RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers because of late symptoms and resistance to chemotherapy and radiation therapy. We have investigated the appearance of c-kit, a stem cell marker, in both normal adult pancreatic tissue and in cancerous tissue. Apart from some very pale staining of islets of Langerhans, normal pancreas was devoid of staining with antibodies to c-kit. In contrast, in cancerous tissue that still preserves the overall integrity of the pancreatic tissue, there was a clear labeling in islets of Langerhans, which seemed to be co-localized with insulin containing ß cells. In other cases, where the pancreatic tissue was completely deteriorated, intensive labeling was clearly evident in remnants of both the exocrine and the endocrine tissues. The duct cells of the adenocarcinoma were moderately but clearly labeled with antibodies to c-kit. In contrast, in metastasis of PDAC, very intensive labeling of c-kit was evident. The location of KRAS, which is strongly associated with PDAC, was also analyzed at the initial stages of the disease, when islets of Langerhans still preserve their integrity to a large extent. KRAS was found exclusively in islets of Langerhans and overlapped in its location with insulin and c-kit expressing cells. It is suggested that the modulation of the expression of c-kit, visualized by antibodies to the oncogene molecule, may play an important role in the formation and progression of PDAC. The absence of c-kit in normal pancreas and its appearance in PDAC is probably due to a mutational event, which probably allows conversion of the ß cells into cancer stem cells (CSC). Co-expression of both c-kit and KRAS, typical markers for CSC with overlapping with insulin in islets of Langerhans, strongly support the notion that ß-cells play a central role in the development of PDAC. The use of specific drugs that can attenuate the kinase activity of c-kit or target KRAS expressing cancer cells should be tested in order to attenuate the progression of this lethal disease.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/enzimologia , Células-Tronco Neoplásicas/enzimologia , Neoplasias Pancreáticas/enzimologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Idoso , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Progressão da Doença , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Pâncreas/enzimologia , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-kit/genéticaRESUMO
Pancreas cancer, is the fourth leading cause of cancer death but its cell of origin is controversial. We compared the localization of stem cells in normal and cancerous pancreas using antibodies to the stem cell markers Nanog and LGR5. Here we show, for the first time, that LGR5 is expressed in normal pancreas, exclusively in the islets of Langerhans and it is co-localized, surprisingly, with Nanog and insulin in clusters of beta cells. In cancerous pancreas Nanog and LGR5 are expressed in the remaining islets and in all ductal cancer cells. We observed insulin staining among the ductal cancer cells, but not in metastases. This indicates that the islet's beta cells, expressing LGR5 and Nanog markers are the initiating cells of pancreas cancer, which migrated from the islets to form the ductal cancerous tissue, probably after mutation and de-differentiation. This discovery may facilitate treatment of this devastating cancer.
Assuntos
Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Receptores Acoplados a Proteínas G/metabolismo , Idoso , Biomarcadores/análise , Biomarcadores/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/metabolismo , Pessoa de Meia-Idade , Proteína Homeobox Nanog , Células-Tronco Neoplásicas/patologia , Ductos Pancreáticos/metabolismo , Valores de ReferênciaRESUMO
One paradigm of cancer development claims that cancer emerges at the niche of tissue stem cells and these cells continue to proliferate in the tumor as cancer stem cells. LGR5, a membrane receptor, was recently found to be a marker of normal colon stem cells in colon polyps and is also expressed in colon cancer stem cells. Nanog, an embryonic stem cell nuclear factor, is expressed in several embryonic tissues, but Nanog expression is not well documented in cancerous stem cells. Our aim was to examine whether both LGR5 and Nanog are expressed in the same clusters of colon stem cells or cancer stem cells, using immunocytochemistry with specific antibodies to each antigen. We analyzed this aspect using paraffin embedded tumor tissue sections obtained from 18 polyps and 36 colon cancer specimens at stages I-IV. Antibodies to LGR5 revealed membrane and cytoplasm immunostaining of scattered labeled cells in normal crypts, with no labeling of Nanog. However, in close proximity to the tumors, staining to LGR5 was much more intensive in the crypts, including that of the epithelial cells. In cancer tissue, positive LGR5 clusters of stem cells were observed mainly in poorly differentiated tumors and in only a few scattered cells in the highly differentiated tumors. In contrast, antibodies to Nanog mainly stained the growing edges of carcinoma cells, leaving the poorly differentiated tumor cells unlabeled, including the clustered stem cells that could be detected even by direct morphological examination. In polyp tissues, scattered labeled cells were immunostained with antibodies to Nanog and to a much lesser extent with antibodies to LGR5. We conclude that expression of LGR5 is probably specific to stem cells of poorly differentiated tumors, whereas Nanog is mainly expressed at the edges of highly differentiated tumors. However, some of the cell layers adjacent to the carcinoma cell layers that still remained undifferentiated, expressed mainly Nanog with only a few cells labeled with antibodies to LGR5. Considering the different sites and pattern of expression in the tumor, our data imply that targeting the clustered stem cells expressing LGR5 in poorly differentiated colon cancer may require different strategies than targeting the stem cells expressing Nanog in the highly differentiated tumors. Alternatively, combined application of specific inhibitory miRNAs to Nanog and to LGR5 expression may assist therapeutically.
