RESUMO
Although neutrophils have been linked to the formation of the pre-metastatic niche, the mechanism of their migration to distant, uninvolved tissues has remained elusive. We report that bone marrow neutrophils from mice with early-stage cancer exhibited much more spontaneous migration than that of control neutrophils from tumor-free mice. These cells lacked immunosuppressive activity but had elevated rates of oxidative phosphorylation and glycolysis, and increased production of ATP, relative to that of control neutrophils. Their enhanced spontaneous migration was mediated by autocrine ATP signaling through purinergic receptors. In ectopic tumor models and late stages of cancer, bone marrow neutrophils demonstrated potent immunosuppressive activity. However, these cells had metabolic and migratory activity indistinguishable from that of control neutrophils. A similar pattern of migration was observed for neutrophils and polymorphonuclear myeloid-derived suppressor cells from patients with cancer. These results elucidate the dynamic changes that neutrophils undergo in cancer and demonstrate the mechanism of neutrophils' contribution to early tumor dissemination.
Assuntos
Quimiotaxia de Leucócito/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Idoso , Animais , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-IdadeRESUMO
Recruitment of monocytic myeloid-derived suppressor cells (MDSCs) and differentiation of tumor-associated macrophages (TAMs) are the major factors contributing to tumor progression and metastasis. We demonstrated that differentiation of TAMs in tumor site from monocytic precursors was controlled by downregulation of the activity of the transcription factor STAT3. Decreased STAT3 activity was caused by hypoxia and affected all myeloid cells but was not observed in tumor cells. Upregulation of CD45 tyrosine phosphatase activity in MDSCs exposed to hypoxia in tumor site was responsible for downregulation of STAT3. This effect was mediated by the disruption of CD45 protein dimerization regulated by sialic acid. Thus, STAT3 has a unique function in the tumor environment in controlling the differentiation of MDSC into TAM, and its regulatory pathway could be a potential target for therapy.
Assuntos
Hipóxia/imunologia , Antígenos Comuns de Leucócito/metabolismo , Macrófagos/imunologia , Monoéster Fosfórico Hidrolases/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Diferenciação Celular , Movimento Celular , Células Cultivadas , Dimerização , Feminino , Antígenos Comuns de Leucócito/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/imunologia , Monoéster Fosfórico Hidrolases/genética , Fator de Transcrição STAT3/genética , Ácidos Siálicos/metabolismo , Microambiente TumoralRESUMO
Cancer metabolism, including in mitochondria, is a disease hallmark and therapeutic target, but its regulation is poorly understood. Here, we show that many human tumors have heterogeneous and often reduced levels of Mic60, or Mitofilin, an essential scaffold of mitochondrial structure. Despite a catastrophic collapse of mitochondrial integrity, loss of bioenergetics, and oxidative damage, tumors with Mic60 depletion slow down cell proliferation, evade cell death, and activate a nuclear gene expression program of innate immunity and cytokine/chemokine signaling. In turn, this induces epithelial-mesenchymal transition (EMT), activates tumor cell movements through exaggerated mitochondrial dynamics, and promotes metastatic dissemination in vivo. In a small-molecule drug screen, compensatory activation of stress response (GCN2) and survival (Akt) signaling maintains the viability of Mic60-low tumors and provides a selective therapeutic vulnerability. These data demonstrate that acutely damaged, "ghost" mitochondria drive tumor progression and expose an actionable therapeutic target in metastasis-prone cancers.
