RESUMO
The landscape of current cancer immunotherapy is dominated by antibodies targeting PD-1/PD-L1 and CTLA-4 that have transformed cancer therapy, yet their efficacy is limited by primary and acquired resistance. The blockade of additional immune checkpoints, especially TIGIT and LAG-3, has been extensively explored, but so far only a LAG-3 antibody has been approved for combination with nivolumab to treat unresectable or metastatic melanoma. Here we report the development of a PDL1 × TIGIT bi-specific antibody (bsAb) GB265, a PDL1 × LAG3 bsAb GB266, and a PDL1 × TIGIT × LAG3 tri-specific antibody (tsAb) GB266T, all with intact Fc function. In in vitro cell-based assays, these antibodies promote greater T cell expansion and tumor cell killing than benchmark antibodies and antibody combinations in an Fc-dependent manner, likely by facilitating T cell interactions (bridging) with cancer cells and monocytes, in addition to blocking immune checkpoints. In animal models, GB265 and GB266T antibodies outperformed benchmarks in tumor suppression. This study demonstrates the potential of a new generation of multispecific checkpoint inhibitors to overcome resistance to current monospecific checkpoint antibodies or their combinations for the treatment of human cancers.
Assuntos
Melanoma , Neoplasias , Animais , Humanos , Neoplasias/terapia , Nivolumabe , Receptores Imunológicos , Imunoterapia , Linfócitos TRESUMO
MiR-145 is downregulated in various cancers including prostate cancer. However, the underlying mechanisms of miR-145 downregulation are not fully understood. Here, we reported that miR-145 was silenced through DNA hypermethylation and p53 mutation status in laser capture microdissected (LCM) prostate cancer and matched adjacent normal tissues. In 22 of 27 (81%) prostate tissues, miR-145 was significantly downregulated in the cancer compared with the normal tissues. Further studies on miR-145 downregulation mechanism showed that miR-145 is methylated at the promoter region in both prostate cancer tissues and 50 different types of cancer cell lines. In seven cancer cell lines with miR-145 hypermethylation, 5-aza-2'-deoxycytidine treatment dramatically induced miR-145 expression. Interestingly, we also found a significant correlation between miR-145 expression and the status of p53 gene in both LCM prostate tissues and 47 cancer cell lines. In 29 cell lines with mutant p53, miR-145 levels were downregulated in 28 lines (97%), whereas in 18 cell lines with wild-type p53 (WT p53), miR-145 levels were downregulated in only 6 lines (33%, P < 0.001). Electrophoretic mobility shift assay showed that p53 binds to the p53 response element upstream of miR-145, but the binding was inhibited by hypermethylation. To further confirm that p53 binding to miR-145 could regulate miR-145 expression, we transfected WT p53 and MUT p53 into PC-3 cells and found that miR-145 is upregulated by WT p53 but not with MUTp53. The apoptotic cells are increased after WT p53 transfection. In summary, this is the first report documenting that downregulation of miR-145 is through DNA methylation and p53 mutation pathways in prostate cancer.
Assuntos
Metilação de DNA , MicroRNAs/genética , Mutação/genética , Hiperplasia Prostática/genética , Neoplasias da Próstata/genética , Proteína Supressora de Tumor p53/genética , Apoptose , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Hibridização In Situ , Lasers , Masculino , Microdissecção , Regiões Promotoras Genéticas/genética , Próstata/metabolismo , Próstata/patologia , Hiperplasia Prostática/patologia , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Cytochrome P450 17 (CYP17) encodes cytochrome P450c17α, an enzyme with 17α-hydroxylase and 17, 20-lyase activities involved in estradiol biosynthesis. Here we examine the role of CYP17 gene in endometrial carcinogenesis. Immunohistochemistry staining of endometrial carcinoma and corresponding uninvolved tissues showed that CYP17 is upregulated in endometrial cancers (15 of 24, 62.5%). To understand the functional significance of this upregulation, we silenced CYP17 gene by introduction of siRNA into endometrial cancer cell line KLE followed by functional studies. Further, to understand the molecular basis of the role of CYP17, we profiled the expression of key pathway-specific genes and identified several components of the apoptosis and invasion pathways that are potentially regulated by CYP17. Silencing of CYP17 caused decreased cell proliferation and induced apoptosis. Significantly, CYP17 depletion leads to downregulation of anti-apoptotic genes B cell lymphoma 2 (Bcl-2) and telomerase reverse transcriptase (TERT), indicating induced apoptosis. Also, attenuation of CYP17 decreased the cellular invasion ability and regulated expression of several invasion pathway components such as melanoma cell adhesion molecule (MCAM), matrix metallopeptidase 2 and 9 (MMP-2 and MMP-9), and tissue inhibitor of metalloproteinase 3 (TIMP3). In conclusion, this is the first report documenting that upregulation of CYP17 in endometrial cancers play a crucial role in endometrial carcinogenesis by targeting multiple components of apoptosis and invasion pathways. Further studies are required to understand the detailed mechanisms underlying CYP17-mediated regulation of these components.
