Insulin and IGF1 signalling pathways in human astrocytes in vitro and in vivo; characterisation, subcellular localisation and modulation of the receptors

AbstractThe signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. Here, we report development of 3-Dimensional culture analyses to separately quantify growth and invasion. We identify that alternate variants of IQSEC1, an ARF GTPase Exchange Factor, act as switches to promote invasion over growth by controlling phosphoinositide metabolism. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P3-AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants promote PI(3,4,5)P3 production to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo. Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PI(3,4,5)P3 signalling, and predicts long-term poor outcome across multiple cancers. IQSEC1-regulated phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis.

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A novel vasculo-angiogenic effect of cilostazol mediated by cross-talk between multiple signalling pathways including the ERK/p38 MAPK signalling transduction cascade

Clinical Science ◽

10.1042/cs20110432 ◽

2012 ◽

Vol 123 (3) ◽

pp. 147-159 ◽

Cited By ~ 15

Author(s):

Ting-Hsing Chao ◽

Shih-Ya Tseng ◽

Yi-Heng Li ◽

Ping-Yen Liu ◽

Chung-Lung Cho ◽

...

Keyword(s):

Protein Kinase ◽

P38 Mapk ◽

Umbilical Vein ◽

Tube Formation ◽

Mitogen Activated Protein Kinase ◽

Signalling Pathways ◽

No Production ◽

Angiogenic Effect

Cilostazol is an anti-platelet agent with vasodilatory activity that acts by increasing intracellular concentrations of cAMP. Recent reports have suggested that cilostazol may promote angiogenesis. In the present study, we have investigated the effect of cilostazol in promoting angiogenesis and vasculogenesis in a hindlimb ischaemia model and have also examined its potential mechanism of action in vitro and in vivo. We found that cilostazol treatment significantly increased colony formation by human early EPCs (endothelial progenitor cells) through a mechanism involving the activation of cAMP/PKA (protein kinase A), PI3K (phosphoinositide 3-kinase)/Akt/eNOS (endothelial NO synthase) and ERK (extracellular-signal-regulated kinase)/p38 MAPK (mitogen-activated protein kinase) signalling pathways. Cilostazol also enhanced proliferation, chemotaxis, NO production and vascular tube formation in HUVECs (human umbilical vein endothelial cells) through activation of multiple signalling pathways downstream of PI3K/Akt/eNOS. Cilostazol up-regulated VEGF (vascular endothelial growth factor)-A165 expression and secretion of VEGF-A in HUVECs through activation of the PI3K/Akt/eNOS pathway. In a mouse hindlimb ischaemia model, recovery of blood flow ratio (ipsilateral/contralateral) 14 days after surgery was significantly improved in cilostazol-treated mice (10 mg/kg of body weight) compared with vehicle-treated controls (0.63±0.07 and 0.43±0.05 respectively, P<0.05). Circulating CD34+ cells were also increased in cilostazol-treated mice (3614±670 compared with 2151±608 cells/ml, P<0.05). Expression of VEGF and phosphorylation of PI3K/Akt/eNOS and ERK/p38 MAPK in ischaemic muscles were significantly enhanced by cilostazol. Our data suggest that cilostazol produces a vasculo-angiogenic effect by up-regulating a broad signalling network that includes the ERK/p38 MAPK, VEGF-A165, PI3K/Akt/eNOS and cAMP/PKA pathways.

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Anti-cancer effects of the aqueous extract of Orostachys japonica A. Berger on 5-fluorouracil-resistant colorectal cancer via MAPK signalling pathways in vitro and in vivo

Journal of Ethnopharmacology ◽

10.1016/j.jep.2021.114412 ◽

2021 ◽

pp. 114412

Author(s):

Jung Woo Kim ◽

Sang Hee Kim ◽

Ramesh Mariappan ◽

Daeun Moon ◽

Jinu Kim ◽

...

Keyword(s):

Colorectal Cancer ◽

Aqueous Extract ◽

Signalling Pathways ◽

Anti Cancer ◽

Mapk Signalling

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Expression and function of the novel proto-oncogene PBF in thyroid cancer: a new target for augmenting radioiodine uptake

Journal of Endocrinology ◽

10.1530/joe-11-0064 ◽

2011 ◽

Vol 210 (2) ◽

pp. 157-163 ◽

Cited By ~ 13

Author(s):

Vicki E Smith ◽

Jayne A Franklyn ◽

Christopher J McCabe

Keyword(s):

