Exacerbation of Liver Tumor Metastasis in twist1a+/xmrk+ Double Transgenic Zebrafish Following Lipopolysaccharide or Dextran Sulphate Sodium Exposure

The poor prognosis for patients with hepatocellular carcinoma (HCC) is related directly to metastasis. The Twist1 gene encodes for a transcription factor essential to embryogenesis. It has also been shown to promote epithelial-to-mesenchymal transition (EMT), invasion, and metastasis; however, there is currently no in vivo evidence that Twist1 plays a role in the metastasis of liver tumors. Zebrafish are increasingly being used as an alternative cancer model. In the current study, an adult-stage zebrafish HCC model was used to examine the synergistic effects of twist1a and xmrk, a well characterized oncogene, during HCC metastasis. We also examined the effects of two inflammatory agents, lipopolysaccharides (LPS) and dextran sulfate sodium (DSS), on the hepatocyte-specific expression of transgenic twist1a and xmrk. The conditional overexpression of twist1a and xmrk was shown to promote liver tumor metastasis in zebrafish, resulting in increased apoptosis and cell proliferation as well as tumor maintenance and propagation independent of the inherent EMT-inducing activity of xmrk. Exposing twist1a+/xmrk+ transgenic zebrafish to LPS or DSS was shown to promote metastasis, indicating that the overexpression of twist1a and xmrk led to crosstalk between the signaling pathways involved in EMT. This study provides important evidence pertaining to the largely overlooked effects of signaling crosstalk between twist1a and xmrk in regulating HCC metastasis. Our results also suggest that the co-expression of twist1a/xmrk in conjunction with exposure to LPS or DSS enhances HCC metastasis, and provides a valuable in vivo platform by which to investigate tumor initiation and metastasis in the study of liver cancer.

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Effect of Lipopolysaccharides on Liver Tumor Metastasis of twist1a/krasV12 Double Transgenic Zebrafish

Biomedicines ◽

10.3390/biomedicines10010095 ◽

2022 ◽

Vol 10 (1) ◽

pp. 95

Author(s):

Jeng-Wei Lu ◽

Liang-In Lin ◽

Yuxi Sun ◽

Dong Liu ◽

Zhiyuan Gong

Keyword(s):

Liver Tumor ◽

Tumor Metastasis ◽

Fluorescent Protein ◽

Epithelial Mesenchymal Transition ◽

In Vivo Model ◽

Transgenic Zebrafish ◽

Bhlh Transcription Factor ◽

Mesenchymal Transition ◽

Green Fluorescent

The poor prognosis of patients diagnosed with hepatocellular carcinoma (HCC) is directly associated with the multi-step process of tumor metastasis. TWIST1, a basic helix-loop-helix (bHLH) transcription factor, is the most important epithelial-mesenchymal transition (EMT) gene involved in embryonic development, tumor progression, and metastasis. However, the role that TWIST1 gene plays in the process of liver tumor metastasis in vivo is still not well understood. Zebrafish can serve as a powerful model for cancer research. Thus, in this study, we crossed twist1a+ and kras+ transgenic zebrafish, which, respectively, express hepatocyte-specific mCherry and enhanced green fluorescent protein (EGFP); they also drive overexpression of their respective transcription factors. This was found to exacerbate the development of metastatic HCC. Fluorescence of mCherry and EGFP-labeled hepatocytes revealed that approximately 37.5% to 45.5% of the twist1a+/kras+ double transgenic zebrafish exhibited spontaneous tumor metastasis from the liver to the abdomen and tail areas, respectively. We also investigated the inflammatory effects of lipopolysaccharides (LPS) on the hepatocyte-specific co-expression of twist1a+ and kras+ in double transgenic zebrafish. Following LPS exposure, co-expression of twist1a+ and kras+ was found to increase tumor metastasis by 57.8%, likely due to crosstalk with the EMT pathway. Our results confirm that twist1a and kras are important mediators in the development of metastatic HCC. Taken together, our in-vivo model demonstrated that co-expression of twist1a+/kras+ in conjunction with exposure to LPS enhanced metastatic HCC offers a useful platform for the study of tumor initiation and metastasis in liver cancer.