Assuntos
Apoptose , Neoplasias do Endométrio/enzimologia , Neoplasias do Endométrio/patologia , Endométrio/enzimologia , Transdução de Sinais , Esteroide 17-alfa-Hidroxilase/fisiologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Endométrio/genética , Endométrio/patologia , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Invasividade Neoplásica , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise Serial de Tecidos , Regulação para CimaRESUMO
Nucleoside diphosphate kinases (Ndks) play an important role in a plethora of regulatory and metabolic functions. Inhibition of the B. anthracis Ndk mRNA results in the formation of nonviable aberrant spores. We report the characterization and crystal structure of the enzyme from B. anthracis nucleoside diphosphate kinase (BaNdk), the first from sporulating bacteria. The enzyme, although from a mesophilic source, is active at extremes of pH (3.5-10.5), temperature (10-95 degrees C) and ionic strength (0.25-4.0M NaCl). It exists as a hexamer that is composed of two SDS-stable trimers interacting in a back-to-back association; mutational analysis confirms that the enzyme is a histidine kinase. The high-resolution crystal structure reported here reveals an unanticipated change in the conformation of residues between 43 and 63 that also regulates substrate entry in other Ndks. A comparative structural analysis involving Ndks from seven mesophiles and three thermophiles has resulted in the delineation of the structure into relatively rigid and flexible regions. The analysis suggests that the larger number of intramolecular hydrogen bonds and to a lesser extent ionic interactions in BaNdk contributes to its high thermostability. Mutational analysis and Molecular Dynamics simulations were used to probe the role of a highly conserved Gly19 (present at the oligomeric interface in most of the Ndks). The results suggest that the mutation leads to a rigidification of those residues that facilitate substrate entry and consequently leads to a large reduction in the kinase activity. Overall, the enzyme characterization helps to understand its apparent adaptation to perform under stress conditions.
Assuntos
Bacillus anthracis/enzimologia , Núcleosídeo-Difosfato Quinase/química , Sequência de Aminoácidos , Bacillus anthracis/metabolismo , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Núcleosídeo-Difosfato Quinase/metabolismo , Conformação Proteica , Alinhamento de Sequência , TemperaturaRESUMO
External and internal changes occurring during the process of germination of Bacillus anthracis spores were observed through atomic force microscopy (AFM) and transmission electron microscopy (TEM), respectively. AFM studies showed that in response to L-alanine (4 mM), as a germinant, the spore germinates into a vegetative cell in 3 hours. The temporal size changes occurring during the germination were gradual but the major change in size was observed between the second and third hour. TEM of spores showed the presence of varied layers, which is in accordance with previous studies. However, the integrity of these layers was lost gradually during the process of germination. The inner spore membrane remains intact even until late stages of germination, whereas the coat, outer spore membrane, and the cortical layers are discarded at the second-hour stage. The results indicate that sequential changes during the germination of a B. anthracis spore are similar to other species of the Bacillus group.
