RESUMO
The AU-rich element RNA-binding protein 1 (AUF1) is an RNA-binding protein, which can both stabilize and destabilize the transcripts of several cancer-related genes. Since epithelial-to-mesenchymal transition (EMT) and the acquisition of cancer stem cell traits are important for cancer onset and progression, we sought to determine the role of AUF1 in these two important processes. We have shown that AUF1 induces EMT and stemness in breast epithelial cells via stabilization of the SNAIL1 and TWIST1 mRNAs, and their consequent upregulation. Indeed, AUF1 binds the transcripts of these two genes at their 3'UTR and reduces their turnover. Ectopic expression of AUF1 also promoted stemness in mammary epithelial cells, and thereby increased the proportion of cancer stem cells. Importantly, breast cancer cells that ectopically express AUF1 were more efficient in forming orthotopic tumor xenografts in nude mice than their corresponding controls with limiting cell inocula. On the other hand, AUF1 downregulation with specific siRNA inhibited EMT and reduced the stemness features in breast cancer cells. Moreover, AUF1 knockdown sensitized breast cancer cells to the killing effect of cisplatin. Together, these findings provide clear evidence that AUF1 is an important inducer of the EMT process through stabilization of SNAIL1 and TWIST1 and the consequent promotion of breast cancer stem cells. Thereby, AUF1 targeted molecules could constitute efficient therapeutics for breast cancer patients.
RESUMO
During aging, cellular plasticity and senescence play important roles in tissue regeneration and the pathogenesis of different diseases, including cancer. We have recently shown that senescent breast luminal cells can activate their adjacent stromal fibroblasts. In the present report, we present clear evidence that these senescence-related active fibroblasts can dedifferentiate proliferating primary human luminal cells to multipotent stem cells in an interleukin-8 (IL-8)-dependent manner. This was confirmed using recombinant IL-8, while the truncated protein was not active. This IL-8-related dedifferentiation of luminal cells was mediated through the STAT3-dependent downregulation of p16INK4A and the microRNA miR-141. Importantly, these in vitro-generated mammary stem cells exhibited high molecular and cellular similarities to human mammary stem cells. They have also shown a long-term mammary gland-reconstituting ability and the capacity to produce milk postdelivery. Thereby, these IL-8-generated mammary stem cells could be of great value for autologous cell therapy procedures and also for biomedical research as well as drug development.
Assuntos
Mama/citologia , Mama/metabolismo , Interleucina-8/metabolismo , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Desdiferenciação Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Senescência Celular/fisiologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Interleucina-6/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fator de Transcrição STAT3/metabolismo , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
Osteosarcoma is the most common malignant bone tumor in children, adolescents, and young adults. This pleiomorphic tumor depends on new blood vessel development, also known as angiogenesis, for tumor growth and metastasis. Therefore, it's of utmost importance to identify the key genes and pathways that regulate this pro-metastatic process in order to develop more efficient therapies. Here, we have shown that the RNA-binding protein AUF1 positively regulates the expression of the pro-angiogenic factor VEGF-A and its positive regulator HIF-1alpha through direct binding and stabilization of their mRNAs. This effect is mediated through the seeding sequence of the AUF1 protein in the VEGF-A and HIF-1alpha 3'UTR sequences. As a consequence, the expression of the 3 genes was highly correlative in various osteosarcoma cell lines, and AUF1 enhanced the pro-angiogenic capabilities of osteosarcoma cells both in vitro and in vivo. Indeed, while inhibition of AUF1 using specific siRNA suppressed the pro-angiogenic effects of osteosarcoma cells, ectopic expression of AUF1 enhanced the pro-angiogenic effect in a VEGF-A-dependent manner. Therefore, in the era of targeted therapy, anti-angiogenic therapies targeting AUF1 could provide effective methods for treating osteosarcoma.