Assuntos
Neoplasias do Colo/patologia , Proteínas de Homeodomínio/metabolismo , Células-Tronco Neoplásicas/patologia , Receptores Acoplados a Proteínas G/metabolismo , Diferenciação Celular , Colo/citologia , Colo/patologia , Neoplasias do Colo/fisiopatologia , Humanos , Proteína Homeobox Nanog , Gradação de TumoresRESUMO
LGR5 and Nanog were recently characterized as stem cell markers in various embryonic, adult and cancer stem cells. However, there are no data on their precise localization in the normal adult ovary, which may be important for the initial steps of development of ovarian cancer, the most lethal gynecological cancer. We analyzed by immunocytochemistry the precise localization of these markers in normal ovary (11 specimens, age range 43-76), in borderline specimens (12 specimens), and in serous ovarian cancer (12 specimens of stage II) which comprises the vast majority (80%) of all ovarian cancer. Surprisingly, we revealed that both Nanog and LGR5 are clearly localized in the epithelial cells of the normal ovary. However, in 5 of 12 ovaries there was no labeling at all, while in 3 ovaries staining of Nanog was more prominent with only weak labeling of LGR5. In addition, we found in 3 of 11 ovaries clear labeling in foci of both LGR5 and Nanog antibodies, with partial overlapping. Occasionally, we also found in the stroma foci labeled by either Nanog or LGR5 antibodies. In general, the stroma area of tissue sections labeled with LGR5 was much greater than that labeled with Nanog. In borderline tumors a significant portion of the specimens (7 of 12) was labeled exclusively with Nanog and not with LGR5. In ovarian carcinomas almost 100% of the cells were exclusively labeled only with Nanog (6 of 12 of the specimens) with no labeling of LGR5. These data may suggest the potential of ovaries from postmenopausal women, which express Nanog, to undergo transformation, since Nanog was shown to be oncogenic. We conclude that Nanog, which probably plays an important role in ovarian embryonic development, may be partially silenced in fertile and post-menopausal women, but is re-expressed in ovarian cancer, probably by epigenetic activation of Nanog gene expression. Expression of Nanog and LGR5 in normal ovaries and in borderline tumors may assist in the early detection and improved prognosis of ovarian cancer. Moreover, targeting of Nanog by inhibitory miRNA or other means may assist in treating this disease.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Homeodomínio/metabolismo , Neoplasias Ovarianas/patologia , Ovário/patologia , Receptores Acoplados a Proteínas G/metabolismo , Adulto , Idoso , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Proteína Homeobox Nanog , Gradação de Tumores , Neoplasias Ovarianas/metabolismo , Ovário/metabolismo , Pós-MenopausaAssuntos
Proteína Supressora de Tumor p53/metabolismo , Zinco/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Mutação , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Tiossemicarbazonas/química , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Vimentin, a mesenchymal marker, is frequently overexpressed in epithelial carcinomas undergoing epithelial to mesenchymal transition (EMT), a condition correlated with invasiveness and poor prognosis. Therefore, vimentin is a potential molecular target for anticancer therapy. Emerging studies in experimental models underscore the functions of homeodomain-interacting protein kinase 2 (HIPK2) as potential oncosuppressor by acting as transcriptional corepressor or catalytic activator of molecules involved in apoptosis and response to antitumor drugs. However, an involvement of HIPK2 in limiting tumor invasion remains to be elucidated. This study, by starting with a microarray analysis, demonstrates that HIPK2 downregulates vimentin expression in invasive, vimentin-positive, MDA-MB-231 breast cancer cells and in the non-invasive MCF7 breast cancer cells subjected to chemical hypoxia, a drive for mesenchymal shift and tumor invasion. At functional level, vimentin downregulation by HIPK2 correlates with inhibition of breast tumor cell invasion. Together, these data show that vimentin is a novel target for HIPK2 repressor function and that HIPK2-mediated vimentin downregulation can contribute to inhibition of breast cancer cells invasion that might be applied in clinical therapy.