RESUMEN

Cancer stem cells (CSCs) are an important player in the resistance of cancers to therapy. In this work, we determined the flavonoids composition and biological action of Aloysia polystachya (AP) extracts in colorectal cancer. The chemical characterization of extracts was performed by HPLC. Assays of cytotoxicity, apoptosis, migration and invasion, metalloproteases activity, clonogenic growth, tumorspheres formation, Hoechts efflux, pluripotency marker expression and sensitization to chemotherapeutic drugs were performed in vitro in human HCT116 and murine CT26 colorectal cancer cells. The AP toxicity and effect in tumor growth administered alone or in combination with 5- Fluorouracile was analyzed in vivo, including histopathological studies. We found that AP extracts induced in vitro the apoptosis of colorectal cancer cell lines decreasing the CSC proportion. Moreover, they were capable to kill 5-Fluorouracile resistant side population cells. At not toxic doses in vivo, AP extracts inhibited tumor growth. Regarding the ability to reduce the CSC population, AP extracts deserves to be investigated as a useful therapy for colorectal cancer treatment.

Asunto(s)

Neoplasias Colorrectales , Células Madre Neoplásicas , Animales , Apoptosis , Muerte Celular , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/tratamiento farmacológico , Humanos , Ratones , Extractos Vegetales/farmacología , Verbenaceae

RESUMEN

RAC3 is a coactivator of steroid receptors and NF-κB. It is usually overexpressed in several tumors, contributes to maintain cancer stem cells and also to induce them when is overexpressed in non-tumoral cells. In this work, we investigated whether the inflammatory cytokine TNF may contribute to the transforming effects of RAC3 overexpression in the non-tumoral HEK293 cell line. The study model included the HEK293 tumoral transformed cell line constitutively overexpressing RAC3 by stable transfection and control non-tumoral cells transfected with an empty vector. The HeLa and T47D tumoral cells that naturally overexpress RAC3 were used as positive control. We found that TNF potentiated RAC3-induced mesenchymal transition, involving an increased E-Cadherin downregulation, Vimentin and SNAIL upregulation and enhanced migratory behavior. Moreover, concerning the molecular mechanisms by which TNF potentiates the RAC3 transforming action, they involve the IKK activation, which in addition induced the ß-Catenin transactivation. Our results demonstrate that although RAC3 overexpression could be a signal strong enough to induce cancer stem cells, the inflammatory microenvironment may be playing a key role contributing to the migratory and invasive phenotype required for metastasis and cancer persistence.

RESUMEN

RAC3 is a transcription coactivator, usually overexpressed in several tumors and required to maintain the pluripotency in normal stem cells. In this work we studied the association between RAC3 overexpression on cancer cell stemness and the capacity of this protein to induce cancer stem properties in non tumoral cells. We performed in vitro and in vivo experiments using two strategies: by overexpressing RAC3 in the non tumoral cell line HEK293 and by silencing RAC3 in the human colorectal epithelial cell line HCT116 by transfection. Furthermore, we analysed public repository microarrays data from human colorectal tumors in different developmental stages. We found that RAC3 overexpression was mainly associated to CD133+ side-population of colon cancer cells and also to early and advanced stages of colon cancer, involving increased expression of mesenchymal and stem markers. In turn, RAC3 silencing induced diminished tumoral properties and cancer stem cells as determined by Hoechst efflux, tumorspheres and clonogenic growth, which correlated with decreased Nanog and OCT4 expression. In non tumoral cells, RAC3 overexpression induced tumoral transformation; mesenchymal phenotype and stem markers expression. Moreover, these transformed cells generated tumors in vivo. Our results demonstrate that RAC3 is required for maintaining and induction of cancer cell stemness.