RESUMO
Increasing evidence supports the critical role of active stromal adipocytes in breast cancer development and spread. However, the mediators and the mechanisms of action are still elusive. We show here that cancer-associated adipocytes (CAAs) isolated from 10 invasive breast carcinomas are proinflammatory and exhibit active phenotypes, including higher proliferative, invasive, and migratory capacities compared to their adjacent tumor-counterpart adipocytes (TCAs). Furthermore, all CAAs secreted higher level of interleukin-8 (IL-8), which is critical in mediating the paracrine procarcinogenic effects of these cells. Importantly, ectopic expression of IL-8 in TCA cells activated them and enhanced their procarcinogenic effects both in vitro, in a STAT3-dependent manner, and in vivo In contrast, inhibition of the IL-8 signaling using specific short hairpin RNA, anti-IL-8 antibody, or reparixin suppressed the active features of CAAs, including their non-cell-autonomous tumor-promoting activities both on breast luminal cells and in orthotopic tumor xenografts in mice. IL-8 played also an important role in enhancing the proangiogenic effects of breast adipocytes. These results provide clear indication that IL-8 plays key roles in the activation of breast CAAs and acts as a major mediator for their paracrine protumorigenic effects. Thus, targeting CAAs by inhibiting the IL-8 pathway could have great therapeutic value.
Assuntos
Adipócitos/imunologia , Neoplasias da Mama/metabolismo , Interleucina-8/imunologia , Adipócitos/patologia , Indutores da Angiogênese/imunologia , Indutores da Angiogênese/metabolismo , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Carcinogênese/imunologia , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Transformação Celular Neoplásica , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Xenoenxertos , Humanos , Células MCF-7 , Camundongos , Invasividade Neoplásica , Neovascularização Patológica/metabolismo , Cultura Primária de Células , Fator de Transcrição STAT3/imunologia , Transdução de Sinais , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
Aging and stress promote senescence, which has intrinsic tumor suppressor functions and extrinsic tumor promoting properties. Therefore, it is of utmost importance to delineate the effects of senescence inducers on the various types of cells that compose the different organs. We show here that primary normal breast luminal (NBL) cells are more sensitive than their corresponding stromal fibroblasts to proliferative as well as oxidative damage-induced senescence. Like fibroblasts, senescent NBL cells secreted elevated amounts of various cytokines, including interleukin-6 (IL-6) and IL-8, and expressed high levels of p16, p21, and p53, while lamin B1 was downregulated. When senescent, luminal cells activated stromal fibroblasts in an IL-8-dependent manner, through the activation of the STAT3 pathway. These myofibroblasts promoted the epithelial-to-mesenchymal transition and the stemness processes in breast cancer cells in a paracrine manner both in vitro and in a breast cancer animal model. These results show the role of senescent breast luminal cells in promoting the inflammatory/carcinogenic microenvironment through the activation of fibroblasts in an IL-8-dependent manner.
Assuntos
Neoplasias da Mama/metabolismo , Interleucina-8/metabolismo , Células Estromais/metabolismo , Neoplasias da Mama/irrigação sanguínea , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Transformação Celular Neoplásica , Senescência Celular/fisiologia , Transição Epitelial-Mesenquimal , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Interleucina-6/metabolismo , Miofibroblastos/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Cultura Primária de Células , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Células Estromais/patologia , Microambiente Tumoral , Proteína Supressora de Tumor p53/metabolismoRESUMO
p16INK4A and p53 are two major tumor suppressor proteins that are both upregulated in response to various cellular stresses and during senescence and aging. p53 is a well-characterized transcription factor, while p16INK4A a cyclin-dependent kinase inhibitor encoded by the CDKN2A gene, and controls the expression of several genes through protein-protein interactions and also via miRNAs. This report demonstrates a p16INK4A-dependent positive regulation of p53 expression, at the protein level, in various human cells as well as in mouse embryonic fibroblasts. p16 suppresses p53 turnover through inhibition of its MDM2-related ubiquitination. This effect occurs through p16-related promotion of the MDM2 mRNA turnover via the p16INK4A downstream effectors miR-141 and miR-146b-5p, which bind specific sites at the 3' untranslated region of the MDM2 mRNA.Implications: The current findings show p16INK4A-dependent stabilization of p53 through miR-141/miR-146b-5p-related posttranscriptional repression of MDM2, thus providing new insights into the complex functional link between p16INK4A and p53. Mol Cancer Res; 16(8); 1299-308. ©2018 AACR.
