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1.
Exp Cell Res ; 411(1): 112731, 2022 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-34270980

RESUMEN

Adhesion of cells to each other and to the extracellular matrix (ECM) are both required for cellular functions. Cell-to-cell adhesion is mediated by cadherins, and their engagement triggers the activation of Stat3, which offers a potent survival signal. Adhesion to the ECM on the other hand, activates FAK which attracts and activates Src, as well as receptor tyrosine kinases (RTKs), the PI3k/Akt and Ras/Erk pathways. However, the effect of cell density upon FAK and Akt activity has not been examined. We now demonstrate that, interestingly, despite being potent Stat3 activators, Src and RTKs are unable to activate Stat3 in sparsely growing (i.e., without cadherin engagement), non-neoplastic cells attached to the ECM. In contrast, cell aggregation (i.e., cadherin engagement in the absence of adhesion to a solid substratum) was found to activate both Stat3 and Akt. Pharmacologic or genetic reduction of FAK activity abolished Akt activity at low densities, indicating that FAK is an important activator of Akt in this setting. Notably, FAK knockout increased cellular sensitivity to the Stat3 inhibitor CPA7, while FAK reintroduction restored resistance to this drug. These findings suggest a complementary role of integrin/FAK/Akt and cadherin/Stat3-mediated pro-survival pathways, which may be of significance during neoplastic transformation and metastasis.

2.
Exp Cell Res ; 404(1): 112601, 2021 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-33957118

RESUMEN

Adhesion of cells to each other and to the extracellular matrix (ECM) are both required for cellular functions. Cell-to-cell adhesion is mediated by cadherins and their engagement triggers the activation of Stat3, which offers a potent survival signal. Adhesion to the ECM on the other hand, activates FAK which attracts and activates Src, as well as receptor tyrosine kinases (RTKs), the PI3k/Akt and Ras/Erk pathways. However, the effect of cell density upon FAK and Akt activity has not been examined. We now demonstrate that, interestingly, despite being potent Stat3 activators, Src and RTKs are unable to activate Stat3 in sparsely growing (i.e., without cadherin engagement), non-neoplastic cells attached to the ECM. In contrast, cell aggregation (i.e., cadherin engagement in the absence of adhesion to a solid substratum) was found to activate both Stat3 and Akt. Pharmacologic or genetic reduction of FAK activity abolished Akt activity at low densities, indicating that FAK is an important activator of Akt in this setting. Notably, FAK knockout increased cellular sensitivity to the Stat3 inhibitor CPA7, while FAK reintroduction restored resistance to this drug. These findings suggest a complementary role of integrin/FAK/Akt and cadherin/Stat3-mediated pro-survival pathways, which may be of significance during neoplastic transformation and metastasis.


Asunto(s)
Cadherinas/metabolismo , Fibroblastos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Adhesión Celular/fisiología , Supervivencia Celular/fisiología , Transformación Celular Neoplásica/metabolismo , Matriz Extracelular/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Humanos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología
3.
Biochem Cell Biol ; 97(5): 638-646, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-30986357

RESUMEN

We recently demonstrated that Cav1 (caveolin-1) is a negative regulator of Stat3 (signal transducer and activator of transcription-3) activity in mouse fibroblasts and human lung carcinoma SHP77 cells. We now examined whether the cellular context may affect their levels as well as the relationship between them, by assessing Cav1 and Stat3-ptyr705 amounts in different cell lines. In MDA-MB-231, A549, and HaCat cells, Cav1 levels were high and Stat3-ptyr705 levels were low, consistent with the notion of a negative effect of endogenous Cav1 on Stat3-ptyr705 levels in these lines. In addition, manipulation of Cav1 levels revealed a negative effect in MCF7 and mouse fibroblast cells, while Cav1 upregulation induced apoptosis in MCF7 cells. In contrast, however, line MRC9 had high Cav1 and high Stat3-ptyr705 levels, indicating that high Cav1 is insufficient to reduce Stat3-ptyr705 levels in this line. MCF7 and LuCi6 cells had very low Cav1 and Stat3-ptyr705 levels, indicating that the low Stat3-ptyr705 can be independent from Cav1 levels altogether. Our results reveal a further level of complexity in the relationship between Cav1 and Stat3-ptyr705 than previously thought. In addition, we demonstrate that in a feedback loop, Stat3 inhibition upregulates Cav1 in HeLa cells but not in other lines tested.