Assuntos
Mitocôndrias/fisiologia , Metástase Neoplásica/fisiopatologia , Neoplasias/genética , Morte Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Transição Epitelial-Mesenquimal , Humanos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/metabolismo , Invasividade Neoplásica/genética , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Processos Neoplásicos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio , Transdução de SinaisRESUMO
Mitochondria are signaling organelles implicated in cancer, but the mechanisms are elusive. Here, we show that Parkin, an E3 ubiquitination (Ub) ligase altered in Parkinson's disease, forms a complex with the regulator of cell motility, Kindlin-2 (K2), at mitochondria of tumor cells. In turn, Parkin ubiquitinates Lys581 and Lys582 using Lys48 linkages, resulting in proteasomal degradation of K2 and shortened half-life from â¼5 h to â¼1.5 h. Loss of K2 inhibits focal adhesion turnover and ß1 integrin activation, impairs membrane lamellipodia size and frequency, and inhibits mitochondrial dynamics, altogether suppressing tumor cell-extracellular matrix interactions, migration, and invasion. Conversely, Parkin does not affect tumor cell proliferation, cell cycle transitions, or apoptosis. Expression of a Parkin Ub-resistant K2 Lys581Ala/Lys582Ala double mutant is sufficient to restore membrane lamellipodia dynamics, correct mitochondrial fusion/fission, and preserve single-cell migration and invasion. In a 3D model of mammary gland developmental morphogenesis, impaired K2 Ub drives multiple oncogenic traits of EMT, increased cell proliferation, reduced apoptosis, and disrupted basal-apical polarity. Therefore, deregulated K2 is a potent oncogene, and its Ub by Parkin enables mitochondria-associated metastasis suppression.
Assuntos
Proteínas de Membrana , Ubiquitina-Proteína Ligases , Movimento Celular , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , HumanosRESUMO
We have previously shown that the αvß6 integrin plays a key role in promoting prostate cancer (PrCa) and it can be transferred to recipient cells via small extracellular vesicles (sEVs). Furthermore, we have reported in a proteomic analysis that αvß6 integrin down-regulation increases the expression of IFIT3 (interferon induced protein with tetratricopeptide repeats 3) in PrCa cells and their derived sEVs. IFIT3 is a protein well known for being an antiviral effector, but recently its role in cancer has also been elucidated. To study the relationship between IFIT3 and STAT1 (signal transducer and activator of transcription 1), an upstream regulator of IFIT3, in PrCa cells and their released sEVs, we used CRISPR/Cas9 techniques to down-regulate the expression of the ß6 integrin subunit, IFIT3 or STAT1. Our results show that IFIT3 and STAT1 are highly expressed in PrCa cells devoid of the ß6 integrin subunit. However, IFIT3 but not STAT1, is present in sEVs derived from PrCa cells lacking the ß6 integrin subunit. We demonstrate that loss of IFIT3 generates sEVs enriched in STAT1 but reduces the levels of STAT1 in the cells. As expected, IFIT3 is not detectable in STAT1 negative cells or sEVs. We thus propose that the observed STAT1 enrichment in sEVs is a compensatory mechanism for the loss of IFIT3. Overall, these results provide new insights into the intrinsic role of IFIT3 as a regulator of STAT1 expression in sEVs and in intercellular communication in PrCa.
