels of BMP7, epithelial-to-mesenchymal transition (EMT)-related proteins, and fibrosis markers. Additionally, the interaction among ZEB1-AS1, miR-216a-5p, and BMP7 was predicted by MiRcode (http://www.mircode.org) and starBase 2.0 (omics_06102, omicX), and confirmed by luciferase reporter assay. ZEB1-AS1 and BMP7 were down-regulated, while miR-216a-5p was highly expressed in kidney tissues of DN patients. Consistently, HG treatment decreased the levels of ZEB1-AS1 and BMP7, whereas HG increased miR-216a-5p expression in HK-2 cells in a time-dependent manner. ZEB1-AS1 upregulation inhibited HG-induced EMT and fibrogenesis. Furthermore, ZEB1-AS1 directly targeted miR-216a-5p, and overexpression of miR-216a-5p restored the inhibitory effects of ZEB1-AS1 overexpression on EMT and fibrogenesis. BMP7 was negatively targeted by miR-216a-5p. In addition, ZEB1-AS1 suppressed HG-induced EMT and fibrogenesis by regulating miR-216a-5p and BMP-7. lncRNA ZEB1-AS1 inhibited high glucose-induced EMT and fibrogenesis via regulating miR-216a-5p/BMP7 axis in diabetic nephropathy, providing a potential target for DN therapy.

Asunto(s)

Proteína Morfogenética Ósea 7/metabolismo , Nefropatías Diabéticas/metabolismo , Transición Epitelial-Mesenquimal/fisiología , ARN Largo no Codificante/fisiología , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Células Cultivadas , Nefropatías Diabéticas/genética , Regulación hacia Abajo , Humanos , MicroARNs/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia Arriba

RESUMEN

The epithelial-mesenchymal transition (EMT) is a complex mechanism in which cells undergo a transition from epithelial to mesenchymal phenotypes (there is also an intermediary hybrid state) in response to microenvironmental alterations and aberrant stimuli triggered by molecules such as TGF-ß. Recent studies in breast cancer progression reported new feedback loops and new participant molecules such as microRNAs 340 and 1199. In this work, we propose a logical model of EMT contemplating the influence of these new published molecules on the regulatory core of EMT. The model results were compared with theoretical and experimental data for the human breast epithelial cell line MCF10A presenting excellent agreement. We propose that the miRNAs 340 and 1199 should be considered phenotypic stability factors of the hybrid state based on the positive feedback loops they form with ZEB1. In addition, the model allows the prediction of phenotype probabilities at the coexistence region. For the tristable dynamics when epithelial, hybrid, and mesenchymal phenotypes coexist, we found that the hybrid state is the most probable, agreeing with experiments. Our results highlight new mechanisms related to the EMT dynamics in response to TGF-ß stimulus in epithelial breast cells and might help the design of therapeutic strategies for breast cancer.

Asunto(s)

Neoplasias de la Mama/genética , Transición Epitelial-Mesenquimal , Retroalimentación Fisiológica , Modelos Teóricos , Neoplasias de la Mama/patología , Línea Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Fenotipo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo

RESUMEN

Diabetic nephropathy (DN) is one of the leading causes of mortality in diabetic patients. Long non-coding RNA zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) plays a crucial role in the development of various diseases, including DN. However, the molecular mechanism of ZEB1-AS1 in DN pathogenesis remains elusive. An in vitro DN model was established by treating HK-2 cells with high glucose (HG). Quantitative polymerase chain reaction (qRT-PCR) was utilized to detect the expression levels of ZEB1-AS1, microRNA-216a-5p (miR-216a-5p), and bone morphogenetic protein 7 (BMP7). Western blot assay was used to evaluate the protein levels of BMP7, epithelial-to-mesenchymal transition (EMT)-related proteins, and fibrosis markers. Additionally, the interaction among ZEB1-AS1, miR-216a-5p, and BMP7 was predicted by MiRcode (http://www.mircode.org) and starBase 2.0 (omics_06102, omicX), and confirmed by luciferase reporter assay. ZEB1-AS1 and BMP7 were down-regulated, while miR-216a-5p was highly expressed in kidney tissues of DN patients. Consistently, HG treatment decreased the levels of ZEB1-AS1 and BMP7, whereas HG increased miR-216a-5p expression in HK-2 cells in a time-dependent manner. ZEB1-AS1 upregulation inhibited HG-induced EMT and fibrogenesis. Furthermore, ZEB1-AS1 directly targeted miR-216a-5p, and overexpression of miR-216a-5p restored the inhibitory effects of ZEB1-AS1 overexpression on EMT and fibrogenesis. BMP7 was negatively targeted by miR-216a-5p. In addition, ZEB1-AS1 suppressed HG-induced EMT and fibrogenesis by regulating miR-216a-5p and BMP-7. lncRNA ZEB1-AS1 inhibited high glucose-induced EMT and fibrogenesis via regulating miR-216a-5p/BMP7 axis in diabetic nephropathy, providing a potential target for DN therapy.