Assuntos
Genes p16/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , Animais , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , TransfecçãoRESUMO
Obesity is increasingly recognized as a risk factor for breast cancer development. However, the molecular basis of obesity-related breast carcinogenesis remains elusive. In this study, we have shown that obesity reduces the level of the tumor suppressor p16INK4A protein in breast adipocytes, which showed active features and strong procarcinogenic potential both in vitro and in orthotopic tumor xenografts compared to mature adipocytes from lean women. Furthermore, obesity triggered epithelial-to-mesenchymal transition (EMT) in breast ductal epithelial cells. Interestingly, specific downregulation of p16INK4A increased the expression/secretion levels of various adipokines, including leptin, and activated breast adipocytes from lean women. Consequently, like breast adipocytes from obese women, p16-deficient adipocytes induced EMT in normal primary breast luminal cells in a leptin-dependent manner and enhanced tumor growth. Additionally, we have shown that p16INK4A negatively controls leptin at the mRNA level through microRNAs 141 and 146b-5p (miR-141 and miR-146b-5p), which bind the leptin mRNA at a specific sequence in the 3' untranslated region (UTR). These results show that obesity activates breast stromal adipocytes through p16 downregulation, which upregulates leptin and promotes procarcinogenic processes.
Assuntos
Adipócitos/metabolismo , Mama/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Obesidade/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Feminino , Fibroblastos/metabolismo , Humanos , Células Estromais/metabolismoRESUMO
p16INK4A and p53 are two important tumor suppressor proteins that play essential roles during cell proliferation and aging through regulating the expression of several genes. Here, we report that p16INK4A and p53 co-regulate a plethora of transcripts. Furthermore, both proteins colocalize in the nucleus of human primary skin fibroblasts and breast luminal cells, and form a heteromer whose level increases in response to genotoxic stress as well as aging of human fibroblasts and various mouse organs. CDK4 is also present in this heteromeric complex, which is formed only in the presence of DNA both in vitro using pure recombinant proteins and in vivo. We have also shown that p16INK4A enhances the binding efficiency of p53 to its cognate sequence presents in the CDKN1A promoter in vitro, and both proteins are present at the promoters of CDKN1A and BAX in vivo. Importantly, the fourth ankyrin repeat of p16INK4A and the C-terminal domain of p53 were necessary for the physical association between these two proteins. The physiologic importance of this association was revealed by the inability of cancer-associated p16INK4A mutants to interact with p53 and to transactivate the expression of its major targets CDKN1A and BAX in the p16-defective U2OS cells expressing either wild-type or mutated p16INK4A . Furthermore, the association between p16INK4A and p53 was capital for their nuclear colocalization, the X-ray-dependent induction of p21 and Bax proteins as well as the induction of apoptosis in various types of cells. Together, these results show DNA-dependent physical interaction between p16INK4A and p53.
Assuntos
Apoptose , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , DNA/metabolismo , Regulação da Expressão Gênica , Proteína Supressora de Tumor p53/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Senescence and epithelial-to-mesenchymal transition (EMT) processes are under the control of common tumor suppressor proteins, EMT transcription factors, and microRNAs. However, the molecular mechanisms that coordinate the functional link between senescence and EMT are still elusive. We have shown here that p16INK4A -related induction of senescence is mediated through miR-141 and miR-146b-5p. These two microRNAs are up-regulated in aging human fibroblast and epithelial cells. Furthermore, miR-141 and miR146b-5p trigger cell cycle arrest at G1 phase and induce senescence in primary human fibroblasts and breast cancer cells in the presence and absence of p16INK4A . Like p16INK4A -induced senescence, miR-141/miR146b-5p-related senescence is not associated with secretory phenotype, and is mediated through the RNA binding protein AUF1. We have further demonstrated that p16INK4A and its downstream miRNA targets inhibit EMT through suppressing the EMT inducer ZEB1 in an AUF1-dependent manner. Indeed, AUF1 binds the mRNA of this gene leading to increase in its level. These results indicate that p16INK4A controls both senescence and EMT through repressing EMT-related transcription factor via miR-141/miR146b-5p and their target AUF1. This sheds more light on the molecular basis of the tumor suppressive functions of p16INK4A , which represses both the proliferative and the migratory/invasive capacities of cells. © 2016 Wiley Periodicals, Inc.