Assuntos
Vesículas Extracelulares/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias da Próstata/metabolismo , Fator de Transcrição STAT1/metabolismo , Linhagem Celular Tumoral , Humanos , MasculinoRESUMO
The role of mitochondria in cancer continues to be debated, and whether exploitation of mitochondrial functions is a general hallmark of malignancy or a tumor- or context-specific response is still unknown. Using a variety of cancer cell lines and several technical approaches, including siRNA-mediated gene silencing, ChIP assays, global metabolomics and focused metabolite analyses, bioenergetics, and cell viability assays, we show that two oncogenic Myc proteins, c-Myc and N-Myc, transcriptionally control the expression of the mitochondrial chaperone TNFR-associated protein-1 (TRAP1) in cancer. In turn, this Myc-mediated regulation preserved the folding and function of mitochondrial oxidative phosphorylation (OXPHOS) complex II and IV subunits, dampened reactive oxygen species production, and enabled oxidative bioenergetics in tumor cells. Of note, we found that genetic or pharmacological targeting of this pathway shuts off tumor cell motility and invasion, kills Myc-expressing cells in a TRAP1-dependent manner, and suppresses primary and metastatic tumor growth in vivo We conclude that exploitation of mitochondrial functions is a general trait of tumorigenesis and that this reliance of cancer cells on mitochondrial OXPHOS pathways could offer an actionable therapeutic target in the clinic.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular/efeitos dos fármacos , Guanidinas/farmacologia , Guanidinas/uso terapêutico , Proteínas de Choque Térmico HSP90/genética , Humanos , Lactamas Macrocíclicas/farmacologia , Lactamas Macrocíclicas/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Nus , Fosforilação Oxidativa , Regiões Promotoras Genéticas , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transcrição GênicaRESUMO
Mitochondria must buffer the risk of proteotoxic stress to preserve bioenergetics, but the role of these mechanisms in disease is poorly understood. Using a proteomics screen, we now show that the mitochondrial unfoldase-peptidase complex ClpXP associates with the oncoprotein survivin and the respiratory chain Complex II subunit succinate dehydrogenase B (SDHB) in mitochondria of tumor cells. Knockdown of ClpXP subunits ClpP or ClpX induces the accumulation of misfolded SDHB, impairing oxidative phosphorylation and ATP production while activating "stress" signals of 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and autophagy. Deregulated mitochondrial respiration induced by ClpXP targeting causes oxidative stress, which in turn reduces tumor cell proliferation, suppresses cell motility, and abolishes metastatic dissemination in vivo. ClpP is universally overexpressed in primary and metastatic human cancer, correlating with shortened patient survival. Therefore, tumors exploit ClpXP-directed proteostasis to maintain mitochondrial bioenergetics, buffer oxidative stress, and enable metastatic competence. This pathway may provide a "drugable" therapeutic target in cancer.
Assuntos
Endopeptidase Clp/metabolismo , Metabolismo Energético , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neoplasias/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Endopeptidase Clp/genética , Feminino , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Pessoa de Meia-Idade , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Metástase Neoplásica , Neoplasias/genética , Neoplasias/patologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteômica/métodos , Interferência de RNA , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Survivina , Transplante HeterólogoRESUMO
Molecular therapies are hallmarks of "personalized" medicine, but how tumors adapt to these agents is not well-understood. Here we show that small-molecule inhibitors of phosphatidylinositol 3-kinase (PI3K) currently in the clinic induce global transcriptional reprogramming in tumors, with activation of growth factor receptors, (re)phosphorylation of Akt and mammalian target of rapamycin (mTOR), and increased tumor cell motility and invasion. This response involves redistribution of energetically active mitochondria to the cortical cytoskeleton, where they support membrane dynamics, turnover of focal adhesion complexes, and random cell motility. Blocking oxidative phosphorylation prevents adaptive mitochondrial trafficking, impairs membrane dynamics, and suppresses tumor cell invasion. Therefore, "spatiotemporal" mitochondrial respiration adaptively induced by PI3K therapy fuels tumor cell invasion, and may provide an important antimetastatic target.
Assuntos
Inibidores Enzimáticos/farmacologia , Mitocôndrias/efeitos dos fármacos , Invasividade Neoplásica , Inibidores de Fosfoinositídeo-3 Quinase , Transporte Biológico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Citoesqueleto/metabolismo , Metabolismo Energético , Humanos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
Protein homeostasis, or proteostasis, is required for mitochondrial function, but its role in cancer is controversial. Here we show that transgenic mice expressing the mitochondrial chaperone TNFR-associated protein 1 (TRAP1) in the prostate develop epithelial hyperplasia and cellular atypia. When examined on a Pten+/- background, a common alteration in human prostate cancer, TRAP1 transgenic mice showed accelerated incidence of invasive prostatic adenocarcinoma, characterized by increased cell proliferation and reduced apoptosis, in situ Conversely, homozygous deletion of TRAP1 delays prostatic tumorigenesis in Pten+/- mice without affecting hyperplasia or prostatic intraepithelial neoplasia. Global profiling of Pten+/--TRAP1 transgenic mice by RNA sequencing and reverse phase protein array reveals modulation of oncogenic networks of cell proliferation, apoptosis, cell motility, and DNA damage. Mechanistically, reconstitution of Pten+/- prostatic epithelial cells with TRAP1 increases cell proliferation, reduces apoptosis, and promotes cell invasion without changes in mitochondrial bioenergetics. Therefore, TRAP1 is a driver of prostate cancer in vivo and an "actionable" therapeutic target.
