RESUMEN
Metastatic prostate cancer remains an incurable lethal disease. Studies indicate that prostate cancer accumulates genomic changes during disease progression and displays the highest levels of chromosomal instability (CIN) across all types of metastatic tumours. CIN, which refers to ongoing chromosomal DNA gain or loss during mitosis, and derived aneuploidy, are known to be associated with increased tumour heterogeneity, metastasis and therapy resistance in many tumour types. Paradoxically, high CIN levels are also proposed to be detrimental to tumour cell survival, suggesting that cancer cells must develop adaptive mechanisms to ensure their survival. In the context of prostate cancer, studies indicate that CIN has a key role in disease progression and might also offer a therapeutic vulnerability that can be pharmacologically targeted. Thus, a comprehensive evaluation of the causes and consequences of CIN in prostate cancer, its contribution to aggressive advanced disease and a better understanding of the acquired CIN tolerance mechanisms can translate into new tumour classifications, biomarker development and therapeutic strategies.
Asunto(s)
Inestabilidad Cromosómica , Neoplasias de la Próstata , Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Progresión de la EnfermedadRESUMEN
Signaling rewiring allows tumors to survive therapy. Here we show that the decrease of the master regulator microphthalmia transcription factor (MITF) in lethal prostate cancer unleashes eukaryotic initiation factor 3B (eIF3B)-dependent translation reprogramming of key mRNAs conferring resistance to androgen deprivation therapy (ADT) and promoting immune evasion. Mechanistically, MITF represses through direct promoter binding eIF3B, which in turn regulates the translation of specific mRNAs. Genome-wide eIF3B enhanced cross-linking immunoprecipitation sequencing (eCLIP-seq) showed specialized binding to a UC-rich motif present in subsets of 5' untranslated regions. Indeed, translation of the androgen receptor and major histocompatibility complex I (MHC-I) through this motif is sensitive to eIF3B amount. Notably, pharmacologic targeting of eIF3B-dependent translation in preclinical models sensitizes prostate cancer to ADT and anti-PD-1 therapy. These findings uncover a hidden connection between transcriptional and translational rewiring promoting therapy-refractory lethal prostate cancer and provide a druggable mechanism that may transcend into effective combined therapeutic strategies. SIGNIFICANCE: Our study shows that specialized eIF3B-dependent translation of specific mRNAs released upon downregulation of the master transcription factor MITF confers castration resistance and immune evasion in lethal prostate cancer. Pharmacologic targeting of this mechanism delays castration resistance and increases immune-checkpoint efficacy. This article is featured in Selected Articles from This Issue, p. 2489.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Neoplasias de la Próstata , Masculino , Humanos , Factores de Transcripción , Antagonistas de Andrógenos/farmacología , Antagonistas de Andrógenos/uso terapéutico , Evasión Inmune , Receptores Androgénicos/genética , Castración , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patologíaRESUMEN
Chromosomal instability (CIN) is a pervasive feature of human cancers involved in tumor initiation and progression and which is found elevated in metastatic stages. CIN can provide survival and adaptation advantages to human cancers. However, too much of a good thing may come at a high cost for tumor cells as excessive degree of CIN-induced chromosomal aberrations can be detrimental for cancer cell survival and proliferation. Thus, aggressive tumors adapt to cope with ongoing CIN and most likely develop unique susceptibilities that can be their Achilles' heel. Determining the differences between the tumor-promoting and tumor-suppressing effects of CIN at the molecular level has become one of the most exciting and challenging aspects in cancer biology. In this review, we summarized the state of knowledge regarding the mechanisms reported to contribute to the adaptation and perpetuation of aggressive tumor cells carrying CIN. The use of genomics, molecular biology, and imaging techniques is significantly enhancing the understanding of the intricate mechanisms involved in the generation of and adaptation to CIN in experimental models and patients, which were not possible to observe decades ago. The current and future research opportunities provided by these advanced techniques will facilitate the repositioning of CIN exploitation as a feasible therapeutic opportunity and valuable biomarker for several types of human cancers.