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Transporte/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Vimentina/metabolismo , Apoptose/fisiologia , Neoplasias da Mama/genética , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Regulação para Baixo , Feminino , Humanos , Invasividade Neoplásica , Proteínas Serina-Treonina Quinases/genética , Transfecção , Vimentina/genéticaRESUMO
Activated p53 is necessary for tumor suppression. Homeodomain-interacting protein kinase-2 (HIPK2) is a positive regulator of functional p53. HIPK2 modulates wild-type p53 activity toward proapoptotic transcription and tumor suppression by the phosphorylation of serine 46. Knock-down of HIPK2 interferes with tumor suppression and sensitivity to chemotherapy. Combined administration of adriamycin and zinc restores activity of misfolded p53 and enables the induction of its proapoptotic and tumor suppressor functions in vitro and in vivo. We therefore looked for a cancer model where HIPK2 expression is low. MMTV-neu transgenic mice overexpressing HER2/neu, develop mammary tumors at puberty with a long latency, showing very low expression of HIPK2. Here we show that whereas these tumors are resistant to adriamycin treatment, a combination of adriamycin and zinc suppresses tumor growth in vivo in these mice, an effect evidenced by the histological features of the mammary tumors. The combined treatment of adriamycin and zinc also restores wild-type p53 conformation and induces proapoptotic transcription activity. These findings may open up new possibilities for the treatment of human cancers via the combination of zinc with chemotherapeutic agents, for a selected group of patients expressing low levels of HIPK2, with an intact p53. In addition, HIPK2 may serve as a new biomarker for tumor aggressiveness.
Assuntos
Antineoplásicos/uso terapêutico , Doxorrubicina/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Proteína Supressora de Tumor p53/fisiologia , Zinco/administração & dosagem , Animais , Apoptose , Sequência de Bases , Primers do DNA , Genes erbB-2 , Neoplasias Mamárias Experimentais/virologia , Vírus do Tumor Mamário do Camundongo/isolamento & purificação , Camundongos , Camundongos Transgênicos , Dobramento de Proteína , Reação em Cadeia da Polimerase em Tempo Real , Proteína Supressora de Tumor p53/metabolismoRESUMO
Hypoxia-inducible factor 1 (HIF-1), the major transcription factor specifically activated during hypoxia, regulates genes involved in critical aspects of cancer biology, including angiogenesis, cell proliferation, glycolysis and invasion. The HIF-1a subunit is stabilized by low oxygen, genetic alteration and cobaltous ions, and its over-expression correlates with drug resistance and increased cancer mortality in various cancer types, therefore representing an important anticancer target. Zinc supplementation has been shown to counteract the hypoxic phenotype in cancer cells, in vitro and in vivo, hence, understanding the molecular pathways modulated by zinc under hypoxia may provide the basis for reprogramming signalling pathways for anticancer therapy. Here we performed genome-wide analyses of colon cancer cells treated with combinations of cobalt, zinc and anticancer drug and evaluated the effect of zinc on gene expression patterns. Using Principal Component Analysis we found that zinc markedly reverted the cobalt-induced changes of gene expression, with reactivation of the drug-induced transcription of pro-apoptotic genes. We conclude that the hypoxia pathway is a potential therapeutic target addressed by zinc that also influences tumor cell response to anticancer drug.
