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To explore whether the p17 protein of oncolytic avian reovirus (ARV) mediates cell migration and invadopodia formation, we applied several molecular biological approaches for studying the involved cellular factors and signal pathways. We found that ARV p17 activates the p53/phosphatase and tensin homolog (PTEN) pathway to suppress the focal adhesion kinase (FAK)/Src signaling and downstream signal molecules, thus inhibiting cell migration and the formation of invadopodia in murine melanoma cancer cell line (B16-F10). Importantly, p17-induced formation of invadopodia could be reversed in cells transfected with the mutant PTENC124A. p17 protein was found to significantly reduce the expression levels of tyrosine kinase substrate 5 (TKs5), Rab40b, non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1), and matrix metalloproteinases (MMP9), suggesting that TKs5 and Rab40b were transcriptionally downregulated by p17. Furthermore, we found that p17 suppresses the formation of the TKs5/NCK1 complex. Coexpression of TKs5 and Rab40b in B16-F10 cancer cells reversed p17-modulated suppression of the formation of invadopodia. This work provides new insights into p17-modulated suppression of invadopodia formation by activating the p53/PTEN pathway, suppressing the FAK/Src pathway, and inhibiting the formation of the TKs5/NCK1 complex.
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Movimiento Celular , Quinasa 1 de Adhesión Focal , Orthoreovirus Aviar , Podosomas , Transducción de Señal , Animales , Ratones , Orthoreovirus Aviar/fisiología , Orthoreovirus Aviar/genética , Línea Celular Tumoral , Podosomas/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Virus Oncolíticos/fisiología , Virus Oncolíticos/genética , Familia-src Quinasas/metabolismo , Familia-src Quinasas/genética , Proteínas Virales/metabolismo , Proteínas Virales/genética , Melanoma Experimental/terapia , Melanoma Experimental/patología , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genéticaRESUMEN
The remodeling of actin cytoskeleton of osteoclasts on the bone matrix is essential for osteoclastic resorption activity. A specific regulator of the osteoclast cytoskeleton, integrin αvß3, is known to provide a key role in the degradation of mineralized bone matrixes. Cilengitide is a potent inhibitor of integrins and is capable of affecting αvß3 receptors, and has anti-tumor and anti-angiogenic and apoptosis-inducing effects. However, its function on osteoclasts is not fully understood. Here, the cilengitide role on nuclear factor κB ligand-receptor activator (RANKL)-induced osteoclasts was explored. Cells were cultured with varying concentrations of cilengitide (0,0.002,0.2 and 20 µM) for 7 days, followed by detected via Cell Counting Kit-8, staining for tartrate resistant acid phosphatase (TRAP), F-actin ring formation, bone resorption assays, adhesion assays, immunoblotting assays, and real-time fluorescent quantitative PCR. Results demonstrated that cilengitide effectively restrained the functionality and formation of osteoclasts in a concentration-dependent manner, without causing any cytotoxic effects. Mechanistically, cilengitide inhibited osteoclast-relevant genes expression; meanwhile, cilengitide downregulated the expression of key signaling molecules associated with the osteoclast cytoskeleton, including focal adhesion kinase (FAK), integrin αvß3 and c-Src. Therefore, this results have confirmed that cilengitide regulates osteoclast activity by blocking the integrin αvß3 signal pathway resulting in diminished adhesion and bone resorption of osteoclasts.
