Src family kinase activity regulates adhesion, spreading and migration of pancreatic endocrine tumour cells

Pancreatic endocrine tumours (PETs) are rare and ‘indolent’ neoplasms that usually develop metastatic lesions and exhibit poor response to standard medical treatments. Few studies have investigated pathways responsible for PET cell growth and invasion and no alternative therapeutic strategies have been proposed. In a recent microarray analysis for genes up-regulated in PETs, we have described the up-regulation of soluble Src family tyrosine kinases in this neoplasia, which may represent potentially promising candidates for therapy. Herein, we have investigated the expression and function of Src family kinases in PETS and PET cell lines. Western blot analysis indicated that Src is highly abundant in the PET cell lines CM and QGP-1. Immunohistochemistry and Western blot analyses showed that Src is up-regulated also in human PET lesions. Pharmacological inhibition of Src family kinases by the specific inhibitor PP2 strongly interfered with adhesion, spreading and migration of PET cell lines. Accordingly, the actin cytoskeleton was profoundly altered after inhibition of Src kinases, whereas even prolonged incubation with PP2 exerted no effect on cell cycle progression and/or apoptosis of PET cells. A transient increase in tyrosine phosphorylation of a subset of proteins was observed in QGP-1 cells adhering to the plate, with a peak at 75 min after seeding, when approximately 80% of cells were attached. Inhibition of Src kinases caused a dramatic reduction in the phosphorylation of proteins with different molecular weight that were isolated from the cell extracts by anti-phosphotyrosine immunoprecipitation or pull-down with the SH2 domain of Src. Among them, the docking protein p130Cas interacted with Src and is a major substrate of the Src kinases in QGP-1 cells undergoing adhesion. Our results suggest that Src kinases play a specific role during adhesion, spreading and migration of PET cells and may indicate therapeutical approaches directed to limiting the metastatic potential of these cells.

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MicroRNA-708 targeting ZNF549 regulates colon adenocarcinoma development through PI3K/AKt pathway

Scientific Reports ◽

10.1038/s41598-020-73929-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Zhidong Zhao ◽

Xianju Qin

Keyword(s):

Cell Proliferation ◽

Western Blot ◽

Cell Lines ◽

Colon Adenocarcinoma ◽

Signal Pathway ◽

Luciferase Reporter ◽

Mesenchymal Transition ◽

Cell Proliferation And Migration ◽

And Migration ◽

Proliferation And Migration

Abstract Colon adenocarcinoma (COAD) is the most common type of gastrointestinal cancer and is still the third leading cause of cancer-related mortality worldwide. Therefore, finding new and promising drugs to eradicate cancer may be a feasible method to treat COAD patients. Cys2-His2 zinc finger proteins (ZFPs) is one of the largest transcription factor family and many of them are highly involved in regulation of cell differentiation, proliferation, apoptosis, and neoplastic transformation. In this study, we identified a tumor-inhibiting factor, ZNF549, which expressed lowly in COAD tissues and COAD cell lines (HT29, HCT116, SW480, LoVo, and SW620). Overexpression of ZNF549 inhibit the ability of COAD cell proliferation and migration. On the contrary, decreasing the ZNF549 expression level promote the ability of COAD cell proliferation and migration. Through bioinformatics analysis, we found that ZNF549 was a potential target of hsa-miR-708-5p (miR-708-5p). Furthermore, we verified the possibility of miR-708-5p targeting the ZNF549 gene, and miR-708-5p inhibited the expression of ZNF549 by luciferase reporter assays, qRT-PCR and western blot assays. Moreover, the relationship between miR-708-5p and phosphatidylinositol 3-kinase/AKt (PI3K/AKt) signal pathway was elucidated. Overexpression and inhibition of miR-708-5p resulted in increased and decreased expression of p-AKt and p-PI3K in HCT116 cells, respectively. RT-qPCR and western blot assays results demonstrated that miR-708-5p regulated COAD cells development by promoting the process of Epithelial-mesenchymal transition (EMT) through PI3K/AKt signaling pathway. In summary, our findings demonstrated that ZNF549, the target gene of miR-708-5p, functions as a tumor suppressor to inhibit COAD cell lines proliferation and migration through regulate the PI3K/AKt signal pathway.

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Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.186 ◽

1995 ◽

Vol 15 (1) ◽

pp. 186-197 ◽

Cited By ~ 135

Author(s):

S Richard ◽

D Yu ◽

K J Blumer ◽

D Hausladen ◽

M W Olszowy ◽

...

