HIF-1 inactivation empowers HIF-2 to drive hypoxia adaptation in aggressive forms of medulloblastoma.

Medulloblastoma (MB) is the most prevalent brain cancer in children. Four subgroups of MB have been identified; of these, Group 3 is the most metastatic. Its genetics and biology remain less clear than the other groups, and it has a poor prognosis and few effective treatments available. Tumor hypoxia and the resulting metabolism are known to be important in the growth and survival of tumors but, to date, have been only minimally explored in MB. Here we show that Group 3 MB tumors do not depend on the canonical transcription factor hypoxia-inducible factor-1α (HIF-1α) to mount an adaptive response to hypoxia. We discovered that HIF-1α is rendered inactive either through post-translational methylation, preventing its nuclear localization specifically in Group 3 MB, or by a low expression that prevents modulation of HIF-target genes. Strikingly, we found that HIF-2 takes over the role of HIF-1 in the nucleus and promotes the activation of hypoxia-dependent anabolic pathways. The exclusion of HIF-1 from the nucleus in Group 3 MB cells enhances the reliance on HIF-2's transcriptional role, making it a viable target for potential anticancer strategies. By combining pharmacological inhibition of HIF-2α with the use of metformin, a mitochondrial complex I inhibitor to block respiration, we effectively induced Group 3 MB cell death, surpassing the effectiveness observed in Non-Group 3 MB cells. Overall, the unique dependence of MB cells, but not normal cells, on HIF-2-mediated anabolic metabolism presents an appealing therapeutic opportunity for treating Group 3 MB patients with minimal toxicity.

GSK3-mediated MAF phosphorylation in multiple myeloma as a potential therapeutic target.

Multiple myeloma (MM) is an incurable haematological malignancy characterised by the proliferation of mature antibody-secreting plasma B cells in the bone marrow. MM can arise from initiating translocations, of which the musculoaponeurotic fibrosarcoma (MAF) family is implicated in ∼5%. MMs bearing Maf translocations are of poor prognosis. These translocations are associated with elevated Maf expression, including c-MAF, MAFB and MAFA, and with t(14;16) and t(14;20) translocations, involving c-MAF and MAFB, respectively. c-MAF is also overexpressed in MM through MEK/ERK activation, bringing the number of MMs driven by the deregulation of a Maf gene close to 50%. Here we demonstrate that MAFB and c-MAF are phosphorylated by the Ser/Thr kinase GSK3 in human MM cell lines. We show that LiCl-induced GSK3 inhibition targets these phosphorylations and specifically decreases proliferation and colony formation of Maf-expressing MM cell lines. Interestingly, bortezomib induced stabilisation of Maf phosphorylation, an observation that could explain, at least partially, the low efficacy of bortezomib for patients carrying Maf translocations. Thus, GSK3 inhibition could represent a new therapeutic approach for these patients.

Altered MENIN expression disrupts the MAFA differentiation pathway in insulinoma.

The protein MENIN is the product of the multiple endocrine neoplasia type I (MEN1) gene. Altered MENIN expression is one of the few events that are clearly associated with foregut neuroendocrine tumours (NETs), classical oncogenes or tumour suppressors being not involved. One of the current challenges is to understand how alteration of MENIN expression contributes to the development of these tumours. We hypothesised that MENIN might regulate factors maintaining endocrine-differentiated functions. We chose the insulinoma model, a paradigmatic example of well-differentiated pancreatic NETs, to study whether MENIN interferes with the expression of v-MAF musculoaponeurotic fibrosarcoma oncogene homologue A (MAFA), a master glucose-dependent transcription factor in differentiated ß-cells. Immunohistochemical analysis of a series of human insulinomas revealed a correlated decrease in both MENIN and MAFA. Decreased MAFA expression resulting from targeted Men1 ablation was also consistently observed in mouse insulinomas. In vitro analyses using insulinoma cell lines showed that MENIN regulated MAFA protein and mRNA levels, and bound to Mafa promoter sequences. MENIN knockdown concomitantly decreased mRNA expression of both Mafa and ß-cell differentiation markers (Ins1/2, Gck, Slc2a2 and Pdx1) and, in parallel, increased the proliferation rate of tumours as measured by bromodeoxyuridine incorporation. Interestingly, MAFA knockdown alone also increased proliferation rate but did not affect the expression of candidate proliferation genes regulated by MENIN. Finally, MENIN variants with missense mutations detected in patients with MEN1 lost the WT MENIN properties to regulate MAFA. Together, our findings unveil a previously unsuspected MENIN/MAFA connection regarding control of the ß-cell differentiation/proliferation balance, which could contribute to tumorigenesis.