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Disrupted osteoblastogenesis or aberrant activation of osteoclastogenesis usually results in the break of bone homeostasis thus causing bone-associated diseases like osteoporosis. Obacunone, as a natural compound present in citrus fruits, has been demonstrated for various biological activities including anti-cancer and anti-inflammatory properties. However, the role of obacunone in regulating osteoclastogenesis has not been elucidated so far. Here, using in vitro cell models of RANKL (Receptor activator of nuclear factor-kB ligand) and M-CSF (Macrophage-colony-stimulating factor)-induced osteoclastogenesis, we showed that obacunone inhibited osteoclast differentiation in RAW264.7 cells and bone marrow macrophages (BMMs), as evidenced by obacunone dose-dependent reduction in numbers of osteoclasts and downregulated expressions of osteoclastogenesis-associated key genes. The anti-osteoclastic properties of obacunone were associated with downregulated expressions of Integrin α1 and attenuated activation of Focal adhesion kinase (FAK) and Steroid receptor coactivator (Src) signaling. Functional Integrin α1 blockade or FAK-Src inhibition suppressed RANKL/M-CSF-induced osteoclastogenesis, while Integrin α1 overexpression or FAK/Src activation partially attenuated obacunone's effects on suppressing RANKL/M-CSF-induced osteoclast differentiation. Furthermore, in vivo administration of obacunone displayed super therapeutic effects in attenuating ovariectomy-induced bone loss in mice, as indicated by decreases in serum biomarkers of bone turnover, restoring of femur fracture maximum force, and reversing of the worsened bone-related parameters in ovariectomized animals. Taken together, these findings demonstrate that obacunone has pharmacological activities to suppress osteoclast differentiation through modulating the Integrin-FAK-Src pathway, and suggest that obacunone is a therapeutic candidate for the treatment and prevention of bone diseases such as osteoporosis.
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Resorción Ósea , Osteoporosis , Receptores de Esteroides , Femenino , Ratones , Animales , Osteogénesis , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Factor Estimulante de Colonias de Macrófagos/farmacología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Integrina alfa1/metabolismo , Diferenciación Celular , Osteoclastos/metabolismo , Osteoporosis/tratamiento farmacológico , Osteoporosis/metabolismo , Receptores de Esteroides/metabolismo , Ligando RANK/metabolismoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Taohong Siwu Decoction (THSWD) is a conventional traditional Chinese prescription aiming at promoting blood circulation and alleviating blood stasis. It is widely prescribed in instances of ischemic strokes, cardiovascular diseases, osteoporosis and bone fracture. However, its molecular functions in bone formation remain uncharacterized. AIM OF STUDY: This study aims to explore the potential effects of THSWD treatment on human bone marrow mesenchymal stem cells (BMSCs) proliferation, osteogenic differentiation, and migration. MATERIALS AND METHODS: BMSCs undergo osteogenic, adipogenic, and chondrogenic differentiation to determine cell stemness. BMSCs were treated with low dose (200 µg/ml), medium dose (400 µg/ml) and high dose (600 µg/ml) THSWD. The cell viability was determined by CCK-8 assays, the osteogenic differentiation ability was determined by alizarin red staining and ALP staining, and cell migration was determined by wound healing and transwell assays. The effect of THSWD on the vascular endothelial growth factor (VEGF)/focal adhesion kinase (FAK) pathway was determined by immunoblotting. RESULTS: THSWD time-dependently and dose-dependently promoted BMSC viability. Moreover, THSWD also promoted BMSC osteogenic differentiation and migration. As opposed to THSWD, VEGF receptor inhibitor Bevacizumab suppressed BMSC osteogenic differentiation and migration. In BMSCs that have been co-treated with THSWD and Bevacizumab, THSWD effects on BMSC functions were partially eliminated by Bevacizumab. Moreover, THSWD treatment boosted VEGF content in the supernatant and was conducive to the phosphorylation of FAK and Src, whereas Bevacizumab exerted opposite effects; similarly, Bevacizumab partially abolished THSWD effects on VEGF and FAK (Tyr397) and Src (Tyr418) phosphorylation. CONCLUSION: THSWD enhances the capacities of BMSCs to proliferate, differentiate, and migrate, possibly through VEGF and the FAK-Src, thereby improving fracture healing.
