Sétha Douc-Rasy

Netrin-1 acts as a survival factor for aggressive neuroblastoma

Neuroblastoma (NB), the most frequent solid tumor of early childhood, is diagnosed as a disseminated disease in >60% of cases, and several lines of evidence support the resistance to apoptosis as a prerequisite for NB progression. We show that autocrine production of netrin-1, a multifunctional laminin-related molecule, conveys a selective advantage in tumor growth and dissemination in aggressive NB, as it blocks the proapoptotic activity of the UNC5H netrin-1 dependence receptors. We show that such netrin-1 up-regulation is a potential marker for poor prognosis in stage 4S and, more generally, in NB stage 4 diagnosed infants. Moreover, we propose that interference with the netrin-1 autocrine loop in malignant neuroblasts could represent an alternative therapeutic strategy, as disruption of this loop triggers in vitro NB cell death and inhibits NB metastasis in avian and mouse models.

ΔN-p73α Accumulates in Human Neuroblastic Tumors

Neuroblastic tumors (NTs), occurring in early childhood, display a wide spectrum of differentiation. Recurrent deletions involving the p73 locus are frequently observed in undifferentiated NTs. To address the question of the possible implication of p73 in neuroblastic differentiation, we investigated the status of the expression of this gene in a panel of differentiated and undifferentiated tumors. Although mutations were not found, p73 transcript profiles differed between undifferentiated and differentiated tumors. The frequency of the transcripts lacking exon 2 (species 1-3) appeared to be higher in undifferentiated than in differentiating and differentiated NTs. In contrast, products from using an alternate promoter (DeltaN-p73) were present in all NTs. In addition, only DeltaN-p73, but not full-length proteins, were detected by immunoblotting, suggesting a greater stability of N-truncated isoforms. Importantly, as in the adrenal medulla, most NTs showed p73-positive immunohistological staining with a cellular distribution and intensity varying according to the neuronal differentiation. Surprisingly, we observed redistribution of p73 from the nucleus to the cytoplasm during neuroblastic differentiation. Our data suggest that, in undifferentiated NTs, a link may exist between the accumulation of DeltaN-p73alpha variants and the nuclear exclusion of p53.

c-myc, p53 and bcl-2, apoptosis-related genes in infiltrating breast carcinomas: evidence of a link between bcl-2 protein over-expression and a lower risk of metastasis and death in operable patients.

Apoptosis is an important physiological process controlled by multiple genes, including c‐myc, p53 and bcl‐2, and its inhibition may lead to the development of human cancers. In this study, we analyzed expression of the c‐myc gene using Northern blot and of the p53 and bcl‐2 proteins by immuno‐histochemistry in 175 breast tumor specimens obtained from patients with operable breast cancer. We evaluated the relation between expression of these 3 genes and (i) the main usual prognostic factors (tumor size, histo‐prognostic grade, hormone receptors and number of positive nodes); (ii) the risk of death and relapse, taking into account these 4 factors, after a mean period of follow‐up of 9.5 years (SD 2 years). Over‐expression of c‐myc, p53 and bcl‐2 was observed in 35%, 23% and 63% of tumors, respectively. Over‐expression of c‐myc was strongly linked to the number of positive nodes (p = 0.0005). p53 protein expression was associated with both high‐grade (p = 0.0001) and hormone receptor‐negative (p = 0.0001) tumors. In contrast, bcl‐2 protein over‐expression was associated with the main favorable prognostic factors and, more particularly, with hormone receptor‐positive tumors (p = 0.0001). Multivariate analysis, using the Cox model, showed that only 2 factors were independently linked to the risk of death: number of positive nodes, which increased the risk (p = 0.0001), and bcl‐2 protein over‐expression, which decreased the risk (p = 0.008). When bcl‐2 over‐expression was studied in relation to nodal status, hormone receptor status and chemo‐ and hormone therapy, no significant difference was observed between different subgroups of patients. bcl‐2 expression was also associated with a significantly lower risk of distant metastasis (p = 0.04). In conclusion, bcl‐2 expression characterizes a particular phenotype of breast cancer with a favorable prognosis, and it may therefore be used as a marker of long‐term survival. Int. J. Cancer (Pred. Oncol.) 84:562–567, 1999.

Expression of C-terminal deleted p53 isoforms in neuroblastoma

The tumor suppressor gene, p53, is rarely mutated in neuroblastomas (NB) at the time of diagnosis, but its dysfunction could result from a nonfunctional conformation or cytoplasmic sequestration of the wild-type p53 protein. However, p53 mutation, when it occurs, is found in NB tumors with drug resistance acquired over the course of chemotherapy. As yet, no study has been devoted to the function of the specific p53 mutants identified in NB cells. This study includes characterization and functional analysis of p53 expressed in eight cell lines: three wild-type cell lines and five cell lines harboring mutations. We identified two transcription-inactive p53 variants truncated in the C-terminus, one of which corresponded to the p53β isoform recently identified in normal tissue by Bourdon et al. [J. C. Bourdon, K. Fernandes, F. Murray-Zmijewski, G. Liu, A. Diot, D. P. Xirodimas, M. K. Saville and D. P. Lane (2005) Genes Dev., 19, 2122–2137]. Our results show, for the first time, that the p53β isoform is the only p53 species to be endogenously expressed in the human NB cell line SK-N-AS, suggesting that the C-terminus truncated p53 isoforms may play an important role in NB tumor development.