Keyword(s):

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Sh3 Domain ◽

Sh2 Domain ◽

Src Family Kinases ◽

Src Family Kinase ◽

Sh3 Domains ◽

Sh2 Domains ◽

Src Family Tyrosine Kinases

src family tyrosine kinases contain two noncatalytic domains termed src homology 3 (SH3) and SH2 domains. Although several other signal transduction molecules also contain tandemly occurring SH3 and SH2 domains, the function of these closely spaced domains is not well understood. To identify the role of the SH3 domains of src family tyrosine kinases, we sought to identify proteins that interacted with this domain. By using the yeast two-hybrid system, we identified p62, a tyrosine-phosphorylated protein that associates with p21ras GTPase-activating protein, as a src family kinase SH3-domain-binding protein. Reconstitution of complexes containing p62 and the src family kinase p59fyn in HeLa cells demonstrated that complex formation resulted in tyrosine phosphorylation of p62 and was mediated by both the SH3 and SH2 domains of p59fyn. The phosphorylation of p62 by p59fyn required an intact SH3 domain, demonstrating that one function of the src family kinase SH3 domains is to bind and present certain substrates to the kinase. As p62 contains at least five SH3-domain-binding motifs and multiple tyrosine phosphorylation sites, p62 may interact with other signalling molecules via SH3 and SH2 domain interactions. Here we show that the SH3 and/or SH2 domains of the signalling proteins Grb2 and phospholipase C gamma-1 can interact with p62 both in vitro and in vivo. Thus, we propose that one function of the tandemly occurring SH3 and SH2 domains of src family kinases is to bind p62, a multifunctional SH3 and SH2 domain adapter protein, linking src family kinases to downstream effector and regulatory molecules.

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The SH3 domain of p56lck is involved in binding to phosphatidylinositol 3'-kinase from T lymphocytes.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7408 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7408-7417 ◽

Cited By ~ 55

Author(s):

L B Vogel ◽

D J Fujita

Keyword(s):

Tyrosine Kinases ◽

Rous Sarcoma Virus ◽

Sh3 Domain ◽

Sh2 Domain ◽

Phosphatidylinositol 3 Kinase ◽

Rous Sarcoma ◽

Cell Extracts ◽

Pi3k Activity ◽

Sarcoma Virus ◽

Chicken Embryo Fibroblasts

Many of the Src-like tyrosine kinases are thought to participate in multiprotein complexes that modulate transmembrane signalling through tyrosine phosphorylation. We have used in vitro binding studies employing bacterially expressed glutathione S-transferase-p56lck fusion proteins and cell extracts to map regions on p56lck that are involved in binding to phosphatidylinositol 3'-kinase (PI3K). Deletions within the SH3 domain of p56lck abolished binding of PI3K activity from T-cell lysates, whereas deletion of the SH2 domain caused only a slight reduction in the level of PI3K activity bound to p56lck sequences. The binding of PI3K from T-cell extracts to p56lck was not blocked by antiphosphotyrosine antibodies, but p56lck-bound PI3K activity was sensitive to phosphatase treatment. The SH3 domain of p56lck also bound the majority of PI3K activity from uninfected chicken embryo fibroblasts. However, a drastically different binding specificity was observed with use of extracts of Rous sarcoma virus v-src-transformed cells, in which the majority of PI3K activity bound to the SH2 domain of p56lck in a phosphotyrosine-dependent manner. These results suggest that are two modes of PI3K binding to p56lck, and presumably to other Src-like tyrosine kinases. In one mode, PI3K from T cells or uninfected chicken embryo fibroblasts binds predominantly to the SH3 domain of p56lck. In the other mode, involving PI3K from Rous sarcoma virus-transformed cells, binding is largely phosphotyrosine dependent and requires the SH2 domain of p56lck.

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DNA synthesis induced by some but not all growth factors requires Src family protein tyrosine kinases.

Molecular and Cellular Biology ◽

10.1128/mcb.15.2.1102 ◽

1995 ◽

Vol 15 (2) ◽

pp. 1102-1109 ◽

Cited By ~ 172

Author(s):

