Biomedical subjects

Zoulika Kherrouche

Publications and source records attributed to Zoulika Kherrouche.

9 recordsLinked to original sources

PubMed2026

Targeting Both Oncogenic Signaling and Dependence Receptor Function is Required to Fully Suppress MET Exon 14 Skipping-Driven tumorigenesis.

Receptor tyrosine kinases (RTKs) classically function as oncogenic drivers that promote survival and proliferation upon ligand binding. A subset of RTKs can also function as dependence receptors, inducing apoptosis in the absence of their ligands. Genetic alterations that enhance RTK signaling are well characterized in cancer and can be targeted with kinase inhibitors, which show limited efficacy in some clinical settings. Elucidation of whether oncogenic mutations can promote tumorigenesis by directly abolishing the pro-apoptotic activity of dependence receptors could help improve strategies to target RTKs. Here, we identified MET exon 14 skipping (METex14Del) as a paradigmatic example of an oncogenic alteration that drives tumorigenesis through genetic inactivation of the dependence receptor function of an RTK. METex14Del removed both the caspase cleavage site and adjacent CBL-binding motif, preventing generation of the pro-apoptotic p40MET fragment while sustaining oncogenic MET signaling. Uncoupling regulatory functions of MET using genome editing showed that loss of apoptosis capacity is a critical determinant of METex14Del-driven tumorigenesis. Combined-but not individual-mutation of the caspase and CBL sites was sufficient to recapitulate resistance to apoptosis and tumor growth induced by METex14Del in HGF-humanized mouse models. Importantly, inducible re-expression of p40MET in METex14Del-expressing cells restored apoptotic sensitivity, decreased tumor formation in vivo, and resensitized tumors to capmatinib. Together, these findings redefine RTKs as receptors with dual oncogenic and tumor-suppressive functions and show that disruption of dependence receptor-mediated apoptosis is an oncogenic mechanism. These results provide a conceptual framework explaining why therapies targeting only RTK signaling may fail and support strategies restoring dependence receptor function to achieve durable tumor suppression.

Journal Article↗

PubMed2006

ASK-1 (apoptosis signal-regulating kinase 1) is a direct E2F target gene.

In the present study, we show that E2Fs (E2 promoter-binding factors) regulate the expression of ASK-1 (apoptosis signal-regulating kinase 1), which encodes a mitogen-activated protein kinase kinase kinase, also known as MAP3K5. Its mRNA expression is cell-cycle-regulated in human T98G cells released from serum starvation. Moreover, overexpression and RNA interference experiments support the requirement of endogenous E2F/DP (E2F dimerization partner) activity for ASK-1 expression. Characterization of the human ASK-1 promoter demonstrates that the -95/+11 region is critical for E2F-mediated up-regulation. Chromatin immunoprecipitation assays show that E2F1-E2F4 are bound in vivo to the ASK-1 promoter in cycling cells, probably through a non-consensus E2F-binding site located 12 bp upstream of the transcription start site. Mutation of this site completely abolishes the ASK-1 promoter response to E2Fs as well as the E2F1 binding in electrophoretic mobility-shift experiments. Our results indicate that E2Fs modulate the expression of ASK-1 and suggest that some of the cellular functions of ASK-1 may be under the control of E2F transcription factors. Moreover, the up-regulation of ASK-1 may also favour the p53-independent E2F1 apoptotic activity.

Binding Sites↗

PubMed2004

The NRF-1/alpha-PAL transcription factor regulates human E2F6 promoter activity.

E2F6 is widely expressed in human tissues and cell lines. Recent studies have demonstrated its involvement in developmental patterning and in the regulation of various genes implicated in chromatin remodelling. Despite a growing number of studies, nothing is really known concerning the E2F6 expression regulation. To understand how cells control E2F6 expression, we analysed the activity of the previously cloned promoter region of the human E2F6 gene. DNase I footprinting, gel electrophoreticmobility shift, transient transfection and site-directed mutagenesis experiments allowed the identification of two functional NRF-1/alpha-PAL (nuclear respiratory factor-1/alpha-palindrome-binding protein)-binding sites within the human E2F6 core promoter region, which are conserved in the mouse and rat E2F6 promoter region. Moreover, ChIP (chromatin immunoprecipitation) analysis demonstrated that overexpressed NRF-1/alpha-PAL is associated in vivo with the E2F6 promoter. Furthermore, overexpression of full-length NRF-1/alpha-PAL enhanced E2F6 promoter activity, whereas expression of its dominant-negative form reduced the promoter activity. Our results indicate that NRF-1/alpha-PAL is implicated in the regulation of basal E2F6 gene expression.