Assuntos
Apoptose , Proliferação de Células , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Neoplasias/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP90/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologiaRESUMO
It is well known that Src tyrosine kinase, insulin-like growth factor 1 receptor (IGF-IR), and focal adhesion kinase (FAK) play important roles in prostate cancer (PrCa) development and progression. Src, which signals through FAK in response to integrin activation, has been implicated in many aspects of tumor biology, such as cell proliferation, metastasis, and angiogenesis. Furthermore, Src signaling is known to crosstalk with IGF-IR, which also promotes angiogenesis. In this study, we demonstrate that c-Src, IGF-IR, and FAK are packaged into exosomes (Exo), c-Src in particular being highly enriched in Exo from the androgen receptor (AR)-positive cell line C4-2B and AR-negative cell lines PC3 and DU145. Furthermore, we show that the active phosphorylated form of Src (SrcpY416 ) is co-expressed in Exo with phosphorylated FAK (FAKpY861 ), a known target site of Src, which enhances proliferation and migration. We further demonstrate for the first time exosomal enrichment of G-protein-coupled receptor kinase (GRK) 5 and GRK6, both of which regulate Src and IGF-IR signaling and have been implicated in cancer. Finally, SrcpY416 and c-Src are both expressed in Exo isolated from the plasma of prostate tumor-bearing TRAMP mice, and those same mice have higher levels of exosomal c-Src than their wild-type counterparts. In summary, we provide new evidence that active signaling molecules relevant to PrCa are enriched in Exo, and this suggests that the Src signaling network may provide useful biomarkers detectable by liquid biopsy, and may contribute to PrCa progression via Exo. J. Cell. Biochem. 118: 66-73, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Exossomos/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Quinases de Receptores Acoplados a Proteína G/metabolismo , Neoplasias da Próstata/metabolismo , Receptores de Somatomedina/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismo , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Exossomos/genética , Exossomos/patologia , Quinase 1 de Adesão Focal/genética , Quinase 5 de Receptor Acoplado a Proteína G/genética , Quinases de Receptores Acoplados a Proteína G/genética , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptor IGF Tipo 1 , Receptores de Somatomedina/genética , Quinases da Família src/genéticaRESUMO
Exosomes, cell-derived vesicles of endosomal origin, are continuously released in the extracellular environment and play a key role in intercellular crosstalk. In this study, we have investigated whether transfer of integrins through exosomes between prostate cancer (PrCa) cells occurs and whether transferred integrins promote cell adhesion and migration. Among others, we have focused on the αvß6 integrin, which is not detectable in normal human prostate but is highly expressed in human primary PrCa as well as murine PrCa in Pten(pc-/-) mice. After confirming the fidelity of the exosome preparations by electron microscopy, density gradient, and immunoblotting, we determined that the αvß6 integrin is actively packaged into exosomes isolated from PC3 and RWPE PrCa cell lines. We also demonstrate that αvß6 is efficiently transferred via exosomes from a donor cell to an αvß6-negative recipient cell and localizes to the cell surface. De novo αvß6 expression in an αvß6-negative recipient cell is not a result of a change in mRNA levels but is a consequence of exosome-mediated transfer of this integrin between different PrCa cells. Recipient cells incubated with exosomes containing αvß6 migrate on an αvß6 specific substrate, latency-associated peptide-TGFß, to a greater extent than cells treated with exosomes in which αvß6 is stably or transiently down-regulated by shRNA or siRNA, respectively. Overall, this study shows that exosomes from PrCa cells may contribute to a horizontal propagation of integrin-associated phenotypes, which would promote cell migration, and consequently, metastasis in a paracrine fashion.