Reexpression of oncoprotein MafB in proliferative ß-cells and Men1 insulinomas in mouse.

MafB, a member of the large Maf transcription factor family, is essential for the embryonic and terminal differentiation of pancreatic α- and ß-cells. However, the role of MafB in the control of adult islet-cell proliferation remains unknown. Considering its oncogenic potential in several other tissues, we investigated the possible alteration of its expression in adult mouse ß-cells under different conditions of proliferation. We found that MafB, in general silenced in these cells, was reexpressed in ∼30% of adaptive ß-cells both in gestational female mice and in mice fed with a high-fat diet. Importantly, reactivated MafB expression was also observed in the early ß-cell lesions and insulinomas that developed in ß-cell specific Men1 mutant mice, appearing in >80% of ß-cells in hyperplasic or dysplastic islets from the mutant mice >4 months of age. Moreover, MafB expression could be induced by glucose stimulation in INS-1 rat insulinoma cells. The induction was further reinforced following Men1 knockdown by siRNA. Furthermore, MafB overexpression in cultured ßTC3 cells enhanced cell foci formation both in culture medium and on soft agar, accompanied with the increased expression of Cyclin B1 and D2. Conversely, MafB downregulation by siRNA transfection reduced BrdU incorporation in INS-1E cells. Taken together, our data reveal that Men1 inactivation leads to MafB reexpression in mouse ß-cells in vivo, and provides evidence that deregulated ectopic MafB expression may have a hitherto unknown role in adult ß-cell proliferation and Men1-related tumorigenesis.

Cell context reveals a dual role for Maf in oncogenesis.

Maf b-Zip transcription factors are involved in both terminal differentiation and oncogenesis. To investigate this apparent contradiction, we used two different primary cell types and performed an extensive analysis of transformation parameters induced by Maf proteins. We show that MafA and c-Maf are potent oncogenes in chicken embryo fibroblasts, while MafB appears weaker. We also provide the first evidence that MafA can confer growth factor independence and promote cell division at low density. Moreover, using MafA as a model, we identified several parameters that are critical for Maf transforming activities. Indeed, MafA ability to induce anchorage-independent cell growth was sensitive to culture conditions. In addition, the transforming activity of MafA was dependent on its phosphorylation state, since mutation on Ser65 impaired its ability to induce growth at low density and anchorage-independent growth. We next examined transforming activity of large Maf proteins in embryonic neuroretina cells, where they are known to induce differentiation. Unlike v-Jun, MafA, MafB and c-Maf did not show oncogenic activity in these cells. Moreover, they counteracted transformation induced by constitutive activation of the Ras/Raf/MEK pathway. Taken together, our results show that Maf proteins could display antagonistic functions in oncogenesis depending on the cellular context, and support a dual role for Maf as both oncogenes and tumor suppressor-like proteins.

TGFbeta influences Myc, Miz-1 and Smad to control the CDK inhibitor p15INK4b.

