RESUMEN
Triple negative breast cancer (TNBC) subtype is characterized with higher EMT/stemness properties and immune suppressive tumor microenvironment (TME). Women with advanced TNBC exhibit aggressive disease and have limited treatment options. Although immune suppressive TME is implicated in driving aggressive properties of basal/TNBC subtype and therapy resistance, effectively targeting it remains a challenge. Minnelide, a prodrug of triptolide currently being tested in clinical trials, has shown anti-tumorigenic activity in multiple malignancies via targeting super enhancers, Myc and anti-apoptotic pathways such as HSP70. Distinct super-enhancer landscape drives cancer stem cells (CSC) in TNBC subtype while inducing immune suppressive TME. We show that Minnelide selectively targets CSCs in human and murine TNBC cell lines compared to cell lines of luminal subtype by targeting Myc and HSP70. Minnelide in combination with cyclophosphamide significantly reduces the tumor growth and eliminates metastasis by reprogramming the tumor microenvironment and enhancing cytotoxic T cell infiltration in 4T1 tumor-bearing mice. Resection of residual tumors following the combination treatment leads to complete eradication of disseminated tumor cells as all mice are free of local and distant recurrences. All control mice showed recurrences within 3 weeks of post-resection while single Minnelide treatment delayed recurrence and one mouse was free of tumor. We provide evidence that Minnelide targets tumor intrinsic pathways and reprograms the immune suppressive microenvironment. Our studies also suggest that Minnelide in combination with cyclophosphamide may lead to durable responses in patients with basal/TNBC subtype warranting its clinical investigation.
RESUMEN
Preserving and regulating cellular homeostasis in the light of changing environmental conditions or developmental processes is of pivotal importance for single cellular and multicellular organisms alike. To counteract an imbalance in cellular homeostasis transcriptional programs evolved, called the heat shock response, unfolded protein response, and integrated stress response, that act cell-autonomously in most cells but in multicellular organisms are subjected to cell-nonautonomous regulation. These transcriptional programs downregulate the expression of most genes but increase the expression of heat shock genes, including genes encoding molecular chaperones and proteases, proteins involved in the repair of stress-induced damage to macromolecules and cellular structures. Sixty-one years after the discovery of the heat shock response by Ferruccio Ritossa, many aspects of stress biology are still enigmatic. Recent progress in the understanding of stress responses and molecular chaperones was reported at the 12th International Symposium on Heat Shock Proteins in Biology, Medicine and the Environment in the Old Town Alexandria, VA, USA from 28th to 31st of October 2023.
Asunto(s)
Proteínas de Choque Térmico , Medicina , Biología , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico/genética , Chaperonas Moleculares/metabolismoRESUMEN
The 90 kilo-Dalton heat shock protein (Hsp90) is a molecular chaperone that facilitates the maturation of nascent polypeptides into their biologically active conformation. Because many of the >400 known client protein substrates are implicated in the development/progression of cancer, it is hypothesized that Hsp90 inhibition will simultaneously shut down numerous oncogenic pathways. Unfortunately, most of the small molecule Hsp90 inhibitors that have undergone clinical evaluation thus far have failed due to various toxicities. Therefore, the disruption of Hsp90 protein-protein interactions with cochaperones and/or client substrates has been proposed as an alternative way to achieve Hsp90 inhibition without such adverse events. The hexadepsipeptide Enniatin A (EnnA) has recently been reported to be one such inhibitor that also manifests immunogenic activity. Herein, we report preliminary structure-activity relationship (SAR) studies to determine the structural features that confer this unprecedented activity for an Hsp90 inhibitor. Our studies find that EnnA's branching moieties are necessary for its activity, but some structural modifications are tolerated.
