RESUMEN
Testosterone is the canonical growth factor of prostate cancer but can paradoxically suppress its growth when present at supraphysiological levels. We have previously demonstrated that the cyclical administration of supraphysiological androgen (SPA), termed bipolar androgen therapy (BAT), can result in tumor regression and clinical benefit for patients with castration-resistant prostate cancer. However, predictors and mechanisms of response and resistance have been ill defined. Here, we show that growth inhibition of prostate cancer models by SPA required high androgen receptor (AR) activity and were driven in part by downregulation of MYC. Using matched sequential patient biopsies, we show that high pretreatment AR activity predicted downregulation of MYC, improved clinical response, and prolonged progression-free and overall survival for patients on BAT. BAT induced strong downregulation of AR in all patients, which is shown to be a primary mechanism of acquired resistance to SPA. Acquired resistance was overcome by alternating SPA with the AR inhibitor enzalutamide, which induced adaptive upregulation of AR and resensitized prostate cancer to SPA. This work identifies high AR activity as a predictive biomarker of response to BAT and supports a treatment paradigm for prostate cancer involving alternating between AR inhibition and activation.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Receptores Androgénicos , Masculino , Humanos , Receptores Androgénicos/metabolismo , Andrógenos/farmacología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Antagonistas de Receptores Androgénicos/farmacología , Antagonistas de Receptores Androgénicos/uso terapéutico , Nitrilos , Testosterona/farmacología , Resistencia a Antineoplásicos , Línea Celular TumoralRESUMEN
Activating variants in the PEST region of NOTCH1 have been associated with aggressive phenotypes in human cancers, including triple-negative breast cancer (TNBC). Previous studies suggested that PEST domain variants in TNBC patients resulted in increased cell proliferation, invasiveness, and decreased overall survival. In this study, we assess the phenotypic transformation of activating NOTCH1 variants and their response to standard of care therapies. AAV-mediated gene targeting was used to isogenically incorporate 3 NOTCH1 variants, including a novel TNBC frameshift variant, in two non-tumorigenic breast epithelial cell lines, MCF10A and hTERT-IMEC. Two different variants at the NOTCH1 A2241 site (A2441fs and A2441T) both demonstrated increased transformative properties when compared to a non-transformative PEST domain variant (S2523L). These phenotypic changes include proliferation, migration, anchorage-independent growth, and MAPK pathway activation. In contrast to previous studies, activating NOTCH1 variants did not display sensitivity to a gamma secretase inhibitor (GSI) or resistance to chemotherapies. This study demonstrates distinct transformative phenotypes are specific to a given variant within NOTCH1 and these phenotypes do not correlate with sensitivities or resistance to chemotherapies or GSIs. Although previous studies have suggested NOTCH1 variants may be prognostic for TNBC, our study does not demonstrate prognostic ability of these variants and suggests further characterization would be required for clinical applications.
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Neoplasias de la Mama Triple Negativas , Línea Celular Tumoral , Proliferación Celular/genética , Inhibidores y Moduladores de Gamma Secretasa , Humanos , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transducción de Señal , Nivel de Atención , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/terapiaRESUMEN
Hypoxia occurs in 90% of solid tumors and is associated with treatment failure, relapse, and mortality. HIF-1α signaling promotes resistance to chemotherapy in cancer cell lines and murine models via multiple mechanisms including the enrichment of breast cancer stem cells (BCSCs). In this work, we utilize a hypoxia fate-mapping system to determine whether triple-negative breast cancer (TNBC) cells that experience hypoxia in the primary tumor are resistant to chemotherapy at sites of metastasis. Using two orthotopic mouse models of TNBC, we demonstrate that cells that experience intratumoral hypoxia and metastasize to the lung and liver have decreased sensitivity to doxorubicin and paclitaxel but not cisplatin or 5-FU. Resistance to therapy leads to metastatic recurrence caused by post-hypoxic cells. We further determined that the post-hypoxic cells that metastasize are enriched in pathways related to cancer stem cell gene expression. Overall, our results show that even when hypoxic cancer cells are reoxygenated in the bloodstream they retain a hypoxia-induced cancer stem cell-like phenotype that persists and promotes resistance and eventually recurrence.
