RESUMEN
Sustained expression of FOXM1 is a hallmark of nearly all human cancers including squamous cell carcinomas of the head and neck (HNSCC). HNSCCs partially preserve the epithelial differentiation program, which recapitulates fetal and adult traits of the tissue of tumor origin but is deregulated by genetic alterations and tumor-supporting pathways. Using shRNA-mediated knockdown, we demonstrate a minimal impact of FOXM1 on proliferation and migration of HNSCC cell lines under standard cell culture conditions. However, FOXM1 knockdown in three-dimensional (3D) culture and xenograft tumor models resulted in reduced proliferation, decreased invasion, and a more differentiated-like phenotype, indicating a context-dependent modulation of FOXM1 activity in HNSCC cells. By ectopic overexpression of FOXM1 in HNSCC cell lines, we demonstrate a reduced expression of cutaneous-type keratin K1 and involucrin as a marker of squamous differentiation, supporting the role of FOXM1 in modulation of aberrant differentiation in HNSCC. Thus, our data provide a strong rationale for targeting FOXM1 in HNSCC. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Proteína Forkhead Box M1/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Animales , Línea Celular Tumoral , Femenino , Proteína Forkhead Box M1/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Ratones Desnudos , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carga TumoralRESUMEN
HPV-positive head and neck squamous cell carcinoma (HNSCC) is increasingly frequent. Management is particularly debated in the case of postsurgical high-risk features, that is, positive surgical margins and extracapsular spread (ECS). In this increasingly complex emerging framework of HNSCC treatment, representative preclinical models are needed to support future clinical trials and advances in personalized medicine. Here, we present an immunocompetent mouse model based on the implantation of mouse tonsil epithelial HPV16-E6/E7-expressing cancer cells into the submental region of the floor-of-the-mouth. Primary tumors were found to replicate the patterns of human HNSCC local invasion and lymphatic dissemination. To study disease progression after surgery, tumors were removed likely leaving behind residual disease. Surgical resection of tumors was followed by a high rate of local recurrences (>90%) within the first 2-3 weeks. While only 50% of mice had lymph node metastases (LNM) at time of primary tumor excision, all mice with recurrent tumors showed evidence of LNM. To study the consecutive steps of LNM progression and distant metastasis development, LNs from tumor-bearing mice were transplanted into naïve recipient mice. Using this approach, transplanted LNs were found to recapitulate all stages and relevant histological features of regional metastasis progression, including ECS and metastatic spread to the lungs. Altogether, we have developed an immunocompetent HPV-positive HNSCC mouse model of postsurgical local recurrence and regional and distant metastasis progression suitable for preclinical studies.
Asunto(s)
Modelos Animales de Enfermedad , Metástasis Linfática/patología , Recurrencia Local de Neoplasia/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Animales , Progresión de la Enfermedad , Ratones , Ratones Endogámicos C57BL , Infecciones por Papillomavirus/complicacionesRESUMEN
PIK3CA, the gene encoding the p110α catalytic subunit of PI3K (phosphoinositide 3-kinase), is mutated in approximately 20% of sporadic CRCs (colorectal cancers), but the role of these mutations in the pathogenesis of CRC remains unclear. In the present study we used a novel mouse model to investigate the role of the Pik3caH1047R mutation, the most common PIK3CA mutation in CRC, during the development and progression of intestinal cancer. Our results demonstrate that Pik3caH1047R, when expressed at physiological levels, is insufficient to initiate intestinal tumorigenesis; however, in the context of Apc (adenomatous polyposis coli) loss, which is observed in 80% of CRCs and by itself results in benign intestinal adenomas, the Pik3caH1047R mutation promotes the development of highly aggressive and invasive adenocarcinomas in both the small and large intestines. The results of the present study show that an activating Pik3ca mutation can act in tandem with Apc loss to drive the progression of gastrointestinal cancer and thus this disease may be susceptible to therapeutic targeting using PI3K pathway inhibitors.
