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1.
EMBO Rep ; 24(11): e56850, 2023 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-37846507

RESUMEN

The remodeling and stiffening of the extracellular matrix (ECM) is a well-recognized modulator of breast cancer progression. How changes in the mechanical properties of the ECM are converted into biochemical signals that direct tumor cell migration and metastasis remain poorly characterized. Here, we describe a new role for the autophagy-inducing serine/threonine kinases ULK1 and ULK2 in mechanotransduction. We show that ULK1/2 activity inhibits the assembly of actin stress fibers and focal adhesions (FAs) and as a consequence impedes cell contraction and migration, independent of its role in autophagy. Mechanistically, we identify PXN/paxillin, a key component of the mechanotransducing machinery, as a direct binding partner and substrate of ULK1/2. ULK-mediated phosphorylation of PXN at S32 and S119 weakens homotypic interactions and liquid-liquid phase separation of PXN, impairing FA assembly, which in turn alters the mechanical properties of breast cancer cells and their response to mechanical stimuli. ULK1/2 and the well-characterized PXN regulator, FAK/Src, have opposing functions on mechanotransduction and compete for phosphorylation of adjacent serine and tyrosine residues. Taken together, our study reveals ULK1/2 as important regulator of PXN-dependent mechanotransduction.


Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Paxillin/metabolismo , Mecanotransducción Celular , Fosforilación , Movimiento Celular , Serina/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo
2.
Bioorg Chem ; 116: 105344, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34598088

RESUMEN

Src kinase activity controls diverse cellular functions, including cell growth, migration, adhesion, and survival. It is de-regulated in several cancers, including breast cancer, where it is highly expressed and phosphorylated. Thus, targeting Src by a small molecule is a feasible strategy for managing different breast cancer types. Several Src kinase inhibitors are available, including the FDA-approved drug (dasatinib). However, they are primarily ATP-competitive inhibitors that have been reported to lack specificity towards Src. We have a long-time interest in discovering protein kinase inhibitors that are non-competitive for ATP. In this project, three groups of 2'-aminospiro[pyrano[3,2-c]quinoline]-3'-carbonitrile derivatives were designed and synthesized, hypothesizing that small molecules with a spiro scaffold appended to a pyrano[3,2-c]quinoline analog could act as non-ATP competitive Src kinase inhibitors. 3b, 3c, and 3d inhibited Src kinase activity with IC50s of 4.9, 5.9, and 0.9 µM, respectively. At the same time, they did not impact the MDM2/p53 interaction in HEK293 cells, which has been reported to be affected by some spirocyclic compounds. 25 µM of 3b, 3c, or 3d did not inhibit the kinase activity of ERK2, JNK1, or p38-alpha in an in-vitro kinase assay. Steady-state kinetic studies for the effect of 3d on the ability of recombinant Src to phosphorylate its substrate (Srctide) revealed a non-ATP competitive inhibition mechanism. 1.6 µM of 3d was enough to diminish Src, Fak, and paxillin phosphorylation in the breast cancer cell lines MDA-MB-231 and MCF7. In the NCI screening, 3d induced broad tumor cytotoxicity for the NCI-60 cell lines, including all the breast cancer cell lines. The potency of 3b, 3c, and 3d to inhibit migration, proliferation, and colony formation of MDA-MB-231 and proliferation of MCF7 cells correlates with their potency to suppress Src kinase activity in the same cell line. Noticeably, the cell growth suppression and apoptosis induction in the tested cell lines can be attributed to the ability of the new derivatives to suppress the ERK and Akt survival pathways downstream of Src.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Desarrollo de Medicamentos , Inhibidores de Proteínas Quinasas/farmacología , Piranos/farmacología , Quinolinas/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HEK293 , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Piranos/síntesis química , Piranos/química , Quinolinas/síntesis química , Quinolinas/química , Relación Estructura-Actividad , Familia-src Quinasas/metabolismo
3.
Exp Cell Res ; 359(2): 394-404, 2017 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-28822708

RESUMEN

Breast cancer is the leading cause of cancer death in women worldwide which is closely related to metastasis. Our previous study has shown that MRTF-A promote the migration of MDA-MB-231 cells and WDR1 promotes breast cancer cell migration. But the exact molecular mechanism on metastasis is still not fully understood, we now report that WDR1 enhanced the effect of MRTF-A induced-MDA-MB-231 cell migration by promoting the expression of the EMT markers and migration markers via RhoA-MRTF-A signaling pathway. Importantly, WDR1 promoted the nuclear importion of MRTF-A by affecting the expression of nuclear transport protein importin. But WDR1 did not affect the expression of MRTF-A. Interestingly, MRTF-A promoted the expression of miR-206 via its promoter CArG box but miR-206 inhibits the migration of breast cancer cells through suppressing the expression of WDR1 and MRTF-A via targeted their 3'UTR. Our data thus provide important and novel insights into MRTF-A-miR-206-WDR1 form feedback loop to regulate breast cancer cell migration.