Assuntos
Bacillus anthracis/fisiologia , Bacillus anthracis/ultraestrutura , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Esporos Bacterianos/fisiologia , Esporos Bacterianos/ultraestruturaRESUMO
PURPOSE: miRNAs can act as oncomirs or tumor-suppressor miRs in cancer. This study was undertaken to investigate the status and role of miR-34b in prostate cancer. EXPERIMENTAL DESIGN: Profiling of miR-34b was carried out in human prostate cancer cell lines and clinical samples by quantitative real-time PCR and in situ hybridization. Statistical analyses were done to assess diagnostic/prognostic potential. Biological significance was elucidated by carrying out a series of experiments in vitro and in vivo. RESULTS: We report that miR-34b is silenced in human prostate cancer and the mechanism is through CpG hypermethylation. miR-34b directly targeted methyltransferases and deacetylases resulting in a positive feedback loop inducing partial demethylation and active chromatin modifications. miR-34b expression could predict overall and recurrence-free survival such that patients with high miR-34b levels had longer survival. Functionally, miR-34b inhibited cell proliferation, colony formation, migration/invasion, and triggered G(0)/G(1) cell-cycle arrest and apoptosis by directly targeting the Akt and its downstream proliferative genes. miR-34b caused a decline in the mesenchymal markers vimentin, ZO1, N-cadherin, and Snail with an increase in E-cadherin expression, thus inhibiting epithelial-to-mesenchymal transition. Finally we showed the antitumor effect of miR-34b in vivo. MiR-34b caused a dramatic decrease in tumor growth in nude mice compared with cont-miR. CONCLUSION: These findings offer new insight into the role of miR-34b in the inhibition of prostate cancer through demethylation, active chromatin modification, and Akt pathways and may provide a rationale for the development of new strategies targeting epigenetic regulation of miRNAs for the treatment of prostate cancer.
Assuntos
Cromatina/genética , Cromatina/metabolismo , MicroRNAs/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Regiões 5' não Traduzidas , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Transformação Celular Neoplásica/genética , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Epigênese Genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Masculino , Camundongos , MicroRNAs/metabolismo , Invasividade Neoplásica/genética , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologiaRESUMO
Ovarian cancer is the fifth most common cancer among women and causes more deaths than any other type of female reproductive cancer. Currently, treatment of ovarian cancer is based on the combination of surgery and chemotherapy. While recurrent ovarian cancer responds to additional chemotherapy treatments, the progression-free interval becomes shorter after each cycle, as chemo-resistance increases until the disease becomes incurable. There is, therefore, a strong need for prognostic and predictive markers to help optimize and personalize treatment in order to improve the outcome of ovarian cancer. An increasing number of studies indicate an essential role for microRNAs in ovarian cancer progression and chemo-resistance. MicroRNAs (miRNAs) are small endogenous non-coding RNAs (~22bp) which are frequently dysregulated in cancer. Typically, miRNAs are involved in crucial biological processes, including development, differentiation, apoptosis and proliferation. Two families of miRNAs, miR-200 and let-7, are frequently dysregulated in ovarian cancer and have been associated with poor prognosis. Both have been implicated in the regulation of epithelial-to-mesenchymal transition, a cellular transition associated with tumor aggressiveness, tumor invasion and chemo-resistance. Moreover, miRNAs also have possible implications for improving cancer diagnosis; for example miR-200 family, let-7 family, miR-21 and miR-214 may be useful in diagnostic tests to help detect ovarian cancer at an early stage. Additionally, the use of multiple target O-modified antagomirs (MTG-AMO) to inhibit oncogenic miRNAs and miRNA replacement therapy for tumor suppressor miRNAs are essential tools for miRNA based cancer therapeutics. In this review we describe the current status of the role miRNAs play in ovarian cancer and focus on the possibilities of microRNA-based therapies and the use of microRNAs as diagnostic tools.