CDK Inhibitors Roscovitine and CR8 Trigger Mcl-1 Down-Regulation and Apoptotic Cell Death in Neuroblastoma Cells

Neuroblastoma (NB), the most frequent extracranial solid tumor of children accounting for nearly 15% of all childhood cancer mortality, displays overexpression of antiapoptotic Bcl-2 and Mcl-1 in aggressive forms of the disease. The clinical phase 2 drug roscovitine (CYC202, seliciclib), a relatively selective inhibitor of cyclin-dependent kinases (CDKs), and CR8, a recently developed and more potent analog, induce concentration-dependent apoptotic cell death of NB cells (average IC(50) values: 24.2 µM and 0.4 µM for roscovitine and CR8, respectively). Both roscovitine and CR8 trigger rapid down-regulation of the short-lived survival factor Mcl-1 in the 9 investigated human NB cell lines. This effect was further analyzed in the human SH-SY5Y NB cell line. Down-regulation of Mcl-1 appears to depend on inhibition of CDKs rather than on interaction of roscovitine and CR8 with their secondary targets. CR8 is an adenosine triphosphate-competitive inhibitor of CDK9, and the structure of a CDK9/cyclin T/CR8 complex is described. Mcl-1 down-regulation occurs both at the mRNA and protein levels. This effect can be accounted for by a reduction in Mcl-1 protein synthesis, under stable Mcl-1 degradation conditions. Mcl-1 down-regulation is accompanied by a transient increase in free Noxa, a proapoptotic factor. Mcl-1 down-regulation occurs independently of the presence or up-regulation of p53 and of the MYCN status. Taken together, these results suggest that the clinical drug roscovitine and its novel analog CR8 induce apoptotic tumor cell death by down-regulating Mcl-1, a key survival factor expressed in all NB cell lines. CDK inhibition may thus constitute a new approach to treat refractory high-risk NB.

Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts.

Ku86 has been shown to be involved in DNA double-strand break (DSB) repair and radiosensitivity in rodents, but its role in human cells is still under investigation. The purpose of this study was to evaluate the radiosensitivity and DSB repair after transfection of a Ku86-antisense in a human fibroblast cell line. Simian virus 40-transformed MRC5V1 human fibroblasts were transfected with a vector (pcDNA3) containing a Ku86-antisense cDNA. The main endpoints were Ku86 protein level, Ku DNA end-binding and DNA protein kinase activity, clonogenic survival, and DSB repair kinetics. After transfection of the Ku86-antisense, decreased Ku86 protein expression, Ku DNA end-binding activity, and DNA protein kinase activity were observed in the uncloned cellular population. The fibroblasts transfected with the Ku86-antisense showed also a radiosensitive phenotype, with a surviving fraction at 2 Gy of 0.29 compared with 0.75 for the control and 20% of unrepaired DSB observed at 24 hours after irradiation compared with 0% for the control. Several clones were also isolated with a decreased level of Ku86 protein, a surviving fraction at 2 Gy between 0.05 and 0.40, and 10–20% of unrepaired DSB at 24 hours. This study is the first to show the implication of Ku86 in DSB repair and in the radiosensitivity of human cells. This investigation strongly suggests that Ku86 could constitute an appealing target for combining gene therapy and radiation therapy.

GALNT9 Gene Expression Is a Prognostic Marker in Neuroblastoma Patients

BACKGROUNDnThe enzymes encoded by the GALNT [UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GALNAC-T)] gene family catalyze the first step of O-glycosylation. Little is known about the link between expression of the genes encoding GALNAC-T enzymes and tumor progression in neuroblastoma, a pediatric cancer that can be classified as either low or high risk. We assessed the expression of genes in the GALNT family in a large cohort of neuroblastoma patients and characterized members of this family that might be used as new prognostic markers.nnnMETHODSnReverse-transcription PCR analysis of 14 GALNT genes with a panel of neuroblastoma cell lines identified the GALNT9 gene as playing a potential role in disease progression. We used the log-rank test and the multivariable Cox proportional hazards model with a cohort of 122 neuroblastoma patients to analyze the relationship between GALNT9 expression and overall survival or disease-free survival.nnnRESULTSnIn the high-risk neuroblastoma experimental model IGR-N-91, GALNT9 expression was present in neuroblasts derived from primary tumors but not in neuroblasts from metastatic bone marrow. Moreover, GALNT9 in neuroblastoma cell lines was expressed in substrate adherent (S)-type cell lines but not in neuronal (N)-type lines. In the tumor cohort, GALNT9 expression was associated with high overall survival, independent of the standard risk-stratification covariates. GALNT9 expression was significantly associated with disease-free survival for patients currently classified as at low risk (P < 0.0007).nnnCONCLUSIONSnGALNT9 expression correlates with both improved overall survival in low- and high-risk groups and an improved clinical outcome (overall and disease-free survival) in low-risk patients. Thus, the GALNT9 expression may be a prognostic marker for personalized therapy.