Transforming growth factor-beta (TGFbeta) is a cytokine that arrests epithelial cell division by switching off the proto-oncogene c-myc and rapidly switching on cyclin-dependent kinase (CDK) inhibitors such as p15INK4b. Gene responses to TGFbeta involve Smad transcription factors that are directly activated by the TGFbeta receptor. Why downregulation of c-myc expression by TGFbeta is required for rapid activation of p15INK4b has remained unknown. Here we provide evidence that TGFbeta signalling prevents recruitment of Myc to the p15INK4b transcriptional initiator by Myc-interacting zinc-finger protein 1 (Miz-1). This relieves repression and enables transcriptional activation by a TGFbeta-induced Smad protein complex that recognizes an upstream p15INK4b promoter region and contacts Miz-1. Thus, two separate TGFbeta-dependent inputs - Smad-mediated transactivation and relief of repression by Myc - keep tight control over p15INK4b activation.

Characterization of a novel quiescence responsive element downregulated by v-Src in the promoter of the neuroretina specific QR1 gene.

The neuroretina is a functional unit of the central nervous system which arises through successive steps of division, growth arrest and differentiation of neuroectodermal precursors. Postmitotic quail neuroretina (QNR) cells are conditionally induced to divide upon infection with temperature sensitive mutants of Rous sarcoma virus (RSV), since QNR cell division can be arrested by either inactivating p60v-Src at the nonpermissive temperature (41 degrees C) or by serum deprivation at 37 degrees C. We are studying the transcriptional control of QR1, a neuroretina specific gene, whose expression is down-regulated in proliferating cells at 37 degrees C and is fully restored when these cells are made quiescent. We previously showed that this quiescence specific upregulation implicates a promoter region named A box, which binds Maf transcription factors. We report the identification of the C box, a second promoter sequence that activates QR1 transcription in non dividing cells. This sequence is able to form two DNA-protein complexes, one of which (C4) is predominantly detected in growth arrested NR cells. We identified the DNA binding site for C4 and described mutations that abolish both C4 binding and promoter activity in quiescent cells. Moreover, we show that a multimerized C box is able to stimulate a heterologous promoter in non dividing cells and constitutes, therefore, a novel quiescence responsive enhancer. Finally, we report that QR1 transcriptional response to cell quiescence requires cooperation between the C box and A box.

Inhibition of the transforming growth factor beta 1 signaling pathway by the AML1/ETO leukemia-associated fusion protein.

The t(8;21) translocation, found in adult acute myelogenous leukemia, results in the formation of an AML1/ETO chimeric transcription factor. AML1/ETO expression leads to alterations in hematopoietic progenitor cell differentiation, although its role in leukemic transformation is not clear. The N-terminal portion of AML1, which is retained in AML1/ETO, contains a region of homology to the FAST proteins, which cooperate with Smads to regulate transforming growth factor beta1 (TGF-beta1) target genes. We have demonstrated the physical association of Smad proteins with AML1 and AML1/ETO by immunoprecipitation and have mapped the region of interaction to the runt homology domain in these AML1 proteins. Using confocal microscopy, we demonstrated that AML1, and ETO and/or AML1/ETO, colocalize with Smads in the nucleus of t(8;21)-positive Kasumi-1 cells, in the presence but not the absence of TGF-beta1. Using transient transfection assays and a reporter gene construct that contains both Smad and AML1 consensus binding sequences, we demonstrated that overexpression of AML1B cooperates with TGF-beta1 in stimulating reporter gene activity, whereas AML1/ETO represses basal promoter activity and blocks the response to TGF-beta1. Considering the critical role of TGF-beta1 in the growth and differentiation of hematopoietic cells, interference with TGF-beta1 signaling by AML1/ETO may contribute to leukemogenesis.

Transforming growth factor beta -inducible independent binding of SMAD to the Smad7 promoter.