Assuntos
Antineoplásicos/farmacologia , Cobalto/farmacologia , Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Zinco/farmacologia , Apoptose/efeitos dos fármacos , Hipóxia Celular , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Doxorrubicina/farmacologia , Estudo de Associação Genômica Ampla , Humanos , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Oxigênio/análise , Análise de Componente Principal , Transdução de SinaisRESUMO
Absence of p53 expression or expression of mutant p53 (mtp53) are common in human cancers and are associated with increased cancer resistance to chemo- and radiotherapy. Therefore, significant efforts towards pharmaceutical reactivation of defective p53 pathways are underway. We previously reported that, in HIPK2 knockdown background, p53 undergoes misfolding with inhibition of DNA binding and transcriptional activities that correlate with increased chemoresistance, and that zinc rescues wild-type p53 activity. Zinc has a crucial role in the biology of p53, in that p53 binds to DNA through a structurally complex domain stabilized by zinc atom. In this study, we explored the role of zinc in p53 reactivation in mutant p53-expressing cancer cells. We found that zinc re-established chemosensitivity in breast cancer SKBR3 (expressing R175H mutation) and glioblastoma U373MG (expressing R273H mutation) cell lines. Biochemical studies showed that zinc partly induced the transition of mutant p53 protein (reactive to conformation-sensitive PAb240 antibody for mutant conformation) into a functional conformation (reactive to conformation-sensitive PAb1620 antibody for wild-type conformation). Zinc-mediated p53 reactivation also reduced the mtp53/p73 interaction restoring both wtp53 and p73 binding to target gene promoters by ChIP assay with in vivo induction of wtp53 target gene expression, which rendered mutant p53 cells more prone to drug killing in vitro. Finally, zinc administration in U373MG tumor xenografts increased drug-induced tumor regression in vivo, which correlated with increased wild-type p53 protein conformation. These results show that the use of zinc might restore drug sensitivity and inhibit tumor growth by reactivating mutant p53.
Assuntos
Antineoplásicos/farmacologia , Proteína Supressora de Tumor p53/química , Zinco/fisiologia , Animais , Antineoplásicos/uso terapêutico , Apoptose , Neoplasias da Mama/tratamento farmacológico , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Camundongos , Camundongos Nus , Mutação , Proteínas Nucleares/metabolismo , Conformação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transplante Heterólogo , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Zinco/farmacologiaRESUMO
BACKGROUND: Hypoxia inducible factor-1α (HIF-1α) is responsible for the majority of HIF-1-induced gene expression changes under hypoxia and for the "angiogenic switch" during tumor progression. HIF-1α is often upregulated in tumors leading to more aggressive tumor growth and chemoresistance, therefore representing an important target for antitumor intervention. We previously reported that zinc downregulated HIF-1α levels. Here, we evaluated the molecular mechanisms of zinc-induced HIF-1α downregulation and whether zinc affected HIF-1α also in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that zinc downregulated HIF-1α protein levels in human prostate cancer and glioblastoma cells under hypoxia, whether induced or constitutive. Investigations into the molecular mechanisms showed that zinc induced HIF-1α proteasomal degradation that was prevented by treatment with proteasomal inhibitor MG132. HIF-1α downregulation induced by zinc was ineffective in human RCC4 VHL-null renal carcinoma cell line; likewise, the HIF-1αP402/P564A mutant was resistant to zinc treatment. Similarly to HIF-1α, zinc downregulated also hypoxia-induced HIF-2α whereas the HIF-1ß subunit remained unchanged. Zinc inhibited HIF-1α recruitment onto VEGF promoter and the zinc-induced suppression of HIF-1-dependent activation of VEGF correlated with reduction of glioblastoma and prostate cancer cell invasiveness in vitro. Finally, zinc administration downregulated HIF-1α levels in vivo, by bioluminescence imaging, and suppressed intratumoral VEGF expression. CONCLUSIONS/SIGNIFICANCE: These findings, by demonstrating that zinc induces HIF-1α proteasomal degradation, indicate that zinc could be useful as an inhibitor of HIF-1α in human tumors to repress important pathways involved in tumor progression, such as those induced by VEGF, MDR1, and Bcl2 target genes, and hopefully potentiate the anticancer therapies.
