RESUMO
Abnormal expression of microRNA miR-214-3p (miR-214) is associated with multiple cancers. In this study, we assessed the effects of CRISPR/Cas9 mediated miR-214 depletion in prostate cancer (PCa) cells and the underlying mechanisms. Knockdown of miR-214 promoted PCa cell proliferation, invasion, migration, epithelial-mesenchymal transition (EMT), and increased resistance to anoikis, a key feature of PCa cells that undergo metastasis. The reintroduction of miR-214 in miR-214 knockdown cells reversed these effects and significantly suppressed cell proliferation, migration, and invasion. These in vitro studies are consistent with the role of miR-214 as a tumor suppressor. Moreover, miR-214 knockout increased tumor growth in PCa xenografts in nude mice supporting its anti-oncogenic role in PCa. Knockdown of miR-214 increased the expression of its target protein, Protein Tyrosine Kinase 6 (PTK6), a kinase shown to promote oncogenic signaling and tumorigenesis in PCa. In addition, miR-214 modulated EMT as exhibited by differential regulation of E-Cadherin, N-Cadherin, and Vimentin both in vitro and in vivo. RNA-seq analysis of miR-214 knockdown cells revealed altered gene expression related to PCa tumor growth pathways, including EMT and metastasis. Collectively, our findings reveal that miR-214 is a key regulator of PCa oncogenesis and is a potential novel therapeutic target for the treatment of the disease.
RESUMO
Prostate cancer (PCa) constitutes a serious health challenge and remains one of the main causes of cancer-related death among men. The more aggressive form of the disease has been attributed to androgen independence, resulting in a lack of response to androgen deprivation therapy and sustained activation of other growth pathways. The scaffold proteins ß-arrestin 1 and 2 (ßarr1 and ßarr2), which are known to mediate G protein-coupled receptor desensitization and internalization, were also shown to modulate prostate tumorigenesis. ßarr1 is significantly overexpressed (>4-fold) in PCa cells relative to ßarr2. In this study, we investigated the effect of ßarr1 overexpression in PCa development and progression using the mouse and human PCa cell xenografts, and autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) models deficient in ß-arrestin depletion of ßarr1 in TRAMP mice (TRAMP/ßarr1-/-) increased PCa growth and decreased overall survival relative to control TRAMP or TRAMP/ßarr2-/- animals. Prostate tissues from TRAMP/ßarr1-/- tumors displayed an increase in androgen receptor (AR) expression, whereas overexpression of ßarr1 in TRAMP-C1 (TRAMP-C1-ßarr1-GFP) which derived from TRAMP decreased AR expression, cell proliferation and tumor growth in nude mice xenografts, relative to control TRAMP-C1-GFP. Knockdown of ßarr1 expression in human MDA PCa 2b cells (MDA PCa 2b-ßarr1-/-) also decreased AR expression cell proliferation and tumor growth relative to control (MDA PCa 2b-Sham) cells. Interestingly, both TRAMP-C1-ßarr1-GFP and MDA PCa 2b-ßarr1-/- xenografts showed a decrease in AKT phosphorylation but an increase in MAPK activation. Altogether, the data indicate that the effect of ßarr1 in modulating AR signaling to regulate PCa aggressiveness is cell and host autonomous.
Assuntos
Carcinogênese/genética , Neoplasias da Próstata/genética , Membro 25 de Receptores de Fatores de Necrose Tumoral/genética , beta-Arrestina 1/genética , beta-Arrestina 2/genética , Animais , Animais Geneticamente Modificados , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Masculino , Camundongos , Neoplasias da Próstata/patologia , Receptores Androgênicos/genética , Transdução de SinaisRESUMO
A field study was carried out on the year-long residual activity of the insect growth regulator (IGR) pyriproxyfen (Nylar 0.5G) in comparison with methoprene (Altosid® XRP Pellets) against mosquito developmental stages in catch basins in northwestern Riverside County, southern California. Pyriproxyfen was applied at 75, 100, 125, 150, 175 g per catch basin and methoprene at 3.5 g per catch basin. A total of 80 catch basins (10 per each treatment and 20 for control) were used. Posttreatment observations of catch basins were carried out at weekly intervals, with all pupal collections reared to adults. Mosquito species composition in this study, consisting mostly of Culex species (693), was predominated by Cx. quinquefasciatus (92.8%), followed by Cx. erythrothorax (5.5%), Cx. tarsalis (1.2%), Cx. stigmatosoma (0.3%), and Cx. thriambus (0.2%). Activity of both IGRs was expressed as percent inhibition of adult emergence (% IAE). Data generated on % IAE showed that, like methoprene, pyriproxyfen provided complete control of mosquitoes at 75, 125, and 175 g per catch basin up to 50 wk posttreatment at the Riverside amusement park, whereas its activity against mosquitoes in catch basins treated with 100 g and 150 g at the Eastvale site was short-lived, up to 48 wk. Water samples, bioassayed against laboratory-reared, 4th-stage larvae of Cx. quinquefasciatus 1-2 wk after the 50-wk-long study, showed evidence of significant % IAE (â¼50) by pyriproxyfen at the 2 higher rates (125 g, 175 g) used at the amusement park. In conclusion, pyriproxyfen can be used to effectively control mosquitoes in catch basins for 48-50 wk, depending on the rate of application.