Asunto(s)
Neoplasias , Humanos , Neoplasias/genética , Neoplasias/terapia , Inestabilidad Cromosómica , Aberraciones Cromosómicas , Genómica , Biomarcadores , Inestabilidad GenómicaRESUMEN
Metastatic prostate cancer (PCa) inevitably acquires resistance to standard therapy preceding lethality. Here, we unveil a chromosomal instability (CIN) tolerance mechanism as a therapeutic vulnerability of therapy-refractory lethal PCa. Through genomic and transcriptomic analysis of patient datasets, we find that castration and chemotherapy-resistant tumors display the highest CIN and mitotic kinase levels. Functional genomics screening coupled with quantitative phosphoproteomics identify MASTL kinase as a survival vulnerability specific of chemotherapy-resistant PCa cells. Mechanistically, MASTL upregulation is driven by transcriptional rewiring mechanisms involving the non-canonical transcription factors androgen receptor splice variant 7 and E2F7 in a circuitry that restrains deleterious CIN and prevents cell death selectively in metastatic therapy-resistant PCa cells. Notably, MASTL pharmacological inhibition re-sensitizes tumors to standard therapy and improves survival of pre-clinical models. These results uncover a targetable mechanism promoting high CIN adaptation and survival of lethal PCa.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Masculino , Humanos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Inestabilidad Cromosómica , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/uso terapéutico , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
The concept of Cancer of Unknown Primary (CUP) has evolved with the advent of medical oncology. CUP can be difficult to diagnose and represents 2 to 5% of new cancers, therefore not exceptionally rare. Within CUPs can be identified a subset of favourable prognosis tumours, however the vast majority of CUP patients belongs to a poor prognosis group. CUP features significant oncological challenges, such as unravelling biological and transversal issues, and most importantly, improving patient's outcomes. In that regard, CUP patients' outcomes regrettably showed minimal improvement for decades and CUP remains a cancer group of very poor prognosis. The biology of CUP has two main hypotheses. One is that CUP is a subgroup of a given primary cancer, where the primary is present but cannot be seen due to its small size. The other, the "true" CUP hypothesis, states that CUP share features that make them a specific entity, whatever their tissue of origin. A true biological signature has not yet been described, but chromosomal instability is a hallmark of poor prognosis CUP group. Precision oncology, despite achieving identifying the putative origin of the CUP, so far failed to globally improve outcomes of patients. Targeting molecular pathways based on molecular analysis in CUP management is under investigation. Immunotherapy has not shown ground-breaking results, to date. Accrual is also a crucial issue in CUP trials. Herein we review CUP history, biological features and remaining questions in CUP biology, the two main approaches of molecular oncology in CUP management, in order to draw perspectives in the enormous challenge of improving CUP patient outcomes.
Asunto(s)
Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/antagonistas & inhibidores , Terapia Molecular Dirigida , Neoplasias Primarias Desconocidas/tratamiento farmacológico , Medicina de Precisión , Biomarcadores de Tumor/genética , Ensayos Clínicos como Asunto , Perfilación de la Expresión Génica , Humanos , Neoplasias Primarias Desconocidas/genética , Neoplasias Primarias Desconocidas/patología , PronósticoRESUMEN
Over the past 5 years, the advent of combination therapeutic strategies has substantially reshaped the clinical management of patients with advanced prostate cancer. However, most of these combination regimens were developed empirically and, despite offering survival benefits, are not enough to halt disease progression. Thus, the development of effective therapeutic strategies that target the mechanisms involved in the acquisition of drug resistance and improve clinical trial design are an unmet clinical need. In this context, we hypothesize that the tumour engineers a dynamic response through the process of cellular rewiring, in which it adapts to the therapy used and develops mechanisms of drug resistance via downstream signalling of key regulatory cascades such as the androgen receptor, PI3K-AKT or GATA2-dependent pathways, as well as initiation of biological processes to revert tumour cells to undifferentiated aggressive states via phenotype switching towards a neuroendocrine phenotype or acquisition of stem-like properties. These dynamic responses are specific for each patient and could be responsible for treatment failure despite multi-target approaches. Understanding the common stages of these cellular rewiring mechanisms to gain a new perspective on the molecular underpinnings of drug resistance might help formulate novel combination therapeutic regimens.
Asunto(s)
Resistencia a Antineoplásicos/fisiología , Neoplasias de la Próstata/tratamiento farmacológico , Transducción de Señal/fisiología , Humanos , MasculinoRESUMEN
The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.