S Roche ◽

M Koegl ◽

M V Barone ◽

M F Roussel ◽

S A Courtneidge

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Dna Synthesis ◽

Tyrosine Kinases ◽

Neutralizing Antibodies ◽

S Phase ◽

Src Family Kinases ◽

Protein Tyrosine Kinases ◽

Src Kinases ◽

Protein Tyrosine

The Src family of protein tyrosine kinases have been implicated in the response of cells to several ligands. These include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and colony stimulating factor type 1 (CSF-1, in macrophages and in fibroblasts engineered to express the receptor). We recently described a microinjection approach which we used to demonstrate that Src family kinases are required for PDGF-induced S phase entry of fibroblasts. We now use this approach to ask whether other ligands also require Src kinases to stimulate cells to replicate DNA. An antibody specific for the carboxy terminus of Src, Fyn, and Yes (anti-cst.1) inhibited Src kinase activity in vitro and caused morphological reversion of Src transformed cells in vivo. Microinjection of this antibody was used to demonstrate that Src kinases were required for both CSF-1 and EGF to drive cells into the S phase. Expression of a kinase-inactive form of Src family kinases also prevented EGF- and CSF-1-stimulated DNA synthesis. However, even though the Src family kinases were necessary for both PDGF- and EGF-induced DNA synthesis in Swiss 3T3 cells, the responses to two other potent growth factors for these cells, lysophosphatidic acid and bombesin, were unaffected by the neutralizing antibodies. Therefore, some but not all growth factors required functional Src family kinases to transmit mitogenic responses.

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Elucidating distinct tumorigenic pathways in nodular versus superficial spreading melanoma.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.8544 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 8544-8544

Author(s):

Joshua Andrew Farhadian ◽

Kathryn E. O'Reilly ◽

Eleazar Vega-Saenz de Miera ◽

Fei Ye ◽

Jiri Zavadil ◽

...

Keyword(s):

Western Blot ◽

Cell Lines ◽

Melanoma Cell ◽

Growth Phase ◽

Superficial Spreading ◽

Superficial Spreading Melanoma ◽

Metastatic Melanoma Cell ◽

And Migration ◽

Melanoma Cell Lines ◽

Proliferation And Migration

8544 Background: Superficial spreading melanoma (SSM) and nodular melanoma (NM), which account for 70% and 20% of all melanoma diagnoses respectively, are believed to represent sequential phases of linear progression from radial to vertical growth. Recent evidence from several groups including ours challenges this linear progression model and suggests that SSM and NM are biologically distinct. In this study, we focused on identifying tumorigenic pathways that are differentially activated in melanoma subtypes and that can be therapeutically exploited. Methods: We performed Protein Pathway Array on SSM/radial growth phase (RGP) and NM/vertical growth phase (VGP) primary melanoma cell lines to determine expression levels of 141 tumorigenic proteins/phospho-proteins. Differential protein expression was determined using the Pavlidis Template Matching algorithm in TIGR Multi Experiment Viewer. Differentially expressed proteins were validated in an expanded panel of RGP, VGP, and metastatic melanoma cell lines using western blot. Constitutively active, overexpressed proteins in VGP melanoma were down-regulated in cell lines using several small molecule inhibitors and the effects on proliferation and migration were assessed. Results: 17 (12%) proteins/phospho-proteins are significantly overexpressed in VGP versus RGP melanoma (p<0.05). Western blot confirmed the results of 5 proteins/phospho-proteins. Each of those 5 was equally overexpressed in VGP and metastatic melanoma cell lines. Phospho-p90 ribosomal s6 kinase (RSK) was prioritized for functional studies based on its role in both the MAPK and AKT signaling cascades, two tumorigenic pathways often dysregulated in melanoma. Constitutive phosphorylation of p90 RSK in VGP, but not RGP melanoma was observed in response to serum starvation. Small molecule inhibition of phospho-p90 RSK attenuated proliferation and migration (p<0.05) in VGP melanoma. Conclusions: Data demonstrate discrete tumorigenic pathways are activated in nodular versus superficial spreading melanoma and suggest that phospho-p90 RSK might be a viable target against nodular melanoma.

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miR-27b Targets HOXB8 to Inhibit Malignant Behaviors of Osteosarcoma

Technology in Cancer Research & Treatment ◽

10.1177/1533033819870791 ◽

2019 ◽

Vol 18 ◽

pp. 153303381987079

Author(s):

Yingyong Wu ◽

Jinyun Peng

Keyword(s):

Cell Proliferation ◽

Western Blot ◽

Cell Lines ◽

Colony Formation ◽

Cell Counting ◽

Osteosarcoma Cell ◽

Polymerase Chain ◽

And Migration ◽

Insight Into

MicroRNAs function as either tumor suppressor or oncogene in human cancers. This study aimed to explore the role of miR-27b in osteosarcoma. Expression of miR-27b or homeobox B8 in osteosarcoma cell lines was analyzed by quantitative real-time polymerase chain reaction and Western blot, respectively. Luciferase activity reporter assay and Western blot were conducted to explore the association between miR-27b and homeobox B8. Cell Counting Kit-8, colony formation assay, and wound-healing assay were performed to investigate the role of miR-27b or homeobox B8 on cell proliferation, colony formation, and cell migration. Expression of miR-27b was significantly reduced, while homeobox B8 was increased in osteosarcoma cell lines. In addition, homeobox B8 was validated as a direct target of homeobox B8. Moreover, miR-27b regulates osteosarcoma cell proliferation, colony formation, and migration through targeting homeobox B8. Taken together, our study provides novel insight into the progression of osteosarcoma, and the miR-27b–homeobox B8 axis identified may be developed as therapeutic targets against hepatocellular carcinoma in the future.

