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Androgen Receptor (AR) activity in prostate stroma is required to maintain prostate homeostasis. This is mediated through androgen-dependent induction and secretion of morphogenic factors that drive epithelial cell differentiation. However, stromal AR expression is lost in aggressive prostate cancer. The mechanisms leading to stromal AR loss and morphogen production are unknown. We identified TGFß1 and TNFα as tumor-secreted factors capable of suppressing AR mRNA and protein expression in prostate stromal fibroblasts. Pharmacological and RNAi approaches identified NF-κB as the major signaling pathway involved in suppressing AR expression by TNFα. In addition, p38α- and p38δ-MAPK were identified as suppressors of AR expression independent of TNFα. Two regions of the AR promoter were responsible for AR suppression through TNFα. FGF10 and Wnt16 were identified as androgen-induced morphogens, whose expression was lost upon TNFα treatment and enhanced upon p38-MAPK inhibition. Wnt16, through non-canonical Jnk signaling, was required for prostate basal epithelial cell survival. These findings indicate that stromal AR loss is mediated by secreted factors within the TME. We identified TNFα/TGFß as two possible factors, with TNFα mediating its effects through NF-κB or p38-MAPK to suppress AR mRNA transcription. This leads to loss of androgen-regulated stromal morphogens necessary to maintain normal epithelial homeostasis.
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FN-kappa B , Neoplasias de la Próstata , Receptores Androgénicos , Células del Estroma , Proteínas Quinasas p38 Activadas por Mitógenos , Masculino , Humanos , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/genética , FN-kappa B/metabolismo , Receptores Androgénicos/metabolismo , Receptores Androgénicos/genética , Células del Estroma/metabolismo , Células del Estroma/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Factor de Necrosis Tumoral alfa/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Regulación Neoplásica de la Expresión Génica , Línea Celular Tumoral , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Proteínas Wnt/metabolismo , Proteínas Wnt/genética , Transducción de Señal , Próstata/patología , Próstata/metabolismoRESUMEN
Lack of adequate models significantly hinders advances in prostate cancer treatment, where resistance to androgen-deprivation therapies and bone metastasis remain as major challenges. Current in vitro models fail to faithfully mimic the complex prostate physiology. In vivo animal models can shed light on the oncogenes involved in prostate cancer development and progression; however, the animal prostate gland is fundamentally different from that of human, and the underlying genetic mechanisms are different. To address this problem, we developed the first in vitro microfluidic human Prostate-Cancer-on-Chip (PCoC) model, where human prostate cancer and stromal fibroblast cells were co-cultivated in two channels separated by a porous membrane under culture medium flow. The established microenvironment enables soluble signaling factors secreted by each culture to locally diffuse through the membrane pores affecting the neighboring culture. We particularly explored the conversion of the stromal fibroblasts into cancer-associated fibroblasts (CAFs) due to the interaction between the 2 cell types. Immunofluorescence microscopy revealed that tumor cells induced CAF biomarkers, αSMA and COL1A1, in stromal fibroblasts. The stromal CAF conversion level was observed to increase along the flow direction in response to diffusion agents, consistent with simulations of solute concentration gradients. The tumor cells also downregulated androgen receptor (AR) expression in stromal fibroblasts, while an adequate level of stromal AR expression is maintained in normal prostate homeostasis. We further investigated tumor invasion into the stroma, an early step in the metastatic cascade, in devices featuring a serpentine channel with orthogonal channel segments overlaying a straight channel and separated by an 8 µm-pore membrane. Both tumor cells and stromal CAFs were observed to cross over into their neighboring channel, and the stroma's role seemed to be proactive in promoting cell invasion. As control, normal epithelial cells neither induced CAF conversion nor promoted cell invasion. In summary, the developed PCoC model allows spatiotemporal analysis of the tumor-stroma dynamic interactions, due to bi-directional signaling and physical contact, recapitulating tissue-level multicellular responses associated with prostate cancer in vivo. Hence, it can serve as an in vitro model to dissect mechanisms in human prostate cancer development and seek advanced therapeutic strategies.
