RESUMEN
Metastatic disease remains the leading cause of death due to cancer, yet the mechanism(s) of metastasis and its timely detection remain to be elucidated. Neutrophil elastase (NE), a serine protease secreted by neutrophils, is a crucial mediator of chronic inflammation and tumor progression. In this study, we used the PyMT model (NE+/+ and NE-/-) of breast cancer to interrogate the tumor-intrinsic and -extrinsic mechanisms by which NE can promote metastasis. Our results showed that genetic ablation of NE significantly reduced lung metastasis and improved metastasis-free survival. RNA-sequencing analysis of primary tumors indicated differential regulation of tumor-intrinsic actin cytoskeleton signaling pathways by NE. These NE-regulated pathways are critical for cell-to-cell contact and motility and consistent with the delay in metastasis in NE-/- mice. To evaluate whether pharmacologic inhibition of NE inhibited pulmonary metastasis and phenotypically mimicked PyMT NE-/- mice, we utilized AZD9668, a clinically available and specific NE inhibitor. We found AZD9668 treated PyMT-NE+/+ mice showed significantly reduced lung metastases, improved recurrence-free, metastasis-free and overall survival, and their tumors showed similar molecular alterations as those observed in PyMT-NE-/- tumors. Finally, we identified a NE-specific signature that predicts recurrence and metastasis in patients with breast cancer. Collectively, our studies suggest that genetic ablation and pharmacologic inhibition of NE reduces metastasis and extends survival of mouse models of breast cancer, providing rationale to examine NE inhibitors as a treatment strategy for the clinical management of patients with metastatic breast cancer.
Asunto(s)
Neoplasias de la Mama , Neoplasias Pulmonares , Piridonas , Sulfonas , Animales , Femenino , Humanos , Ratones , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Elastasa de Leucocito/genética , Neoplasias Pulmonares/patologíaRESUMEN
Salivary gland cancers (SGCs) are rare yet aggressive malignancies with significant histological heterogeneity, which has made prediction of prognosis and development of targeted therapies challenging. In majority of patients, local recurrence and/or distant metastasis are common and systemic treatments have minimal impact on survival. Therefore, identification of novel targets for treatment that can also be used as predictors of recurrence for multiple histological subtypes of SGCs is an area of unmet need. In this study, we developed a novel transgenic mouse model of SGC, efficiently recapitulating the major histological subtype (adenocarcinomas of the parotid gland) of human SGC. CDK2 knock out (KO) mice crossed with MMTV-low molecular weight forms of cyclin E (LMW-E) mice generated the transgenic mouse models of SGC, which arise in the parotid region of the salivary gland, similar to the common site of origin seen in human SGCs. To identify the CDK2 independent catalytic partner(s) of LMW-E, we used LMW-E expressing cell lines in mass spectrometric analysis and subsequent biochemical validation in pull down assays. These studies revealed that in the absence of CDK2, LMW-E preferentially binds to CDK5. Molecular targeting of CDK5, using siRNA, resulted in inhibition of cell proliferation of human SGCs overexpressing LMW-E. We also provide clinical evidence of significant association of LMW-E/CDK5 co-expression and decreased recurrence free survival in human SGC. Immunohistochemical analysis of LMW-E and CDK5 in 424 patients representing each of the four major histological subtypes of human salivary cancers (Aci, AdCC, MEC, and SDC) revealed that LMW-E and CDK5 are concordantly (positive/positive or negative/negative) expressed in 70% of these patients. The co-expression of LMW-E/CDK5 (both positive) robustly predicts the likelihood of recurrence, regardless of the histological classification of these tumors. Collectively, our results suggest that CDK5 is a novel and targetable biomarker for the treatment of patients with SGC presenting with LMW-E overexpressing tumors.
