RESUMO
Partial epithelial-mesenchymal transition (p-EMT) has recently been identified as a hybrid state consisting of cells with both epithelial and mesenchymal characteristics and is associated with the migration, metastasis, and chemoresistance of cancer cells. Here, we describe the induction of p-EMT in starved colorectal cancer (CRC) cells and identify a p-EMT gene signature that can predict prognosis. Functional characterisation of starvation-induced p-EMT in HCT116, DLD1, and HT29 cells showed changes in proliferation, morphology, and drug sensitivity, supported by in vivo studies using the chorioallantoic membrane model. An EMT-specific quantitative polymerase chain reaction (qPCR) array was used to screen for deregulated genes, leading to the establishment of an in silico gene signature that was correlated with poor disease-free survival in CRC patients along with the CRC consensus molecular subtype CMS4. Among the significantly deregulated p-EMT genes, a triple-gene signature consisting of SERPINE1, SOX10, and epidermal growth factor receptor (EGFR) was identified. Starvation-induced p-EMT was characterised by increased migratory potential and chemoresistance, as well as E-cadherin processing and internalisation. Both gene signature and E-cadherin alterations could be reversed by the proteasomal inhibitor MG132. Spatially resolving EGFR expression with high-resolution immunofluorescence imaging identified a proliferation stop in starved CRC cells caused by EGFR internalisation. In conclusion, we have gained insight into a previously undiscovered EMT mechanism that may become relevant when tumour cells are under nutrient stress, as seen in early stages of metastasis. Targeting this process of tumour cell dissemination might help to prevent EMT and overcome drug resistance. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Assuntos
Neoplasias Colorretais , Humanos , Neoplasias Colorretais/patologia , Proliferação de Células , Transição Epitelial-Mesenquimal/genética , Receptores ErbB , Linhagem Celular Tumoral , Caderinas/genética , Caderinas/metabolismo , Movimento CelularRESUMO
BACKGROUND: The role of ATF2 in colon cancer (CC) is controversial. Recently, we reported that low ATF2 expression is characteristic of highly invasive tumors, suggesting that ATF2 might also be involved in therapy resistance. 5-Fluorouracil (5-FU) is the best-known chemotherapeutic drug for CC, but drug resistance affects its curative effect. To date, the role of ATF2 in the 5-FU response remains elusive. METHODS/RESULTS: For our study, we had available HCT116 cells (wild-type p53) and HT29 colon tumor cells (mutant p53) and their corresponding CRISPRâCas9-generated ATF2-KO clones. We observed that loss of ATF2 triggered dose- and time-dependent 5-FU resistance in HCT116 cells by activating the DNA damage response (DDR) pathway with high p-ATRThr1989 and p-Chk1Ser317 levels accompanied by an increase in the DNA damage marker γ-H2AX in vitro and in vivo using the chicken chorioallantoic membrane (CAM) model. Chk1 inhibitor studies causally displayed the link between DDR and drug resistance. There were contradictory findings in HT29 ATF2-KO cells upon 5-FU exposure with low p-Chk1Ser317 levels, strong apoptosis induction, but no effects on DNA damage. In ATF2-silenced HCT116 p53-/- cells, 5-FU did not activate the DDR pathway. Co-immunoprecipitation and proximity ligation assays revealed that upon 5-FU treatment, ATF2 binds to ATR to prevent Chk1 phosphorylation. Indeed, in silico modelling showed reduced ATR-Chk1 binding when ATF2 was docked into the complex. CONCLUSIONS: We demonstrated a novel ATF2 scaffold function involved in the DDR pathway. ATF2-negative cells are highly resistant due to effective ATR/Chk1 DNA damage repair. Mutant p53 seems to overwrite the tumor suppressor function of ATF2.