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Given the central role of the androgen receptor (AR) in prostate cancer cell biology, AR-targeted therapies have been the backbone of prostate cancer treatment for over 50 years. New data indicate that AR is expressed in additional cell types within the tumor microenvironment. Moreover, targeting AR for the treatment of prostate cancer has established side effects such as bone complications and an increased risk of developing cardiometabolic disease, indicating broader roles for AR. With the advent of novel technologies, such as single-cell approaches and advances in preclinical modeling, AR has been identified to have clinically significant functions in other cell types. In this mini-review, we describe new cancer cell-extrinsic roles for AR within the tumor microenvironment as well as systemic effects that collectively impact prostate cancer progression and patient outcomes.
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Neoplasias de la Próstata Resistentes a la Castración , Neoplasias de la Próstata , Humanos , Masculino , Antagonistas de Receptores Androgénicos , Huesos/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Receptores Androgénicos/metabolismo , Microambiente TumoralRESUMEN
Distinguishing key factors that drive the switch from indolent to invasive disease will make a significant impact on guiding the treatment of prostate cancer (PCa) patients. Here, we identify a novel signaling pathway linking hypoxia and PIM1 kinase to the actin cytoskeleton and cell motility. An unbiased proteomic screen identified Abl-interactor 2 (ABI2), an integral member of the wave regulatory complex (WRC), as a PIM1 substrate. Phosphorylation of ABI2 at Ser183 by PIM1 increased ABI2 protein levels and enhanced WRC formation, resulting in increased protrusive activity and cell motility. Cell protrusion induced by hypoxia and/or PIM1 was dependent on ABI2. In vivo smooth muscle invasion assays showed that overexpression of PIM1 significantly increased the depth of tumor cell invasion, and treatment with PIM inhibitors significantly reduced intramuscular PCa invasion. This research uncovers a HIF-1-independent signaling axis that is critical for hypoxia-induced invasion and establishes a novel role for PIM1 as a key regulator of the actin cytoskeleton.
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Actinas , Proteínas Adaptadoras Transductoras de Señales , Neoplasias de la Próstata , Proteínas Proto-Oncogénicas c-pim-1 , Humanos , Masculino , Actinas/genética , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular Tumoral , Hipoxia , Proteómica , Proteínas Proto-Oncogénicas c-pim-1/genética , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Transducción de Señal , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Invasividad NeoplásicaRESUMEN
Angiogenesis is essential for the sustained growth of solid tumors. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of angiogenesis and constitutive activation of HIF-1 is frequently observed in human cancers. Therefore, understanding the mechanisms governing the activation of HIF-1 is critical for successful therapeutic targeting of tumor angiogenesis. Herein, we establish a new regulatory mechanism responsible for the constitutive activation of HIF-1α in cancer, irrespective of oxygen tension. PIM1 kinase directly phosphorylates HIF-1α at threonine 455, a previously uncharacterized site within its oxygen-dependent degradation domain. This phosphorylation event disrupts the ability of prolyl hydroxylases to bind and hydroxylate HIF-1α, interrupting its canonical degradation pathway and promoting constitutive transcription of HIF-1 target genes. Moreover, phosphorylation of the analogous site in HIF-2α (S435) stabilizes the protein through the same mechanism, indicating post-translational modification within the oxygen-dependent degradation domain as a mechanism of regulating the HIF-α subunits. In vitro and in vivo models demonstrate that expression of PIM1 is sufficient to stabilize HIF-1α and HIF-2α in normoxia and stimulate angiogenesis in a HIF-1-dependent manner. CRISPR mutants of HIF-1α (Thr455D) promoted increased tumor growth, proliferation, and angiogenesis. Moreover, HIF-1α-T455D xenograft tumors were refractory to the anti-angiogenic and cytotoxic effects of PIM inhibitors. These data identify a new signaling axis responsible for hypoxia-independent activation of HIF-1 and expand our understanding of the tumorigenic role of PIM1 in solid tumors.