RESUMEN
Cyclin E and its co-activator, phospho-cyclin-dependent kinase 2 (p-CDK2), regulate G1 to S phase transition and their deregulation induces oncogenesis. Immunohistochemical assessments of these proteins in cancer have been reported but were based only on their nuclear expression. However, the oncogenic forms of cyclin E (low molecular weight cyclin E or LMW-E) in complex with CDK2 are preferentially mislocalized to the cytoplasm. Here, we used separate nuclear and cytoplasmic scoring systems for both cyclin E and p-CDK2 expression to demonstrate altered cellular accumulation of these proteins using immunohistochemical analysis. We examined the specificity of different cyclin E antibodies and evaluated their concordance between immunohistochemical and Western blot analyses in a panel of 14 breast cell lines. Nuclear versus cytoplasmic staining of cyclin E readily differentiated full-length from LMW-E, respectively. We also evaluated the expression of cyclin E and p-CDK2 in 1676 breast carcinoma patients by immunohistochemistry. Cytoplasmic cyclin E correlated strongly with cytoplasmic p-CDK2 (P < 0.0001), high tumor grade, negative estrogen/progesterone receptor status, and human epidermal growth factor receptor 2 positivity (all P < 0.0001). In multivariable analysis, cytoplasmic cyclin E plus phosphorylated CDK2 (as one variable) predicted breast cancer recurrence-free and overall survival. These results suggest that cytoplasmic cyclin E and p-CDK2 can be readily detected with immunohistochemistry and used as clinical biomarkers for aggressive breast cancer.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias de la Mama/patología , Ciclina E/análisis , Quinasa 2 Dependiente de la Ciclina/análisis , Adulto , Anciano , Anciano de 80 o más Años , Animales , Western Blotting , Línea Celular Tumoral , Núcleo Celular/química , Núcleo Celular/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Femenino , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Análisis de Matrices TisularesRESUMEN
Expression of cyclin E proteolytic cleavage products, low-molecular weight cyclin E (LMW-E), is associated with poor clinical outcome in patients with breast cancer and it enhances tumorigenecity in mouse models. Here we report that LMW-E expression in human mammary epithelial cells induces an epithelial-to-mesenchymal transition phenotype, increases the CD44(hi)/CD24(lo) population, enhances mammosphere formation, and upregulates aldehyde dehydrogenase expression and activity. We also report that breast tumors expressing LMW-E have a higher proportion of CD44(hi)/CD24(lo) tumor cells as compared with tumors expressing only full-length cyclin E. In order to explore how LMW-E enriches cancer stem cells in breast tumors, we conducted a protein microarray analysis that identified the histone acetyltransferase (HAT) Hbo1 as a novel cyclin E/CDK2 substrate. The LMW-E/CDK2 complex phosphorylated Hbo1 at T88 without affecting its HAT activity. When coexpressed with LMW-E/CDK2, wild-type Hbo1 promoted enrichment of cancer stem-like cells (CSC), whereas the T88 Hbo1 mutant reversed the CSC phenotype. Finally, doxorubicin and salinomycin (a CSC-selective cytotoxic agent) synergized to kill cells expressing LMW-E, but not full-length cyclin E. Collectively, our results suggest that the heightened oncogenecity of LMW-E relates to its ability to promote CSC properties, supporting the design of therapeutic strategies to target this unique function.
Asunto(s)
Neoplasias de la Mama/patología , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Transición Epitelial-Mesenquimal , Histona Acetiltransferasas/metabolismo , Células Madre Neoplásicas/patología , Antibacterianos/farmacología , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Antígeno CD24/genética , Antígeno CD24/metabolismo , Proliferación Celular/efectos de los fármacos , Ciclina E/genética , Quinasa 2 Dependiente de la Ciclina/genética , Doxorrubicina/farmacología , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Histona Acetiltransferasas/antagonistas & inhibidores , Histona Acetiltransferasas/genética , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Inmunoprecipitación , Peso Molecular , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Fosforilación , Análisis por Matrices de Proteínas , Piranos/farmacología , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Elastase-mediated cleavage of cyclin E generates low molecular weight cyclin E (LMW-E) isoforms exhibiting enhanced CDK2-associated kinase activity and resistance to inhibition by CDK inhibitors p21 and p27. Approximately 27% of breast cancers express high LMW-E protein levels, which significantly correlates with poor survival. The objective of this study was to identify the signaling pathway(s) deregulated by LMW-E expression in breast cancer patients and to identify pharmaceutical agents to effectively target this pathway. Ectopic LMW-E expression in nontumorigenic human mammary epithelial cells (hMECs) was sufficient to generate xenografts with greater tumorigenic potential than full-length cyclin E, and the tumorigenicity was augmented by in vivo passaging. However, cyclin E mutants unable to interact with CDK2 protected hMECs from tumor development. When hMECs were cultured on Matrigel, LMW-E mediated aberrant acinar morphogenesis, including enlargement of acinar structures and formation of multi-acinar complexes, as denoted by reduced BIM and elevated Ki67 expression. Similarly, inducible expression of LMW-E in transgenic mice generated hyper-proliferative terminal end buds resulting in enhanced mammary tumor development. Reverse-phase protein array assay of 276 breast tumor patient samples and cells cultured on monolayer and in three-dimensional Matrigel demonstrated that, in terms of protein expression profile, hMECs cultured in Matrigel more closely resembled patient tissues than did cells cultured on monolayer. Additionally, the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E-expressing patient samples, and activation of this pathway was associated with poor disease-specific survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (a pan kinase inhibitor targeting b-Raf) effectively prevented aberrant acinar formation in LMW-E-expressing cells by inducing G1/S cell cycle arrest. LMW-E requires CDK2-associated kinase activity to induce mammary tumor formation by disrupting acinar development. The b-Raf-ERK1/2-mTOR signaling pathway is aberrantly activated in breast cancer and can be suppressed by combination treatment with roscovitine plus either rapamycin or sorafenib.
