RESUMEN

Autophagy is an evolutionarily conserved process that enables catabolic and degradative pathways. These pathways commonly depend on vesicular transport controlled by Rabs, small GTPases inactivated by TBC/RabGAPs. The Rac1 effector TBC/RabGAP Armus (TBC1D2A) is known to inhibit Rab7, a key regulator of lysosomal function. However, the precise coordination of signaling and intracellular trafficking that regulates autophagy is poorly understood. We find that overexpression of Armus induces the accumulation of enlarged autophagosomes, while Armus depletion significantly delays autophagic flux. Upon starvation-induced autophagy, Rab7 is transiently activated. This spatiotemporal regulation of Rab7 guanosine triphosphate/guanosine diphosphate cycling occurs by Armus recruitment to autophagosomes via interaction with LC3, a core autophagy regulator. Interestingly, autophagy potently inactivates Rac1. Active Rac1 competes with LC3 for interaction with Armus and thus prevents its appropriate recruitment to autophagosomes. The precise coordination between Rac1 and Rab7 activities during starvation suggests that Armus integrates autophagy with signaling and endocytic trafficking.

Asunto(s)

Autofagia , Proteínas Activadoras de GTPasa/metabolismo , Queratinocitos/patología , Proteínas de Unión al GTP rab/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Secuencia de Aminoácidos , Medios de Cultivo/metabolismo , Activación Enzimática , Técnica del Anticuerpo Fluorescente , Proteínas Activadoras de GTPasa/genética , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Humanos , Recién Nacido , Queratinocitos/metabolismo , Lisosomas/genética , Lisosomas/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Fagosomas/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Transducción de Señal , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7 , Proteína de Unión al GTP rac1/genética

RESUMEN

BACKGROUND: Cell-cell adhesion and intracellular trafficking are regulated by signaling pathways from small GTPases of the Rho, Arf, and Rab subfamilies. How signaling from distinct small GTPases are integrated in a given process is poorly understood. RESULTS: We find that a TBC/RabGAP protein, Armus, integrates signaling between Arf6, Rac1, and Rab7 during junction disassembly. Armus binds specifically to activated Rac1 and its C-terminal TBC/RabGAP domain inactivates Rab7. Thus, Armus is a novel Rac1 effector and a bona fide GAP for Rab7 in vitro and in vivo, a unique and previously unreported combination. Arf6 activation efficiently disrupts cell-cell contacts and is known to activate Rac1 and Rab7. Arf6-induced E-cadherin degradation is efficiently blocked by expression of Armus C-terminal domain or after Armus RNAi. Coexpression of Arf6 with dominant-negative Rab7 or Rac1 also inhibits junction disassembly. Importantly, Armus RabGAP expression also prevents EGF-induced scattering in keratinocytes, a process shown here to require Arf6, Rac1, and Rab7 function. To our knowledge, this is the first report to demonstrate a molecular and functional link between Rac1 and Rab7. CONCLUSIONS: Our data indicate that active Rac1 recruits Armus to locally inactivate Rab7 and facilitate E-cadherin degradation in lysosomes. Thus, the integration of Rac1 and Rab7 activities by Armus provides an important regulatory node for E-cadherin turnover and stability of cell-cell contacts.

Asunto(s)

Cadherinas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de Unión al GTP rab/antagonistas & inhibidores , Proteína de Unión al GTP rac1/metabolismo , Animales , Células COS , Proteínas Portadoras/genética , Comunicación Celular , Células Cultivadas , Chlorocebus aethiops , Proteínas Activadoras de GTPasa/genética , Humanos , Uniones Intercelulares/metabolismo , Queratinocitos/metabolismo , Modelos Biológicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Proteínas de Unión a GTP rab7

RESUMEN

The tyrosine kinase receptor, HER2 is a crucial prognostic marker and therapeutic target for breast cancer; however, the downstream targets and biological effectors of HER2 remain unclear. We investigated the relationship between HER2 and the transcription factor FoxM1 in breast cancer. HER2 and FoxM1 expression levels were compared in breast carcinoma cell lines, paraffin-embedded breast cancer patient samples and at the mRNA level in purified breast epithelial cells. To further examine the relationship between HER2 and FoxM1 expression, we either overexpressed or siRNA-mediated depleted endogenous HER2 in breast cancer cell lines. Additionally, a mammary epithelium-targeted HER2 (neu) transgenic mouse model was also used to assess the effect of HER2 on FoxM1 levels. Furthermore, the effect of the HER2-tyrosine kinase inhibitor lapatinib on FoxM1 in HER2 positive breast cancer cells was investigated. HER2 protein levels directly correlated with FoxM1 expression in both breast carcinoma cell lines and paraffin-embedded breast cancer patient samples. Moreover, in purified breast epithelial cells, overexpression of HER2 was associated with high levels of FoxM1 mRNA, suggesting that the upregulation of FoxM1 expression is at least partially mediated transcriptionally. Furthermore, overexpression or ablation of endogenous HER2 resulted in parallel changes in FoxM1 expression. Critically, mammary epithelium-targeted HER2 mouse tumours also resulted in increased FoxM1 expression, suggesting that HER2 directed FoxM1 expression occurs in vivo and may be a critical downstream effector of HER2-targeting therapies. Indeed, treatment of breast cancer cells with lapatinib reduced FoxM1 expression at protein, mRNA and gene promoter levels. Moreover, analysis of normal and breast cancer patient samples revealed that elevated FoxM1 expression at protein and mRNA levels correlated with breast cancer development, but not significantly with cancer progression and survival. Our results indicate that the HER2 receptor regulates the expression of the FoxM1 transcription factor, which has a role in breast cancer development.