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Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias/etiología , Neoplasias/metabolismo , Neovascularización Patológica/etiología , Neovascularización Patológica/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Mutación , Neoplasias/patología , Fosforilación , Unión Proteica , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-pim-1/genéticaRESUMEN
BACKGROUND: The extensive alveolar capillary network of the lungs is an attractive route for administration of several agents. One key functional attribute is the rapid onset of systemic action due to the absence of first-pass metabolism. METHODS: Here we applied a combinatorial approach for ligand-directed pulmonary delivery as a unique route for systemic targeting in vaccination. FINDINGS: We screened a phage display random peptide library in vivo to select, identify, and validate a ligand (CAKSMGDIVC) that specifically targets and is internalized through its receptor, α3ß1 integrin, on the surface of cells lining the lung airways and alveoli and mediates CAKSMGDIVC-displaying phage binding and systemic delivery without compromising lung homeostasis. As a proof-of-concept, we show that the pulmonary delivery of targeted CAKSMGDIVC-displaying phage particles in mice and non-human primates elicit a systemic and specific humoral response. CONCLUSIONS: This broad methodology blueprint represents a robust and versatile platform tool enabling new ligand-receptor discovery with many potential translational applications. FUNDING: Cancer Center Support Grants to the University of Texas M.D. Anderson Cancer Center (CA016672), University of New Mexico Comprehensive Cancer Center (CA118100), Rutgers Cancer Institute of New Jersey (CA072720), research awards from the Gillson Longenbaugh Foundation, and National Institutes of Health (NIH) grant no. 1R01CA226537.
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Bacteriófagos , Pulmón , Animales , Bacteriófagos/genética , Proteínas Portadoras/metabolismo , Ligandos , Pulmón/metabolismo , Ratones , Primates/metabolismo , Estados Unidos , VacunaciónRESUMEN
[This corrects the article DOI: 10.18632/oncotarget.27589.].
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The androgen receptor (AR) is a major driver of prostate cancer development and progression. Men who develop advanced prostate cancer often have long-term cancer control when treated with androgen-deprivation therapies (ADT). Still, their disease inevitably becomes resistant to ADT and progresses to castration-resistant prostate cancer (CRPC). ADT involves potent competitive AR antagonists and androgen synthesis inhibitors. Resistance to these types of treatments emerges, primarily through the maintenance of AR signaling by ligand-independent activation mechanisms. There is a need to find better ways to block AR to overcome CRPC. In the findings reported here, we demonstrate that the nuclear scaffold protein, nucleolin (NCL), suppresses the expression of AR. NCL binds to a G-rich region in the AR promoter that forms a G-quadruplex (G4) structure. Binding of NCL to this G4-element is required for NCL to suppress AR expression, specifically in AR-expressing tumor cells. Compounds that stabilize G4 structures require NCL to associate with the G4-element of the AR promoter in order to decrease AR expression. A newly discovered G4 compound that suppresses AR expression demonstrates selective killing of AR-expressing tumor cells, including CRPC lines. Our findings raise the significant possibility that G4-stabilizing drugs can be used to increase NCL transcriptional repressor activity to block AR expression in prostate cancer. Our studies contribute to a clearer understanding of the mechanisms that control AR expression, which could be exploited to overcome CRPC.