Assuntos
Neoplasias do Colo , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Fluoruracila/farmacologia , Dano ao DNA , Fator 2 Ativador da Transcrição/genéticaRESUMO
In cancer, the activating transcription factor 2 (ATF2) has pleiotropic functions in cellular responses to growth stimuli, damage, or inflammation. Due to only limited studies, the significance of ATF2 in colorectal cancer (CRC) is not well understood. We report that low ATF2 levels correlated with worse prognosis and tumor aggressiveness in CRC patients. NanoString gene expression and ChIP analysis confirmed trophoblast cell surface antigen 2 (TROP2) as a novel inhibitory ATF2 target gene. This inverse correlation was further observed in primary human tumor tissues. Immunostainings revealed that high intratumoral heterogeneity for ATF2 and TROP2 expression was sustained also in liver metastasis. Mechanistically, our in vitro data of CRISPR/Cas9-generated ATF2 knockout (KO) clones revealed that high TROP2 levels were critical for cell de-adhesion and increased cell migration without triggering EMT. TROP2 was enriched in filopodia and displaced Paxillin from adherens junctions. In vivo imaging, micro-computer tomography, and immunostainings verified that an ATF2KO/TROP2high status triggered tumor invasiveness in in vivo mouse and chicken xenograft models. In silico analysis provided direct support that ATF2low/TROP2high expression status defined high-risk CRC patients. Finally, our data demonstrate that ATF2 acts as a tumor suppressor by inhibiting the cancer driver TROP2. Therapeutic TROP2 targeting might prevent particularly the first steps in metastasis, i.e., the de-adhesion and invasion of colon cancer cells.
Assuntos
Fator 2 Ativador da Transcrição , Antígenos de Neoplasias , Neoplasias Colorretais , Fator 2 Ativador da Transcrição/genética , Fator 2 Ativador da Transcrição/metabolismo , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral/metabolismo , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Camundongos , Regulação para CimaRESUMO
In the last decade, the chicken chorioallantoic membrane (CAM) assay has been re-discovered in cancer research to study the molecular mechanisms of anti-cancer drug effects. Literature about the CAM assay as an alternative in vivo cancer xenograft model according to the 3R principles has exploded in the last 3 years. Following a summary of the basic knowledge about the chicken embryo, we compare advantages and disadvantages with the classical mouse xenograft model, exemplify established and innovative imaging techniques that are used in the CAM model, and give examples of its successful utilization for studying major hallmarks of cancer such as angiogenesis, proliferation, invasion, and metastasis.
Assuntos
Membrana Corioalantoide , Preparações Farmacêuticas , Animais , Bioensaio , Embrião de Galinha , Galinhas , Camundongos , Neovascularização Patológica/tratamento farmacológicoRESUMO
Peritumoral budding and intratumoral budding (ITB) are important prognostic factors for colorectal cancer patients. Scientists worldwide have investigated the role of budding in tumor progression and its prognosis, but guidelines for reliably identifying tumor buds based on morphology are lacking. In this study, next-generation tissue microarray (ngTMA®) construction was used for tumor bud evaluation, and highly detailed rule-out annotation was used for tumor definition in pancytokeratin-stained tissue sections. Initially, tissues of 245 colon cancer patients were evaluated with high interobserver reliability, and a concordance of 96% was achieved. It was shown that high ITB scores were associated with poor distant metastasis-free survival (p = 0.006 with a cut-off of ≥10 buds). This cut-off was defined as the best maximum value from one of two/three ngTMA® cores (0.6 mm diameter). ITB in 30 cases of mucinous, medullary, and signet ring cell carcinoma was analyzed for the subsequent determination of differences in tumor bud analyses between those subtypes. In conclusion, blinded randomized punched cores in the tumor center can be useful for ITB detection. It can be assumed that this method is suitable for its adoption in clinical routines.