SMAD proteins can mediate transforming growth factor beta (TGF-beta)-inducible transcriptional responses. Whereas SMAD can recognize specific DNA sequences, it is usually recruited to a promoter through interaction with a DNA-binding partner. In an effort to search for TGF-beta-inducible genes, we used a subtractive screening method and identified human Smad7, which can antagonize TGF-beta signaling and is rapidly up-regulated by TGF-beta. In this report, we show that TGF-beta can stabilize Smad7 mRNA and activate Smad7 transcription. The Smad7 promoter is the first TGF-beta responsive promoter identified in vertebrates that contains the 8-bp palindromic SMAD-binding element (SBE), an optimal binding site previously identified by a PCR-based selection from random oligonucleotides by using recombinant Smad3 and Smad4. We demonstrate that on TGF-beta treatment, endogenous SMAD complex can bind to a Smad7 promoter DNA as short as 14 or 16 bp that contains the 8-bp SBE in gel mobility shift and supershift assays. Our studies provide strong evidence that SMAD proteins can bind to a natural TGF-beta responsive promoter independent of other sequencespecific transcription factors. We further show that, whereas recombinant Smad3 binds to the SBE, endogenous or even transfected Smad3 cannot bind to the SBE in the absence of Smad4. These findings have important implications in the identification of target genes of the TGF-beta/SMAD signaling pathways.

Physical and functional interaction of SMADs and p300/CBP.

SMADs are transforming growth factor beta (TGF-beta) receptor substrates and mediators of TGF-beta transcriptional responses. Here we provide evidence that the coactivators p300 and CBP interact with Smads 1 through 4. The biological relevance of this interaction is shown in vivo by overexpression of the adenovirus E1A protein and mutant forms of E1A that lack p300-binding sites. Wild-type E1A, but not the mutants, inhibits SMAD-dependent transcriptional responses to TGF-beta. E1A also inhibits the intrinsic transactivating function of the Smad4 MH2 domain. In addition, overexpression of p300 enhances SMAD-dependent transactivation. Our results suggest a role for p300/CBP in SMAD-mediated transcriptional activation and provide an explanation for the observed ability of E1A to interfere with TGF-beta action.

mafA, a novel member of the maf proto-oncogene family, displays developmental regulation and mitogenic capacity in avian neuroretina cells.

Transcription factors of the Maf proto-oncogene family have been shown to participate in the regulation of several differentiation specific genes. We previously reported that a member(s) of this family is involved in the regulation of the neuroretina specific gene, QR1, through a promoter region, designated the A box, that is closely related to the Maf recognition element (MARE). We undertook an identification of Maf family genes expressed in the quail neuroretina (QNR) and we report the isolation of mafA, a gene encoding a novel member of the large Maf proteins subgroup. Expression of this gene is developmentally regulated in the neuroretina. MafA is able to bind to MARE sequence and to heterodimerize with v-Maf, MafB, Jun and Fos, but not with the small MafF and MafK proteins. Accordingly, it is able to transactivate the QR1 promoter A box. We also show that increased expression of mafA induces sustained proliferation of postmitotic QNR cells.

Dual role of the Smad4/DPC4 tumor suppressor in TGFbeta-inducible transcriptional complexes.

Upon ligand binding, the receptors of the TGFbeta family phosphorylate Smad proteins, which then move into the nucleus where they activate transcription. To carry out this function, the receptor-activated Smads 1 and 2 require association with the product of deleted in pancreatic carcinoma, locus 4 (DPC4), Smad4. We investigated the step at which Smad4 is required for transcriptional activation. Smad4 is not required for nuclear translocation of Smads 1 or 2, or for association of Smad2 with a DNA binding partner, the winged helix protein FAST-1. Receptor-activated Smad2 takes Smad4 into the nucleus where they form a complex with FAST-1 that requires these three components to activate transcription. Smad4 contributes two functions: Through its amino-terminal domain, Smad4 promotes binding of the Smad2/Smad4/FAST-1 complex to DNA; through its carboxy-terminal domain, Smad4 provides an activation function required for Smad1 or Smad2 to stimulate transcription. The dual function of Smad4 in transcriptional activation underscores its central role in TGFbeta signaling.

QRI, a retina-specific gene, encodes an extracellular matrix protein exclusively expressed during neural retina differentiation.