RESUMEN

BACKGROUND: Lung cancer is the most frequently diagnosed cancer and the leading cause of cancer-related deaths worldwide. Up to 80% of cancer patients are classified as non-small-cell lung cancer (NSCLC) and cisplatin remains as the gold standard chemotherapy treatment, despite its limited efficacy due to both intrinsic and acquired resistance. The CK2 is a Ser/Thr kinase overexpressed in various types of cancer, including lung cancer. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to CK2 substrates thus preventing the enzyme activity. The aim of this work was to analyze the effects of CIGB-300 treatment targeting CK2-dependent signaling pathways in NSCLC cell lines and whether it may help improve current chemotherapy treatment. METHODS: The human NSCLC cell lines NCI-H125 and NIH-A549 were used. Tumor spheroids were obtained through the hanging-drop method. A cisplatin resistant A549 cell line was obtained by chronic administration of cisplatin. Cell viability, apoptosis, immunoblotting, immunofluorescence and luciferase reporter assays were used to assess CIGB-300 effects. A luminescent assay was used to monitor proteasome activity. RESULTS: We demonstrated that CIGB-300 induces an anti-proliferative response both in monolayer- and three-dimensional NSCLC models, presenting rapid and complete peptide uptake. This effect was accompanied by the inhibition of the CK2-dependent canonical NF-κB pathway, evidenced by reduced RelA/p65 nuclear levels and NF-κB protein targets modulation in both lung cancer cell lines, as well as conditionally reduced NF-κB transcriptional activity. In addition, NF-κB modulation was associated with enhanced proteasome activity, possibly through its α7/C8 subunit. Neither the peptide nor a classical CK2 inhibitor affected cytoplasmic ß-CATENIN basal levels. Given that NF-κB activation has been linked to cisplatin-induced resistance, we explored whether CIGB-300 could bring additional therapeutic benefits to the standard cisplatin treatment. We established a resistant cell line that showed higher p65 nuclear levels after cisplatin treatment as compared with the parental cell line. Remarkably, the cisplatin-resistant cell line became more sensitive to CIGB-300 treatment. CONCLUSIONS: Our data provide new insights into CIGB-300 mechanism of action and suggest clinical potential on current NSCLC therapy.

RESUMEN

Protein kinase C (PKC) is a family of serine/threonine kinases that regulate diverse cellular functions including cell death, proliferation, and survival. Recent studies have reported that PKCδ, are involved in apoptosis or autophagy induction. In the present study we focused on how PKCδ regulates proliferation and cancer stem cell (CSC) properties of the hormone-independent mammary cancer cell line LM38-LP, using pharmacological and genetic approaches. We found that pharmacological inhibition of PKCδ, by Rottlerin treatment, impairs in vitro LM38-LP proliferation through cell cycle arrest, inducing the formation of cytoplasmic-vacuoles. Using immunofluorescence we confirmed that Rottlerin treatment induced the apparition of LC3 dots in cell cytoplasm, and increased autophagy flux. On the other side, the same treatment increased CSC growth rate and self-renewal. Furthermore, Rottlerin pre-treatment induced in CSC the development of a "grape-like" morphology when they are growing in 3D cultures (Matrigel), usually associated with a malignant phenotype, as well as an increase in the number of experimental lung metastasis when these cells were inoculated in vivo. The PKCδ knockdown, by RNA interference, induced autophagy and increased CSC number, indicating that these effects are indeed exerted through a PKCδ dependent pathway. Finally, the increase in the number of mammospheres could be reversed by a 3MA treatment, suggesting that autophagy mechanism is necessary for the increased of CSC self-renewal induced by PKCδ inhibition. Here we demonstrated that PKCδ activity exerts a dual role through the autophagy mechanism, decreasing proliferative capacity of mammary tumor cells but also regulating tumor stem cell self-renewal.