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Effect of GPC1 on epithelial-to-mesenchymal transition and stemness and interaction with ITGB1 in gastric cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e15580 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e15580-e15580

Author(s):

Shiqing Wang ◽

Zhenzhen Wu ◽

Minyu Zhou ◽

Wangjun Liao

Keyword(s):

Gastric Cancer ◽

Western Blot ◽

Poor Prognosis ◽

Tumor Metastasis ◽

Epithelial To Mesenchymal Transition ◽

Underlying Mechanism ◽

Mesenchymal Transition ◽

Normal Tissues ◽

Epithelial Marker

e15580 Background: Gastric cancer is the fourth frequently diagnosed cancer worldwide and tumor metastasis plays an important role in its poor prognosis. EMT, which occurs during embryonic development and carcinoma progression, contributes to metastasis and regulates stemness. GPC1, a member of the heparan sulfate proteoglycans family, is overexpressed in many types of cancer, and associates with metastasis and tumor progression. However, it is unknown that the functions of GPC1 and its underlying mechanism in gastric cancer. Methods: Immunohistochemistry analysis was performed in 245 GC tissues to explore the relationship between GPC1 and tumor metastasis. Transwell migration and wound-healing assay were conducted to evaluate the effects of GPC1 on migration and invasiveness. Epithelial marker and mesenchymal markers expression in GC cells were detected by Western blot and immunofluorescence. GPC1 expression induced by TGF-β was detected by Western Blot. After GPC1 was knock downed, the metastatic effect and EMT markers induced by TGF-β were detected. Co-Immunoprecipitation was used to detect the interaction between ITGB1 and GPC1. Results: The expression of GPC1 was higher in GC tissues than in adjacent normal tissues and associated with poor prognosis. Knocking down GPC1 suppressed EMT and stemness which reduced metastasis in vivo and vitro by inhibiting Erk1/2 MAPK and FAK/Akt pathways. TGF-β stimulation significantly upregulated the expression of GPC1 whereas knocking down of GPC1 reversed TGF-β-mediated EMT and stemness. Moreover, we found that knocking down GPC1 could downregulate ITGB1 which was known to promote EMT in gastric cancer. Overexpression of ITGB1 counteracted the influence of knocking down GPC1 on EMT and stemness. Furthermore, GPC1 formed a complex with ITGB1, which indicated that the ability of GPC1 to stimulate EMT is ITGB1-dependent. Conclusions: We conclude that GPC1, through its interaction with ITGB1, plays an important role in regulating TGF-β-mediated EMT and stemness, and could be a potential future therapeutic target to prevent progression of gastric cancer.

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Arrestin Domain Containing 3 Reverses Epithelial to Mesenchymal Transition and Chemo-Resistance of TNBC Cells by Up-Regulating Expression of miR-200b

Cells ◽

10.3390/cells8070692 ◽

2019 ◽

Vol 8 (7) ◽

pp. 692 ◽

Cited By ~ 6

Author(s):

Young Hwa Soung ◽

Heesung Chung ◽

Cecilia Yan ◽

Jingfang Ju ◽

Jun Chung

Keyword(s):