Asunto(s)
Células Madre Neoplásicas/patología , Sarcoma/patología , Animales , Diferenciación Celular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Xenoinjertos , Humanos , RatonesRESUMEN
Nuclear pore complexes (NPCs) regulate nuclear-cytoplasmic transport, transcription, and genome integrity in eukaryotic cells. However, their functional roles in cancer remain poorly understood. We interrogated the evolutionary transcriptomic landscape of NPC components, nucleoporins (Nups), from primary to advanced metastatic human prostate cancer (PC). Focused loss-of-function genetic screen of top-upregulated Nups in aggressive PC models identified POM121 as a key contributor to PC aggressiveness. Mechanistically, POM121 promoted PC progression by enhancing importin-dependent nuclear transport of key oncogenic (E2F1, MYC) and PC-specific (AR-GATA2) transcription factors, uncovering a pharmacologically targetable axis that, when inhibited, decreased tumor growth, restored standard therapy efficacy, and improved survival in patient-derived pre-clinical models. Our studies molecularly establish a role of NPCs in PC progression and give a rationale for NPC-regulated nuclear import targeting as a therapeutic strategy for lethal PC. These findings may have implications for understanding how NPC deregulation contributes to the pathogenesis of other tumor types.
Asunto(s)
Factor de Transcripción E2F1/metabolismo , Glicoproteínas de Membrana/metabolismo , Poro Nuclear/fisiología , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular , Carcinogénesis , Núcleo Celular/metabolismo , Proliferación Celular , Factor de Transcripción GATA2/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Membrana Nuclear , Proteínas de Complejo Poro Nuclear , Transducción de SeñalRESUMEN
Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.
Asunto(s)
Neoplasias de la Próstata/tratamiento farmacológico , Taxoides/farmacología , Antineoplásicos/farmacología , Docetaxel , Resistencia a Antineoplásicos , Humanos , Masculino , Neoplasias de la Próstata/patologíaRESUMEN
PP2A comprising B56 regulatory subunit isoforms (PP2AB56) is a serine/threonine phosphatase essential for mitosis. At the kinetochore, PP2AB56 both stabilizes microtubule binding and promotes silencing of the spindle assembly checkpoint (SAC) through its association with the SAC protein BubR1. Cells depleted of the B56 regulatory subunits of PP2A are delayed in activation of Cdc20-containing APC/C (APC/CCdc20), which is an essential step for mitotic exit. It has been hypothesized that this delay arises from increased production of the mitotic checkpoint complex (MCC), an APC/CCdc20 inhibitor formed at unattached kinetochores through SAC signaling. In contrast to this prediction, we show that depletion of B56 subunits does not increase the amount or stability of the MCC. Rather, delays in APC/CCdc20 activation in B56-depleted cells correlate with impaired Cdc20 binding to APC/C. Stimulation of APC/CCdc20 assembly does not require binding between PP2AB56 and BubR1, and thus this contribution of PP2AB56 towards mitotic exit is distinct from its functions at kinetochores. PP2AB56 associates with APC/C constitutively in a BubR1-independent manner. A mitotic phosphorylation site on Cdc20, known to be a substrate of PP2AB56, modulates APC/CCdc20 assembly. These results elucidate the contributions of PP2AB56 towards completion of mitosis.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas Cdc20/metabolismo , Mitosis , Proteína Fosfatasa 2/metabolismo , Puntos de Control del Ciclo Celular , Células HeLa , Humanos , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
The spindle assembly checkpoint kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1's error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers in vivo and reduced the efficiency of the Ska complex's conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation.
Asunto(s)
Segregación Cromosómica/fisiología , Cinetocoros/metabolismo , Metaloproteínas/metabolismo , Microtúbulos/metabolismo , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/metabolismo , Anafase/fisiología , Aurora Quinasa B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Humanos , Puntos de Control de la Fase M del Ciclo Celular/genéticaRESUMEN
Human leukocyte antigen class I (HLA-I) down-regulation has been reported in many human cancers to be associated with poor clinical outcome. However, its connection to tumor-initiating cells (TICs) remains unknown. In this study, we report that HLA-I is down-regulated in a subpopulation of cells that have high tumor initiating capacity in different types of human sarcomas. Detailed characterization revealed their distinct molecular profiles regarding proliferation, apoptosis and stemness programs. Notably, these TICs can be induced to differentiate along distinct mesenchymal lineages, including the osteogenic pathway. The retinoic acid receptor signaling pathway is overexpressed in HLA-1 negative TICs. All-trans retinoic acid treatment successfully induced osteogenic differentiation of this subpopulation, in vitro and in vivo, resulting in significantly decreased tumor formation. Thus, our findings indicate down-regulated HLA-I is a shared feature of TICs in a variety of human sarcomas, and differentiation therapy strategies may specifically target undifferentiated TICs and inhibit tumor formation.