Neural retina development results from growth arrest of neuroectodermal precursors and differentiation of postmitotic cells. The QRI gene is specifically expressed in Müller retinal glial cells. Its expression coincides with the stage of withdrawal from the cell cycle and establishment of differentiation and is repressed upon induction of retinal cell proliferation by the v-src gene product. In this report, we show that the QR1 gene encodes several glycosylated proteins that are secreted and can either associate with the extracellular matrix or remain diffusible in the medium. By using pulse-chase experiments, the 100-103 kDa forms seem to appear first and are specifically incorporated into the extracellular matrix, whereas the 108 and 60 kDa polypeptides appear later and are detected as soluble forms in the culture medium. We also report that expression of the QR1 gene is developmentally regulated in the chicken. Its mRNA is first detectable at embryonic day 10, reaches a maximal level at embryonic day 15 and is no longer detected at embryonic day 18. Immunolocalization of the QR1 protein in chicken retina sections during development shows that expression of the protein parallels the differentiation pattern of post-miotic cells (in particular Müller cells and rods), corresponding to the two differentiation gradients in the retina: from the ganglion cell layer to the inner nuclear layer and outer nuclear layer, and from the optic nerve to the iris. At embryonic day 10, expression of the QR1 protein(s) is restricted to the optic nerve region and the inner nuclear layer, colocalizing with Müller cell bodies. As development proceeds, QR1 protein localization spreads towards the iris and towards the outer nuclear layer, following Müller cell elongations towards the photoreceptors. Between embryonic days 16 and 18, the QR1 protein is no longer detectable in the optic nerve region and is concentrated around the basal segment of the photoreceptors in the peripheral retina. Our results suggest a role for the QR1 gene product in the process of growth arrest and establishment of photoreceptor differentiation.

Transcriptional stimulation of the retina-specific QR1 gene upon growth arrest involves a Maf-related protein.

The avian neural retina (NR) is derived from proliferating neuroectodermal precursors which differentiate after terminal mitosis and become organized in cell strata. Proliferation of postmitotic NR cells can be induced by infection with Rous sarcoma virus (RSV) and requires the expression of a functional v-Src protein. QR1 is a retina-specific gene expressed exclusively at the stage of growth arrest and differentiation during retinal development. In NR cells infected with tsPA101, an RSV mutant conditionally defective in pp60v-src mitogenic capacity, QR1 expression is downregulated in proliferating cells at 37 degrees C and is fully restored when the cells become quiescent as a result of pp60v-src inactivation at 41 degrees C. We were able to arrest proliferation of tsPA101-infected quail NR cells expressing an active v-Src protein by serum starvation at 37 degrees C. This allowed us to investigate the role of cell growth in regulating QR1 transcription. We report that QR1 transcription is stimulated in growth-arrested cells at 37 degrees C compared with that in proliferating cells maintained at the same temperature. Growth arrest-dependent stimulation of QR1 transcription requires the integrity of the A box, a previously characterized cis-acting element responsible for QR1 transcriptional stimulation upon v-Src inactivation and during retinal differentiation. We also show that formation of the C1 complex on the A box is increased upon growth arrest by serum starvation in the presence of an active v-Src oncoprotein. Thus, the C1 complex represents an important link between cell cycle and developmental control of QR1 gene transcription during NR differentiation and RSV infection. By using antibodies directed against different Maf proteins of the leucine zipper family and competition with Maf consensus site-containing oligonucleotides in a gel shift assay, we show that the C1 complex is likely to contain a Maf-related protein. We also show that a purified bacterially expressed v-Maf protein is able to bind the A box and that the level of a 43-kDa Maf-related protein is increased upon growth arrest in infected retinal cells. Moreover, ectopic expression of c-mafI, c-mafII, and mafB cDNAs in quiescent tsPA101-infected quail NR cells is able to stimulate transcription of a QR1 reporter gene through the A box. Therefore, QR1 appears to be the first target gene for a Maf-related protein(s) in the NR.

Developmental control of transcription of a retina-specific gene, QR1, during differentiation: involvement of factors from the POU family.