RESUMEN
Low response rates and immune-related adverse events limit the remarkable impact of cancer immunotherapy. To improve clinical outcomes, preclinical studies have shown that combining immunotherapies with N-terminal Hsp90 inhibitors resulted in improved efficacy, even though induction of an extensive heat shock response (HSR) and less than optimal dosing of these inhibitors limited their clinical efficacy as monotherapies. We discovered that the natural product Enniatin A (EnnA) targets Hsp90 and destabilizes its client oncoproteins without inducing an HSR. EnnA triggers immunogenic cell death in triple-negative breast cancer (TNBC) syngeneic mouse models and exhibits superior antitumor activity compared to Hsp90 N-terminal inhibitors. EnnA reprograms the tumor microenvironment (TME) to promote CD8+ T cell-dependent antitumor immunity by reducing PD-L1 levels and activating the chemokine receptor CX3CR1 pathway. These findings provide strong evidence for transforming the immunosuppressive TME into a more tumor-hostile milieu by engaging Hsp90 with therapeutic agents involving novel mechanisms of action.
RESUMEN
Heat shock protein 90 (Hsp90) and its co-chaperones promote cancer, and targeting Hsp90 holds promise for cancer treatment. Most of the efforts to harness this potential have focused on targeting the Hsp90 N-terminus ATP binding site. Although newer-generation inhibitors have shown improved efficacy in aggressive cancers, induction of the cellular heat shock response (HSR) by these inhibitors is thought to limit their clinical efficacy. Therefore, Hsp90 inhibitors with novel mechanisms of action and that do not trigger the HSR would be advantageous. Here, we investigated the mechanism by which capsaicin inhibits Hsp90. Through mutagenesis, chemical modifications, and proteomic studies, we show that capsaicin binds to the N-terminus of Hsp90 and inhibits its ATPase activity. Consequently, capsaicin and its analogs inhibit Hsp90 ATPase-dependent progesterone receptor reconstitution in vitro. Capsaicin did not induce the HSR, instead, it promoted the degradation of Hsp70 through the lysosome-autophagy pathway. Remarkably, capsaicin did not induce degradation of the constitutively expressed cognate Hsc70, indicating selectivity for Hsp70. Combined treatments of capsaicin and the Hsp90 inhibitor 17-AAG improved the anti-tumor efficacy of 17-AAG in cell culture and tridimensional tumor spheroid growth assays using breast and prostate cancer models. Consistent with this, in silico docking studies revealed that capsaicin binding to the ATP binding site of Hsp90 was distinct from classical N-terminus Hsp90 inhibitors, indicating a novel mechanism of action. Collectively, these findings support the use of capsaicin as a chemical scaffold to develop novel Hsp90 N-terminus inhibitors as well as its ability to be a potential cancer co-therapeutic.
Asunto(s)
Capsaicina , Neoplasias de la Próstata , Masculino , Humanos , Capsaicina/farmacología , Proteómica , Proteínas HSP70 de Choque Térmico , Proteínas HSP90 de Choque Térmico , Lisosomas , Adenosina Trifosfatasas , Adenosina TrifosfatoRESUMEN
The triggering receptor expressed on myeloid cell 1 (TREM1) plays a critical role in development of chronic inflammatory disorders and the inflamed tumor microenvironment (TME) associated with most solid tumors. We examined whether loss of TREM1 signaling can abrogate the immunosuppressive TME and enhance cancer immunity. To investigate the therapeutic potential of TREM1 in cancer, we used mice deficient in Trem1 and developed a novel small molecule TREM1 inhibitor, VJDT. We demonstrated that genetic or pharmacological TREM1 silencing significantly delayed tumor growth in murine melanoma (B16F10) and fibrosarcoma (MCA205) models. Single-cell RNA-Seq combined with functional assays during TREM1 deficiency revealed decreased immunosuppressive capacity of myeloid-derived suppressor cells (MDSCs) accompanied by expansion in cytotoxic CD8+ T cells and increased PD-1 expression. Furthermore, TREM1 inhibition enhanced the antitumorigenic effect of anti-PD-1 treatment, in part, by limiting MDSC frequency and abrogating T cell exhaustion. In patient-derived melanoma xenograft tumors, treatment with VJDT downregulated key oncogenic signaling pathways involved in cell proliferation, migration, and survival. Our work highlights the role of TREM1 in cancer progression, both intrinsically expressed in cancer cells and extrinsically in the TME. Thus, targeting TREM1 to modify an immunosuppressive TME and improve efficacy of immune checkpoint therapy represents what we believe to be a promising therapeutic approach to cancer.