Asunto(s)
Diferenciación Celular , Células Madre Neoplásicas/patología , Sarcoma/patología , Sarcoma/terapia , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinogénesis/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Antígenos HLA/inmunología , Humanos , Células Madre Neoplásicas/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Fenotipo , Sarcoma/genética , Tretinoina/farmacologíaRESUMEN
Advanced prostate cancer is a classic example of the intractability and consequent lethality that characterizes metastatic carcinomas. Novel treatments have improved the survival of men with prostate cancer; however, advanced prostate cancer invariably becomes resistant to these therapies and ultimately progresses to a lethal metastatic stage. Consequently, detailed knowledge of the molecular mechanisms that control prostate cancer cell survival and progression towards this lethal stage of disease will benefit the development of new therapeutics. The transcription factor endothelial transcription factor GATA-2 (GATA2) has been reported to have a key role in driving prostate cancer aggressiveness. In addition to being a pioneer transcription factor that increases androgen receptor (AR) binding and activity, GATA2 regulates a core subset of clinically relevant genes in an AR-independent manner. Functionally, GATA2 overexpression in prostate cancer increases cellular motility and invasiveness, proliferation, tumorigenicity, and resistance to standard therapies. Thus, GATA2 has a multifaceted function in prostate cancer aggressiveness and is a highly attractive target in the development of novel treatments against lethal prostate cancer.
Asunto(s)
Biomarcadores de Tumor/fisiología , Factor de Transcripción GATA2/fisiología , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/mortalidad , Humanos , Masculino , Invasividad Neoplásica/diagnóstico , Invasividad Neoplásica/genética , Neoplasias de la Próstata/diagnósticoRESUMEN
Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.
Asunto(s)
Células Neoplásicas Circulantes/patología , Neoplasias de la Próstata/patología , Trasplante Heterólogo/métodos , Animales , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/sangre , Recuento de Células , Citometría de Flujo , Xenoinjertos , Humanos , Antígenos Comunes de Leucocito/análisis , Antígenos Comunes de Leucocito/sangre , Masculino , Ratones , Neoplasias de la Próstata/sangreRESUMEN
Elucidating the determinants of aggressiveness in lethal prostate cancer may stimulate therapeutic strategies that improve clinical outcomes. We used experimental models and clinical databases to identify GATA2 as a regulator of chemotherapy resistance and tumorigenicity in this context. Mechanistically, direct upregulation of the growth hormone IGF2 emerged as a mediator of the aggressive properties regulated by GATA2. IGF2 in turn activated IGF1R and INSR as well as a downstream polykinase program. The characterization of this axis prompted a combination strategy whereby dual IGF1R/INSR inhibition restored the efficacy of chemotherapy and improved survival in preclinical models. These studies reveal a GATA2-IGF2 aggressiveness axis in lethal prostate cancer and identify a therapeutic opportunity in this challenging disease.
Asunto(s)
Factor de Transcripción GATA2/genética , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Animales , Antígenos CD/genética , Proliferación Celular , Resistencia a Antineoplásicos/genética , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Masculino , Ratones , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptor de Insulina/genética , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.
Asunto(s)
Técnicas Citológicas/métodos , Células Madre Neoplásicas/patología , Neoplasias de la Próstata/patología , Citometría de Flujo/métodos , Humanos , Masculino , Células Madre Neoplásicas/química , Neoplasias de la Próstata/química , Neoplasias de la Próstata Resistentes a la Castración/química , Neoplasias de la Próstata Resistentes a la Castración/patología , Coloración y Etiquetado/métodosRESUMEN
The spindle assembly checkpoint (SAC) delays anaphase until all chromosomes are bioriented on the mitotic spindle. Under current models, unattached kinetochores transduce the SAC by catalyzing the intramitotic production of a diffusible inhibitor of APC/C(Cdc20) (the anaphase-promoting complex/cyclosome and its coactivator Cdc20, a large ubiquitin ligase). Here we show that nuclear pore complexes (NPCs) in interphase cells also function as scaffolds for anaphase-inhibitory signaling. This role is mediated by Mad1-Mad2 complexes tethered to the nuclear basket, which activate soluble Mad2 as a binding partner and inhibitor of Cdc20 in the cytoplasm. Displacing Mad1-Mad2 from nuclear pores accelerated anaphase onset, prevented effective correction of merotelic errors, and increased the threshold of kinetochore-dependent signaling needed to halt mitosis in response to spindle poisons. A heterologous Mad1-NPC tether restored Cdc20 inhibitor production and normal M phase control. We conclude that nuclear pores and kinetochores both emit "wait anaphase" signals that preserve genome integrity.
