Hedgehog-mediated regulation of PPARγ controls metabolic patterns in neural precursors and shh-driven medulloblastoma

Sonic hedgehog (Shh) signaling is critical during development and its aberration is common across the spectrum of human malignancies. In the cerebellum, excessive activity of the Shh signaling pathway is associated with the devastating pediatric brain tumor medulloblastoma. We previously demonstrated that exaggerated de novo lipid synthesis is a hallmark of Shh-driven medulloblastoma and that hedgehog signaling inactivates the Rb/E2F tumor suppressor complex to promote lipogenesis. Indeed, such Shh-mediated metabolic reprogramming fuels tumor progression, in an E2F1- and FASN-dependent manner. Here, we show that the nutrient sensor PPARγ is a key component of the Shh metabolic network, particularly its regulation of glycolysis. Our data show that in primary cerebellar granule neural precursors (CGNPs), proposed medulloblastoma cells-of-origin, Shh stimulation elicits a marked induction of PPARγ alongside major glycolytic markers. This is also documented in the actively proliferating Shh-responsive CGNPs in the developing cerebellum, and PPARγ expression is strikingly elevated in Shh-driven medulloblastoma in vivo. Importantly, pharmacological blockade of PPARγ and/or Rb inactivation inhibits CGNP proliferation, drives medulloblastoma cell death and extends survival of medulloblastoma-bearing animals in vivo. This coupling of mitogenic Shh signaling to a major nutrient sensor and metabolic transcriptional regulator define a novel mechanism through which Shh signaling engages the nutrient sensing machinery in brain cancer, controls the cell cycle, and regulates the glycolytic index. This also reveals a dominant role of Shh in the etiology of glucose metabolism in medulloblastoma and underscores the function of the Shh → E2F1 → PPARγ axis in altering substrate utilization patterns in brain cancers in favor of tumor growth. These findings emphasize the value of PPARγ downstream of Shh as a global therapeutic target in hedgehog-dependent and/or Rb-inactivated tumors

p16INK4A Positively Regulates Cyclin D1 and E2F1 through Negative Control of AUF1

/pRB/E2F pathway, a key regulator of the critical G1 to S phase transition of the cell cycle, is universally disrupted in human cancer. However, the precise function of the different members of this pathway and their functional interplay are still not well defined. -dependent manner, and several of these genes are also members of the AUF1 and E2F1 regulons. We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis. is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1

In vitro hypoxia-conditioned colon cancer cell lines derived from HCT116 and HT29 exhibit altered apoptosis susceptibility and a more angiogenic profile in vivo

Hypoxia is an important selective force in the clonal evolution of tumours. Through HIF-1 and other transcription factors combined with tumour-specific genetic alterations, hypoxia is a dominant factor in the angiogenic phenotype. Cellular adaptation to hypoxia is an important requirement of tumour progression independent of angiogenesis. The adaptive changes, insofar as they alter hypoxia-induced apoptosis, are likely to determine responsiveness to antiangiogenic strategies. To investigate this adaptation of tumour cells to hypoxia, we recreated in vitro the in vivo situation of chronic intermittent exposure to low-oxygen levels. The colon carcinoma cell lines HT29 and HCT116 were subjected to 40 episodes of sublethal hypoxia (4 h) three times a week. The resulting two hypoxia-conditioned cell lines have been maintained in culture for more than 2 years. In both cell lines changes in doubling times occurred: in HT29 an increase, and in HCT116 a decrease. Cell survival in response to hypoxia and to DNA damage differed strikingly in the two cell lines. The HT29 hypoxia-conditioned cells were more resistant than the parental line to a 24 h hypoxic challenge, while those from HCT116 surprisingly were more sensitive. Sensitivity to cisplatin in vitro was also significantly different for the hypoxia-conditioned compared with the parental lines, suggesting a change in pathways leading to apoptosis following DNA damage signaling. The growth of both conditioned cell lines in vivo as xenografts in immunodeficient (SCID) mice was more rapid than their parental lines, and was accompanied in each by evidence of enhanced vascular proliferation as a consequence of the hypoxia-conditioning. Thus the changes in apoptotic susceptibility were independent of altered angiogenesis. The derivation of these lines provides a model for events within hypoxic regions of colon cancers, and for the acquisition of resistance and sensitivity characteristics that may have therapeutic implications for the use of antiangiogenesis drugs