Transcriptional Control ◽

Epithelial To Mesenchymal Transition ◽

Target Genes ◽

Therapeutic Potential ◽

Synergistic Effects ◽

Metastatic Potential ◽

Metastasis Suppressor ◽

Mesenchymal Transition

Our previous studies demonstrated the importance of arrestin domain containing 3 (ARRDC3), a metastasis suppressor, in inhibiting invasive and metastatic potential of triple negative breast cancer (TNBC) in vitro and in vivo. However, little is known about ARRDC3 mediated transcriptional control and its target genes that are implicated in its metastatic suppressing activity. In this study, we used miRNA array and subsequent functional analyses to identify miRNAs whose expression are significantly regulated by ARRDC3 in TNBC cells. We identified miR-200b as a major target gene of ARRDC3. miR-200b played an essential role in mediating ARRDC3 dependent reversal of EMT phenotypes and chemo-resistance to DNA damaging agents in TNBC cells. Expression of miR-200b also increased the expression of ARRDC3 as well in TNBC cells, suggesting a positive feedback loop between these two molecules. In addition, we combined the therapeutic powers of miR-200b and 5-fluorourancil (5-FU) into a single compound (5-FU-miR-200b) to maximize the synergistic effects of these compounds. Chemically modified miR-200b (5-FU-miR-200b mimic) was more effective in inhibiting metastatic potentials of TNBC cells than unmodified miR-200b and does not require transfection reagents, implying its therapeutic potential in TNBC. Our studies showed the importance of therapeutic targeting ARRDC3/miR-200b pathway in TNBC.

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Operative ubiquitin-specific protease 22 deubiquitination confers a more invasive phenotype to cholangiocarcinoma

Cell Death and Disease ◽

10.1038/s41419-021-03940-0 ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Yu Tian ◽

Bo Tang ◽

Chengye Wang ◽

Yan Wang ◽

Jiakai Mao ◽

...

Keyword(s):

Tumour Growth ◽

Molecular Mechanisms ◽

Epithelial To Mesenchymal Transition ◽

Sirtuin 1 ◽

Mesenchymal Transition ◽

Specific Protease ◽

Ubiquitin Specific Protease ◽

Paired Tumour

AbstractOncogenic ubiquitin-specific protease 22 (USP22) is implicated in a variety of tumours; however, evidence of its role and underlying molecular mechanisms in cholangiocarcinoma (CCA) development remains unknown. We collected paired tumour and adjacent non-tumour tissues from 57 intrahepatic CCA (iCCA) patients and evaluated levels of the USP22 gene and protein by qPCR and immunohistochemistry. Both the mRNA and protein were significantly upregulated, correlated with the malignant invasion and worse OS of iCCA. In cell cultures, USP22 overexpression increased CCA cell proliferation and mobility, and induced epithelial-to-mesenchymal transition (EMT). Upon an interaction, USP22 deubiquitinated and stabilized sirtuin-1 (SIRT1), in conjunction with Akt/ERK activation. In implantation xenografts, USP22 overexpression stimulated tumour growth and metastasis to the lungs of mice. Conversely, the knockdown by USP22 shRNA attenuated the tumour growth and invasiveness in vitro and in vivo. Furthermore, SIRT1 overexpression reversed the USP22 functional deficiency, while the knockdown acetylated TGF-β-activated kinase 1 (TAK1) and Akt. Our present study defines USP22 as a poor prognostic predictor in iCCA that cooperates with SIRT1 and facilitates tumour development.

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Roles of microRNAs in Regulating Cancer Stemness in Head and Neck Cancers

Cancers ◽

10.3390/cancers13071742 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1742

Author(s):

Melysa Fitriana ◽

Wei-Lun Hwang ◽

Pak-Yue Chan ◽

Tai-Yuan Hsueh ◽

Tsai-Tsen Liao

Keyword(s):

