RESUMO
Obesity can initiate, promote, and maintain systemic inflammation via metabolic reprogramming of macrophages that encircle adipocytes, termed crown-like structures (CLS). In breast cancer the presence of CLS has been correlated to high body mass index (BMI), larger mammary adipocyte size and postmenopausal status. However, the prognostic significance of CLS in HER2 + breast cancer is still unknown. We investigated the prognostic significance of CLS in a cohort of 69 trastuzumab-naïve and 117 adjuvant trastuzumab-treated patients with primary HER2 + breast cancer. Immunohistochemistry of tumour blocks was performed for CLS and correlated to clinical outcomes. CLS were more commonly found at the adipose-tumour border (B-CLS) (64.8% of patients). The presence of multiple B-CLS was associated with reduced time to metastatic disease (TMD) in trastuzumab treated patients with BMI ≥ 25 kg/m2 but not those with BMI < 25 kg/m2. Phenotypic analysis showed the presence of CD32B + B-CLS was strongly correlated to BMI ≥ 25 kg/m2 and reduced TMD in trastuzumab treated patients. Multivariable analysis suggested that CD32B + B-CLS positive tumours are associated with shorter TMD in trastuzumab-treated patients (HR 4.2 [95%CI, (1.01-17.4). This study indicates adipose-tumour border crown-like structures that are CD32B + potentially represent a biomarker for improved personalisation of treatment in HER2-overexpressed breast cancer patients.
Assuntos
Neoplasias da Mama , Tecido Adiposo/metabolismo , Mama/patologia , Neoplasias da Mama/patologia , Feminino , Humanos , Prognóstico , Receptor ErbB-2/metabolismo , Trastuzumab/metabolismo , Trastuzumab/uso terapêuticoRESUMO
Adaptive responses to hypoxia are mediated by the hypoxia-inducible factor (HIF) family of transcription factors. These responses include the upregulation of glycolysis to maintain ATP production. This also generates acidic metabolites, which require HIF-induced carbonic anhydrase IX (CAIX) for their neutralisation. C-terminal binding proteins (CtBPs) are coregulators of gene transcription and couple glycolysis with gene transcription due to their regulation by the glycolytic coenzyme NADH. Here, we find that experimental manipulation of glycolysis and CtBP function in breast cancer cells through multiple complementary approaches supports a hypothesis whereby the expression of known HIF-inducible genes, and CAIX in particular, adapts to available glucose in the microenvironment through a mechanism involving CtBPs. This novel pathway promotes the survival of stem cell-like cancer (SCLC) cells in hypoxia.
Assuntos
Oxirredutases do Álcool/metabolismo , Neoplasias da Mama/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glicólise , NAD/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Multimerização Proteica , Oxirredutases do Álcool/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Hipóxia Celular/genética , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Células MCF-7 , NAD/genética , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/patologia , Proteínas do Tecido Nervoso/genéticaRESUMO
High rates of glycolysis in cancer cells are a well-established characteristic of many human tumors, providing rapidly proliferating cancer cells with metabolites that can be used as precursors for anabolic pathways. Maintenance of high glycolytic rates depends on the lactate dehydrogenase-catalyzed regeneration of NAD+ from GAPDH-generated NADH because an increased NADH:NAD+ ratio inhibits GAPDH. Here, using human breast cancer cell models, we identified a pathway in which changes in the extramitochondrial-free NADH:NAD+ ratio signaled through the CtBP family of NADH-sensitive transcriptional regulators to control the abundance and activity of p53. NADH-free forms of CtBPs cooperated with the p53-binding partner HDM2 to suppress p53 function, and loss of these forms in highly glycolytic cells resulted in p53 accumulation. We propose that this pathway represents a "glycolytic stress response" in which the initiation of a protective p53 response by an increased NADH:NAD+ ratio enables cells to avoid cellular damage caused by mismatches between metabolic supply and demand.