Asunto(s)

Neoplasias de la Mama/enzimología , Factores de Transcripción Forkhead/metabolismo , Receptor ErbB-2/metabolismo , Transducción de Señal , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/genética , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Lapatinib , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Adhesión en Parafina , Regiones Promotoras Genéticas , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Interferencia de ARN , ARN Mensajero/metabolismo , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transcripción Genética , Transfección , Regulación hacia Arriba

RESUMEN

Gefitinib (Iressa) is a specific and effective epidermal growth factor receptor inhibitor. An understanding of the downstream cellular targets of gefitinib will allow the discovery of biomarkers for predicting outcomes and monitoring anti-epidermal growth factor receptor therapies and provide information for overcoming gefitinib resistance. In this study, we investigated the role and regulation of FOXM1 in response to gefitinib treatment in breast cancer. Using the gefitinib-sensitive breast carcinoma cell lines BT474 and SKBR3 as well as the resistant lines MCF-7, MDA-MB-231, and MDA-MB-453, we showed that gefitinib represses the expression of the transcription factor FOXM1 in sensitive, but not resistant, cells. FOXM1 repression by gefitinib is associated with FOXO3a activation and is mediated at the transcriptional level and gene promoter level. These results were verified by immunohistochemical staining of biopsy samples from primary breast cancer patients obtained from a gefitinib neoadjuvant study. We also showed that ectopic expression of an active FOXO3a represses FOXM1 expression, whereas knockdown of FOXO3a expression using small interfering RNA can up-regulate FOXM1 and its downstream targets polo-like kinase, cyclin B1, and CDC25B and rescue sensitive BT474 cells from gefitinib-induced cell proliferative arrest. These results suggest that gefitinib represses FOXM1 expression via FOXO3a in breast cancer. We further showed that overexpression of a wild-type FOXM1 or a constitutively active FOXM1, DeltaN-FOXM1, abrogates the cell death induced by gefitinib, indicating that FOXM1 has a functional role in mediating the gefitinib-induced proliferative arrest and in determining sensitivity to gefitinib. In summary, our study defined FOXM1 as a cellular target and marker of gefitinib activity in breast cancer.

Asunto(s)

Neoplasias de la Mama/genética , Carcinoma/genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/fisiología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Quinazolinas/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma/metabolismo , Carcinoma/patología , Estudios de Casos y Controles , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Proteína Forkhead Box M1 , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/antagonistas & inhibidores , Factores de Transcripción Forkhead/metabolismo , Gefitinib , Humanos , ARN Interferente Pequeño/farmacología , Células Tumorales Cultivadas

RESUMEN

Using the doxorubicin-sensitive K562 cell line and the resistant derivative lines KD30 and KD225 as models, we found that acquisition of multidrug resistance (MDR) is associated with enhanced FOXO3a activity and expression of ABCB1 (MDR1), a plasma membrane P-glycoprotein that functions as an efflux pump for various anticancer agents. Furthermore, induction of ABCB1 mRNA expression on doxorubicin treatment of naive K562 cells was also accompanied by increased FOXO3a activity. Analysis of transfected K562, KD30, and KD225 cells in which FOXO3a activity can be induced by 4-hydroxytamoxifen showed that FOXO3a up-regulates ABCB1 expression at protein, mRNA, and gene promoter levels. Conversely, silencing of endogenous FOXO3a expression in KD225 cells inhibited the expression of this transport protein. Promoter analysis and chromatin immunoprecipitation assays showed that FOXO3a regulation of ABCB1 expression involves binding of this transcription factor to the proximal promoter region. Moreover, activation of FOXO3a increased ABCB1 drug efflux potential in KD30 cells, whereas silencing of FOXO3a by siRNA significantly reduced ABCB1 drug efflux ability. Together, these findings suggest a novel mechanism that can contribute towards MDR, involving FOXO3a as sensor for the cytotoxic stress induced by anticancer drugs. Although FOXO3a may initially trigger a program of cell cycle arrest and cell death in response to doxorubicin, sustained FOXO3a activation promotes drug resistance and survival of cells by activating ABCB1 expression.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Antineoplásicos/farmacología , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Factores de Transcripción Forkhead/genética , Subfamilia B de Transportador de Casetes de Unión a ATP , Secuencia de Bases , Inmunoprecipitación de Cromatina , Cartilla de ADN , Citometría de Flujo , Proteína Forkhead Box O3 , Humanos , Células K562 , Fosforilación , Reacción en Cadena de la Polimerasa