RESUMEN
Intratumor heterogeneity is an important mediator of poor outcomes in many cancers, including breast cancer. Genetic subclones frequently contribute to this heterogeneity; however, their growth dynamics and interactions remain poorly understood. PIK3CA and HER2 alterations are known to coexist in breast and other cancers. Herein, we present data that describe the ability of oncogenic PIK3CA mutant cells to induce the proliferation of quiescent HER2 mutant cells through a cell contact-mediated mechanism. Interestingly, the HER2 cells proliferated to become the major subclone over PIK3CA counterparts both in vitro and in vivo. Furthermore, this phenotype was observed in both hormone receptor-positive and -negative cell lines, and was dependent on the expression of fibronectin from mutant PIK3CA cells. Analysis of human tumors demonstrated similar HER2:PIK3CA clonal dynamics and fibronectin expression. Our study provides insight into nonrandom subclonal architecture of heterogenous tumors, which may aid the understanding of tumor evolution and inform future strategies for personalized medicine.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Comunicación Celular/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Técnicas de Cocultivo , Femenino , Fibronectinas/antagonistas & inhibidores , Fibronectinas/genética , Fibronectinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Frecuencia de los Genes , Técnicas de Inactivación de Genes , Humanos , Inmunohistoquímica , Células MCF-7 , Mutación , Fenotipo , Receptor ErbB-2/genéticaRESUMEN
PRX302 is a highly potent, mutant bacterial pore-forming biologic protoxin engineered for selective activation by PSA, a serine protease expressed by benign and malignant prostate epithelial cells. Although being developed as a local therapy for benign prostatic hyperplasia and localized prostate cancer, PRX302 cannot be administered systemically as a treatment for metastatic disease due to binding to ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored proteins, which leads to poor accumulation within the tumor microenvironment. To overcome this limitation, poly-lactic-co-glycolic acid (PLGA) microparticles encapsulating the protoxin were developed, which are known to accumulate in the liver, a major site of metastasis for prostate cancer and other solid tumors. A highly sensitive and reproducible sandwich ELISA to quantify PRX302 released from microparticles was developed. Utilizing this assay, PRX302 release from different microparticle formulations was assessed over multiple days. Hemolysis assays documented PSA-dependent pore formation and lytic potential (i.e., function) of the released protoxin. MTT assays demonstrated that conditioned supernatant from PRX302-loaded, but not blank (i.e., unloaded), PLGA microparticles was highly cytotoxic to PC3 and DU145 human prostate cancer cells in the presence of exogenous PSA. Microparticle encapsulation prevented PRX302 from immediately interacting with GPI-anchored proteins as demonstrated in a competition assay, which resulted in an increased therapeutic index and significant antitumor efficacy following a single dose of PRX302-loaded microparticles in a preclinical model of prostate cancer liver metastasis with no obvious toxicity. These results document that PRX302 released from PLGA microparticles demonstrate in vivo antitumor efficacy in a clinically relevant preclinical model of metastatic prostate cancer.
Asunto(s)
Antineoplásicos/farmacología , Productos Biológicos/farmacología , Composición de Medicamentos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/secundario , Neoplasias de la Próstata Resistentes a la Castración/patología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Toxinas Bacterianas/metabolismo , Productos Biológicos/administración & dosificación , Productos Biológicos/química , Línea Celular Tumoral , Modelos Animales de Enfermedad , Composición de Medicamentos/métodos , Hemólisis/efectos de los fármacos , Humanos , Masculino , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Unión Proteica , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Estrogen receptor-alpha (ER) is a therapeutic target of ER-positive (ER+) breast cancers. Although ER signaling is complex, many mediators of this pathway have been identified. Specifically, phosphorylation of ER at serine 118 affects responses to estrogen and therapeutic ligands and has been correlated with clinical outcomes in ER+ breast cancer patients. We hypothesized that a newly described germline variant (S118P) at this residue would drive cellular changes consistent with breast cancer development and/or hormone resistance. METHODS: Isogenic human breast epithelial cell line models harboring ER S118P were developed via genome editing and characterized to determine the functional effects of this variant. We also examined the frequency of ER S118P in a case-control study (N = 536) of women with and without breast cancer with a familial risk. RESULTS: In heterozygous knock-in models, the S118P variant demonstrated no significant change in proliferation, migration, MAP Kinase pathway signaling, or response to the endocrine therapies tamoxifen and fulvestrant. Further, there was no difference in the prevalence of S118P between women with and without cancer relative to population registry databases. CONCLUSIONS: This study suggests that the ER S118P variant does not affect risk for breast cancer or hormone therapy resistance. Germline screening and modification of treatments for patients harboring this variant are likely not warranted.