Asunto(s)
Melanoma , Células Supresoras de Origen Mieloide , Humanos , Animales , Ratones , Receptor Activador Expresado en Células Mieloides 1/genética , Receptor Activador Expresado en Células Mieloides 1/metabolismo , Células Mieloides/patología , Línea Celular Tumoral , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Linfocitos T Citotóxicos/patología , Modelos Animales de Enfermedad , Microambiente TumoralRESUMEN
BACKGROUND: Activating the Stimulator of Interferon Genes (STING) adaptor incites antitumor immunity against immunogenic tumors in mice, prompting clinical trials to test STING activators. However, STING signaling in the tumor microenvironment (TME) during development of Lewis lung carcinoma (LLC) suppresses antitumor immunity to promote tumor growth. We hypothesized that local immune balance favoring suppression of antitumor immunity also attenuates antitumor responses following STING activation. The purpose of this study was to evaluate how STING activation impacts antitumor responses in mice bearing LLC tumors. METHODS: Mice bearing established LLC tumors were treated with synthetic cyclic diadenyl monophosphate (CDA) to activate STING. Mice were monitored to assess LLC tumor growth, survival and protective antitumor immunity. Transcriptional and metabolic analyses were used to identify pathways responsive to CDA, and mice were co-treated with CDA and drugs that disrupt these pathways. RESULTS: CDA slowed LLC tumor growth but most CDA-treated mice (77%) succumbed to tumor growth. No evidence of tumor relapse was found in surviving CDA-treated mice at experimental end points but mice were not immune to LLC challenge. CDA induced rapid increase in immune regulatory pathways involving programmed death-1 (PD-1), indoleamine 2,3 dioxygenase (IDO) and cyclooxygenase-2 (COX2) in the TME. PD-1 blockade enhanced antitumor responses to CDA and increased mouse survival but mice did not eliminate primary tumor burdens. Two IDO inhibitor drugs had little or no beneficial effects on antitumor responses to CDA. A third IDO inhibitor drug synergized with CDA to enhance tumor control and survival but mice did not eliminate primary tumor burdens. In contrast, co-treatments with CDA and the COX2-selective inhibitor celecoxib controlled tumor growth, leading to uniform survival without relapse, and mice acquired resistance to LLC re-challenge and growth of distal tumors not exposed directly to CDA. Thus, mice co-treated with CDA and celecoxib acquired stable and systemic antitumor immunity. CONCLUSIONS: STING activation incites potent antitumor responses and boosts local immune regulation to attenuate antitumor responses. Blocking STING-responsive regulatory pathways synergizes with CDA to enhance antitumor responses, particularly COX2 inhibition. Thus, therapy-induced resistance to STING may necessitate co-treatments to disrupt regulatory pathways responsive to STING in patients with cancer.
Asunto(s)
Inmunoterapia/métodos , Proteínas de la Membrana/uso terapéutico , Neoplasias/tratamiento farmacológico , Animales , Humanos , Inmunidad , Masculino , Proteínas de la Membrana/farmacología , Ratones , Neoplasias/patologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Although clinically apparent metastasis is associated with late stages of cancer development, micro-metastatic dissemination may be an early event. However, the fate of these early disseminated tumor cells (DTC) remains elusive. We show that despite their capacity to disseminate into secondary organs, 4T1 tumor models develop overt metastasis while EMT6-tumor bearing mice clear DTCs shed from primary tumors as well as those introduced by intravenous (IV) injection. Following the surgical resection of primary EMT6 tumors, mice do not develop detectable metastasis and reject IV-injected tumor cells. In contrast, these cells readily grow and metastasize in immuno-deficient athymic or Rag2-/- mice, an effect mimicked by CD8+ T-cell depletion in immunocompetent mice. Furthermore, recombinant G-CSF or adoptive transfer of granulocytic-MDSCs isolated from 4T1 tumor-bearing mice, induce metastasis by suppressing CD8+ T-cells in EMT6-primed mice. Our studies support the concept of immune surveillance providing molecular insights into the immune mechanisms during tumor progression.
