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Aberrant ErbB2 receptor tyrosine kinase activation in breast cancer is strongly linked to an invasive disease. The molecular basis of ErbB2-driven invasion is largely unknown. We show that cysteine cathepsins B and L are elevated in ErbB2 positive primary human breast cancer and function as effectors of ErbB2-induced invasion in vitro. We identify Cdc42-binding protein kinase beta, extracellular regulated kinase 2, p21-activated protein kinase 4, and protein kinase C alpha as essential mediators of ErbB2-induced cysteine cathepsin expression and breast cancer cell invasiveness. The identified signaling network activates the transcription of cathepsin B gene (CTSB) via myeloid zinc finger-1 transcription factor that binds to an ErbB2-responsive enhancer element in the first intron of CTSB. This work provides a model system for ErbB2-induced breast cancer cell invasiveness, reveals a signaling network that is crucial for invasion in vitro, and defines a specific role and targets for the identified serine-threonine kinases.
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Neoplasias de la Mama/patología , Catepsina B/genética , Catepsina B/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Receptor ErbB-2/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Catepsina L/genética , Catepsina L/metabolismo , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa de Distrofia Miotónica , Invasividad Neoplásica , Regiones Promotoras Genéticas , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteína Proto-Oncogénica c-ets-1/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Receptor ErbB-2/genética , Elementos de Respuesta , Transducción de Señal , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismoRESUMEN
Misregulation of acid-base transport plays central roles in cancer development. We previously demonstrated the strong up-regulation of the Na(+),HCO3(-) cotransporter NBCn1 (SLC4A7) in MCF-7 breast cancer cells by a truncated, constitutively active ErbB2 (HER2) receptor, ΔNErbB2, and showed that NBCn1 expression and activity are increased in breast cancer tissue from patients. Here, we present the first in-depth characterization of an SLC4A7 promoter and identify its minimal ΔNErbB2-sensitive region. Inhibition or siRNA-mediated knockdown of PI3K, Akt1, ERK1/2, or Src decreased the NBCn1 protein level in ΔNErbB2-expressing MCF-7 cells by ~50, 60, 30 and 35%, respectively. Further, knockdown of the transcription factor Krüppel-like factor 4 (KLF4) reduced NBCn1 protein expression by ~40%, and KLF4 overexpression increased NBCn1 expression by 50-80%. In contrast, knockdown of the closely related transcription factor specificity protein 1 (Sp1) or transfection with dominant-negative Sp1 increased NBCn1 expression by ~35 and ~50%, respectively. NBCn1 expression was also increased by stimulation of full-length ErbB1, -2, and -3 receptors in SKBr3 cells (1.5- and 2-fold by NRG1 or EGF, respectively) or after their exogenous expression in MCF-7 cells. Finally, stimulation with NRG1 or EGF more than doubled acid extrusion capacity in SKBr3 cells. In conclusion, NBCn1 is strongly upregulated by ErbB receptor signaling in a manner involving opposite effects of KLF4 and Sp1, transcription factors with central roles in cancer development. ErbB-induced up-regulation of NBCn1-mediated acid extrusion may play important physiological and pathophysiological roles in the breast epithelium and other tissues with high ErbB receptor levels.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Familia-src Quinasas/metabolismo , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/genética , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Proteínas Proto-Oncogénicas c-akt/genética , Receptor ErbB-2/genética , Simportadores de Sodio-Bicarbonato/genética , Intercambiadores de Sodio-Hidrógeno/genética , Familia-src Quinasas/genéticaRESUMEN
Patient-derived organoids (PDOs) are ideal ex vivo model systems to study cancer progression and drug resistance mechanisms. Here, we present a protocol for measuring drug efficacy in three-dimensional (3D) high-grade serous ovarian cancer PDO cultures through quantification of cytotoxicity using propidium iodide incorporation in dead cells. We also provide detailed steps to analyze proliferation of PDOs using the Ki67 biomarker. We describe steps for sample processing, immunofluorescent staining, high-throughput confocal imaging, and image-based quantification for 3D cultures. For complete details on the use and execution of this protocol, please refer to Lahtinen et al. (2023).1.