Animals↗

PubMed2004

Roles of stem cell factor/c-Kit and effects of Glivec/STI571 in human uveal melanoma cell tumorigenesis.

The B-Raf(V599E)-mediated constitutive activation of ERK1/2 is involved in establishing the transformed phenotype of some uveal melanoma cells (Calipel, A., Lefevre, G., Pouponnot, C., Mouriaux, F., Eychene, A., and Mascarelli, F. (2003) J. Biol. Chem. 278, 42409-42418). We have shown that stem cell factor (SCF) is involved in the proliferation of normal uveal melanocytes and that c-Kit is expressed in 75% of primary uveal melanomas. This suggests that the acquisition of autonomous growth during melanoma progression may involve the SCF/c-Kit axis. We used six human uveal melanoma tumor-derived cell lines and normal uveal melanocytes to characterize the SCF/c-Kit system and to assess its specific role in transformation. We investigated the possible roles of activating mutations in c-KIT, the overexpression of this gene, and ligand-dependent c-Kit overactivation in uveal melanoma cell tumorigenesis. Four cell lines (92.1, SP6.5, Mel270, and TP31) expressed both SCF and c-Kit, and none harbored the c-KIT mutations in exons 9, 11, 13, and 17 that have been shown to induce SCF-independent c-Kit activation. Melanoma cell proliferation was strongly inhibited by small interfering RNA-mediated depletion of c-Kit in these cells, despite the presence of (V599E)B-Raf in SP6.5 and TP31 cells. We characterized the signaling pathways involved in SCF/c-Kit-mediated cell growth and survival in normal and tumoral melanocytes and found that constitutive ERK1/2 activation played a key role in both the SCF/c-Kit autocrine loop and the gain of function of (V599E)B-Raf for melanoma cell proliferation and transformation. We also provide the first evidence that Glivec/STI571, a c-Kit tyrosine kinase inhibitor, could be used to treat uveal melanomas.

Base Sequence↗

PubMed2004

Human E2F6 is alternatively spliced to generate multiple protein isoforms.

E2F6 protein belongs to the family of the E2F transcription factors. Here, we showed that the human E2F6 gene contains nine exons distributed along 20.4kbp of genomic DNA on chromosome 2 leading to the transcription of six alternatively spliced E2F6 mRNAs that encode four different E2F6 proteins. Moreover, we identified an E2F6 pseudogene localized on chromosome 22 completely spliced and devoid of exons 2, 3, and 4, and part of exons 1 and 5. Definition of the transcriptional initiation site and sequence analysis show that the gene contains a TATA less, CAAT less, GC-rich promoter with multiple transcription start sites. Regulatory elements necessary for basal transcription reside within a 134bp fragment as determined by transient transfection experiments.

Alternative Splicing↗

PubMed2003

Microphthalmia transcription factor analysis in posterior uveal melanomas.

PURPOSE: The protein encoded by the Microphthalmia gene (MITF) is a transcription factor essential for the development and survival of melanocytes. It serves as a master regulator in modulating extracellular signals. Because of its central role in melanocytes survival and to assess its potential use as a histopathological marker for melanoma, MITF expression was examined in human choroidal melanomas. METHODS: Fifty-seven paraffin-embedded sections of choroidal melanoma specimens and 1 choroidal melanoma cell line were analyzed using immunochemistry and RT-PCR. Normal choroids and normal choroidal melanocyte cells were used as control. RESULTS: Sixty-five percent of the tumoral specimens stained positively for MITF with a predominant nuclear pattern of reactivity. MITF-M and MITF-A isoforms were detected by RT-PCR in all specimens examined. Using a chimeric protein resulting from the fusion of each Mitf protein with the GFP, Mitf-M exhibited an exclusive nuclear staining whereas Mitf-A exhibited a mixed nuclear and cytoplasmic staining. No correlation between MITF-positivity and parameters such as cell type, largest tumor diameter, sclera invasion, mitotic figures was observed. In contrast, a significant negative association was found between MITF staining and the pigmentation (p=0.02) and a positive correlation between MITF staining and the proliferative marker Ki67 was found (p=0.02). CONCLUSION: MITF may be implicated in choroidal melanoma pigmentation and proliferation. Further analysis should provide new insights into the mechanisms underlying the molecular and cellular changes of choroidal melanomas.