Asunto(s)
Adenocarcinoma/genética , Proteína de la Poliposis Adenomatosa del Colon/deficiencia , Regulación Neoplásica de la Expresión Génica , Neoplasias Intestinales/genética , Mutación/genética , Invasividad Neoplásica , Fosfatidilinositol 3-Quinasas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Fosfatidilinositol 3-Quinasa Clase I , Progresión de la Enfermedad , Femenino , Técnicas de Sustitución del Gen , Neoplasias Intestinales/metabolismo , Neoplasias Intestinales/patología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Fosfatidilinositol 3-Quinasas/biosíntesisRESUMEN
Elevated peripheral blood and tumor-infiltrating neutrophils are often associated with a poor patient prognosis. However, therapeutic strategies to target these cells are difficult to implement due to the life-threatening risk of neutropenia. In a genetically engineered mouse model of lung adenocarcinoma, tumor-associated neutrophils (TAN) demonstrate tumor-supportive capacities and have a prolonged lifespan compared to circulating neutrophils. Here, we show that tumor cell-derived GM-CSF triggers the expression of the anti-apoptotic Bcl-xL protein and enhances neutrophil survival through JAK/STAT signaling. Targeting Bcl-xL activity with a specific BH3 mimetic, A-1331852, blocked the induced neutrophil survival without impacting their normal lifespan. Specifically, oral administration with A-1331852 decreased TAN survival and abundance, and reduced tumor growth without causing neutropenia. We also show that G-CSF, a drug used to combat neutropenia in patients receiving chemotherapy, increased the proportion of young TANs and augmented the anti-tumor effect resulting from Bcl-xL blockade. Finally, our human tumor data indicate the same role for Bcl-xL on pro-tumoral neutrophil survival. These results altogether provide preclinical evidence for safe neutrophil targeting based on their aberrant intra-tumor longevity.
Asunto(s)
Neoplasias Pulmonares , Neutropenia , Animales , Humanos , Ratones , Envejecimiento , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína bcl-X , Línea Celular Tumoral , Neoplasias Pulmonares/patología , Neutropenia/tratamiento farmacológico , Neutropenia/metabolismo , Neutropenia/patología , Neutrófilos/metabolismoRESUMEN
High-risk human papillomavirus (HPV) encoding E6/E7-HPV oncogenes are responsible for a subgroup of head and neck squamous-cell carcinoma (HNSCC) and thus therapeutic E7-vaccines may be used to control HPV+HNSCC tumors. Herein we investigated the effects of an optimized nanoparticle-conjugated E7 long-peptide vaccine adjuvanted with CpG (NP-E7LP) in an orthotopic immunocompetent mouse model of HPV+HNSCC which is based on injection of HPV16 E6/E7-expressing mEERL95-cells into the submental space. In absence of surgery, vaccination performed before or after tumor-cell injection decreased tumor growth or prolonged mice survival only marginally, despite the high numbers of vaccine-induced circulating E7-specific IFN-γ-secreting CD8+ T-cells. This contrasts with the high-efficacy of NP-E7LP-vaccination reported in the genital and subcutaneous HPV16-E6/E7-expressing TC-1 models. Our data show that in a direct comparison, NP-E7LP-vaccination fully controlled TC-1, but not mEERL95, tumors subcutaneously growing in the flanks. Immune-cell infiltration was 10-fold higher in TC-1-tumors, than in mEERL95-tumors, suggesting that vaccine-induced CD8+ T-cells can only poorly infiltrate mEERL95-tumors. Indeed, immunofluorescence staining of orthotopic mEERL95-tumors showed that CD3+ T-cells are preferentially located peritumorally. However, when NP-E7LP-vaccination was performed after mEERL95-cell injection, but before resection of primary tumors, no postsurgical recurrence was observed and 100% of the mice survived until the experimental endpoint (day 70) in the NP-E7LP-vaccinated group. In contrast, we observed a 60% recurrence rate and only 35% survival in PBS-vaccinated mice. This suggests that removal of the primary tumor modified the tumor microenvironment, allowing a therapeutic effect of the vaccine-induced anti-tumor response. E7-vaccination combined with surgery may thus benefit patients with HPV+HNSCC.