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Células Madre Mesenquimatosas , Factor A de Crecimiento Endotelial Vascular , Humanos , Proteína-Tirosina Quinasas de Adhesión Focal , Osteogénesis , Bevacizumab/farmacología , Diferenciación Celular , Factores de Crecimiento Endotelial Vascular , Curación de Fractura , Proliferación Celular , Células de la Médula Ósea , Células CultivadasRESUMEN
Historically, immunoglobulin (Ig) has been known as an antibody and is expressed only in B lineage cells; importantly, Ig light chains are conjugated to heavy chains to form intact Igs. However, in this study, we found a free Igκ light chain with a unique Vκ4-1/Jκ3 rearrangement (Vκ4-1/Jκ3-FLC) that was widely expressed in different non-B lineages and was overexpressed in cancer cells. Further study indicated that Vκ4-1/Jκ3-FLC was hydrophobic, formed obvious insoluble deposits in the extracellular matrix (ECM) and existed in free form. Functional analyses demonstrated that Vκ4-1/Jκ3-FLC promoted the proliferation, migration and metastasis of colon cancer cells in vitro and in vivo. Mechanistically, Vκ4-1/Jκ3-FLC bound to integrin ß1 and activated the FAK and Src pathways. More importantly, specific antibodies against the variable region of Vκ4-1/Jκ3-FLC significantly inhibited the growth of colon cancer tumors. Our findings suggested that Vκ4-1/Jκ3-FLC is a novel ECM protein and integrin ß1 ligand and that it is involved in cancer progression and is a potential therapeutic target in cancer, particularly colon cancer.
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Neoplasias del Colon , Integrina beta1 , Neoplasias del Colon/genética , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Cadenas kappa de Inmunoglobulina , Integrina beta1/genética , Integrina beta1/metabolismoRESUMEN
BACKGROUND: Tetraspanins are members of the 4-transmembrane protein superfamily (TM4SF) that function by recruiting many cell surface receptors and signaling proteins into tetraspanin-enriched microdomains (TEMs) that play vital roles in the regulation of key cellular processes including adhesion, motility, and proliferation. Tetraspanin7 (Tspan7) is a member of this superfamily that plays documented roles in hippocampal neurogenesis, synaptic transmission, and malignant transformation in certain tumor types. How Tspan7 influences the onset or progression of osteosarcoma (OS), however, remains to be defined. Herein, this study aimed to explore the relationship between Tspan7 and the malignant progression of OS, and its underlying mechanism of action. METHODS: In this study, the levels of Tspan7 expression in human OS cell lines were evaluated via qRT-PCR and western blotting. The effect of Tspan7 on proliferation was examined using CCK-8 and colony formation assays, while metastatic role of Tspan7 was assessed by functional assays both in vitro and in vivo. In addition, mass spectrometry and co-immunoprecipitation were performed to verify the interaction between Tspan7 and ß1 integrin, and western blotting was used to explore the mechanisms of Tspan7 in OS progresses. RESULTS: We found that Tspan7 is highly expressed in primary OS tumors and OS cell lines. Downregulation of Tspan7 significantly suppressed OS growth, metastasis, and attenuated epithelial-mesenchymal transition (EMT), while its overexpression had the opposite effects in vitro. Furthermore, it exhibited reduced OS pulmonary metastases in Tspan7-deleted mice comparing control mice in vivo. Additionally, we proved that Tspan7 interacted with ß1 integrin to facilitate OS metastasis through the activation of integrin-mediated downstream FAK-Src-Ras-ERK1/2 signaling pathway. CONCLUSION: In summary, this study demonstrates for the first time that Tspan7 promotes OS metastasis via interacting with ß1 integrin and activating the FAK-Src-Ras-ERK1/2 pathway, which could provide rationale for a new therapeutic strategy for OS.
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BACKGROUND: Gastric cancer (GC) is one of the most common cancers causing a poor prognosis worldwide. HOXA13, as a member of the homeobox (HOX) family, is involved in the regulation of cancer progression and has attracted increasing attention, as a potential novel target for anticancer strategies. However, the significance of HOXA13 in GC remains unclear. This article aims to explore the potential mechanism of HOXA13 in GC progression. METHODS: Quantitative real-time PCR was carried out to detect the expression of HOXA13 and FN1 and the correlation between HOXA13 and FN1 in GC tissues. In vitro assays were conducted to investigate the role of HOXA13 and FN1 in the malignant phenotypes of GC cells and the function of HOXA13 in the activation of the FAK/Src axis in GC cells. Coimmunoprecipitation was performed to reveal the relationship between ITGA5, ITGB1 and FN1 in GC cells. A dual luciferase assay was performed to assess miR-449a-targeted regulation of HOXA13 expression. RESULTS: Quantitative real-time PCR verified that HOXA13 was elevated and positively correlated with FN1 in GC. In vitro and in vivo assays demonstrated that high expression of HOXA13 promoted GC progression, especially metastasis. Mechanistically, rescue experiments, chromatin immunoprecipitation and dual luciferase assays revealed that HOXA13 directly bound to the FN1 promoter region to enhance the activation of the FAK/Src axis, leading to GC cell proliferation and metastasis. Furthermore, the result of a dual luciferase assay suggested that HOXA13 was directly targeted by miR-449a. CONCLUSIONS: Our results show that HOXA13 is a positive regulator of the FAK/Src axis mediated by FN1 in GC and promotes GC progression. Thus, targeting HOXA13, together with FN1, may provide a novel prospective anticancer strategy.