Thyroid Cancer ◽

Current Data ◽

Subcellular Localisation ◽

Sodium Iodide Symporter ◽

Iodide Uptake ◽

Radioiodine Uptake ◽

Novel Target ◽

And Function

Pituitary tumor-transforming gene (PTTG)-binding factor (PBF; PTTG1IP) was initially identified through its interaction with the human securin, PTTG. Like PTTG, PBF is upregulated in multiple endocrine tumours including thyroid cancer. PBF is believed to induce the translocation of PTTG into the cell nucleus where it can drive tumourigenesis via a number of different mechanisms. However, an independent transforming ability has been demonstrated both in vitro and in vivo, suggesting that PBF is itself a proto-oncogene. Studied in only a limited number of publications to date, PBF is emerging as a protein with a growing repertoire of roles. Recent data suggest that PBF possesses a complex multifunctionality in an increasing number of tumour settings. For example, PBF is upregulated by oestrogen and mediates oestrogen-stimulated cell invasion in breast cancer cells. In addition to a possible role in the induction of thyroid tumourigenesis, PBF overexpression in thyroid cancers inhibits iodide uptake. PBF has been shown to repress sodium iodide symporter (NIS) activity by transcriptional regulation of NIS expression through the human NIS upstream enhancer and further inhibits iodide uptake via a post-translational mechanism of NIS governing subcellular localisation. This review discusses the current data describing PBF expression and function in thyroid cancer and highlights PBF as a novel target for improving radioiodine uptake and thus prognosis in thyroid cancer.

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BSCI-12. Inhibition of melanoma brain metastasis by targeting miR-146a

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab071.011 ◽

2021 ◽

Vol 3 (Supplement_3) ◽

pp. iii3-iii3

Author(s):

Jiwei Wang ◽

Emma Rigg ◽

Taral R Lunavat ◽

Wenjing Zhou ◽

Zichao Feng ◽

...

Keyword(s):

Brain Metastasis ◽

Tumor Cells ◽

Cell Lines ◽

Western Blots ◽

Cell Growth And Migration ◽

Human Astrocytes ◽

And Migration ◽

The Brain

Abstract Background Melanoma has the highest propensity of any cancer to metastasize to the brain, with late-stage patients developing brain metastasis (MBM) in 40% of cases. Survival of patients with MBM is around 8 months with current therapies, illustrating the need for new treatments. MBM development is likely caused by molecular interactions between tumor cells and the brain, constituting the brain metastatic niche. miRNAs delivered by exosomes released by the primary tumor cells may play a role in niche establishment, yet the mechanisms are poorly understood. Here, the aim was to identify miRNAs released by exosomes from melanomas, which may be important in niche establishment and MBM progression. Materials and Methods miRNAs from exosomes collected from human astrocytes, melanocytes, and MBM cell lines were profiled to determine differential expression. Functional in vitro validation was performed by cell growth and migration assays, cytokine arrays, qPCR and Western blots. Functional in vivo studies were performed after miR knockdown in MBM cell lines. An in silico docking study was performed to determine drugs that potentially inhibit transcription of miR-146a to impede MBM development. Results miR-146a was the most upregulated miRNA in exosomes from MBM cells and was highly expressed in human and animal MBM samples. miR-146a mimics activated human astrocytes, shown by increased proliferation and migration, elevated expression of GFAP in vitro and in mouse brain tumor samples, and increased cytokine production. In animal studies, knockdown of miR-146a in MBM cells injected intracardially into mice reduced BM burden and increased animal survival. Based on the docking studies, deserpidine was found to be an effective inhibitor of MBM growth in vitro and in vivo. Conclusions MiR-146a may play an important role in MBM development, and deserpidine is a promising candidate for clinical use.

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P-444 Presence of pharmacological inhibitors of the PI3K/PTEN/Akt and mTOR signalling pathways during cryopreservation and organotypic cultures of murine ovaries limits early primordial follicle depletion

Human Reproduction ◽

10.1093/humrep/deab127.006 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

C Terren ◽

M Nisolle ◽

C Munaut

Keyword(s):

