The Mdm2 and Mdm4 oncoproteins are key negative regulators of the p53 tumor suppressor. However, their physiological contributions to the regulation of p53 stability and activity remain highly controversial. Here, we combined a p53 knock-in allele, in which p53 is silenced by a transcriptional stop element flanked by loxP sites, with the mdm2-and mdm4-null alleles. This approach allows Cre-mediated conditional p53 expression in tissues in vivo and cells in vitro lacking Mdm2, Mdm4, or both. Using this strategy, we show that Mdm2 and Mdm4 are essential in a nonredundant manner for preventing p53 activity in the same cell type, irrespective of the proliferation͞differentiation status of the cells. Although Mdm2 prevents accumulation of the p53 protein, Mdm4 contributes to the overall inhibition of p53 activity independent of Mdm2. We propose a model in which Mdm2 is critical for the regulation of p53 levels and Mdm4 is critical for the fine-tuning of p53 transcriptional activity, both proteins acting synergistically to keep p53 in check.cre͞lox

The p53 tumour suppressor functions as a transcriptional activator, and several p53-inducible genes that play a critical proapoptotic role have been described. Moreover, p53 regulates the expression of various proteins participating in autoregulatory feedback loops, including proteins that negatively control p53 stability (Mdm2 and Pirh2) or modulate stress-induced phosphorylation of p53 on Ser-46 (p53DINP1 or Wip1), a key event for p53-induced apoptosis. Here, we describe a new systematic analysis of p53 targets using oligonucleotide chips, and report the identification of dapk1 as a novel p53 target. We demonstrate that dapk1 mRNA levels increase in a p53-dependent manner in various cellular settings. Both human and mouse dapk1 genomic loci contain DNA sequences that bind p53 in vitro and in vivo. Since dapk1 encodes a serine/threonine kinase previously shown to suppress oncogene-induced transformation by activating a p19 ARF /p53-dependent apoptotic checkpoint, our results suggest that Dapk1 participates in a new positive feedback loop controlling p53 activation and apoptosis.

Small nucleolar RNAs (snoRNAs) are emerging as a novel class of proto-oncogenes and tumor suppressors; their involvement in tumorigenesis remains unclear. The box C/D snoRNAs U3 and U8 are upregulated in breast cancers. Here we characterize the function of human U3 and U8 in ribosome biogenesis, nucleolar structure, and tumorigenesis. We show in breast (MCF-7) and lung (H1944) cancer cells that U3 and U8 are required for pre-rRNA processing reactions leading, respectively, to synthesis of the small and large ribosomal subunits. U3 or U8 depletion triggers a remarkably potent p53-dependent anti-tumor stress response involving the ribosomal proteins uL5 (RPL11) and uL18 (RPL5). Interestingly, the nucleolar structure is more sensitive to perturbations in lung cancer than in breast cancer cells. We reveal in a mouse xenograft model that the tumorigenic potential of cancer cells is reduced in the case of U3 suppression and totally abolished upon U8 depletion. Tumors derived from U3-knockdown cells displayed markedly lower metabolic volume and activity than tumors derived from aggressive control cancer cells. Unexpectedly, metabolic tracer uptake by U3-suppressed tumors appeared more heterogeneous, indicating distinctive tumor growth properties that may reflect non-conventional regulatory functions of U3 (or fragments derived from it) in mRNA metabolism.

Myoferlin is a multiple C2-domain-containing protein that regulates membrane repair, tyrosine kinase receptor function and endocytosis in myoblasts and endothelial cells. Recently it has been reported as overexpressed in several cancers and shown to contribute to proliferation, migration and invasion of cancer cells. We have previously demonstrated that myoferlin regulates epidermal growth factor receptor activity in breast cancer. In the current study, we report a consistent overexpression of myoferlin in triple-negative breast cancer cells (TNBC) over cells originating from other breast cancer subtypes. Using a combination of proteomics, metabolomics and electron microscopy, we demonstrate that myoferlin depletion results in marked alteration of endosomal system and metabolism. Mechanistically, myoferlin depletion caused impaired vesicle traffic that led to a misbalance of saturated/unsaturated fatty acids. This provoked mitochondrial dysfunction in TNBC cells. As a consequence of the major metabolic stress, TNBC cells rapidly triggered AMP activated protein kinase-mediated metabolic reprogramming to glycolysis. This reduced their ability to balance between oxidative phosphorylation and glycolysis, rendering TNBC cells metabolically inflexible, and more sensitive to metabolic drug targeting in vitro. In line with this, our in vivo findings demonstrated a significantly reduced capacity of myoferlin-deficient TNBC cells to metastasise to lungs. The significance of this observation was further supported by clinical data, showing that TNBC patients whose tumors overexpress myoferlin have worst distant metastasis-free and overall survivals. This novel insight into myoferlin function establishes an important link between vesicle traffic, cancer metabolism and progression, offering new diagnostic and therapeutic concepts to develop treatments for TNBC patients.