An immunohistochemical perspective of PPARβ and one of its putative targets PDK1 in normal ovaries, benign and malignant ovarian tumours

Peroxisome proliferator-activated receptor β (PPARβ) is a member of the nuclear hormone receptor family and is a ligand-activated transcription factor with few known molecular targets including 3-phosphoinositide-dependent protein kinase 1(PDK1). In view of the association of PPARβ and PDK1 with cancer, we have examined the expression of PPARβ and PDK1 in normal ovaries and different histological grades of ovarian tumours. Normal ovaries, benign, borderline, grades 1, 2 and 3 ovarian tumours of serous, muciuous, endometrioid, clear cell and mixed subtypes were analysed by immunohistochemistry for PPARβ and PDK1 expression. All normal ovarian tissues, benign, borderline and grade 1 tumours showed PPARβ staining localised in the epithelium and stroma. Staining was predominantly nuclear, but some degree of cytoplasmic staining was also evident. Approximately 20% of grades 2 and 3 tumours lacked PPARβ staining, whereas the rest displayed some degree of nuclear and cytoplasmic staining of the scattered epithelium and stroma. The extent of epithelial and stromal PPARβ staining was significantly different among the normal and the histological grades of tumours (χ2=59.25, d.f.=25, P<0.001; χ2=64.48, d.f.=25, P<0.001). Significantly different staining of PPARβ was observed in the epithelium and stroma of benign and borderline tumours compared with grades 1, 2 and 3 tumours (χ2=11.28, d.f.=4, P<0.05; χ2=16.15, d.f.=4, P<0.005). In contrast, PDK1 immunostaining was absent in 9 out of 10 normal ovaries. Weak staining for PDK1 was observed in one normal ovary and 40% of benign ovarian tumours. All borderline and malignant ovarian tumours showed positive cytoplasmic and membrane PDK1 staining. Staining of PDK1 was confined to the epithelium and the blood vessels, and no apparent staining of the stroma was evident. Significantly different PDK1 staining was observed between the benign/borderline and malignant ovarian tumours (χ2=22.45, d.f.=5, P<0.001). In some borderline and high-grade tumours, staining of the reactive stroma was also evident. Our results suggest that unlike the colon, the endometrial, head and neck carcinomas, overexpression of PPARβ does not occur in ovarian tumours. However, overexpression of PDK1 was evident in borderline and low- to high-grade ovarian tumours and is consistent with its known role in tumorigenesis

Combination of adenoviral virotherapy and temozolomide chemotherapy eradicates malignant glioma through autophagic and apoptotic cell death in vivo

Conditionally replicative adenoviruses (CRAds) represent a novel treatment strategy for malignant glioma. Recent studies suggest that the cytopathic effect elicited by these vectors is mediated through autophagy, a form of programmed cell death. Likewise, temozolomide (TMZ), a chemotherapeutic agent used for the treatment of malignant gliomas, also triggers autophagic cell death. In this study, we examined the potential to combine the two treatments in the setting of experimental glioma. In vitro, pretreatment with TMZ followed by CRAd-Surivin-pk7 enhanced cytotoxicity against a panel of glioma cell lines. Western blot analysis showed increased expression of BAX and p53, decreased expression of BCL2 and elevated level of APG5. Treatment with TMZ followed by CRAd-Survivin-pk7 (CRAd-S-pk7) led to a significant over-expression of autophagy markers, acidic vesicular organelles and light-chain 3 (LC3). These results were further evaluated in vivo, in which 90% of the mice with intracranial tumours were long-term survivors (>100 days) after treatment with TMZ and CRAd-S-pk7 (P<0.01). Analysis of tumours ex vivo showed expression of both LC3 and cleaved Caspase-3, proving that both autophagy and apoptosis are responsible for cell death in vivo. These results suggest that combination of chemovirotherapy offers a powerful tool against malignant glioma and should be further explored in the clinical setting

