Phosphorylation of HIC1 (Hypermethylated in Cancer 1) Ser694 by ATM is essential for DNA repair.

The tumor suppressor gene HIC1 (Hypermethylated in Cancer 1) encodes a transcriptional repressor involved in the DNA-damage response. A SUMOylation increase on HIC1 Lysine314 favors the direct transcriptional repression of SIRT1 and thus the P53-dependent apoptotic response to irreparable DNA double strand breaks (DSBs). HIC1 is also essential for DSBs repair but in a SUMOylation-independent manner. Here, we show that repairable DSBs induced by a 1 h Etoposide treatment results in three specific posttranslational modifications (PTMs) of HIC1. Two of these PTMs, phosphorylation of Serine 694 and Acetylation of Lysine 623 are located in the conserved HIC1 C-terminal region located downstream of the Zinc Finger DNA-binding domain. By contrast, phosphorylation of Serine 285 found in the poorly conserved central region is unique to the human protein. We showed that Ser694 phosphorylation is mediated mainly by the PIKK kinase ATM and is essential for the DNA repair activity of HIC1 as demonstrated by the lack of efficiency of the S694A point mutant in Comet assays. Thus, our results provide the first evidence for a functional role of the conserved HIC1 C-terminal region as a novel ATM substrate that plays an essential role in the cellular HIC1-mediated cellular response to repairable DSBs.

Evidence of a compensatory regulation of colonic O-GlcNAc transferase and O-GlcNAcase expression in response to disruption of O-GlcNAc homeostasis.

O-GlcNAcylation is a post-translational modification of thousands of intracellular proteins that dynamically regulates many fundamental cellular processes. Cellular O-GlcNAcylation levels are regulated by a unique couple of enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which adds and removes the GlcNAc residue, respectively. Maintenance of O-GlcNAc homeostasis is essential to ensure optimal cellular function and disruption of this homeostasis has been linked to the etiology of several human diseases including cancer. The mechanisms through which the cell maintains O-GlcNAc homeostasis are not fully understood but several studies have suggested that a reciprocal regulation of OGT and OGA expression could be one of them. In this study, we investigated the putative regulation of OGT and OGA expression in response to disruption in O-GlcNAc homeostasis in colon. We provide in vitro and in vivo evidences that in colon cells, modulation of O-GlcNAcylation levels leads to a compensatory regulation of OGT and OGA expression in an attempt to restore basal O-GlcNAcylation levels. Our results also suggests that the regulation of colonic OGA expression in response to changes in O-GlcNAc homeostasis occurs mostly at the transcriptional level whereas OGT regulation seems to rely mainly on post-transcriptional mechanisms.

DNA double-strand breaks lead to activation of hypermethylated in cancer 1 (HIC1) by SUMOylation to regulate DNA repair.

HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene frequently epigenetically silenced in human cancers. HIC1 encodes a transcriptional repressor involved in the regulation of growth control and DNA damage response. We previously demonstrated that HIC1 can be either acetylated or SUMOylated on lysine 314. This deacetylation/SUMOylation switch is governed by an unusual complex made up of SIRT1 and HDAC4 which deacetylates and thereby favors SUMOylation of HIC1 by a mechanism not yet fully deciphered. This switch regulates the interaction of HIC1 with MTA1, a component of the NuRD complex and potentiates the repressor activity of HIC1. Here, we show that HIC1 silencing in human fibroblasts impacts the repair of DNA double-strand breaks whereas ectopic expression of wild-type HIC1, but not of nonsumoylatable mutants, leads to a reduced number of γH2AX foci induced by etoposide treatment. In this way, we demonstrate that DNA damage leads to (i) an enhanced HDAC4/Ubc9 interaction, (ii) the activation of SIRT1 by SUMOylation (Lys-734), and (iii) the SUMO-dependent recruitment of HDAC4 by SIRT1 which permits the deacetylation/SUMOylation switch of HIC1. Finally, we show that this increase of HIC1 SUMOylation favors the HIC1/MTA1 interaction, thus demonstrating that HIC1 regulates DNA repair in a SUMO-dependent way. Therefore, epigenetic HIC1 inactivation, which is an early step in tumorigenesis, could contribute to the accumulation of DNA mutations through impaired DNA repair and thus favor tumorigenesis.

Loss of Hypermethylated in Cancer 1 (HIC1) in breast cancer cells contributes to stress-induced migration and invasion through ß-2 adrenergic receptor (ADRB2) misregulation.

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 ß-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 ß-2 adrenergic receptor.

Identification of p21 (CIP1/WAF1) as a direct target gene of HIC1 (Hypermethylated In Cancer 1).

The tumor suppressor gene HIC1 (Hypermethylated In Cancer 1) encodes a transcriptional repressor involved in the regulation of growth control and DNA damage response. We previously demonstrated that p57Kip2; a member of the CIP/KIP family of CDK (cyclin dependent kinase) inhibitors (CKI); is a direct target gene of HIC1 in quiescent cells. Here we show that ectopic expression of HIC1 in MDA-MB-231 cells or its overexpression in BJ-Tert fibroblasts induces decreased mRNA and protein expression of p21 (CIP1/WAF1) another member of this CKI family that plays essential roles in the p53-mediated DNA damage response. Conversely, knock-down of endogenous HIC1 in BJ-Tert through RNA interference up-regulates p21 in basal conditions and further potentiates this CKI in response to apoptotic etoposide-induced DNA damage. Through promoter luciferase activity and chromatin immunoprecipitation (ChIP), we demonstrate that HIC1 is a direct transcriptional repressor of p21. Thus, our results further demonstrate that HIC1 is a key player in the regulation of the DNA damage response.

Molecular dissection of the interaction between HIC1 and SIRT1.

HIC1 (Hypermethylated in Cancer 1) is a tumor suppressor gene frequently epigenetically silenced in human cancers. HIC1 encodes a transcriptional repressor involved in the regulation of growth control, cell survival and DNA damage response. The deacetylase SIRT1 regulates the repressive capacity of HIC1 in several fashions. First SIRT1 interacts with the BTB/POZ domain of HIC1 to form a transcriptional repression complex that prevents the transcription of SIRT1 itself. SIRT1 is also responsible of the deacetylation of the lysine 314 of HIC1 that allows its subsequent SUMOylation which in turn favors its interaction with the NuRD complex. To better understand the interplay between HIC1 and SIRT1, we performed co-immunoprecipitation experiments to define the domains essential for the HIC1/SIRT1 interaction. We demonstrated that the isolated four last zinc fingers of HIC1 were capable to interact with SIRT1 and that the amino-acids 610-677 of SIRT1 encompassing the ESA region of the deacetylase were crucial for the HIC1/SIRT1 interaction and HIC1 deacetylation. Finally we demonstrated that this interaction mainly depends on CKII-mediated phosphorylation of SIRT1 serine 659/661 which occurs upon DNA damage. Therefore, our results demonstrate that the activating acetylation to SUMOylation switch of HIC1 is favored by genotoxic stresses to regulate the DNA damage response.

Istradefylline protects from cisplatin-induced nephrotoxicity and peripheral neuropathy while preserving cisplatin antitumor effects.

Cisplatin is a potent chemotherapeutic drug that is widely used in the treatment of various solid cancers. However, its clinical effectiveness is strongly limited by frequent severe adverse effects, in particular nephrotoxicity and chemotherapy-induced peripheral neuropathy. Thus, there is an urgent medical need to identify novel strategies that limit cisplatin-induced toxicity. In the present study, we show that the FDA-approved adenosine A2A receptor antagonist istradefylline (KW6002) protected from cisplatin-induced nephrotoxicity and neuropathic pain in mice with or without tumors. Moreover, we also demonstrate that the antitumoral properties of cisplatin were not altered by istradefylline in tumor-bearing mice and could even be potentiated. Altogether, our results support the use of istradefylline as a valuable preventive approach for the clinical management of patients undergoing cisplatin treatment.

Parvovirus H-1 induces cytopathic effects in breast carcinoma-derived cultures.

Parvovirus H-1 (H-1 PV) preferentially replicates in malignant cells resulting in their death by cytolysis. It has often been considered a potential candidate for use in novel anticancer therapy. To evaluate its potential in a model of natural tumors, we assayed in vitro the effect exerted by H-1 PV on short-term cultures derived from breast tumor samples freshly excised from patients. Our results show that H-1 PV effectively kills tumor-derived cells, whereas normal tissue-derived cells showed no H-1 PV-induced cytopathic effects (CPE). We also determined that the H-1 PV sensitivity (up to 67% sensitive cultures) is related with the quantities of virus assayed. We further examined the expression and phosphorylation state of the parvoviral nonstructural protein 1 (NS1), known to be associated with parvoviruses-induced CPE. Both appear to be impaired in normal tissue-derived cells and resistant cultures. Finally, we show that H-1 PV sensitivity in cultures correlates significantly with higher tumor grades (Nottingham combined histologic grade 2 or 3). This report confirms that H-1 PV can efficiently induce CPE in primary breast tumor cells in vitro. It identifies tumor characteristics representing potential criteria for recruiting patients for clinical evaluation of H-1 PV antitumor effects.

Ectopic expression of H-1 parvovirus NS1 protein induces alterations in actin filaments and cell death in human normal MRC-5 and transformed MRC-5 SV2 cells.

When grown in human cell lines, oncolytic H-1 parvovirus (H-1PV) replication preferentially occurs in transformed cells, which ultimately die upon infection. H-1PV-induced cytotoxicity is mainly due to P4 promoter-driven NS1 protein expression. Infection of untransformed cells generally does not induce deleterious effects because the P4 promoter is not activated. Here, we show that ectopic CMV-driven NS1 protein expression in normal human MRC-5 cells results in alterations of actin filaments and cell death, and both effects are prevented by a serine 473 mutation. The same substitution preserves actin filaments of transfected MRC-5 SV2 cells, that are MRC-5 transformed counterparts, but does not impair NS1-induced cytotoxicity.

HIC1 (Hypermethylated in Cancer 1) modulates the contractile activity of prostate stromal fibroblasts and directly regulates CXCL12 expression.

HIC1 (Hypermethylated In Cancer 1) a tumor suppressor gene located at 17p13.3, is frequently deleted or epigenetically silenced in many human tumors. HIC1 encodes a transcriptional repressor involved in various aspects of the DNA damage response and in complex regulatory loops with P53 and SIRT1. HIC1 expression in normal prostate tissues has not yet been investigated in detail. Here, we demonstrated by immunohistochemistry that detectable HIC1 expression is restricted to the stroma of both normal and tumor prostate tissues. By RT-qPCR, we showed that HIC1 is poorly expressed in all tested prostate epithelial lineage cell types: primary (PrEC), immortalized (RWPE1) or transformed androgen-dependent (LnCAP) or androgen-independent (PC3 and DU145) prostate epithelial cells. By contrast, HIC1 is strongly expressed in primary PrSMC and immortalized (WMPY-1) prostate myofibroblastic cells. HIC1 depletion in WPMY-1 cells induced decreases in α-SMA expression and contractile capability. In addition to SLUG, we identified stromal cell-derived factor 1/C-X-C motif chemokine 12 (SDF1/CXCL12) as a new HIC1 direct target-gene. Thus, our results identify HIC1 as a tumor suppressor gene which is poorly expressed in the epithelial cells targeted by the tumorigenic process. HIC1 is expressed in stromal myofibroblasts and regulates CXCL12/SDF1 expression, thereby highlighting a complex interplay mediating the tumor promoting activity of the tumor microenvironment. Our studies provide new insights into the role of HIC1 in normal prostatic epithelial-stromal interactions through direct repression of CXCL12 and new mechanistic clues on how its loss of function through promoter hypermethylation during aging could contribute to prostatic tumors.

O-GlcNAcylation Links Nutrition to the Epigenetic Downregulation of UNC5A during Colon Carcinogenesis.

While it is now accepted that nutrition can influence the epigenetic modifications occurring in colorectal cancer (CRC), the underlying mechanisms are not fully understood. Among the tumor suppressor genes frequently epigenetically downregulated in CRC, the four related genes of the UNC5 family: UNC5A, UNC5B, UNC5C and UNC5D encode dependence receptors that regulate the apoptosis/survival balance. Herein, in a mouse model of CRC, we found that the expression of UNC5A, UNC5B and UNC5C was diminished in tumors but only in mice subjected to a High Carbohydrate Diet (HCD) thus linking nutrition to their repression in CRC. O-GlcNAcylation is a nutritional sensor which has enhanced levels in CRC and regulates many cellular processes amongst epigenetics. We then investigated the putative involvement of O-GlcNAcylation in the epigenetic downregulation of the UNC5 family members. By a combination of pharmacological inhibition and RNA interference approaches coupled to RT-qPCR (Reverse Transcription-quantitative Polymerase Chain Reaction) analyses, promoter luciferase assay and CUT&RUN (Cleavage Under Target & Release Using Nuclease) experiments, we demonstrated that the O-GlcNAcylated form of the histone methyl transferase EZH2 (Enhancer of Zeste Homolog 2) represses the transcription of UNC5A in human colon cancer cells. Collectively, our data support the hypothesis that O-GlcNAcylation could represent one link between nutrition and epigenetic downregulation of key tumor suppressor genes governing colon carcinogenesis including UNC5A.

hPCL3S promotes proliferation and migration of androgen-independent prostate cancer cells.

Polycomb repressive complex 2 (PRC2) allows the deposition of H3K27me3. PRC2 facultative subunits modulate its activity and recruitment such as hPCL3/PHF19, a human ortholog of Drosophila Polycomb-like protein (PCL). These proteins contain a TUDOR domain binding H3K36me3, two PHD domains and a "Winged-helix" domain involved in GC-rich DNA binding. The human PCL3 locus encodes the full-length hPCL3L protein and a shorter isoform, hPCL3S containing the TUDOR and PHD1 domains only. In this study, we demonstrated by RT-qPCR analyses of 25 prostate tumors that hPCL3S is frequently up-regulated. In addition, hPCL3S is overexpressed in the androgen-independent DU145 and PC3 cells, but not in the androgen-dependent LNCaP cells. hPCL3S knockdown decreased the proliferation and migration of DU145 and PC3 whereas its forced expression into LNCaP increased these properties. A mutant hPCL3S unable to bind H3K36me3 (TUDOR-W50A) increased proliferation and migration of LNCaP similarly to wt hPCL3S whereas inactivation of its PHD1 domain decreased proliferation. These effects partially relied on the up-regulation of genes known to be important for the proliferation and/or migration of prostate cancer cells such as S100A16, PlexinA2, and Spondin1. Collectively, our results suggest hPCL3S as a new potential therapeutic target in castration resistant prostate cancers.

HIC1 (hypermethylated in cancer 1) SUMOylation is dispensable for DNA repair but is essential for the apoptotic DNA damage response (DDR) to irreparable DNA double-strand breaks (DSBs).

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, TGFßR3 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.

Protein Kinase C-Mediated Phosphorylation of BCL11B at Serine 2 Negatively Regulates Its Interaction with NuRD Complexes during CD4+ T-Cell Activation.

The transcription factor BCL11B/CTIP2 is a major regulatory protein implicated in various aspects of development, function and survival of T cells. Mitogen-activated protein kinase (MAPK)-mediated phosphorylation and SUMOylation modulate BCL11B transcriptional activity, switching it from a repressor in naive murine thymocytes to a transcriptional activator in activated thymocytes. Here, we show that BCL11B interacts via its conserved N-terminal MSRRKQ motif with endogenous MTA1 and MTA3 proteins to recruit various NuRD complexes. Furthermore, we demonstrate that protein kinase C (PKC)-mediated phosphorylation of BCL11B Ser2 does not significantly impact BCL11B SUMOylation but negatively regulates NuRD recruitment by dampening the interaction with MTA1 or MTA3 (MTA1/3) and RbAp46 proteins. We detected increased phosphorylation of BCL11B Ser2 upon in vivo activation of transformed and primary human CD4(+) T cells. We show that following activation of CD4(+) T cells, BCL11B still binds to IL-2 and Id2 promoters but activates their transcription by recruiting P300 instead of MTA1. Prolonged stimulation results in the direct transcriptional repression of BCL11B by KLF4. Our results unveil Ser2 phosphorylation as a new BCL11B posttranslational modification linking PKC signaling pathway to T-cell receptor (TCR) activation and define a simple model for the functional switch of BCL11B from a transcriptional repressor to an activator during TCR activation of human CD4(+) T cells.