The tumor suppressor gene HIC1 (Hypermethylated in Cancer 1) that is epigenetically silenced in many human tumors and is essential for mammalian development encodes a sequence-specific ...transcriptional repressor. The few genes that have been reported to be directly regulated by HIC1 include ATOH1, FGFBP1, SIRT1, and E2F1. HIC1 is thus involved in the complex regulatory loops modulating p53-dependent and E2F1-dependent cell survival and stress responses. We performed genome-wide expression profiling analyses to identify new HIC1 target genes, using HIC1-deficient U2OS human osteosarcoma cells infected with adenoviruses expressing either HIC1 or GFP as a negative control. These studies identified several putative direct target genes, including CXCR7, a G-protein-coupled receptor recently identified as a scavenger receptor for the chemokine SDF-1/CXCL12. CXCR7 is highly expressed in human breast, lung, and prostate cancers. Using quantitative reverse transcription-PCR analyses, we demonstrated that CXCR7 was repressed in U2OS cells overexpressing HIC1. Inversely, inactivation of endogenous HIC1 by RNA interference in normal human WI38 fibroblasts results in up-regulation of CXCR7 and SIRT1. In silico analyses followed by deletion studies and luciferase reporter assays identified a functional and phylogenetically conserved HIC1-responsive element in the human CXCR7 promoter. Moreover, chromatin immunoprecipitation (ChIP) and ChIP upon ChIP experiments demonstrated that endogenous HIC1 proteins are bound together with the C-terminal binding protein corepressor to the CXCR7 and SIRT1 promoters in WI38 cells. Taken together, our results implicate the tumor suppressor HIC1 in the transcriptional regulation of the chemokine receptor CXCR7, a key player in the promotion of tumorigenesis in a wide variety of cell types.
The tumor suppressor gene HIC1 (Hypermethylated In Cancer 1) encodes a transcriptional repressor mediating the p53-dependent apoptotic response to irreparable DNA double-strand breaks (DSBs) through ...direct transcriptional repression of SIRT1. HIC1 is also essential for DSB repair as silencing of endogenous HIC1 in BJ-hTERT fibroblasts significantly delays DNA repair in functional Comet assays. HIC1 SUMOylation favours its interaction with MTA1, a component of NuRD complexes. In contrast with irreparable DSBs induced by 16-hours of etoposide treatment, we show that repairable DSBs induced by 1 h etoposide treatment do not increase HIC1 SUMOylation or its interaction with MTA1. Furthermore, HIC1 SUMOylation is dispensable for DNA repair since the non-SUMOylatable E316A mutant is as efficient as wt HIC1 in Comet assays. Upon induction of irreparable DSBs, the ATM-mediated increase of HIC1 SUMOylation is independent of its effector kinase Chk2. Moreover, irreparable DSBs strongly increase both the interaction of HIC1 with MTA1 and MTA3 and their binding to the SIRT1 promoter. To characterize the molecular mechanisms sustained by this increased repression potential, we established global expression profiles of BJ-hTERT fibroblasts transfected with HIC1-siRNA or control siRNA and treated or not with etoposide. We identified 475 genes potentially repressed by HIC1 with cell death and cell cycle as the main cellular functions identified by pathway analysis. Among them, CXCL12, EPHA4, TGFβR3 and TRIB2, also known as MTA1 target-genes, were validated by qRT-PCR analyses. Thus, our data demonstrate that HIC1 SUMOylation is important for the transcriptional response to non-repairable DSBs but dispensable for DNA repair.