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The androgen receptor (AR) is the major driver of prostate cancer growth and survival. However, almost all patients relapse with castration-resistant disease (CRPC) when treated with anti-androgen therapy. In CRPC, AR is often aberrantly activated independent of androgen. Targeting survival pathways downstream of AR could be a viable strategy to overcome CRPC. Surprisingly, little is known about how AR drives prostate cancer survival. Furthermore, CRPC tumors in which Pten is lost are also resistant to eradication by PI3K inhibitors. We sought to identify the mechanism by which AR drives tumor survival in CRPC to identify ways to overcome resistance to PI3K inhibition. We found that integrins α6ß1 and Bnip3 are selectively elevated in CRPC downstream of AR. While integrin α6 promotes survival and is a direct transcriptional target of AR, the ability of AR to induce Bnip3 is dependent on adhesion to laminin and integrin α6ß1-dependent nuclear translocation of HIF1α. Integrins α6ß1 and Bnip3 were found to promote survival of CRPC cells selectively on laminin through the induction of autophagy and mitophagy. Furthermore, blocking Bnip3 or integrin α6ß1 restored sensitivity to PI3K inhibitors in Pten-negative CRPC. We identified an AR driven pathway that cooperates with laminin and hypoxia to drive resistance to PI3K inhibitors. These findings can help explain in part why PI3K inhibitors have failed in clinical trials to overcome AR-dependent CRPC.
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Integrina alfa6beta1/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Proteínas Proto-Oncogénicas/metabolismo , Receptores Androgénicos/genética , Animales , Humanos , Masculino , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptores Androgénicos/metabolismo , Análisis de SupervivenciaRESUMEN
Prostate cancer metastases primarily localize in the bone where they induce a unique osteoblastic response. Elevated Notch activity is associated with high-grade disease and metastasis. To address how Notch affects prostate cancer bone lesions, we manipulated Notch expression in mouse tibia xenografts and monitored tumor growth, lesion phenotype, and the bone microenvironment. Prostate cancer cell lines that induce mixed osteoblastic lesions in bone expressed 5-6 times more Notch3, than tumor cells that produce osteolytic lesions. Expression of active Notch3 (NICD3) in osteolytic tumors reduced osteolytic lesion area and enhanced osteoblastogenesis, while loss of Notch3 in osteoblastic tumors enhanced osteolytic lesion area and decreased osteoblastogensis. This was accompanied by a respective decrease and increase in the number of active osteoclasts and osteoblasts at the tumor-bone interface, without any effect on tumor proliferation. Conditioned medium from NICD3-expressing cells enhanced osteoblast differentiation and proliferation in vitro, while simultaneously inhibiting osteoclastogenesis. MMP-3 was specifically elevated and secreted by NICD3-expressing tumors, and inhibition of MMP-3 rescued the NICD3-induced osteoblastic phenotypes. Clinical osteoblastic bone metastasis samples had higher levels of Notch3 and MMP-3 compared with patient matched visceral metastases or osteolytic metastasis samples. We identified a Notch3-MMP-3 axis in human prostate cancer bone metastases that contributes to osteoblastic lesion formation by blocking osteoclast differentiation, while also contributing to osteoblastogenesis. These studies define a new role for Notch3 in manipulating the tumor microenvironment in bone metastases.
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Neoplasias Óseas/genética , Metaloproteinasa 3 de la Matriz/genética , Neoplasias de la Próstata/genética , Receptor Notch3/genética , Animales , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Diferenciación Celular/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/genética , Xenoinjertos , Humanos , Masculino , Ratones , Metástasis de la Neoplasia , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoclastos/metabolismo , Osteoclastos/patología , Osteogénesis/genética , Neoplasias de la Próstata/patología , Transducción de Señal/genéticaRESUMEN
Bone-metastatic castration-resistant prostate cancer (CRPC) is lethal due to inherent resistance to androgen deprivation therapy, chemotherapy, and targeted therapies. Despite the fact that a majority of CRPC patients (approximately 70%) harbor a constitutively active PI3K survival pathway, targeting the PI3K/mTOR pathway has failed to increase overall survival in clinical trials. Here, we identified two separate and independent survival pathways induced by the bone tumor microenvironment that are hyperactivated in CRPC and confer resistance to PI3K inhibitors. The first pathway involves integrin α6ß1-mediated adhesion to laminin and the second involves hypoxia-induced expression of PIM kinases. In vitro and in vivo models demonstrate that these pathways transduce parallel but independent signals that promote survival by reducing oxidative stress and preventing cell death. We further demonstrate that both pathways drive resistance to PI3K inhibitors in PTEN-negative tumors. These results provide preclinical evidence that combined inhibition of integrin α6ß1 and PIM kinase in CRPC is required to overcome tumor microenvironment-mediated resistance to PI3K inhibitors in PTEN-negative tumors within the hypoxic and laminin-rich bone microenvironment.