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Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/epidemiología , Receptor alfa de Estrógeno/genética , Mutación de Línea Germinal , Adulto , Anciano , Neoplasias de la Mama/genética , Estudios de Casos y Controles , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Receptor alfa de Estrógeno/metabolismo , Femenino , Fulvestrant/uso terapéutico , Variación Genética , Humanos , Incidencia , Células MCF-7 , Persona de Mediana Edad , Fosforilación , Análisis de Supervivencia , Tamoxifeno/uso terapéutico , Resultado del TratamientoRESUMEN
Prostate cancer is the most diagnosed malignancy and the second leading cause of cancer-related death in American men. While localized therapy is highly curative, treatments for metastatic prostate cancer are largely palliative. Thus, new innovative therapies are needed to target metastatic tumors. Prostate-Specific Antigen (PSA) is a chymotrypsin-like protease with a unique substrate specificity that is secreted by both normal and malignant prostate epithelial cells. Previous studies demonstrated the presence of high levels (µM-mM) of enzymatically active PSA is present in the extracellular fluid of the prostate cancer microenvironment. Because of this, PSA is an attractive target for a protease activated pro-toxin therapeutic strategy. Because prostate cancers typically grow very slowly, a strategy employing a proliferation-independent cytotoxic payload is preferred. Recently, it was shown that the human protease Granzyme B (GZMB), at low micromolar concentrations in the extracellular space, can cleave an array of extracellular matrix (ECM) proteins thus perturbing cell growth, signaling, motility, and integrity. It is also well established that other human proteases such as trypsin can induce similar effects. Because both enzymes require N-terminal proteolytic activation, we propose to convert these proteins into PSA-activated cytotoxins. In this study, we examine the enzymatic and cell targeting parameters of these PSA-activated cytotoxic serine proteases. These pro-enzymes were activated robustly by PSA and induced ECM damage that led to the death of prostate cancer cells in vitro thus supporting the potential use of this strategy as means to target metastatic prostate cancers.
RESUMEN
The GATA3 transcription factor is one of the most frequently mutated genes in breast cancer. Heterozygous mutations, mostly frameshifts, are seen in 15% of estrogen receptor positive breast cancers, the subtype in which these mutations are almost exclusively found. Mouse studies have shown that Gata3 is critical for breast development and that GATA3 gene dosage affects breast tumor progression. Human patient data have shown that high Gata3 expression, a feature of luminal subtype breast cancers, is associated with a better prognosis. Although the frequency of GATA3 mutation suggests an important role in breast cancer development or progression, there is little understanding of how mutations in GATA3 affect its function in luminal breast epithelial cells and what gene expression changes result as a consequence of the mutations. Here, using gene editing, we have created two sets of isogenic human luminal breast cancer cell lines with and without a hotspot truncating GATA3 mutation. GATA3 mutation enhanced tumor growth in vivo but did not affect sensitivity to clinically used hormonal therapies or chemotherapeutic agents. We identified genes with upregulated and downregulated expression in GATA3 mutant cells, a subset of which was concordantly differentially expressed in GATA3 mutant primary luminal breast cancers. Addback of mutant GATA3 recapitulated mutation-specific gene expression changes and enhanced soft agar colony formation, suggesting a gain of function for the mutant protein.