Assuntos
Inibidor de Quinase Dependente de Ciclina p18/genética , Células Epiteliais/citologia , Fibroblastos/citologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , MicroRNAs/genética , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p18/metabolismo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Humanos , Regulação para CimaRESUMO
BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive histological subtype with limited treatment options and very poor prognosis following progression after standard chemotherapeutic regimens. Therefore, novel molecules and therapeutic options are urgently needed for this category of patients. Recently, we have identified PAC as a curcumin analogue with potent anti-cancer features. METHODS: HPLC was used to evaluate the stability of PAC and curcumin in PBS and also in circulating blood. Cytotoxicity/apoptosis was assessed in different breast cancer cell lines using propidium iodide/annexinV associated with flow cytometry. Furthermore, immunoblotting analysis determined the effects of PAC on different oncogenic proteins and pathways. Additionally, the real time xCELLigence RTCA technology was applied to investigate the effect of PAC on the cellular proliferation, migration and invasion capacities. RESULTS: PAC is more stable than curcumin in PBS and in circulating blood. Furthermore, we have shown differential sensitivity of estrogen receptor-alfa positive (ERα(+)) and estrogen receptor alfa negative (ERα(-)) breast cancer cells to PAC, which down-regulated ERα in both cell types. This led to complete disappearance of ERα in ERα(-) cells, which express very low level of this receptor. Interestingly, specific down-regulation of ERα in receptor positive cells increased the apoptotic response of these cells to PAC, confirming that ERα inhibits PAC-dependent induction of apoptosis, which could be mediated through ERα down-regulation. Additionally, PAC inhibited the proliferation and suppressed the epithelial-to-mesenchymal transition process in breast cancer cells, with higher efficiency on the TNBC subtype. This effect was also observed in vivo on tumor xenografts. Additionally, PAC suppressed the expression/secretion of 2 important cytokines IL-6 and MCP-1, and consequently inhibited the paracrine procarcinogenic effects of breast cancer cells on breast stromal fibroblasts. CONCLUSION: These results indicate that PAC could be considered as important candidate for future therapeutic options against the devastating TNBC subtype.
Assuntos
Antineoplásicos/administração & dosagem , Compostos de Benzilideno/administração & dosagem , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Piperidonas/administração & dosagem , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Compostos de Benzilideno/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Curcumina/administração & dosagem , Curcumina/análogos & derivados , Curcumina/farmacologia , Estabilidade de Medicamentos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-6/metabolismo , Camundongos , Piperidonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Colorectal cancer (CRC) is a major cause of cancer morbidity and mortality worldwide. Although response rates and overall survival have been improved in recent years, resistance to multiple drug combinations is inevitable. Therefore, the development of more efficient drugs, with fewer side effects is urgently needed. To this end, we have investigated in the present report the effect of PAC, a novel cucumin analogue, on CRC cells both in vitro and in vivo. We have shown that PAC induces apoptosis, mainly via the internal mitochondrial route, and inhibits cell proliferation through delaying the cell cycle at G2/M phase. Interestingly, the pro-apoptotic effect was mediated through STAT3-dependent down-regulation of cyclin D1 and its downstream target survivin. Indeed, change in the expression level of cyclin D1 modulated the expression of survivin and the response of CRC cells to PAC. Furthermore, using the ChIP assay, we have shown PAC-dependent reduction in the binding of STAT3 to the cyclin D1 promoter in vivo. Additionally, PAC suppressed the epithelial-to-mesenchymal process through down-regulating the mesenchymal markers (N-cadherin, vimentin and Twist1) and inhibiting the invasion/migration abilities of the CRC cells via repressing the pro-migration/invasion protein kinases AKT and ERK1/2. In addition, PAC inhibited tumor growth and repressed the JAK2/STAT3, AKT/mTOR and MEK/ERK pathways as well as their common downstream effectors cyclin D1 and survivin in humanized CRC xenografts. Collectively, these results indicate that PAC has potent anti-CRC effects, and therefore could constitute an effective alternative chemotherapeutic agent, which may consolidate the adjuvant treatment of colon cancer.