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BACKGROUND: The development of lethal cancer metastasis depends on the dynamic interactions between cancer cells and the tumor microenvironment, both of which are embedded in the extracellular matrix (ECM). The acquisition of resistance to detachment-induced apoptosis, also known as anoikis, is a critical step in the metastatic cascade. Thus, a more in-depth and systematic analysis is needed to identify the key drivers of anoikis resistance. METHODS: Genome-wide CRISPR/Cas9 knockout screen was used to identify critical drivers of anoikis resistance using SKOV3 cell line and found protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) as a candidate. Quantitative real-time PCR (qRT-PCR) and immune-histochemistry (IHC) were used to measure differentially expressed PCMT1 in primary tissues and metastatic cancer tissues. PCMT1 knockdown/knockout and overexpression were performed to investigate the functional role of PCMT1 in vitro and in vivo. The expression and regulation of PCMT1 and integrin-FAK-Src pathway were evaluated using immunoprecipitation followed by mass spectrometry (IP-MS), western blot analysis and live cell imaging. RESULTS: We found that PCMT1 enhanced cell migration, adhesion, and spheroid formation in vitro. Interestingly, PCMT1 was released from ovarian cancer cells, and interacted with the ECM protein LAMB3, which binds to integrin and activates FAK-Src signaling to promote cancer progression. Strikingly, treatment with an antibody against extracellular PCMT1 effectively reduced ovarian cancer cell invasion and adhesion. Our in vivo results indicated that overexpression of PCMT1 led to increased ascites formation and distant metastasis, whereas knockout of PCMT1 had the opposite effect. Importantly, PCMT1 was highly expressed in late-stage metastatic tumors compared to early-stage primary tumors. CONCLUSIONS: Through systematically identifying the drivers of anoikis resistance, we uncovered the contribution of PCMT1 to focal adhesion (FA) dynamics as well as cancer metastasis. Our study suggested that PCMT1 has the potential to be a therapeutic target in metastatic ovarian cancer.
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Sistemas CRISPR-Cas/genética , Neoplasias Ováricas/genética , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/metabolismo , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Desnudos , Persona de Mediana Edad , Metástasis de la Neoplasia , Neoplasias Ováricas/patologíaRESUMEN
Metastasis is the leading cause of death in breast cancer patients. Osthole, as an active compound detected in the traditional Chinese medicine Wenshen Zhuanggu Formula, has shown a promising anti-metastatic activity in human breast cancer cells, but the underlying mechanisms remain ambiguous. In this study we elucidated the anti-metastatic mechanisms of osthole in highly metastatic breast cancer cells and a zebrafish xenograft model. We showed that the expression of integrin α3 (ITGα3) and integrin ß5 (ITGß5) was upregulated in highly metastatic MDA-MB-231, MDA-MB-231BO breast cancer cell lines but was downregulated in poorly metastatic MCF-7 breast cancer cell line, which might be the key targets of osthole's anti-metastatic action. Furthermore, we showed that knockdown of ITGα3 and ITGß5 attenuated breast cancer cell migration and invasion possibly via suppression of FAK/Src/Rac1 pathway, whereas overexpression of ITGα3 and ITGß5 caused the opposite effects. Consistently, osthole significantly inhibited breast cancer metastasis by downregulating ITGα3/ITGß5 signaling in vitro and in vivo. These results provide new evidence that osthole may be developed as a candidate therapeutic drug for metastatic breast cancer.
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Neoplasias de la Mama , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Cumarinas/farmacología , Cumarinas/uso terapéutico , Femenino , Humanos , Invasividad Neoplásica/prevención & control , Pez CebraRESUMEN
[This corrects the article DOI: 10.3389/fcell.2021.607001.].