Growth Factor ◽

Head And Neck ◽

Cancer Cells ◽

Signaling Pathways ◽

Epithelial To Mesenchymal Transition ◽

Survival Rates ◽

Growth Factor Receptor ◽

Cancer Stemness ◽

Mesenchymal Transition

Head and neck squamous cell carcinomas (HNSCCs) are epithelial malignancies with 5-year overall survival rates of approximately 40–50%. Emerging evidence indicates that a small population of cells in HNSCC patients, named cancer stem cells (CSCs), play vital roles in the processes of tumor initiation, progression, metastasis, immune evasion, chemo-/radioresistance, and recurrence. The acquisition of stem-like properties of cancer cells further provides cellular plasticity for stress adaptation and contributes to therapeutic resistance, resulting in a worse clinical outcome. Thus, targeting cancer stemness is fundamental for cancer treatment. MicroRNAs (miRNAs) are known to regulate stem cell features in the development and tissue regeneration through a miRNA–target interactive network. In HNSCCs, miRNAs act as tumor suppressors and/or oncogenes to modulate cancer stemness and therapeutic efficacy by regulating the CSC-specific tumor microenvironment (TME) and signaling pathways, such as epithelial-to-mesenchymal transition (EMT), Wnt/β-catenin signaling, and epidermal growth factor receptor (EGFR) or insulin-like growth factor 1 receptor (IGF1R) signaling pathways. Owing to a deeper understanding of disease-relevant miRNAs and advances in in vivo delivery systems, the administration of miRNA-based therapeutics is feasible and safe in humans, with encouraging efficacy results in early-phase clinical trials. In this review, we summarize the present findings to better understand the mechanical actions of miRNAs in maintaining CSCs and acquiring the stem-like features of cancer cells during HNSCC pathogenesis.

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Inhibition of Lysine 63 Ubiquitination Prevents the Progression of Renal Fibrosis in Diabetic DBA/2J Mice

International Journal of Molecular Sciences ◽

10.3390/ijms22105194 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5194

Author(s):

Paola Pontrelli ◽

Francesca Conserva ◽

Rossella Menghini ◽

Michele Rossini ◽

Alessandra Stasi ◽

...

Keyword(s):

Diabetic Nephropathy ◽

Epithelial To Mesenchymal Transition ◽

Selective Inhibition ◽

Collagen I ◽

Protein Accumulation ◽

Mesenchymal Transition ◽

Collagen Iii ◽

Proximal Tubular Epithelial Cells

Diabetic nephropathy (DN) is the most frequent cause of end-stage renal disease. Tubulointerstitial accumulation of lysine 63 (K63)-ubiquitinated (Ub) proteins is involved in the progression of DN fibrosis and correlates with urinary miR-27b-3p downregulation. We explored the renoprotective effect of an inhibitor of K63-Ub (NSC697923), alone or in combination with the ACE-inhibitor ramipril, in vitro and in vivo. Proximal tubular epithelial cells and diabetic DBA/2J mice were treated with NSC697923 and/or ramipril. K63-Ub protein accumulation along with α-SMA, collagen I and III, FSP-1, vimentin, p16INK4A expression, SA-α Gal staining, Sirius Red, and PAS staining were measured. Finally, we measured the urinary albumin to creatinine ratio (uACR), and urinary miR-27b-3p expression in mice. NSC697923, both alone and in association with ramipril, in vitro and in vivo inhibited hyperglycemia-induced epithelial to mesenchymal transition by significantly reducing K63-Ub proteins, α-SMA, collagen I, vimentin, FSP-1 expression, and collagen III along with tubulointerstitial and glomerular fibrosis. Treated mice also showed recovery of urinary miR-27b-3p and restored expression of p16INK4A. Moreover, NSC697923 in combination with ramipril demonstrated a trend in the reduction of uACR. In conclusion, we suggest that selective inhibition of K63-Ub, when combined with the conventional treatment with ACE inhibitors, might represent a novel treatment strategy to prevent the progression of fibrosis and proteinuria in diabetic nephropathy and we propose miR-27b-3p as a biomarker of treatment efficacy.

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DDRE-07. DIPG HARBOUR ALTERATIONS TARGETABLE BY MEK INHIBITORS, WITH ACQUIRED RESISTANCE MECHANISMS OVERCOME BY COMBINATORIAL INHIBITION

Neuro-Oncology ◽

10.1093/neuonc/noaa215.252 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii62-ii62

Author(s):

Elisa Izquierdo ◽

Diana Carvalho ◽

Alan Mackay ◽

Sara Temelso ◽

Jessica K R Boult ◽

...