Cytotoxicity and global transcriptional responses induced by zinc oxide nanoparticles NM 110 in PMA-differentiated THP-1 cells

Despite a wide production and use of zinc oxide nanoparticles (ZnONP), their toxicological study is only of limited number and their impact at a molecular level is seldom addressed. Thus, we have used, as a model, zinc oxide nanoparticle NM110 (ZnO110NP) exposure to PMA-differentiated THP-1 macrophages. The cell viability was studied at the cellular level using WST-1, LDH and Alamar Blue® assays, as well as at the molecular level by transcriptomic analysis. Exposure of cells to ZnO110NP for 24 h decreased their viability in a dose-dependent manner with mean inhibitory concentrations (IC50) of 8.1 μg/mL. Transcriptomic study of cells exposed to two concentrations of ZnO110NP: IC50 and a quarter of it (IC50/4) for 4 h showed that the expressions of genes involved in metal metabolism are perturbed. In addition, expression of genes acting in transcription regulation and DNA binding, as well as clusters of genes related to protein synthesis and structure were altered. It has to be noted that the expressions of metallothioneins genes (MT1, MT2) and genes of heat-shock proteins genes (HSP) were strongly upregulated for both conditions. These genes might be used as an early marker of exposure to ZnONP. On the contrary, at IC50 exposure, modifications of gene expression involved in inflammation, apoptosis and mitochondrial suffering were noted indicating a less specific cellular response. Overall, this study brings a resource of transcriptional data for ZnONP toxicity for further mechanistic studies

CD36-dependent Regulation of Muscle FoxO1 and PDK4 in the PPAR{delta}/{beta}-mediated Adaptation to Metabolic Stress

The transcription factor FoxO1 contributes to the metabolic adaptation to fasting by suppressing muscle oxidation of glucose, sparing it for glucose-dependent tissues. Previously, we reported that FoxO1 activation in C(2)C(12) muscle cells recruits the fatty acid translocase CD36 to the plasma membrane and increases fatty acid uptake and oxidation. This, together with FoxO1 induction of lipoprotein lipase, would promote the reliance on fatty acid utilization characteristic of the fasted muscle. Here, we show that CD36-mediated fatty acid uptake, in turn, up-regulates protein levels and activity of FoxO1 as well as its target PDK4, the negative regulator of glucose oxidation. Increased fatty acid flux or enforced CD36 expression in C(2)C(12) cells is sufficient to induce FoxO1 and PDK4, whereas CD36 knockdown has opposite effects. In vivo, CD36 loss blunts fasting induction of FoxO1 and PDK4 and the associated suppression of glucose oxidation. Importantly, CD36-dependent regulation of FoxO1 is mediated by the nuclear receptor PPARdelta/beta. Loss of PPARdelta/beta phenocopies CD36 deficiency in blunting fasting induction of muscle FoxO1 and PDK4 in vivo. Expression of PPARdelta/beta in C(2)C(12) cells, like that of CD36, robustly induces FoxO1 and suppresses glucose oxidation, whereas co-expression of a dominant negative PPARdelta/beta compromises FoxO1 induction. Finally, several PPRE sites were identified in the FoxO1 promoter, which was responsive to PPARdelta/beta. Agonists of PPARdelta/beta were sufficient to confer responsiveness and transactivate the heterologous FoxO1 promoter but not in the presence of dominant negative PPARdelta/beta. Taken together, our findings suggest that CD36-dependent FA activation of PPARdelta/beta results in the transcriptional regulation of FoxO1 as well as PDK4, recently shown to be a direct PPARdelta/beta target. FoxO1 in turn can regulate CD36, lipoprotein lipase, and PDK4, reinforcing the action of PPARdelta/beta to increase muscle reliance on FA. The findings could have implications in the chronic abnormalities of fatty acid metabolism associated with obesity and diabetes