Asunto(s)
Neoplasias de la Mama/metabolismo , Caveolina 1/metabolismo , Neoplasias Pulmonares/metabolismo , Factor de Transcripción STAT3/metabolismo , Tirosina/metabolismo , Animales , Caveolina 1/antagonistas & inhibidores , Células Cultivadas , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C
4.
Exp Cell Res ; 361(1): 112-125, 2017 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-29031557

RESUMEN

It was previously demonstrated that differentiation of some established breast epithelial cell lines requires confluence and stimulation with hydrocortisone, insulin and prolactin inducers. We and others previously demonstrated that E-cadherin engagement, which is favored under conditions of confluence, increases the levels and activity of the Rac small GTPase. To investigate the functional relationship between the transforming ability of Rac and its role as an integral component of the differentiative E-cadherin signaling pathway, we introduced a mutationally activated form of Rac, RacV12, into the mouse breast epithelium-derived cell line, HC11. Our results demonstrate that the strength of the Rac signal is key for the outcome of the differentiation process; cRac1 is critically required for differentiation, and at low levels, mutationally activated RacV12 is able to increase differentiation, presumably reinforcing the E-cadherin/Rac differentiative signal. However, high RacV12 expression blocked differentiation concomitant with E-cadherin downregulation, while inducing neoplastic transformation. Therefore, the intensity of the Rac signal is a central determinant in the balance between cell proliferation vs differentiation, two fundamentally opposed processes, a finding which could also have important therapeutic implications.


Asunto(s)
Cadherinas/metabolismo , Diferenciación Celular , Células Epiteliales/citología , Glándulas Mamarias Animales/citología , Proteínas de Unión al GTP rac/metabolismo , Animales , Células Cultivadas , Células Epiteliales/metabolismo , Femenino , Glándulas Mamarias Animales/metabolismo , Ratones , Transducción de Señal
6.
Protein Expr Purif ; 129: 1-8, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27590918

RESUMEN

STAT5B, a ubiquitious transcription factor, has been implicated in the onset and progression of several cancers. Since the inhibition of STAT activity holds significant therapeutic potential, there is a need to develop high-throughput biophysical screening platforms to rapidly identify high affinity binders of STATs. Biophysical assays would benefit from the efficient and cost-effective production of high purity, full-length STAT proteins. Herein, we have sampled a large region of protein expression and purification space that has substantially increased recombinant STAT5B protein yields from Escherichia coli. The identity of STAT5B was confirmed by Western blotting analysis, while the results of a fluorescence polarization assay indicated that the purified protein is correctly folded and functional. A thermal shift assay was employed to assess the effect of various osmolytes on the stability of the protein. The protein expression conditions identified in this study allowed for more efficient and higher recovery of soluble STAT5B protein, which will enable a broad range of biophysical studies and facilitate high-throughput STAT5B drug screening.


Asunto(s)
Escherichia coli/metabolismo , Expresión Génica , Factor de Transcripción STAT5 , Escherichia coli/genética , Humanos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Factor de Transcripción STAT5/biosíntesis , Factor de Transcripción STAT5/química , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/aislamiento & purificación , Solubilidad
7.
Bioorg Med Chem Lett ; 26(18): 4542-4547, 2016 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-27520940

RESUMEN

Protein conjugation with ubiquitin and ubiquitin-like small molecules, such as UFM1, is important for promoting cancer cell survival and proliferation. Herein, the development of the first selective micromolar inhibitor of the UBA5 E1 enzyme that initiates UFM1 protein conjugation is described. This organometallic inhibitor incorporates adenosine and zinc(II)cyclen within its core scaffold and inhibits UBA5 noncompetitively and selectively over other E1 enzymes and a panel of human kinases. Furthermore, this compound selectively impedes the cellular proliferation (above 50µM) of cancer cells containing higher levels of UBA5. This inhibitor may be used to further probe the intracellular role of the UFM1 pathway in disease progression.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Línea Celular Tumoral , Inhibidores Enzimáticos/química , Humanos
8.
Exp Cell Res ; 336(2): 223-31, 2015 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-26187405