Asunto(s)
Alphapapillomavirus , Vacunas contra el Cáncer , Neoplasias de Cabeza y Cuello , Nanopartículas , Vacunas contra Papillomavirus , Animales , Linfocitos T CD8-positivos , Humanos , Ratones , Ratones Endogámicos C57BL , Recurrencia Local de Neoplasia/prevención & control , Papillomaviridae , Microambiente Tumoral , VacunaciónRESUMEN
PURPOSE: The incidence of human papillomavirus-associated head and neck squamous cell carcinoma (HPV+-HNSCC) is rising worldwide and although current therapeutic modalities are efficient in the majority of patients, there is a high rate of treatment failures. Thus, novel combination approaches are urgently needed to achieve better disease control in patients with HPV+-HNSCC. We investigated the safety and therapeutic efficacy of a novel fibroblast activation protein (FAP)-targeted CD40 agonist (FAP-CD40) in combination with local hypofractionated radiation in a syngeneic HPV+-HNSCC model. EXPERIMENTAL DESIGN: Using an established orthotopic model, we treated tumor-bearing mice with local hypofractionated radiotherapy (2 × 6 Gy) alone or in combination with a systemic administration of the FAP-CD40 antibody. Following up the mice, we evaluated the changes in the tumor microenvironment (TME) by immunofluorescence, FACS, and NanoString RNA analysis. RESULTS: The suboptimal radiotherapy regimen chosen failed to control tumors in the treated mice. The FAP-CD40 administered in monotherapy transiently controlled tumor growth, whereas the combined therapy induced durable complete responses in more than 80% of the tumor-bearing mice. This notable efficacy relied on the radiotherapy-induced remodeling of the TME and activation of the CD8+ T-cell-cDC1 axis and was devoid of the systemic toxicity frequently associated with CD40-targeted therapy. Moreover, the robust immunologic memory developed effectively prevented tumor relapses, a common feature in patients with HNSCC. CONCLUSIONS: Our study provides proof of concept, as well as mechanistic insights of the therapeutic efficacy of a bispecific FAP-CD40 combined with local radiotherapy in a FAP+-HNSCC model increasing overall survival and inducing long-term antitumor immunity.
Asunto(s)
Antígenos CD40/agonistas , Endopeptidasas/efectos de los fármacos , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/radioterapia , Neoplasias de Cabeza y Cuello/virología , Proteínas de la Membrana/efectos de los fármacos , Papillomaviridae/aislamiento & purificación , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/radioterapia , Carcinoma de Células Escamosas de Cabeza y Cuello/virología , Animales , Terapia Combinada , RatonesRESUMEN
Local recurrence after surgery for head and neck squamous cell carcinoma (HNSCC) remains a common event associated with a dismal prognosis. Improving this outcome requires a better understanding of cancer cell populations that expand from postsurgical minimal residual disease (MRD). Therefore, we assessed clonal dynamics in a surgical model of barcoded HNSCC growing in the submental region of immunodeficient mice. Clonal substitution and massive reduction of clonal heterogeneity emerged as hallmarks of local recurrence, as the clones dominating in less heterogeneous recurrences were scarce in their matched primary tumors. These lineages were selected by their ability to persist after surgery and competitively expand from MRD. Clones enriched in recurrences exhibited both private and shared genetic features and likely originated from ancestors shared with clones dominating in primary tumors. They demonstrated high invasiveness and epithelial-to-mesenchymal transition, eventually providing an attractive target for obtaining better local control for these tumors.