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Despite sustained effort, the prognosis of lung cancer remains poor and the therapeutic responses are limited. Cell movement ability is a prerequisite for lung cancer metastasis, which involves focal adhesion kinase (FAK)-mediated cell migration and invasion via complex formation with Src. Hence, FAK-Src signaling might be an effective target for anti-cancer treatment. ß-elemene, the major component of elemene extracted from Curcuma Rhizoma, exhibits broad-spectrum anti-tumor properties. However, the role of ß-elemene in lung cancer cell motility and its possible mechanism remain unknown. Herein, the role of ß-elemene in the migration and invasion of two non-small cell lung cancer (NSCLC) cell lines was investigated by performing wound-healing and Transwell assays. The mRNA expression levels of genes associated with motility, including RhoA, Rac1, Cac42, matrix metalloprotease (MMP)2 and MMP9, were examined by reverse transcription-quantitative polymerase chain reaction. To determine whether ß-elemene acts through FAK-Src signaling, western blotting was performed and the levels of phosphorylated FAK and Src were detected. The results indicated that ß-elemene inhibited the migration and invasion of A549 and NCI-H1299 (H1299) cells, while the motility-associated genes were de-regulated following exposure to ß-elemene. Furthermore, ß-elemene decreased the activity of FAK and Src. Overall, these results suggest that ß-elemene potentially inhibits NSCLC through FAK-Src signaling.
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Bone marrow mesenchymal stem cells (BMSCs) are beneficial to repair the damaged liver. Tumor-derived extracellular vesicles (EV) are notorious in tumor metastasis. But the mechanism underlying hepatoma cell-derived EVs in BMSCs and liver cancer remains unclear. We hypothesize that hepatoma cell-derived EVs compromise the effects of BMSCs on the metastasis of liver cancer. The differentially expressed microRNAs (miRNAs) were screened. HepG2 cells were transfected with miR-181d-5p mimic or inhibitor, and the EVs were isolated and incubated with BMSCs to evaluate the differentiation of BMSCs into fibroblasts. Hepatoma cells were cultured with BMSCs conditioned medium (CM) treated with HepG2-EVs to assess the malignant behaviors of hepatoma cells. The downstream genes and pathways of miR-181d-5p were analyzed and their involvement in the effect of EVs on BMSC differentiation was verified through functional rescue experiments. The nude mice were transplanted with BMSCs-CM or BMSCs-CM treated with HepG2-EVs, and then tumor growth and metastasis in vivo were assessed. HepG2-EVs promoted fibroblastic differentiation of BMSCs, and elevated levels of α-SMA, vimentin, and collagen in BMSCs. BMSCs-CM treated with HepG2-EVs stimulated the proliferation, migration, invasion and epithelial-mesenchymal-transition (EMT) of hepatoma cells. miR-181d-5p was the most upregulated in HepG2-EVs-treated BMSCs. miR-181d-5p targeted SOCS3 to activate the FAK/Src pathway and SOCS3 overexpression inactivated the FAK/Src pathway. Reduction of miR-181d-5p in HepG2-EVs or SOCS3 overexpression reduced the differentiation of BMSCs into fibroblasts, and compromised the promoting effect of HepG2-EVs-treated BMSCs-CM on hepatoma cells. In vivo, HepG2-EVs-treated BMSCs facilitated liver cancer growth and metastasis. In conclusion, HepG2-EVs promote the differentiation of BMSCs, and promote liver cancer metastasis through the delivery of miR-181d-5p and the SOCS3/FAK/Src pathway.
