RESUMO
Despite strong preclinical data, the therapeutic benefit of the RANKL inhibitor, denosumab, in breast cancer patients, beyond the bone, is unclear. Aiming to select patients who may benefit from denosumab, we hereby analyzed RANK and RANKL protein expression in more than 2,000 breast tumors (777 estrogen receptor-negative, ER- ) from four independent cohorts. RANK protein expression was more frequent in ER- tumors, where it associated with poor outcome and poor response to chemotherapy. In ER- breast cancer patient-derived orthoxenografts (PDXs), RANKL inhibition reduced tumor cell proliferation and stemness, regulated tumor immunity and metabolism, and improved response to chemotherapy. Intriguingly, tumor RANK protein expression associated with poor prognosis in postmenopausal breast cancer patients, activation of NFKB signaling, and modulation of immune and metabolic pathways, suggesting that RANK signaling increases after menopause. Our results demonstrate that RANK protein expression is an independent biomarker of poor prognosis in postmenopausal and ER- breast cancer patients and support the therapeutic benefit of RANK pathway inhibitors, such as denosumab, in breast cancer patients with RANK+ ER- tumors after menopause.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Neoplasias da Mama/patologia , Denosumab/farmacologia , Denosumab/uso terapêutico , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/uso terapêutico , Pós-Menopausa , Ligante RANK , Transdução de SinaisRESUMO
Most breast cancers exhibit low immune infiltration and are unresponsive to immunotherapy. We hypothesized that inhibition of the receptor activator of nuclear factor-κB (RANK) signaling pathway may enhance immune activation. Here we report that loss of RANK signaling in mouse tumor cells increases leukocytes, lymphocytes, and CD8+ T cells, and reduces macrophage and neutrophil infiltration. CD8+ T cells mediate the attenuated tumor phenotype observed upon RANK loss, whereas neutrophils, supported by RANK-expressing tumor cells, induce immunosuppression. RANKL inhibition increases the anti-tumor effect of immunotherapies in breast cancer through a tumor cell mediated effect. Comparably, pre-operative single-agent denosumab in premenopausal early-stage breast cancer patients from the Phase-II D-BEYOND clinical trial (NCT01864798) is well tolerated, inhibits RANK pathway and increases tumor infiltrating lymphocytes and CD8+ T cells. Higher RANK signaling activation in tumors and serum RANKL levels at baseline predict these immune-modulatory effects. No changes in tumor cell proliferation (primary endpoint) or other secondary endpoints are observed. Overall, our preclinical and clinical findings reveal that tumor cells exploit RANK pathway as a mechanism to evade immune surveillance and support the use of RANK pathway inhibitors to prime luminal breast cancer for immunotherapy.
Assuntos
Neoplasias da Mama/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunidade , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais , Adulto , Animais , Neoplasias da Mama/sangue , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Denosumab/farmacologia , Denosumab/uso terapêutico , Feminino , Humanos , Terapia de Imunossupressão , Imunoterapia , Mediadores da Inflamação/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Modelos Biológicos , Células Mieloides/imunologia , Estadiamento de Neoplasias , Neutrófilos/imunologia , Ligante RANK/sangue , Ligante RANK/metabolismoRESUMO
AIMS: Biodegradable polymeric microneedles containing atorvastatin calcium were developed in order to improve the percutaneous absorption of the drug, useful for the treatment of hypercholesterolemia. BACKGROUND: The use of physical enhancers like microneedles have shown good results to increase the delivery of drugs through the skin, the use of microneedles has very important advantages for transdermal drug delivery, for example, they are painless, easy to use and safe, they increase time interval of drug activity, dose, and reductions in adverse reactions, they also offer, the facility to remove the system instantly of the skin. OBJECTIVE: Develop polymer microneedles loaded with a calcium atorvastatin and evaluate them by Differential Scanning Calorimetry (DSC), Scanning Electron Microscopy (SEM), bioadhesion, postwetting- bioadhesion, breaking strength, drug release test and in vitro percutaneous absorption studies to demonstrate the use of microneedles atorvastatin is able to cross the skin. METHODS: The microneedles were made with poly (methyl vinyl ether-alt-maleic acid) as biodegradable polymer using the technique of casting in solution in a mold. After solidification these microneedles were characterized by Differential Scanning Calorimetry (DSC), Scanning Electron Microscopy (SEM), bioadhesion, post-wetting-bioadhesion, breaking strength, drug release test and in vitro percutaneous absorption studies. RESULTS: In general, the performances were satisfactory for optimal formulation in terms of DSC with no interactions between drug and excipients, SEM shows microneedles with a conical shape, bioadhesion of 1570 g.f, post wetting-bioadhesion of 1503.4 g.f, breaking strength of 1566.7g.f that is sufficient to disrupt Stratum corneum, good drug release and a flux of 33.4 µg/cm2*h with a tLag of 15.14 h for the in vitro percutaneous absorption. CONCLUSION: The results indicate that it is possible to generate microneedles to increase the percutaneous absorption of calcium atorvastatin transdermally, with the potential to be used as an alternative to the oral route for the treatment of dyslipidemias.