Assuntos
Antígenos de Neoplasias/biossíntese , Exossomos/química , Expressão Gênica , Integrinas/biossíntese , Transfecção/métodos , Animais , Antígenos de Neoplasias/genética , Linhagem Celular , Movimento Celular/genética , Humanos , Integrinas/genética , Masculino , Camundongos , Camundongos Knockout , Metástase Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Comunicação Parácrina/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Active Stat5a/b predicts early recurrence and disease-specific death in prostate cancer (PC), which both typically are caused by development of metastatic disease. Herein, we demonstrate that Stat5a/b induces epithelial-to-mesenchymal transition (EMT) of PC cells, as shown by Stat5a/b regulation of EMT marker expression (Twist1, E-cadherin, N-cadherin, vimentin, and fibronectin) in PC cell lines, xenograft tumors in vivo, and patient-derived PCs ex vivo using organ explant cultures. Jak2-Stat5a/b signaling induced functional end points of EMT as well, indicated by disruption of epithelial cell monolayers and increased migration and adhesion of PC cells to fibronectin. Knockdown of Twist1 suppressed Jak2-Stat5a/b-induced EMT properties of PC cells, which were rescued by re-introduction of Twist1, indicating that Twist1 mediates Stat5a/b-induced EMT in PC cells. While promoting EMT, Jak2-Stat5a/b signaling induced stem-like properties in PC cells, such as sphere formation and expression of cancer stem cell markers, including BMI1. Mechanistically, both Twist1 and BMI1 were critical for Stat5a/b induction of stem-like features, because genetic knockdown of Twist1 suppressed Stat5a/b-induced BMI1 expression and sphere formation in stem cell culture conditions, which were rescued by re-introduction of BMI1. By using human prolactin knock-in mice, we demonstrate that prolactin-Stat5a/b signaling promoted metastases formation of PC cells in vivo. In conclusion, our data support the concept that Jak2-Stat5a/b signaling promotes metastatic progression of PC by inducing EMT and stem cell properties in PC cells.
Assuntos
Transição Epitelial-Mesenquimal , Janus Quinase 2/metabolismo , Neoplasias da Próstata/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Animais , Caderinas/metabolismo , Humanos , Masculino , Camundongos , Células-Tronco Neoplásicas/patologia , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/patologia , Recidiva , Transdução de Sinais/fisiologia , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Transforming growth factor (TGF) ß1 activity depends on a complex signalling cascade that controls expression of several genes. Among others, TGFß1 regulates expression of matrix metalloproteinases (MMPs) through activation of Smads. In the present study, we demonstrate for the first time that the αvß6 integrin interacts with TGFß receptor II (TßRII) through the ß6 cytoplasmic domain and promotes Smad3 activation in prostate cancer (PrCa) cells. Another related αv integrin, αvß5, as well as the αvß6/3 integrin, which contains a chimeric form of ß6 with a ß3 cytoplasmic domain, do not associate with TßRII and fail to show similar responses. We provide evidence that αvß6 is required for up-regulation of MMP2 by TGFß1 through a Smad3-mediated transcriptional programme in PrCa cells. The functional relevance of these results is underscored by the finding that αvß6 modulates cell migration in an MMP2-dependent manner on an αvß6-specific ligand, latency-associated peptide (LAP)-TGFß. Overall, these mechanistic studies establish that expression of a single integrin, αvß6, is sufficient to promote activation of Smad3, regulation of MMP2 levels and consequent catalytic activity, as well as cell migration. Our study describes a new TGFß1-αvß6-MMP2 signalling pathway that, given TGFß1 pro-metastatic activity, may have profound implications for PrCa therapy.