Assuntos
Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Zinco/farmacologia , Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Progressão da Doença , Glioblastoma/metabolismo , Humanos , Técnicas In Vitro , Masculino , Neoplasias da Próstata/metabolismo , Complexo de Endopeptidases do Proteassoma/farmacologia , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
Homeodomain-interacting protein kinase-2 (HIPK2) is a crucial regulator of p53 apoptotic function by phosphorylating serine 46 (Ser46) in response to DNA damage. In tumors with wild-type p53, its tumor suppressor function is often impaired by MDM2 overexpression that targets p53 for proteasomal degradation. Likewise, MDM2 targets HIPK2 for protein degradation impairing p53-apoptotic function. Here we report that zinc antagonised MDM2-induced HIPK2 degradation as well as p53 ubiquitination. The zinc inhibitory effect on MDM2 activity leads to HIPK2-induced p53Ser46 phosphorylation and p53 pro-apoptotic transcriptional activity. These results suggest that zinc derivatives are potential molecules to target the MDM2-induced HIPK2/p53 inhibition.
Assuntos
Apoptose/fisiologia , Proteínas de Transporte/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Transporte/genética , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Genes p53 , Células HeLa , Humanos , Modelos Biológicos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/genética , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Ubiquitinação/efeitos dos fármacos , Zinco/farmacologiaRESUMO
Oncosuppressor p53 is often inactivated by either mutations or deregulation of regulatory proteins. These include the homeodomain-interacting protein kinase 2 (HIPK2) that, by phosphorylating p53 at Ser46 modulates p53 response to DNA damage by inducing pro-apoptotic transcription. There is compelling evidence that HIPK2 is also involved in the response to hypoxia by acting as co-suppressor of hypoxia inducible factor 1α (HIF-1α), a major factor in cancer progression that activates the transcription of genes involved in angiogenesis, glucose metabolism and invasion. Hence conditions that induce HIPK2 deregulation would end up in a multifactorial response leading to tumor chemoresistance by affecting p53 activity on one hand and to angiogenesis and cell proliferation by affecting HIF-1 activity on the other hand. For these reasons, HIPK2 protein is a promising target for anti-cancer therapies. HIPK2 can be inhibited by hypoxia. In this respect, we have recently shown that hypoxia-driven HIPK2 downregulation is not irreversible. We found that, zinc supplementation reactivates the hypoxia-inhibited HIPK2, leading to repression of the HIF-1 pathway and restoration of p53Ser46 apoptotic activity. Here, we discuss about these findings and the potential relevance of zinc supplementation to chemotherapy in cancer treatment. The results will be also discussed in light of recent findings showing that cancer treatment with antiangiogenic agents may result in hypoxia and selection of cancer cells with increased tumor aggressiveness and metastasis.
Assuntos
Proteínas de Transporte/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Proteínas de Transporte/genética , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Modelos Biológicos , Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Reprogramming of differentiated cells into induced pluripotent cells (iPS) was accomplished in 2006 by expressing four, or less, embryonic stem cell (ESC)-specific transcription factors. Due to the possible danger of DNA damage and the potential tumorigenicity associated with such DNA damage, attempts were made to minimize DNA integration by the vectors involved in this process without complete success. Here we present a method of using RNA transfection as a tool for reprogramming human fibroblasts to iPS. We used RNA synthesized in vitro from cDNA of the same reprogramming four transcription factors. After transfection of the RNA, we show intracellular expression and nuclear localization of the respective proteins in at least 70% of the cells. We used five consecutive transfections to support continuous protein expression resulting in the formation of iPS colonies that express alkaline phosphatase and several ESC markers and that can be expanded. This method completely avoids DNA integration and may be developed to replace the use of DNA vectors in the formation of iPS.