RESUMEN
BACKGROUND/PURPOSE: The combined contributions of oncogenes and tumor suppressor genes toward carcinogenesis remain poorly understood. Elucidation of cancer gene cooperativity can provide new insights leading to more effective use of therapies. EXPERIMENTAL DESIGN/METHODS: We used somatic cell genome editing to introduce singly and in combination PIK3CA mutations (E545K or H1047R) with TP53 alterations (R248W or knockout), to assess any enhanced cancerous phenotypes. The non-tumorigenic human breast epithelial cell line, MCF10A, was used as the parental cell line, and resultant cells were assessed via various in vitro assays, growth as xenografts, and drug sensitivity assays using targeted agents and chemotherapies. RESULTS: Compared to single-gene-targeted cells and parental controls, cells with both a PIK3CA mutation and TP53 alteration had increased cancerous phenotypes including cell proliferation, soft agar colony formation, aberrant morphology in acinar formation assays, and genomic heterogeneity. Cells also displayed varying sensitivities to anti-neoplastic drugs, although all cells with PIK3CA mutations showed a relative increased sensitivity to paclitaxel. All cell lines remained non-tumorigenic. CONCLUSIONS: This cell line panel provides a resource for further elucidating cooperative genetic mediators of carcinogenesis and response to therapies.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Fosfatidilinositol 3-Quinasa Clase I/genética , Mutación , Fenotipo , Proteína p53 Supresora de Tumor/genética , Animales , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Centrómero/genética , Variaciones en el Número de Copia de ADN , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Amplificación de Genes , Edición Génica , Técnicas de Inactivación de Genes , Inestabilidad Genómica , Genotipo , Humanos , Ratones , Paclitaxel/farmacologíaRESUMEN
BACKGROUND: We have previously documented a paradoxical anti-tumor effect when castration-resistant prostate cancer patients were treated with intermittent, high-dose testosterone (i.e., Bipolar Androgen Therapy; BAT). Because, an adaptive increase in androgen receptor expression following chronic androgen deprivation therapy (ADT) may underlie this effect, we tested whether men with hormone-sensitive (HS) prostate cancer (PC) would also respond to BAT if given following a 6-month ADT lead-in. METHODS: Asymptomatic HS PC patients with low metastatic burden or non-metastatic biochemically recurrent disease were enrolled. Following 6-month of ADT, those with a PSA <4 ng/ml went on to receive alternating 3-month cycles of BAT and ADT. BAT was administered as intramuscular testosterone (T) cypionate or enanthate 400 mg on Days (D) 1, 29, and 57. ADT was continued throughout the study to allow rapid cycling from near castrate to supraphysiologic range T following T injections. The primary endpoint was the percent of patients with a PSA <4 ng/ml after 18 months. Secondary endpoints included radiographic response and quality of life (QoL). RESULTS: Twenty-nine of 33 patients received BAT following the ADT lead-in. The primary endpoint was met, with 17/29 men (59%, 90% confidence interval: 42-74%) having a PSA <4 ng/ml at 18 months. Ten patients receiving BAT had RECIST evaluable disease, and eight (80%) objective responses were observed (four complete; four partial). Three patients progressed per RECIST criteria and three had unconfirmed progression on bone scan. Men treated with 6-month of ADT had improved QoL following the first cycle of BAT as measured by the SF-36, FACT-P, and IIEF surveys. CONCLUSIONS: BAT demonstrated preliminary efficacy in men with HS PC following 6-month of ADT. BAT may improve QoL in men treated with ADT. Prostate 76:1218-1226, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Antagonistas de Andrógenos/uso terapéutico , Neoplasias de la Próstata Resistentes a la Castración/diagnóstico , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Testosterona/uso terapéutico , Anciano , Humanos , Masculino , Persona de Mediana Edad , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/sangre , Calidad de VidaRESUMEN
BACKGROUND: There is an urgent need to develop new agents for treating metastatic prostate cancer to overcome multiple drug resistance to the current standard targeted cancer therapy. Emetine is a highly cytotoxic natural product protein synthesis inhibitor, which is toxic to all cell types. Its cytotoxicity can be blocked by derivatizing its N-2' position. Thus emetine can be selectively delivered to cancer cells in the region of metastatic cancer as a prodrug that will be activated by an enzyme selectively overexpressed within the metastatic tumor microenvironment. In this work, we convert emetine to a prodrug activatable by the fibroblast activation protein (FAP), a serine protease overexpressed by the carcinoma associated fibroblasts. METHOD: By using