Assuntos
Antígenos de Neoplasias/metabolismo , Regulação Enzimológica da Expressão Gênica , Integrinas/metabolismo , Metaloproteinase 2 da Matriz/biossíntese , Fator de Crescimento Transformador beta1/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Humanos , MasculinoRESUMO
In tumor cells, two factors are abnormally increased that contribute to metastatic bone disease: Runx2, a transcription factor that promotes expression of metastasis related and osteolytic genes; and IL-11, a secreted osteolytic cytokine. Here, we addressed a compelling question: Does Runx2 regulate IL-11 gene expression? We find a positive correlation between Runx2, IL-11 and TGFß1, a driver of the vicious cycle of metastatic bone disease, in prostate cancer (PC) cell lines representing early (LNCaP) and late (PC3) stage disease. Further, like Runx2 knockdown, IL-11 knockdown significantly reduced expression of several osteolytic factors. Modulation of Runx2 expression results in corresponding changes in IL-11 expression. The IL-11 gene has Runx2, AP-1 sites and Smad binding elements located on the IL-11 promoter. Here, we demonstrated that Runx2-c-Jun as well as Runx2-Smad complexes upregulate IL-11 expression. Functional studies identified a significant loss of IL-11 expression in PC3 cells in the presence of the Runx2-HTY mutant protein, a mutation that disrupts Runx2-Smad signaling. In response to TGFß1 and in the presence of Runx2, we observed a 30-fold induction of IL-11 expression, accompanied by increased c-Jun binding to the IL-11 promoter. Immunoprecipitation and in situ co-localization studies demonstrated that Runx2 and c-Jun form nuclear complexes in PC3 cells. Thus, TGFß1 signaling induces two independent transcriptional pathways - AP-1 and Runx2. These transcriptional activators converge on IL-11 as a result of Runx2-Smad and Runx2-c-Jun interactions to amplify IL-11 gene expression that, together with Runx2, supports the osteolytic pathology of cancer induced bone disease.
Assuntos
Neoplasias Ósseas/genética , Neoplasias Ósseas/secundário , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Interleucina-11/genética , Neoplasias da Próstata/genética , Fator de Crescimento Transformador beta1/farmacologia , Sítios de Ligação , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Interleucina-11/química , Interleucina-11/metabolismo , Masculino , Complexos Multiproteicos/metabolismo , Regiões Promotoras Genéticas , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Smad/metabolismo , Regulação para CimaRESUMO
Prostate cancer has heterogeneous growth patterns, and its prognosis is the poorest when it progresses to a neuroendocrine phenotype. Using bioinformatic analysis, we evaluated RNA expression of neuroendocrine genes in a panel of five different cancer types: prostate adenocarcinoma, breast cancer, kidney chromophobe, kidney renal clear cell carcinoma and kidney renal papillary cell carcinoma. Our results show that specific neuroendocrine genes are significantly dysregulated in these tumors, suggesting that they play an active role in cancer progression. Among others, synaptophysin (SYP), a conventional neuroendocrine marker, is upregulated in prostate adenocarcinoma (PRAD) and breast cancer (BRCA). Our analysis shows that SYP is enriched in small extracellular vesicles (sEVs) derived from plasma of PRAD patients, but it is absent in sEVs derived from plasma of healthy donors. Similarly, classical sEV markers are enriched in sEVs derived from plasma of prostate cancer patients, but weakly detectable in sEVs derived from plasma of healthy donors. Overall, our results pave the way to explore new strategies to diagnose these diseases based on the neuroendocrine gene expression in patient tumors or plasma sEVs.
