RESUMO
Depending on the circumstance, FOXO (Forkhead O) (FOXO1, FOXO3, and FOXO4) transcription factors activate the expression of markedly different sets of genes to produce different phenotypic effects. For example, distinct FOXO-regulated transcriptional programs stimulate cell death or enhance organism life span. To gain insight into how FOXOs select specific genes for regulation, we performed a screen for genes that modify FOXO activation of TRAIL, a death receptor ligand capable of inducing extrinsic apoptosis. We discovered that the bZIP transcriptional repressor NFIL3 (nuclear factor interleukin 3-regulated) hindered FOXO transcription factor access to chromatin at the TRAIL promoter by binding to nearby DNA and recruiting histone deacetylase-2 (HDAC2) to reduce histone acetylation. In the same manner, NFIL3 repressed expression of certain FOXO targets--e.g., FAS, GADD45α (growth arrest and DNA damage-inducible, α), and GADD45ß--but not others. NFIL3, which we found to be overexpressed in different cancers, supported tumor cell survival largely through repression of TRAIL and antagonized hydrogen peroxide-induced cell death. Moreover, its expression in cancer was associated with lower patient survival. Therefore, NFIL3 alters cancer cell behavior and FOXO function by acting on chromatin to restrict the menu of FOXO target genes. Targeting of NFIL3 could be of therapeutic benefit for cancer patients.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Apoptose/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Sítios de Ligação , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Cromatina/metabolismo , Proteína Forkhead Box O1 , Células HEK293 , Histona Desacetilases/metabolismo , Humanos , Estimativa de Kaplan-Meier , Prognóstico , Regiões Promotoras Genéticas , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
Basal-like breast cancers (BBCs) are enriched for increased EGFR expression and decreased expression of PTEN. We found that treatment with metformin and erlotinib synergistically induced apoptosis in a subset of BBC cell lines. The drug combination led to enhanced reduction of EGFR, AKT, S6 and 4EBP1 phosphorylation, as well as prevented colony formation and inhibited mammosphere outgrowth. Our data with other compounds suggested that biguanides combined with EGFR inhibitors have the potential to outperform other targeted drug combinations and could be employed in other breast cancer subtypes, as well as other tumor types, with activated EGFR and PI3K signaling. Analysis of BBC cell line alterations led to the hypothesis that loss of PTEN sensitized cells to the drug combination which was confirmed using isogenic cell line models with and without PTEN expression. Combined metformin and erlotinib led to partial regression of PTEN-null and EGFR-amplified xenografted MDA-MB-468 BBC tumors with evidence of significant apoptosis, reduction of EGFR and AKT signaling, and lack of altered plasma insulin levels. Combined treatment also inhibited xenografted PTEN null HCC-70 BBC cells. Measurement of trough plasma drug levels in xenografted mice and a separately performed pharmacokinetics modeling study support possible clinical translation.
Assuntos
Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Sinergismo Farmacológico , Metformina/farmacologia , Quinazolinas/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Feminino , Citometria de Fluxo , Humanos , Hipoglicemiantes/farmacologia , Técnicas Imunoenzimáticas , Camundongos , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A virtual screening procedure was applied to identify new tankyrase inhibitors. Through pharmacophore screening of a compounds collection from the SPECS database, the methoxy[l]benzothieno[2,3-c]quinolin-6(5H)-one scaffold was identified as nicotinamide mimetic able to inhibit tankyrase activity at low micromolar concentration. In order to improve potency and selectivity, tandem structure-based and scaffold hopping approaches were carried out over the new scaffold leading to the discovery of the 2-(phenyl)-3H-benzo[4,5]thieno[3,2-d]pyrimidin-4-one as powerful chemotype suitable for tankyrase inhibition. The best compound 2-(4-tert-butyl-phenyl)-3H-benzo[4,5]thieno[3,2-d]pyrimidin-4-one (23) displayed nanomolar potencies (IC50s TNKS-1 = 21 nM and TNKS-2 = 29 nM) and high selectivity when profiled against several other PARPs. Furthermore, a striking Wnt signaling, as well as cell growth inhibition, was observed assaying 23 in DLD-1 cancer cells.
Assuntos
Inibidores Enzimáticos/farmacologia , Tanquirases/antagonistas & inibidores , Inibidores Enzimáticos/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Breast cancer can be classified into different molecular subtypes with varying clinical and pathological characteristics. The basal-like breast cancer subtype represents one of the most aggressive and lethal types of breast cancer, and due to poor mechanistic understanding, it lacks targeted therapy. Many basal-like breast cancer patient samples display alterations of established drivers of cancer development, including elevated expression of EGFR, p53 inactivating mutations and loss of expression of the tumor suppressor PTEN; however, their contribution to human basal-like breast cancer pathogenesis remains ill-defined. Using non-transformed human mammary epithelial cells, we set out to determine whether altering EGFR, p53 and PTEN in different combinations could contribute to basal-like breast cancer progression through transformation of cells. Altering PTEN in combination with either p53 or EGFR in contrast to any of the single alterations caused increased growth of transformed colonies in soft agar. Concomitantly modifying all three genes led to the highest rate of cellular proliferation and the greatest degree of anchorage-independent colony formation. Results from our effort to engineer a model of BBC expressing alterations of EGFR, p53 and PTEN suggest that these changes are cooperative and likely play a causal role in basal-like breast cancer pathogenesis. Consideration should be given to targeting EGFR and restoring p53 and PTEN signaling simultaneously as a strategy for treatment of this subtype of breast cancer.