Asunto(s)
Inmunidad , Neoplasias/inmunología , Neoplasias/patología , Animales , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Subgrupos Linfocitarios/inmunología , Ratones , Modelos Biológicos , Invasividad Neoplásica , Metástasis de la Neoplasia , Análisis de Supervivencia , Cola (estructura animal)/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/cirugía , Venas/patologíaRESUMEN
Cumulative evidence suggests that the heat shock protein 90 (Hsp90) co-chaperone UNC-45 myosin chaperone A (UNC45A) contributes to tumorigenesis and that its expression in cancer cells correlates with proliferation and metastasis of solid tumors. However, the molecular mechanism by which UNC45A regulates cancer cell proliferation remains largely unknown. Here, using siRNA-mediated gene silencing and various human cells, we report that UNC45A is essential for breast cancer cell growth, but is dispensable for normal cell proliferation. Immunofluorescence microscopy, along with gene microarray and RT-quantitative PCR analyses, revealed that UNC45A localizes to the cancer cell nucleus, where it up-regulates the transcriptional activity of the glucocorticoid receptor and thereby promotes expression of the mitotic kinase NIMA-related kinase 7 (NEK7). We observed that UNC45A-deficient cancer cells exhibit extensive pericentrosomal material disorganization, as well as defects in centrosomal separation and mitotic chromosome alignment. Consequently, these cells stalled in metaphase and cytokinesis and ultimately underwent mitotic catastrophe, phenotypes that were rescued by heterologous NEK7 expression. Our results identify a key role for the co-chaperone UNC45A in cell proliferation and provide insight into the regulatory mechanism. We propose that UNC45A represents a promising new therapeutic target to inhibit cancer cell growth in solid tumor types.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinogénesis/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Quinasas Relacionadas con NIMA/biosíntesis , Proteínas de Neoplasias/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinogénesis/genética , Carcinogénesis/patología , Femenino , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Células MCF-7 , Mitosis/genética , Quinasas Relacionadas con NIMA/genética , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Células PC-3RESUMEN
TNFα is a pleiotropic cytokine which fuels tumor cell growth, invasion, and metastasis in some malignancies, while in others it induces cytotoxic cell death. However, the molecular mechanism by which TNFα exerts its diverse effects on breast cancer subtypes remains elusive. Using in vitro assays and mouse xenografts, we show here that TNFα contributes to the aggressive properties of triple negative breast cancer (TNBC) cell lines via upregulation of TNFAIP3(A20). In a striking contrast, TNFα induces a potent cytotoxic cell death in luminal (ER+) breast cancer cell lines which fail to upregulate A20 expression. Overexpression of A20 not only protects luminal breast cancer cell lines from TNFα-induced cell death via inducing HSP70-mediated anti-apoptotic pathway but also promotes a robust EMT/CSC phenotype by activating the pStat3-mediated inflammatory signaling. Furthermore, A20 overexpression in luminal breast cancer cells induces aggressive metastatic properties in mouse xenografts via generating a permissive inflammatory microenvironment constituted by granulocytic-MDSCs. Collectively, our results reveal a mechanism by which A20 mediates pleiotropic effects of TNFα playing role in aggressive behaviors of TNBC subtype while its deficiency results in TNFα-induced apoptotic cell death in luminal breast cancer subtype.
Asunto(s)
Neoplasias de la Mama/patología , Regulación Neoplásica de la Expresión Génica , Pleiotropía Genética , Proteínas de Neoplasias/fisiología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Apoptosis/fisiología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Femenino , Proteínas del Choque Térmico HSP72/antagonistas & inhibidores , Proteínas del Choque Térmico HSP72/fisiología , Xenoinjertos , Humanos , Inflamación , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica/genética , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Nucleósidos de Purina/farmacología , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Factor de Transcripción STAT3/fisiología , Transducción de Señal , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/biosíntesis , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
D-Ring-seco-limonoids (tetranortriterpenoids), such as gedunin and xylograninâ B display anti-cancer activity, acting via inhibition of Hsp90 and/or associated chaperon machinery (e.g., p23). Despite this, these natural products have received relatively little attention, both in terms of an enabling synthetic approach (which would allow access to derivatives), and as a consequence their structure-activity relationship (SAR). Disclosed herein is a generally applicable synthetic route to the BCD ring system of the seco-D-ring double bond containing limonoids. Furthermore, cell based assays revealed the first skeletal fragment that exhibited inhibition of the p23 enzyme at a level which was equipotent to that of gedunin, despite being much less structurally complex.