RESUMEN

Gap junctions are channels that connect the cytoplasm of adjacent cells. Oncogenes such as the middle Tumor antigen of polyoma virus (mT) are known to suppress gap junctional, intercellular communication (GJIC). mT associates with and is tyrosine-phosphorylated by cSrc family members. Specific mT phosphotyrosines provide docking sites for the phosphotyrosine binding domain of Shc (mT-tyr250) or the SH2 domain of the regulatory subunit of the phosphatidylinositol-3 kinase (PI3k, mT-tyr315). Binding results in the activation of their downstream signaling cascades, Ras/Raf/Erk and PI3 kinase/Akt, respectively, both of which are needed for full neoplastic transformation. To examine the effect of mT-initiated pathways upon gap junctional communication, GJIC was quantitated in rat liver epithelial T51B cells expressing mT-mutants, using a novel technique of in situ electroporation. The results demonstrate for the first time that, although even low levels of wild-type mT are sufficient to interrupt gap junctional communication, GJIC suppression still requires an intact tyr-250 site, that is activation of the Ras pathway. In sharp contrast, activation of the PI3k pathway is not required for GJIC suppression, indicating that GJIC suppression is independent of full neoplastic conversion and the concomitant morphological changes. Interestingly, expression of a constitutively active, myristylated form of the catalytic subunit of PI3k, p110, or the constitutively active mutants E545K and H1047R increased GJIC, while pharmacological inhibition of PI3k eliminated communication. Therefore, although PI3k is growth promoting and in an activated form it can act as an oncogene, it actually plays a positive role upon gap junctional, intercellular communication.


Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Comunicación Celular/genética , Uniones Comunicantes/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Androstadienos/farmacología , Animales , Sitios de Unión/genética , Dominio Catalítico , Línea Celular , Transformación Celular Neoplásica/genética , Cromonas/farmacología , Electroporación , Hígado/citología , Morfolinas/farmacología , Mutación/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Unión Proteica/genética , Ratas , Transducción de Señal , Wortmanina
9.
J Med Chem ; 66(24): 16658-16679, 2023 12 28.
Artículo en Inglés | MEDLINE | ID: mdl-38060537

RESUMEN

Histone deacetylases (HDACs) have emerged as powerful epigenetic modifiers of histone/non-histone proteins via catalyzing the deacetylation of ε-N-acetyl lysines. The dysregulated activity of these Zn2+-dependent hydrolases has been broadly implicated in disease, notably cancer. Clinically, the recurring dose-limiting toxicities of first-generation HDACi sparked a paradigm shift toward safer isoform-specific molecules. With pervasive roles in aggressive diseases, there remains a need for novel approaches to target these enzymes. Herein, we report the discovery of YSR734, a first-in-class covalent HDACi, with a 2-aminobenzanilide Zn2+ chelate and a pentafluorobenzenesulfonamide electrophile. This class I selective proof of concept modified HDAC2Cys274 (catalytic domain), with nM potency against HDAC1-3, sub-µM activity in MV4-11 cells, and limited cytotoxicity in MRC-9 fibroblasts. In C2C12 myoblasts, YSR734 activated muscle-specific biomarkers myogenin/Cav3, causing potent differentiation into myotubes (applications in Duchenne Muscular Dystrophy). Current efforts are focused on improving in vivo ADME toward a preclinical covalent HDACi.


Asunto(s)
Leucemia Mieloide Aguda , Distrofia Muscular de Duchenne , Humanos , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Isoformas de Proteínas/metabolismo , Histona Desacetilasas/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico
10.
BMC Cancer ; 12: 605, 2012 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-23244248