Assuntos
Anticolesterolemiantes/administração & dosagem , Atorvastatina/administração & dosagem , Plásticos Biodegradáveis/química , Portadores de Fármacos/química , Maleatos/química , Polietilenos/química , Administração Cutânea , Animais , Anticolesterolemiantes/farmacocinética , Atorvastatina/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos , Humanos , Técnicas In Vitro , Agulhas , Pele/metabolismo , Absorção CutâneaRESUMO
Taxanes are the mainstay of treatment in triple-negative breast cancer (TNBC), with de novo and acquired resistance limiting patient's survival. To investigate the genetic basis of docetaxel resistance in TNBC, exome sequencing was performed on matched TNBC patient-derived xenografts (PDX) sensitive to docetaxel and their counterparts that developed resistance in vivo upon continuous drug exposure. Most mutations, small insertions/deletions, and copy number alterations detected in the initial TNBC human metastatic samples were maintained after serial passages in mice and emergence of resistance. We identified a chromosomal amplification of chr12p in a human BRCA1-mutated metastatic sample and the derived chemoresistant PDX, but not in the matched docetaxel-sensitive PDX tumor. Chr12p amplification was validated in a second pair of docetaxel-sensitive/resistant BRCA1-mutated PDXs and after short-term docetaxel treatment in several TNBC/BRCA1-mutated PDXs and cell lines, as well as during metastatic recurrence in a patient with BRCA1-mutated breast cancer who had progressed on docetaxel treatment. Analysis of clinical data indicates an association between chr12p amplification and patients with TNBC/basal-like breast cancer, a BRCA1 mutational signature, and poor survival after chemotherapy. Detection of chr12p amplification in a cohort of TNBC PDX models was associated with an improved response to carboplatin. Our findings reveal tumor clonal dynamics during chemotherapy treatments and suggest that a preexisting population harboring chr12p amplification is associated with the emergence of docetaxel resistance and carboplatin responsiveness in TNBC/BRCA1-mutated tumors. SIGNIFICANCE: Chr12p copy number gains indicate rapid emergence of resistance to docetaxel and increased sensitivity to carboplatin, therefore sequential docetaxel/carboplatin treatment could improve survival in TNBC/BRCA1 patients. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/16/4258/F1.large.jpg.
Assuntos
Carboplatina/farmacologia , Cromossomos Humanos Par 12 , Docetaxel/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Animais , Proteína BRCA1/genética , Linhagem Celular Tumoral , Exoma , Feminino , Humanos , Camundongos , Mutação , Resultado do Tratamento , Neoplasias de Mama Triplo Negativas/mortalidade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RANK expression is associated with poor prognosis in breast cancer even though its therapeutic potential remains unknown. RANKL and its receptor RANK are downstream effectors of the progesterone signaling pathway. However, RANK expression is enriched in hormone receptor negative adenocarcinomas, suggesting additional roles for RANK signaling beyond its hormone-dependent function. Here, to explore the role of RANK signaling once tumors have developed, we use the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT), which mimics RANK and RANKL expression patterns seen in human breast adenocarcinomas. Complementary genetic and pharmacologic approaches demonstrate that therapeutic inhibition of RANK signaling drastically reduces the cancer stem cell pool, decreases tumor and metastasis initiation, and enhances sensitivity to chemotherapy. Mechanistically, genome-wide expression analyses show that anti-RANKL therapy promotes lactogenic differentiation of tumor cells. Moreover, RANK signaling in tumor cells negatively regulates the expression of Ap2 transcription factors, and enhances the Wnt agonist Rspo1 and the Sca1-population, enriched in tumor-initiating cells. In addition, we found that expression of TFAP2B and the RANK inhibitor, OPG, in human breast cancer correlate and are associated with relapse-free tumors. These results support the use of RANKL inhibitors to reduce recurrence and metastasis in breast cancer patients based on its ability to induce tumor cell differentiation. Cancer Res; 76(19); 5857-69. ©2016 AACR.