RESUMO
Tumor recurrence in prostate cancer has been attributed to the presence of CD44-expressing tumor-initiating cells. In this study, we report that miR-708 is a key negative regulator of this CD44(+) subpopulation of prostate cancer cells, with important implications for diagnosis and prognosis of this disease. miR-708 was underexpressed in CD44(+) cells from prostate cancer xenografts. Reconstitution of miR-708 in prostate cancer cell lines or CD44(+) prostate cancer cells led to decreased tumorigenicity in vitro. Intratumoral delivery of synthetic miR-708 oligonucleotides triggered regression of established tumors in a murine xenograft model of human prostate cancer. Conversely, miR-708 silencing in a purified CD44(-) population of prostate cancer cells promoted tumor growth. Functional studies validated CD44 to be a direct target of miR-708 and also identified the serine/threonine kinase AKT2 as an additional target. Clinically, low miR-708 expression was associated significantly with poor survival outcome, tumor progression, and recurrence in patients with prostate cancer. Together, our findings suggest that reduced miR-708 expression leads to prostate cancer initiation, progression, and development by regulating the expression of CD44 as well as AKT2. miR-708 therefore may represent a novel therapeutic target or diagnostic and prognostic biomarker in prostate cancer.
Assuntos
Receptores de Hialuronatos/metabolismo , MicroRNAs/fisiologia , Células-Tronco Neoplásicas/metabolismo , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Biomarcadores Tumorais/análise , Progressão da Doença , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos SCID , Invasividade Neoplásica/genética , Transplante de Neoplasias , Neoplasias da Próstata/metabolismo , Recidiva , Transplante HeterólogoRESUMO
The miRNAs have great potential as biomarkers and therapeutic agents owing to their ability to control multiple genes and potential to influence cellular behavior. Here, we identified that miR-23b is a methylation-silenced tumor suppressor in prostate cancer. We showed that miR-23b expression is controlled by promoter methylation and has great promise as a diagnostic and prognostic biomarker in prostate cancer. High levels of miR-23b expression are positively correlated with higher overall and recurrence-free survival in patients with prostate cancer. Furthermore, we elucidated the tumor suppressor role of miR-23b using in vitro and in vivo models. We showed that proto-oncogene Src kinase and Akt are direct targets of miR-23b. Increased expression of miR-23b inhibited proliferation, colony formation, migration/invasion, and triggered G(0)-G(1) cell-cycle arrest and apoptosis in prostate cancer. Overexpression of miR-23b inhibited epithelial-to-mesenchymal transition (EMT) causing a decline in mesenchymal markers Vimentin and Snail and increasing the epithelial marker, E-cadherin. Depletion of Src by RNA interference conferred similar functional effects as that of miR-23b reconstitution. miR-23b expression caused a dramatic decrease in tumor growth in nude mice and attenuated Src expression in excised tumors compared with a control miR. These findings suggest that miR-23b is a methylation-silenced tumor suppressor that may be a useful biomarker in prostate cancer. Loss of miR-23b may confer proliferative advantage and promote prostate cancer migration and invasion, and reexpression of miR-23b may contribute to the epigenetic therapy for prostate cancer.
Assuntos
Carcinoma/diagnóstico , Metilação de DNA/genética , Repressão Epigenética/genética , Genes src/genética , MicroRNAs/fisiologia , Neoplasias da Próstata/diagnóstico , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/fisiologia , Carcinoma/genética , Carcinoma/patologia , Carcinoma/terapia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor/fisiologia , Terapia Genética , Humanos , Masculino , Camundongos , Camundongos Nus , MicroRNAs/genética , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Proto-Oncogene Mas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: MicroRNA-21 is up-regulated in a variety of cancers like, breast, colorectal, lung, head and neck etc. However, the regulation of miR-21 in renal cell carcinoma (RCC) has not yet been studied systematically. METHODS AND RESULTS: We measured miR-21 levels in 54 pairs of kidney cancers and their normal matched tissues by real-time PCR. The expression level of miR-21 was correlated with 5 year survival and the pathological stage. Functional studies were done after inhibiting miR-21 in RCC cell lines. We studied in vitro and in vivo effects of the chemo preventive agent genistein on miR-21 expression. In 48 cases (90%), miR-21 was increased. All patients with low miR-21 expression survived 5 years, while with high miR-21 expression, only 50% survived. Higher expression of miR-21 is associated with an increase in the stage of renal cancer. Functional studies after inhibiting miRNA-21 in RCC cell lines show cell cycle arrest, induction of apoptosis and reduced invasive and migratory capabilities. Western blot analysis showed an increase in the expression of p21 and p38 MAP kinase genes and a reduction in cyclin E2. Genistein inhibited the expression of miR-21 in A-498 cells and in the tumors formed after injecting genistein treated A-498 cells in nude mice besides inhibiting tumor formation. CONCLUSIONS: The current study shows a clear correlation between miR-21 expression and clinical characteristics of renal cancer. Thus we believe that miR-21 can be used as a tumor marker and its inhibition may prove to be useful in controlling cancers with up-regulated miR-21.