an iterative structure-activity relationship strategy, several peptidyl emetine prodrug analogs (1-11) were synthesized by chemical derivatization of emetine at its N-2' position and tested for in-vitro activation by FAP. The lead prodrug 11 is made up of a DPPIV activatable prodrug precursor 10 (Ala-Pro-PABC-Emetine) coupled to FAP substrate (Ala-Ser-Gly-Pro-Ala-Gly-Pro). Activation assays of the prodrugs were performed in purified FAP, DPPIV, FBS, and human serum and were analyzed by LCMS. In vitro cytotoxicity assays of these prodrugs are carried out in prostate (LNCaP, PC3) and breast (MCF7 and MDA-MB-231) cancer cell lines. The prodrugs are also tested in normal immortalized human prostatic epithelial cell line (PrEC). RESULTS: The lead FAP activated emetine prodrug 11 is activated to emetine in tandem by FAP and DPPIV in about 70% conversion within 24 hr. In prostate and breast cancer cell lines treated with prodrug 11, it is found to be equipotent with emetine in the presence of FAP and DPPIV. However, in the PrEC cell line grown in serum free media, prodrug 11 is more than 200-fold less cytotoxic than emetine in the absence of FAP and DPPIV. CONCLUSION: This FAP activated prodrug of cytotoxic agent emetine further shows the crucial role of the N-2' position of emetine in controlling its cytotoxicity. Significantly reduced toxicity observed in the PrEC cell line in the absence of FAP and DPPIV shows that prodrug 11 could be systemically delivered to regions of metastatic prostate cancer or other solid tumor for activation by cancer selective enzymes within the cancer microenvironment, such as FAP that is overexpressed by the carcinoma-associated fibroblasts. The two-step tandem enzymatic activation of prodrug 11 by FAP and DPPIV is a strategy for overcoming steric hindrance.
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Antineoplásicos/uso terapéutico , Dipeptidil Peptidasa 4/metabolismo , Diseño de Fármacos , Emetina/uso terapéutico , Gelatinasas/metabolismo , Proteínas de la Membrana/metabolismo , Profármacos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Serina Endopeptidasas/metabolismo , Línea Celular Tumoral , Endopeptidasas , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Relación Estructura-ActividadRESUMEN
Ki-67 expression is correlated with cell proliferation and is a prognostic marker for various cancers; however, its function is unknown. Here we demonstrate that genetic disruption of Ki-67 in human epithelial breast and colon cancer cells depletes the cancer stem cell niche. Ki-67 null cells had a proliferative disadvantage compared to wildtype controls in colony formation assays and displayed increased sensitivity to various chemotherapies. Ki-67 null cancer cells showed decreased and delayed tumor formation in xenograft assays, which was associated with a reduction in cancer stem cell markers. Immunohistochemical analyses of human breast cancers revealed that Ki-67 expression is maintained at equivalent or greater levels in metastatic sites of disease compared to matched primary tumors, suggesting that maintenance of Ki-67 expression is associated with metastatic/clonogenic potential. These results elucidate Ki-67's role in maintaining the cancer stem cell niche, which has potential diagnostic and therapeutic implications for human malignancies.
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Neoplasias de la Mama/metabolismo , Neoplasias del Colon/metabolismo , Antígeno Ki-67/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Transducción de SeñalRESUMEN
Recurrent human epidermal growth factor receptor 2 (HER2) missense mutations have been reported in human cancers. These mutations occur primarily in the absence of HER2 gene amplification such that most HER2-mutant tumors are classified as "negative" by FISH or immunohistochemistry assays. It remains unclear whether nonamplified HER2 missense mutations are oncogenic and whether they are targets for HER2-directed therapies that are currently approved for the treatment of HER2 gene-amplified breast cancers. Here we functionally characterize HER2 kinase and extracellular domain mutations through gene editing of the endogenous loci in HER2 nonamplified human breast epithelial cells. In in vitro and in vivo assays, the majority of HER2 missense mutations do not impart detectable oncogenic changes. However, the HER2 V777L mutation increased biochemical pathway activation and, in the context of a PIK3CA mutation, enhanced migratory features in vitro. However, the V777L mutation did not alter in vivo tumorigenicity or sensitivity to HER2-directed therapies in proliferation assays. Our results suggest the oncogenicity and potential targeting of HER2 missense mutations should be considered in the context of cooperating genetic alterations and provide previously unidentified insights into functional analysis of HER2 mutations and strategies to target them.