Assuntos
Regulação Neoplásica da Expressão Gênica , Glicólise , NAD/metabolismo , Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Aerobiose , Linhagem Celular Tumoral , Humanos , NAD/genética , Neoplasias/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Glycolysis and hypoxia are key regulators of human embryonic stem cell (hESC) self-renewal, but how changes in metabolism affect gene expression is poorly understood. C-terminal binding proteins (CTBPs) are glycolytic sensors that through NADH binding link the metabolic state of the cell to its gene expression, by acting as transcriptional corepressors, or coactivators. However, the role of CTBPs in hESCs has not previously been investigated. A direct interaction between hypoxia-inducible factor 2α (HIF-2α) and the CTBP proximal promoters in hESCs cultured only under hypoxia was demonstrated. Decreasing the rate of flux through glycolysis in hESCs maintained under hypoxia resulted in a reduction of CTBPs, OCT4, SOX2, and NANOG, but also in the expression of HIF-2α. Silencing CTBP expression resulted in the loss of pluripotency marker expression demonstrating that CTBPs are involved in hESC maintenance. These data suggest that under hypoxia, glycolysis regulates self-renewal through HIF-2α and the induction of the metabolic sensors CTBPs.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Autorrenovação Celular/genética , Proteínas Correpressoras/genética , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas Correpressoras/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Glicólise , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Modelos Biológicos , Oxigênio/metabolismo , Regiões Promotoras GenéticasRESUMO
BACKGROUND: Metabolic changes in tumour cells are used in clinical imaging and may provide potential therapeutic targets. Human papillomavirus (HPV) status is important in classifying head and neck cancers (HNSCC), identifying a distinct clinical phenotype; metabolic differences between these HNSCC subtypes remain poorly understood. METHODS: We used RNA sequencing to classify the metabolic expression profiles of HPV+ve and HPV-ve HNSCC, performed a meta-analysis on FDG-PET imaging characteristics and correlated results with in vitro extracellular flux analysis of HPV-ve and HPV+ve HNSCC cell lines. The monocarboxylic acid transporter-1 (MCT1) was identified as a potential metabolic target and tested in functional assays. RESULTS: Specific metabolic profiles were associated with HPV status, not limited to carbohydrate metabolism. There was dominance of all energy pathways in HPV-negative disease, with elevated expression of genes associated with glycolysis and oxidative phosphorylation. In vitro analysis confirmed comparative increased rates of oxidative phosphorylation and glycolysis in HPV-negative cell lines. PET SUV(max) scores however were unable to reliably differentiate between HPV-positive and HPV-negative tumours. MCT1 expression was significantly increased in HPV-negative tumours, and inhibition suppressed tumour cell invasion, colony formation and promoted radiosensitivity. CONCLUSION: HPV-positive and negative HNSCC have different metabolic profiles which may have potential therapeutic applications.
Assuntos
Transportadores de Ácidos Monocarboxílicos/genética , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Simportadores/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Glicólise/genética , Humanos , Transportadores de Ácidos Monocarboxílicos/isolamento & purificação , Transportadores de Ácidos Monocarboxílicos/metabolismo , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Fosforilação Oxidativa , Papillomaviridae/genética , Papillomaviridae/metabolismo , Infecções por Papillomavirus/diagnóstico por imagem , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Tomografia por Emissão de Pósitrons , Tolerância a Radiação , Análise de Sequência de RNA , Carcinoma de Células Escamosas de Cabeça e Pescoço/diagnóstico por imagem , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Simportadores/isolamento & purificação , Simportadores/metabolismoRESUMO
Altered flux through major metabolic pathways is a hallmark of cancer cells and provides opportunities for therapy. Stem cell-like cancer (SCLC) cells can cause metastasis and therapy resistance. They possess metabolic plasticity, theoretically enabling resistance to therapies targeting a specific metabolic state. The C-terminal binding protein (CtBP) transcriptional regulators are potential therapeutic targets in highly glycolytic cancer cells, as they are activated by the glycolytic coenzyme nicotinamide adenine dinucleotide (NADH). However, SCLC cells commonly exist in an oxidative state with low rates of glycolysis. Metformin inhibits complex I of the mitochondrial electron transport chain; it can kill oxidative SCLC cells and has anti-cancer activity in patients. SCLC cells can acquire resistance to metformin through increased glycolysis. Given the potential for long-term metformin therapy, we have studied acquired metformin resistance in cells from the claudin-low subtype of breast cancer. Cells cultured for 8 weeks in sub-IC50 metformin concentration proliferated comparably to untreated cells and exhibited higher rates of glucose uptake. SCLC cells were enriched in metformin-adapted cultures. These SCLC cells acquired sensitivity to multiple methods of inhibition of CtBP function, including a cyclic peptide inhibitor of NADH-induced CtBP dimerization. Single-cell mRNA sequencing identified a reprogramming of epithelial-mesenchymal and stem cell gene expression in the metformin-adapted SCLC cells. These SCLC cells demonstrated an acquired dependency on one of these genes, Tenascin C. Thus, in addition to acquisition of sensitivity to glycolysis-targeting therapeutic strategies, the reprograming of gene expression in the metformin-adapted SCLC cells renders them sensitive to potential therapeutic approaches not directly linked to cell metabolism.
Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Antineoplásicos Alquilantes/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Metformina/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Oxirredutases do Álcool/metabolismo , Animais , Antineoplásicos Alquilantes/uso terapêutico , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Glicólise , Humanos , Concentração Inibidora 50 , Redes e Vias Metabólicas/efeitos dos fármacos , Metformina/uso terapêutico , Camundongos , NAD/metabolismo , Células-Tronco Neoplásicas/patologia , Análise de Sequência de RNA , Análise de Célula Única , Esferoides Celulares , Tenascina/antagonistas & inibidores , Tenascina/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Altered glycolysis is a characteristic of many cancers, and can also be associated with changes in stem cell-like cancer (SCLC) cell populations. We therefore set out to directly examine the effect of glycolysis on SCLC cell phenotype, using a model where glycolysis is stably reduced by adapting the cells to a sugar source other than glucose. Restricting glycolysis using this approach consistently resulted in cells with increased oncogenic potential; including an increase in SCLC cells, proliferation in 3D matrigel, invasiveness, chemoresistance, and altered global gene expression. Tumorigenicity in vivo was also markedly increased. SCLC cells exhibited increased dependence upon alternate metabolic pathways. They also became c-KIT dependent, indicating that their apparent state of maturation is regulated by glycolysis. Single-cell mRNA sequencing identified altered networks of metabolic-, stem- and signaling- gene expression within SCLC-enriched populations in response to glycolytic restriction. Therefore, reduced glycolysis, which may occur in niches within tumors where glucose availability is limiting, can promote tumor aggressiveness by increasing SCLC cell populations, but can also introduce novel, potentially exploitable, vulnerabilities in SCLC cells.
RESUMO
Tumors carrying hereditary mutations in BRCA1, which attenuate the BRCA1 DNA damage repair pathway, are more susceptible to dual treatment with PARP inhibitors and DNA damaging therapeutics. Conversely, breast cancer tumors with nonmutated functional BRCA1 are less sensitive to PARP inhibition. We describe a method that triggers susceptibility to PARP inhibition in BRCA1-functional tumor cells. BRCA1 exon 11 is a key for the function of BRCA1 in DNA damage repair. Analysis of the BRCA1 exon 11 splicing mechanism identified a key region within this exon which, when deleted, induced exon 11 skipping. An RNA splice-switching oligonucleotide (SSO) developed to target this region was shown to artificially stimulate skipping of exon 11 in endogenous BRCA1 pre-mRNA. SSO transfection rendered wild-type BRCA1 expressing cell lines more susceptible to PARP inhibitor treatment, as demonstrated by a reduction in cell survival at all SSO concentrations tested. Combined SSO and PARP inhibitor treatment increased γH2AX expression indicating that SSO-dependent skipping of BRCA1 exon 11 was able to promote DSBs and therefore synthetic lethality. In conclusion, this SSO provides a new potential therapeutic strategy for targeting BRCA1-functional breast cancer by enhancing the effect of PARP inhibitors.