Assuntos
Antineoplásicos/uso terapêutico , Colo/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Curcumina/uso terapêutico , Ciclina D1/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Reto/efeitos dos fármacos , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Curcumina/análogos & derivados , Curcumina/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Células HCT116 , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Camundongos Nus , Reto/metabolismo , Reto/patologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The development and spread of mammary carcinomas require synergetic interplay between tumor cells and their microenvironment through paracrine secretions, which are still not well defined. We have shown here that interleukin-6 (IL-6), either recombinant or secreted from highly invasive breast cancer cells, down-regulates the tumor suppressor proteins p16(INK4A), p21(WAF1), and p53 and activates breast stromal fibroblasts in a paracrine manner. The formation of myofibroblasts requires p16(INK4A) down-regulation and the activation of the JAK2/STAT3 pathway. Indeed, the transcription factor STAT3 positively controls the expression of the three major myofibroblast markers, SDF-1, α-smooth muscle actin (α-SMA), and TGF-ß1, and mediates IL-6-related down-regulation of p16(INK4A), p21(WAF1), and p53 as well as the activation of stromal fibroblasts. Importantly, these effects were mediated through STAT3-dependent up-regulation of the mRNA-binding protein AUF1, whose promoter contains three canonical STAT3 binding sites. AUF1 binds the SDF-1, α-SMA, TGF-ß1, and IL-6 mRNAs and reduces their turnover. Consequently, specific AUF1 down-regulation inhibits IL-6-dependent activation of breast stromal fibroblasts, whereas AUF1 ectopic expression of p37(AUF1) activated these cells and enhanced their paracrine induction of epithelial-to-mesenchymal transition in breast cancer cells, which shows a non-cell-autonomous oncogenic function of AUF1. Together, these results demonstrate a major role of IL-6 in activating breast stromal fibroblasts through STAT3-dependent AUF1 induction.
Assuntos
Fibroblastos/citologia , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Células Estromais/citologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Meios de Cultivo Condicionados/química , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Fibroblastos/metabolismo , Ribonucleoproteína Nuclear Heterogênea D0 , Humanos , Miofibroblastos/metabolismo , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
miR-141 and miR-146b-5p are two important tumor suppressor microRNAs, which control several cancer-related genes and processes. In the present report, we have shown that these microRNAs bind specific sites at the 3'-untranslated region (UTR) of the mRNA-binding protein AUF1, leading to its down-regulation. This inverse correlation between the levels of these microRNAs and AUF1 has been identified in various osteosarcoma cell lines. Additionally, we present clear evidence that AUF1 promotes mesenchymal features in osteosarcoma cells and that miR-141 and miR-146b-5p suppress this prometastatic process through AUF1 repression. Indeed, both microRNAs suppressed the invasion/migration and proliferation abilities of osteosarcoma cells through inhibiting the AKT protein kinase in an AUF1-dependent manner. We have also shown that AUF1 binds to and stabilizes the mRNA of the AKT activator phosphoinositide-dependent kinase-1 (PDK1). Furthermore, miR-141 and miR-146b-5p positively regulate the epithelial markers (E-cadherin and Epcam) and repress the mesenchymal markers (N-cadherin, Vimentin, Twist2, and ZEB1). These effects were mediated via the repression of the epithelial-to-mesenchymal inducer ZEB1 through targeting AUF1, which binds the 3'-UTR of the ZEB1 mRNA and reduces its turnover. These results indicate that at least some tumor suppressor functions of miR-141 and miR-146b-5p are mediated through the repression of the oncogenic potentials of AUF1. Therefore, these 3'-UTR-directed post-transcriptional gene expression regulators constitute promising new targets for diagnostic and/or therapeutic interventions.