Asunto(s)
Retroalimentación Fisiológica , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Proteínas de Microfilamentos/genética , Transactivadores/genética , Regiones no Traducidas 3' , Secuencia de Bases , Sitios de Unión , Movimiento Celular , Células Epiteliales/metabolismo , Células Epiteliales/patología , Transición Epitelial-Mesenquimal , Femenino , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Células MCF-7 , MicroARNs/metabolismo , Proteínas de Microfilamentos/metabolismo , Regiones Promotoras Genéticas , Transducción de Señal , Transactivadores/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo
4.
Tumour Biol ; 37(6): 8249-58, 2016 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-26718214

RESUMEN

Adenosine monophosphate-activated protein kinase (AMPK) acts as a fuel gauge that maintains energy homeostasis in both normal and cancerous cells, and has emerged as a tumor suppressor. The present study aims to delineate the functional relationship between AMPK and transforming growth factor beta (TGF-ß). Our results showed that expression of liver kinase B1 (LKB1), an upstream kinase of AMPK, impeded TGF-ß-induced Smad phosphorylation and their transcriptional activity in breast cancer cells, whereas knockdown of LKB1 or AMPKα1 subunit by short hairpin RNA (shRNA) enhanced the effect of TGF-ß. Furthermore, AMPK activation reduced the promoter activity of TGF-ß1. In accordance, type 2 diabetic patients taking metformin displayed a trend of reduction of serum TGF-ß1, as compared with those without metformin. A significant reduction of serum TGF-ß1 was found in mice after treatment with metformin. These results suggest that AMPK inhibits the transcription of TGF-ß1, leading to reduction of its concentration in serum. Finally, metformin suppressed epithelial-to-mesenchymal transition of mammary epithelial cells. Taken together, our study demonstrates that AMPK exerts multiple actions on TGF-ß signaling and supports that AMPK can serve as a therapeutic drug target for breast cancer.


Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Neoplasias de la Mama/tratamiento farmacológico , Hipoglucemiantes/farmacología , Metformina/farmacología , Proteínas Serina-Treonina Quinasas/genética , Factor de Crecimiento Transformador beta1/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Anciano , Animales , Western Blotting , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Cicatrización de Heridas
5.
IUBMB Life ; 67(3): 202-17, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25854163

RESUMEN

Breast cancer is the leading cause of cancer death in women worldwide which is closely related to metastasis. But the exact molecular mechanism of metastasis is still not fully understood. We now report that both MRTF-A and STAT3 play important roles in migration of MDA-MB-231 breast cancer cells. Moreover, MRTF-A and STAT3 synergistically increased MDA-MB-231 cell migration by promoting the expression of migration markers urokinase-type plasminogen activator (uPA) and osteopontin (OPN) and inhibiting the expression of breast cancer metastasis suppressor 1 (BRMS1). Luciferase reporter assays demonstrated that MRTF-A and STAT3 do not affect transcription of the BRMS1 promoter. Instead, we identified a newly molecular mechanism by which MRTF-A and STAT3 synergistically controlled MDA-MB-231 cell migration by recruiting DNMT1 to hypermethylate the promoter of BRMS1 and thus affect the expression of BRMS1. Interestingly, physical interaction between MRTF-A and STAT3 synergistically promotes the transactivity of DNMT1 by binding to the GAS element within the DNMT1 promoter. Our data thus provide important and novel insights into the roles of MRTF-A and STAT3 in regulating MDA-MB-231 cell migration.


Asunto(s)
Neoplasias de la Mama/patología , Proteínas Represoras/genética , Factor de Transcripción STAT3/metabolismo , Transactivadores/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Osteopontina/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Factor de Transcripción STAT3/genética , Transactivadores/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo
6.
Biomedicines ; 10(5)2022 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-35625882

RESUMEN

The development of peptide inhibitors against intracellular targets depends upon the dual challenge of achieving a high affinity and specificity for the target and maintaining cellular permeability for biological activity. Previous efforts to develop bicyclic peptides targeted to the Grb7 signalling protein implicated in HER2+ve cancer progression have resulted in improved affinity. However, these same peptides demonstrated a lowered activity due to their decreased ability to penetrate cell membranes. Here, we report the testing of a new series of bicyclic G7 peptides designed to possess improved bioactivity. We discovered that the incorporation of two amino acids (Phe-Pro, Phe-Trp or Phe-Arg) within the bicyclic peptide framework maintains an enhanced binding affinity for the Grb7-SH2 domain compared to that of the first-generation monocyclic peptide G7-18NATE. Structure determination using X-ray crystallography revealed that the mode of binding by the expanded bicyclic G7 peptide is analogous to that of G7-18NATE. Interestingly, while the bicyclic peptide containing Phe-Trp did not display the highest affinity for Grb7-SH2 in the series, it was the most potent inhibitor of HER2+ve SKBR3 breast cancer cell migration when coupled to Penetratin. Together, this demonstrates that peptide flexibility as well as the amino acid tryptophan can play important roles in the uptake of peptides into the cell.