Assuntos
Processamento Alternativo , Neoplasias da Mama/genética , Genes BRCA1 , Oligonucleotídeos/genética , Inibidores de Poli(ADP-Ribose) Polimerases/química , Apoptose/efeitos dos fármacos , Benzimidazóis/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Éxons , Feminino , Humanos , Células MCF-7 , Mutação , Oligonucleotídeos/química , Poli(ADP-Ribose) Polimerases/metabolismo , Reação em Cadeia da Polimerase , Precursores de RNARESUMO
We describe the development of a small-molecule mimic of Xaa-trans-Pro dipeptide in poly-l-proline type II helix conformation, based upon a (3R,6S,9S)-2-oxo-1-azabicyclo[4.3.0]nonane core structure. Stereoselective synthesis of the mimic from l-pyroglutamic acid is achieved in twelve linear steps and 9.9% yield. Configurational and conformational analyses are conducted using a combination of (1)H NMR spectroscopy, X-ray crystallography and circular dichroism spectroscopy; and evaluation of the mimic as a promising surrogate dipeptide, in a protein-protein interaction between the SH3 domain of human Fyn kinase (Fyn SH3) and peptidomimetics of its biological ligand, are conducted by (1)H-(15)N HSQC NMR titration experiments.
Assuntos
Compostos Azabicíclicos/síntese química , Dipeptídeos/química , Peptídeos/química , Peptidomiméticos/síntese química , Sequência de Aminoácidos , Compostos Azabicíclicos/química , Compostos Azabicíclicos/farmacologia , Cristalografia por Raios X , Dipeptídeos/síntese química , Dipeptídeos/farmacologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/farmacologia , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas c-fyn/química , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Domínios de Homologia de srcRESUMO
Interactions between cancer cells and cancer-associated fibroblasts (CAFs) play an important role in tumour development and progression. In this study we investigated the functional role of CAFs in oesophageal adenocarcinoma (EAC). We used immunochemistry to analyse a cohort of 183 EAC patients for CAF markers related to disease mortality. We characterized CAFs and normal oesophageal fibroblasts (NOFs) using western blotting, immunofluorescence and gel contraction. Transwell assays, 3D organotypic culture and xenograft models were used to examine the effects on EAC cell function and to dissect molecular mechanisms regulating invasion. Most EACs (93%) contained CAFs with a myofibroblastic (α-SMA-positive) phenotype, which correlated significantly with poor survival [p = 0.016; HR 7. 1 (1.7-29.4)]. Primary CAFs isolated from EACs have a contractile, myofibroblastic phenotype and promote EAC cell invasion in vitro (Transwell assays, p ≤ 0.05; organotypic culture, p < 0.001) and in vivo (p ≤ 0.05). In vitro, this pro-invasive effect is modulated through the matricellular protein periostin. Periostin is secreted by CAFs and acts as a ligand for EAC cell integrins αvß3 and αvß5, promoting activation of the PI3kinase-Akt pathway. In patient samples, periostin expression at the tumour cell-stromal interface correlates with poor overall and disease-free survival. Our study highlights the importance of the tumour stroma in EAC progression. Paracrine interaction between CAF-secreted periostin and EAC-expressed integrins results in PI3 kinase-Akt activation and increased tumour cell invasion. Most EACs contain a myofibroblastic CAF-rich stroma; this may explain the aggressive, highly infiltrative nature of the disease, and suggests that stromal targeting may produce therapeutic benefit in EAC patients.
Assuntos
Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Moléculas de Adesão Celular/farmacologia , Movimento Celular/efeitos dos fármacos , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/patologia , Esôfago/patologia , Fibroblastos/patologia , Actinas/metabolismo , Adenocarcinoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/metabolismo , Células Cultivadas , Estudos de Coortes , Modelos Animais de Doenças , Neoplasias Esofágicas/metabolismo , Feminino , Xenoenxertos , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Taxa de Sobrevida , Microambiente TumoralRESUMO
Click DNA ligation promises an alternative to the current enzymatic approaches for DNA assembly, with the ultimate goal of using efficient chemical reactions for the total chemical synthesis and assembly of genes and genomes. Such an approach would enable the incorporation of various chemically modified bases throughout long stretches of DNA, a feat not possible with current polymerase-based methods. An unequivocal requirement for this approach is the biocompatibility of the resulting triazole-linked DNA. The correct function of this unnatural DNA linker in human cells is demonstrated here by using a click-linked gene encoding the fluorescent protein mCherry. Reverse transcription of mRNA isolated from these cells and subsequent sequencing of the mCherry cDNA shows error-free transcription. Nucleotide excision repair (NER) is shown to not play a role in the observed biocompatibility by using a NER-deficient human cell line. This is the first example of a non-natural DNA linker being functional in a eukaryotic cell.