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Movimiento Celular/genética , Mutación Missense/genética , Neoplasias/genética , Receptor ErbB-2/genética , Transducción de Señal/genética , Western Blotting , Línea Celular Tumoral , Proliferación Celular/fisiología , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo , Marcación de Gen , Células HEK293 , Humanos , Immunoblotting , Lapatinib , Quinazolinas , Quinolinas , TiazolesRESUMEN
The tumor protein 53 (TP53) tumor suppressor gene is the most frequently somatically altered gene in human cancers. Here we show expression of N-Myc down-regulated gene 1 (NDRG1) is induced by p53 during physiologic low proliferative states, and mediates centrosome homeostasis, thus maintaining genome stability. When placed in physiologic low-proliferating conditions, human TP53 null cells fail to increase expression of NDRG1 compared with isogenic wild-type controls and TP53 R248W knockin cells. Overexpression and RNA interference studies demonstrate that NDRG1 regulates centrosome number and amplification. Mechanistically, NDRG1 physically associates with γ-tubulin, a key component of the centrosome, with reduced association in p53 null cells. Strikingly, TP53 homozygous loss was mutually exclusive of NDRG1 overexpression in over 96% of human cancers, supporting the broad applicability of these results. Our study elucidates a mechanism of how TP53 loss leads to abnormal centrosome numbers and genomic instability mediated by NDRG1.
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Proteínas de Ciclo Celular/metabolismo , Centrosoma/ultraestructura , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Aneuploidia , Animales , Mama/metabolismo , Línea Celular , Proliferación Celular , Centrosoma/metabolismo , Femenino , Genoma , Heterocigoto , Homeostasis , Homocigoto , Humanos , Hibridación Fluorescente in Situ , Ratones , Ratones Noqueados , Neoplasias/patología , Fenotipo , Interferencia de ARN , Tubulina (Proteína)/metabolismoRESUMEN
Multikinase inhibitors (MKIs) targeting VEGF receptors and other receptor tyrosine kinases have shown considerable activity in clinical trials of thyroid cancer. Thyroid cancer frequently exhibits activation of the RAS/RAF/MEK/ERK pathway. In other types of cancer, paradoxical ERK activation has emerged as a potential resistance mechanism to RAF-inhibiting drugs including MKIs such as sorafenib and pazopanib. We therefore queried whether the MEK inhibitor trametinib, could augment the activity of pazopanib in thyroid cancer cell lines. Trametinib potently inhibited growth in vitro (GI50 1.1-4.8 nM), whereas pazopanib had more limited in vitro activity, as anticipated (GI50 1.4-7.1 µM). We observed progressive upregulation of ERK activity with pazopanib treatment, an effect abrogated by trametinib. For xenografts (bearing either KRASG12R or BRAFV600E mutations), the combination of trametinib and pazopanib led to sustained shrinkage in tumor volume by 50% or more, compared to pre-treatment baseline. Trametinib also was highly effective as a single agent, compared to pazopanib alone. These preclinical findings support the evaluation of trametinib, alone or in combination with pazopanib or other kinase inhibitors, in thyroid cancer clinical trials. We highlight the importance of pharmacodynamic assessment of the ERK pathway for patients enrolled in trials involving MKIs.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Tiroides/tratamiento farmacológico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Indazoles , Sistema de Señalización de MAP Quinasas , Ratones Desnudos , Piridonas/administración & dosificación , Pirimidinas/administración & dosificación , Pirimidinonas/administración & dosificación , Sulfonamidas/administración & dosificación , Neoplasias de la Tiroides/patología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tamoxifen is effective for treating estrogen receptor-alpha (ER) positive breast cancers. However, few molecular mediators of tamoxifen resistance have been elucidated. Here we describe a previously unidentified gene, MACROD2 that confers tamoxifen resistance and estrogen independent growth. We found MACROD2 is amplified and overexpressed in metastatic tamoxifen-resistant tumors. Transgene overexpression of MACROD2 in breast cancer cell lines results in tamoxifen resistance, whereas RNAi-mediated gene knock down reverses this phenotype. MACROD2 overexpression also leads to estrogen independent growth in xenograft assays. Mechanistically, MACROD2 increases p300 binding to estrogen response elements in a subset of ER regulated genes. Primary breast cancers and matched metastases demonstrate MACROD2 expression can change with disease evolution, and increased expression and amplification of MACROD2 in primary tumors is associated with worse overall survival. These studies establish MACROD2 as a key mediator of estrogen independent growth and tamoxifen resistance, as well as a potential novel target for diagnostics and therapy.