Asunto(s)

Autofagia , Neoplasias Pulmonares/enzimología , Neoplasias Mamarias Experimentales/enzimología , Células Madre Neoplásicas/fisiología , Proteína Quinasa C-delta/metabolismo , Acetofenonas/farmacología , Animales , Antineoplásicos/farmacología , Benzopiranos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Autorrenovación de las Células , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Trasplante de Neoplasias , Proteína Quinasa C-delta/antagonistas & inhibidores , Proteína Quinasa C-delta/genética , Inhibidores de Proteínas Quinasas/farmacología

RESUMEN

PURPOSE: Breast cancer is the leading cause of death among women worldwide. The exact role of luminal epithelial (LEP) and myoephitelial (MEP) cells in breast cancer development is as yet unclear, as also how retinoids may affect their behaviour. Here, we set out to evaluate whether retinoids may differentially regulate cell type-specific processes associated with breast cancer development using the bi-cellular LM38-LP murine mammary adenocarcinoma cell line as a model. MATERIALS AND METHODS: The bi-cellular LM38-LP murine mammary cell line was used as a model throughout all experiments. LEP and MEP subpopulations were separated using inmunobeads, and the expression of genes known to be involved in epithelial to mysenchymal transition (EMT) was assessed by qPCR after all-trans retinoic acid (ATRA) treatment. In vitro invasive capacities of LM38-LP cells were evaluated using 3D Matrigel cultures in conjunction with confocal microscopy. Also, in vitro proliferation, senescence and apoptosis characteristics were evaluated in the LEP and MEP subpopulations after ATRA treatment, as well as the effects of ATRA treatment on the clonogenic, adhesive and invasive capacities of these cells. Mammosphere assays were performed to detect stem cell subpopulations. Finally, the orthotopic growth and metastatic abilities of LM38-LP monolayer and mammosphere-derived cells were evaluated in vivo. RESULTS: We found that ATRA treatment modulates a set of genes related to EMT, resulting in distinct gene expression signatures for the LEP or MEP subpopulations. We found that the MEP subpopulation responds to ATRA by increasing its adhesion to extracellular matrix (ECM) components and by reducing its invasive capacity. We also found that ATRA induces apoptosis in LEP cells, whereas the MEP compartment responded with senescence. In addition, we found that ATRA treatment results in smaller and more organized LM38-LP colonies in Matrigel. Finally, we identified a third subpopulation within the LM38-LP cell line with stem/progenitor cell characteristics, exhibiting a partial resistance to ATRA. CONCLUSIONS: Our results show that the luminal epithelial (LEP) and myoephitelial (MEP) mammary LM38-P subpopulations respond differently to ATRA, i.e., the LEP subpopulation responds with increased cell cycle arrest and apoptosis and the MEP subpopulation responds with increased senescence and adhesion, thereby decreasing its invasive capacity. Finally, we identified a third subpopulation with stem/progenitor cell characteristics within the LM38-LP mammary adenocarcinoma cell line, which appears to be non-responsive to ATRA.

Asunto(s)

Adenocarcinoma/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Neoplasias Mamarias Animales/tratamiento farmacológico , Tretinoina/farmacología , Carga Tumoral/efectos de los fármacos , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Receptor alfa de Estrógeno/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/metabolismo , Ratones Endogámicos BALB C , Microscopía Fluorescente , Modelos Biológicos , Receptores de Ácido Retinoico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

It has been established that retinoids exert some of their effects on cell differentiation and malignant phenotype reversion through the interaction with different members of the protein kinase C (PKC) family. Till nowadays the nature and extension of this interaction is not well understood. Due to the cytostatic and differentiating effects of retinoids, in the present study we propose to evaluate whether the crosstalk between the retinoid system and the PKC pathway could become a possible target for breast cancer treatment. We could determine that ATRA (all-trans retinoic) treatment showed a significant growth inhibition due to (G1 or G2) cell cycle arrest both in LM3 and SKBR3, a murine and human mammary cell line respectively. ATRA also induced a remarkable increase in PKCα and PKCδ expression and activity. Interestingly, the pharmacological inhibition of these two PKC isoforms prevented the activation of retinoic acid receptors (RARs) by ATRA, indicating that both PKC isoforms are required for RARs activation. Moreover, PKCδ inhibition also impaired ATRA-induced RARα translocation to the nucleus. In vivo assays revealed that a combined treatment using ATRA and PKCα inhibitors prevented lung metastatic dissemination in an additive way. Our results clearly indicate that ATRA modulates the expression and activity of different PKCs. Besides inducing cell arrest, the activity of both PKC is necessary for the induction of the retinoic acid system. The combined ATRA and PKCα inhibitors could be an option for the hormone-independent breast cancer treatment.