Assuntos
DNA/síntese química , DNA/genética , Transcrição Gênica , Linhagem Celular , Química Click , DNA/química , Reparo do DNA , Humanos , Proteínas Luminescentes/genética , Biologia Sintética , Triazóis/química , Proteína Vermelha FluorescenteRESUMO
INTRODUCTION: Breast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice. METHODS: More than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer 'stem' cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account. RESULTS: The 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working. CONCLUSIONS: With resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.
Assuntos
Neoplasias da Mama , Pesquisa , Pesquisa Translacional Biomédica , Animais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/etiologia , Neoplasias da Mama/terapia , Feminino , HumanosRESUMO
Identification of direct modulators of transcription factor protein-protein interactions is a key challenge for ligand discovery that promises to significantly advance current approaches to cancer therapy. Here, we report an inhibitor of NADH-dependent dimerization of the C-terminal binding protein (CtBP) transcriptional repressor, identified by screening genetically encoded cyclic peptide libraries of up to 64 million members. CtBP dimers form the core of transcription complexes associated with epigenetic regulation of multiple genes that control many characteristics of cancer cells, including proliferation, survival and migration. CtBP monomers also have distinct and critical cellular function, thus current experimental tools that deplete all forms of a targeted protein (e.g. siRNA) do not allow the cellular consequences of this metabolically regulated transcription factor to be deciphered. The most potent inhibitor from our screen (cyclo-SGWTVVRMY) is demonstrated to disrupt CtBP dimerization in vitro and in cells. This compound is used as a chemical tool to establish that the NADH-dependent dimerization of CtBPs regulates the maintenance of mitotic fidelity in cancer cells. Treatment of highly glycolytic breast cancer cell lines with the identified inhibitor significantly reduced their mitotic fidelity, proliferation and colony forming potential, whereas the compound does not affect mitotic fidelity of cells with lower glycolytic flux. This work not only links the altered metabolic state of transformed cells to a key determinant of the tumor cell phenotype, but the uncovered compound also serves as the starting point for the development of potential therapeutic agents that target tumors by disrupting the CtBP chromatin-modifying complex.
RESUMO
Aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is a bifunctional homodimeric enzyme that catalyzes the last two steps of de novo purine biosynthesis. Homodimerization of ATIC, a protein-protein interaction with an interface of over 5000 Å(2), is required for its aminoimidazole carboxamide ribonucleotide (AICAR) transformylase activity, with the active sites forming at the interface of the interacting proteins. Here, we report the development of a small-molecule inhibitor of AICAR transformylase that functions by preventing the homodimerization of ATIC. The compound is derived from a previously reported cyclic hexapeptide inhibitor of AICAR transformylase (with a K(i) of 17 µM), identified by high-throughput screening. The active motif of the cyclic peptide is identified as an arginine-tyrosine dipeptide, a capped analogue of which inhibits AICAR transformylase with a K(i) value of 84 µM. A library of nonnatural analogues of this dipeptide was designed, synthesized, and assayed. The most potent compound inhibits AICAR transformylase with a K(i) value of 685 nM, a 25-fold improvement in activity from the parent cyclic peptide. The potential for this AICAR transformylase inhibitor in cancer therapy was assessed by studying its effect on the proliferation of a model breast cancer cell line. Using a nonradioactive proliferation assay and live cell imaging, a dose-dependent reduction in cell numbers and cell division rates was observed in cells treated with our ATIC dimerization inhibitor.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Inibidores Enzimáticos/farmacologia , Peptídeos Cíclicos/farmacologia , Fosforribosilaminoimidazolcarboxamida Formiltransferase/química , Multimerização Proteica/efeitos dos fármacos , Antineoplásicos/síntese química , Antineoplásicos/química , Domínio Catalítico/efeitos dos fármacos , Contagem de Células , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Células MCF-7 , Estrutura Molecular , Peso Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Fosforribosilaminoimidazolcarboxamida Formiltransferase/antagonistas & inibidores , Fosforribosilaminoimidazolcarboxamida Formiltransferase/metabolismo , Relação Estrutura-AtividadeRESUMO