Assuntos
Técnicas de Cultura de Células , Transdiferenciação Celular , Reprogramação Celular , Fibroblastos/citologia , Células-Tronco Pluripotentes/citologia , RNA Mensageiro/genética , Humanos , Fatores de Transcrição/genética , TransfecçãoRESUMO
HIPK2 is a stress-induced kinase and a transcriptional corepressor that functionally cooperates with p53 to suppress cancer. Activation of the p53 proapoptotic function requires a cascade of phosphorylations and acetylations, and HIPK2 takes part in both modifications in that it phosphorylates p53 Ser46 and induces p53 Lys382 acetylation. Here, to further investigate the role of HIPK2 in p53 activation, we started with the finding that HIPK2 inhibition upregulated Nox1, a homolog of the catalytic subunit of the superoxide-generating NADPH oxidase, involved in tumor progression and ROS production. We found that Nox1 inhibited p53 Lys382 acetylation, which is a target of SIRT1 deacetylase, and impaired p53 proapoptotic transcriptional activity. By the use of either small interfering RNAs to target SIRT1 or the SIRT1 inhibitor nicotinamide we found that Nox1-dependent inhibition of p53 transcriptional activity was SIRT1-dependent. Thus, Nox1 was unable to inhibit p53 when coexpressed with a SIRT1 deacetylase-defective mutant (SIRT1HY), suggesting a link between Nox1 and SIRT1 activity. Finally, recovery of HIPK2 function downregulated Nox1 expression with rescue of p53 Lys382 acetylation and p53 activity. Together, our findings indicate that Nox1 upregulation may activate SIRT1 and inhibit p53 and that Lys382 is important for p53 proapoptotic function.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias Pulmonares/metabolismo , NADPH Oxidases/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Acetilação/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma/genética , Carcinoma/patologia , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Clonagem Molecular , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , NADPH Oxidase 1 , NADPH Oxidases/genética , Niacinamida/farmacologia , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Sirtuína 1/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Activation of p53-mediated gene transcription is a critical cellular response to DNA damage and involves a phosphorylation-acetylation cascade of p53. The discovery of differences in the response to different agents raises the question whether some of the p53 oncosuppressor functions might be exerted by different posttranslational modifications. Stress-induced homeodomain-interacting protein kinase-2 (HIPK2) phosphorylates p53 at serine-46 (Ser46) for p53 apoptotic activity; p53 acetylation at different C-terminus lysines including p300-mediated lysine-382 (Lys382) is also required for full activation of p53 transcriptional activity. The purpose of the current study was to evaluate the interplay among HIPK2, p300, and p53 in p53 acetylation and apoptotic transcriptional activity in response to drug by using siRNA interference, p300 overexpression or deacetylase inhibitors, in cancer cells. RESULTS: Knockdown of HIPK2 inhibited both adriamycin-induced Ser46 phosphorylation and Lys382 acetylation in p53 protein; however, while combination of ADR and zinc restored Ser46 phosphorylation it did not recover Lys382 acetylation. Chromatin immunoprecipitation studies showed that HIPK2 was required in vivo for efficient p300/p53 co-recruitment onto apoptotic promoters and that both p53 modifications at Ser46 and Lys382 were necessary for p53 apoptotic transcription. Thus, p53Lys382 acetylation in HIPK2 knockdown as well as p53 apoptotic activity in response to drug could be rescued by p300 overexpression. Similar effect was obtained with the Sirt1-inhibitor nicotinamide. Interestingly trichostatin A (TSA), the inhibitor of histone deacetylase complexes (HDAC) did not have effect, suggesting that Sirt1 was the deacetylase involved in p53 deacetylation in HIPK2 knockdown. CONCLUSION: These results reveal a novel role for HIPK2 in activating p53 apoptotic transcription. Our results indicate that HIPK2 may regulate the balance between p53 acetylation and deacetylation, by stimulating on one hand co-recruitment of p300 and p53Lys382 on apoptotic promoters and on the other hand by inhibiting Sirt1 deacetylase activity. We attempted to reactivate p53 apoptotic transcriptional activity by rescuing both Ser46 and Lys382 modification in response to drug. Our data propose combination strategies for the treatment of tumors with dysfunctional p53 and/or HIPK2 that include classical chemotherapy with pharmacological or natural agents such as Sirt1-deacetylase inhibitors or zinc, respectively.