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The tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post-ER. Moreover, CD82 is essential for TLR9-dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9-dependent NF-κB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.-Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E.-M., Becker, C. E., Dagher, Z., Kim, Y.-M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG-dependent TLR9 signaling.
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Núcleo Celular/inmunología , Proteína Kangai-1/inmunología , Macrófagos/inmunología , Oligodesoxirribonucleótidos/farmacología , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 9/inmunología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/inmunología , Animales , Núcleo Celular/genética , Citocinas/genética , Citocinas/inmunología , Retículo Endoplásmico/genética , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/patología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Proteína Kangai-1/genética , Macrófagos/patología , Ratones , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/inmunología , Células RAW 264.7 , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptor Toll-Like 9/genéticaRESUMEN
Human prostate cancer confined to the gland is indolent (low-risk), but tumors outside the capsule are aggressive (high-risk). Extracapsular extension requires invasion within and through a smooth muscle-structured environment. Because integrins respond to biomechanical cues, we used a gene editing approach to determine if a specific region of laminin-binding α6ß1 integrin was required for smooth muscle invasion both in vitro and in vivo. Human tissue specimens showed prostate cancer invasion through smooth muscle and tumor coexpression of α6 integrin and E-cadherin in a cell-cell location and α6 integrin in a cell-extracellular matrix (ECM) distribution. Prostate cancer cells expressing α6 integrin (DU145 α6WT) produced a 3D invasive network on laminin-containing Matrigel and invaded into smooth muscle both in vitro and in vivo. In contrast, cells without α6 integrin (DU145 α6KO) and cells expressing an integrin mutant (DU145 α6AA) did not produce invasive networks, could not invade muscle both in vitro and in vivo, and surprisingly formed 3D cohesive clusters. Using electric cell-substrate impedance testing, cohesive clusters had up to a 30-fold increase in normalized resistance at 400 Hz (cell-cell impedance) as compared with the DU145 α6WT cells. In contrast, measurements at 40,000 Hz (cell-ECM coverage) showed that DU145 α6AA cells were two-fold decreased in normalized resistance and were defective in restoring resistance after a 1 µmol/L S1P challenge as compared with the DU145 α6WT cells. The results suggest that gene editing of a specific α6 integrin extracellular region, not required for normal tissue function, can generate a new biophysical cancer phenotype unable to invade the muscle, presenting a new therapeutic strategy for metastasis prevention in prostate cancer. SIGNIFICANCE: This study shows an innovative strategy to block prostate cancer metastasis and invasion in the muscle through gene editing of a specific α6 integrin extracellular region.
