RESUMEN
Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; Ki<0.5 nM; cellular IC50 2-6 nM). Treatment of tumor cells in vitro with PF-7006 modulates expected Mps1-dependent biology as demonstrated by molecular and phenotypic measures (reduced pHH3-Ser10 levels, shorter duration of mitosis, micro-nucleation, and apoptosis). Tumor-bearing mice treated with PF-7006 exhibit tumor growth inhibition concomitant with pharmacodynamic modulation of a downstream biomarker (pHH3-Ser10). Unfortunately, efficacy only occurs at drug exposures that cause dose-limiting body weight loss, gastrointestinal toxicities, and neutropenia. Mps1 inhibitor toxicities may be mitigated by inducing G1 cell cycle arrest in Rb1-competent cells with the cyclin-dependent kinase-4/6 inhibitor palbociclib. Using an isogenic cellular model system, PF-7006 is shown to be selectively cytotoxic to Rb1-deficient cells relative to Rb1-competent cells (also a measure of kinase selectivity). Human bone marrow cells pretreated with palbociclib have decreased PF-7006-dependent apoptosis relative to cells without palbociclib pretreatment. Collectively, this study raises a concern that single agent therapies inhibiting Mps1 will not be well-tolerated clinically but may be when combined with a selective CDK4/6 drug.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/metabolismo , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Histonas/metabolismo , Humanos , Intestino Delgado/patología , Ratones , Ratones SCID , Mitosis/efectos de los fármacos , Fosforilación , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/toxicidad , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Piridinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Trasplante HeterólogoRESUMEN
Breast cancer patients with tumors lacking the three diagnostic markers (ER, PR, and HER2) are classified as triple-negative (primarily basal-like) and have poor prognosis because there is no disease-specific therapy available. To address this unmet medical need, gene expression analyses using more than a thousand breast cancer samples were conducted, which identified elevated centromere protein E (CENP-E) expression in the basal-a molecular subtype relative to other subtypes. CENP-E, a mitotic kinesin component of the spindle assembly checkpoint, is shown to be induced in basal-a tumor cell lines by the mitotic spindle inhibitor drug docetaxel. CENP-E knockdown by inducible shRNA reduces basal-a breast cancer cell viability. A potent, selective CENP-E inhibitor (PF-2771) was used to define the contribution of CENP-E motor function to basal-like breast cancer. Mechanistic evaluation of PF-2771 in basal-a tumor cells links CENP-E-dependent molecular events (e.g., phosphorylation of histone H3 Ser-10; phospho-HH3-Ser10) to functional outcomes (e.g., chromosomal congression defects). Across a diverse panel of breast cell lines, CENP-E inhibition by PF-2771 selectively inhibits proliferation of basal breast cancer cell lines relative to premalignant ones and its response correlates with the degree of chromosomal instability. Pharmacokinetic-pharmacodynamic efficacy analysis in a basal-a xenograft tumor model shows that PF-2771 exposure is well correlated with increased phospho-HH3-Ser10 levels and tumor growth regression. Complete tumor regression is observed in a patient-derived, basal-a breast cancer xenograft tumor model treated with PF-2771. Tumor regression is also observed with PF-2771 in a taxane-resistant basal-a model. Taken together, CENP-E may be an effective therapeutic target for patients with triple-negative/basal-a breast cancer.
Asunto(s)
Antineoplásicos/farmacología , Benzamidas/farmacología , Proteínas Cromosómicas no Histona/genética , Glicina/análogos & derivados , Neoplasias Basocelulares/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Proteínas Cromosómicas no Histona/metabolismo , Femenino , Expresión Génica , Glicina/farmacología , Humanos , Estimación de Kaplan-Meier , Ratones SCID , Neoplasias Basocelulares/tratamiento farmacológico , Neoplasias Basocelulares/mortalidad , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/mortalidad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Evaluating the effects of novel drugs on appropriate tumor models has become crucial for developing more effective therapies that target highly tumorigenic and drug-resistant cancer stem cell (CSC) populations. In this study, we demonstrate that a subset of cancer cells with CSC properties may be enriched into tumor spheroids under stem cell conditions from a non-small cell lung cancer cell line. Treating these CSC-like cells with gemcitabine alone and a combination of gemcitabine and the novel CHK1 inhibitor PF-00477736 revealed that PF-00477736 enhances the anti-proliferative effect of gemcitabine against both the parental and the CSC-like cell populations. However, the CSC-like cells exhibited resistance to gemcitabine-induced apoptosis. Collectively, the spheroid-forming CSC-like cells may serve as a model system for understanding the mechanism underlying the drug resistance of CSCs and for guiding the development of better therapies that can inhibit tumor growth and eradicate CSCs.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos , Células Madre Neoplásicas/efectos de los fármacos , Esferoides Celulares/efectos de los fármacos , Animales , Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Benzodiazepinonas/administración & dosificación , Biomarcadores de Tumor/metabolismo , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Sinergismo Farmacológico , Femenino , Humanos , Ratones , Ratones Desnudos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Pirazoles/administración & dosificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Células Tumorales Cultivadas , GemcitabinaRESUMEN