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Sequential phosphorylation of protein band 3 by Syk and Lyn tyrosine kinases in intact human erythrocytes: identification of primary and secondary phosphorylation sites

Blood ◽

10.1182/blood.v96.4.1550 ◽

2000 ◽

Vol 96 (4) ◽

pp. 1550-1557 ◽

Cited By ~ 86

Author(s):

Anna Maria Brunati ◽

Luciana Bordin ◽

Giulio Clari ◽

Peter James ◽

Manfredo Quadroni ◽

...

Keyword(s):

Tyrosine Kinases ◽

Transmembrane Protein ◽

Specific Inhibitor ◽

Protein Band ◽

Band 3 ◽

Sh2 Domain ◽

Human Erythrocytes ◽

Red Cell ◽

Red Cell Membranes ◽

Peptide Maps

Abstract Treatment of intact human erythrocytes with pervanadate induces Tyr (Y)-phosphorylation of the transmembrane protein band 3; in parallel, the activity of the immunoprecipitated tyrosine kinases Syk and Lyn is increased. When erythrocytes are incubated with pervanadate together with PP1, a specific inhibitor of Src kinases, including Lyn, the Y-phosphorylation of band 3 is only partially reduced. Indeed, the PP1-resistant phosphorylation of band 3 precedes and is a prerequisite for its coimmunoprecipitation with Lyn, which interacts with the phosphoprotein via the SH2 domain of the enzyme, as proven by binding competition experiments. Upon recruitment to primarily phosphorylated band 3, Lyn catalyzes the secondary phosphorylation of the transmembrane protein. These data are consistent with the view that band 3 is phosphorylated in intact erythrocytes by both PP1-resistant (most likely Syk) and PP1-inhibited (most likely Lyn) tyrosine kinases according to a sequential phosphorylation process. Similar radiolabeled peptide maps are obtained by tryptic digestion of32P-band 3 isolated from either pervanadate-treated erythrocytes or red cell membranes incubated with exogenous Syk and Lyn. It has also been demonstrated by means of mass spectrometry that the primary phosphorylation of band 3 occurs at Y8 and Y21, while the secondary phosphorylation affects Y359 and Y904.

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PD-1 Blockade Delays Tumor Growth by Inhibiting an Intrinsic SHP2/Ras/MAPK Signalling in Thyroid Cancer Cells

10.21203/rs.3.rs-100301/v1 ◽

2020 ◽

Author(s):

Federica Liotti ◽

Narender Kumar ◽

Nella Prevete ◽

Maria Marotta ◽

Daniela Sorriento ◽

...

Keyword(s):

Cell Proliferation ◽

Thyroid Cancer ◽

Western Blot ◽

Cancer Cells ◽

Cell Lines ◽

Cancer Immunity ◽

Anti Cancer ◽

Mapk Signalling ◽

And Migration ◽

Proliferation And Migration

Abstract Background: The programmed cell death-1 (PD-1) receptor and its ligands PD-L1 and PD-L2 are immune checkpoints that suppress anti-cancer immunity. Typically, cancer cells express the PD-Ls that bind PD-1 on immune cells, inhibiting their activity. Recently, PD-1 expression has also been found in cancer cells. Here, we analysed expression and functions of PD-1 in thyroid cancer (TC).Methods: PD-1 expression was evaluated by immunohistochemistry on human TC samples and by RT-PCR; western blot and FACS on TC cell lines. Proliferation and migration of TC cells in culture were assessed by BrdU incorporation and Boyden chamber assays. Biochemical studies were performed by western blot, immunoprecipitation, pull-down and phosphatase assays. TC cell tumorigenicity was assessed by xenotransplants in nude mice.Results: Human TC specimens (47%), but not normal thyroids, displayed PD-1 expression in epithelial cells, which significantly correlated with tumour stage and lymph-node metastasis. PD-1 was also constitutively expressed on TC cell lines. PD-1 overexpression/stimulation promoted TC cell proliferation and migration. Accordingly, PD-1 genetic/pharmacologic inhibition caused the opposite effects. Mechanistically, PD-1 recruited the SHP2 phosphatase to the plasma membrane and potentiated its phosphatase activity. SHP2 enhanced Ras activation by dephosphorylating its inhibitory tyrosine 32, thus triggering the MAPK cascade. SHP2, BRAF and MEK were necessary for PD-1-mediated biologic functions. PD-1 inhibition decreased, while PD-1 enforced expression facilitated, TC cell xenograft growth in mice by affecting tumour cell proliferation.Conclusions: PD-1 circuit blockade in TC, besides restoring anti-cancer immunity, could also directly impair TC cell growth by inhibiting the SHP2/Ras/MAPK signalling pathway.