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Comunicación Celular , Edición Génica , Integrina alfa6/genética , Neoplasias de los Músculos/patología , Neoplasias de la Próstata/patología , Animales , Apoptosis , Adhesión Celular , Movimiento Celular , Proliferación Celular , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Femenino , Humanos , Integrina alfa6/química , Integrina alfa6/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias de los Músculos/genética , Neoplasias de los Músculos/metabolismo , Invasividad Neoplásica , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tetraspanins are a family of proteins possessing four transmembrane domains that help in lateral organization of plasma membrane proteins. These proteins interact with each other as well as other receptors and signaling proteins, resulting in functional complexes called "tetraspanin microdomains." Tetraspanins, including CD82, play an essential role in the pathogenesis of fungal infections. Dectin-1, a receptor for the fungal cell wall carbohydrate ß-1,3-glucan, is vital to host defense against fungal infections. The current study identifies a novel association between tetraspanin CD82 and Dectin-1 on the plasma membrane of Candida albicans-containing phagosomes independent of phagocytic ability. Deletion of CD82 in mice resulted in diminished fungicidal activity, increased C. albicans viability within macrophages, and decreased cytokine production (TNF-α, IL-1ß) at both mRNA and protein level in macrophages. Additionally, CD82 organized Dectin-1 clustering in the phagocytic cup. Deletion of CD82 modulates Dectin-1 signaling, resulting in a reduction of Src and Syk phosphorylation and reactive oxygen species production. CD82 knockout mice were more susceptible to C. albicans as compared with wild-type mice. Furthermore, patient C. albicans-induced cytokine production was influenced by two human CD82 single nucleotide polymorphisms, whereas an additional CD82 single nucleotide polymorphism increased the risk for candidemia independent of cytokine production. Together, these data demonstrate that CD82 organizes the proper assembly of Dectin-1 signaling machinery in response to C. albicans.
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Candida albicans/fisiología , Candidiasis/metabolismo , Membrana Celular/metabolismo , Proteína Kangai-1/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/inmunología , Fagosomas/metabolismo , Animales , Candidiasis/inmunología , Línea Celular , Predisposición Genética a la Enfermedad , Humanos , Inmunidad Celular , Interleucina-1beta/metabolismo , Proteína Kangai-1/genética , Lectinas Tipo C/genética , Microdominios de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Polimorfismo de Nucleótido Simple , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We used a myeloid-specific Cre to conditionally delete CD82 in mouse osteoclasts and their precursors. In contrast to global loss of CD82 (gKO), conditional loss of CD82 (cKO) in osteoclasts does not affect cortical bone, osteoblasts, or adipocytes. CD82 loss results in greater trabecular volume and trabecular number but reduced trabecular space in 6-month old male mice. Though this trend is present in females it did not reach significance; whereas there was an increase in osteoclast numbers and eroded surface area only in female cKO mice. In vitro, there is an increase in osteoclast fusion and defects in actin assembly in both gKO and cKO mice, irrespective of sex. This is accompanied by altered osteoclast morphology and decreased release of CTX in vitro. Integrin αvß3 expression is reduced, while integrin ß1 is increased. Signaling to Src, Syk, and Vav are also compromised. We further discovered that expression of Clec2 and its ligand, Podoplanin, molecules that also signal to Syk and Vav, are increased in differentiated osteoclasts. Loss of CD82 reduces their expression. Thus, CD82 is required for correct assembly of the cytoskeleton and to limit osteoclast fusion, both needed for normal osteoclast function.
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The laminin-binding integrins, α3ß1 and α6ß1, are needed for tumor metastasis and their surface expression is regulated by endocytic recycling. ß1 integrins share the Rab11 recycling machinery, but the trafficking of α3ß1 and α6ß1 are distinct by an unknown mechanism. Using a mouse PDX tumor model containing human metastatic prostate cancer, Rab11 family interacting protein 5 (Rab11-FIP5) was identified as a lead candidate for α6ß1 trafficking. Rab11-FIP5 and its membrane-binding domain were required for α6ß1 recycling, without affecting the other laminin-binding integrin (i.e., α3ß1) or unrelated membrane receptors like CD44, transferrin receptor, or E-cadherin. Depletion of Rab11-FIP5 resulted in the intracellular accumulation of α6ß1 in the Rab11 recycling compartment, loss of cell migration on laminin, and an unexpected loss of α6ß1 recycling in cell-cell locations. Taken together, these data demonstrate that α6ß1 is distinct from α3ß1 via Rab11-FIP5 recycling and recycles in an unexpected cell-cell location.Implications: Rab11-FIP5-dependent α6ß1 integrin recycling may be selectively targeted to limit migration of prostate cancer cells into laminin-rich tissues. Mol Cancer Res; 16(8); 1319-31. ©2018 AACR.