Assuntos
Adenocarcinoma Mucinoso/patologia , Carcinoma Medular/patologia , Carcinoma de Células em Anel de Sinete/patologia , Neoplasias do Colo/patologia , Adenocarcinoma Mucinoso/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Medular/metabolismo , Carcinoma de Células em Anel de Sinete/metabolismo , Neoplasias do Colo/metabolismo , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Análise de Sobrevida , Análise Serial de TecidosRESUMO
In contrast to the continuous increase in survival rates for many cancer entities, colorectal cancer (CRC) and pancreatic cancer are predicted to be ranked among the top 3 cancer-related deaths in the European Union by 2025. Especially, fighting metastasis still constitutes an obstacle to be overcome in CRC and pancreatic cancer. As described by Fearon and Vogelstein, the development of CRC is based on sequential mutations leading to the activation of proto-oncogenes and the inactivation of tumour suppressor genes. In pancreatic cancer, genetic alterations also attribute to tumour development and progression. Recent findings have identified new potentially important transcription factors in CRC, among those the activating transcription factor 2 (ATF2). ATF2 is a basic leucine zipper protein and is involved in physiological and developmental processes, as well as in tumorigenesis. The mutation burden of ATF2 in CRC and pancreatic cancer is rather negligible; however, previous studies in other tumours indicated that ATF2 expression level and subcellular localisation impact tumour progression and patient prognosis. In a tissue- and stimulus-dependent manner, ATF2 is activated by upstream kinases, dimerises and induces target gene expression. Dependent on its dimerisation partner, ATF2 homodimers or heterodimers bind to cAMP-response elements or activator protein 1 consensus motifs. Pioneering work has been performed in melanoma in which the dual role of ATF2 is best understood. Even though there is increasing interest in ATF2 recently, only little is known about its involvement in CRC and pancreatic cancer. In this review, we summarise the current understanding of the underestimated 'cancer gene chameleon' ATF2 in apoptosis, epithelial-to-mesenchymal transition and microRNA regulation and highlight its functions in CRC and pancreatic cancer. We further provide a novel ATF2 3D structure with key phosphorylation sites and an updated overview of all so-far available mouse models to study ATF2 in vivo.
Assuntos
Fator 2 Ativador da Transcrição/genética , Neoplasias/genética , Neoplasias/patologia , Animais , Apoptose/genética , Progressão da Doença , Transição Epitelial-Mesenquimal/genética , Humanos , MicroRNAs/genética , Mutação/genéticaRESUMO
PURPOSE: Gastrointestinal stromal tumors (GISTs) are the most common soft tissue tumors of the GI tract. Studies have been published reporting additional neoplasms in GIST patients. This study aimed to evaluate possible associations of mutation type, morphology, and clinical aspects of GISTs. METHODS: All cases of GIST were identified from our pathology files. Coding exons of KIT and PDGFRA in GISTs with additional malignancies were sequenced. RESULTS: A total of 70 of 188 (37%) retrieved patients with confirmed diagnosis of GIST showed at least one additional malignant neoplasm. Fifty of these GISTs were located in the stomach (71%), 8 in the small intestine (11%), 5 in the colon/rectum (7%), and 7 cases (6.2%) were of undetermined sites of origin. The distribution of identified mutations was similar to that described in GISTs without secondary malignancies. A total of 37 of 57 cases (65%) showed mutations in the KIT gene exon 11, 3 (5%) cases in exon 9, and 1 (2%) case in exon 13. Nine tumors (16%) had mutations of the PDGFRA gene. KIT and PDGFRA wild-type status were found in seven cases (12%). Most of the secondary neoplasms were located within the GI tract (34%), in the urogenital system (24%), or the breast/female genital tract (20%). CONCLUSION: This study confirms the high rate of additional malignant tumors in GIST patients. GIST features in these cases are very similar to those with sole GIST.
Assuntos
Neoplasias Gastrointestinais/genética , Tumores do Estroma Gastrointestinal/genética , Mutação/genética , Segunda Neoplasia Primária/genética , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Idoso , Idoso de 80 Anos ou mais , Feminino , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Segunda Neoplasia Primária/patologia , Estudos RetrospectivosRESUMO
Hepatocellular carcinoma (HCC) is one of the most common causes for cancer-related death worldwide with rapidly increasing incidence and mortality rates. As for other types of cancers, also in HCC cancer stem cells (CSCs) are thought to be responsible for tumour initiation, progression and therapy failure. However, as rare subpopulations of tumour tissue, CSCs are difficult to isolate, thus making the development of suitable and reliable model systems necessary. In our study, we generated HepG2 subclones with enriched CSC potential by application of the spheroid formation method and subsequent single-cell cloning. Analyses in several 2D and 3D cell culture systems as well as a panel of functional assays both in vitro and in vivo revealed that the generated subclones displayed characteristic and sustained features of tumour initiating cells as well as highly aggressive properties related to tumour progression and metastasis. These characteristics could clearly be correlated with the expression of CSC markers that might have prognostic value in the clinical HCC setting. Therefore, we conclude that our CSC enriched HepG2 clones certainly represent suitable model systems to study the role of CSCs during HCC initiation, progression and drug resistance.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/patologia , Carcinoma Hepatocelular/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Células-Tronco Neoplásicas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Death-associated protein kinase (DAPK) is a serine-threonine kinase with tumor suppressor function. Previously, we demonstrated that tumor necrosis factor (TNF) induced DAPK-mediated apoptosis in colorectal cancer. However, the protein-protein interaction network associated with TNF-DAPK signaling still remains unclear. We identified HSF1 as a new DAPK phosphorylation target in response to low concentrations of TNF and verified a physical interaction between DAPK and HSF1 both in vitro and in vivo. We show that HSF1 binds to the DAPK promoter. Transient overexpression of HSF1 protein led to an increase in DAPK mRNA level and consequently to an increase in the amount of apoptosis. By contrast, treatment with a DAPK-specific inhibitor as well as DAPK knockdown abolished the phosphorylation of HSF1 at Ser230 (pHSF1(Ser230)). Furthermore, translational studies demonstrated a positive correlation between DAPK and pHSF1(Ser230) protein expression in human colorectal carcinoma tissues. Taken together, our data define a novel link between DAPK and HSF1 and highlight a positive-feedback loop in DAPK regulation under mild inflammatory stress conditions in colorectal tumors. For the first time, we show that under TNF the pro-survival HSF1 protein can be redirected to a pro-apoptotic program.