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Imagenología Tridimensional , Neoplasias Ováricas , Humanos , Femenino , Neoplasias Ováricas/diagnóstico por imagen , Neoplasias Ováricas/tratamiento farmacológico , Muerte Celular , Organoides , Proliferación CelularRESUMEN
Cancer cells are dependent on cholesterol, and they possess strictly controlled cholesterol homeostasis mechanisms. These allow them to smoothly switch between cholesterol synthesis and uptake to fulfill their needs and to adapt environmental changes. Here we describe a mechanism of how cancer cells employ oncogenic growth factor signaling to promote uptake and utilization of extracellular cholesterol via Myeloid Zinc Finger 1 (MZF1)-mediated Niemann Pick C1 (NPC1) expression and upregulated macropinocytosis. Expression of p95ErbB2, highly oncogenic, standard-treatment resistant form of ErbB2 mobilizes lysosomes and activates EGFR, invasion and macropinocytosis. This is connected to a metabolic shift from cholesterol synthesis to uptake due to macropinocytosis-enabled flow of extracellular cholesterol. NPC1 increase facilitates extracellular cholesterol uptake and is necessary for the invasion of ErbB2 expressing breast cancer spheroids and ovarian cancer organoids, indicating a regulatory role for NPC1 in the process. The ability to obtain cholesterol as a byproduct of increased macropinocytosis allows cancer cells to direct the resources needed for the energy-consuming cholesterol synthesis towards other activities such as invasion. These results demonstrate that macropinocytosis is not only an alternative energy source for cancer cells but also an efficient way to provide building material, such as cholesterol, for its macromolecules and membranes.
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Colesterol , Péptidos y Proteínas de Señalización Intracelular , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Colesterol/metabolismo , Transporte Biológico , Proteína Niemann-Pick C1/metabolismoRESUMEN
Ovarian high-grade serous carcinoma (HGSC) is typically diagnosed at an advanced stage, with multiple genetically heterogeneous clones existing in the tumors long before therapeutic intervention. Herein we integrate clonal composition and topology using whole-genome sequencing data from 510 samples of 148 patients with HGSC in the prospective, longitudinal, multiregion DECIDER study. Our results reveal three evolutionary states, which have distinct features in genomics, pathways, and morphological phenotypes, and significant association with treatment response. Nested pathway analysis suggests two evolutionary trajectories between the states. Experiments with five tumor organoids and three PI3K inhibitors support targeting tumors with enriched PI3K/AKT pathway with alpelisib. Heterogeneity analysis of samples from multiple anatomical sites shows that site-of-origin samples have 70% more unique clones than metastatic tumors or ascites. In conclusion, these analysis and visualization methods enable integrative tumor evolution analysis to identify patient subtypes using data from longitudinal, multiregion cohorts.
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Cistadenocarcinoma Seroso , Neoplasias de las Trompas Uterinas , Neoplasias Ováricas , Femenino , Humanos , Neoplasias Ováricas/patología , Fosfatidilinositol 3-Quinasas/genética , Estudios Prospectivos , Cistadenocarcinoma Seroso/metabolismo , Neoplasias de las Trompas Uterinas/genéticaRESUMEN
Ovarian cancer is the deadliest gynecological cancer, the high-grade serous ovarian carcinoma (HGSC) being its most common and most aggressive form. Despite the latest therapeutical advancements following the introduction of vascular endothelial growth factor receptor (VEGFR) targeting angiogenesis inhibitors and poly-ADP-ribose-polymerase (PARP) inhibitors to supplement the standard platinum- and taxane-based chemotherapy, the expected overall survival of HGSC patients has not improved significantly from the five-year rate of 42%. This calls for the development and testing of more efficient treatment options. Many oncogenic kinase-signaling pathways are dysregulated in HGSC. Since small-molecule kinase inhibitors have revolutionized the treatment of many solid cancers due to the generality of the increased activation of protein kinases in carcinomas, it is reasonable to evaluate their potential against HGSC. Here, we present the latest concluded and on-going clinical trials on kinase inhibitors in HGSC, as well as the recent work concerning ovarian cancer patient organoids and xenograft models. We discuss the potential of kinase inhibitors as personalized treatments, which would require comprehensive assessment of the biological mechanisms underlying tumor spread and chemoresistance in individual patients, and their connection to tumor genome and transcriptome to establish identifiable subgroups of patients who are most likely to benefit from a given therapy.
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Docetaxel (DTX) was the first chemotherapeutic agent to demonstrate significant efficacy in the treatment of men with metastatic castration-resistant prostate cancer. However, response to DTX is generally short-lived, and relapse eventually occurs due to emergence of drug-resistance. We previously established two DTX-resistant prostate cancer cell lines, LNCaPR and C4-2BR, derived from the androgen-dependent LNCaP cell line, and from the LNCaP lineage-derived androgen-independent C4-2B sub-line, respectively. Using an unbiased drug screen, we identify itraconazole (ITZ), an oral antifungal drug, as a compound that can efficiently re-sensitize drug-resistant LNCaPR and C4-2BR prostate cancer cells to DTX treatment. ITZ can re-sensitize multiple DTX-resistant cell models, not only in prostate cancer derived cells, such as PC-3 and DU145, but also in docetaxel-resistant breast cancer cells. This effect is dependent on expression of ATP-binding cassette (ABC) transporter protein ABCB1, also known as P-glycoprotein (P-gp). Molecular modeling of ITZ bound to ABCB1, indicates that ITZ binds tightly to the inward-facing form of ABCB1 thereby inhibiting the transport of DTX. Our results suggest that ITZ may provide a feasible approach to re-sensitization of DTX resistant cells, which would add to the life-prolonging effects of DTX in men with metastatic castration-resistant prostate cancer.
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PURPOSE: Most HER2 positive invasive cancers are either intrinsic non-responsive or develop resistance when treated with 1st line HER2 targeting drugs. Both 1st and 2nd line treatments of HER2 positive cancers are aimed at targeting the HER2 receptor directly, thereby strongly limiting the treatment options of HER2/ErbB2 inhibition resistant invasive cancers. METHODS: We used phenotypic high throughput microscopy screening to identify efficient inhibitors of ErbB2-induced invasion using 1st line HER2 inhibitor trastuzumab- and pertuzumab-resistant, p95-ErbB2 expressing breast cancer cells in conjunction with the Prestwick Chemical Library®. The screening entailed a drug's ability to inhibit ErbB2-induced, invasion-promoting positioning of lysosomes at the cellular periphery, a phenotype that defines their invasiveness. In addition, we used high throughput microscopy and biochemical assays to assess the effects of the drugs on lysosomal membrane permeabilization (LMP) and autophagy, two features connected to cancer treatment. Using 2nd line HER2 inhibitor lapatinib resistant 3-dimensional model systems, we assessed the effects of the drugs on ErbB2 positive breast cancer spheroids and developed a high-throughput invasion assay for HER2 positive ovarian cancer organoids for further evaluation. RESULTS: We identified Auranofin, Colchicine, Monensin, Niclosamide, Podophyllotoxin, Quinacrine and Thiostrepton as efficient inhibitors of invasive growth of 2nd line HER2 inhibitor lapatinib resistant breast cancer spheroids and ovarian cancer organoids. We classified these drugs into four groups based on their ability to target lysosomes by inducing autophagy and/or LMP, i.e., drugs inducing early LMP, early autophagy with late LMP, late LMP, or neither. CONCLUSIONS: Our results indicate that targetable lysosome-engaging cellular pathways downstream of ErbB2 contribute to invasion. They support lysosomal trafficking as an attractive target for therapy aiming at preventing the spreading of cancer cells. Since these drugs additionally possess anti-inflammatory activities, they could serve as multipurpose drugs simultaneously targeting infection/inflammation and cancer spreading.
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Antiinflamatorios/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Lisosomas/efectos de los fármacos , Receptor ErbB-2/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Antineoplásicos/uso terapéutico , Autofagia/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Lapatinib/uso terapéutico , Lisosomas/metabolismo , Células MCF-7 , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Invasividad NeoplásicaRESUMEN
Anti-cancer treatments have never been so numerous and so efficient [...].
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Antineoplásicos Inmunológicos/uso terapéutico , Manejo de la Enfermedad , Inmunoterapia/métodos , Terapia Molecular Dirigida/métodos , Neoplasias/terapia , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/inmunología , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunoconjugados/uso terapéutico , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/patología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/inmunología , Esferoides Celulares/patologíaRESUMEN
Over 90% of cancer deaths are due to cancer cells metastasizing into other organs. Invasion is a prerequisite for metastasis formation. Thus, inhibition of invasion can be an efficient way to prevent disease progression in these patients. This could be achieved by targeting the molecules regulating invasion. One of these is an oncogenic transcription factor, Myeloid Zinc Finger 1 (MZF1). Dysregulated transcription factors represent a unique, increasing group of drug targets that are responsible for aberrant gene expression in cancer and are important nodes driving cancer malignancy. Recent studies report of a central involvement of MZF1 in the invasion and metastasis of various solid cancers. In this review, we summarize the research on MZF1 in cancer including its function and role in lysosome-mediated invasion and in the expression of genes involved in epithelial to mesenchymal transition. We also discuss possible means to target it on the basis of the current knowledge of its function in cancer.
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Factores de Transcripción de Tipo Kruppel/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Dedos de Zinc , Animales , Humanos , Factores de Transcripción de Tipo Kruppel/química , Modelos Biológicos , Terapia Molecular Dirigida , Invasividad NeoplásicaRESUMEN
Active Ras oncogene is expressed in approximately 30% of human cancers. Yet, very little is known about the molecular mechanisms responsible for its transforming potential. Here, we show that H-Ras-mediated transformation requires isoform 2 of the c-Jun-NH(2)-terminal kinase (JNK). H-Ras-transduced JNK2-deficient (Jnk2-/-) murine embryonic fibroblasts (MEFs) were severely inhibited in colony formation and growth in soft agar in vitro as well as in tumor formation in immunodeficient mice as compared with corresponding Jnk1-/- and wild-type MEFs. Accordingly, the RNA interference-based depletion of JNK2 form wild-type MEFs also resulted in defective Ras transformation. The extra barrier against H-Ras transformation in Jnk2-/- MEFs was not due to their inability to inactivate p53 signaling because all JNK2-deficient MEF lines had lost p19(Arf). Furthermore, expression of the E6 protein of the human papilloma virus failed to overcome the transformation defect. It could, however, be overcome by coexpression of H-Ras with the SV40 large T antigen or c-Myc. Surprisingly, the H-Ras-transduced JNK2-deficient MEFs exhibited higher activity of activator protein-1 and higher levels of c-Jun expression compared with H-Ras-transduced JNK1-deficient or wild-type cells, indicating that the key target of JNK2 during Ras transformation was divergent from activator protein-1. These results clearly show that a single kinase, JNK2, could control Ras transformation and thus point out a vulnerable control point that may prove important for the tumor development in general.
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Transformación Celular Neoplásica/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Factor de Transcripción AP-1/metabolismo , Proteínas ras/metabolismo , Animales , Transformación Celular Neoplásica/genética , Regulación hacia Abajo , Femenino , Genes ras , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Proteína Quinasa 9 Activada por Mitógenos/deficiencia , Proteína Quinasa 9 Activada por Mitógenos/genética , Células 3T3 NIH , Fosforilación , Interferencia de ARN , Transducción de Señal , Transducción Genética , Proteínas ras/genéticaRESUMEN
Inducible gene expression systems are favored over stable expression systems in a wide variety of basic and applied research areas, including functional genomics, gene therapy, tissue engineering, biopharmaceutical protein production and drug discovery. This is because they are mostly reversible and thus more flexible to use. Furthermore, compared to constitutive expression, they generally exhibit a higher efficiency and have fewer side effects, such as cell death and delayed growth or development. Empowered by decades of development of inducible gene expression systems, researchers can now efficiently activate or suppress any gene, temporarily and quantitively at will, depending on experimental requirements and designs. Here, we review a number of most commonly used mammalian inducible expression systems and provide basic standards and criteria for the selection of the most suitable one.
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Expresión Génica , Mamíferos/genética , Activación Transcripcional , Animales , Genes Reporteros , Humanos , Regiones Promotoras GenéticasRESUMEN
HER2/ErbB2 activation turns on transcriptional processes that induce local invasion and lead to systemic metastasis. The early transcriptional changes needed for ErbB2-induced invasion are poorly understood. Here, we link ErbB2 activation to invasion via ErbB2-induced, SUMO-directed phosphorylation of a single serine residue, S27, of the transcription factor myeloid zinc finger-1 (MZF1). Utilizing an antibody against MZF1-pS27, we show that the phosphorylation of S27 correlates significantly (p < 0.0001) with high-level expression of ErbB2 in primary invasive breast tumors. Phosphorylation of MZF1-S27 is an early response to ErbB2 activation and results in increased transcriptional activity of MZF1. It is needed for the ErbB2-induced expression of MZF1 target genes CTSB and PRKCA, and invasion of single-cells from ErbB2-expressing breast cancer spheroids. The phosphorylation of MZF1-S27 is preceded by poly-SUMOylation of K23, which can make S27 accessible to efficient phosphorylation by PAK4. Based on our results, we suggest for an activation mechanism where phosphorylation of MZF1-S27 triggers MZF1 dissociation from its transcriptional repressors such as the CCCTC-binding factor (CTCF). Our findings increase understanding of the regulation of invasive signaling in breast cancer by uncovering a detailed biological mechanism of how ErbB2 activation can rapidly lead to its invasion-promoting target gene expression and invasion.
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Neoplasias de la Mama/metabolismo , Factor de Unión a CCCTC/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Lisosomas/metabolismo , Receptor ErbB-2/metabolismo , Serina/metabolismo , Quinasas p21 Activadas/metabolismo , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Humanos , Factores de Transcripción de Tipo Kruppel/química , Factores de Transcripción de Tipo Kruppel/metabolismo , Células MCF-7 , Modelos Moleculares , Invasividad Neoplásica , Fosforilación , Sumoilación , Transcripción Genética , Regulación hacia ArribaRESUMEN
The c-Jun N-terminal kinase (JNK) signalling pathway has an established role in cellular stress signalling, cell survival and tumorigenesis. Here, we demonstrate that inhibition of JNK signalling results in partial delocalization of the RNA helicase DDX21 from the nucleolus to the nucleoplasm, increased nucleolar mobility of DDX21 and inhibition of rRNA processing. Furthermore, our results show that JNK signalling regulates DDX21 phosphorylation and protein expression. In conclusion, the results presented in this study reveal a previously unidentified cellular role for JNK signalling in the regulation of nucleolar functions. Based on these results, we propose that JNK-mediated effects on nucleolar homeostasis and rRNA processing should be considered when interpreting cellular phenotypes observed in JNK-deficient cell and animal models.
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Nucléolo Celular/enzimología , ARN Helicasas DEAD-box/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Transporte Activo de Núcleo Celular , Animales , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Ratones , Ratones Noqueados , Fosforilación , Biosíntesis de Proteínas , ARN Ribosómico/metabolismo , Transducción de SeñalRESUMEN
Cancer cells utilize lysosomes for invasion and metastasis. Myeloid Zinc Finger1 (MZF1) is an ErbB2-responsive transcription factor that promotes invasion of breast cancer cells via upregulation of lysosomal cathepsins B and L. Here we identify let-7 microRNA, a well-known tumor suppressor in breast cancer, as a direct negative regulator of MZF1. Analysis of primary breast cancer tissues reveals a gradual upregulation of MZF1 from normal breast epithelium to invasive ductal carcinoma and a negative correlation between several let-7 family members and MZF1 mRNA, suggesting that the inverse regulatory relationship between let-7 and MZF1 may play a role in the development of invasive breast cancer. Furthermore, we show that MZF1 regulates lysosome trafficking in ErbB2-positive breast cancer cells. In line with this, MZF1 depletion or let-7 expression inhibits invasion-promoting anterograde trafficking of lysosomes and invasion of ErbB2-expressing MCF7 spheres. The results presented here link MZF1 and let-7 to lysosomal processes in ErbB2-positive breast cancer cells that in non-cancerous cells have primarily been connected to the transcription factor EB. Identifying MZF1 and let-7 as regulators of lysosome distribution in invasive breast cancer cells, uncouples cancer-associated, invasion-promoting lysosomal alterations from normal lysosomal functions and thus opens up new possibilities for the therapeutic targeting of cancer lysosomes.
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Normal functioning of the nervous system requires precise regulation of dendritic shape and synaptic connectivity. Here, we report a severe impairment of dendritic structures in the cerebellum and motor cortex of c-Jun N-terminal kinase 1 (JNK1)-deficient mice. Using an unbiased screen for candidate mediators, we identify the dendrite-specific high-molecular-weight microtubule-associated protein 2 (MAP2) as a JNK substrate in the brain. We subsequently show that MAP2 is phosphorylated by JNK in intact cells and that MAP2 proline-rich domain phosphorylation is decreased in JNK1-/- brain. We developed compartment-targeted JNK inhibitors to define whether a functional relationship exists between the physiologically active, cytosolic pool of JNK and dendritic architecture. Using these, we demonstrate that cytosolic, but not nuclear, JNK determines dendritic length and arbor complexity in cultured neurons. Moreover, we confirm that MAP2-dependent process elongation is enhanced after activation of JNK. Using JNK1-/- neurons, we reveal a dominant role for JNK1 over ERK in regulating dendritic arborization, whereas ERK only regulates dendrite shape under conditions in which JNK activity is low (JNK1-/- neurons). These results reveal a novel antagonism between JNK and ERK, potentially providing a mechanism for fine-tuning the dendritic arbor. Together, these data suggest that JNK phosphorylation of MAP2 plays an important role in defining dendritic architecture in the brain.
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Citosol/enzimología , Dendritas/ultraestructura , Proteínas Asociadas a Microtúbulos/fisiología , Proteína Quinasa 8 Activada por Mitógenos/fisiología , Animales , Células COS , Diferenciación Celular , Núcleo Celular/enzimología , Células Cultivadas/enzimología , Células Cultivadas/ultraestructura , Corteza Cerebelosa/citología , Corteza Cerebelosa/enzimología , Corteza Cerebelosa/crecimiento & desarrollo , Chlorocebus aethiops , Ratones , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 8 Activada por Mitógenos/deficiencia , Proteína Quinasa 8 Activada por Mitógenos/genética , Corteza Motora/citología , Corteza Motora/enzimología , Neuronas/ultraestructura , Fosforilación , Prosencéfalo/citología , Prosencéfalo/enzimología , Procesamiento Proteico-Postraduccional , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/fisiología , TransfecciónRESUMEN
Tumorigenesis is associated with several changes that alter the cellular susceptibility to programmed cell death. Here, we show that immortalization and transformation sensitize cells in particular to the cysteine cathepsin-mediated lysosomal death pathway. Spontaneous immortalization increased the susceptibility of wild-type murine embryonic fibroblasts (MEFs) to tumor necrosis factor (TNF)-mediated cytotoxicity >1000-fold, whereas immortalized MEFs deficient for lysosomal cysteine protease cathepsin B (CathB) retained the resistant phenotype of primary cells. This effect was specific for cysteine cathepsins, because also lack of cathepsin L (a lysosomal cysteine protease), but not that of cathepsin D (a lysosomal aspartyl protease) or caspase-3 (the major executioner protease in classic apoptosis) inhibited the immortalization-associated sensitization of MEFs to TNF. Oncogene-driven transformation of immortalized MEFs was associated with a dramatic increase in cathepsin expression and additional sensitization to the cysteine cathepsin-mediated death pathway. Importantly, exogenous expression of CathB partially reversed the resistant phenotype of immortalized CathB-deficient MEFs, and the inhibition of CathB activity by pharmacological inhibitors or RNA interference attenuated TNF-induced cytotoxicity in immortalized and transformed wild-type cells. Thus, tumorigenesis-associated changes in lysosomes may counteract cancer progression and enhance therapeutic responses by sensitizing cells to programmed cell death.
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Apoptosis/efectos de los fármacos , Transformación Celular Neoplásica , Resistencia a Antineoplásicos , Embrión de Mamíferos/citología , Fibroblastos/efectos de los fármacos , Lisosomas/enzimología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Antineoplásicos/farmacología , Caspasa 3 , Inhibidores de Caspasas , Caspasas/genética , Caspasas/fisiología , Catepsina B/antagonistas & inhibidores , Catepsina B/genética , Catepsina B/fisiología , Catepsina D/antagonistas & inhibidores , Catepsina D/genética , Catepsina D/fisiología , Catepsina L , Catepsinas/antagonistas & inhibidores , Catepsinas/genética , Catepsinas/fisiología , Cisteína Endopeptidasas , Citocromos c/metabolismo , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Fibroblastos/citología , Fibroblastos/enzimología , Genes ras/fisiología , Genes src/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , Interferencia de ARN , Transducción de Señal , TransfecciónRESUMEN
SCAN domains in zinc-finger transcription factors are crucial mediators of protein-protein interactions. Up to 240 SCAN-domain encoding genes have been identified throughout the human genome. These include cancer-related genes, such as the myeloid zinc finger 1 (MZF1), an oncogenic transcription factor involved in the progression of many solid cancers. The mechanisms by which SCAN homo- and heterodimers assemble and how they alter the transcriptional activity of zinc-finger transcription factors in cancer and other diseases remain to be investigated. Here, we provide the first description of the conformational ensemble of the MZF1 SCAN domain cross-validated against NMR experimental data, which are probes of structure and dynamics on different timescales. We investigated the protein-protein interaction network of MZF1 and how it is perturbed in different cancer types by the analyses of high-throughput proteomics and RNASeq data. Collectively, we integrated many computational approaches, ranging from simple empirical energy functions to all-atom microsecond molecular dynamics simulations and network analyses to unravel the effects of cancer-related substitutions in relation to MZF1 structure and interactions.
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Overexpression and activation of ErbB2 receptor tyrosine kinase in breast cancer is strongly linked to an aggressive disease with high potential for invasion and metastasis. In addition to inducing very aggressive, metastatic cancer, ErbB2 activation mediates processes such as increased cancer cell proliferation and survival and is needed for normal physiological activities, such as heart function and development of the nervous system. How does ErbB2 activation make cancer cells invasive and when? Comprehensive understanding of the cellular mechanisms leading to ErbB2-induced malignant processes is necessary for answering these questions. Here we present current knowledge about the invasion-promoting function of ErbB2 and the mechanisms involved in it. Obtaining detailed information about the "bad" behavior of ErbB2 can facilitate development of novel treatments against ErbB2-positive cancers.
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Cell migration and invasion require increased plasma membrane dynamics and ability to navigate through dense stroma, thereby exposing plasma membrane to tremendous physical stress. Yet, it is largely unknown how metastatic cancer cells acquire an ability to cope with such stress. Here we show that S100A11, a calcium-binding protein upregulated in a variety of metastatic cancers, is essential for efficient plasma membrane repair and survival of highly motile cancer cells. Plasma membrane injury-induced entry of calcium into the cell triggers recruitment of S100A11 and Annexin A2 to the site of injury. We show that S100A11 in a complex with Annexin A2 helps reseal the plasma membrane by facilitating polymerization of cortical F-actin and excision of the damaged part of the plasma membrane. These data reveal plasma membrane repair in general and S100A11 and Annexin A2 in particular as new targets for the therapy of metastatic cancers.