Assuntos
Apoptose/genética , Proteínas de Transporte/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Acetilação/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Proteína p300 Associada a E1A/metabolismo , Deleção de Genes , Técnicas de Silenciamento de Genes , Humanos , Ácidos Hidroxâmicos/farmacologia , Lisina/metabolismo , Niacinamida/farmacologia , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Sirtuína 1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Zinco/farmacologia , Proteína X Associada a bcl-2/metabolismoRESUMO
BACKGROUND: The tumor suppressor homeodomain-interacting protein kinase-2 (HIPK2) by phosphorylating serine 46 (Ser46) is a crucial regulator of p53 apoptotic function. HIPK2 is also a transcriptional co-repressor of hypoxia-inducible factor-1alpha (HIF-1alpha) restraining tumor angiogenesis and chemoresistance. HIPK2 can be deregulated in tumors by several mechanisms including hypoxia. Here, we sought to target hypoxia by restoring HIPK2 function and suppressing HIF-1alpha, in order to provide evidence for the involvement of both HIPK2 and p53 in counteracting hypoxia-induced chemoresistance. METHODOLOGY/PRINCIPAL FINDINGS: Upon exposure of colon and lung cancer cells to hypoxia, by either low oxygen or cobalt, HIPK2 function was impaired allowing for increased HIF-1alpha expression and inhibiting the p53-apoptotic response to drug. Cobalt suppressed HIPK2 recruitment onto HIF-1alpha promoter. Hypoxia induced expression of the p53 target MDM2 that downregulates HIPK2, thus MDM2 inhibition by siRNA restored the HIPK2/p53Ser46 response to drug. Zinc supplementation to hypoxia-treated cells increased HIPK2 protein stability and nuclear accumulation, leading to restoration of HIPK2 binding to HIF-1alpha promoter, repression of MDR1, Bcl2, and VEGF genes, and activation of the p53 apoptotic response to drug. Combination of zinc and ADR strongly suppressed tumor growth in vivo by inhibiting HIF-1 pathway and upregulating p53 apoptotic target genes. CONCLUSIONS/SIGNIFICANCE: We show here for the first time that hypoxia-induced HIPK2 deregulation was counteracted by zinc that restored HIPK2 suppression of HIF-1 pathway and reactivated p53 apoptotic response to drug, underscoring the potential use of zinc supplementation in combination with chemotherapy to address hypoxia and improve tumor treatment.
Assuntos
Proteínas de Transporte/metabolismo , Hipóxia Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos/farmacologia , Cobalto/farmacologia , Doxorrubicina/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-mdm2/genética , Transcrição Gênica/efeitos dos fármacos , Zinco/farmacologiaRESUMO
The maintenance of p53 transactivation activity is important for p53 apoptotic function. We have shown that stable knockdown of HIPK2 induces p53 misfolding with inhibition of p53 target gene transcription. In this study we established a lentiviral-based system for doxycyclin (Dox)-induced conditional interference of HIPK2 expression to evaluate the molecular mechanisms involved in p53 deregulation. We found that HIPK2 knockdown induced metallothionein 2A (MT2A) upregulation as assessed by RT-PCR analysis, increased promoter acetylation, and increased promoter luciferase activity. The MT2A upregulation correlated with resistance to Adriamycin (ADR)-driven apoptosis and with p53 inhibition. Thus, acute knockdown of HIPK2 (HIPK2i) induced misfolded p53 protein in MCF7 breast cancer cells and inhibited p53 DNA-binding and transcription activities in response to ADR treatment. Previous works show that MT may modulate p53 activity through zinc exchange. Here, we found that inhibition of MT2A expression by siRNA in the HIPK2i cells restored p53 transcription activity. Similarly zinc supplementation to HIPK2i cells restored p53 transcription activity and drug-induced apoptosis. These data support the notion that MT2A is involved in p53 deregulation and strengthen the possibility that combination of chemotherapy and zinc might be useful to treat tumors with inactive wtp53.