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo D/química , Proteínas de Homeodomínio/metabolismo , MicroRNAs/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/metabolismo , Regiões 3' não Traduzidas , Sequência de Bases , Proliferação de Células , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/fisiologia , Humanos , Mesoderma/citologia , Dados de Sequência Molecular , Metástase Neoplásica , Neoplasias/metabolismo , Prognóstico , Ligação Proteica , Homologia de Sequência do Ácido Nucleico , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Ultraviolet (UV) light is a carcinogenic agent that upregulates the expression of several genes involved in various cellular processes, including cell cycle checkpoints and apoptosis. The universal cyclin-dependent kinase inhibitor p21(WAF1/Cip1) plays major roles in these processes, and the level of its corresponding message increases several times in response to UV-induced DNA damage. This upregulation is mainly posttranscriptional owing to HuR-dependent mRNA stabilization. Since the protein kinase Atr plays major roles during the cellular response to UV damage, we sought to investigate its possible implication in the stabilization of the p21(WAF1/Cip1) coding mRNA. We have shown that the UV-dependent accumulation of the CDKN1A mRNA is indeed under the control of the Atr protein kinase. Upon UV damage, Atr allows nuclear-cytoplasmic shuttling of the HuR protein, which binds the CDKN1A mRNA and reduces its turnover. This ATR-dependent effect is mediated through UV-related phosphorylation/inactivation of the Cdk1 protein kinase by Atr, which leads to the dissociation of HuR from Cdk1. Indeed, inhibition or shRNA specific knockdown of CDK1 in ATR-deficient cells enhanced the cytoplasmic level of HuR and restored the CDKN1A mRNA upregulation in response to UV damage. These results show that ATR stabilizes the CDKN1A message in response to UV damage through Cdk1-related cytoplasmic accumulation of HuR.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Quinases Ciclina-Dependentes/genética , Proteínas ELAV/genética , RNA Mensageiro/genética , Regulação para Cima/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteína Quinase CDC2 , Proteínas de Ciclo Celular/genética , Linhagem Celular , Citoplasma/genética , Dano ao DNA/genética , Proteína Semelhante a ELAV 1 , Humanos , Fosforilação/genética , Ativação Transcricional/genética , Raios UltravioletaRESUMO
p16(INK4a) is a tumor suppressor protein involved in several stress-related cellular responses, including apoptosis. Recent lines of evidence indicate that p16(INK4a) is also a modulator of gene expression. However, the molecular mechanisms underlying this novel function are still obscure. Here, we present clear evidence that p16(INK4a) modulates the levels of various microRNAs, with marked positive effect on miR-141 and miR-146b-5p. This effect is mediated through the formation of the p16-CDK4-Sp1 heterocomplex, which binds to Sp1 consensus-binding motifs present in the promoters of miR-141 and miR-146b-5p, and it enables their transcription. In addition, we have shown that p16(INK4a) interacts with Sp1 through the fourth ankyrin repeat, which is crucial for Sp1 binding to the miR-141 and miR-146b-5p promoters and their transcriptional activation. The physiological importance of this association was revealed by the inability of cancer-related p16(INK4a) mutants to interact with Sp1. Moreover, we have shown p16-CDK4-Sp1-dependent up-regulation of miR-141 and miR-146b-5p following UV light-induced DNA damage and the role of these two microRNAs in mediating p16-related induction of apoptosis in response to this genotoxic stress. Together, these results indicate that p16(INK4a) associates with CDK4 not only to inhibit the cell cycle but also to enable the transcription of two important onco-microRNAs, which act as downstream effectors.