Asunto(s)
Modelos Anatómicos , Recurrencia Local de Neoplasia/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/cirugía , Animales , Biomarcadores de Tumor/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Linaje de la Célula , Proliferación Celular , Células Clonales , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal , Femenino , Humanos , Masculino , Ratones Desnudos , Modelos Estadísticos , Células Madre Neoplásicas/patología , Neprilisina/metabolismo , Fenotipo , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
NDRG1 is a hypoxia-inducible protein, whose modulated expression is associated with the progression of human cancers. Here, we reveal that NDRG1 is markedly upregulated in the cytoplasm and on the membrane in human hepatocellular carcinoma (HCC). We demonstrate further that hypoxic stress increases the cytoplasmic expression of NDRG1 in vitro, but does not result in its localization on the plasma membrane. However, grown within an HCC-xenograft in vivo, cells express NDRG1 in the cytoplasm and on the plasma membrane. In conclusion, hypoxia is a potent inducer of NDRG1 in HCCs, albeit requiring additional stimuli within the tumour microenvironment for its recruitment to the membrane.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Hepáticas/metabolismo , Secuencia de Aminoácidos , Animales , Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Membrana Celular/metabolismo , Citoplasma/metabolismo , Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Hepáticas/genética , Masculino , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Trasplante de Neoplasias , Estructura Terciaria de Proteína , Trasplante HeterólogoRESUMEN
PIK3CA, the gene coding for the p110α subunit of phosphoinositide 3-kinase, is frequently mutated in a variety of human tumors including breast cancers. To better understand the role of mutant PIK3CA in the initiation and/or progression of breast cancer, we have generated mice with a conditional knock-in of the common activating mutation, Pik3ca(H1047R), into one allele of the endogenous gene in the mammary gland. These mice developed a ductal anaplasia and hyperplasia by 6 weeks of age characterized by multi-layering of the epithelial lining of the mammary ducts and expansion of the luminal progenitor (Lin(-); CD29(lo); CD24(+); CD61(+)) cell population. The Pik3ca(H1047R) expressing mice eventually develop mammary tumors with 100% penetrance but with a long latency (>12 months). This is significantly longer than has been reported for transgenic models where expression of the mutant Pik3ca is driven by an exogenous promoter. Histological analysis of the tumors formed revealed predominantly ERα-positive fibroadenomas, carcinosarcomas and sarcomas. In vitro induction of Pik3ca(H1047R) in immortalized mammary epithelial cells also resulted in tumor formation when injected into the mammary fat pad of immunodeficient recipient mice. This novel model, which reproduces the scenario of a heterozygous somatic mutation occurring in the endogenous PIK3CA gene, will thus be a valuable tool for investigating the role of Pik3ca(H1047R) mutation in mammary tumorigenesis both in vivo and in vitro.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Glándulas Mamarias Animales/enzimología , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Mutación , Fosfatidilinositol 3-Quinasas/genética , Alelos , Animales , Secuencia de Bases , Fosfatidilinositol 3-Quinasa Clase I , Femenino , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Sustitución del Gen , Hiperplasia/enzimología , Hiperplasia/genética , Glándulas Mamarias Animales/crecimiento & desarrollo , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/patología , Ratones , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas/genéticaRESUMEN
Sirtuins and hypoxia-inducible transcription factors (HIF) have well-established roles in regulating cellular responses to metabolic and oxidative stress. Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2. Here we investigated the role of SIRT1, a NAD+-dependent deacetylase, in the regulation of HIF-1 activity in hypoxic conditions. Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes. In hypoxic models in vitro and in in vivo models of systemic hypoxia and xenograft tumor growth, knockdown of SIRT1 protein with shRNA or inhibition of its activity with small molecule inhibitors impaired the accumulation of HIF-1α protein and the transcriptional increase of its target genes. In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α. Taken together, our data suggest that HIF-1α and SIRT1 proteins interact in HCC cells and that HIF-1α is a target of SIRT1 deacetylase activity. Moreover, SIRT1 is necessary for HIF-1α protein accumulation and activation of HIF-1 target genes under hypoxic conditions.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Sirtuina 1/metabolismo , Activación Transcripcional , Animales , Benzamidas/farmacología , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Hipoxia de la Célula , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Humanos , Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Noqueados , Ratones Desnudos , Naftalenos/farmacología , Naftoles/farmacología , Unión Proteica , Pirimidinonas/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirtuina 1/antagonistas & inhibidores , Sirtuina 1/genética , Trasplante Heterólogo , Carga Tumoral/efectos de los fármacosRESUMEN
Mutations in the gene encoding the p110α subunit of PI3K (PIK3CA) that result in enhanced PI3K activity are frequently observed in human cancers. To better understand the role of mutant PIK3CA in the initiation or progression of tumorigenesis, we generated mice in which a PIK3CA mutation commonly detected in human cancers (the H1047R mutation) could be conditionally knocked into the endogenous Pik3ca locus. Activation of this mutation in the mouse ovary revealed that alone, Pik3caH1047R induced premalignant hyperplasia of the ovarian surface epithelium but no tumors. Concomitantly, we analyzed several human ovarian cancers and found PIK3CA mutations coexistent with KRAS and/or PTEN mutations, raising the possibility that a secondary defect in a co-regulator of PI3K activity may be required for mutant PIK3CA to promote transformation. Consistent with this notion, we found that Pik3caH1047R mutation plus Pten deletion in the mouse ovary led to the development of ovarian serous adenocarcinomas and granulosa cell tumors. Both mutational events were required for early, robust Akt activation. Pharmacological inhibition of PI3K/mTOR in these mice delayed tumor growth and prolonged survival. These results demonstrate that the Pik3caH1047R mutation with loss of Pten is enough to promote ovarian cell transformation and that we have developed a model system for studying possible therapies.