Assuntos
Neoplasias Renais/genética , Neoplasias Renais/mortalidade , MicroRNAs/biossíntese , Regulação para Cima , Animais , Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Genisteína/farmacologia , Humanos , Neoplasias Renais/patologia , Camundongos , MicroRNAs/análise , Prognóstico , Taxa de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MicroRNAs (miRNAs) are non-protein-coding sequences that can function as oncogenes or tumor suppressor genes. This study documents the tumor suppressor role of miR-1280 in bladder cancer. Quantitative real-time PCR and in situ hybridization analyses showed that miR-1280 is significantly down-regulated in bladder cancer cell lines and tumors compared to a non-malignant cell line or normal tissue samples. To decipher the functional significance of miR-1280 in bladder cancer, we ectopically over-expressed miR-1280 in bladder cancer cell lines. Over-expression of miR-1280 had antiproliferative effects and impaired colony formation of bladder cancer cell lines. FACS (fluorescence activated cell sorting) analysis revealed that re-expression of miR-1280 in bladder cancer cells induced G2-M cell cycle arrest and apoptosis. Our results demonstrate that miR-1280 inhibited migration and invasion of bladder cancer cell lines. miR-1280 also attenuated ROCK1 and RhoC protein expression. Luciferase reporter assays demonstrated that oncogene ROCK1 is a direct target of miR-1280 in bladder cancer. This study also indicates that miR-1280 may be of diagnostic and prognostic importance in bladder cancer. For instance, ROC analysis showed that miR-1280 expression can distinguish between malignant and normal bladder cancer cases and Kaplan-Meier analysis revealed that patients with miR-1280 high expression had higher overall survival compared to those with low miR-1280 expression. In conclusion, this is the first study to document that miR-1280 functions as a tumor suppressor by targeting oncogene ROCK1 to invasion/migration and metastasis. Various compounds are currently being used as ROCK1 inhibitors; therefore restoration of tumor suppressor miR-1280 might be therapeutically useful either alone or in combination with these compounds in the treatment of bladder cancer.
Assuntos
MicroRNAs/metabolismo , Neoplasias da Bexiga Urinária/genética , Quinases Associadas a rho/metabolismo , Apoptose/genética , Apoptose/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Immunoblotting , Hibridização In Situ , Técnicas In Vitro , Reação em Cadeia da Polimerase em Tempo Real , Quinases Associadas a rho/genéticaRESUMO
BACKGROUND: miR-23b is located on chromosome number 9 and plays different roles in different organs especially with regards to cancer development. However, the functional significance of miR-23b-3p in renal cell carcinoma (RCC) has not been reported. METHODS AND RESULTS: We measured miR-23b-3p levels in 29 pairs of renal cell carcinoma and their normal matched tissues using real-time PCR. The expression level of miR-23b-3p was correlated with the 5 year survival rate of renal cancer patients. In 15 cases (52%), miR-23b-3p expression was found to be high. All patients with moderate to low miR-23b-3p expression survived 5 years, while those with high miR-23b-3p expression, only 50% survived. After knocking down miRNA-23b-3p expression in RCC cell lines, there was an induction of apoptosis and reduced invasive capabilities. MiR-23b-3p was shown to directly target PTEN gene through 3'UTR reporter assays. Inhibition of miR-23b-3p induces PTEN gene expression with a concomitant reduction in PI3-kinase, total Akt and IL-32. Immunohistochemistry showed the lack of PTEN protein expression in cancerous regions of tissue samples where the expression of miR-23b-3p was high. We studied the in vitro effects of the dietary chemo preventive agent genistein on miR-23b-3p expression and found that it inhibited expression of miR-23b-3p in RCC cell lines. CONCLUSIONS: The current study shows that miR-23b-3p is an oncogenic miRNA and inhibits PTEN tumor suppressor gene in RCC. Therefore, inhibition of miR-23b-3p may be a useful therapeutic target for the treatment of renal cell carcinoma.