Assuntos
Culex , Inseticidas , Metoprene , Controle de Mosquitos , Piridinas , Animais , California , Culex/crescimento & desenvolvimento , Feminino , Larva/crescimento & desenvolvimento , Masculino , Óvulo/crescimento & desenvolvimento , Pupa/crescimento & desenvolvimentoRESUMO
Prostate cancer (PCa) is a leading cause of cancer death among men, with greater prevalence of the disease among the African American population in the USA. Activator of G-protein signaling 3 (AGS3/G-protein signaling modulator 1) was shown to be overexpressed in prostate adenocarcinoma relative to the prostate gland. In this study, we investigated the correlation between AGS3 overexpression and PCa malignancy. Immunoblotting analysis and real-time quantitative-PCR showed increase in AGS3 expression in the metastatic cell lines LNCaP (~3-fold), MDA PCa 2b (~2-fold), DU 145 (~2-fold) and TRAMP-C1 (~20-fold) but not in PC3 (~1-fold), relative to control RWPE-1. Overexpression of AGS3 in PC3, LNCaP and MDA PCa 2b enhanced tumor growth. AGS3 contains seven tetratricopeptide repeats (TPR) and four G-protein regulatory (GPR) motifs. Overexpression of the TPR or the GPR motifs in PC3 cells had no effect in tumor growth. Depletion of AGS3 in the TRAMP-C1 cells (TRAMP-C1-AGS3-/-) decreased cell proliferation and delayed wound healing and tumor growth in both C57BL/6 (~3-fold) and nude mice xenografts, relative to control TRAMP-C1 cells. TRAMP-C1-AGS3-/- tumors also exhibited a marked increase (~5-fold) in both extracellular signal-regulated kinase (ERK) 1/2 and P38 mitogen-activated protein kinase (MAPK) activation, which correlated with a significant increase (~3-fold) in androgen receptor (AR) expression, relative to TRAMP-C1 xenografts. Interestingly, overexpression of AGS3 in TRAMP-C1-AGS3-/- cells inhibited ERK activation and AR overexpression as compared with control TRAMP-C1 cells. Taken together, the data indicate that the effect of AGS3 in prostate cancer development and progression is probably mediated via a MAPK/AR-dependent pathway.
Assuntos
Carcinogênese/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Neoplasias da Próstata/patologia , Animais , Linhagem Celular Tumoral , Progressão da Doença , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias da Próstata/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The IL-8 (CXCL8) receptors CXCR1 and CXCR2 couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. We recently showed that CXCR1 couples predominantly to the G protein-coupled receptor kinase (GRK)2, whereas CXCR2 interacts with GRK6 to regulate cellular responses. In addition to G protein-coupled receptors, GRKs displayed a more diverse protein/protein interaction in cells. In this study, we sought to identify GRK6 binding partner(s) that may influence CXCL8 activities, using RBL-2H3 cells stably expressing CXCR1 (RBL-CXCR1) or CXCR2 (RBL-CXCR2), as well as human and murine neutrophils. Our data demonstrated that, upon CXCR2 activation, GRK6 interacts with activator of G protein signaling (AGS)3 and Gαi2 to form a GRK6/AGS3/Gαi2 complex. This complex is time dependent and peaked at 2-3 min postactivation. GTPγS pretreatment blocked GRK6/AGS3/Gαi2 formation, suggesting that this assembly depends on G protein activation. Surprisingly, CXCR2 activation induced AGS3 phosphorylation in a PKC-dependent, but GRK6-independent, fashion. Overexpression of AGS3 in RBL-CXCR2 significantly inhibited CXCL8-induced Ca(2+) mobilization, phosphoinositide hydrolysis, and chemotaxis. In contrast, short hairpin RNA inhibition of AGS3 enhanced CXCL8-induced Ca(2+) mobilization, receptor resistance to desensitization, and recycling to the cell surface, with no effect on receptor internalization. Interestingly, RBL-CXCR2-AGS3(-/-) cells displayed a significant increase in CXCR2 expression on the cell surface but decreased ERK1/2 and P38 MAPK activation. Taken together, these results indicate that GRK6 complexes with AGS3-Gαi2 to regulate CXCR2-mediated leukocyte functions at different levels, including downstream effector activation, receptor trafficking, and expression at the cell membrane.