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Extracellular matrix (ECM) plays an important role in tissue repair, cell proliferation, and differentiation. Our previous study showed that collagen I and collagen V differently regulate the proliferation of rat pancreatic ß cells (INS-1 cells) through opposite influences on the nuclear translocation of ß-catenin. In this study, we investigated the ß-catenin pathway in INS-1 cells on dishes coated with collagen I or V. We found that nuclear translocation of the transcription factor Yes-associated protein (YAP) was enhanced by collagen I and suppressed by collagen V, but had no effect on INS-1 cell proliferation. Morphologically, INS-1 cells on collagen V-coated dishes showed stronger cell-to-cell adhesion, while the cells on collagen I-coated dishes showed weaker cell-to-cell adhesion in comparison with the cells on non-coated dishes. E-cadherin played an inhibitory role in the proliferation of INS-1 cells cultured on collagen I or collagen V coated dishes via regulation of the nuclear translocation of ß-catenin. Integrin ß1 was enhanced with collagen I, while it was repressed with collagen V. The integrin ß1 pathway positively regulated the cell proliferation. Inhibition of integrin ß1 pathway restored the protein level of E-cadherin and inhibited the nuclear translocation of ß-catenin in the cells on collagen I-coated dishes, but no effect was observed in the cells on collagen V-coated dishes. In conclusion, collagen I enhances the proliferation of INS-1 cells via the integrin ß1 and E-cadherin/ß-catenin signaling pathway. In INS-1 cells on collagen V-coated dishes, both integrin ß1 and E-cadherin/ß-catenin signal pathways are involved in the inhibition of proliferation.
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Integrina beta1 , beta Catenina , Animales , Cadherinas/metabolismo , Cadherinas/farmacología , Proliferación Celular , Colágeno/farmacología , Colágeno Tipo I/metabolismo , Integrina beta1/metabolismo , Integrina beta1/farmacología , Ratas , beta Catenina/metabolismoRESUMEN
BPDE (benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide), a metabolite of environmental carcinogenic BaP, weakens the migration and invasion of human villous trophoblast cells and may further induce miscarriage. However, the underlying mechanisms remain largely unknown. In this study, we identified that in trophoblast Swan 71 and HTR-8/SVneo cells, miR-hz02 upregulates the level of lnc-HZ02, which inhibits the expression of an RNA-binding protein HuR. HuR could interact with FAK mRNA and promote its mRNA stability, thus upregulating the FAK level and the FAK/SRC/PI3K/AKT pathway, and finally maintaining the normal migration and invasion of trophoblast cells. If trophoblast cells are exposed to BPDE, both miR-hz02 and lnc-HZ02 are upregulated, which reduce the level of HuR, weaken the interactions of HuR with FAK mRNA, downregulate FAK level and the FAK/SRC/PI3K/AKT pathway, and finally inhibit cell migration and invasion. This study provides a novel scientific understanding of the dysfunctions of human trophoblast cells.
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7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/toxicidad , Regulación hacia Abajo/efectos de los fármacos , Quinasa 1 de Adhesión Focal/metabolismo , MicroARNs/biosíntesis , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , ARN Largo no Codificante/biosíntesis , Trofoblastos/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Línea Celular Transformada , Humanos , Trofoblastos/patologíaRESUMEN
Ophiopogonin D, a steroidal glycoside extracted from the Traditional Chinese Medicine Ophiopogon japonicus, shows anti-tumor property in several lines of cancers; however, its effect on triple-negative breast cancer (TNBC) has not been investigated. In this study, the anti-metastatic effect of Ophiopogonin D in TNBC cells as well as the underlying mechanism in such process was explored. Ophiopogonin D dose-dependently decreased cell proliferation of MDA-MB-231 cells. Meanwhile, Ophiopogonin D significantly inhibited TGF-ß1-induced metastatic behavior of MDA-MB-231 cells, including EMT, anoikis resistance as well as migration and invasion, via suppressing MMP-9 activity. Mechanically, Ophiopogonin D achieved its effect through efficiently abolishing ITGB1 expression, thus reducing the phosphorylation of FAK, Src and AKT, as well as upregulating nuclear ß-catenin. ITGB1 overexpression partly recovered Ophiopogonin D's inhibitory effect on metastatic behavior via activating MMP-9. These results demonstrated that Ophiopogonin D could suppress TGF-ß1-mediated metastatic behavior of MDA-MB-231 cells by regulating ITGB1/FAK/Src/AKT/ß-catenin/MMP-9 signaling axis, which might provide new insight for the control of TNBC metastasis.