RESUMEN
Overexpression of the MYC oncogene is a key feature of many human malignancies including Burkitt lymphoma. While MYC is widely regarded to be a promising therapeutic target, a clinically effective MYC inhibitor is still elusive. Here, we report an alternative strategy, targeting MYC indirectly through inhibition of the HSP90 machinery. We found that inhibition of HSP90 function reduces MYC expression in human Burkitt lymphoma through suppression of MYC transcription and destabilization of MYC protein, thereby diminishing the proliferation of tumor cells. Consistently, treatment of Burkitt lymphoma cell lines with HSP90 inhibitors (17-AAG or 17-DMAG) was accompanied by downregulation of canonical MYC target genes. Combination treatment with 17-DMAG and the proteasome inhibitor, MG-132, led to accumulation of MYC protein, indicating that upon HSP90 inhibition, MYC is degraded by the proteasome. Using co-immunoprecipitation, we furthermore demonstrated a direct interaction between MYC and HSP90, indicating that MYC is an HSP90 client protein in Burkitt lymphoma. Together, we report here the use of HSP90 inhibitors as an alternative approach to target the MYC oncogene and its network in Burkitt lymphoma.
RESUMEN
It is widely accepted that dynamic and reversible tumour cell plasticity is required for metastasis, however, in vivo steps and molecular mechanisms are poorly elucidated. We demonstrate here that monocytic (mMDSC) and granulocytic (gMDSC) subsets of myeloid-derived suppressor cells infiltrate in the primary tumour and distant organs with different time kinetics and regulate spatiotemporal tumour plasticity. Using co-culture experiments and mouse transcriptome analyses in syngeneic mouse models, we provide evidence that tumour-infiltrated mMDSCs facilitate tumour cell dissemination from the primary site by inducing EMT/CSC phenotype. In contrast, pulmonary gMDSC infiltrates support the metastatic growth by reverting EMT/CSC phenotype and promoting tumour cell proliferation. Furthermore, lung-derived gMDSCs isolated from tumour-bearing animals enhance metastatic growth of already disseminated tumour cells. MDSC-induced 'metastatic gene signature' derived from murine syngeneic model predicts poor patient survival in the majority of human solid tumours. Thus spatiotemporal MDSC infiltration may have clinical implications in tumour progression.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Granulocitos/metabolismo , Monocitos/metabolismo , Células Supresoras de Origen Mieloide/metabolismo , Neoplasias/genética , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Células Cultivadas , Femenino , Humanos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Heat shock protein 90 (Hsp90) is a molecular chaperone that orchestrates the folding and stability of proteins that regulate cellular signaling, proliferation and inflammation. We have previously shown that Hsp90 controls the production of reactive oxygen species by modulating the activity of Noxes1-3 and 5, but not Nox4. The goal of the current study was to define the regions on Nox5 that bind Hsp90 and determine how Hsp90 regulates enzyme activity. In isolated enzyme activity assays, we found that Hsp90 inhibitors selectively decrease superoxide, but not hydrogen peroxide, production. The addition of Hsp90 alone only modestly increases Nox5 enzyme activity but in combination with the co-chaperones, Hsp70, HOP, Hsp40, and p23 it robustly stimulated superoxide, but not hydrogen peroxide, production. Proximity ligation assays reveal that Nox5 and Hsp90 interact in intact cells. In cell lysates using a co-IP approach, Hsp90 binds to Nox5 but not Nox4, and the degree of binding can be influenced by calcium-dependent stimuli. Inhibition of Hsp90 induced the degradation of full length, catalytically inactive and a C-terminal fragment (aa398-719) of Nox5. In contrast, inhibition of Hsp90 did not affect the expression levels of N-terminal fragments (aa1-550) suggesting that Hsp90 binding maintains the stability of C-terminal regions. In Co-IP assays, Hsp90 was bound only to the C-terminal region of Nox5. Further refinement using deletion analysis revealed that the region between aa490-550 mediates Hsp90 binding. Converse mapping experiments show that the C-terminal region of Nox5 bound to the M domain of Hsp90 (aa310-529). In addition to Hsp90, Nox5 bound other components of the foldosome including co-chaperones Hsp70, HOP, p23 and Hsp40. Silencing of HOP, Hsp40 and p23 reduced Nox5-dependent superoxide. In contrast, increased expression of Hsp70 decreased Nox5 activity whereas a mutant of Hsp70 failed to do so. Inhibition of Hsp90 results in the loss of higher molecular weight complexes of Nox5 and decreased interaction between monomers. Collectively these results show that the C-terminal region of Nox5 binds to the M domain of Hsp90 and that the binding of Hsp90 and select co-chaperones facilitate oligomerization and the efficient production of superoxide.