Assuntos
Quinases de Receptores Acoplados a Proteína G/imunologia , Inibidores de Dissociação do Nucleotídeo Guanina/imunologia , Complexos Multiproteicos/imunologia , Receptores de Interleucina-8B/imunologia , Animais , Cálcio/imunologia , Sinalização do Cálcio/genética , Sinalização do Cálcio/imunologia , Linhagem Celular , Membrana Celular/genética , Membrana Celular/imunologia , Quinases de Receptores Acoplados a Proteína G/genética , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/imunologia , Regulação da Expressão Gênica/fisiologia , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Humanos , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Fosforilação/genética , Fosforilação/imunologia , Transporte Proteico/fisiologia , Receptores de Interleucina-8B/genéticaRESUMO
G protein-coupled receptor kinases (GRKs) phosphorylate the activated form of G protein-coupled receptors leading to receptor desensitization and downregulation. We have recently shown that the chemokine receptor, CXCR2, couples to GRK6 to regulate cellular responses including chemotaxis, angiogenesis, and wound healing. In this study, we investigate the role of GRK6 in tumorigenesis using murine models of human lung cancer. Mice deficient in GRK6 (GRK6(-/-)) exhibited a significant increase in Lewis lung cancer growth and metastasis relative to control littermates (GRK6(+/+)). GRK6 deletion had no effect on the expression of proangiogenic chemokine or vascular endothelial growth factor, but upregulated matrix metalloproteinase (MMP)-2 and MMP-9 release, tumor-infiltrating PMNs, and microvessel density. Because ß-arrestin-2-deficient (ßarr2(-/-)) mice exhibited increased Lewis lung cancer growth and metastasis similar to that of GRK6(-/-), we developed a double GRK6(-/-)/ßarr2(-/-) mouse model. Surprisingly, GRK6(-/-)/ßarr2(-/-) mice exhibited faster tumor growth relative to GRK6(-/-) or ßarr2(-/-) mice. Treatment of the mice with anti-CXCR2 Ab inhibited tumor growth in both GRK6(-/-) and GRK6(-/-)/ßarr2(-/-) animals. Altogether, the results indicate that CXCR2 couples to GRK6 to regulate angiogenesis, tumor progression, and metastasis. Deletion of GRK6 increases the activity of the host CXCR2, resulting in greater PMN infiltration and MMP release in the tumor microenvironment, thereby promoting angiogenesis and metastasis. Because GRK6(-/-)/ßarr2(-/-) showed greater tumor growth relative to GRK6(-/-) or ßarr2(-/-) mice, the data further suggest that CXCR2 couples to different mechanisms to mediate tumor progression and metastasis.
Assuntos
Arrestinas/genética , Carcinoma Pulmonar de Lewis/metabolismo , Quinases de Receptores Acoplados a Proteína G/genética , Quinases de Receptores Acoplados a Proteína G/metabolismo , Neutrófilos/imunologia , Receptores de Interleucina-8B/metabolismo , Animais , Arrestinas/deficiência , Arrestinas/metabolismo , Linhagem Celular Tumoral , Quimiotaxia , Progressão da Doença , Regulação para Baixo , Quinases de Receptores Acoplados a Proteína G/deficiência , Genótipo , Neoplasias Pulmonares/metabolismo , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Camundongos , Camundongos Knockout , Metástase Neoplásica , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Compostos de Fenilureia/farmacologia , Fosforilação , Receptores de Interleucina-8B/antagonistas & inibidores , Transdução de Sinais , Microambiente Tumoral , Regulação para Cima , Cicatrização , beta-Arrestina 2 , beta-ArrestinasRESUMO
Inorganic arsenic (iAs) is an environmental toxicant currently poisoning millions of people worldwide, and chronically exposed individuals are susceptible to arsenicosis or arsenic poisoning. Using a state-of-the-art technique to map the methylomes of our study subjects, we identified a large interactome of hypermethylated genes that are enriched for their involvement in arsenic-associated diseases, such as cancer, heart disease, and diabetes. Notably, we have uncovered an arsenic-induced tumor suppressorome, a complex of 17 tumor suppressors known to be silenced in human cancers. This finding represents a pivotal clue in unraveling a possible epigenetic mode of arsenic-induced disease.