Multicellular tumour spheroid (MCTS) cultures are excellent model systems for simulating the development and microenvironmental conditions of in vivo tumour growth. Many documented cell lines can generate differentiated MCTS when cultured in suspension or in a non-adhesive environment. While physiological and biochemical properties of MCTS have been extensively characterized, insight into the events and conditions responsible for initiation of these structures is lacking. MCTS are formed by only a small subpopulation of cells during surface-associated growth but the processes responsible for this differentiation are poorly understood and have not been previously studied experimentally. Analysis of gene expression within spheroids has provided clues but to date it is not known if the observed differences are a cause or consequence of MCTS growth. One mechanism linked to tumourigenesis in a number of cancers is genetic instability arising from impaired DNA mismatch repair (MMR). This study aimed to determine the role of MMR in MCTS initiation and development. Using surface-associated N2a and CHLA-02-ATRT culture systems we have investigated the impact of impaired MMR on MCTS growth. Analysis of the DNA MMR genes MLH1 and PMS2 revealed both to be significantly down-regulated at the mRNA level compared with non-spheroid-forming cells. By using small interfering RNA (siRNA) against these genes we show that silencing of MLH1 and PMS2 enhances both MCTS initiation and subsequent expansion. This effect was prolonged over several passages following siRNA transfection. Down-regulation of DNA MMR can contribute to tumour initiation and progression in N2a and CHLA-02-ATRT MCTS models. Studies of surface-associated MCTS differentiation may have broader applications in studying events in the initiation of cancer foci.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Adenosina Trifosfatases/genética , Neoplasias Encefálicas/patologia , Reparo de Erro de Pareamento de DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Neuroblastoma/patologia , Proteínas Nucleares/genética , Esferoides Celulares/patologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Adenosina Trifosfatases/antagonistas & inibidores , Animais , Neoplasias Encefálicas/genética , Enzimas Reparadoras do DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Endonuclease PMS2 de Reparo de Erro de Pareamento , Proteína 1 Homóloga a MutL , Neuroblastoma/genética , Proteínas Nucleares/antagonistas & inibidores , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
BACKGROUND: Cyclin-dependent kinases 2, 4 and 6 (Cdk2, Cdk4, Cdk6) are closely structurally homologous proteins which are classically understood to control the transition from the G1 to the S-phases of the cell cycle by combining with their appropriate cyclin D or cyclin E partners to form kinase-active holoenzymes. Deregulation of Cdk4 is widespread in human cancer, CDK4 gene knockout is highly protective against chemical and oncogene-mediated epithelial carcinogenesis, despite the continued presence of CDK2 and CDK6; and overexpresssion of Cdk4 promotes skin carcinogenesis. Surprisingly, however, Cdk4 kinase inhibitors have not yet fulfilled their expectation as 'blockbuster' anticancer agents. Resistance to inhibition of Cdk4 kinase in some cases could potentially be due to a non-kinase activity, as recently reported with epidermal growth factor receptor. RESULTS: A search for a potential functional site of non-kinase activity present in Cdk4 but not Cdk2 or Cdk6 revealed a previously-unidentified loop on the outside of the C'-terminal non-kinase domain of Cdk4, containing a central amino-acid sequence, Pro-Arg-Gly-Pro-Arg-Pro (PRGPRP). An isolated hexapeptide with this sequence and its cyclic amphiphilic congeners are selectively lethal at high doses to a wide range of human cancer cell lines whilst sparing normal diploid keratinocytes and fibroblasts. Treated cancer cells do not exhibit the wide variability of dose response typically seen with other anticancer agents. Cancer cell killing by PRGPRP, in a cyclic amphiphilic cassette, requires cells to be in cycle but does not perturb cell cycle distribution and is accompanied by altered relative Cdk4/Cdk1 expression and selective decrease in ATP levels. Morphological features of apoptosis are absent and cancer cell death does not appear to involve autophagy. CONCLUSION: These findings suggest a potential new paradigm for the development of broad-spectrum cancer specific therapeutics with a companion diagnostic biomarker and a putative functional site for kinase-unrelated activities of Cdk4.