Assuntos
Carcinoma de Células Renais/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , MicroRNAs/genética , PTEN Fosfo-Hidrolase/genética , Apoptose/genética , Sequência de Bases , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/mortalidade , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Genisteína/metabolismo , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/mortalidade , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Interferência de RNARESUMO
Genistein has been shown to suppress the growth of several cancers through modulation of various pathways. However, the effects of genistein on the regulation of oncogenic microRNA-151 (miR-151) have not been reported. In this study, we investigated whether genistein could alter the expression of oncogenic miR-151 and its target genes that are involved in the progression and metastasis of prostate cancer (PCa). Real-time RT-PCR showed that the expression of miR-151 was higher in PC3 and DU145 cells compared with RWPE-1 cells. Treatment of PC3 and DU145 cells with 25 µM genistein down-regulated the expression of miR-151 compared with vehicle control. Inhibition of miR-151 in PCa cells by genistein significantly inhibited cell migration and invasion. In-silico analysis showed that several genes (CASZ1, IL1RAPL1, SOX17, N4BP1 and ARHGDIA) suggested to have tumor suppressive functions were target genes of miR-151. Luciferase reporter assays indicated that miR-151 directly binds to specific sites on the 3'UTR of target genes. Quantitative real-time PCR analysis showed that the mRNA expression levels of the five target genes in PC3 and DU145 were markedly changed with miR-151 mimics and inhibitor. Kaplan-Meier curves and log-rank tests revealed that high expression levels of miR-151 had an adverse effect on survival rate. This study suggests that genistein mediated suppression of oncogenic miRNAs can be an important dietary therapeutic strategy for the treatment of PCa.
Assuntos
Antineoplásicos/farmacologia , Genisteína/farmacologia , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Oncogenes/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Regiões 3' não Traduzidas/genética , Idoso , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Biologia Computacional , Progressão da Doença , Técnicas de Silenciamento de Genes , Genes Reporter/genética , Vetores Genéticos/genética , Humanos , Luciferases/genética , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Regulação para Cima/efeitos dos fármacosRESUMO
ARHI is an imprinted tumor suppressor gene and is downregulated in various malignancies. However, ARHI expression, function, and mechanisms of action in prostate cancer have not been reported. Here, we report that ARHI mRNA and protein levels were downregulated in prostate cancer tissues compared with adjacent normal tissues. Overexpression of ARHI inhibited cell proliferation, colony formation, invasion, and induced apoptosis. Further studies on a new mechanism of ARHI downregulation showed a significant inverse relationship between ARHI and miR-221 and 222, which were upregulated in prostate cancer cell lines. Transfection of miR-221 and 222 inhibitors into PC-3 cells caused a significant induction of ARHI expression. A direct interaction of miR-221 or 222 with a target site on the 3'UTR of ARHI was confirmed by a dual luciferase pMIR-REPORT assay. Finally, we also found that genistein upregulates ARHI by downregulating miR-221 and 222 in PC-3 cells. In conclusion, ARHI is a tumor suppressor gene downregulated in prostate cancer, and overexpression of ARHI can inhibit cell proliferation, colony formation, and invasion. This study demonstrates for the first time that prostate cancer cells have decreased level of ARHI which could be caused by direct targeting of 3'UTR of ARHI by miR221/222. Genistein, a potential nontoxic chemopreventive agent, restores expression of ARHI and may be an important dietary therapeutic agent for treating prostate cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genisteína/farmacologia , MicroRNAs/genética , Neoplasias da Próstata/genética , Proteínas rho de Ligação ao GTP/genética , Anticarcinógenos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Neoplasias Ósseas/secundário , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Metilação