Assuntos
Apoptose , Neoplasias Colorretais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Quinases Associadas com Morte Celular/metabolismo , Retroalimentação Fisiológica , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Sequência de Bases , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Simulação por Computador , Proteínas de Ligação a DNA/química , Proteínas Quinases Associadas com Morte Celular/química , Proteínas Quinases Associadas com Morte Celular/genética , Retroalimentação Fisiológica/efeitos dos fármacos , Feminino , Fatores de Transcrição de Choque Térmico , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Fatores de Transcrição/química , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Biomarkers are widely used in clinical diagnosis, prognosis and therapy monitoring. Here, we developed a protocol for the efficient and selective enrichment of small and low concentrated biomarkers from human serum, involving a 95% effective depletion of high-abundant serum proteins by partial denaturation and enrichment of low-abundant biomarkers by size exclusion chromatography. The recovery of low-abundance biomarkers was above 97%. Using this protocol, we quantified the tumour markers DcR3 and growth/differentiation factor (GDF)15 from 100 µl human serum by isotope dilution mass spectrometry, using (15) N metabolically labelled and concatamerized fingerprint peptides for the both proteins. Analysis of three different fingerprint peptides for each protein by liquid chromatography electrospray ionization mass spectrometry resulted in comparable concentrations in three healthy human serum samples (DcR3: 27.23 ± 2.49 fmol/ml; GDF15: 98.11 ± 0.49 fmol/ml). In contrast, serum levels were significantly elevated in tumour patients for DcR3 (116.94 ± 57.37 fmol/ml) and GDF15 (164.44 ± 79.31 fmol/ml). Obtained data were in good agreement with ELISA and qPCR measurements, as well as with literature data. In summary, our protocol allows the reliable quantification of biomarkers, shows a higher resolution at low biomarker concentrations than antibody-based strategies, and offers the possibility of multiplexing. Our proof-of-principle studies in patient sera encourage the future analysis of the prognostic value of DcR3 and GDF15 for colon cancer patients in larger patient cohorts.
Assuntos
Fator 15 de Diferenciação de Crescimento/sangue , Membro 6b de Receptores do Fator de Necrose Tumoral/sangue , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Aminoácidos , Biomarcadores/sangue , Proteínas Sanguíneas/metabolismo , Cromatografia em Gel , Fator 15 de Diferenciação de Crescimento/química , Humanos , Imunoprecipitação , Limite de Detecção , Dados de Sequência Molecular , Mapeamento de Peptídeos , Desnaturação Proteica , Proteólise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Membro 6b de Receptores do Fator de Necrose Tumoral/químicaRESUMO
The plant secondary metabolite gallotannin (GT) is the simplest hydrolyzable tannin shown to have anti-carcinogenic properties in several cell lines and to inhibit tumor development in different animal models. Here, we determined if GT induces senescence and DNA damage and investigated the involvement of p53 and p21 in this response. Using HCT116 human colon cancer cells wildtype for p53(+/+) /p21(+/+) and null for p53(+/+) /p21(-/-) or p53(-/-) /p21(+/+) , we found that GT induces senescence independently of p21 and p53. GT was found to increase the production of reactive oxygen species (ROS) by altering the redox balance in the cell, mainly by reducing the levels of glutathione and superoxide dismutase (SOD). Using the key antioxidants N-acetyl cysteine, dithiothreitol, SOD, and catalase, we showed that ROS were partially involved in the senescence response. Furthermore, GT-induced cell cycle arrest in S-phase in all HCT116 cell lines. At later time points, we noticed that p53 and p21 null cells escaped complete arrest and re-entered cell cycle provoking higher rates of multinucleation. The senescence induction by GT was irreversible and was accompanied by significant DNA damage as evidenced by p-H2AX staining. Our findings indicate that GT is an interesting anti colon cancer agent which warrants further study.