Assuntos
Quinase 4 Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fator de Transcrição Sp1/metabolismo , Sequência de Bases , Ciclo Celular , Linhagem Celular , Quinase 4 Dependente de Ciclina/química , Inibidor p16 de Quinase Dependente de Ciclina/química , Dano ao DNA , Humanos , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Estabilidade de RNA , Fator de Transcrição Sp1/química , Ativação Transcricional/efeitos da radiação , Raios Ultravioleta/efeitos adversosRESUMO
BACKGROUND: p16(INK4a) and p21(WAF1) are two independent cyclin-dependent kinase inhibitors encoded by the CDKN2A and CDKN1A genes, respectively. p16(INK4a) and p21(WAF1) are similarly involved in various anti-cancer processes, including the regulation of the critical G1 to S phase transition of the cell cycle, senescence and apoptosis. Therefore, we sought to elucidate the molecular mechanisms underlying the link between these two important tumor suppressor proteins. METHODOLOGY/PRINCIPAL FINDINGS: We have shown here that the p16(INK4a) protein positively controls the expression of p21(WAF1) in both human and mouse cells. p16(INK4a) stabilizes the CDKN1A mRNA through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by quantitative RT-PCR indicated that endogenous AUF1 binds to the CDKN1A mRNA in a p16(INK4A)-dependent manner. Furthermore, while AUF1 down-regulation increased the expression level of the CDKN1A mRNA, the concurrent knockdown of AUF1 and CDKN2A, using specific silencing RNAs, restored the normal expression of the gene. Moreover, we used EGFP reporter fused to the CDKN2A AU-rich element (ARE) to demonstrate that p16(INK4A) regulation of the CDKN1A mRNA is AUF1- and ARE-dependent. Furthermore, ectopic expression of p16(INK4A) in p16(INK4A)-deficient breast epithelial MCF-10A cells significantly increased the level of p21(WAF1), with no effect on cell proliferation. In addition, we have shown direct correlation between p16(INK4a) and p21(WAF1) levels in various cancer cell lines. CONCLUSION/SIGNIFICANCE: These findings show that p16(INK4a) stabilizes the CDKN1A mRNA in an AUF1-dependent manner, and further confirm the presence of a direct link between the 2 important cancer-related pathways, pRB/p16(INK4A) and p14(ARF)/p53/p21(WAF1).
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação da Expressão Gênica , Animais , Linhagem Celular Tumoral , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Humanos , Camundongos , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Transdução de SinaisRESUMO
Activated cancer-associated fibroblasts (CAFs) or myofibroblasts not only facilitate tumor growth and spread but also affect tumor response to therapeutic agents. Therefore, it became clear that efficient therapeutic regimens should also take into account the presence of these supportive cells and inhibit their paracrine effects. To this end, we tested the effect of low concentrations of curcumin, a pharmacologically safe natural product, on patient-derived primary breast CAF cells. We have shown that curcumin treatment upregulates p16(INK4A) and other tumor suppressor proteins while inactivates the JAK2/STAT3 pathway. This reduced the level of alpha-smooth muscle actin (α-SMA) and the migration/invasion abilities of these cells. Furthermore, curcumin suppressed the expression/secretion of stromal cell-derived factor-1 (SDF-1), interleukin-6 (IL-6), matrix metalloproteinase-2 (MMP-2), MMP-9, and transforming growth factor-ß, which impeded their paracrine procarcinogenic potential. Intriguingly, these effects were sustained even after curcumin withdrawal and cell splitting. Therefore, using different markers of senescence [senescence-associated ß-galactosidase (SA-ß-gal) activity, Ki-67 and Lamin B1 levels, and bromodeoxyuridine incorporation], we have shown that curcumin markedly suppresses Lamin B1 and triggers DNA damage-independent senescence in proliferating but not quiescent breast stromal fibroblasts. Importantly, this curcumin-related senescence was p16(INK4A)-dependent and occurred with no associated inflammatory secretory phenotype. These results indicate the possible inactivation of cancer-associated myofibroblasts and present the first indication that curcumin can trigger DNA damage-independent and safe senescence in stromal fibroblasts.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/patologia , Curcumina/farmacologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Fibroblastos/efeitos dos fármacos , Actinas/metabolismo , Neoplasias da Mama/metabolismo , Movimento Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Fibroblastos/citologia , Fibroblastos/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Antígeno Ki-67/metabolismo , Lamina Tipo B/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fator de Transcrição STAT3/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Survivin, an important anti-apoptotic protein, is highly expressed in most cancers, which generally arise in cells of older individuals. We have shown here accumulation of survivin and phospho-survivin in aged normal human skin fibroblasts and mice organs. This age-related accumulation of survivin was due to protein stabilization through association with the molecular chaperone Hsp90 protein, which was also up-regulated during aging. Interestingly, Hsp90 binds preferentially to phospho-survivin, which explains its higher stability. In addition, we provide clear evidence that aged cells exhibit apoptosis resistance when challenged with UV light, cisplatin, γ-rays or H2O2 as compared to their younger counterparts. In response to γ-rays and H2O2, the levels of Bcl-2 and both forms of survivin were up-regulated in old cells, but not in their corresponding young ones. This repression of survivin and phospho-survivin in young cells is p53 dependent. Importantly, survivin inhibition/down-regulation with flavopiridol or specific shRNAs increased the apoptotic response of old fibroblasts to various genotoxic agents, and restored the pro-apoptotic Bax/Bcl2 ratio and the increase in the levels of cleaved caspase-3 and PARP in old cells. These results show the role of survivin in the age-dependent resistance of human fibroblasts, and provide new insights into the molecular mechanisms that underlie the complex relationship between aging, apoptosis, and cancer.