Assuntos
Neoplasias Mamárias Experimentais/prevenção & controle , Recidiva Local de Neoplasia/prevenção & controle , Receptor Ativador de Fator Nuclear kappa-B/antagonistas & inibidores , Transdução de Sinais/fisiologia , Animais , Apoptose/efeitos dos fármacos , Ataxina-1/análise , Diferenciação Celular/efeitos dos fármacos , Docetaxel , Feminino , Humanos , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/efeitos dos fármacos , Ligante RANK/antagonistas & inibidores , Ligante RANK/farmacologia , Receptor Ativador de Fator Nuclear kappa-B/fisiologia , Taxoides/farmacologia , Fator de Transcrição AP-2/fisiologiaRESUMO
A role of endothelial cells in the survival of CLL cells during extravasation is presently unknown. Herein we show that CLL cells but not normal B cells can receive apoptotic signals through physical contact with TNF-α activated endothelium impairing survival in transendothelial migration (TEM) assays. In addition, the CLL cells of patients having lymphadenopathy (LApos) show a survival advantage during TEM that can be linked to increased expression of α4 and αL integrin chains. Within this context, ephrinA4 expressed on the surface of CLL cells sequestrates integrins and inactivates them resulting in reduced adhesion and inhibition of apoptotic/survival signals through them. In agreement, ephrinA4 silencing resulted in increased survival of CLL cells of LApos patients but not LA neg patients. Similarly was observed when a soluble ephrinA4 isoform was added to TEM assays strongly suggesting that accumulation of this isoform in the serum of LApos patients could contribute to CLL cells dissemination and survival in vivo. In supporting, CLL lymphadenopathies showed a preferential accumulation of apoptotic CLL cells around high endothelial venules lacking ephrinA4. Moreover, soluble ephrinA4 isolated from sera of patients increased the number and viability of CLL cells recovered from the lymph nodes of adoptively transferred mice. Finally, we present evidence suggesting that soluble ephrinA4 mediated survival during TEM could enhance a transcellular TEM route of the CLL cells. Together these findings point to an important role of ephrinA4 in the nodal dissemination of CLL cells governing extravasation and survival.
Assuntos
Apoptose , Antígeno CD11a/metabolismo , Sobrevivência Celular , Efrina-A4/metabolismo , Integrina alfa4/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Linfócitos B/fisiologia , Células Cultivadas , Técnicas de Cocultura , Endotélio/metabolismo , Efrina-A4/sangue , Efrina-A4/genética , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Linfonodos/patologia , Metástase Linfática , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Isoformas de Proteínas/sangue , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Organismos Livres de Patógenos Específicos , Migração Transendotelial e Transepitelial , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Prolactin and progesterone both orchestrate the proliferation and differentiation of the mammary gland during gestation. Differentiation of milk secreting alveoli depends on the presence of prolactin receptor, the downstream Jak2-Stat5 pathway and the transcription factor Elf5. A strict regulation of Rank signaling is essential for the differentiation of the mammary gland and in particular for alveolar commitment. Impaired alveologenesis and lactation failure are observed in both, knockout and Rank overexpressing mice; however, the underlying molecular mechanism responsible for these phenotypes remains largely unknown. Using genome-wide expression analyses and functional studies, we show here that Rankl (RL) exposure leads to impaired secretory differentiation of alveolar cells not only in MMTV-RANK but also in wild-type (WT) mammary acini. Conversely, pharmacological blockage of Rank signaling at midgestation in WT mice leads to precocious and exacerbated lactogenesis. Mechanistically, RL negatively regulates Stat5 phosphorylation and Elf5 expression at the onset of lactogenesis. Continuous RL exposure leads to the expansion of basal and bipotent cells in WT and MMTV-RANK acini. Overall, we demonstrate that enhanced Rank signaling impairs secretory differentiation during pregnancy by inhibition of the prolactin/p-Stat5 pathway.
Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Prolactina/genética , Ligante RANK/genética , Fator de Transcrição STAT5/genética , Fatores de Transcrição/genética , Animais , Proliferação de Células/genética , Proteínas de Ligação a DNA/biossíntese , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Janus Quinase 2/biossíntese , Janus Quinase 2/genética , Lactação/genética , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Knockout , Gravidez , Progesterona/genética , Progesterona/metabolismo , Prolactina/metabolismo , Ligante RANK/biossíntese , Fator de Transcrição STAT5/biossíntese , Transdução de Sinais , Fatores de Transcrição/biossínteseRESUMO
OBJECTIVES: The radiopharmacokinetic parameters of the therapeutic radiopharmaceutical (188)Re-lanreotide were compared in rats implanted with hepatocarcinoma tumours (n= 18) and healthy rats (n= 18). METHODS: Rats were injected with approximately 1.8 MBq (188)Re-lanreotide (0.1 ml) via the tail vein and blood samples were obtained. The activity per gram of tissue (%IA/g) was calculated and the radiopharmacokinetic parameters determined. Data were fitted using a two-compartment model. KEY FINDINGS: Significant differences were found between healthy and hepatoma rats for beta elimination half-life (22.56 vs 48.14 h); transference constants K(10) (k(e)) (6.44 vs 3.05 h(-1)) and K(12) (2.76 vs 7.09 h(-1)); volume of distribution (2.06 vs 5.45 ml); mean residence time (66.58 vs 95.50 h) and apparent volume of distribution at steady state (131.30 vs 810.37 ml). The tumour/organ ratios after 24 h were 11.20 for tumour/muscle, 8.00 for tumour/liver and 7.72 for tumour/bone. The scintigraphic images obtained therefore had high resolution. CONCLUSIONS: (188)Re-lanreotide had a prolonged beta elimination half-life and increased volume of distribution in rats with hepatocellular carcinoma. This may be beneficial in the diagnosis and therapy of metastatic lesions in patients with cancer.
Assuntos
Antineoplásicos/farmacocinética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Peptídeos Cíclicos/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Rênio/farmacocinética , Somatostatina/análogos & derivados , Animais , Antineoplásicos/uso terapêutico , Osso e Ossos , Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/radioterapia , Meia-Vida , Fígado , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/radioterapia , Masculino , Músculos , Peptídeos Cíclicos/uso terapêutico , Radioisótopos/farmacocinética , Radioisótopos/uso terapêutico , Cintilografia , Compostos Radiofarmacêuticos/uso terapêutico , Ratos , Ratos Wistar , Valores de Referência , Rênio/uso terapêutico , Somatostatina/farmacocinética , Somatostatina/uso terapêutico , Distribuição TecidualRESUMO
A better knowledge of the molecular mechanisms that govern leukocyte trafficking is of major relevance for the clinics. Both normal and pathologic extravasation of lymphocytes are a fine-tuned spatio-temporal event of migratory path-finding likely regulated by molecular guidance cues underlying cell movements in other systems. We have recently reported that members of the Eph family of receptor tyrosine kinases, namely EphA2 and one of its ligands, ephrin-A4 (EFNA4) can mediate in the traffic of chronic lymphocytic leukemia (CLL) cells and presumably of normal B cells between the blood and the tissues. The importance of EphA2-EFNA4 interactions at the endothelium-lymphocyte interface during TEM could rely on their attractive/repulsive properties. In the present work, we expand on those results by including additional insights and new suggestions for future studies that discuss the relevance of these molecules in overall cell adhesion dynamic events.