RESUMEN

Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) that causes growth delay in cancer cell lines and human tumor xenografts expressing high levels of EGFR. An understanding of the downstream cellular targets of gefitinib will allow the discovery of biomarkers for predicting outcomes and monitoring anti-EGFR therapies and provide information for key targets for therapeutic intervention. In this study, we investigated the role of FOXO3a in gefitinib action and resistance. Using two gefitinib-sensitive (i.e., BT474 and SKBR3) as well as three other resistant breast carcinoma cell lines (i.e., MCF-7, MDA-MB-231, and MDA-MB-453), we showed that gefitinib targets the transcription factor FOXO3a to mediate cell cycle arrest and cell death in sensitive breast cancer cells. In the sensitive cells, gefitinib treatment causes cell cycle arrest predominantly at the G(0)-G(1) phase and apoptosis, which is associated with FOXO3a dephosphorylation at Akt sites and nuclear translocation, whereas in the resistant cells, FOXO3a stays phosphorylated and remains in the cytoplasm. The nuclear accumulation of FOXO3a in response to gefitinib was confirmed in tumor tissue sections from breast cancer patients presurgically treated with gefitinib as monotherapy. We also showed that knockdown of FOXO3a expression using small interfering RNA (siRNA) can rescue sensitive BT474 cells from gefitinib-induced cell-proliferative arrest, whereas reintroduction of active FOXO3a in resistant MDA-MB-231 cells can at least partially restore cell-proliferative arrest and sensitivity to gefitinib. These results suggest that the FOXO3a dephosphorylation and nuclear localization have a direct role in mediating the gefitinib-induced proliferative arrest and in determining sensitivity to gefitinib.

Asunto(s)

Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Factores de Transcripción Forkhead/efectos de los fármacos , Quinazolinas/farmacología , Western Blotting , Línea Celular Tumoral , Proliferación Celular , Receptores ErbB/antagonistas & inhibidores , Proteína Forkhead Box O3 , Fase G1/efectos de los fármacos , Gefitinib , Silenciador del Gen , Humanos , Inmunohistoquímica , ARN Interferente Pequeño , Fase de Descanso del Ciclo Celular/efectos de los fármacos

RESUMEN

In this study, we have identified the Forkhead transcription factor FoxM1 as a physiological regulator of estrogen receptor alpha (ERalpha) expression in breast carcinoma cells. Our survey of a panel of 16 different breast cell lines showed a good correlation (13/16) between FoxM1 expression and expression of ERalpha at both protein and mRNA levels. We have also demonstrated that ectopic expression of FoxM1 in two different estrogen receptor-positive breast cancer cell lines, MCF-7 and ZR-75-30, led to up-regulation of ERalpha expression at protein and transcript levels. Furthermore, treatment of MCF-7 cells with the MEK inhibitor U0126, which blocks ERK1/2-dependent activation of FoxM1, also repressed ERalpha expression. Consistent with this, silencing of FoxM1 expression in MCF-7 cells using small interfering RNA resulted in the almost complete abrogation of ERalpha expression. We also went on to show that FoxM1 can activate the transcriptional activity of human ERalpha promoter primarily through two closely located Forkhead response elements located at the proximal region of the ERalpha promoter. Chromatin immunoprecipitation and biotinylated oligonucleotide pulldown assays have allowed us to confirm these Forkhead response elements as important for FoxM1 binding. Further co-immunoprecipitation experiments showed that FoxO3a and FoxM1 interact in vivo. Together with the chromatin immunoprecipitation and biotinylated oligonucleotide pulldown data, the co-immunoprecipitation results also suggest the possibility that FoxM1 and FoxO3a cooperate to regulate ERalpha gene transcription.

Asunto(s)

Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/biosíntesis , Factores de Transcripción Forkhead/fisiología , Transcripción Genética , Biotinilación , Ciclo Celular , Línea Celular Tumoral , Receptor alfa de Estrógeno/genética , Proteína Forkhead Box M1 , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/química , Regulación Neoplásica de la Expresión Génica , Humanos , Modelos Genéticos , Oligonucleótidos/química , ARN Mensajero/metabolismo , Regulación hacia Arriba