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Model of Human Tongue Squamous Cell Lines Stably Transfected with Human Papillomavirus (HPV)16 E6 and E7 Genes and Biological Characteristic Analysis

BioMed Research International ◽

10.1155/2021/9968691 ◽

2021 ◽

Vol 2021 ◽

pp. 1-12

Author(s):

ZiLian Lan ◽

Ziyao Jia ◽

Hengyuan Guo ◽

Zhaoshou Yang ◽

Zifan Yang ◽

...

Keyword(s):

Human Papillomavirus ◽

Oral Cancer ◽

Western Blot ◽

Cell Lines ◽

Squamous Cell ◽

Characteristic Analysis ◽

Migration Ability ◽

Human Tongue ◽

And Migration ◽

Proliferation And Migration

Background. Human tongue squamous cell carcinoma (TSCC) is the most common oral cancer with the highest human papillomavirus (HPV) infection rate in oral cancer. The purpose of this study was to research the correlation between HPV and TSCC. Method. Plasmid pEGFP/HPV16 E6E7 and plasmid pEGFP/no HPV16 E6E7 were constructed. TSCC cell lines SCC9 and SCC15 were infected by liposome transfection and would be highly selected by antibiotic. Fluorescence imaging, PCR, and Western blot were used to detect the expression of HPV16 E6E7 in cells. The biological characteristics were detected by CCK-8, wound healing assay, qRT-PCR, and Western blot. Result. TSCC cell lines transfected with HPV16 E6E7 gene were successfully established and identified. And the proliferation and migration ability of the TSCC cell lines infected with HPV16 E6E7 gene were significantly stronger than that of the blank group. Conclusion. TSCC cell lines infected with HPV16 E6E7 with significantly higher ability of proliferation and migration were more malignant than those not infected with HPV16 E6E7.

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VSNL1 Promotes Gastric Cancer Cell Proliferation and Migration by Regulating P2X3/P2Y2 Receptors and Is a Clinical Indicator of Poor Prognosis in Gastric Cancer Patients

Gastroenterology Research and Practice ◽

10.1155/2020/7241942 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9

Author(s):

Qiao-Qiong Dai ◽

Yuan-Yu Wang ◽

Yu-peng Jiang ◽

Li Li ◽

Hui-Ju Wang

Keyword(s):

Gastric Cancer ◽

Western Blot ◽

Cell Lines ◽

Colony Formation ◽

Lentiviral Vector ◽

Prognostic Significance ◽

Migration And Invasion ◽

And Migration ◽

Gastric Cancer Patients ◽

Proliferation And Migration

Purpose. The aim of this study was to investigate the role of Visinin Like 1 (VSNL1) in the proliferation and migration of gastric cancer (GC) cells as well as its clinical prognostic significance. Methods. To this end, we evaluated VSNL1 expression in GC tissues and cell lines by real-time PCR and immunohistochemistry. To further explore the effects of VSNL1, a lentiviral vector expressing a short hairpin RNA (shRNA) against VSNL1 was constructed and transduced into the GC cell lines BGC-823 and SGC-7901. The interference efficiency of VSNL1-shRNA was determined by western blot. The effects of VSNL1 on the migration and invasion of GC cells as well as the expression of P2X3/P2Y2 were explored using MTS, colony formation, migration, and western blot assays. Results. VSNL1 mRNA and protein levels were increased in GC tissues and cell lines. Furthermore, VSNL1 expression was positively correlated with Lauren’s classification, lymph node metastasis, distant metastasis, TNM stage, and prognosis. VSNL1 expression was inversely correlated with the 5-year survival rate of GC patients. VSNL1 expression was markedly reduced in cells transduced with lentivirus expressing shRNA against VSNL1, and inhibiting VSNL1 expression significantly suppressed cell growth, migration, and colony formation and reduced the expression of P2X3/P2Y2. Conclusion. VSNL1 may promote the proliferation and migration of GC cells by regulating P2X3 and P2Y2 expression. VSNL1 plays important roles in GC development and metastasis and may be correlated with patient prognosis.

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