Keyword(s):

Drug Exposure ◽

Mapk Pathway ◽

Synergistic Effects ◽

Acquired Resistance ◽

Mek Inhibitor ◽

Resistance Mechanisms ◽

Genetic Alterations ◽

Mesenchymal Transition

Abstract The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In the era of precision medicine, targeted therapies represent an exciting treatment opportunity, yet resistance can rapidly emerge, playing an important role in treatment failure. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling (methylation BeadArray, exome, RNAseq, phospho-proteomics) linked to drug screening in newly-established patient-derived models of DIPG in vitro and in vivo. We identified a high degree of in vitro sensitivity to the MEK inhibitor trametinib (GI50 16-50nM) in samples, which harboured genetic alterations targeting the MAPK pathway, including the non-canonical BRAF_G469V mutation, and those affecting PIK3R1 and NF1. However, treatment of PDX models and of a patient with trametinib at relapse failed to elicit a significant response. We generated trametinib-resistant clones (62-188-fold, GI50 2.4–5.2µM) in the BRAF_G469V model through continuous drug exposure, and identified acquired mutations in MEK1/2 (MEK1_K57N, MEK1_I141S and MEK2_I115N) with sustained pathway up-regulation. These cells showed the hallmarks of mesenchymal transition, and expression signatures overlapping with inherently trametinib-insensitive primary patient-derived cells that predicted an observed sensitivity to dasatinib. Combinations of trametinib with dasatinib and the downstream ERK inhibitor ulixertinib showed highly synergistic effects in vitro. These data highlight the MAPK pathway as a therapeutic target in DIPG, and show the importance of parallel resistance modelling and rational combinatorial treatments likely to be required for meaningful clinical translation.

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KCNN4 promotes the progression of lung adenocarcinoma by activating the AKT and ERK signaling pathways

Cancer Biomarkers ◽

10.3233/cbm-201045 ◽

2021 ◽

pp. 1-15

Author(s):

Ping Xu ◽

Xiao Mo ◽

Ruixue Xia ◽

Long Jiang ◽

Chengfei Zhang ◽

...

Keyword(s):

Potassium Channels ◽

Lung Adenocarcinoma ◽

Potassium Channel ◽

Signaling Pathways ◽

Epithelial To Mesenchymal Transition ◽

Tumor Biology ◽

Erk Signaling ◽

Mesenchymal Transition

BACKGROUND: Potassium channels, encoded by more than seventy genes, are cell excitability transmembrane proteins and become evident to play essential roles in tumor biology. OBJECTIVE: The deregulation of potassium channel genes has been related to cancer development and patient prognosis. The objective of this study is to understand the role of potassium channels in lung cancer. METHODS: We examined all potassium channel genes and identified that KCNN4 is the most significantly overexpressed one in lung adenocarcinoma. The role and mechanism of KCNN4 in lung adenocarcinoma were further investigated by in vitro cell and molecular assay and in vivo mouse xenograft models. RESULTS: We revealed that the silencing of KCNN4 significantly inhibits cell proliferation, migration, invasion, and tumorigenicity of lung adenocarcinoma. Further studies showed that knockdown of KCNN4 promotes cell apoptosis, induces cell cycle arrested in the S phase, and is associated with the epithelial to mesenchymal transition (EMT) process. Most importantly, we demonstrated that KCNN4 regulates the progression of lung adenocarcinoma through P13K/AKT and MEK/ERK signaling pathways. The use of inhibitors that targeted AKT and ERK also significantly inhibit the proliferation and metastasis of lung adenocarcinoma cells. CONCLUSIONS: This study investigated the function and mechanism of KCNN4 in lung adenocarcinoma. On this basis, this means that KCNN4 can be used as a tumor marker for lung adenocarcinoma and is expected to become an important target for a potential drug.