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismo , Animales , Sitios de Unión , Western Blotting , Células COS , Chlorocebus aethiops , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Inmunoprecipitación , NADPH Oxidasa 5 , Unión Proteica , ARN Interferente Pequeño , TransfecciónRESUMEN
The UCS family of proteins regulates cellular functions through their interactions with myosin. Here we show that one member of this family, UNC45A, is also a novel centrosomal protein. UNC45A is required for cellular proliferation of cancer cell in vitro and for tumor growth in vivo through its ability to bind and regulate ChK1 nuclear-cytoplasmic localization in an Hsp90-independent manner. Immunocytochemical and biochemical fractionation studies revealed that UNC45A and ChK1 co-localize to the centrosome. Inhibition of UNC45A expression reduced ChK1 activation and its tethering to the centrosome, events required for proper centrosome function. Lack of UNC45A caused the accumulation of multi-nucleated cells, consistent with a defect in Chk1 regulation of centrosomes. These findings identify a novel centrosomal function for UNC45A and its role in cell proliferation and tumorigenesis.
Asunto(s)
Centrosoma/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Quinasas/metabolismo , Carcinogénesis/genética , Carcinogénesis/metabolismo , Proliferación Celular/fisiología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Chaperonas Moleculares/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
Hsp90 and its co-chaperones are known to be important for cancer cell survival. The N-terminal inhibitors of Hsp90 that are in ongoing clinical trials as antitumor agents have unfortunately shown disappointing efficacies in the clinic. Thus, novel inhibitors of the Hsp90 machine with a different mechanism of action are urgently needed. We report here the development of a novel high-throughput screening assay platform to identify small-molecule inhibitors of Hsp90 and its co-chaperones. This assay quantitatively measures the ability of Hsp90 and its co-chaperones to refold/protect the progesterone receptor, a physiological client of Hsp90, in a 96-well plate format. We screened the National Institutes of Health clinical collection drug library and identified capsaicin as a hit molecule. Capsaicin is a Food and Drug Administration-approved drug for topical use in pain management. Cell survival assays showed that capsaicin selectively kills cancer cells and destabilizes several Hsp90 client proteins. Thus, our data may explain the seemingly pleotropic effect of capsaicin.
Asunto(s)
Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Chaperonas Moleculares/metabolismo , Receptores de Progesterona/metabolismo , Animales , Antineoplásicos/farmacología , Capsaicina/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Unión Proteica , Receptores de Progesterona/antagonistas & inhibidores , Receptores de Progesterona/química , Reproducibilidad de los Resultados , Bibliotecas de Moléculas PequeñasRESUMEN
Chemical investigation of the EtOAc extract of the fungus Chaetomium aureum, an endophyte of the Moroccan medicinal plant Thymelaea lythroides, afforded one new resorcinol derivative named chaetorcinol, together with five known metabolites. The structures of the isolated compounds were determined on the basis of one- and two-dimensional NMR spectroscopy and high-resolution mass spectrometry as well as by comparison with the literature. All compounds were tested for their activity towards the Hsp90 chaperoning machine in vitro using the progesterone receptor (PR) and rabbit reticulocyte lysate (RRL). Among the isolated compounds, only sclerotiorin efficiently inhibited the Hsp90 machine chaperoning activity. However, sclerotiorin showed no cytotoxic effect on breast cancer Hs578T, MDA-MB-231 and prostate cancer LNCaP cell lines. Interestingly, deacetylation of sclerotiorin increased its cytotoxicity toward the tested cell lines over a period of 48 h.