The neurogene BTG2TIS21/PC3 is transactivated by ΔNp73α via p53 specifically in neuroblastoma cells

The p53 gene and its homologue p73 are rarely mutated in neuroblastoma. In recent studies, we showed that overexpression of ΔNp73α, an isoform lacking the N-terminal transactivation (TA) domain, surprisingly induces p53 protein accumulation in the wild-type (wt) p53 human neuroblastoma line SH-SY5Y. As can be expected owing to its dominant-negative effect, ΔNp73α inhibits Waf1/p21 gene expression, but equally importantly, it upregulates BTG2TIS21/PC3, another p53 target gene. This effect is not observed in neuroblastoma cells that express a mutated p53. To better understand the ΔNp73-mediated transactivation of the BTG2TIS21/PC3 gene we performed luciferase assays with two reporter plasmids harboring long and short BTG2 promoter sequences in three human neuroblastoma cell lines and one breast cancer cell line. Our results demonstrate that BTG2TIS21/PC3 transactivation by ΔNp73α depends on both p53 status (as it is not observed in a p53–/– neuroblastoma cell line) and cellular context (as it occurs in a p53+/+ neuroblastoma cell line but not in a p53+/+ breast tumor cell line). The fact that ΔNp73α may either inhibit or stimulate wt-p53 transcriptional activity, depending on both the p53 target gene and the cellular context, was confirmed by real-time quantitative PCR. Moreover, transactivation of the BTG2TIS21/PC3 promoter requires a complete ΔNp73α C-terminus sequence as it is not observed with ΔNp73β, which lacks most of the C-terminal domain. We have previously shown that ΔNp73α is the only p73 isoform expressed in undifferentiated neuroblastoma tumors. In light of all these findings, we propose that ΔNp73α not only acts as an inhibitor of p53/TAp73 functions in neuroblastoma tumors, but also cooperates with wt-p53 in playing a physiological role through the activation of BTG2TIS21/PC3 gene expression.

Loss of the Malignant Phenotype of Human Neuroblastoma Cells by a Catalytically Inactive Dominant-Negative hTERT Mutant

Telomerase, a ribonucleoprotein complex mainly composed of the reverse transcriptase catalytic subunit (human telomerase reverse transcriptase, hTERT) and the RNA component (hTR), is a key enzyme of cancer progression. That aggressive stage 4-neuroblastoma expressed high levels of telomerase activity, whereas favorable tumors had no or little telomerase expression and activity, prompted us to investigate the role of this enzyme in this tumor model of altered proliferation, neuronal differentiation, and apoptosis. A human MYCN-amplified neuroblastoma cell line (IGR-N-91) was engineered to stably express either the normal hTERT protein (WT-hTERT) or a catalytically inactive dominant-negative mutant of this protein (DN-hTERT). We showed that DN-hTERT expression inhibited the endogenous hTERT in the malignant neuroblasts without telomere shortening nor loss of in vitro proliferative capacity. Importantly, DN-hTERT expression induced major changes in cell morphology of neuroblasts that switched them from a neuronal to a substrate adherent phenotype, which was more prone to apoptosis and lost their tumorigenic properties in nude mice. These biologic effects arose from modifications in the expression of genes involved in both apoptosis and neuroblastoma biology. Taken together these results highlighted the functional relevance of noncanonical functions of hTERT in the determination of neuroblast cell fate. Therefore, our results envision new therapeutic strategies for metastatic neuroblastoma therapeutic management. Mol Cancer Ther; 11(11); 2384–93. ©2012 AACR.

ATP-independent DNA topoisomerase II as potential drug target in trypanosomes

Two categories of trypanosomal type II topoisomerases have been isolated from trypanosomes: one is unique since it is able to realize DNA topoisomerization reactions in the absence of ATP, in contrast to the other enzyme and mammalian topoisomerase II. The biochemical properties of ATP‐independent topoisomerase II from Trypanosoma cruzi are described in this report. The enzyme can decatenate trypanosome kinetoplast DNA networks, catenate supercoiled DNA molecules, unknot P4 phage DNA, and cleave double‐stranded DNA. The enzyme is inhibited by various classes of drugs and is more sensitive than mammalian topoisomerase II. Therefore, trypanosome ATP‐independent topoisomerase II provides a potential target for chemotherapy.