7.
Cell Calcium ; 97: 102435, 2021 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-34167050

RESUMEN

The Transient Receptor Potential Vanilloid type 2 (TRPV2) channel is highly selective for Ca2+ and can be activated by lipids, such as LysoPhosphatidylCholine (LPC). LPC analogues, such as the synthetic alkyl-ether-lipid edelfosine or the endogenous alkyl-ether-lipid Platelet Activating Factor (PAF), modulates ion channels in cancer cells. This opens the way to develop alkyl-ether-lipids for the modulation of TRPV2 in cancer. Here, we investigated the role of 2-Acetamido-2-Deoxy-l-O-Hexadecyl-rac-Glycero-3-PhosphatidylCholine (AD-HGPC), a new alkyl-ether-lipid (LPC analogue), on TRPV2 trafficking and its impact on Ca2+ -dependent cell migration. The effect of AD-HGPC on the TRPV2 channel and tumour process was further investigated using calcium imaging and an in vivo mouse model. Using molecular and pharmacological approaches, we dissected the mechanism implicated in alkyl-ether-lipids sensitive TRPV2 trafficking. We found that TRPV2 promotes constitutive Ca2+ entry, leading to migration of highly metastatic breast cancer cell lines through the PI3K/Akt-Girdin axis. AD-HGPC addresses the functional TRPV2 channel in the plasma membrane through Golgi stimulation and PI3K/Akt/Rac-dependent cytoskeletal reorganization, leading to constitutive Ca2+ entry and breast cancer cell migration (without affecting the development of metastasis), in a mouse model. We describe, for the first time, the biological role of a new alkyl-ether-lipid on TRPV2 channel trafficking in breast cancer cells and highlight the potential modulation of TRPV2 by alkyl-ether-lipids as a novel avenue for research in the treatment of metastatic cancer.

8.
Cell Signal ; 66: 109488, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31785332

RESUMEN

Despite improvements in cancer early detection and treatment, metastatic breast cancer remains deadly. Current therapeutic approaches have very limited efficacy in patients with triple negative breast cancer. Among the many mechanisms associated that contribute to cancer progression, signaling through the CXCL12-CXCR4 is an essential step in cancer cell migration. We previously demonstrated the formation of CXCL12-CXCL4 heterodimers (Carlson et al., 2013). Here, we investigated whether CXCL12-CXCL4 heterodimers alter tumor cell migration. CXCL12 alone dose-dependently promoted the MDA-MB 231 cell migration (p < .05), which could be prevented by blocking the CXCR4 receptor. The addition of CXCL4 inhibited the CXCL12-induced cell migration (p < .05). Using NMR spectroscopy, we identified the CXCL4-CXCL12 binding interface. Moreover, we generated a CXCL4-derived peptide homolog of the binding interface that mimicked the activity of native CXCL4 protein. These results confirm the formation of CXCL12-CXCL4 heterodimers and their inhibitory effects on the migration of breast tumors cells. These findings suggest that specific peptides mimicking heterodimerization of CXCL12 might prevent breast cancer cell migration.


Asunto(s)
Adenocarcinoma/metabolismo , Quimiocina CXCL12/metabolismo , Factor Plaquetario 4/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Adenocarcinoma/patología , Línea Celular Tumoral , Movimiento Celular , Femenino , Humanos , Multimerización de Proteína , Neoplasias de la Mama Triple Negativas/patología
9.
J Control Release ; 289: 79-93, 2018 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-30149048