Assuntos
Senescência Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA/efeitos dos fármacos , Taninos Hidrolisáveis/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Antioxidantes/metabolismo , Catalase/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Ditiotreitol/metabolismo , Células HCT116 , Humanos , Espécies Reativas de Oxigênio/metabolismo , Fase S/efeitos dos fármacos , Superóxido Dismutase/metabolismoRESUMO
Gastrokines (GKNs) were originally described as stomach-specific tumor suppressor genes. Recently, we identified GKN1 in extravillous trophoblasts (EVT) of human placenta. GKN1 treatment reduced the migration of the trophoblast cell line JEG-3. GKN2 is known to inhibit the proliferation, migration and invasion of gastric cancer cells and may interact with GKN1. Recently, GKN2 was detected in the placental yolk sac of mice. We therefore aimed to further characterize placental GKN2 expression. By immunohistochemistry, healthy first-trimester placenta showed ubiquitous staining for GKN2 at its early gestational stage. At later gestational stages, a more differentiated expression pattern in EVT and villous cytotrophoblasts became evident. In healthy third-trimester placenta, only EVT retained strong GKN2 immunoreactivity. In contrast, HELLP placentas showed a tendency of increased levels of GKN2 expression with a more prominent GKN2 staining in their syncytiotrophoblast. Choriocarcinoma cell lines did not express GKN2. Besides its trophoblastic expression, we found human GKN2 in fibrotic villi, in amniotic membrane and umbilical cord. GKN2 co-localized with smooth muscle actin in villous myofibroblasts and with HLA-G and GKN1 in EVT. In the rodent placenta, GKN2 was specifically located in the spongiotrophoblast layer. Thus, the gestational age-dependent and compartment-specific expression pattern of GKN2 points to a role for placental development. The syncytial expression of GKN2 in HELLP placentas might represent a reduced state of functional differentiation of the syncytiotrophoblast. Moreover, the specific GKN2 expression in the rodent spongiotrophoblast layer (equivalent to human EVT) might suggest an important role in EVT physiology.
Assuntos
Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Trofoblastos/metabolismo , Adulto , Âmnio/metabolismo , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Vilosidades Coriônicas/metabolismo , Feminino , Síndrome HELLP/metabolismo , Humanos , Imuno-Histoquímica , Músculo Liso Vascular/metabolismo , Hormônios Peptídicos/genética , Hormônios Peptídicos/metabolismo , Placenta/metabolismo , Doenças Placentárias/genética , Doenças Placentárias/metabolismo , Gravidez , Primeiro Trimestre da Gravidez/metabolismo , Terceiro Trimestre da Gravidez/metabolismo , Ratos , Cordão Umbilical/metabolismoRESUMO
BACKGROUND: BRAF mutation frequencies in melanoma subtypes have clinical implications and offer pathogenetic clues. OBJECTIVES: To characterize BRAF mutation status in melanoma of unknown primary (MUP) patients, in histological melanoma subtypes and by localization of primary tumors. METHODS: In 179 patients with stage IV metastatic melanoma, BRAF mutation status, histological subtype and localization of primary (except for 29 MUP patients) were analyzed. RESULTS: BRAF mutations were found in 44.3%, of which 80.5% were BRAF V600E and 19.5% showed non-V600E BRAF mutations. BRAF mutation frequency depended on histological subtype (57.4% superficial spreading melanoma, 54.7% nodular melanoma, 11.1% mucosal melanoma, 28.6% acral lentiginous melanoma) and concerning non-V600E BRAF mutations on localization of primary. In MUP the BRAF mutation pattern resembled superficial spreading and nodular melanomas. CONCLUSION: BRAF mutation frequencies depend on histological subtype and localization of primary melanoma. Non-V600E BRAF mutations mostly occur in patients with primaries on 'head and neck' as well as 'trunk' but not on 'extremities'.