Asunto(s)
Transformación Celular Neoplásica/genética , Mutación , Neoplasias Ováricas/genética , Fosfohidrolasa PTEN/deficiencia , Fosfatidilinositol 3-Quinasas/genética , Animales , Fosfatidilinositol 3-Quinasa Clase I , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias Ováricas/patología , Ovario/anatomía & histología , Ovario/patología , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Tasa de SupervivenciaRESUMEN
AIM: The mTOR-inhibitor rapamycin has shown antitumor activity in various tumors. Bedside observations have suggested that rapamycin may be effective as a treatment for colorectal carcinomatosis. METHODS: We established an orthotopic syngenic model by transplanting CT26 peritoneal tumors in Balb/C mice and an orthotopic xenograft model by transplanting SW620 peritoneal tumors in nu/nu mice. Expression levels of tissue inhibitor of matrix-metalloproteinases 1 (TIMP-1) in the tumor and serum was determined by enzyme-linked immunosorbent assay. RESULTS: Rapamycin significantly suppressed growth of syngenic and xenografted peritoneal tumors. The effect was similar with intraperitoneal or oral rapamycin administration. Tumor suppression was further enhanced when rapamycin was combined with 5-fluorouracil and/or oxaliplatin. The combination treatment showed no acute toxicity. TIMP-1 serum levels correlated well (CC = 0.75; P < 0.01) with rapamycin treatment. CONCLUSIONS: Rapamycin suppressed advanced stage colorectal cancer, even with oral administration. Combining rapamycin with current chemotherapy regimens significantly increased antitumor efficacy without apparent toxicity. The treatment efficacy correlated with serum TIMP-1 levels, suggesting its potential as a surrogate marker in future clinical trials.
Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias Colorrectales/tratamiento farmacológico , Fluorouracilo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Fluorouracilo/administración & dosificación , Humanos , Ratones , Compuestos Organoplatinos/administración & dosificación , Oxaliplatino , Neoplasias Peritoneales/tratamiento farmacológico , Sirolimus/administración & dosificación , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Hypermethylated in Cancer 1 (HIC1) gene encodes a zinc finger transcriptional repressor that cooperates with p53 to suppress cancer development. We and others recently showed that HIC1 is a transcriptional target of p53. To identify additional transcriptional regulators of HIC1, we screened a set of transcription factors for regulation of a human HIC1 promoter reporter. We found that E2F1 strongly activates the full-length HIC1 promoter reporter. Promoter deletions and mutations identified two E2F responsive elements in the HIC1 core promoter region. Moreover, in vivo binding of E2F1 to the HIC1 promoter was shown by chromatin immunoprecipitation assays in human TIG3 fibroblasts expressing tamoxifen-activated E2F1. In agreement, activation of E2F1 in TIG3-E2F1 cells markedly increased HIC1 expression. Interestingly, expression of E2F1 in the p53(-/-) hepatocellular carcinoma cell line Hep3B led to an increase of endogenous HIC1 mRNA, although bisulfite genomic sequencing of the HIC1 promoter revealed that the region bearing the two E2F1 binding sites is hypermethylated. In addition, endogenous E2F1 induced by etoposide treatment bound to the HIC1 promoter. Moreover, inhibition of E2F1 strongly reduced the expression of etoposide-induced HIC1. In conclusion, we identified HIC1 as novel E2F1 transcriptional target in DNA damage responses.