RESUMEN

BACKGROUND: Neoplastic transformation of cultured cells by a number of oncogenes such as src suppresses gap junctional, intercellular communication (GJIC); however, the role of Src and its effector Signal transducer and activator of transcription-3 (Stat3) upon GJIC in non small cell lung cancer (NSCLC) has not been defined. Immunohistochemical analysis revealed high Src activity in NSCLC biopsy samples compared to normal tissues. Here we explored the potential effect of Src and Stat3 upon GJIC, by assessing the levels of tyr418-phosphorylated Src and tyr705-phosphorylated Stat3, respectively, in a panel of NSCLC cell lines. METHODS: Gap junctional communication was examined by electroporating the fluorescent dye Lucifer yellow into cells grown on a transparent electrode, followed by observation of the migration of the dye to the adjacent, non-electroporated cells under fluorescence illumination. RESULTS: An inverse relationship between Src activity levels and GJIC was noted; in five lines with high Src activity GJIC was absent, while two lines with extensive GJIC (QU-DB and SK-LuCi6) had low Src levels, similar to a non-transformed, immortalised lung epithelial cell line. Interestingly, examination of the mechanism indicated that Stat3 inhibition in any of the NSCLC lines expressing high endogenous Src activity levels, or in cells where Src was exogenously transduced, did not restore GJIC. On the contrary, Stat3 downregulation in immortalised lung epithelial cells or in the NSCLC lines displaying extensive GJIC actually suppressed junctional permeability. CONCLUSIONS: Our findings demonstrate that although Stat3 is generally growth promoting and in an activated form it can act as an oncogene, it is actually required for gap junctional communication both in nontransformed lung epithelial cells and in certain lung cancer lines that retain extensive GJIC.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Comunicación Celular/fisiología , Uniones Comunicantes/fisiología , Neoplasias Pulmonares/metabolismo , Factor de Transcripción STAT3/fisiología , Adhesión Celular , Conexinas/metabolismo , Regulación hacia Abajo , Humanos , Inmunohistoquímica , Fosforilación , Factor de Transcripción STAT3/antagonistas & inhibidores , Células Tumorales Cultivadas , Familia-src Quinasas/metabolismo
11.
Exp Cell Res ; 317(13): 1787-95, 2011 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-21619876

RESUMEN

The signal transducer and activator of transcription-3 (Stat3) is a member of the STAT family of cytoplasmic transcription factors. Overactivation of Stat3 is detected with high frequency in human cancer and is considered a molecular abnormality that supports the tumor phenotype. Despite concerted investigative efforts, the molecular mechanisms leading to the aberrant Stat3 activation and Stat3-mediated transformation and tumorigenesis are still not clearly defined. Recent evidence reveals a crosstalk close relationship between Stat3 signaling and members of the Rho family of small GTPases, including Rac1, Cdc42 and RhoA. Specifically, Rac1, acting in a complex with the MgcRacGAP (male germ cell RacGAP), promotes tyrosine phosphorylation of Stat3 by the IL6-receptor family/Jak kinase complex, as well as its translocation to the nucleus. Studies have further revealed that the mutational activation of Rac1 and Cdc42 results in Stat3 activation, which occurs in part through the upregulation of IL6 family cytokines that in turn stimulates Stat3 through the Jak kinases. Interestingly, evidence also shows that the engagement of cadherins, cell to cell adhesion molecules, specifically induces a striking increase in Rac1 and Cdc42 protein levels and activity, which in turn results in Stat3 activation. In this review we integrate recent findings clarifying the role of the Rho family GTPases in Stat3 activation in the context of malignant progression.


Asunto(s)
Neoplasias/metabolismo , Factor de Transcripción STAT3/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Citocinas/metabolismo , Humanos , Modelos Biológicos
12.
Cancer Gene Ther ; 29(10): 1502-1513, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35411090

RESUMEN

We previously demonstrated that engagement of cadherins, cell to cell adhesion molecules, triggers a dramatic increase in levels and activity of the Rac/Cdc42 small GTPases, which is followed by secretion of IL6 family cytokines and activation of their common receptor, gp130, in an autocrine manner. This results in phosphorylation of the Signal Transducer and Activator of Transcription-3 (Stat3) on tyrosine-705, which then dimerizes, migrates to the nucleus, and activates transcription of genes involved in cell division and survival. In the present report we demonstrate that, in mouse Balb/c3T3 fibroblasts, mutationally activated Src527F also increases Rac levels, leading to secretion of IL6 family cytokines and gp130 activation, which triggers the Stat3-ptyr705 increase. Interestingly, our results also demonstrate that cadherin-11 is required to preserve gp130 levels for IL6 family signaling. At the same time, however, activated Src527F downregulates cadherin-11, in a quantitative manner. As a result, Src527F expression to intermediate levels allows sufficient cadherin-11, hence gp130 levels for Stat3 activation, as expected. However, expressed to high levels, Src527F eliminates cadherin-11, hence gp130 signaling, thus abolishing Stat3-ptyr705 stimulation. Taken together, these data establish for the first time a loop between Src, cadherin-11, gp130, and Stat3 activation. This fine balance between Src527F and cadherin-11 levels which is required for Stat3 activation and cellular survival could have significant therapeutic implications.