Assuntos
Neoplasias de Cabeça e Pescoço/genética , Melanoma/genética , Neoplasias Primárias Desconhecidas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Extremidades , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Melanoma/patologia , Melanoma/secundário , Mucosa , Taxa de Mutação , Estadiamento de Neoplasias , Neoplasias Primárias Desconhecidas/patologia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/secundário , TroncoRESUMO
Crocin, a bioactive molecule of saffron, inhibited proliferation of both HCT116 wild-type and HCT116 p53(-/-) cell lines at a concentration of 10 mM. Flow cytometric analysis of cell cycle distribution revealed that there was an accumulation of HCT116 wild-type cells in G1 (55.9%, 56.1%) compared to the control (30.4%) after 24 and 48 h of crocin treatment, respectively. However, crocin induced only mild G2 arrest in HCT116 p53(-/-) after 24 h. Crocin induced inefficient autophagy in HCT116 p53(-/-) cells, where crocin induced the formation of LC3-II, which was combined with a decrease in the protein levels of Beclin 1 and Atg7 and no clear p62 degradation. Autophagosome formation was not detected in HCT116 p53(-/-) after crocin treatment predicting a nonfunctional autophagosome formation. There was a significant increase of p62 after treating the cells with Bafilomycin A1 (Baf) and crocin compared to crocin exposure alone. Annexin V staining showed that Baf-pretreatment enhanced the induction of apoptosis in HCT116 wild-type cells. Baf-exposed HCT116 p53(-/-) cells did not, however, show any enhancement of apoptosis induction despite an increase in the DNA damage-sensor accumulation, γH2AX indicating that crocin induced an autophagy-independent classical programmed cell death.
Assuntos
Apoptose/efeitos dos fármacos , Carotenoides/toxicidade , Proteína Supressora de Tumor p53/genética , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia , Proteína Beclina-1 , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Células HCT116 , Humanos , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismoRESUMO
BACKGROUND: Thymoquinone (TQ) was shown to reduce tumor growth in several cancer models both in vitro and in vivo. So far only a few targets of TQ, including protein kinases have been identified. Considering that kinases are promising candidates for targeted anticancer therapy, we studied the complex kinase network regulated by TQ. METHODS: Novel kinase targets influenced by TQ were revealed by in silico analysis of peptide array data obtained from TQ-treated HCT116wt cells. Western blotting and kinase activity assays were used to determine changes in kinase expression patterns in colorectal cancer cells (HCT116wt, DLD-1, HT29). To study the viability/apoptotic effects of combining the PAK1 inhibitor IPA-3 and TQ, crystal violet assay and AnnexinV/PI staining were employed. Interactions between PAK1 and ERK1/2 were investigated by co-immunoprecipitation and modeled by docking studies. Transfection with different PAK1 mutants unraveled the role of TQ-induced changes in PAK1 phosphorylation and TQ's effects on PAK1 scaffold function. RESULTS: Of the 104 proteins identified, 50 were upregulated ≥ 2 fold by TQ and included molecules in the AKT-MEK-ERK1/2 pathway. Oncogenic PAK1 emerged as an interesting TQ target. Time-dependent changes in two PAK1 phosphorylation sites generated a specific kinase profile with early increase in pPAK(Thr212) followed by late increase in pPAK(Thr423). TQ induced an increase of pERK1/2 and triggered the early formation of an ERK1/2-PAK1 complex. Modeling confirmed that TQ binds in the vicinity of Thr212 accompanied by conformational changes in ERK2-PAK1 binding. Transfecting the cells with the non-phosphorylatable mutant T212A revealed an increase of pPAK(Thr423) and enhanced apoptosis. Likewise, an increase in apoptosis was observed in cells transfected with both the kinase-dead K299R mutant and PAK1 siRNA. Using structural modeling we suggest that TQ interferes also with the kinase domain consequently disturbing its interaction with pPAK(Thr423), finally inhibiting MEK-ERK1/2 signaling and disrupting its prosurvival function. pERK1/2 loss was also validated in vivo. CONCLUSIONS: Our study shows for the first time that the small molecule TQ directly binds to PAK1 changing its conformation and scaffold function. Because TQ affects the central RAF/MEK/ERK1/2 pathway, the combination of TQ with targeted therapies is worth considering for future anticancer treatments.