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Integrina alfa5beta1/metabolismo , Neoplasias de la Próstata/genética , Proteínas de Unión al GTP rab/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/metabolismoRESUMEN
CD82 is a widely expressed member of the tetraspanin family of transmembrane proteins known to control cell signaling, adhesion, and migration. Tetraspanin CD82 is induced over 9-fold during osteoclast differentiation in vitro; however, its role in bone homeostasis is unknown. A globally deleted CD82 mouse model was used to assess the bone phenotype. Based on microCT and 4-point bending tests, CD82-deficient bones are smaller in diameter and weaker, but display no changes in bone density. Histomorphometry shows a decrease in size, erosion perimeter, and number of osteoclasts in situ, with a corresponding increase in trabecular surface area, specifically in male mice. Male-specific alterations are observed in trabecular structure by microCT and in vitro differentiated osteoclasts are morphologically abnormal. Histomorphometry did not reveal a significant reduction in osteoblast number; however, dynamic labeling reveals a significant decrease in bone growth. Consistent with defects in OB function, OB differentiation and mineralization are defective in vitro, whereas adipogenesis is enhanced. There is a corresponding increase in bone marrow adipocytes in situ. Thus, combined defects in both osteoclasts and osteoblasts can account for the observed bone phenotypes, and suggests a role for CD82 in both bone mesenchyme and myeloid cells.
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Adipogénesis/fisiología , Desarrollo Óseo/fisiología , Médula Ósea/metabolismo , Huesos/metabolismo , Proteína Kangai-1/deficiencia , Animales , Diferenciación Celular/fisiología , Femenino , Masculino , Ratones , Ratones Noqueados , Osteoblastos/metabolismo , Osteoclastos/metabolismoRESUMEN
Many pathways dysregulated in prostate cancer are also involved in epithelial differentiation. To better understand prostate tumor initiation, we sought to investigate specific genes and mechanisms required for normal basal to luminal cell differentiation. Utilizing human prostate basal epithelial cells and an in vitro differentiation model, we tested the hypothesis that regulation of NOTCH3 by the p38 MAPK family (hereafter p38-MAPK), via MYC, is required for luminal differentiation. Inhibition (SB202190 and BIRB796) or knockdown of p38α (also known as MAPK14) and/or p38δ (also known as MAPK13) prevented proper differentiation. Additionally, treatment with a γ-secretase inhibitor (RO4929097) or knockdown of NOTCH1 and/or NOTCH3 greatly impaired differentiation and caused luminal cell death. Constitutive p38-MAPK activation through MKK6(CA) increased NOTCH3 (but not NOTCH1) mRNA and protein levels, which was diminished upon MYC inhibition (10058-F4 and JQ1) or knockdown. Furthermore, we validated two NOTCH3 enhancer elements through a combination of enhancer (e)RNA detection (BruUV-seq) and luciferase reporter assays. Finally, we found that the NOTCH3 mRNA half-life increased during differentiation or upon acute p38-MAPK activation. These results reveal a new connection between p38-MAPK, MYC and NOTCH signaling, demonstrate two mechanisms of NOTCH3 regulation and provide evidence for NOTCH3 involvement in prostate luminal cell differentiation.