In Vitro Culture ◽

Organotypic Culture ◽

Primordial Follicle ◽

Slow Freezing ◽

Signalling Pathways ◽

Primordial Follicle Activation ◽

Mtor Signalling ◽

Follicle Activation

Abstract Study question Which signalling pathways are implicated in primordial follicle activation induced by cryopreservation and/or organotypic culture? Is it possible to limit this activation through pharmacological inhibitors? Summary answer Our findings provide support for the hypothesis that mTOR and PI3K inhibitors might represent an attractive tool to delay cryopreservation- and culture-induced primordial follicle activation. What is known already Cryopreservation of ovarian tissue containing immature primordial follicles followed by auto-transplantation (OTCTP) is the only option available to preserve the fertility of prepubertal patients or patients requiring urgent therapy for aggressive malignancies. However, a major obstacle in this process is follicular loss immediately after grafting, possibly due to slow neovascularization, apoptosis and/or massive follicular recruitment. In vitro and in vivo studies indicate that the PI3K/PTEN/Akt and mTOR signalling pathways are involved in follicle activation. The transplantation process seems to be the major cause of primordial follicle activation after OTCTP but information about how cryopreservation itself impacts follicle activation is sparse. Study design, size, duration Whole murine ovaries (4-8-weeks old) were cryopreserved by slow freezing and exposed to LY294002 (a powerful PI3K inhibitor) or rapamycin (a specific mTOR inhibitor) during cryopreservation and/or organotypic in vitro culture for a 24 h or 2 days. Participants/materials, setting, methods Western Blot and immunofluorescence analyses were used to determine the activation of PI3K/PTEN/Akt and mTOR signalling pathways in murine ovaries cryopreserved and/or organotypically cultured with/without inhibitors.Follicles were quantified according to their maturation degree on H&E stained histological sections. Main results and the role of chance Ratio of phosphorylated Akt or rps6 to total proteins (p-Akt/Akt and p-rps6/rps6) was increased in slow-frozen murine ovaries compared to control fresh ovaries, indicating an activation of the PI3K/PTEN/Akt and mTOR signalling pathways. The use of pharmacological inhibitors of follicle signalling pathways (LY294002 (25µM) and rapamycin (1µM)) during the cryopreservation process decreased p-Akt/Akt and p-rps6/rps6 ratios. In vitro organotypic culture for 24 h increased only the activation of the PI3K/PTEN/Akt pathway, as shown by increased p-Akt/Akt ratio in fresh ovaries cultured for 24 h compared to fresh non-cultured ovaries. This activation can be counteracted by cryopreservation of murine ovaries with rapamycin followed by in vitro culture for 24 h in the presence of LY294002. Follicle density quantifications indicated that when cryopreserved ovaries were maintained in culture for 2 days, a decrease of primordial follicle density concomitant with an increase of secondary and more mature follicles were found in comparison to slow-frozen/thawed ovaries without culture. Supplementation of the culture medium with LY294002 and rapamycin for 24 h or 2 days preserved primordial follicle densities compared to ovaries cultured without inhibitors. Limitations, reasons for caution This study is an in vitro study using murine ovaries. To analyze the efficiency of LY294002 and rapamycin to limit cryopreservation and transplantation induced follicle recruitment, these inhibitors should be tested in an in vivo model. Furthermore, these findings will need to be confirmed with human samples. Wider implications of the findings We showed for the first-time that the sequential use of pharmacological inhibitors, rapamycin during the slow freezing process followed by organotypic culture supplemented with LY294002, is effective to limit early primordial follicle depletion. Trial registration number /

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P16.08 Inhibition of melanoma brain metastasis by targeting miR-146a

Neuro-Oncology ◽

10.1093/neuonc/noab180.199 ◽

2021 ◽

Vol 23 (Supplement_2) ◽

pp. ii57-ii57

Author(s):

J Wang ◽

E K Rigg ◽

T R Lunavat ◽

W Zhou ◽

Z Feng ◽

...

Keyword(s):

Brain Metastasis ◽

Tumor Cells ◽

Cell Lines ◽

Western Blots ◽

Cell Growth And Migration ◽

Human Astrocytes ◽

And Migration ◽

The Brain

Abstract BACKGROUND Melanoma has the highest propensity of any cancer to metastasize to the brain, with late-stage patients developing brain metastasis (MBM) in 40% of cases. Survival of patients with MBM is around 8 months with current therapies, illustrating the need for new treatments. MBM development is likely caused by molecular interactions between tumor cells and the brain, constituting the brain metastatic niche. miRNAs delivered by exosomes released from the primary tumor cells may play a role in niche establishment, yet the mechanisms are poorly understood. Here, the aim was to identify miRNAs released by exosomes from melanomas, which may be important in niche establishment and MBM progression. MATERIAL AND METHODS miRNAs in exosomes collected from human astrocytes, melanocytes, and MBM cell lines were profiled to determine differential expression. Functional in vitro validation was performed by cell growth and migration assays, cytokine arrays, qPCR and Western blots. Functional in vivo studies were performed after miR knockdown in MBM cell lines. An in silico docking study was performed to determine drugs that potentially inhibit transcription of miR-146a to impede MBM development. RESULTS miR-146a was the most upregulated miRNA in exosomes from MBM cells and was highly expressed in human and animal MBM samples. miR-146a mimics activated human astrocytes, shown by increased proliferation and migration, elevated expression of GFAP in vitro and in mouse brain tumor samples, and increased cytokine production. In animal studies, knockdown of miR-146 in MBM cells injected intracardially into mice reduced BM burden and increased animal survival. Based on the docking studies, deserpidine was found to be an effective inhibitor of MBM growth in vitro and in vivo. CONCLUSION miR-146a may play an important role in MBM development, and deserpidine is a promising candidate for clinical use.