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Neoplasias de la Mama/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Resistencia a Antineoplásicos , Estrógenos/metabolismo , Hidrolasas/metabolismo , Tamoxifeno/farmacología , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Epigénesis Genética , Femenino , Eliminación de Gen , Dosificación de Gen , Humanos , Datos de Secuencia Molecular , Trasplante de Neoplasias , Fenotipo , Pronóstico , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores de Estrógenos/metabolismo , Transgenes , Resultado del TratamientoRESUMEN
Tasquinimod, an orally active quinoline-3-carboxamide, binds with high affinity to HDAC4 and S100A9 in cancer and infiltrating host cells within compromised tumor microenvironment inhibiting adaptive survival pathways needed for an angiogenic response. Clinical trials document that as low as 0.5-1mg tasquinimod/day is therapeutic against castrate resistant metastatic prostate cancer. Tasquinimod is metabolized via cytochrome P4503A4, but ketoconazole at a dose which completely inhibits CYP3A metabolism does not affect tasquinimod's ability to inhibit endothelial "sprouting" in vitro or anti-cancer efficacy against human prostate cancer xenografts in vivo. Tasquinimod's potency is facilitated by its reversible binding (Kd < 35 µM) to the IIA subdomain of albumin (Sudlow's site I). As blood vessels within the compromised cancer microenvironment are characterized by a higher degree of leakiness than those in normal tissues, this results in an enhanced uptake of tasquinimod bound to albumin in cancer tissue via a tumor specific process known as the "enhanced permeability and retention" (i.e., EPR) effect. Thus, despite plasma levels of < 1 µM, the EPR effect results in intracellular drug concentrations of 2-3 µM, levels several-fold higher than needed for inhibition of endothelial sprouting (IC50 ~ 0.5 µM) or for inhibition of HDAC4 and S100A9 mediated tumor growth.
Asunto(s)
Inhibidores de la Angiogénesis/farmacocinética , Neoplasias de la Próstata Resistentes a la Castración/irrigación sanguínea , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Quinolinas/farmacocinética , Albúmina Sérica/metabolismo , Microambiente Tumoral , Administración Oral , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/sangre , Animales , Disponibilidad Biológica , Biotransformación , Permeabilidad Capilar , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Desnudos , Neovascularización Fisiológica/efectos de los fármacos , Orquiectomía , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Unión Proteica , Quinolinas/administración & dosificación , Quinolinas/sangre , Quinolonas , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: As carcinoma progresses, the stroma undergoes a variety of phenotypic changes, including the presence of carcinoma-associated fibroblasts (CAFs) that express fibroblast activation protein (FAP). FAP is a post-prolyl endopeptidase whose expression in a healthy adult is largely restricted to the cancer-associated stroma. FAP-targeted prodrugs with a 100-fold greater therapeutic window over the parent compound were previously generated. METHODS: Prodrugs and non-cleavable controls were incubated in the presence of FAP. Plasma and tumor half-lives (t1/2) of the full-length and active forms of the prodrugs were determined using LCMS. Biodistribution studies of prodrug activation were performed. Histopathological analysis of tissues from treated animals were compared to vehicle-treated controls. Toxicity and efficacy studies were performed in human breast (MDA-MB-231 and MCF-7) and prostate (LNCaP) cancer xenografts models. RESULTS: These FAP-activated prodrugs have a significantly slower clearance from tumor tissue than the circulation (â¼12 vs. â¼4.5 hr). Micromolar concentrations of active drug persist in the tumor. Active drug is detected in non-target tissues; however, histopathologic evaluation reveals no evidence of drug-induced toxicity. A FAP-activated prodrug (ERGETGP-S12ADT) inhibits tumor growth in multiple human breast and prostate cancer xenograft models. The anti-tumor effect is comparable to that observed with docetaxel, but results in significantly less toxicity. CONCLUSION: FAP-activated prodrugs are a viable strategy for the management of prostate and other cancers. These prodrugs exhibit less toxicity than a commonly used chemotherapeutic agent. Further refinement of the FAP cleavage site for greater specificity may reduce prodrug activation in non-target tissues and enhance clinical benefit.