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Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas c-myc/genética , Receptor Notch3/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Diferenciación Celular , Línea Celular Transformada , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Genes Reporteros , Células HEK293 , Semivida , Humanos , Imidazoles/farmacología , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Naftalenos/farmacología , Cultivo Primario de Células , Próstata/citología , Próstata/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Pirazoles/farmacología , Piridinas/farmacología , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor Notch3/antagonistas & inhibidores , Receptor Notch3/metabolismo , Transducción de Señal , Tiazoles/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Short hairpin RNA (shRNA) is an established and effective tool for stable knock down of gene expression. Lentiviral vectors can be used to deliver shRNAs, thereby providing the ability to infect most mammalian cell types with high efficiency, regardless of proliferation state. Furthermore, the use of inducible promoters to drive shRNA expression allows for more thorough investigations into the specific timing of gene function in a variety of cellular processes. Moreover, inducible knockdown allows the investigation of genes that would be lethal or otherwise poorly tolerated if constitutively knocked down. Lentiviral inducible shRNA vectors are readily available, but unfortunately the process of cloning, screening, and testing shRNAs can be time-consuming and expensive. Therefore, we sought to refine a popular vector (Tet-pLKO-Puro) and streamline the cloning process with efficient protocols so that researchers can more efficiently utilize this powerful tool. METHODS: First, we modified the Tet-pLKO-Puro vector to make it easy ("EZ") for molecular cloning (EZ-Tet-pLKO-Puro). Our primary modification was to shrink the stuffer region, which allows vector purification via polyethylene glycol precipitation thereby avoiding the need to purify DNA through agarose. In addition, we generated EZ-Tet-pLKO vectors with hygromycin or blasticidin resistance to provide greater flexibility in cell line engineering. Furthermore, we provide a detailed guide for utilizing these vectors, including shRNA design strategy and simplified screening methods. RESULTS: Notably, we emphasize the importance of loop sequence design and demonstrate that the addition of a single mismatch in the loop stem can greatly improve shRNA efficiency. Lastly, we display the robustness of the system with a doxycycline titration and recovery time course and provide a cost/benefit analysis comparing our system with purchasing pre-designed shRNA vectors. CONCLUSIONS: Our aim was twofold: first, to take a very useful shRNA vector and make it more amenable for molecular cloning and, secondly, to provide a streamlined protocol and rationale for cost-effective design, cloning, and screening of shRNAs. With this knowledge, anyone can take advantage of this powerful tool to inducibly knockdown any gene of their choosing.
Asunto(s)
Clonación Molecular/métodos , Técnicas de Silenciamiento del Gen/métodos , Vectores Genéticos/genética , Lentivirus/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Doxiciclina/farmacología , Diseño de Fármacos , Vectores Genéticos/química , Vectores Genéticos/efectos de los fármacos , Transfección/métodosRESUMEN
BACKGROUND: How prostate epithelial cells differentiate and how dysregulation of this process contributes to prostate tumorigenesis remain unclear. We recently identified a Myc target and chromatin reader protein, ING4, as a necessary component of human prostate luminal epithelial cell differentiation, which is often lost in primary prostate tumors. Furthermore, loss of ING4 in the context of oncogenic mutations is required for prostate tumorigenesis. Identifying the gene targets of ING4 can provide insight into how its loss disrupts differentiation and leads to prostate cancer. METHODS: Using a combination of RNA-Seq, a best candidate approach, and chromatin immunoprecipitation (ChIP), we identified Miz1 as a new ING4 target. ING4 or Miz1 overexpression, shRNA knock-down, and a Myc-binding mutant were used in a human in vitro differentiation assay to assess the role of Miz1 in luminal cell differentiation. RESULTS: ING4 directly binds the Miz1 promoter and is required to induce Miz1 mRNA and protein expression during luminal cell differentiation. Miz1 mRNA was not induced in shING4 expressing cells or tumorigenic cells in which ING4 is not expressed. Miz1 dependency on ING4 was unique to differentiating luminal cells; Miz1 mRNA expression was not induced in basal cells. Although Miz1 is a direct target of ING4, and its overexpression can drive luminal cell differentiation, Miz1 was not required for differentiation. CONCLUSIONS: Miz1 is a newly identified ING4-induced target gene which can drive prostate luminal epithelial cell differentiation although it is not absolutely required. Prostate 77:49-59, 2017. © 2016 Wiley Periodicals, Inc.