Assuntos
Adenocarcinoma , Neoplasias da Próstata , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Sinaptofisina/metabolismo , Sinaptofisina/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Perfilação da Expressão Gênica/métodosRESUMO
It is known that small extracellular vesicles (sEVs) are released from cancer cells and contribute to cancer progression via crosstalk with recipient cells. We have previously reported that sEVs expressing the αVß3 integrin, a protein upregulated in aggressive neuroendocrine prostate cancer (NEPrCa), contribute to neuroendocrine differentiation (NED) in recipient cells. Here, we examine the impact of αVß3 expression on sEV protein content, density and function. sEVs used in this study were isolated by iodixanol density gradients and characterized by nanoparticle tracking analysis, immunoblotting and single vesicle analysis. Our proteomic profile of sEVs containing αVß3 shows downregulation of typical effectors involved in apoptosis and necrosis and an upregulation of tumour cell survival factors compared to control sEVs. We also show that the expression of αVß3 in sEVs causes a distinct reposition of EV markers (Alix, CD81, CD9) to a low-density sEV subpopulation. This low-density reposition is independent of extracellular matrix (ECM) protein interactions with sEVs. This sEV subset contains αVß3 and an αVß3 downstream effector, NgR2, a novel marker for NEPrCa. We show that sEVs containing αVß3 are loaded with higher amounts of NgR2 as compared to sEVs that do not express αVß3. Mechanistically, we demonstrate that sEVs containing NgR2 do not affect the sEV marker profile, but when injected in vivo intratumorally, they promote tumour growth and induce NED. We show that sEVs expressing NgR2 increase the activation of focal adhesion kinase (FAK), a known promoter of cancer cell proliferation, in recipient cells. We also show that NgR2 mimics the effect of sEVs containing αVß3 since it displays increased growth of NgR2 transfectants in vivo, as compared to control cells. Overall, our results describe the changes that occur in cargo, density and functions of cancer cell-derived sEVs containing the αVß3 integrin and its effector, NgR2, without affecting the sEV tetraspanin profiles.
Assuntos
Vesículas Extracelulares , Integrina alfaVbeta3 , Neoplasias da Próstata , Masculino , Integrina alfaVbeta3/metabolismo , Humanos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/genética , Vesículas Extracelulares/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos , Carcinogênese/metabolismoRESUMO
Protein folding quality control does not occur randomly in cells, but requires the action of specialized molecular chaperones compartmentalized in subcellular microenvironments and organelles. Fresh experimental evidence has now linked a mitochondrial-specific Heat Shock Protein-90 (Hsp90) homolog, Tumor Necrosis Factor Receptor-Associated Protein-1 (TRAP-1) to pleiotropic signaling circuitries of organelle integrity and cellular homeostasis. TRAP-1-directed compartmentalized protein folding is broadly exploited in cancer and neurodegenerative diseases, presenting new opportunities for therapeutic intervention in humans. This article is part of a Special Issue entitled: Heat Shock Protein 90 (Hsp90).
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismo , Microambiente Celular/fisiologia , Humanos , Dobramento de ProteínaRESUMO
This study was carried out to dissect the mechanism by which ß1 integrins promote resistance to radiation. For this purpose, we conditionally ablated ß1 integrins in the prostatic epithelium of transgenic adenocarcinoma of mouse prostate (TRAMP) mice. The ability of ß1 to promote resistance to radiation was also analyzed by using an inhibitory antibody to ß1 , AIIB2, in a xenograft model. The role of ß1 integrins and of a ß1 downstream target, c-Jun amino-terminal kinase 1 (JNK1), in regulating radiation-induced apoptosis in vivo and in vitro was studied. We show that ß1 integrins promote prostate cancer (PrCa) progression and resistance to radiation in vivo. Mechanistically, ß1 integrins are shown here to suppress activation of JNK1 and, consequently apoptosis, in response to irradiation. Downregulation of JNK1 is necessary to preserve the effect of ß1 on resistance to radiation in vitro and in vivo. Finally, given the established crosstalk between ß1 integrins and type1 insulin-like growth factor receptor (IGF-IR), we analyzed the ability of IGF-IR to modulate ß1 integrin levels. We report that IGF-IR regulates the expression of ß1 integrins, which in turn confer resistance to radiation in PrCa cells. In conclusion, this study demonstrates that ß1 integrins mediate resistance to ionizing radiation through inhibition of JNK1 activation.