Asunto(s)
Interleucina-6 , Factor de Transcripción STAT3 , Animales , Ratones , Cadherinas/genética , Cadherinas/metabolismo , Receptor gp130 de Citocinas/genética , Receptor gp130 de Citocinas/metabolismo , Citocinas/metabolismo , Fibroblastos/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Fosforilación , Factor de Transcripción STAT3/metabolismo , Tirosina/metabolismo , Genes src , Proteínas de Unión al GTP rac/metabolismo
13.
J Med Chem ; 65(4): 3193-3217, 2022 02 24.
Artículo en Inglés | MEDLINE | ID: mdl-35119267

RESUMEN

Histone deacetylase 6 (HDAC6) has been targeted in clinical studies for anticancer effects due to its role in oncogenic transformation and metastasis. Through a second-generation structure-activity relationship (SAR) study, the design, and biological evaluation of the selective HDAC6 inhibitor NN-390 is reported. With nanomolar HDAC6 potency, >200-550-fold selectivity for HDAC6 in analogous HDAC isoform functional assays, potent intracellular target engagement, and robust cellular efficacy in cancer cell lines, NN-390 is the first HDAC6-selective inhibitor to show therapeutic potential in metastatic Group 3 medulloblastoma (MB), an aggressive pediatric brain tumor often associated with leptomeningeal metastases and therapy resistance. MB stem cells contribute to these patients' poor clinical outcomes. NN-390 selectively targets this cell population with a 44.3-fold therapeutic margin between patient-derived Group 3 MB cells in comparison to healthy neural stem cells. NN-390 demonstrated a 45-fold increased potency over HDAC6-selective clinical candidate citarinostat. In summary, HDAC6-selective molecules demonstrated in vitro therapeutic potential against Group 3 MB.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Histona Desacetilasa 6/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/farmacología , Meduloblastoma/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Simulación por Computador , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Células Madre Neoplásicas/efectos de los fármacos , Relación Estructura-Actividad
14.
Exp Cell Res ; 316(5): 875-86, 2010 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-19852956

RESUMEN

Rac1 (Rac) is a member of the Rho family of small GTPases which controls cell migration by regulating the organization of actin filaments. Previous results suggested that mutationally activated forms of the Rho GTPases can activate the Signal Transducer and Activator of Transcription-3 (Stat3), but the exact mechanism is a matter of controversy. We recently demonstrated that Stat3 activity of cultured cells increases dramatically following E-cadherin engagement. To better understand this pathway, we now compared Stat3 activity levels in mouse HC11 cells before and after expression of the mutationally activated Rac1 (Rac(V12)), at different cell densities. The results revealed for the first time a dramatic increase in protein levels and activity of both the endogenous Rac and Rac(V12) with cell density, which was due to inhibition of proteasomal degradation. In addition, Rac(V12)-expressing cells had higher Stat3, tyrosine-705 phosphorylation and activity levels at all densities, indicating that Rac(V12) is able to activate Stat3. Further examination of the mechanism of Stat3 activation showed that Rac(V12) expression caused a surge in mRNA of Interleukin-6 (IL6) family cytokines, known potent Stat3 activators. Knockdown of gp130, the common subunit of this family reduced Stat3 activity, indicating that these cytokines may be responsible for the Stat3 activation by Rac(V12). The upregulation of IL6 family cytokines was required for cell migration and proliferation induced by Rac(V12), as shown by gp130 knockdown experiments, thus demonstrating that the gp130/Stat3 axis represents an essential effector of activated Rac for the regulation of key cellular functions.


Asunto(s)
Movimiento Celular/fisiología , Proliferación Celular , Receptor gp130 de Citocinas/metabolismo , Factor de Transcripción STAT3/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Línea Celular , Receptor gp130 de Citocinas/genética , Citocinas/genética , Citocinas/metabolismo , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Interleucina-6/metabolismo , Quinasas Janus/metabolismo , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Factor de Transcripción STAT3/genética , Transducción de Señal/fisiología , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/genética
15.
J Med Chem ; 64(5): 2691-2704, 2021 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-33576627

RESUMEN

Histone deacetylase 6 (HDAC6) is involved in multiple regulatory processes, ranging from cellular stress to intracellular transport. Inhibition of aberrant HDAC6 activity in several cancers and neurological diseases has been shown to be efficacious in both preclinical and clinical studies. While selective HDAC6 targeting has been pursued as an alternative to pan-HDAC drugs, identifying truly selective molecular templates has not been trivial. Herein, we report a structure-activity relationship study yielding TO-317, which potently binds HDAC6 catalytic domain 2 (Ki = 0.7 nM) and inhibits the enzyme function (IC50 = 2 nM). TO-317 exhibits 158-fold selectivity for HDAC6 over other HDAC isozymes by binding the catalytic Zn2+ and, uniquely, making a never seen before direct hydrogen bond with the Zn2+ coordinating residue, His614. This novel structural motif targeting the second-sphere His614 interaction, observed in a 1.84 Å resolution crystal structure with drHDAC6 from zebrafish, can provide new pharmacophores for identifying enthalpically driven, high-affinity, HDAC6-selective inhibitors.