RESUMEN

Overexpression of RhoC protein in breast cancer patients has been linked to increased cancer cell invasion, migration, and metastases. Suppressing RhoC expression in aggressive breast cancer cells using silencing RNA (siRNA) molecules is a viable strategy to inhibit the metastatic spread of breast cancer. In this report, we describe the synthesis of a series of asymmetric pH-sensitive, membrane-destabilizing polymers engineered to complex anti-RhoC siRNA molecules forming "smart" nanoparticles. Using ß-CD as the particle core, polyethylene glycol (PEG) chains were conjugated to the primary face via non-cleavable bonds and amphiphilic polymers incorporating hydrophobic and cationic monomers were grafted to the secondary face via acid-labile linkages. We investigated the effect of PEG molecular weight (2 & 5 kDa) on transfection capacity and serum stability of the formed particles. We evaluated the efficacy of EPPT1 peptides presented on the free tips of the PEG brush to function as a targeting ligand against underglycosylated MUC1 (uMUC1) receptors overexpressed on the surface of metastatic breast cancer cells. Results show that "smart" nanoparticles successfully delivered anti-RhoC siRNA into the cytoplasm of aggressive SUM149 and MDA-MB-231 breast cancer cells, which resulted in a dose-dependent inhibition of cell migration and invasion. Further, EPPT1-targeted nanoparticles demonstrate a synergistic inhibition of cell migration and invasion imparted via RhoC knockdown and EPPT1-mediated signaling via the uMUC1 receptor.


Asunto(s)
Neoplasias de la Mama/terapia , Nanocápsulas/química , Invasividad Neoplásica/prevención & control , Oligopéptidos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Proteína rhoC de Unión a GTP/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Movimiento Celular , Proliferación Celular , Liberación de Fármacos , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen/métodos , Humanos , Mucina-1/metabolismo , Oligopéptidos/química , Polietilenglicoles/química , Polimerizacion , Transfección , Microambiente Tumoral , beta-Ciclodextrinas/química , Proteína rhoC de Unión a GTP/metabolismo
10.
Horm Cancer ; 8(1): 16-27, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-28050799

RESUMEN

Thyroid hormones (TH) play a fundamental role in diverse processes, including cellular movement. Cell migration requires the integration of events that induce changes in cell structure towards the direction of migration. These actions are driven by actin remodeling and stabilized by the development of adhesion sites to extracellular matrix via transmembrane receptors linked to the actin cytoskeleton. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that promotes cell migration and invasion through the control of focal adhesion turnover. In this work, we demonstrate that the thyroid hormone triiodothyronine (T3) regulates actin remodeling and cell movement in breast cancer T-47D cells through the recruitment of FAK. T3 controls FAK phosphorylation and translocation at sites where focal adhesion complexes are assembled. This process is triggered via rapid signaling to integrin αV/ß3, Src, phosphatidylinositol 3-OH kinase (PI3K), and FAK. In addition, we established a cellular model with different concentration of T3 levels: normal, absence, and excess in T-47D breast cancer cells. We found that the expression of Src, FAK, and PI3K remained at normal levels in the excess of T3 model, while it was significantly reduced in the absence model. In conclusion, these results suggest a novel role for T3 as an important modulator of cell migration, providing a starting point for the development of new therapeutic strategies for breast cancer treatment.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Quinasa 1 de Adhesión Focal/metabolismo , Triyodotironina/metabolismo , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Femenino , Adhesiones Focales/metabolismo , Humanos , Integrina alfaVbeta3/metabolismo , Invasividad Neoplásica , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Transporte de Proteínas , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Transducción de Señal
11.
Cell Signal ; 26(11): 2370-80, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25038455

RESUMEN

Breast cancer is the leading cause of cancer death in women worldwide which is closely related to metastasis. But the exact molecular mechanism on metastasis is still not fully understood; we now report that both MRTF-A and STAT3 play important role in breast cancer migration of MDA-MB-231 cells. Moreover, MRTF-A and STAT3 synergistically increased MDA-MB-231 cell migration by promoting the expression of migration markers Myl-9 and Cyr-61. Importantly, we identified a detailed molecular mechanism of MDA-MB-231 cell migration controlled via physical interaction between MRTF-A and STAT3, which synergistically promote the transactivity of the migration marker Myl-9 and Cyr-61 by CArG box binding. Interestingly, the two signaling pathways RhoA-MRTF-A and JAK-STAT3 across talk to regulate MDA-MB-231 cell migration. Our data thus provide important and novel insights into the roles of MRTF-A and STAT3 in regulating MDA-MB-231 cell migration.


Asunto(s)
Biomarcadores de Tumor/biosíntesis , Neoplasias de la Mama/metabolismo , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Factor de Transcripción STAT3/biosíntesis , Transactivadores/biosíntesis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proteína 61 Rica en Cisteína/biosíntesis , Proteína 61 Rica en Cisteína/genética , Femenino , Humanos , Cadenas Ligeras de Miosina/biosíntesis , Cadenas Ligeras de Miosina/genética , Proteínas de Neoplasias/genética , Elementos de Respuesta , Factor de Transcripción STAT3/genética , Transactivadores/genética , Activación Transcripcional/genética
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