Assuntos
Envelhecimento/genética , Apoptose/genética , Dano ao DNA/genética , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Proteínas Inibidoras de Apoptose/genética , RNA/genética , Proteínas Repressoras/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Modelos Animais de Doenças , Feminino , Fibroblastos/patologia , Citometria de Fluxo , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Proteínas Inibidoras de Apoptose/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , RNA/metabolismo , Proteínas Repressoras/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SurvivinaRESUMO
The universal cyclin-dependent kinase inhibitor p21(WAF1/Cip1) promotes cell cycle arrest and inhibits apoptosis in response to UV-induced DNA damage. Since the protein kinase ATR plays a major role in the cellular response to these carcinogenic lesions, we investigated the possible role of ATR in the modulation of p21(WAF1/Cip1) expression in response to UVC radiation. We have shown that p21(WAF1/Cip1) is up-regulated in human fibroblast and epithelial cells, but only in response to low UV fluences and low passage cells. Importantly, this up-regulation is ATR-dependent. In fact, in ATR-deficient or caffeine-treated cells UV light rather down-regulated the p21(WAF1/Cip1) protein through SKP2-dependent ubiquitination and degradation via the proteasomal pathway. Furthermore, we present evidence that ATR inhibits apoptosis in response to low fluences of UV light, through inhibiting the cleavage of caspase 3 and PARP as well as the repression of the proapoptotic proteins BAX and BAK. Interestingly, ATR is also required for the stability of the p21(WAF1/Cip1) protein in absence of genotoxic stress. Together, these results indicate that during the cellular response to low UVC fluences the ATR protein kinase up-regulates p21(WAF1/Cip1) and inhibits apoptosis. © 2011 Wiley Periodicals, Inc.
Assuntos
Apoptose/efeitos da radiação , Proteínas de Ciclo Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Raios Ultravioleta , Regulação para Cima/efeitos da radiação , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Ciclo Celular , Relação Dose-Resposta à Radiação , Eletroforese em Gel de Poliacrilamida , Humanos , Ubiquitinação , Regulação para Cima/fisiologiaRESUMO
BACKGROUND: The cyclin-D/CDK4,6/p16(INK4a)/pRB/E2F pathway, a key regulator of the critical G1 to S phase transition of the cell cycle, is universally disrupted in human cancer. However, the precise function of the different members of this pathway and their functional interplay are still not well defined. METHODOLOGY/PRINCIPAL FINDINGS: We have shown here that the tumor suppressor p16(INK4a) protein positively controls the expression of cyclin D1 and E2F1 in both human and mouse cells. p16(INK4a) stabilizes the mRNAs of the corresponding genes through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by RT-PCR indicated that endogenous AUF1 binds to the cyclin D1 and E2F1 mRNAs. Furthermore, AUF1 down-regulation increased the expression levels of these genes, while concurrent silencing of AUF1 and p16(INK4a), using specific siRNAs, restored normal expression of both cyclinD1 and E2F1. Besides, we have shown the presence of functional AU-rich elements in the E2F1 3'UTR, which contributed to p16/AUF1-mediated regulation of E2F1 post-transcriptional events in vivo. Importantly, genome-wide gene expression microarray analysis revealed the presence of a large number of genes differentially expressed in a p16(INK4a) -dependent manner, and several of these genes are also members of the AUF1 and E2F1 regulons. We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis. CONCLUSION/SIGNIFICANCE: These findings show that the cyclin-dependent kinase inhibitor p16( INK4a) is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1.