Asunto(s)
Neoplasias de la Mama/genética , Transformación Celular Neoplásica , Ciclina E , Quinasa 2 Dependiente de la Ciclina , Isoformas de Proteínas , Proteínas Proto-Oncogénicas B-raf , Células Acinares/citología , Células Acinares/metabolismo , Animales , Bencenosulfonatos/farmacología , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Sistema de Señalización de MAP Quinasas/genética , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Ratones , Ratones Desnudos , Invasividad Neoplásica/genética , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Pronóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Purinas/farmacología , Piridinas/farmacología , Estudios Retrospectivos , Roscovitina , Sirolimus/farmacología , Sorafenib , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Expression of low molecular weight (LMW) isoforms of cyclin E is a strong predictor of poor outcome in patients with breast cancer. The purpose of this study was to examine the expression of full-length and LMW cyclin E in bladder cancer cell lines and patient tumors. We used western blotting, immunoprecipitation and kinase assays to examine the expression and activity of key cell cycle-regulatory proteins in various human bladder cell lines, both tumorigenic and non-tumorigenic. We also analyzed cyclin E expression, kinase activity and immune complex binding partners in 43 tissue samples from grade 2 and 3 transitional cell carcinomas. Cyclin E was overexpressed and LMW isoforms were present only in bladder cancer cells. Overexpression of LMW isoforms of cyclin E and increased cyclin E kinase activity were both significantly associated with tumorigenicity of the bladder cell lines (p = 0.005 and 0.022, respectively). Binding of the cyclin-dependent kinase inhibitors p21 and p27 to LMW cyclin E did not inhibit the kinase activity of cyclin E and cyclin-dependent kinase 2 in primary tumor samples overexpressing LMW cyclin E. Full-length and LMW cyclin E were significantly overexpressed in grade 3 tumors compared with grade 2 tumors (p = 0.004). Finally, LMW cyclin E levels were significantly associated with a non-papillary growth pattern (p = 0.031) and invasiveness (p = 0.021) of the bladder tumors and poor overall survival (p = 0.06). These results suggest that LMW cyclin E can be used as a new prognostic marker for bladder cancer.