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antineoplásicos/farmacocinética , Gelatinasas/farmacocinética , Proteínas de la Membrana/farmacocinética , Profármacos/farmacocinética , Neoplasias de la Próstata/tratamiento farmacológico , Serina Endopeptidasas/farmacocinética , Adenocarcinoma/patología , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Endopeptidasas , Gelatinasas/efectos adversos , Gelatinasas/uso terapéutico , Humanos , Masculino , Proteínas de la Membrana/efectos adversos , Proteínas de la Membrana/uso terapéutico , Ratones , Profármacos/efectos adversos , Profármacos/uso terapéutico , Neoplasias de la Próstata/patología , Serina Endopeptidasas/efectos adversos , Serina Endopeptidasas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Activating mutations in the phosphoinositide-3-kinase (PI3K)/AKT/mTOR pathway are present in the majority of breast cancers and therefore are a major focus of drug development and clinical trials. Pathway mutations have been proposed as predictive biomarkers for efficacy of PI3K-targeted therapies. However, the precise contribution of distinct PI3K pathway mutations to drug sensitivity is unknown. EXPERIMENTAL DESIGN: We describe the creation of a physiologic human luminal breast cancer cell line model to study the phenotype of these mutations using the MCF-7 cell line. We used somatic cell gene targeting to "correct" PIK3CA E545K-mutant alleles in MCF-7 cells to wild-type sequence. The AKT1 E17K hotspot mutation was knocked in on this wild-type background. RESULTS: Loss of mutant PIK3CA dramatically reduced phosphorylation of AKT proteins and several known AKT targets, but other AKT target proteins and downstream effectors of mTOR were not affected. PIK3CA wild-type cells exhibited reduced proliferation in vitro and in vivo. Knockin of the AKT1 E17K hotspot mutation on this PIK3CA wild-type background restored pathway signaling, proliferation, and tumor growth in vivo. PIK3CA, but not AKT1 mutation, increased sensitivity to the PI3K inhibitor GDC-0941 and the allosteric AKT inhibitor MK-2206. CONCLUSIONS: AKT1 E17K is a bona fide oncogene in a human luminal breast cancer context. Distinct PI3K pathway mutations confer differential sensitivity to drugs targeting the pathway at different points and by distinct mechanisms. These findings have implications for the use of tumor genome sequencing to assign patients to targeted therapies.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Mutación , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/efectos de los fármacos , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Femenino , Xenoinjertos , Humanos , Células MCF-7 , Ratones , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genéticaRESUMEN
Prostate-specific antigen (PSA) is a serine protease produced at high levels by normal and malignant prostate epithelial cells that is used extensively as a biomarker in the clinical management of prostate cancer. To better understand PSA's role in prostate cancer progression, we prepared a library of peptidyl boronic acid-based inhibitors. To enhance selectivity for PSA vs other serine proteases, we modified the P1 site of the inhibitors to incorporate a bromopropylglycine group. This allowed the inhibitors to participate in halogen bond formation with the serine found at the bottom of the specificity pocket. The best of these Ahx-FSQn(boro)Bpg had PSA Ki of 72 nM and chymotrypsin Ki of 580 nM. In vivo studies using PSA-producing xenografts demonstrated that candidate inhibitors had minimal effect on growth but significantly altered serum levels of PSA. Biodistribution of (125)I labeled peptides showed low levels of uptake into tumors compared to other normal tissues.