Asunto(s)
Histona Desacetilasa 6/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Sulfonamidas/farmacología , Animales , Dominio Catalítico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Histona Desacetilasa 6/metabolismo , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/metabolismo , Inhibidores de Histona Desacetilasas/farmacocinética , Humanos , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/metabolismo , Ácidos Hidroxámicos/farmacocinética , Masculino , Ratones Endogámicos BALB C , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/metabolismo , Sulfonamidas/farmacocinética , Pez Cebra , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/metabolismo
16.
J Med Chem ; 64(12): 8486-8509, 2021 06 24.
Artículo en Inglés | MEDLINE | ID: mdl-34101461

RESUMEN

Epigenetic targeting has emerged as an efficacious therapy for hematological cancers. The rare and incurable T-cell prolymphocytic leukemia (T-PLL) is known for its aggressive clinical course. Current epigenetic agents such as histone deacetylase (HDAC) inhibitors are increasingly used for targeted therapy. Through a structure-activity relationship (SAR) study, we developed an HDAC6 inhibitor KT-531, which exhibited higher potency in T-PLL compared to other hematological cancers. KT-531 displayed strong HDAC6 inhibitory potency and selectivity, on-target biological activity, and a safe therapeutic window in nontransformed cell lines. In primary T-PLL patient cells, where HDAC6 was found to be overexpressed, KT-531 exhibited strong biological responses, and safety in healthy donor samples. Notably, combination studies in T-PLL patient samples demonstrated KT-531 synergizes with approved cancer drugs, bendamustine, idasanutlin, and venetoclax. Our work suggests HDAC inhibition in T-PLL could afford sufficient therapeutic windows to achieve durable remission either as stand-alone or in combination with targeted drugs.


Asunto(s)
Antineoplásicos/uso terapéutico , Inhibidores de Histona Desacetilasas/uso terapéutico , Ácidos Hidroxámicos/uso terapéutico , Leucemia Prolinfocítica de Células T/tratamiento farmacológico , Sulfonamidas/uso terapéutico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Apoptosis/efectos de los fármacos , Clorhidrato de Bendamustina/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Sinergismo Farmacológico , Histona Desacetilasa 6/metabolismo , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/farmacocinética , Humanos , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/farmacocinética , Masculino , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Pirrolidinas/farmacología , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/farmacocinética , Sulfonamidas/farmacología , para-Aminobenzoatos/farmacología
17.
J Vis Exp ; (156)2020 02 27.
Artículo en Inglés | MEDLINE | ID: mdl-32176212

RESUMEN

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation. The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins ß-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed "domes". The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce ß-casein and WAP. Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.


Asunto(s)
Diferenciación Celular , Células Epiteliales/citología , Glándulas Mamarias Animales/citología , Animales , Cadherinas/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Transformación Celular Neoplásica , Medios de Cultivo/química , Células Epiteliales/metabolismo , Femenino , Glándulas Mamarias Animales/crecimiento & desarrollo , Ratones , Proteínas de la Leche/metabolismo , Esferoides Celulares/citología , Esferoides Celulares/metabolismo , Proteínas de Unión al GTP rac/metabolismo
18.
Biochem Cell Biol ; 87(6): 835-43, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19935869

RESUMEN

Cells in normal tissues or in tumors have extensive opportunities for adhesion to their neighbors and the importance of cell to cell contact in the study of fundamental cellular processes is beginning to emerge. In this review, we discuss recent evidence of dramatic changes in the activity of an important signal transducer found to be profoundly affected by cell to cell adhesion, the signal transducer and activator of transcription-3 (Stat3). Direct cadherin engagement, growth of cells to postconfluence, or formation of multicellular aggregates were found to induce a striking increase in the levels of Stat3 activity, Rac1/Cdc42, and members of the IL6 receptor family in different settings. This activation was specific to Stat3, in that the levels of the extracellular signal regulated kinase (Erk1/2), a signal transducer often coordinately activated with Stat3 by a number of growth factors or oncogenes, remained unaffected by cell density. Density-dependent Stat3 activation may play a key role in survival, and could contribute to the establishment of cell polarity. It is clear that at any given time the total Stat3 activity levels in a cell are the sum of the effects of cell to cell adhesion plus the conventional Stat3 activating factors present.