Assuntos
Integrina beta1/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/radioterapia , Tolerância a Radiação/fisiologia , Animais , Apoptose/fisiologia , Apoptose/efeitos da radiação , Caspase 3/metabolismo , Linhagem Celular Tumoral , Humanos , Integrina beta1/genética , Masculino , Camundongos , Camundongos Knockout , Camundongos Nus , Camundongos Transgênicos , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/genética , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Transplante HeterólogoRESUMO
Highly aggressive, metastatic, neuroendocrine prostate cancer, which typically develops from prostate cancer cells acquiring resistance to androgen deprivation therapy, is associated with limited treatment options and hence poor prognosis. We have previously demonstrated that the αVß3 integrin is over-expressed in neuroendocrine prostate cancer. We now show that LM609, a monoclonal antibody that specifically targets the human αVß3 integrin, hinders the growth of neuroendocrine prostate cancer patient-derived xenografts in vivo. Our group has recently identified a novel αVß3 integrin binding partner, NgR2, responsible for regulating the expression of neuroendocrine markers and for inducing neuroendocrine differentiation in prostate cancer cells. Through in vitro functional assays, we here demonstrate that NgR2 is crucial in promoting cell adhesion to αVß3 ligands. Moreover, we describe for the first time co-fractionation of αVß3 integrin and NgR2 in small extracellular vesicles derived from metastatic prostate cancer patients' plasma. These prostate cancer patient-derived small extracellular vesicles have a functional impact on human monocytes, increasing their adhesion to fibronectin. The monocytes incubated with small extracellular vesicles do not show an associated change in conventional polarization marker expression and appear to be in an early stage that may be defined as "adhesion competent". Overall, these findings allow us to better understand integrin-directed signaling and cell-cell communication during cancer progression. Furthermore, our results pave the way for new diagnostic and therapeutic perspectives for patients affected by neuroendocrine prostate cancer.
Assuntos
Neoplasias da Próstata , Masculino , Humanos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Antagonistas de Androgênios , Transdução de Sinais , Anticorpos Monoclonais , Integrinas , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Linhagem Celular TumoralRESUMO
Despite the findings that ß1 integrins play a vital role in the regulation of cell proliferation and survival, the mechanisms through which they operate and lead to cancer progression remain elusive. Previously, our laboratory has shown that ß(1A) integrins support insulin-like growth factor 1 (IGFI)-mediated mitogenic and transforming activities. Here, we report that ß(1A) integrins regulate basal levels of IGF-IR, although they are not critical for maintaining cancer cell morphology. Upon transfection of ß(1A) siRNA and consequent downregulation of IGF-IR, we show inhibition of anchorage-independent growth of prostate cancer cells, a function which is dependent on IGF-IR expression. In addition, we demonstrate that IGFI-mediated activation of androgen receptor (AR), known to occur in prostate cancer cells, requires expression of ß(1A) integrins as evaluated by luciferase reporter assays and immunoblotting analysis. Since ß(1A) integrin levels are increased by R1881 or dihydrotestosterone (DHT), our results imply that ß(1A) integrins support an androgen-enhanced feedback loop that regulates the expression of IGF-IR. ß(1A) integrins also regulate inducible levels of IGF-IR in cells stimulated by androgen or by a combination of androgen and IGFI, as evaluated by flow cytometric analysis and immunoblotting. Furthermore, upon transfection of ß(1A) siRNA and consequent downregulation of IGF-IR, neither activation of AKT, an effector of IGF-IR, nor AR levels are affected. We conclude that ß(1A) integrin expression is critical for maintaining the regulatory crosstalk between IGF-IR and AR.