Asunto(s)

Neoplasias de la Mama/metabolismo , Proteína Quinasa C-alfa/metabolismo , Proteína Quinasa C-delta/metabolismo , Receptores de Ácido Retinoico/metabolismo , Tretinoina/metabolismo , Animales , Neoplasias de la Mama/tratamiento farmacológico , Diferenciación Celular/efectos de los fármacos , Nucléolo Celular/efectos de los fármacos , Nucléolo Celular/metabolismo , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos BALB C , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Proteína Quinasa C-alfa/antagonistas & inhibidores , Proteína Quinasa C-delta/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Células Tumorales Cultivadas

RESUMEN

OBJECTIVE: This study aimed to investigate whether the overexpression of protein kinase C ß1 (PKCß1) is able to modulate the malignant phenotype displayed by the human ductal pancreatic carcinoma cell line PANC1. METHODS: PKCß1 overexpression was achieved using a stable transfection approach. PANC1-PKCß1 and control cells were analyzed both in vitro and in vivo. RESULTS: PANC1-PKCß1 cells displayed a lower growth capacity associated with the down-regulation of the MEK/ERK pathway and cyclin expression. Furthermore, PKCß1 overexpression was associated with an enhancement of cell adhesion to fibronectin and with reduced migratory and invasive phenotypes. In agreement with these results, PANC1-PKCß1 cells showed an impaired ability to secrete proteolytic enzymes. We also found that PKCß1 overexpressing cells were more resistant to cell death induced by serum deprivation, an event associated with G0/G1 arrest and the modulation of PI3K/Akt and NF-κB pathways. Most notably, the overexpression of PKCß1 completely abolished the ability of PANC1 cells to induce tumors in nude mice. CONCLUSIONS: Our results established an important role for PKCß1 in PANC1 cells suggesting it would act as a suppressor of tumorigenic behavior in pancreatic cancer.

Asunto(s)

Carcinoma Ductal Pancreático/enzimología , Carcinoma Ductal Pancreático/etiología , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/etiología , Proteína Quinasa C beta/metabolismo , Animales , Carcinoma Ductal Pancreático/patología , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Xenoinjertos , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Trasplante de Neoplasias , Neoplasias Pancreáticas/patología , Péptido Hidrolasas/metabolismo , Proteína Quinasa C beta/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia Arriba

RESUMEN

The protein kinase C (PKC) family of serine/threonine kinases has been intensively studied in cancer since their discovery as major receptors for the tumor-promoting phorbol esters. The contribution of each individual PKC isozyme to malignant transformation is only partially understood, but it is clear that each PKC plays different role in cancer progression. PKC deregulation is a common phenomenon observed in breast cancer, and PKC expression and localization are usually dynamically regulated during mammary gland differentiation and involution. In fact, the overexpression of several PKCs has been reported in malignant human breast tissue and breast cancer cell lines. In this review, we summarize the knowledge available on the specific roles of PKC isoforms in the development, progression, and metastatic dissemination of mammary cancer. We also discuss the role of PKC isoforms as therapeutic targets, and their potential as markers for prognosis or treatment response.