Assuntos
Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Receptores ErbB/genética , Neoplasia de Células Basais/genética , PTEN Fosfo-Hidrolase/genética , Proteína Supressora de Tumor p53/genética , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Camundongos , Modelos Biológicos , Neoplasia de Células Basais/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais , Transplante Heterólogo , Ensaio Tumoral de Célula-Tronco , Proteína Supressora de Tumor p53/metabolismoRESUMO
Recent insights into the role of the von-Hippel Lindau (VHL) tumor suppressor gene in hereditary and sporadic clear-cell renal cell carcinoma (ccRCC) have led to new treatments for patients with metastatic ccRCC, although virtually all patients eventually succumb to the disease. We performed an integrated, genome-wide analysis of copy-number changes and gene expression profiles in 90 tumors, including both sporadic and VHL disease-associated tumors, in hopes of identifying new therapeutic targets in ccRCC. We identified 14 regions of nonrandom copy-number change, including 7 regions of amplification (1q, 2q, 5q, 7q, 8q, 12p, and 20q) and 7 regions of deletion (1p, 3p, 4q, 6q, 8p, 9p, and 14q). An analysis aimed at identifying the relevant genes revealed VHL as one of three genes in the 3p deletion peak, CDKN2A and CDKN2B as the only genes in the 9p deletion peak, and MYC as the only gene in the 8q amplification peak. An integrated analysis to identify genes in amplification peaks that are consistently overexpressed among amplified samples confirmed MYC as a potential target of 8q amplification and identified candidate oncogenes in the other regions. A comparison of genomic profiles revealed that VHL disease-associated tumors are similar to a subgroup of sporadic tumors and thus more homogeneous overall. Sporadic tumors without evidence of biallelic VHL inactivation fell into two groups: one group with genomic profiles highly dissimilar to the majority of ccRCC and a second group with genomic profiles that are much more similar to tumors with biallelic inactivation of VHL.
Assuntos
Carcinoma de Células Renais/genética , Dosagem de Genes , Perfilação da Expressão Gênica , Neoplasias Renais/genética , Doença de von Hippel-Lindau/genética , Carcinoma de Células Renais/complicações , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Hibridização Genômica Comparativa , Regulação Neoplásica da Expressão Gênica , Frequência do Gene , Estudo de Associação Genômica Ampla , Humanos , Rim/patologia , Neoplasias Renais/complicações , Neoplasias Renais/patologia , Metástase Neoplásica , Polimorfismo de Nucleotídeo Único , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Doença de von Hippel-Lindau/complicações , Doença de von Hippel-Lindau/patologiaRESUMO
Basal-like breast cancer (BBC) is a subtype of breast cancer with poor prognosis. Inherited mutations of BRCA1, a cancer susceptibility gene involved in double-strand DNA break (DSB) repair, lead to breast cancers that are nearly always of the BBC subtype; however, the precise molecular lesions and oncogenic consequences of BRCA1 dysfunction are poorly understood. Here we show that heterozygous inactivation of the tumor suppressor gene Pten leads to the formation of basal-like mammary tumors in mice, and that loss of PTEN expression is significantly associated with the BBC subtype in human sporadic and BRCA1-associated hereditary breast cancers. In addition, we identify frequent gross PTEN mutations, involving intragenic chromosome breaks, inversions, deletions and micro copy number aberrations, specifically in BRCA1-deficient tumors. These data provide an example of a specific and recurrent oncogenic consequence of BRCA1-dependent dysfunction in DNA repair and provide insight into the pathogenesis of BBC with therapeutic implications. These findings also argue that obtaining an accurate census of genes mutated in cancer will require a systematic examination for gross gene rearrangements, particularly in tumors with deficient DSB repair.
Assuntos
Neoplasias da Mama/genética , Reparo do DNA , Neoplasias Mamárias Animais/genética , PTEN Fosfo-Hidrolase/genética , Animais , Linhagem Celular Tumoral , Feminino , Genes BRCA1 , Genes Supressores de Tumor , Humanos , Camundongos , MutaçãoRESUMO
Molecular mechanisms underlying prostate and urothelial development remain unclear. This situation presents major limitations in identifying the cell type(s) and molecular events involved in the development of prostate and bladder cancer. It has been shown that mice lacking the basal cell marker p63 present several epithelial defects, including epidermis and prostate buds agenesis and urothelial abnormalities. Here, we use the p63-/- mouse as a tool to define cell lineages in the prostate epithelium and urothelium. By complementing p63-/- blastocysts with p63+/+ beta-galactosidase (beta-gal)-positive ES cells, we show that secretory cells of the prostate originate from p63-positive basal progenitor cells. Importantly, our urogenital sinus transplantation studies demonstrate that p63 prevents intestinal differentiation of the urogenital sinus endoderm and is therefore required to maintain commitment to the prostate cell lineage. Finally, in contrast with the prostate findings, analysis of the urothelium from rescued p63-/- chimeras shows that umbrella (superficial) cells can develop and be maintained independently from p63-positive basal and intermediate cells.