Adult↗

PubMed2003

Expression of the c-kit receptor in choroidal melanomas.

The KIT gene encodes c-kit, a transmembrane receptor that has tyrosine kinase activity and plays a role in haematopoiesis, gametogenesis and melanogenesis. The c-kit protein is found in normal cutaneous and choroidal melanocytes, and there is evidence that expression is lost in melanoma. Expression of c-kit was analysed in 57 paraffin-embedded sections of choroidal melanoma specimens and three choroidal melanoma cell lines using immunochemistry and Western blotting. Of the tumour specimens, 75% stained positively for c-kit with a membrane pattern of reactivity. Of the six patients who underwent proton beam therapy before enucleation, five tumours exhibited no c-kit immunoreactivity and the other tumour demonstrated weak staining. Of the three melanoma cell lines used, c-kit expression was observed in only one. No correlations between c-kit positivity and parameters such as cell type, largest macroscopic tumour dimension, scleral invasion or pigmentation were observed. In contrast, a significant positive association was found between c-kit staining and mitotic activity (P = 0.02). However, c-kit expression did not significantly influence survival when evaluated by univariate analysis. In conclusion, c-kit is expressed in most choroidal melanoma tumours. Further analysis should provide new insights into the mechanisms underlying the molecular and cellular changes in choroidal melanomas.

Blotting, Western↗

PubMed2002

Hepatocyte growth factor/scatter factor activates the ETS1 transcription factor by a RAS-RAF-MEK-ERK signaling pathway.

Hepatocyte growth factor/scatter factor (HGF/SF) induces scattering and morphogenesis of epithelial cells through the activation of the MET tyrosine kinase receptor. Although the activated MET receptor recruits a number of signaling proteins, little is known of the downstream signaling pathways activated by HGF/SF. In this study, we wished to examine the signaling pathway leading to activation of the ETS1 transcription factor. Using in vitro and in vivo kinase assays, we found that HGF/SF activates the ERK1 MAP kinase, leading to the phosphorylation of the threonine 38 residue of ETS1 within a putative MAP kinase phosphorylation site (PLLT38P). This threonine residue was neither phosphorylated by JNK1, nor by p38 MAP kinases and was required for the induction of transcriptional activity of ETS1 by HGF/SF. Using kinase and transcription assays, we further demonstrated that phosphorylation and activation of ETS1 occurs downstream of a RAS-RAF-MEK-ERK pathway. The functional involvement of this pathway in HGF/SF action was demonstrated using U0126, a pharmacological inhibitor of MEK, which blocked phosphorylation and activation of ETS1, RAS-dependent transcriptional responses, cell scattering and morphogenesis. These data demonstrated that ETS1 is a downstream target of HGF/SF acting through a RAS-RAF-MEK-ERK pathway and provides a signaling pathway leading to the regulation of gene expression by HGF/SF.

Animals↗

PubMed1999

Atypical nucleoprotein complexes mediate CRE-dependent regulation of the early promoter of minute virus of mice.

The P4 promoter of the parvovirus minute virus of mice (MVMp) directs transcription of the genes encoding non-structural proteins. We have previously shown that functional upstream CRE elements contribute to both the ras oncogene-dependent activation of promoter P4 and its down-modulation by known activators of cyclic AMP-dependent protein kinase A (PKA). In the present work, the nucleoprotein complexes formed with the P4 CRE elements were characterized with regard to their polypeptide constituents and the nucleotides taking part in the interaction. Atypical interactions, both at the protein-protein and protein-DNA level, were observed, which may be a reflection of the divergence of the parvoviral CREs from the usual consensus. The CRE-mediated regulation of promoter P4 by PKA and Ras is discussed in light of these findings.

Animals↗