Asunto(s)

Neoplasias de la Mama/enzimología , Proteína Quinasa C/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Isoenzimas/metabolismo , Fenotipo , Proteína Quinasa C/química , Proteína Quinasa C/genética , Estructura Terciaria de Proteína

RESUMEN

Doxorubicin is an anti-tumor antibiotic widely used in the management of cancer patients. Its main mechanism of action involves the generation of DNA damage and the inhibition of topoisomerase II, promoting apoptosis. AD 198 is a novel doxorubicin analog devoid of DNA binding and topoisomerase II inhibitory capacities. It has been proposed that AD 198 induces apoptosis by activating protein kinase C delta (PKCδ); a PKC isoform described as growth inhibitory in a large number of cell types. We have previously demonstrated that PKCδ overexpression in NMuMG cells induced the opposite effect, promoting proliferation and cell survival. In this study, we found that PKCδ overexpression confers an enhanced cell death resistance against AD 198 cytotoxic effect and against AD 288, another doxorubicin analog that preserves its mechanism of action. These resistances involve PKCδ-mediated activation of two well-known survival pathways: Akt and NF-κB. While the resistance against AD 198 could be abrogated upon the inhibition of either Akt or NF-κB pathways, only NF-κB inhibition could revert the resistance to AD 288. Altogether, our results indicate that PKCδ increases cell death resistance against different apoptosis inductors, independently of their mechanism of action, through a differential modulation of Akt and NF-κB pathways. Our study contributes to a better understanding of the mechanisms involved in PKCδ-induced resistance and may greatly impact in the rationale design of isozyme-specific PKC modulators as therapeutic agents.

Asunto(s)

Doxorrubicina/análogos & derivados , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Proteína Quinasa C-delta/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , ADN-Topoisomerasas de Tipo II/química , Femenino , Perfilación de la Expresión Génica , Neoplasias Mamarias Animales/metabolismo , Ratones , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fracciones Subcelulares

RESUMEN

Autophagy is a catabolic process responsible for the degradation and recycling of long-lived proteins and organelles by lysosomes. This degradative pathway sustains cell survival during nutrient deprivation, but in some circumstances, autophagy leads to cell death. Thereby, autophagy can serve as tumor suppressor, as the reduction in autophagic capacity causes malignant transformation and spontaneous tumors. On the other hand, this process also functions as a protective cell-survival mechanism against environmental stress causing resistance to antineoplastic therapies. Although autophagy inhibition, combined with anticancer agents, could be therapeutically beneficial in some cases, autophagy induction by itself could lead to cell death in some apoptosis-resistant cancers, indicating that autophagy induction may also be used as a therapy. This paper summarizes the most important findings described in the literature about autophagy and also discusses the importance of this process in clinical settings.

RESUMEN

Neural cell adhesion molecule (NCAM) is involved in cell growth, migration, and differentiation. Its expression and/or polysialylation appear to be deregulated in many different cancer types. We employed the lung tumor cell line LP07, syngeneic in BALB/c mice to investigate the role of NCAM in malignant progression. LP07 cells express the three main NCAM isoforms, all of them polysialylated. This cells line, pretreated with an anti-NCAM antibody and inoculated intravenously (i.v.) into syngeneic mice, developed less and smaller lung metastases. In vitro studies showed that NCAM bound antibody inhibited cell growth, mainly due to an increase in apoptosis, associated with a decrease of cyclin D1 and enhanced expression of active caspase 3 and caspase 9. Anti-NCAM-treated LP07 cells showed impairment in their ability to migrate and adhere to several extracellular matrix components. Secreted uPA activity was also reduced. NCAM-140 knocked-down by siRNA in LP07 cells pretreated or not with anti-NCAM showed an impaired metastasizing ability upon i.v. inoculation into mice. These results suggest that anti-NCAM treatment could be mimicking homophilic trans-interactions and NCAM-140 knocked-down impairs heterophilic interactions, both leading to inhibition of metastatic dissemination. The involvement of NCAM in lung tumor progression was confirmed in human NSCLC tumors. Sixty percent of the cases expressed NCAM at tumor cell level. A multivariate analysis indicated that NCAM expression was associated with a shorter overall survival in this homogeneous series of Stages I and II NSCLC patients. NCAM may be able to modulate mechanisms involved in lung carcinoma progression and represents an attractive target to control metastatic progression.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Modelos Animales de Enfermedad , Neoplasias Pulmonares/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Adhesión Celular/genética , Adhesión Celular/inmunología , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/inmunología , Proliferación Celular , Regulación hacia Abajo , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia/fisiopatología , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/metabolismo