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Antineoplásicos/farmacología , Saponinas/farmacología , Espirostanos/farmacología , Neoplasias de la Mama Triple Negativas , Anoicis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Integrina beta1/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Cicatrización de Heridas/efectos de los fármacos , beta Catenina/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Paraspeckle protein 1 (PSPC1) overexpression in cancers is known to be the pro-metastatic switch of tumor progression associated with poor prognosis of cancer patients. However, the detail molecular mechanisms to facilitate cancer cell migration remain elusive. Here, we conducted integrated analysis of human phospho-kinase antibody array, transcriptome analysis with RNA-seq, and proteomic analysis of protein pulldown to study the molecular detail of PSPC1-potentiated phenotypical transformation, adhesion, and motility in human hepatocellular carcinoma (HCC) cells. We found that PSPC1 overexpression re-assembles and augments stress fiber formations to promote recruitment of focal adhesion contacts at the protruding edge to facilitate cell migration. PSPC1 activated focal adhesion-associated kinases especially FAK/Src signaling to enhance cell adhesion and motility toward extracellular matrix (ECM). Integrated transcriptome and gene set enrichment analysis indicated that PSPC1 modulated receptor tyrosine kinase IGF1R involved in the focal adhesion pathway and induction of diverse integrins expression. Knockdown IGF1R expression and treatment of IGF1R inhibitor suppressed PSPC1-induced cell motility. Interestingly, knockdown PSPC1-interacted paraspeckle components including NONO, FUS, and the lncRNA Neat1 abolished PSPC1-activated IGF1R expression. Together, PSPC1 overexpression induced focal adhesion formation and facilitated cell motility via activation of IGF1R signaling. PSPC1 overexpression in tumors could be a potential biomarker of target therapy with IGF1R inhibitor for improvement of HCC therapy.
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Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/fisiopatología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/fisiopatología , Proteínas de Unión al ARN/fisiología , Receptor IGF Tipo 1/fisiología , Carcinoma Hepatocelular/genética , Adhesión Celular/genética , Adhesión Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/fisiología , Quimiotaxis , Adhesiones Focales/genética , Adhesiones Focales/fisiología , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Hepáticas/genética , Proteómica , Motivo de Reconocimiento de ARN , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Receptor IGF Tipo 1/antagonistas & inhibidores , Receptor IGF Tipo 1/genética , Transducción de SeñalRESUMEN
Epithelial to mesenchymal transition (EMT) is a fundamental biological process that occurs during development and tumorigenesis. The Rho family of GTPases (Rho-family) is a well-characterized regulator of actin cytoskeleton that gives rise to EMT-associated cell activities. Meanwhile, there are in total at least 66 different Rho-GTPase-activating proteins (Rho-GAPs), which, as an upstream regulator, inactivate specific members of the Rho-family in a cell context-dependent manner. However, molecular roles of individual Rho-GAPs are poorly understood, particularly regarding their involvements in EMT. Here, based on comprehensive screening on the whole Rho-GAP family, we identified specific Rho-GAPs that are responsible for the maintenance of epithelial cell phenotypes, suppressing EMT in human mammary epithelial cells. Specifically, we revealed that at least two Rho-GAPs, that is, ARHGAP4 and SH3BP1, critically regulate the cell morphology. Among them, we focused on ARHGAP4 and demonstrated with multidisciplinary approaches that this specific Rho-GAP regulates epithelial/mesenchymal-selective marker expression, cell proliferation, migration, 3D morphogenesis, and focal adhesion/stress fiber-driven physical force generation in a manner reminiscent of the EMT process. Furthermore, we identified Septin9 with proteomic analyses as a negative regulator of ARHGAP4, which promotes the occurrence of EMT by activation of the FAK/Src signaling pathway. These findings shed light on the novel Rho-GAP-associated pathway in the EMT process under development and tumorigenesis.
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Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Septinas/metabolismo , Citoesqueleto de Actina/metabolismo , Adhesión Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Adhesiones Focales/metabolismo , GTP Fosfohidrolasas/metabolismo , Humanos , Células MCF-7 , Morfogénesis/fisiología , Proteómica/métodos , Transducción de Señal/fisiologíaRESUMEN
Maternal embryo leucine zipper kinase (MELK) has a higher expression level in a variety of cancers and involved in progression of colorectal cancer. The MELK expression levels in colorectal cancer tissues and cells were detected by RT-qPCR. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and transwell assays were used to examine the effect of the MELK konckdown on the proliferation, migration and invasion of colorectal cancer cells. Western blot analysis was used to detect the protein level of MELK and the downstream signaling pathways related proteins. Our findings indicated that MELK expression in colorectal cancer tissues was significantly higher than that in para-carcinoma tissues. Knockdown of MELK with shRNA had strong inhibition effects on the proliferation, migration and invasion of colorectal cancer cells. MELK knockdown could also decrease the phosphorylation level of AKT through FAK/Src pathway. Our results indicated downregulation of MELK retarded the progression of CRC by inhibition of the phosphorylation level of AKT through inactivating FAK/Src pathways. Therefore, MELK has the potential to be explored as a new therapeutic target and knockdown can be used as a potential treatment strategy for colorectal cancer.