Assuntos
Benzoquinonas/farmacologia , Neoplasias Colorretais/química , Dissulfetos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Naftóis/farmacologia , Quinases Ativadas por p21/química , Quinases Ativadas por p21/metabolismo , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HT29 , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Fosforilação/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Quinases Ativadas por p21/genéticaRESUMO
Imbalanced cell death is a common phenomenon in many human diseases, including cancer. DAPK's essential function is in promoting apoptosis. DAPK interacts with stress-induced receptors through its death domain to initiate an apoptosis cascade. In addition, DAPK phosphorylates multiple cytosolic substrates and can mediate transfer of signaling pathways to the effector caspases. A series of studies demonstrated that, depending on stimuli, DAPK expression is regulated on both the transcriptional and posttranscriptional levels. Silencing of DAPK due to hypermethylation of its promoter was reported in many types of cancer. STAT3 and p52-NFkB transcription factors have been shown to down-regulate DAPK expression. In contrast, p53, C/EBP-ß and Smad transcription factors bind to their specific response elements within the DAPK promoter and induce its transcription. Post-transcriptionally, DAPK undergoes alternative splicing, which results in the production of two functionally different isoforms. Moreover, miRNA 103 and miRNA 107 recently were shown to inhibit DAPK in colorectal cancer. Here we summarize our recent knowledge about transcriptional regulation of DAPK expression.
Assuntos
Proteínas Quinases Associadas com Morte Celular/genética , Regulação da Expressão Gênica , Processamento Alternativo , Animais , Apoptose , Metilação de DNA , Proteínas Quinases Associadas com Morte Celular/metabolismo , Epigênese Genética , Humanos , MicroRNAs/genética , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Death-associated protein kinase (DAPK) undergoes activation in response to various death stimuli, and they have been associated with an increase in DAPK catalytic activity. One of the most prominent features of DAPK-induced cell death is the effect on the cytoskeleton, including loss of matrix attachment, and membrane blebbing. One known cytoskeletal-associated substrate of DAPK is the myosin-II light chain, phosphorylated by DAPK on Ser(19), thus stabilizing actin stress fibres. Moreover, paxillin, a component of focal adhesions, was found to be localized in close proximity to the tips of the DAPK-positive filaments, indicating that stress fibres containing DAPK extend to focal contacts. Forced expression of DAPK in multiple cell types results in morphological changes such as cell rounding, membrane blebbing, shrinking and detachment. During directed migration, DAPK functions as a potent inhibitor of cell polarization, as evidenced by its perturbation of the formation of static protrusion at the leading edge. Furthermore, DAPK inhibits random migration by suppressing directional persistence. One of the studies considered DAPK as an anoikis inducer. Others showed that DAP-kinase inhibits the activities of cell surface integrins by converting them into an inactive conformation. Biochemical experiments have established the DAPK binding to Syntaxin1 and its subsequent phosphorylation at Ser(188) in a Ca(2+) dependent manner. This phosphorylation event has been shown to decrease the binding of Syntaxin to MUNC18-1, a protein critically involved in synaptic vesicle docking. Here, we have investigated the structural interactions that modulate DAPK phosphorylation with Syntaxin and its functional role in binding to the MUNC18-1 to regulate vesicle docking. This review will summarize our current knowledge of the role of DAPK on cytoskeleton reorganization and report the mechanisms that regulate these changes.