Medulloblastoma (MB), a primitive neuroectodermal tumor, is the most common malignant childhood brain tumor and remains incurable in about a third of patients. Currently, survivors carry a ...significant burden of late treatment effects. The p53 tumor suppressor protein plays a crucial role in influencing cell survival in response to cellular stress and while the p53 pathway is considered a key determinant of anti-tumor responses in many tumors, its role in cell survival in MB is much less well defined. Herein, we report that the experimental drug VMY-1-103 acts through induction of a partial DNA damage-like response as well induction of non-survival autophagy. Surprisingly, the genetic or chemical silencing of p53 significantly enhanced the cytotoxic effects of both VMY and the DNA damaging drug, doxorubicin. The inhibition of p53 in the presence of VMY revealed increased late stage apoptosis, increased DNA fragmentation and increased expression of genes involved in apoptosis, including CAPN12 and TRPM8, p63, p73, BIK, EndoG, CIDEB, P27Kip1 and P21cip1. These data provide the groundwork for additional studies on VMY as a therapeutic drug and support further investigations into the intriguing possibility that targeting p53 function may be an effective means of enhancing clinical outcomes in MB.
Children diagnosed with brain tumors have the lowest overall survival of all pediatric cancers. Recent molecular studies have resulted in the discovery of recurrent driver mutations in many pediatric ...brain tumors. However, despite these molecular advances, the clinical outcomes of high grade tumors, including H3K27M diffuse midline glioma (H3K27M DMG), remain poor. To address the paucity of tissue for biological studies, we have established a comprehensive protocol for the coordination and processing of donated specimens at postmortem. Since 2010, 60 postmortem pediatric brain tumor donations from 26 institutions were coordinated and collected. Patient derived xenograft models and cell cultures were successfully created (76% and 44% of attempts respectively), irrespective of postmortem processing time. Histological analysis of mid-sagittal whole brain sections revealed evidence of treatment response, immune cell infiltration and the migratory path of infiltrating H3K27M DMG cells into other midline structures and cerebral lobes. Sequencing of primary and disseminated tumors confirmed the presence of oncogenic driver mutations and their obligate partners. Our findings highlight the importance of postmortem tissue donations as an invaluable resource to accelerate research, potentially leading to improved outcomes for children with aggressive brain tumors.
The transcriptional repressor HIC1 (Hypermethylated in Cancer 1) is a tumor suppressor gene inactivated in many human cancers including breast carcinomas. In this study, we show that HIC1 is a direct ...transcriptional repressor of β-2 adrenergic receptor (ADRB2). Through promoter luciferase activity, chromatin immunoprecipitation (ChIP) and sequential ChIP experiments, we demonstrate that ADRB2 is a direct target gene of HIC1, endogenously in WI-38 cells and following HIC1 re-expression in breast cancer cells. Agonist-mediated stimulation of ADRB2 increases the migration and invasion of highly malignant MDA-MB-231 breast cancer cells but these effects are abolished following HIC1 re-expression or specific down-regulation of ADRB2 by siRNA treatment. Our results suggest that early inactivation of HIC1 in breast carcinomas could predispose to stress-induced metastasis through up-regulation of the β-2 adrenergic receptor.
The transcriptional repressor HIC1 is a tumor suppressor gene epigenetically silenced in many human tumors.
We identified β-2 adrenergic receptor (ADRB2) as a new direct target gene of HIC1.
Early inactivation of HIC1 in breast carcinomas could predispose to stress-induced metastasis through up-regulation of the β-2 adrenergic receptor.
Characterization of HIC1 target genes may explain how its inactivation contributes to tumorigenesis.
The tumor suppressor gene hypermethylated in cancer 1 (HIC1), which encodes a transcriptional repressor, is epigenetically silenced in many human tumors. Here, we show that ectopic expression of HIC1 ...in the highly malignant MDA-MB-231 breast cancer cell line severely impairs cell proliferation, migration, and invasion in vitro. In parallel, infection of breast cancer cell lines with a retrovirus expressing HIC1 also induces decreased mRNA and protein expression of the tyrosine kinase receptor EphA2. Moreover, chromatin immunoprecipitation (ChIP) and sequential ChIP experiments demonstrate that endogenous HIC1 proteins are bound, together with the MTA1 corepressor, to the EphA2 promoter in WI38 cells. Taken together, our results identify EphA2 as a new direct target gene of HIC1. Finally, we observe that inactivation of endogenous HIC1 through RNA interference in normal breast epithelial cells results in the up-regulation of EphA2 and is correlated with increased cellular migration. To conclude, our results involve the tumor suppressor HIC1 in the transcriptional regulation of the tyrosine kinase receptor EphA2, whose ligand ephrin-A1 is also a HIC1 target gene. Thus, loss of the regulation of this Eph pathway through HIC1 epigenetic silencing could be an important mechanism in the pathogenesis of epithelial cancers.
The tumor suppressor gene HIC1 epigenetically silenced in many human cancers encodes a transcriptional repressor.
We identified the receptor tyrosine kinase EphA2 as a new HIC1 direct target gene.
HIC1 directly represses a gene implicated in cell adhesion and migration.
Loss of HIC1 contributes to epithelial tumorigenesis through deregulation of the EphA2 signaling pathway.
Diffuse intrinsic pontine glioma (DIPG) is a leading cause of brain tumor-related death in children. DIPG is not surgically resectable, resulting in a paucity of tissue available for molecular ...studies. As such, tumor biology is poorly understood, and, currently, there are no effective treatments. In the absence of frozen tumor specimens, body fluids--such as cerebrospinal fluid (CSF), serum, and urine--can serve as more readily accessible vehicles for detecting tumor-secreted proteins. We analyzed a total of 76 specimens, including CSF, serum, urine, and normal and tumor brainstem tissue. Protein profiling of CSF from patients with DIPG was generated by mass spectrometry using an LTQ-Orbitrap-XL and database search using the Sequest algorithm. Quantitative and statistical analyses were performed with ProteoIQ and Partek Genomics Suite. A total of 528 unique proteins were identified, 71% of which are known secreted proteins. CSF proteomic analysis revealed selective upregulation of Cyclophillin A (CypA) and dimethylarginase 1 (DDAH1) in DIPG (n = 10), compared with controls (n = 4). Protein expression was further validated with Western blot analysis and immunohistochemical assays using CSF, brain tissue, serum, and urine from DIPG and control specimens. Immunohistochemical staining showed selective upregulation of secreted but not cytosolic CypA and DDAH1 in patients with DIPG. In this study, we present the first comprehensive protein profile of CSF specimens from patients with DIPG to demonstrate selective expression of tumor proteins potentially involved in brainstem gliomagenesis. Detection of secreted CypA and DDAH1 in serum and urine has potential clinical application, with implications for assessing treatment response and detecting tumor recurrence in patients with DIPG.
PTC299 is a novel, orally-bioavailable small molecule that selectively inhibits vascular endothelial growth factor receptor protein synthesis at the post-transcriptional level. Based on promising ...preclinical results, we conducted a pediatric phase I study to estimate the maximum tolerated dose, describe dose-limiting toxicities (DLT) and characterize the pharmacokinetic profile of PTC299 in children with recurrent CNS tumors. PTC299 was administered orally twice or three times daily, depending on the regimen. Four regimens were evaluated using the rolling 6 design, starting with 1.2 mg/kg/dose twice daily and escalating to 2 mg/kg/dose three times daily. Pharmacokinetic studies were performed during the first two courses. Twenty-seven children (14 male, median age 11.2, range 5.5–21 years) with recurrent brain tumors were treated; 21 were fully evaluable for toxicity assessment. Therapy was well-tolerated, and the only DLT was grade 3 hyponatremia. Grade three and grade four toxicities were uncommon in subsequent cycles. Median AUC
0–Tlast
values at the 2 mg/kg were similar to those observed in adults. The study was terminated while patients were being treated at the highest planned dose, due to hepatotoxicity encountered in the ongoing adult phase I studies. No complete or partial responses were observed. Two patients with low-grade gliomas were noted to have minor responses, and at the time of the study’s closure, 5 children with low-grade gliomas had been on therapy for 8 or more courses (range 8–16). PTC299 was well-tolerated at the highest dose level tested (2 mg/kg/dose TID) in children with recurrent brain tumors and prolonged disease stabilization was seen in children with low-grade gliomas.
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