Asunto(s)
Ciclina E/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Invasividad Neoplásica , Estadificación de Neoplasias , Pronóstico , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
Cyclin E activates Cdk2, controls centrosome duplication, and regulates histone gene transcription. Cyclin E is deregulated in cancer and appears as low-molecular-weight (LMW) isoforms that correlate strongly with decreased survival in breast cancer patients. Transgenic mice overexpressing LMW-cyclin E have increased incidence of mammary tumors and distant metastasis when compared with mice that had full-length cyclin E. To specifically test the requirement for Cdk2 in LMW-cyclin E-mediated mammary tumorigenesis, we generated transgenic mice, which expressed LMW-cyclin E in a Cdk2-deficient background. We found that mammary gland development proceeds relatively normally in these animals, indicating that Cdk2 kinase activity is largely dispensable for this process. However, Cdk2-deficient mice were completely resistant to LMW-cyclin E-mediated mammary tumors. Cdk2 wild-type or heterozygous mice succumbed to mammary tumors with mean latencies of 16 and 19.5 months, respectively, but Cdk2 nullizygous littermates did not display tumors through 24 months. Similarly, continuous administration of two different Cdk inhibitors significantly delayed LMW-cyclin E-induced mammary tumor progression. Triple transgenic mice generated in a p53 heterozygous background also displayed no tumors. Finally, we found that Cdk2 silencing induced cell death in LMW-overexpressing breast cancer cell lines, but not in cell lines lacking LMW expression. Our findings establish a requirement for Cdk2 in LMW-cyclin E-mediated mammary tumorigenesis, arguing that human breast tumors overexpressing LMW-cyclin E are prime candidates for anti-Cdk2 therapy.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Animales , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Ciclina E/genética , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/genética , Femenino , Silenciador del Gen , Humanos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/prevención & control , Ratones , Ratones Noqueados , Isoformas de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , RoscovitinaRESUMEN
PURPOSE: Low-molecular-weight cyclin E (LMW-E) in breast cancer cells induces genomic instability and resistance to inhibition by p21, p27, and fulvestrant therapy. Here, we sought to determine if LMW-E renders breast cancer cells unresponsive to aromatase inhibitors (AI), elucidate the mechanism of such resistance, and ascertain if inhibitors of LMW-E-associated kinase activity could overcome this resistance. EXPERIMENTAL DESIGN: The antiproliferative effects of the AIs were examined in aromatase-overexpressing MCF-7/Ac1 cells in the presence or absence of full-length cyclin E and LMW-E. Inhibition of LMW cyclin E kinase activity by roscovitine [a cyclin-dependent kinase (CDK) inhibitor] was examined in letrozole-unresponsive MCF-7/Ac1 cells. The role of LMW-E and CDK2 in mediating recurrence following AI treatment was also assessed in breast cancer patients. RESULTS: Overexpression of LMW-E in postmenopausal patients was associated with a poor prognosis. Letrozole, but not exemestane or anastrozole, mediated a pronounced G(1) arrest in MCF-7/Ac1 cells. Androstenedione-induced G(1) exit correlated with increased cyclin E-associated kinase activity and increased CDK2 levels. Letrozole treatment inhibited cyclin E-CDK2 kinase activity by preventing the androstenedione-induced increase in CDK2. LMW-E bypassed this effect and rendered the cells resistant to letrozole inhibition. Roscovitine blocked the androstenedione-induced increase in CDK2, and LMW-E overexpression could not bypass this effect. Lastly, breast cancer patients whose tumors overexpress LMW-E were not responsive to AI treatment. CONCLUSIONS: Roscovitine treatment can reverse intrinsic or acquired resistance to letrozole due to LMW-E expression in breast cancer cells. These data support the clinical investigation of CDK2 inhibitor therapy for postmenopausal women with estrogen receptor-positive, LMW-E-expressing breast cancer.
Asunto(s)
Inhibidores de la Aromatasa/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Ciclina E/metabolismo , Purinas/farmacología , Inhibidores de la Aromatasa/farmacología , Ciclo Celular , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Fase G1 , Humanos , Letrozol , Peso Molecular , Nitrilos/farmacología , Posmenopausia , Pronóstico , Roscovitina , Triazoles/farmacologíaRESUMEN
In tumor cells, cyclin E deregulation results in the appearance of five low molecular weight (LMW) isoforms. When overexpressed in breast cancer cells, these forms of cyclin E induce genomic instability, resistance to inhibition by p21 and p27, and resistance to antiestrogen therapy. Additionally, the LMW forms of cyclin E strongly correlate with decreased survival in patients with breast cancer. However, the oncologic role of the LMW forms of cyclin E in breast cancer tumorigenesis is yet to be determined. To this end, we generated transgenic mice expressing full-length cyclin E alone (M46A), full-length and the EL4 isoforms (EL1/EL4), or the EL2/3 isoforms of cyclin E (T1) under the control of the mouse mammary tumor virus promoter. Compared with full-length cyclin E, LMW cyclin E overexpression induces delayed mammary growth during the pubertal phase and abnormal cell morphology during lactation. Both primary mammary tumor formation and metastasis were markedly enhanced in LMW cyclin E transgenic mice. LMW cyclin E overexpression in mammary epithelial cells of mice is sufficient by itself to induce mammary adenocarcinomas in 34 of 124 (27%) animals compared with 7 of 67 (10.4%) mice expressing only the full-length cyclin E (P < 0.05). In addition, metastasis was seen in 25% of LMW cyclin E tumor-bearing animals compared with only 8.3% of tumors in the full-length cyclin E background (P < 0.05). Moreover, LMW cyclin E overexpression selects for inactivation of p53 by loss of heterozygosity and spontaneous and frequent inactivation of ARF. Therefore, LMW cyclin E overexpression strongly selects for spontaneous inactivation of the ARF-p53 pathway in vivo, canceling its protective checkpoint function and accelerating progression to malignancy.