Assuntos
Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/química , Neoplasias/fisiopatologia , Peptídeos/farmacologia , Sequência de Aminoácidos , Antineoplásicos/química , Autofagia/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Fibroblastos/efeitos dos fármacos , Humanos , Modelos Moleculares , Neoplasias/enzimologia , Peptídeos/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteômica , Homologia de Sequência de Aminoácidos , Telomerase/genética , Telomerase/metabolismoRESUMO
BACKGROUND INFORMATION: CtBPs [C-terminal (of E1A) binding protein] have roles in the nucleus as transcriptional co-repressors, and in the cytoplasm in the maintenance of vesicular membranes. CtBPs are expressed from two genes, CTBP1 and CTBP2, mRNA products of which are alternatively spliced at their 5'-ends to generate distinct protein isoforms. Extensive molecular and cellular analyses have identified CtBPs as regulators of pathways critical for tumour initiation, progression and response to therapy. However, little is known of the expression or regulation of CtBP isoforms in human cancer, nor of the relative contributions of CTBP1 and CTBP2 to the tumour cell phenotype. RESULTS: Expression of CtBP proteins and CTBP1 and CTBP2 mRNA splice forms in breast cancer cell lines and tumour tissue was examined. CtBP1 proteins are identifiable as a single band on Western blots and are ubiquitously detectable in breast tumour samples, by both Western blotting and immunohistochemistry. CtBP1 is present in six of six breast cancer cell lines, although it is barely detectable in SKBr3 cells due to reduced CTBP1 mRNA expression. In the cell lines, the predominant CTBP1 mRNA splice form encodes CtBP1-S protein; in tumours, both major CTBP1 mRNA splice forms are variably expressed. CtBP2 proteins are ubiquitously expressed in all lines and tumour samples. The predominant CTBP2 mRNA encodes CtBP2-L, although an alternatively spliced form that encodes CtBP2-S, previously unidentified in humans, is expressed at low abundance. Both CtBP2-L and CtBP2-S are readily detectable as two distinct bands on Western blots; here we show that the CTBP2-L mRNA is translated from two AUG codons to generate both CtBP2-L and CtBP2-S. We have also identified an autoregulatory feedback mechanism whereby CtBP protein abundance is maintained in proliferating breast cancer cells through the post-transcriptional regulation of CtBP2. This feedback is disrupted by UV-C radiation or exposure to cisplatin. Finally, we demonstrate that CtBP1 and CtBP2 both have p53-dependent and -independent roles in suppressing the sensitivity of breast cancer cells to mechanistically diverse cancer chemotherapeutic agents. CONCLUSIONS: These studies support recent evidence that CtBP family proteins represent potential targets for therapeutic strategies for the treatment of cancer in general, and breast cancer in particular.
Assuntos
Oxirredutases do Álcool/metabolismo , Neoplasias da Mama/genética , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Proteínas do Tecido Nervoso/metabolismo , Oxirredutases do Álcool/genética , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proteínas Correpressoras , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Proteínas do Tecido Nervoso/genética , Splicing de RNARESUMO
BACKGROUND INFORMATION: Carcinoma of the oesophagus is the sixth leading cause of cancer death in the western world and is associated with a 5-year survival of less than 15%. Recent evidence suggests that stromal-epithelial interactions are fundamental in carcinogenesis. The advent of co-culture techniques permits the investigation of cross-talk between the stroma and epithelium in a physiological setting. We have characterized a histologically representative oesophageal organotypic model and have used it to compare the most commonly used squamous oesophageal cell line, HET-1A, with primary oesophageal squamous cells for use in studies of the oesophageal epithelium in vitro. RESULTS: When grown in an organotypic culture with normal fibroblasts, the oesophageal carcinoma cell lines OE21 (squamous) and OE19 (adenocarcinoma) morphologically resembled the tumour of origin with evidence of stromal invasion and mucus production, respectively. However, HET-1A cells, which were derived from normal squamous oesophageal cells, appeared dysplastic and failed to display evidence of squamous differentiation. By comparison with primary oesophageal epithelial cells, the HET-1A cells were highly proliferative and did not express the epithelial markers E-cadherin or CK5/6 (casein kinase 5/6), or the stratified epithelial marker ΔNp63, but did express the mesenchymal markers vimentin and N-cadherin. CONCLUSION: Studies of epithelial carcinogenesis will benefit from culture systems which allow manipulation of the stromal and epithelial layers independently. We have developed an organotypic culture using primary oesophageal squamous cells and fibroblasts in which a stratified epithelium with a proliferative basal layer that stains strongly for ΔNp63 develops. This model will be suitable for the study of the molecular events in the development of Barrett's oesophagus. The most commonly used normal oesophageal squamous cell line, HET-1A, does not have the characteristics of normal oesophageal squamous cells and should not be used in models of the normal oesophageal epithelium. Until more representative cell lines are available, future studies in oesophageal cancer will be reliant on the availability and manipulation of primary tissue.