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Neoplasias Colorrectales/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Desnudos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
Studies have found that miR-665 acted as a tumor suppressor or an oncogene in different malignancies. miR-665 expression was elevated in gastric adenocarcinoma tissues; however, its role and mechanism in this disease are not fully clarified. The expression of miR-665 and its target gene was detected in human gastric adenocarcinoma tissues and cells. Moreover, we analyzed the effects of miR-665 on the proliferation, migration, and epithelial-mesenchymal transition (EMT) of gastric adenocarcinoma cells as well as tumor growth in vivo. The mechanisms of miR-665 in gastric adenocarcinoma were investigated by using molecular biology techniques. We found miR-665 was upregulated and suppressor of cytokine signaling 3 (SOCS3) was downregulated in gastric adenocarcinoma tissues and cells. Elevated miR-665 was positively correlated with tumor size, lymph node metastasis, invasion depth, TNM stage, and poor differentiation in gastric adenocarcinoma patients. Overexpression of miR-665 promoted, whereas knockdown of miR-665 suppressed the proliferation, migration, and EMT of gastric adenocarcinoma cells. Furthermore, we demonstrated that miR-665 functioned through targeting SOCS3, followed by activation of the FAK/Src signaling pathway in gastric adenocarcinoma cells. miR-665 antagomir inhibited tumor growth as well as the activation of the FAK/Src pathway but increased SOCS3 expression in nude mice. In addition, miR-665 expression was negatively regulated by long noncoding RNA maternally expressed gene 3 (MEG3). In conclusion, miR-665 acted as an oncogene in gastric adenocarcinoma by inhibiting SOCS3 followed by activation of the FAK/Src pathway and it was negatively modulated by MEG3. miR-665 may be a promising therapeutic target for the treatment of gastric adenocarcinoma.
Asunto(s)
Adenocarcinoma/enzimología , Quinasa 1 de Adhesión Focal/metabolismo , MicroARNs/metabolismo , ARN Largo no Codificante/metabolismo , Neoplasias Gástricas/enzimología , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Activación Enzimática , Transición Epitelial-Mesenquimal , Femenino , Quinasa 1 de Adhesión Focal/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones Desnudos , MicroARNs/genética , Persona de Mediana Edad , ARN Largo no Codificante/genética , Transducción de Señal , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Proteína 3 Supresora de la Señalización de Citocinas/genética , Carga TumoralRESUMEN
Human breast cancer cell line, MDA-MB-231, is highly invasive and aggressive, compared to less invasive cell line, MCF-7. To explore the genes that might influence the malignancy of MDA-MB-231, DNA microarray analysis was performed. The results showed that G0/G1 switch 2 (G0S2) was one of the most highly expressed genes among the genes upregulated in MDA-MB-231. Although G0S2 acts as a direct inhibitor of adipose triglyceride lipase, action of G0S2 in cancer progression is not yet understood. To investigate whether G0S2 affects invasiveness of MDA-MB-231 cells, G0S2 expression was inhibited using siRNA, which led to decreased cell proliferation, migration, and invasion of MDA-MB-231 cells. Consequently, G0S2 inhibition inactivated integrinregulated FAK-Src signaling, which promoted Hippo signaling and inactivated ERK1/2 signaling. In addition, G0S2 downregulation decreased ß-catenin expression, while E-cadherin expression was increased. It was demonstrated for the first time that G0S2 mediates the Hippo pathway and induces epithelial to mesenchymal transition (EMT). Taken together, our results suggest that G0S2 is a major factor contributing to cell survival and metastasis of MDA-MB-231 cells.