Developmental control of gene expression often results from the coupling of growth arrest with the establishment of differentiation programs. QR1 is a gene specifically expressed in retinas during the late phase of embryogenesis. At this stage neuroectodermal precursors have reached terminal mitosis and are undergoing differentiation into distinct cell types. Transcription of the QR1 gene is tightly regulated during retinal development: this gene is expressed between embryonic day 9 (ED9) and ED17 and is completely repressed at hatching in quail. Moreover, QR1 transcription is downregulated when postmitotic neural retina cells are induced to proliferate by pp60v-src. We studied the stage-dependent transcriptional control of this gene during quail neural retina (QNR) cell development. Transient transfection experiments with QR1/CAT constructs at various stages of development showed that a region located between -935 and -1265 bp upstream of the transcription start site is necessary to promote transcription in retina cells during the late phase of embryonal development (QNR9, corresponding to ED9). By in vivo footprinting assays we identified at least two elements that are occupied by DNA-protein complexes in QNR cells: the A and B boxes. The A box allows formation of several biochemically distinct complexes: C1, C2, C3, and C4. Formation of the C2 complex mainly during early stages (ED7) and of C2, C3, and C4 complexes during postnatal life correlates with repression of QR1 transcription, whereas the C1 complex is strongly induced at ED11 when the QR1 gene is expressed. We previously showed that C1 was involved in downregulation of QR1 transcription by pp60v-src. Several complexes are also formed on the B box. We show that these complexes are exclusively present in neural tissues and that they involve members of the POU family of transcription factors. Mutations of each one of the two regions which abolish the binding of the C1 factor(s) on the A box and of the POU factor(s) on the B box also prevent stimulation of QR1 transcription in QNR9. Therefore, both elements appear to be required for the stage-specific transcription of the QR1 gene. We also show that the regulatory region from position -1265 to position -935 is able to confer stage-specific transcription upon a heterologous promoter (thymidine kinase). Indeed, this region stimulates transcription in differentiating retinas (QNR9) and represses transcription in terminally differentiated retinas (QNR17, corresponding to postnatal life). Our results suggest that cell growth regulation and developmental control are coordinated through the A and B boxes in regulating QR1 transcription during retinal differentiation.

Transcriptional downregulation of the retina-specific QR1 gene by pp60v-src and identification of a novel v-src-responsive unit.

The embryonic avian neuroretina (NR) is part of the central nervous system and is composed of various cell types: photoreceptors and neuronal and Müller (glial) cells. These cells are derived from proliferating neuroectodermal precursors which differentiate after terminal mitosis and become organized in cell strata. Proliferation of differentiating NR cells can be induced by infection with Rous sarcoma virus (RSV) and requires the expression of a functional v-src gene. To understand the mechanisms involved in the regulation of neural cell growth and differentiation, we studied the transcriptional regulation of QR1, a gene specifically expressed in postmitotic NR cells. Transcription of this gene is detected primarily in Müller cells and is strongly downregulated by the v-src gene product. Moreover, QR1 expression takes place only during the late phase of retinal development and is shut off abruptly at hatching. We have isolated a promoter region(s) of the QR1 gene that confers v-src responsiveness. By transfection of QR1-CAT constructs into quail NR cells infected with the temperature-sensitive mutant of RSV, PA101, we have identified a v-src-responsive region located between -1208 and -1161 upstream of the transcription initiation site. This sequence is able to form two DNA-protein complexes, C1 and C2. Formation of complex C2 is specifically induced in cells expressing an active v-src product, while formation of C1 is detected mainly in nonproliferating quail NR cells upon pp60v-src inactivation. C1 is also a target for regulation during development. We have identified the DNA binding site for the C1 complex, a repeated GCTGAC sequence, and shown that mutations in this element abolish binding of this factor as well as transcription of the gene at the nonpermissive temperature. Neither formation of C1 nor that of C2 seems to involve factors known to be targeted in the pp60v-src cascade. Our data suggest that C1 could be a novel target for both developmental control and oncogene-induced cell growth regulation.