Deregulation of the phosphoinositide 3-kinase (PI3K) signaling pathway such as by PTEN loss or PIK3CA mutation occurs frequently in human cancer and contributes to resistance to antitumor therapies. Inhibition of key signaling proteins in the pathway therefore represents a valuable targeting strategy for diverse cancers. PF-04691502 is an ATP-competitive PI3K/mTOR dual inhibitor, which potently inhibited recombinant class I PI3K and mTOR in biochemical assays and suppressed transformation of avian fibroblasts mediated by wild-type PI3K γ, δ, or mutant PI3Kα. In PIK3CA-mutant and PTEN-deleted cancer cell lines, PF-04691502 reduced phosphorylation of AKT T308 and AKT S473 (IC(50) of 7.5-47 nmol/L and 3.8-20 nmol/L, respectively) and inhibited cell proliferation (IC(50) of 179-313 nmol/L). PF-04691502 inhibited mTORC1 activity in cells as measured by PI3K-independent nutrient stimulated assay, with an IC(50) of 32 nmol/L and inhibited the activation of PI3K and mTOR downstream effectors including AKT, FKHRL1, PRAS40, p70S6K, 4EBP1, and S6RP. Short-term exposure to PF-04691502 predominantly inhibited PI3K, whereas mTOR inhibition persisted for 24 to 48 hours. PF-04691502 induced cell cycle G(1) arrest, concomitant with upregulation of p27 Kip1 and reduction of Rb. Antitumor activity was observed in U87 (PTEN null), SKOV3 (PIK3CA mutation), and gefitinib- and erlotinib-resistant non-small cell lung carcinoma xenografts. In summary, PF-04691502 is a potent dual PI3K/mTOR inhibitor with broad antitumor activity. PF-04691502 has entered phase I clinical trials.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piridonas/farmacología , Pirimidinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Animales , Antineoplásicos/uso terapéutico , Unión Competitiva , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Ratones , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
New approaches to prevent and reverse UV damage are needed to combat rising sunlight-induced skin cancer rates. Mouse studies have shown that oral or topical caffeine promotes elimination of UV-damaged keratinocytes through apoptosis and markedly inhibits subsequent skin cancer development. This potentially important therapeutic effect has not been studied in human skin cells. Here, we use primary human keratinocytes to examine which of several caffeine effects mediates this process. In these cells, caffeine more than doubled apoptosis after 75 mJ cm(-2) of ultraviolet light B (UVB). Selectively targeting two of caffeine's known effects did not alter UVB-induced apoptosis: inhibition of ataxia-telangiectasia mutated and augmentation of cyclic AMP levels. In contrast, siRNA against ataxia-telangiectasia and Rad3-related (ATR) doubled apoptosis after UV through a p53-independent mechanism. Caffeine did not further augment apoptosis after UVB in cells in which ATR had been specifically depleted, suggesting that a key target of caffeine in this effect is ATR. Inhibition of a central ATR target, checkpoint kinase 1 (Chk1), through siRNA or a new and highly specific inhibitor (PF610666) also augmented UVB-induced apoptosis. These data suggest that a relevant target of caffeine is the ATR-Chk1 pathway and that inhibiting ATR or Chk1 might have promise in preventing or reversing UV damage.
Asunto(s)
Apoptosis , Cafeína/farmacología , Proteínas de Ciclo Celular/metabolismo , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Rayos Ultravioleta/efectos adversos , Adulto , Anciano , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Apoptosis/efectos de la radiación , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Células Cultivadas , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Epidérmicas , Femenino , Humanos , Queratinocitos/citología , Inhibidores de Fosfodiesterasa/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Checkpoints are present in all phases of the cell cycle and are regarded as the gatekeepers maintaining the integrity of the genome. Many conventional agents used to treat cancer impart damage to the genome and activate cell cycle checkpoints. Many tumors are defective in the tumor suppressor p53 and therefore lack a functional G(1) checkpoint. In these tumors, however, the S-G(2) checkpoints remain intact and, in response to DNA damage, arrest cell cycle progression allowing time for DNA repair. Checkpoint kinase 1 (Chk1) is a key element in the DNA damage response pathway and plays a crucial role in the S-G(2)-phase checkpoints. Inhibiting Chk1 represents a therapeutic strategy for creating a "synthetic lethal" response by overriding the last checkpoint defense of tumor cells against the lethal damage induced by DNA-directed chemotherapeutic agents. Chk1 inhibition is consistent with emerging targeted therapies aiming to exploit molecular differences between normal and cancer cells. Adding a Chk1 inhibitor to DNA-damaging cytotoxic therapy selectively targets tumors with intrinsic checkpoint defects while minimizing toxicity in checkpoint-competent normal cells. PF-00477736 was identified as a potent, selective ATP-competitive small-molecule inhibitor that inhibits Chk1 with a K(i) of 0.49 nM. PF-00477736 abrogates cell cycle arrest induced by DNA damage and enhances cytotoxicity of clinically important chemotherapeutic agents, including gemcitabine and carboplatin. In xenografts, PF-00477736 enhanced the antitumor activity of gemcitabine in a dose-dependent manner. PF-00477736 combinations were well tolerated with no exacerbation of side effects commonly associated with cytotoxic agents.
Asunto(s)
Benzodiazepinonas/farmacología , Daño del ADN , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/efectos de los fármacos , Pirazoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cromatografía Liquida , Desoxicitidina/análogos & derivados , Desoxicitidina/antagonistas & inhibidores , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Histonas/metabolismo , Humanos , Masculino , Ratones , Ratones Desnudos , Fosforilación , Ratas , Ratas Sprague-Dawley , Fase S/efectos de los fármacos , Espectrometría de Masas en Tándem , GemcitabinaRESUMEN
The cocrystal structure of a library hit was used to design a novel series of CHK1 inhibitors. The new series retained the critical hydrogen-bonding groups of the resorcinol moiety for binding but lacked the phenolic anilide moiety. The newly designed compounds exhibited similar enzymatic activity, while demonstrating increased cellular potency. Compound 10c, showing no single agent effect, potentiated the antiproliferative effect of Gemcitabine in both prostate and breast cancer cell lines.