Assuntos
Efrinas/metabolismo , Linfócitos/citologia , Linfócitos/metabolismo , Receptores da Família Eph/metabolismo , Transdução de Sinais , Migração Transendotelial e Transepitelial , Animais , Efrina-A4/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/enzimologia , Linfonodos/patologia , Receptor EphA2/metabolismoRESUMO
Chronic lymphocytic leukemia (CLL) cell migration into lymphoid tissues is an important aspect of the pathobiology of this disease. Here, we investigated the role of ephrin-A4 (EFNA4) in the transendothelial migration (TEM) capacity of CLL and normal B cells through interacting with endothelial EphA2 (erythropoietin-producing hepatocellular carcinoma). CLL cells showed a remarkable impairment in the adhesion to and transmigration through human umbilical vein endothelial cell (HUVEC) monolayers, correlating with their higher EFNA4 expression. In vitro, TEM was mediated by EFNA4 binding to endothelial EphA2 receptor, which is highly expressed in tumor necrosis factor-alpha-activated HUVECs as well as in the CD31(+) endothelial cells of human lymph nodes. The pretreatment of CLL cells with EphA2 homodimers further impaired their adhesion to and transmigration through HUVEC monolayers, whereas pretreatment of HUVECs with EFNA4 homodimers improved those phenomena in both CLL and normal B cells, suggesting that EFNA4 signaling negatively contributed to TEM. In fact, EFNA4 signaling into CLL cells significantly reduced their adhesion to intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and several extracellular matrix molecules and impaired CCL-19-mediated TEM and chemotaxis. Our results suggest that EFNA4-EphA2 interactions are involved in CLL cell trafficking between blood and the tissues and therefore may become a therapeutic target in the future.
Assuntos
Quimiotaxia de Leucócito/fisiologia , Endotélio/metabolismo , Efrina-A4/biossíntese , Leucemia Linfocítica Crônica de Células B/metabolismo , Adesão Celular/fisiologia , Quimiocina CCL19/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Microscopia Confocal , Receptor EphA2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Increasing information relates some Eph receptors and their ligands, ephrins (EFN), with the immune system. Herein, we found that normal B-cells from peripheral blood (PB) and lymph nodes (LN) showed a differential expression of certain Eph/EFN members, some of them being modulated upon in vitro stimulation including EFNA1, EFNA4, EphB6 and EphA10. In contrast, PB CLL B-cells showed a more heterogeneous Eph/EFN profile than their normal PB B-cell counterparts, expressing Eph/EFN members frequently found within the LN and activated B-cells, specially EFNA4, EphB6 and EphA10. Two of them, EphB6 and EFNA4 were further related with the clinical course of CLL patients. EphB6 expression correlated with a high content of ZAP-70 mRNA and a poor prognosis. High serum levels of a soluble EFNA4 isoform positively correlated with increasing peripheral blood lymphocyte counts and lymphadenopathy. These findings suggest that Eph/EFN might be relevant in normal B-cell biology and could represent new potential prognostic markers and therapeutic targets for CLL.
Assuntos
Linfócitos B/patologia , Efrinas/genética , Leucemia Linfocítica Crônica de Células B/genética , Receptores da Família Eph/genética , Biomarcadores Tumorais/análise , Estudos de Casos e Controles , Efrina-A4/genética , Perfilação da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Prognóstico , Receptores Proteína Tirosina Quinases/genética , Proteína-Tirosina Quinase ZAP-70/genéticaRESUMO
Radiolabeled peptides, like the somatostatin analogs, have been used for peptide receptor-mediated radionuclide therapy (PRMRT) in metastatic neuroendocrine tumors. The eight amino acid peptide 3-(2-naphthalenyl)-D-alanyl-L-cysteinyl-L-tyrosyl-D-tryptophyl-L-lysyl-L-valyl-L-cysteinyl-L-threoninamide,cyclic(2-->7)-disulfide (9Cl) (lanreotide) was found to bind to the five somatostatin tumor receptors. Lanreotide has been labeled via the bifunctional chelating agent, DOTA, to (111)In, and (90)Y. A direct labeling method was used to label lanreotide with (188)Re. Athymic mice with implanted human cancer tumors (uterine-cervix, renal, and neuroblastoma) were injected with radiochemically pure (188)Re-lanreotide (1.11 MBq). The percent injected activity (%IA/g) from serial blood samples was the input data for the WinNonlin computer program to obtain radiopharmacokinetic parameters. The organs' percent injected activity per gram of tissue (%IA/g) was extrapolated to the weights of a 70 kg male model organs and the number of nuclear transitions (N) were the input for the OLINDA/EXM program to obtain dosimetry estimates. Induced uterine-cervix tumors (HeLa cells) show a mean 2.4 %IA/g uptake up to 24 h and the tumor/blood ratio was over 1.85 (1.5-24 h post-injection) confirming (188)Re-lanreotide remains bound to the tumor. The estimated tumor absorbed dose was 460 mGy/MBq. Human effective dose was 0.0182 mSv/MBq. Therefore, (188)Re-lanreotide is a good candidate for PRMRT and a clinical trial is being planned in order to acquire individual dosimetric data.