Assuntos
Metalotioneína/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteína Supressora de Tumor p53/metabolismo , Zinco/metabolismo , Apoptose/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , DNA de Neoplasias/metabolismo , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lentivirus , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Regulação para Cima/efeitos dos fármacos , Zinco/farmacologiaRESUMO
Activating mutations in FGFR3 cause achondroplasia and thanatophoric dysplasia, the most common human skeletal dysplasias. In these disorders, spinal canal and foramen magnum stenosis can cause serious neurologic complications. Here, we provide evidence that FGFR3 and MAPK signaling in chondrocytes promote synchondrosis closure and fusion of ossification centers. We observed premature synchondrosis closure in the spine and cranial base in human cases of homozygous achondroplasia and thanatophoric dysplasia as well as in mouse models of achondroplasia. In both species, premature synchondrosis closure was associated with increased bone formation. Chondrocyte-specific activation of Fgfr3 in mice induced premature synchondrosis closure and enhanced osteoblast differentiation around synchondroses. FGF signaling in chondrocytes increases Bmp ligand mRNA expression and decreases Bmp antagonist mRNA expression in a MAPK-dependent manner, suggesting a role for Bmp signaling in the increased bone formation. The enhanced bone formation would accelerate the fusion of ossification centers and limit the endochondral bone growth. Spinal canal and foramen magnum stenosis in heterozygous achondroplasia patients, therefore, may occur through premature synchondrosis closure. If this is the case, then any growth-promoting treatment for these complications of achondroplasia must precede the timing of the synchondrosis closure.
Assuntos
Acondroplasia/metabolismo , Desenvolvimento Ósseo , Sistema de Sinalização das MAP Quinases , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Displasia Tanatofórica/metabolismo , Acondroplasia/genética , Acondroplasia/fisiopatologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Células Cultivadas , Condrócitos/metabolismo , Condrogênese , Humanos , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Displasia Tanatofórica/genética , Displasia Tanatofórica/fisiopatologiaRESUMO
p73 has been identified as a structural and functional homolog of the tumor suppressor p53. The transcriptional coactivator Yes-associated protein (YAP) has been demonstrated to interact with and to enhance p73-dependent apoptosis in response to DNA damage. Here, we show the existence of a proapoptotic autoregulatory feedback loop between p73, YAP, and the promyelocytic leukemia (PML) tumor suppressor gene. We demonstrate that PML is a direct transcriptional target of p73/YAP, and we show that PML transcriptional activation by p73/YAP is under the negative control of the proto-oncogenic Akt/PKB kinase. Importantly, we find that PML and YAP physically interact through their PVPVY and WW domains, respectively, causing PML-mediated sumoylation and stabilization of YAP. Hence, we determine a mechanistic pathway in response to DNA damage that could have relevant implications for the treatment of human cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Proteínas de Ligação a DNA/metabolismo , Retroalimentação Fisiológica , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular , Linhagem Celular , Cisplatino/farmacologia , Retroalimentação Fisiológica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Modelos Biológicos , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteína da Leucemia Promielocítica , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Ubiquitina/metabolismo , Proteínas de Sinalização YAPRESUMO
We examined the microRNA profiles of Glioblastoma stem (CD133+) and non-stem (CD133-) cell populations and found up-regulation of several miRs in the CD133- cells, including miR-451, miR-486, and miR-425, some of which may be involved in regulation of brain differentiation. Transfection of GBM cells with the above miRs inhibited neurosphere formation and transfection with the mature miR-451 dispersed neurospheres, and inhibited GBM cell growth. Furthermore, transfection of miR-451 combined with Imatinib mesylate treatment had a cooperative effect in dispersal of GBM neurospheres. In addition, we identified a target site for SMAD in the promoter region of miR-451 and showed that SMAD3 and 4 activate such a promoter-luciferase construct. Transfection of SMAD in GBM cells inhibited their growth, suggesting that SMAD may drive GBM stem cells to differentiate to CD133- cells through up-regulation of miR-451 and reduces their tumorigenicity. Identification of additional miRs and target genes that regulate GBM stem cells may provide new potential drugs for therapy.