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 farmacológicoRESUMEN
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/metabolismoRESUMEN
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-ActividadRESUMEN
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.
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/farmacologíaRESUMEN
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/fisiologíaRESUMEN
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/metabolismoRESUMEN
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/metabolismoRESUMEN
BACKGROUND/AIM: The differentiation of the mouse breast epithelial cell line HC11 is known to require confluence as well as the addition of hydrocortisone, insulin and prolactin. MATERIALS AND METHODS: Since confluence, which triggers the engagement of the cell-to-cell adhesion molecule E-cadherin, induces a dramatic increase in the activity of signal transducer and activator of transcription-3 (Stat3), we examined the role of Stat3 in HC11 cell differentiation. RESULTS: Stat3 inhibition abolished differentiation, indicating that Stat3 activity is critically required. However, expression of the mutationally activated form of Stat3 (Stat3C), rather than promoting, it was found to block cell differentiation, even when expressed in low levels, and in the absence of full neoplastic conversion. CONCLUSION: The strength of the E-cadherin/Stat3 signal is key for the outcome of the differentiation process.
Asunto(s)
Células Epiteliales/citología , Glándulas Mamarias Animales/citología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Animales , Cadherinas/metabolismo , Diferenciación Celular , Femenino , Glándulas Mamarias Animales/metabolismo , Ratones , Mutación , Fosforilación , Transducción de Señal , Tirosina/metabolismoRESUMEN
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 CRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, incurable cancer with a 20% 1 year survival rate. While standard-of-care therapy can prolong life in a small fraction of cases, PDAC is inherently resistant to current treatments, and novel therapies are urgently required. Histone deacetylase (HDAC) inhibitors are effective in killing pancreatic cancer cells in in vitro PDAC studies, and although there are a few clinical studies investigating combination therapy including HDAC inhibitors, no HDAC drug or combination therapy with an HDAC drug has been approved for the treatment of PDAC. We developed an inhibitor of HDACs, AES-135, that exhibits nanomolar inhibitory activity against HDAC3, HDAC6, and HDAC11 in biochemical assays. In a three-dimensional coculture model, AES-135 kills low-passage patient-derived tumor spheroids selectively over surrounding cancer-associated fibroblasts and has excellent pharmacokinetic properties in vivo. In an orthotopic murine model of pancreatic cancer, AES-135 prolongs survival significantly, therefore representing a candidate for further preclinical testing.
Asunto(s)
Benzamidas/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Hidrocarburos Fluorados/farmacología , Ácidos Hidroxámicos/química , Neoplasias Pancreáticas/tratamiento farmacológico , Sulfonamidas/farmacología , Animales , Apoptosis/efectos de los fármacos , Benzamidas/química , Benzamidas/farmacocinética , Benzamidas/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacocinética , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Hidrocarburos Fluorados/química , Hidrocarburos Fluorados/farmacocinética , Hidrocarburos Fluorados/uso terapéutico , Ratones , Neoplasias Pancreáticas/patología , Sulfonamidas/química , Sulfonamidas/farmacocinética , Sulfonamidas/uso terapéuticoRESUMEN
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.
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 SeñalRESUMEN
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 , SolubilidadRESUMEN
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 , HumanosRESUMEN
Both cell-cell and cell-matrix adhesion are important for epithelial cell differentiation and function. Classical cadherins mediate cell to cell interactions and are potent activators of the signal transducer and activator of transcription (Stat3), thereby offering survival signaling. While the epithelial (E)-cadherin is required for cells to remain tightly associated within differentiated epithelial tissues, cadherin-11 promotes invasion and metastasis, preferentially to the bone. Cell adhesion to the extracellular matrix is mediated through the integrin receptors that bind to the focal adhesion kinase (FAK)/Src complex, thus activating downstream effectors such as Ras/Erk1/2 and PI3k/Akt, but not Stat3. Therefore, at high densities of cultured cells or in epithelial tissues, co-ordinate activation of the complementary cadherin/Stat3 and integrin/FAK pathways can greatly enhance survival and growth of tumor cells. In neoplastically transformed cells on the other hand, a variety of oncogenes including activated Src or receptor tyrosine kinases, activate both pathways. Still, most single-agent therapies directed against these signaling pathways have proven disappointing in the clinic. Combined targeting of the Src/FAK and Stat3 pathways with inhibitory drugs would be expected to have greater efficacy in inhibiting tumor cell survival, and enhancing sensitivity to conventional cytotoxic drugs for treatment of metastatic disease.
Asunto(s)
Matriz Extracelular/metabolismo , Neoplasias/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Cadherinas/metabolismo , Adhesión Celular , Supervivencia Celular , Humanos , Modelos Biológicos , Metástasis de la Neoplasia , Neoplasias/patologíaRESUMEN
Brain metastases (BM) represent the most common tumor to affect the adult central nervous system. Despite the increasing incidence of BM, likely due to consistently improving treatment of primary cancers, BM remain severely understudied. In this study, we utilized patient-derived stem cell lines from lung-to-brain metastases to examine the regulatory role of STAT3 in brain metastasis initiating cells (BMICs). Annotation of our previously described BMIC regulatory genes with protein-protein interaction network mapping identified STAT3 as a novel protein interactor. STAT3 knockdown showed a reduction in BMIC self-renewal and migration, and decreased tumor size in vivo. Screening of BMIC lines with a library of STAT3 inhibitors identified one inhibitor to significantly reduce tumor formation. Meta-analysis identified the oncomir microRNA-21 (miR-21) as a target of STAT3 activity. Inhibition of miR-21 displayed similar reductions in BMIC self-renewal and migration as STAT3 knockdown. Knockdown of STAT3 also reduced expression of known downstream targets of miR-21. Our studies have thus identified STAT3 and miR-21 as cooperative regulators of stemness, migration and tumor initiation in lung-derived BM. Therefore, STAT3 represents a potential therapeutic target in the treatment of lung-to-brain metastases.
Asunto(s)
Neoplasias Encefálicas/secundario , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/patología , MicroARNs/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Genes Reguladores , Humanos , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos NOD , Ratones SCID , Metástasis de la Neoplasia , Trasplante de Neoplasias , Mapeo de Interacción de Proteínas , Proteómica , ARN Interferente Pequeño/metabolismo , Células Madre/citologíaRESUMEN
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 , WortmaninaRESUMEN
We herein report the design and synthesis of the first nanomolar binding inhibitor of STAT5 protein. Lead compound 13a, possessing a phosphotyrosyl-mimicking salicylic acid group, potently and selectively binds to STAT5 over STAT3, inhibits STAT5-SH2 domain complexation events in vitro, silences activated STAT5 in leukemic cells, as well as STAT5's downstream transcriptional targets, including MYC and MCL1, and, as a result, leads to apoptosis. We believe 13a represents a useful probe for interrogating STAT5 function in cells as well as being a potential candidate for advanced preclinical trials.