de DNA/efeitos dos fármacos , Citometria de Fluxo , Genes Supressores de Tumor , Humanos , Técnicas Imunoenzimáticas , Luciferases/metabolismo , Masculino , Invasividade Neoplásica , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaio Tumoral de Célula-Tronco , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The Src family of protein kinases (SFK) plays key roles in regulating fundamental cellular processes, including cell growth, differentiation, cell shape, migration, and survival, and specialized cell signals in various malignancies. The pleiotropic functions of SFKs in cancer make them promising targets for intervention. Here, we sought to investigate the role of microRNA-205 (miR-205) in inhibition of Src-mediated oncogenic pathways in renal cancer. We report that expression of miR-205 was significantly suppressed in renal cancer cell lines and tumors when compared with normal tissues and a nonmalignant cell line and is correlated inversely with the expression of SFKs. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3'-UTR (untranslated region) sequences complementary to either Src, Lyn, or Yes, which was abolished by mutations in these 3'-UTR regions. Overexpression of miR-205 in A498 cells reduced Src, Lyn, and Yes expression, both at mRNA and protein levels. Proliferation of renal cancer cells was suppressed by miR-205, mediated by the phospho-Src-regulated ERK1/2 pathway. Cell motility factor FAK (focal adhesion kinase) and STAT3 activation were also inhibited by miR-205. Transient and stable overexpression of miR-205 in A498 cells resulted in induction of G0/G1 cell-cycle arrest and apoptosis, as indicated by decreased levels of cyclin D1 and c-Myc, suppressed cell proliferation, colony formation, migration, and invasion in renal cancer cells. miR-205 also inhibited tumor cell growth in vivo. This is the first study showing that miR-205 inhibits proto-oncogenic SFKs, indicating a therapeutic potential of miR-205 in the treatment of renal cancer.
Assuntos
Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , MicroRNAs/biossíntese , Quinases da Família src/antagonistas & inibidores , Apoptose/fisiologia , Carcinoma de Células Renais/enzimologia , Carcinoma de Células Renais/genética , Ciclo Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Regulação para Baixo , Humanos , Neoplasias Renais/enzimologia , Neoplasias Renais/genética , Sistema de Sinalização das MAP Quinases , MicroRNAs/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-yes/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Quinases raf/metabolismo , Proteínas ras/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
The minichromosome maintenance (MCM) gene family is essential for DNA replication and is frequently upregulated in various cancers. Here, we examined the role of MCM2 in prostate cancer and the effect of microRNA-1296 (miR-1296), genistein, and trichostatin A (TSA) on the MCM complex. Profiling results showed that expression of MCM genes was higher in tumor samples. Genistein and TSA significantly downregulated the expression of all MCM genes. Genistein, TSA, and small interfering RNA duplexes caused a significant decrease in the S phase of the cell cycle. There was also downregulation of CDT1, CDC7, and CDK2 genes, which govern loading of the MCM complex on chromatin. We also found that miR-1296 was significantly downregulated in prostate cancer samples. In PC3 cells, inhibition of miR-1296 upregulated both MCM2 mRNA and protein, whereas overexpression caused a significant decrease in MCM2 mRNA, protein, and the S phase of the cell cycle. MCM genes are excellent anticancer drug targets because they are essential DNA replication factors that are highly expressed in cancer cells. This is the first report showing anti-MCM effect by miR-1296, genistein, and TSA. TSA is undergoing clinical trials as a prostate cancer treatment but has high toxicity. Genistein, a natural, nontoxic dietary isoflavone, may be an advantageous therapeutic agent for treating prostate cancer. The use of RNA interference is currently being implemented as a gene-specific approach for molecular medicine. The specific downregulation of oncogenes by miR may contribute to novel therapeutic approaches in the treatment of prostate cancer.