Asunto(s)

Humanos , Nefropatías Diabéticas/metabolismo , Proteína Morfogenética Ósea 7/metabolismo , Transición Epitelial-Mesenquimal/fisiología , ARN Largo no Codificante/fisiología , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Regulación hacia Abajo , Regulación hacia Arriba , Células Cultivadas , MicroARNs/metabolismo , Nefropatías Diabéticas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

One of the factors promoting tumoral progress is the abnormal activation of the epithelial-mesenchymal transition (EMT) program which has been associated with chemoresistance in tumoral cells. The transcription factor zinc finger E-box-binding homeobox 1 (ZEB1), a key EMT activator, has recently been related to docetaxel resistance, the main chemotherapeutic used in advanced prostate cancer treatment. The mechanisms involved in this protective effect are still unclear. In a previous work, we demonstrated that ZEB1 expression induced an EMT-like phenotype in prostate cancer cell lines. In this work, we used prostate cancer cell lines 22Rv1 and DU145 to study the effect of ZEB1 modulation on docetaxel resistance and its possible mechanisms. The results showed that ZEB1 overexpression conferred to 22Rv1 cell resistance to docetaxel while its silencing made DU145 cells more sensitive to it. Analysis of resistance markers showed no presence of ATP-binding cassette subfamily B member 1 (MDR1) and no changes in breast cancer resistance protein (BCRP) or ATP-binding cassette subfamily C member 10 (MRP7). However, a correlation between ZEB1, multidrug resistance-associated protein 1 (MRP1), and ATP-binding cassette subfamily C member 4 (MRP4) expression was observed. MRP4 inhibition, using MK571, resensitized cells with ZEB1 overexpression to docetaxel treatment. In addition, modulation of ZEB1 and subsequent change in MRP4 expression correlated with a lower apoptotic response to docetaxel, characterized by lower B-cell lymphoma 2 (Bcl2), high BCL2-associated X protein (Bax), and high active caspase 3 expression. The response to docetaxel in our model seems to be mediated mainly by activation of the apoptotic death program. Our results showed that modulation of MRP4 could be a mediator of ZEB1-related resistance to docetaxel in prostate cancer, making it a possible marker for chemotherapy response in patients who do not express MDR1.

Asunto(s)

Antineoplásicos/uso terapéutico , Docetaxel/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Western Blotting , Línea Celular Tumoral , Resistencia a Antineoplásicos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Silenciador del Gen , Humanos , Masculino , Neoplasias de la Próstata/metabolismo

RESUMEN

Prostate cancer (PCa) is the second most common type of male malignancy worldwide. The transcription factor zinc finger E­box binding homeobox 1 (ZEB1) is associated with epithelial­mesenchymal transition and is also involved in regulation of androgen receptor (AR) expression, the main ligands of which are testosterone and dihydrotestosterone (DHT). These androgens are synthesized through the steroidogenic pathway within the prostate, and their synthesis is altered in PCa. The present study aimed to determine the ZEB1­induced alterations in androgen synthesis and AR expression in the DU145 PCa cell line. Reverse transcription­quantitative polymerase chain reaction, western blotting and immunocytochemistry were used to determine the mRNA and protein expression levels, and cellular localization of steroidogenic pathway enzymes in the DU145 cell line in response to ZEB1 silencing. Furthermore, the concentrations of testosterone and DHT were detected in cell culture medium using ELISA. ZEB1­silenced cells exhibited an increase in testosterone and DHT production, an increase in AR expression and an alteration in the steroidogenic pathway. In particular, steroidogenic acute regulatory protein and 5α­reductase 2 expression levels were decreased, whereas cytochrome P450 family 17 subfamily A member 1, 5α­reductase 1, aldo­keto reductase family 1 member D1 and aldo­keto reductase family 1 member C2 expression levels were increased. In conclusion, the present study provided novel information regarding the regulation of intratumoral androgen production in PCa, which is relevant for the progression of the disease to a castration­resistant form.

Asunto(s)

Dihidrotestosterona/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Testosterona/biosíntesis , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/fisiología , Línea Celular Tumoral , Dihidrotestosterona/análisis , Silenciador del Gen , Humanos , Masculino , Próstata/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/química , Receptores Androgénicos/metabolismo , Testosterona/análisis , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética

RESUMEN

Syndecan 1 (SDC-1) is a cell surface proteoglycan with a significant role in cell adhesion, maintaining epithelial integrity. SDC1 expression is inversely related to aggressiveness in prostate cancer (PCa). During epithelial to mesenchymal transition (EMT), loss of epithelial markers is mediated by transcriptional repressors such as SNAIL, SLUG, or ZEB1/2 that bind to E-box promoter sequences of specific genes. The effect of these repressors on SDC-1 expression remains unknown. Here, we demonstrated that SNAIL, SLUG and ZEB1 expressions are increased in advanced PCa, contrarily to SDC-1. SNAIL, SLUG and ZEB1 also showed an inversion to SDC-1 in prostate cell lines. ZEB1, but not SNAIL or SLUG, represses SDC-1 as demonstrated by experiments of ectopic expression in epithelial prostate cell lines. Inversely, expression of ZEB1 shRNA in PCa cell line increased SDC-1 expression. The effect of ZEB1 is transcriptional since ectopic expression of this gene represses SDC-1 promoter activity and ZEB1 binds to the SDC-1 promoter as detected by ChIP assays. An epigenetic mark associated to transcription repression H3K27me3 was bound to the same sites that ZEB1. In conclusion, this study identifies ZEB1 as a key repressor of SDC-1 during PCa progression and point to ZEB1 as a potentially diagnostic marker for PCa.

Asunto(s)

Neoplasias de la Próstata/genética , Sindecano-1/genética , Factores de Transcripción/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Adhesión Celular/genética , Línea Celular Tumoral , Epigénesis Genética/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Células PC-3 , Regiones Promotoras Genéticas/genética , Factores de Transcripción de la Familia Snail/genética , Transcripción Genética/genética

RESUMEN

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.

Asunto(s)

Enfermedades de los Perros/metabolismo , Neoplasias Mamarias Animales/metabolismo , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Actinas/metabolismo , Animales , Western Blotting/veterinaria , Línea Celular Tumoral , Enfermedades de los Perros/patología , Perros , Femenino , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Animales/patología , Invasividad Neoplásica , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Factor de Transcripción STAT3/efectos de los fármacos , Factor de Transcripción STAT3/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo