RESUMEN
The generation and release of membrane-enclosed packets from cancer cells, called extracellular vesicles (EVs), play important roles in propagating transformed phenotypes, including promoting cell survival. EVs mediate their effects by transferring their contents, which include specific proteins and nucleic acids, to target cells. However, how the cargo and function of EVs change in response to different stimuli remains unclear. Here, we discovered that treating highly aggressive MDAMB231 breast cancer cells with paclitaxel (PTX), a chemotherapy that stabilizes microtubules, causes them to generate a specific class of EV, namely exosomes, that are highly enriched with the cell survival protein and cancer marker, Survivin. Treating MDAMB231 cells with a variety of other chemotherapeutic agents, and inhibitors that block cell growth and survival, did not have the same effect as PTX, with the exception of nocodazole, another inhibitor of microtubule dynamics. Exosomes isolated from PTX-treated MDAMB231 cells strongly promoted the survival of serum-starved and PTX-treated fibroblasts and SKBR3 breast cancer cells, an effect that was ablated when Survivin was knocked-down from these vesicles using siRNA. These findings underscore how the enrichment of a specific cargo in exosomes promotes cell survival, as well as can potentially serve as a marker of PTX resistance.
RESUMEN
Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), have emerged as a major form of intercellular communication, playing important roles in several physiological processes and diseases, including cancer. EVs generated by cancer cells contain a variety of proteins and RNA species that can be transferred between cancer cells as well as between cancer and non-transformed (normal) cells, thereby impacting a number of aspects of cancer progression. Here we show how oncogenic transformation influences the biogenesis and function of EVs using a mouse embryonic fibroblast (MEF) cell line that can be induced to express an oncogenic form of diffuse B cell lymphoma (Dbl). Although MEFs induced to express onco-Dbl generated a similar amount of MVs as uninduced control cells, we found that MVs isolated from onco-Dbl-transformed cells contain a unique signaling protein, the ubiquitously expressed non-receptor tyrosine kinase focal adhesion kinase. The addition of MVs isolated from MEFs expressing onco-Dbl to cultures of fibroblasts strongly promoted their survival and induced their ability to grow under anchorage-independent conditions, outcomes that could be reversed by knocking down focal adhesion kinase and depleting it from the MVs or by inhibiting its kinase activity using a specific inhibitor. We then showed the same to be true for MVs isolated from aggressive MDAMB231 breast cancer cells. Together, these findings demonstrate that the induction of oncogenic transformation gives rise to MVs, which uniquely contain a signaling protein kinase that helps propagate the transformed phenotype and thus may offer a specific diagnostic marker of malignant disease.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Micropartículas Derivadas de Células/metabolismo , Exosomas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Micropartículas Derivadas de Células/genética , Embrión de Mamíferos/metabolismo , Exosomas/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Ratones , Células 3T3 NIH , Neoplasias/genética , Proteínas Proto-Oncogénicas/genéticaRESUMEN
The Hippo pathway regulates contact inhibition of cell proliferation and, ultimately, organ size in diverse multicellular organisms. Inactivation of the Hippo pathway promotes nuclear localization of the transcriptional coactivator Yap1, a Hippo pathway effector, and can cause cancer. Here, we show that deletion of αE (α epithelial) catenin in the hair follicle stem cell compartment resulted in the development of skin squamous cell carcinoma in mice. Tumor formation was accelerated by simultaneous deletion of αE-catenin and the tumor suppressor-encoding gene p53. A small interfering RNA screen revealed a functional connection between αE-catenin and Yap1. By interacting with Yap1, αE-catenin promoted its cytoplasmic localization, and Yap1 showed constitutive nuclear localization in αE-catenin-null cells. We also found an inverse correlation between αE-catenin abundance and Yap1 activation in human squamous cell carcinoma tumors. These findings identify αE-catenin as a tumor suppressor that inhibits Yap1 activity and sequesters it in the cytoplasm.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinoma de Células Escamosas/metabolismo , Núcleo Celular/metabolismo , Fosfoproteínas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , alfa Catenina/metabolismo , Transporte Activo de Núcleo Celular/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Núcleo Celular/genética , Proliferación Celular , Células HEK293 , Humanos , Ratones , Ratones Desnudos , Ratones Transgénicos , Fosfoproteínas/genética , Factores de Transcripción , Proteína p53 Supresora de Tumor/genética , Proteínas Señalizadoras YAP , alfa Catenina/genéticaRESUMEN
During development and regeneration, proliferation of tissue-specific stem cells is tightly controlled to produce organs of a predetermined size. The molecular determinants of this process remain poorly understood. Here, we investigate the function of Yap1, the transcriptional effector of the Hippo signaling pathway, in skin biology. Using gain- and loss-of-function studies, we show that Yap1 is a critical modulator of epidermal stem cell proliferation and tissue expansion. Yap1 mediates this effect through interaction with TEAD transcription factors. Additionally, our studies reveal that α-catenin, a molecule previously implicated in tumor suppression and cell density sensing in the skin, is an upstream negative regulator of Yap1. α-catenin controls Yap1 activity and phosphorylation by modulating its interaction with 14-3-3 and the PP2A phosphatase. Together, these data identify Yap1 as a determinant of the proliferative capacity of epidermal stem cells and as an important effector of a "crowd control" molecular circuitry in mammalian skin.