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1.
Sci Rep ; 9(1): 5695, 2019 04 05.
Artículo en Inglés | MEDLINE | ID: mdl-30952903

RESUMEN

Skp2 is a crucial component of SCFSkp2 E3 ubiquitin ligase and is often overexpressed in various types of cancer, including prostate cancer (PCa). The epithelial-to-mesenchymal transition (EMT) is involved in PCa progression. The acquisition of a mesenchymal phenotype that results in a cancer stem cell (CSC) phenotype in PCa was described. Therefore, we aimed to investigate the expression and localization of Skp2 in clinical samples from patients with PCa, the association of Skp2 with EMT status, and the role of Skp2 in prostate CSC. We found that nuclear expression of Skp2 was increased in patients with PCa compared to those with benign hyperplasia, and correlated with high Gleason score in PCa patients. Increased Skp2 expression was observed in PCa cell lines with mesenchymal and CSC-like phenotype compared to their epithelial counterparts. Conversely, the CSC-like phenotype was diminished in cells in which SKP2 expression was silenced. Furthermore, we observed that Skp2 downregulation led to the decrease in subpopulation of CD44+CD24- cancer stem-like cells. Finally, we showed that high expression levels of both CD24 and CD44 were associated with favorable recurrence-free survival for PCa patients. This study uncovered the Skp2-mediated CSC-like phenotype with oncogenic functions in PCa.


Asunto(s)
Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Células Madre Neoplásicas/fisiología , Neoplasias de la Próstata/genética , Proteínas Quinasas Asociadas a Fase-S/genética , Animales , Antígeno CD24/genética , Línea Celular Tumoral , Humanos , Receptores de Hialuranos/genética , Masculino , Ratones , Ratones Desnudos , Clasificación del Tumor , Células Madre Neoplásicas/metabolismo , Células PC-3 , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/fisiopatología , Ensayos Antitumor por Modelo de Xenoinjerto
2.
Carcinogenesis ; 39(11): 1411-1418, 2018 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-30010814

RESUMEN

The cell surface glycoprotein Trop-2 is commonly overexpressed in carcinomas and represents an exceptional antigen for targeted therapy. Here, we provide evidence that surface Trop-2 expression is functionally connected with an epithelial phenotype in breast and prostate cell lines and in patient tumor samples. We further show that Trop-2 expression is suppressed epigenetically or through the action of epithelial-to-mesenchymal transition transcription factors and that deregulation of Trop-2 expression is linked with cancer progression and poor patient prognosis. Moreover, our data suggest that the cancer plasticity-driven intratumoral heterogeneity in Trop-2 expression may significantly contribute to response and resistance to therapies targeting Trop-2-expressing cells.


Asunto(s)
Antígenos de Neoplasias/metabolismo , Neoplasias de la Mama/patología , Carcinoma/patología , Moléculas de Adhesión Celular/metabolismo , Células Epiteliales/metabolismo , Neoplasias de la Próstata/patología , Animales , Antígenos CD/biosíntesis , Antígenos de Neoplasias/genética , Neoplasias de la Mama/mortalidad , Cadherinas/biosíntesis , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Metilación de ADN/genética , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal/fisiología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Neoplasias de la Próstata/mortalidad , Ensayos Antitumor por Modelo de Xenoinjerto
3.
Cytometry A ; 93(9): 941-951, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-28383825

RESUMEN

The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-ß1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (EpCAM) identification of fibroblasts from breast and prostate tumor tissues is advised. © 2017 International Society for Advancement of Cytometry.


Asunto(s)
Biomarcadores/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Fibroblastos/metabolismo , Gelatinasas/metabolismo , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Endopeptidasas , Molécula de Adhesión Celular Epitelial/metabolismo , Células Epiteliales/metabolismo , Femenino , Humanos , Antígenos Comunes de Leucocito/metabolismo , Masculino , Células PC-3 , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Neoplasias de la Próstata/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo
4.
Cytometry A ; 93(2): 239-248, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29220555

RESUMEN

Complex analysis of cellular responses after experimental treatment is important for screening, mechanistic understanding of treatment effects, and the identification of sensitive and resistant cell phenotypes. Modern multicolor flow cytometry has demonstrated its power for such analyses. Here, we introduce a multiparametric protocol for complex analysis of cytokinetics by the simultaneous detection of seven fluorescence parameters. This analysis includes the detection of two surface markers for immunophenotyping, analysis of proliferation based on the cell cycle and the measurement of incorporated nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) in newly synthesized DNA, analysis of DNA damage using an anti-phospho-histone H2A.X (Ser139) antibody, and determination of cell death using a fixable viability probe and intracellular detection of caspase-3 activation. To demonstrate the applicability of this protocol for the analysis of heterogeneous and complex cell responses, we used different treatments and model cell lines. We demonstrated that this protocol has the potential to provide complex and simultaneous analysis of cytokinetics and analyze the heterogeneity of the response at the single-cell level. © 2017 International Society for Advancement of Cytometry.