Asunto(s)
Adenocarcinoma/secundario , Ciclina E/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/patología , Proteína p53 Supresora de Tumor/genética , Adenocarcinoma/genética , Animales , Apoptosis , Western Blotting , Femenino , Silenciador del Gen , Humanos , Técnicas para Inmunoenzimas , Etiquetado Corte-Fin in Situ , Pérdida de Heterocigocidad , Neoplasias Pulmonares/genética , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutación , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
In this review, we focus on pathways intersecting through p53 and cyclin E, highlighting how oncogenic effects of cyclin E deregulation, especially overexpression of shortened or low molecular weight (LMW) forms of cyclin E protein, are amplified by loss of regulatory control through p53 to promote tumor development. Expression of cyclin E protein promotes progression into S-phase, an activity opposed by p53-regulated activation of checkpoint controls or apoptosis. Loss of p53 function is an escape hatch by which tumor cells, initiated by a number of means including cyclin E deregulation, can avoid cell cycle arrest or cell death and progress through further stages of unchecked deregulation and growth. To determine how this escape hatch is opened and, ultimately, how to close it, we must understand the networks of normal signaling and processing in a cell and where they intersect.
Asunto(s)
Neoplasias de la Mama/metabolismo , Ciclina E/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis , Neoplasias de la Mama/genética , Neoplasias de la Mama/terapia , Ciclo Celular/fisiología , Ciclina E/química , Ciclina E/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Femenino , Humanos , Peso Molecular , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p14ARF Supresora de Tumor/genética , Proteína p14ARF Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Cyclin E, a key mediator of transition during the G1/S cellular division phase, is deregulated in a wide variety of human cancers. Our group recently reported that overexpression and generation of low-molecular-weight (LMW) isoforms of cyclin E were associated with poor clinical outcome among breast cancer patients. However, the link between LMW cyclin E biology in mediating a tumorigenic phenotype and clinical outcome is unknown. To address this gap in knowledge, we assessed the role of LMW isoforms in breast cancer cells; we found that these forms of cyclin E induced genomic instability and resistance to p21, p27, and antiestrogens in breast cancer. These findings suggest that high levels of LMW isoforms of cyclin E not only can predict failure to endocrine therapy but also are true prognostic indicators because of their influence on cell proliferation and genetic instability.
Asunto(s)
Neoplasias de la Mama/metabolismo , Ciclina E/metabolismo , Animales , Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , División Celular , Línea Celular Tumoral , Inestabilidad Cromosómica , Ciclina E/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Resistencia a Antineoplásicos , Antagonistas de Estrógenos/farmacología , Ratones , Pronóstico , Isoformas de Proteínas , Transfección , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The deregulated expression of cyclin E as measured by the overexpression of its low molecular weight (LMW) isoforms is a powerful predictor of poor outcome in patients with breast cancer. The mechanism by which these LMW forms give tumor cells a growth advantage is not known and is the subject of this article. In this article, we provide the pathobiological mechanisms of how these LMW forms are involved in disease progression. Specifically, we show that overexpression of the LMW forms of cyclin E but not the full-length form in MCF-7 results in (a) their hyperactivity because of increased affinity for cdk2 and resistance to inhibition by the cyclin-dependent kinase inhibitors p21 and p27, (b) resistance to the growth inhibiting effects of antiestrogens, and (c) chromosomal instability. Lastly, tumors from breast cancer patients overexpressing the LMW forms of cyclin E are polyploid in nature and are resistant to endocrine therapy. Collectively, the biochemical and functional differences between the full-length and the LMW isoforms of cyclin E provide a molecular mechanism for the poor clinical outcome observed in breast cancer patients harboring tumors expressing high levels of the LMW forms of cyclin E. These properties of the LMW forms cyclin E suggest that they are not just surrogate markers of poor outcome but bona fide mediators of aggressive disease and potential therapeutic targets for patients whose tumors overexpress these forms.