Asunto(s)
Cadherinas/metabolismo , Supervivencia Celular/fisiología , Factor de Transcripción STAT3/metabolismo , Animales , Comunicación Autocrina/fisiología , Cadherinas/genética , Línea Celular , Interleucina-6/metabolismo , Janus Quinasa 1/metabolismo , Factor de Transcripción STAT3/genética , Transducción de Señal/fisiología , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo
19.
Exp Hematol ; 36(11): 1449-60, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18687517

RESUMEN

OBJECTIVE: The (8;21)(q22;q22) chromosomal translocation, which involves AML1 gene on chromosome 21 and the ETO gene on chromosome 8, generates an AML1/ETO fusion. AML1/ETO is associated with 15% of acute myeloid leukemia (AML) cases. The fusion gene is a dominant inhibitor of myeloid-specific genes, notably AML1, CCAAT/enhancer-binding protein-alpha (C/EBPalpha), and myeloperoxidase (MPO). In this study, we investigated the role of antiapoptosis gene survivin as a target of AML1/ETO-related leukemia. MATERIALS AND METHODS: Through the combination of reporter assays, electrophoretic mobility shift assay, quantitative real-time polymerase chain reaction analysis, and short hairpin RNA (shRNA)-mediated knockdown of genes, we showed that survivin is a critical target of AML1/ETO. Biological studies were performed in cell lines and primary human CD 34(+) cells. RESULTS: In this study, we have shown that ectopic expression of AML1/ETO induces survivin gene expression in both a cell line model and in the primary human hematopoietic CD34(+) cells. Reporter assays demonstrate that ectopically expressed AML1/ETO activates survivin promoter. Endogenous AML1/ETO derived from the Kasumi-1 cell line nuclear extract binds physically to the AML1 core enhancer-binding sequence, TGTGGT, derived from the survivin promotor. Knockdown of survivin expression by shRNA in ectopically expressed AML1/ETO myeloid leukemia cell lines restores expression of C/EBPalpha, granulocyte colony-stimulating factor receptor, and MPO genes, which leads to their growth arrest and granulocytic differentiation. CONCLUSIONS: Our results demonstrate that survivin gene acts as a critical mediator of AML1/ETO-induced late oncogeneic events.


Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Granulocitos/citología , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/fisiología , Secuencia de Bases , Proteína alfa Potenciadora de Unión a CCAAT/fisiología , Diferenciación Celular , Humanos , Proteínas Inhibidoras de la Apoptosis , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Interferente Pequeño/farmacología , Proteína 1 Compañera de Translocación de RUNX1 , Survivin , Transcripción Genética
20.
Cancers (Basel) ; 11(2)2019 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-30717267

RESUMEN

Gap junctional, intercellular communication (GJIC) is interrupted in cells transformed by oncogenes such as activated Src. The Src effector, Ras, is required for this effect, so that Ras inhibition restores GJIC in Src-transformed cells. Interestingly, the inhibition of the Src effector phosphatidyl-inositol-3 kinase (PI3k) or Signal Transducer and Activator of Transcription-3 (Stat3) pathways does not restore GJIC. In the contrary, inhibition of PI3k or Stat3 in non-transformed rodent fibroblasts or epithelial cells or certain human lung carcinoma lines with extensive GJIC inhibits communication, while mutational activation of PI3k or Stat3 increases GJIC. Therefore, it appears that oncogenes such as activated Src have a dual role upon GJIC; acting as inhibitors of communication through the Ras pathway, and as activators through activation of PI3k or Stat3. In the presence of high Src activity the inhibitory functions prevail so that the net effect is gap junction closure. PI3k and Stat3 constitute potent survival signals, so that their inhibition in non-transformed cells triggers apoptosis which, in turn, has been independently demonstrated to suppress GJIC. The interruption of gap junctional communication would confine the apoptotic event to single cells and this might be essential for the maintenance of tissue integrity. We hypothesize that the GJIC activation by PI3k or Stat3 may be linked to their survival function.

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