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PL3.6 Targeting GSK-3 activity promotes mitotic catastrophe via centrosome destabilisation and enhances the effect of radiotherapy in glioma models

Neuro-Oncology ◽

10.1093/neuonc/noz126.009 ◽

2019 ◽

Vol 21 (Supplement_3) ◽

pp. iii4-iii4

Author(s):

A Bruning-Richardson ◽

H Sanganee ◽

S Barry ◽

D Tams ◽

T Brend ◽

...

Keyword(s):

Cell Lines ◽

Cancer Progression ◽

Combination Treatment ◽

Single Agent ◽

Drug Penetration ◽

In Vivo Models ◽

Human Astrocytes ◽

Normal Human

Abstract BACKGROUND Targeting kinases as regulators of cellular processes that drive cancer progression is a promising approach to improve patient outcome in GBM management. The glycogen synthase kinase 3 (GSK-3) plays a role in cancer progression and is known for its pro-proliferative activity in gliomas. The anti-proliferative and cytotoxic effects of the GSK-3 inhibitor AZD2858 were assessed in relevant in vitro and in vivo glioma models to confirm GSK-3 as a suitable target for improved single agent or combination treatments. MATERIAL AND METHODS The immortalised cell line U251 and the patient derived cell lines GBM1 and GBM4 were used in in vitro studies including MTT, clonogenic survival, live cell imaging, immunofluorescence microscopy and flow cytometry to assess the cytotoxic and anti-proliferative effects of AZD2858. Observed anti-proliferative effects were investigated by microarray technology for the identification of target genes with known roles in cell proliferation. Clinical relevance of targeting GSK-3 with the inhibitor either for single agent or combination treatment strategies was determined by subcutaneous and orthotopic in vivo modelling. Whole mount mass spectroscopy was used to confirm drug penetration in orthotopic tumour models. RESULTS AZD2858 was cytotoxic at low micromolar concentrations and at sub-micromolar concentrations (0.01 - 1.0 μM) induced mitotic defects in all cell lines examined. Prolonged mitosis, centrosome disruption/duplication and cytokinetic failure leading to cell death featured prominently among the cell lines concomitant with an observed S-phase arrest. No cytotoxic or anti-proliferative effect was observed in normal human astrocytes. Analysis of the RNA microarray screen of AZD2858 treated glioma cells revealed the dysregulation of mitosis-associated genes including ASPM and PRC1, encoding proteins with known roles in cytokinesis. The anti-proliferative and cytotoxic effect of AZD2858 was also confirmed in both subcutaneous and orthotopic in vivo models. In addition, combination treatment with AZD2858 enhanced clinically relevant radiation doses leading to reduced tumour volume and improved survival in orthotopic in vivo models. CONCLUSION GSK-3 inhibition with the small molecule inhibitor AZD2858 led to cell death in glioma stem cells preventing normal centrosome function and promoting mitotic failure. Normal human astrocytes were not affected by treatment with the inhibitor at submicromolar concentrations. Drug penetration was observed alongside an enhanced effect of clinical radiotherapy doses in vivo. The reported aberrant centrosomal duplication may be a direct consequence of failed cytokinesis suggesting a role of GSK-3 in regulation of mitosis in glioma. GSK-3 is a promising target for combination treatment with radiation in GBM management and plays a role in mitosis-associated events in glioma biology.

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A Review of the Role of Neurotensin and Its Receptors in Colorectal Cancer

Gastroenterology Research and Practice ◽

10.1155/2017/6456257 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8 ◽

Cited By ~ 17

Author(s):

Shengyang Qiu ◽

Gianluca Pellino ◽

Francesca Fiorentino ◽

Shahnawaz Rasheed ◽

Ara Darzi ◽

...

Keyword(s):

Colorectal Cancer ◽

Animal Studies ◽

In Vivo Studies ◽

Signalling Pathways ◽

Diagnostic Biomarker ◽

Gi Tract ◽

Cellular Receptors

Neurotensin (NTS) is a physiologically occurring hormone which affects the function of the gastrointestinal (GI) tract. In recent years, NTS, acting through its cellular receptors (NTSR), has been implicated in the carcinogenesis of several cancers. In colorectal cancer (CRC), a significant body of evidence, from in vitro and in vivo studies, is available which elucidates the molecular biology of NTS/NTSR signalling and the resultant growth of CRC cells. There is growing clinical data from human studies which corroborate the role NTS/NTSR plays in the development of human CRC. Furthermore, blockade and modulation of the NTS/NTSR signalling pathways appears to reduce CRC growth in cell cultures and animal studies. Lastly, NTS/NTSR also shows potential of being utilised as a diagnostic biomarker for cancers as well as targets for functional imaging. We summarise the existing evidence and understanding of the role of NTS and its receptors in CRC.

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