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina E/metabolismo , Ciclinas/metabolismo , Estradiol/análogos & derivados , Moduladores de los Receptores de Estrógeno/farmacología , Inestabilidad Genómica , Proteínas Supresoras de Tumor/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Quinasas CDC2-CDC28/antagonistas & inhibidores , Quinasas CDC2-CDC28/metabolismo , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , División Celular/fisiología , Línea Celular Tumoral , Ciclina E/biosíntesis , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Ciclinas/genética , Estradiol/farmacología , Femenino , Fulvestrant , Fase G1/efectos de los fármacos , Fase G1/fisiología , Humanos , Persona de Mediana Edad , Peso Molecular , Poliploidía , Isoformas de Proteínas , Transfección , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genéticaRESUMEN
Cyclin E in complex with CDK2 is a positive regulator of the G1 to S phase transition of the cell cycle and is responsible for cells passing the restriction point, committing the cell to another round of cell division. Cyclin E is overexpressed and proteolytically cleaved into low molecular weight (LMW) isoforms in breast cancer cell lines and tumor tissues compared to normal cells and tissues. These alterations in cyclin E are linked to poor prognosis in breast cancer patients. In order to evaluate the biological effects of the LMW cyclin E, immortalized mammary epithelial cells, 76NE6, were stably transfected with each of the three cyclin E constructs. Our results reveal that the LMW forms of cyclin E (T1 and T2) are biologically functional, as their overexpression in the immortalized cells increases the ability of these cells to enter S and G2/M phase by 2 fold over full length or vector-alone transfected cells, concomitant with an increased rate of cell proliferation. In addition, these LMW isoforms are biochemically hyperactive, shown by their ability to phosphorylate substrates such as histone H1 4 fold more in cells transfected with T1 or T2 versus cells transfected with the full length form. These results suggest that overexpression of the LMW forms of cyclin E is mitogenic, stimulating the cells to progress through the cell cycle much more efficiently than the full length cyclin E.
Asunto(s)
Ciclo Celular/fisiología , Ciclina E/metabolismo , Proteínas Musculares , Empalme Alternativo/genética , Empalme Alternativo/fisiología , Animales , Neoplasias de la Mama/metabolismo , Quinasas CDC2-CDC28/metabolismo , Clonación Molecular , Ciclina E/genética , Quinasa 2 Dependiente de la Ciclina , Fase G1/fisiología , Histonas/metabolismo , Humanos , Proteínas de Microfilamentos/metabolismo , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fase S/fisiología , Células Tumorales CultivadasRESUMEN
The progression of a cell through the cell cycle is promoted by cyclin dependent kinases (CDKs), which are positively regulated by cyclins and negatively regulated by CDK inhibitors. D type cyclins interact with CDK4 and CDK6 to drive the progression of a cell through early/mid-G(1)in response to mitogen stimulation. The association of cyclin E with CDK2 forms an active complex in late G(1) that directs entry into S-phase. S-phase progression is directed by the cyclin A/CDK2 complex, and the complex of cyclin A with Cdc2 (also known as CDK1) is important for G(2) phase. Lastly, cyclin B/CDK1 complex is necessary for the entry into mitosis. To date only one class of substrates have been identified for cyclinD-CDK4 and -CDK6 complexes, those belonging to pRb family of proteins, whereas the list of cyclin E-CDK2 substrates continues to lengthen. The tight regulation of cyclin E both at the transcriptional level and by ubiquitin-mediated proteolysis indicates that it has a major role for the control of G(1)/S transition. The recent identification of key substrates for cyclin E-CDK2 complex has increased our appreciation of how cyclin E overexpression seen in many human cancers can lead to genomic instability, a feature that leads the tumor to a more aggressive state. In breast cancer, the identification of low molecular weight (LMW) forms of cyclin E generated specifically in tumors due to elastase mediated amino-terminal proteolytic processing opens new possibilities for a targeted treatment of breast cancer. These truncated forms of cyclin E have an increased cyclin E-CDK2 kinase activity, which correlates in vivo with accelerated entry into S phase. Characterization of the biochemical properties of these LMW forms of cyclin E, in terms of substrate specificity, extent of their inhibition by the CDK inhibitors of the Cip/Kip family, their sensitivity to degradation, as well as elucidating their biological activities in the whole animal, should help us to better understand their role in breast cancer oncogenesis and help provide novels agents to target them.