Asunto(s)
Apoptosis/fisiología , Proliferación Celular/fisiología , Daño del ADN/fisiología , Citometría de Flujo/métodos , Inmunofenotipificación/métodos , Línea Celular Tumoral , Humanos
5.
Tumour Biol ; 39(10): 1010428317727479, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-29025359

RESUMEN

A broad spectrum of tumors develop resistance to classic chemotherapy, necessitating the discovery of new therapies. One successful strategy exploits the synthetic lethality between poly(ADP-ribose) polymerase 1/2 proteins and DNA damage response genes, including BRCA1, a factor involved in homologous recombination-mediated DNA repair, and CDK12, a transcriptional kinase known to regulate the expression of DDR genes. CHK1 inhibitors have been shown to enhance the anti-cancer effect of DNA-damaging compounds. Since loss of BRCA1 increases replication stress and leads to DNA damage, we tested a hypothesis that CDK12- or BRCA1-depleted cells rely extensively on S-phase-related CHK1 functions for survival. The silencing of BRCA1 or CDK12 sensitized tumor cells to CHK1 inhibitors in vitro and in vivo. BRCA1 downregulation combined with CHK1 inhibition induced excessive amounts of DNA damage, resulting in an inability to complete the S-phase. Therefore, we suggest CHK1 inhibition as a strategy for targeting BRCA1- or CDK12-deficient tumors.


Asunto(s)
Proteína BRCA1/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Neoplasias Colorrectales/genética , Quinasas Ciclina-Dependientes/genética , Animales , Proteína BRCA1/antagonistas & inhibidores , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Daño del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Células HCT116 , Humanos , Ratones , Poli(ADP-Ribosa) Polimerasa-1/genética , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , Ensayos Antitumor por Modelo de Xenoinjerto
6.
Mol Cancer ; 13: 113, 2014 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-24884804

RESUMEN

BACKGROUND: Tumor heterogeneity and the plasticity of cancer cells present challenges for effective clinical diagnosis and therapy. Such challenges are epitomized by neuroendocrine transdifferentiation (NED) and the emergence of neuroendocrine-like cancer cells in prostate tumors. This phenomenon frequently arises from androgen-depleted prostate adenocarcinoma and is associated with the development of castration-resistant prostate cancer and poor prognosis. RESULTS: In this study, we showed that NED was evoked in both androgen receptor (AR)-positive and AR-negative prostate epithelial cell lines by growing the cells to a high density. Androgen depletion and high-density cultivation were both associated with cell cycle arrest and deregulated expression of several cell cycle regulators, such as p27Kip1, members of the cyclin D protein family, and Cdk2. Dual inhibition of Cdk1 and Cdk2 using pharmacological inhibitor or RNAi led to modulation of the cell cycle and promotion of NED. We further demonstrated that the cyclic adenosine 3', 5'-monophosphate (cAMP)-mediated pathway is activated in the high-density conditions. Importantly, inhibition of cAMP signaling using a specific inhibitor of adenylate cyclase, MDL-12330A, abolished the promotion of NED by high cell density. CONCLUSIONS: Taken together, our results imply a new relationship between cell cycle attenuation and promotion of NED and suggest high cell density as a trigger for cAMP signaling that can mediate reversible NED in prostate cancer cells.


Asunto(s)
Transdiferenciación Celular , Células Neuroendocrinas/patología , Neoplasias de la Próstata/patología , Andrógenos/farmacología , Proteína Quinasa CDC2 , Recuento de Células , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Transdiferenciación Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Células Epiteliales/patología , Humanos , Inmunohistoquímica , Masculino , Células Neuroendocrinas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Receptores Androgénicos/metabolismo , Transducción de Señal/efectos de los fármacos
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