Assuntos
Esôfago de Barrett/patologia , Carcinoma de Células Escamosas/patologia , Células Epiteliais/patologia , Neoplasias Esofágicas/patologia , Adenocarcinoma/patologia , Antígenos CD/biossíntese , Caderinas/biossíntese , Caseína Quinases/biossíntese , Técnicas de Cocultura , Células Epiteliais/metabolismo , Esôfago/citologia , Humanos , Proteínas de Membrana/biossíntese , Vimentina/biossínteseRESUMO
eIF4E is over-expressed in many tumours, including a high proportion of breast cancers. eIF4E is an oncogene, and signalling pathways which promote eIF4E activity represent potential targets for therapeutic intervention in cancer. MNKs phosphorylate eIF4E on serine 209, a modification that can be required for eIF4E-dependent cell transformation. There is therefore a clear requirement to determine the role of MNKs in the proliferation and survival of cells from the major human tumours, such as breast cancer. Phosphorylated eIF4E protein was readily detectable in some breast tumour samples, but was below the limits of detection in others. Of 6 breast cancer cell lines representing the major sub-types of breast cancer, phosphorylated eIF4E was readily detectable in 5 of them, with MCF-7 cells displaying markedly lower levels. Long term colony forming assays demonstrated that all the five lines with high levels of phosphorylated eIF4E were highly sensitive to a MNK inhibitor. In short term assays, a range of responses was revealed. MCF-7 cells were insensitive in both assays. The anti-proliferative effects of the MNK inhibitor in breast cancer cells are primarily cytostatic, rather than cytotoxic, and are potentially due to the inhibition of cyclin D1 synthesis. Our data provide evidence that the sensitivity of breast cancer cells to MNK inhibition may correlate with baseline levels of eIF4E phosphorylation, and suggest that a proportion of breast cancers could be sensitive to inhibiting MNK kinase activity, and that the presence of phosphorylated eIF4E could provide a biomarker for the identification of responsive tumours.
Assuntos
Proliferação de Células , Fator de Iniciação 4E em Eucariotos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Compostos de Anilina/farmacologia , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Ciclina D1/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Purinas/farmacologiaRESUMO
The p53 regulatory network is critically involved in preventing the initiation of cancer. In unstressed cells, p53 is maintained at low levels and is largely inactive, mainly through the action of its two essential negative regulators, HDM2 and HDMX. p53 abundance and activity are up-regulated in response to various stresses, including DNA damage and oncogene activation. Active p53 initiates transcriptional and transcription-independent programs that result in cell cycle arrest, cellular senescence, or apoptosis. p53 also activates transcription of HDM2, which initially leads to the degradation of HDMX, creating a positive feedback loop to obtain maximal activation of p53. Subsequently, when stress-induced post-translational modifications start to decline, HDM2 becomes effective in targeting p53 for degradation, thus attenuating the p53 response. To date, no clear function for HDMX in this critical attenuation phase has been demonstrated experimentally. Like HDM2, the HDMX gene contains a promoter (P2) in its first intron that is potentially inducible by p53. We show that p53 activation in response to a plethora of p53-activating agents induces the transcription of a novel HDMX mRNA transcript from the HDMX-P2 promoter. This mRNA is more efficiently translated than that expressed from the constitutive HDMX-P1 promoter, and it encodes a long form of HDMX protein, HDMX-L. Importantly, we demonstrate that HDMX-L cooperates with HDM2 to promote the ubiquitination of p53 and that p53-induced HDMX transcription from the P2 promoter can play a key role in the attenuation phase of the p53 response, to effectively diminish p53 abundance as cells recover from stress.