RESUMEN

OBJECTIVE: Our objective was to study the role of protein kinase C delta (PKCdelta) in the progression of human pancreatic carcinoma. METHODS: Protein kinase C delta expression in human ductal carcinoma (n = 22) was studied by immunohistochemistry. We analyzed the effect of PKCdelta overexpression on in vivo and in vitro properties of human ductal carcinoma cell line PANC1. RESULTS: Human ductal carcinomas showed PKCdelta overexpression compared with normal counterparts. In addition, in vitro PKCdelta-PANC1 cells showed increased anchorage-independent growth and higher resistance to serum starvation and to treatment with cytotoxic drugs. Using pharmacological inhibitors, we determined that phosphatidylinositol-3-kinase and extracellular receptor kinase pathways were involved in the proliferation of PKCdelta-PANC1. Interestingly, PKCdelta-PANC1 cells showed a less in vitro invasive ability and an impairment in their ability to migrate and to secrete the proteolytic enzyme matrix metalloproteinase-2. In vivo experiments indicated that PKCdelta-PANC1 cells were more tumorigenic, as they developed tumors with a significantly lower latency and a higher growth rate with respect to the tumors generated with control cells. Besides, only PKCdelta-PANC1 cells developed lung metastasis. CONCLUSION: Our results showed that the overexpression of PKCdelta in PANC1 cells induced a more malignant phenotype in vivo, probably through the modulation of cell proliferation and survival, involving phosphatidylinositol-3-kinase and extracellular receptor kinase signaling pathways.

Asunto(s)

Carcinoma Ductal Pancreático/patología , Neoplasias Experimentales/patología , Neoplasias Pancreáticas/patología , Proteína Quinasa C-delta/metabolismo , Anciano , Animales , Western Blotting , Carcinoma Ductal Pancreático/enzimología , Carcinoma Ductal Pancreático/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero/farmacología , Progresión de la Enfermedad , Femenino , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Masculino , Ratones , Persona de Mediana Edad , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neoplasias Experimentales/enzimología , Neoplasias Experimentales/genética , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C-delta/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Trasplante Heterólogo

RESUMEN

In this paper we investigated whether protein kinase C (PKC) beta1 and PKCepsilon, members of the classical and novel PKC family, respectively, induce phenotypic alterations that could be associated with tumor progression and metastatic dissemination in a murine model of breast cancer. Stable overexpression of PKCbeta1 in LM3 cells altered their ability to proliferate, adhere, and survive, and impaired their tumorigenicity and metastatic capacity. Moreover, PKCbeta1 induced the re-expression of fibronectin, an extracellular matrix glycoprotein which loss has been associated with the acquisition of a transformed phenotype in different cell models, and exerted an important inhibition on proteases production, effects that probably impact on LM3 invasiveness and dissemination. Conversely, PKCepsilon overexpression enhanced LM3 survival, anchorage-independent growth, and caused a significant increase in spontaneous lung metastasis. Our results suggest PKCbeta1 functions as an inhibitory protein for tumor growth and metastasis dissemination whereas PKCepsilon drives metastatic dissemination without affecting primary tumor growth.