Assuntos
Citoesqueleto/metabolismo , Proteínas Quinases Associadas com Morte Celular/metabolismo , Animais , Anoikis , Apoptose , Autofagia , Movimento Celular , Polaridade Celular , Citoesqueleto/ultraestrutura , Humanos , Modelos Moleculares , Proteínas Munc18/metabolismo , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Sintaxina 1/metabolismoRESUMO
The TNF-IL-6-STAT3 pathway plays a crucial role in promoting ulcerative colitis-associated carcinoma (UCC). To date, the negative regulation of STAT3 is poorly understood. Interestingly, intestinal epithelial cells of UCC in comparison to ulcerative colitis show high expression levels of anti-inflammatory death-associated protein kinase (DAPK) and low levels of pSTAT3. Accordingly, epithelial DAPK expression was enhanced in STAT3(IEC-KO) mice. To unravel a possible regulatory mechanism, we used an in vitro TNF-treated intestinal epithelial cell model. We identified a new function of DAPK in suppressing TNF-induced STAT3 activation as DAPK siRNA knockdown and treatment with a DAPK inhibitor potentiated STAT3 activation, IL-6 mRNA expression, and secretion. DAPK attenuated STAT3 activity directly by physical interaction shown in three-dimensional structural modeling. This model suggests that DAPK-induced conformational changes in the STAT3 dimer masked its nuclear localization signal. Alternatively, pharmacological inactivation of STAT3 led to an increase in DAPK mRNA and protein levels. Chromatin immunoprecipitation showed that STAT3 restricted DAPK expression by promoter binding, thereby reinforcing its own activation by inducing IL-6. This novel negative regulation principle might balance TNF-induced inflammation and seems to play an important role in the inflammation-associated transformation process as confirmed in an AOM+DSS colon carcinogenesis mouse model. DAPK as a negative regulator of STAT3 emerges as therapeutic option in the treatment of ulcerative colitis and UCC.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Epiteliais/enzimologia , Intestinos/patologia , Fator de Transcrição STAT3/metabolismo , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Colite Ulcerativa/enzimologia , Colite Ulcerativa/patologia , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Proteínas Quinases Associadas com Morte Celular , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Ligação de Hidrogênio/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Inflamação/patologia , Interleucina-6/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Fator de Transcrição STAT3/química , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Cinnamic acid and its derivatives represent attractive building blocks for the development of pharmacological tools. A series of piperoniloyl and cinnamoyl-based amides (6-9 a-f) have been synthesized and assayed against a wide panel of colorectal cancer (CRC) cells, with the aim of finding promising anticancer agents. Among all twenty-four synthesized molecules, 7a, 7e-f, 9c, and 9f displayed the best antiproliferative activity. The induced G1 cell cycle arrest and the increase in apoptotic cell death was seen in FACS analysis and western Blotting in the colon tumor cell lines HCT116, SW480, LoVo, and HT29, but not in the nontumor cell line HCEC. In particular, 9f overcame the resistance of HT29 cells, which have a mutant p53 and BRAF. Furthermore, 9f, amide of piperonilic acid with the 3,4-dichlorobenzyl substituent upregulated p21, which is involved in cell cycle arrest as well as in apoptosis induction. Cinnamic acid derivatives might be potential anticancer compounds, useful for the development of promising anti-CRC agents.
Assuntos
Antineoplásicos , Neoplasias Colorretais , Humanos , Proliferação de Células , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Cinamatos/farmacologia , Linhagem Celular Tumoral , Apoptose , Neoplasias Colorretais/tratamento farmacológicoRESUMO
Pancreatic cancer is a malignant tumor of the digestive system. It is highly aggressive, easily metastasizes, and extremely difficult to treat. This study aimed to analyze the genes that might regulate pancreatic cancer migration to provide an essential basis for the prognostic assessment of pancreatic cancer and individualized treatment. A CRISPR knockout library directed against 915 murine genes was transfected into TB 32047 cell line to screen which gene loss promoted cell migration. Next-generation sequencing and PinAPL.py- analysis was performed to identify candidate genes. We then assessed the effect of serine/threonine kinase 11 (STK11) knockout on pancreatic cancer by wound-healing assay, chick agnosia (CAM) assay, and orthotopic mouse pancreatic cancer model. We performed RNA sequence and Western blotting for mechanistic studies to identify and verify the pathways. After accelerated Transwell migration screening, STK11 was identified as one of the top candidate genes. Further experiments showed that targeted knockout of STK11 promoted the cell migration and increased liver metastasis in mice. Mechanistic analyses revealed that STK11 knockout influences blood vessel morphogenesis and is closely associated with the enhanced expression of phosphodiesterases (PDEs), especially PDE4D, PDE4B, and PDE10A. PDE4 inhibitor Roflumilast inhibited STK11-KO cell migration and tumor size, further demonstrating that PDEs are essential for STK11-deficient cell migration. Our findings support the adoption of therapeutic strategies, including Roflumilast, for patients with STK11-mutated pancreatic cancer in order to improve treatment efficacy and ultimately prolong survival.