Asunto(s)
Chaetomium/química , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Resorcinoles/química , Resorcinoles/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , ConejosRESUMEN
Adenosine provides anti-inflammatory effects in cardiovascular disease via the activation of adenosine A2A receptors; however, the physiological effect of adenosine could be limited due to its phosphorylation by adenosine kinase. We hypothesized that inhibition of adenosine kinase exacerbates extracellular adenosine levels to reduce renal inflammation and injury in streptozotocin-induced diabetes. Diabetes was induced in male C57BL/6 mice by daily injection of streptozotocin (50mg/kg/day, i.p. for 5 days). Control and diabetic mice were then treated with the adenosine kinase inhibitor ABT702 (1.5mg/kg, i.p. two times a week for 8 weeks, n=7-8/group) or the vehicle (5% DMSO). ABT702 treatment reduced blood glucose level in diabetic mice (â¼20%; P<0.05). ABT702 also reduced albuminuria and markers of glomerular injury, nephrinuria and podocalyxin excretion levels, in diabetic mice. Renal NADPH oxidase activity and urinary thiobarbituric acid reactive substances (TBARS) excretion, indices of oxidative stress, were also elevated in diabetic mice and ABT702 significantly reduced these changes. ABT702 increased renal endothelial nitric oxide synthase expression (eNOS) and nitrate/nitrite excretion levels in diabetic mice. In addition, the diabetic mice displayed an increase in renal macrophage infiltration, in association with increased renal NFκB activation. Importantly, treatment with ABT702 significantly reduced all these inflammatory parameters (P<0.05). Furthermore, ABT702 decreased glomerular permeability and inflammation and restored the decrease in glomerular occludin expression in vitro in high glucose treated human glomerular endothelial cells. Collectively, the results suggest that the reno-protective effects of ABT702 could be attributed to the reduction in renal inflammation and oxidative stress in diabetic mice.
Asunto(s)
Adenosina Quinasa/antagonistas & inhibidores , Antiinflamatorios/farmacología , Antioxidantes/farmacología , Diabetes Mellitus Experimental/metabolismo , Riñón/efectos de los fármacos , Morfolinas/farmacología , Pirimidinas/farmacología , Adenosina Quinasa/metabolismo , Animales , Antiinflamatorios/uso terapéutico , Antioxidantes/uso terapéutico , Glucemia/análisis , Línea Celular , Dextranos/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/patología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Riñón/metabolismo , Riñón/patología , Masculino , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Morfolinas/uso terapéutico , NADPH Oxidasas/metabolismo , Nitratos/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Nitritos/metabolismo , Proteinuria/tratamiento farmacológico , Proteinuria/metabolismo , Proteinuria/patología , Pirimidinas/uso terapéutico , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Factor de Transcripción ReIA/metabolismoRESUMEN
Steroid hormone receptors are ligand-dependent transcription factors that require the ordered assembly of multichaperone complexes for transcriptional activity. Although heat shock protein (Hsp) 90 and Hsp70 are key players in this process, multiple Hsp70- and Hsp90-associated cochaperones associate with receptor-chaperone complexes to regulate receptor folding and activation. Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) was recently characterized as an Hsp70 and Hsp90-associated cochaperone that specifically regulates androgen receptor activity. However, the specificity of SGTA for additional members of the steroid hormone receptor superfamily and the mechanism by which SGTA regulates receptor activity remain unclear. Here we report that SGTA associates with and specifically regulates the androgen, glucocorticoid, and progesterone receptors and has no effect on the mineralocorticoid and estrogen receptors in both yeast and mammalian cell-based reporter assays. In both systems, SGTA knockdown/deletion enhances receptor activity, whereas SGTA overexpression suppresses receptor activity. We demonstrate that SGTA binds directly to Hsp70 and Hsp90 in vitro with similar affinities yet predominately precipitates with Hsp70 from cell lysates, suggesting a role for SGTA in early, Hsp70-mediated folding. Furthermore, SGTA expression completely abrogates the regulation of receptor function by FKBP52 (52-kDa FK506-binding protein), which acts at a later stage of the chaperone cycle. Taken together, our data suggest a role for SGTA at distinct steps in the chaperone-dependent modulation of androgen, glucocorticoid, and progesterone receptor activity.