Asunto(s)

Neoplasias Mamarias Experimentales/enzimología , Invasividad Neoplásica/patología , Proteína Quinasa C-epsilon/metabolismo , Proteína Quinasa C/metabolismo , Animales , Western Blotting , Adhesión Celular/fisiología , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica/genética , Proteína Quinasa C/genética , Proteína Quinasa C beta , Proteína Quinasa C-epsilon/genética , Transfección

RESUMEN

In previous studies we have determined that protein kinase C (PKC) delta, a widely expressed member of the novel PKC serine-threonine kinases, induces in vitro changes associated with the acquisition of a malignant phenotype in NMuMG murine mammary cells. In this study we show that PKCdelta overexpression significantly decreases urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) production, two proteases associated with migratory and invasive capacities. This effect is markedly enhanced by treatment with phorbol 12-myristate 13-acetate (PMA). On the other hand, depletion of PKCdelta using RNAi led to a marked increase in both uPA and MMP-9 secretion, suggesting a physiological role for PKCdelta in controlling protease secretion. The MEK-1 inhibitor PD98059 reverted the characteristic pattern of proteases secretion and phospho-ERK1/2 up-regulation observed in PKCdelta overexpressors, suggesting that the PKCdelta effect is mediated by the MEK/ERK pathway. Our results suggest a dual role for PKCdelta in murine mammary cell cancer progression. While this kinase clearly promotes mitogenesis and favors malignant transformation, it also down-modulates the secretion of proteases probably limiting metastatic dissemination.

Asunto(s)

Sistema de Señalización de MAP Quinasas , Glándulas Mamarias Animales/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteína Quinasa C-delta/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Línea Celular , Movimiento Celular , Transformación Celular Neoplásica , Regulación hacia Abajo , Flavonoides/farmacología , Ratones , Péptido Hidrolasas/metabolismo , Transfección

RESUMEN

Protein kinase C (PKC) delta, a member of the novel family of PKC serine-threonine kinases, has been implicated in negative regulation of proliferation and apoptosis in a large number of cell types, including breast cancer cell lines, and postulated as a tumor suppressor gene. In this study we show that in murine NMuMG mammary cells PKCdelta promotes a mitogenic response. Overexpression of PKCdelta in NMuMG cells leads to a significant increase in [3H]-tymidine incorporation and cell proliferation, as well as enhanced extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) activation. Activation of PKCdelta with a phorbol ester leads to elevated cyclin D1 expression and an hyperphosphorylated Rb state. Surprisingly, ectopic expression of PKCdelta conferred anchorage-independent growth capacity to NMuMG cells. PKCdelta overexpressors showed enhanced resistance to apoptotic stimuli, such as serum deprivation or doxorubicin treatment, an effect that correlates with hyperactivation of the Akt survival pathway. Our results provide evidence for a role of PKCdelta as a positive modulator of proliferative and survival signals in immortalized mammary cells. The fact that PKCdelta exerts differential responses depending on the cell context not only highlights the necessity to carefully understand the signaling events controlled by this PKC in each cell type but also suggests that we should be cautious in considering this kinase a target for cancer therapy.

Asunto(s)

Glándulas Mamarias Animales/citología , Proteína Quinasa C-delta/genética , Proteína Quinasa C-delta/metabolismo , Animales , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero , Doxorrubicina/farmacología , Femenino , Insulina/farmacología , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/enzimología , Ratones , Ratones Noqueados , Proteína Quinasa C-delta/deficiencia , Transfección

RESUMEN

In the present work we used a murine mammary cancer model of two related adenocarcinomas with different lung metastasizing abilities, to compare their global gene expression profiles. Clontech Atlas mouse cDNA microarrays of primary cultured tumor cells were employed to identify genes that are modulated in the more metastatic variant MM3 relative to its parental tumor M3. A total of 88 from 1,176 genes were differentially expressed in MM3 primary cultures, most of them (n=86) were upregulated. Genes were grouped according to their functions as associated with signal transduction and transcription regulation (e.g. Stat1 and Zfp 92), with cell adhesion and motility (cadherin 1, fibronectin), with invasion and angiogenesis (uPA, 72 kDa MMP2), with the regulation of cell proliferation and cell death (cyclins G and A2, TNF), and also included growth factors and receptors, oncogenes and tumor suppressors genes (p107, TGFbeta2, TBR-I, PDGFR). Only 2 genes, TTF1 and fibronectin (FN), showed a significant downregulation. Notably FN expression, loss of which has been associated with a malignant phenotype, was reduced about 19-fold in the more metastatic MM3 cells. Previously known differences in expression patterns associated with the metastatic capacity of MM3 and M3 adenocarcinomas, including downregulation of FN or upregulated expression of TGFbeta and proteases, were confirmed by the array data. The fact that FN was one of the only two genes significantly down-regulated out of the 1,176 genes analyzed stresses the hypothesis that FN may behave as an important metastasis suppressor gene in mammary cancer.