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Molecular mechanisms underpinning sarcomas and implications for current and future therapy

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-021-00647-8 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Victoria Damerell ◽

Michael S. Pepper ◽

Sharon Prince

Keyword(s):

Molecular Mechanisms ◽

Epithelial To Mesenchymal Transition ◽

Treatment Strategies ◽

Mesenchymal Transition ◽

Mesenchymal To Epithelial Transition ◽

Cells Of Origin ◽

Novel Treatment Strategies ◽

Future Therapy

AbstractSarcomas are complex mesenchymal neoplasms with a poor prognosis. Their clinical management is highly challenging due to their heterogeneity and insensitivity to current treatments. Although there have been advances in understanding specific genomic alterations and genetic mutations driving sarcomagenesis, the underlying molecular mechanisms, which are likely to be unique for each sarcoma subtype, are not fully understood. This is in part due to a lack of consensus on the cells of origin, but there is now mounting evidence that they originate from mesenchymal stromal/stem cells (MSCs). To identify novel treatment strategies for sarcomas, research in recent years has adopted a mechanism-based search for molecular markers for targeted therapy which has included recapitulating sarcomagenesis using in vitro and in vivo MSC models. This review provides a comprehensive up to date overview of the molecular mechanisms that underpin sarcomagenesis, the contribution of MSCs to modelling sarcomagenesis in vivo, as well as novel topics such as the role of epithelial-to-mesenchymal-transition (EMT)/mesenchymal-to-epithelial-transition (MET) plasticity, exosomes, and microRNAs in sarcomagenesis. It also reviews current therapeutic options including ongoing pre-clinical and clinical studies for targeted sarcoma therapy and discusses new therapeutic avenues such as targeting recently identified molecular pathways and key transcription factors.

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Phosphorylation of Serine 11 and Serine 92 as New Positive Regulators of Human Snail1 Function: Potential Involvement of Casein Kinase-2 and the cAMP-activated Kinase Protein Kinase A

Molecular Biology of the Cell ◽

10.1091/mbc.e09-06-0504 ◽

2010 ◽

Vol 21 (2) ◽

pp. 244-253 ◽

Cited By ~ 47

Author(s):

Matthew Reid MacPherson ◽

Patricia Molina ◽

Serhiy Souchelnytskyi ◽

Christer Wernstedt ◽

Jorge Martin-Pérez ◽

...

Keyword(s):

Nuclear Export ◽

Tumor Metastasis ◽

Epithelial Mesenchymal Transition ◽

Cop9 Signalosome ◽

Mesenchymal Transition ◽

Depth Analysis ◽

Positive Regulators

Snail1 is a major factor for epithelial-mesenchymal transition (EMT), an important event in tumor metastasis and in other pathologies. Snail1 is tightly regulated at transcriptional and posttranscriptional levels. Control of Snail1 protein stability and nuclear export by GSK3β phosphorylation is important for Snail1 functionality. Stabilization mechanisms independent of GSK3β have also been reported, including interaction with LOXL2 or regulation of the COP9 signalosome by inflammatory signals. To get further insights into the role of Snail1 phosphorylation, we have performed an in-depth analysis of in vivo human Snail1 phosphorylation combined with mutational studies. We identify new phosphorylation sites at serines 11, 82, and 92 and confirmed previously suggested phosphorylations at serine 104 and 107. Serines 11 and 92 participate in the control of Snail1 stability and positively regulate Snail1 repressive function and its interaction with mSin3A corepressor. Furthermore, serines 11 and 92 are required for Snail1-mediated EMT and cell viability, respectively. PKA and CK2 have been characterized as the main kinases responsible for in vitro Snail1 phosphorylation at serine 11 and 92, respectively. These results highlight serines 11 and 92 as new players in Snail1 regulation and suggest the participation of CK2 and PKA in the modulation of Snail1 functionality.

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