Asunto(s)
Anafase , Proteínas de Ciclo Celular/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Proteínas Mad2/metabolismo , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Transporte Activo de Núcleo Celular , Proteínas de Ciclo Celular/genética , Dimerización , Células HCT116 , Células HeLa , Humanos , Interfase , Cinetocoros/metabolismo , Mitosis , Proteínas Nucleares/genéticaRESUMEN
Acquired resistance to Docetaxel precedes fatality in hormone-refractory prostate cancer (HRPC). However, strategies that target Docetaxel resistant cells remain elusive. Using in vitro and in vivo models, we identified a subpopulation of cells that survive Docetaxel exposure. This subpopulation lacks differentiation markers and HLA class I (HLAI) antigens, while overexpressing the Notch and Hedgehog signaling pathways. These cells were found in prostate cancer tissues and were related to tumor aggressiveness and poor patient prognosis. Notably, targeting Notch and Hedgehog signaling depleted this population through inhibition of the survival molecules AKT and Bcl-2, suggesting a therapeutic strategy for abrogating Docetaxel resistance in HRPC. Finally, these cells exhibited potent tumor-initiating capacity, establishing a link between chemotherapy resistance and tumor progression.
Asunto(s)
Proteínas Hedgehog/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Notch/metabolismo , Taxoides/farmacología , Animales , Apoptosis , Línea Celular Tumoral , Progresión de la Enfermedad , Docetaxel , Resistencia a Antineoplásicos , Proteínas Hedgehog/genética , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Datos de Secuencia Molecular , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Receptores Notch/genética , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Animal cells initiate cytokinesis in parallel with anaphase onset, when an actomyosin ring assembles and constricts through localized activation of the small GTPase RhoA, giving rise to a cleavage furrow. Furrow formation relies on positional cues provided by anaphase spindle microtubules (MTs), but how such cues are generated remains unclear. Using chemical genetics to achieve both temporal and spatial control, we show that the self-organized delivery of Polo-like kinase 1 (Plk1) to the midzone and its local phosphorylation of a MT-bound substrate are critical for generating this furrow-inducing signal. When Plk1 was active but unable to target itself to this equatorial landmark, both cortical RhoA recruitment and furrow induction failed to occur, thus recapitulating the effects of anaphase-specific Plk1 inhibition. Using tandem mass spectrometry and phosphospecific antibodies, we found that Plk1 binds and directly phosphorylates the HsCYK-4 subunit of centralspindlin (also known as MgcRacGAP) at the midzone. At serine 157, this modification creates a major docking site for the tandem BRCT repeats of the Rho GTP exchange factor Ect2. Cells expressing only a nonphosphorylatable form of HsCYK-4 failed to localize Ect2 at the midzone and were severely impaired in cleavage furrow formation, implying that HsCYK-4 is Plk1's rate-limiting target upstream of RhoA. Conversely, tethering an inhibitor-resistant allele of Plk1 to HsCYK-4 allowed furrows to form despite global inhibition of all other Plk1 molecules in the cell. Our findings illuminate two key mechanisms governing the initiation of cytokinesis in human cells and illustrate the power of chemical genetics to probe such regulation both in time and space.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , División Celular/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Anafase/fisiología , Animales , Proteínas de Ciclo Celular/genética , División Celular/genética , Línea Celular , Proteínas Activadoras de GTPasa/genética , Humanos , Immunoblotting , Inmunoprecipitación , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/fisiología , Serina/metabolismo , Serina/fisiología , Huso Acromático/metabolismo , Spodoptera , Espectrometría de Masas en Tándem , Quinasa Tipo Polo 1RESUMEN
The decision to enter mitosis is mediated by a network of proteins that regulate activation of the cyclin B-Cdk1 complex. Within this network, several positive feedback loops can amplify cyclin B-Cdk1 activation to ensure complete commitment to a mitotic state once the decision to enter mitosis has been made. However, evidence is accumulating that several components of the feedback loops are redundant for cyclin B-Cdk1 activation during normal cell division. Nonetheless, defined feedback loops become essential to promote mitotic entry when normal cell cycle progression is perturbed. Recent data has demonstrated that at least three Plk1-dependent feedback loops exist that enhance cyclin B-Cdk1 activation at different levels. In this review, we discuss the role of various feedback loops that regulate cyclin B-Cdk1 activation under different conditions, the timing of their activation, and the possible identity of the elusive trigger that controls mitotic entry in human cells.