Asunto(s)

Adenocarcinoma/metabolismo , Fibronectinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Experimentales/metabolismo , Metástasis de la Neoplasia/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Western Blotting , Regulación hacia Abajo , Femenino , Fibronectinas/genética , Expresión Génica , Perfilación de la Expresión Génica , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos

RESUMEN

In this paper, we investigated whether protein kinase C-zeta (PKC zeta), a member of the atypical PKC family, induces phenotypic alterations associated with malignant transformation and tumor progression in mammary cells. The stable overexpression of PKC zeta in immortalized mammary epithelial cells (NMuMG), activates the mitogenic extracellular signal-regulated kinase (ERK) pathway, enhanced clonal cell growth and exerts profound effects on proteases secretion. The effect on proteases expression seems to be specific for urokinase-type plasminogen activator and metalloproteinase-9 (MMP-9) because no modulation in MMP-2 and MMP-3 production could be detected. In addition, our experiments demonstrated that PKC zeta overexpression markedly altered the adhesive, spreading, and migratory abilities of NMuMG cells. The overexpression of this enzyme was not sufficient to confer an anchorage-independent growth capacity. An extensive mutational analysis of PKC zeta revealed that the effects observed in NMuMG cells were strictly dependent on the kinase (catalytic) domain of the enzyme. Taken together, these results suggest that in mammary cells PKC zeta modulates several of the critical events involved in tumor development and dissemination through the activation of mitogen activated protein kinase (MAPK)/ERK pathway.

Asunto(s)

Movimiento Celular/fisiología , Glándulas Mamarias Animales/metabolismo , Péptido Hidrolasas/metabolismo , Proteína Quinasa C/metabolismo , Animales , División Celular/fisiología , Supervivencia Celular/fisiología , Femenino , Glándulas Mamarias Animales/citología , Metaloproteinasas de la Matriz/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo

RESUMEN

We studied the expression of TGF-beta/T(beta)R system and its biological role in tumor development, in M3 and MM3 murine mammary adenocarcinomas with different metastasizing capability and in LM3 and LMM3 derived cell lines. All the studied cells secreted TGF-beta(s) and expressed T(beta)Rs. While the proliferation of the poorly metastatic M3 cells was significantly inhibited by 4 ng/ml TGF-beta(s), the highly metastatic MM3 cells were only slightly inhibited in response to the highest dose used. LM3 and LMM3 cells, highly invasive and metastatic, were totally refractory to TGF-beta antiproliferative effect. The role of TGF-beta in modulating key proteolytic cascades in tumor progression was also studied. TGF-beta(s) enhanced metalloproteinases production in all the studied cells while induced a stimulatory net effect on plasmin system activity only in the more metastatic cells. Our results in this murine mammary tumor lineage support the concept that dissociation of TGF-beta regulated growth control versus proteolytic enzyme pathways promotes tumor dissemination.

Asunto(s)

Adenocarcinoma/inmunología , Adenocarcinoma/fisiopatología , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Animales/inmunología , Neoplasias Mamarias Animales/fisiopatología , Metástasis de la Neoplasia/fisiopatología , Factor de Crecimiento Transformador beta/farmacología , Animales , Diferenciación Celular , División Celular , Progresión de la Enfermedad , Metaloproteasas/farmacología , Ratones , Células Tumorales Cultivadas