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Summary Concerted alteration of immune and metabolic homeostasis underlies several inflammation-related pathologies, ranging from metabolic syndrome to infectious diseases. Here, we explored the coordination of nucleic acid-dependent inflammatory responses and metabolic homeostasis. We reveal that the STING (stimulator of interferon genes) protein regulates metabolic homeostasis through inhibition of the fatty acid desaturase 2 (FADS2) rate-limiting enzyme in polyunsaturated fatty acid (PUFA) desaturation. STING ablation and agonist-mediated degradation increased FADS2-associated desaturase activity and led to accumulation of PUFA derivatives that drive thermogenesis. STING agonists directly activated FADS2-dependent desaturation, promoting metabolic alterations. PUFAs in turn inhibited STING, thereby regulating antiviral responses and contributing to resolving STING-associated inflammation. Thus, we have unveiled a negative regulatory feedback loop between STING and FADS2 that fine-tunes inflammatory responses. Our results highlight the role of metabolic alterations in human pathologies associated with aberrant STING activation and STING-targeting therapies.

Cancer research is increasingly dependent of patient-derived xenograft model (PDX). However, a major point of concern regarding the PDX model remains the replacement of the human stroma with murine counterpart. In the present work we aimed at clarifying the significance of the human-to-murine stromal replacement for the fidelity of colorectal cancer (CRC) and liver metastasis (CRC-LM) PDX model. We have conducted a comparative metabolic analysis between 6 patient tumors and corresponding PDX across 4 generations. Metabolic signatures of cancer cells and stroma were measured separately by MALDI-imaging, while metabolite changes in entire tumors were quantified using mass spectrometry approach. Measurement of glucose metabolism was also conducted in vivo using [F]-fluorodeoxyglucose (FDG) and positron emission tomography (PET). In CRC/CRC-LM PDX model, human stroma was entirely replaced at the second generation. Despite this change, MALDI-imaging demonstrated that the metabolic profiles of both stromal and cancer cells remained stable for at least four generations in comparison to the original patient material. On the tumor level, profiles of 86 water-soluble metabolites as well as 93 lipid mediators underlined the functional stability of the PDX model. In vivo PET measurement of glucose uptake (reflecting tumor glucose metabolism) supported the ex vivo observations. Our data show for the first time that CRC/CRC-LM PDX model maintains the functional stability at the metabolic level despite the early replacement of the human stroma by murine cells. The findings demonstrate that human cancer cells actively educate murine stromal cells during PDX development to adopt the human-like phenotype.

Idiopathic pulmonary fibrosis is characterized by a progressive and irreversible respiratory failure. Validated noninvasive methods able to assess disease activity are essential for prognostic purposes as well as for the evaluation of emerging antifibrotic treatments. Methods: C57BL/6 mice were used in a murine model of pulmonary fibrosis induced by an intratracheal instillation of bleomycin (control mice were instilled with a saline solution). At different times after instillation, PET/CT with 18 F-FDG-or 18 F-4-fluorobenzamido-Nethylamino-maleimide ( 18 F-FBEM)-labeled leukocytes was performed to assess metabolic activity and leukocyte recruitment, respectively. Results: In bleomycin-treated mice, a higher metabolic activity was measured on 18 F-FDG PET/CT scans from day 7 to day 24 after instillation, with a peak of activity measured at day 14. Of note, lung mean standardized uptake values correlated with bleomycin doses, histologic score of fibrosis, lung hydroxyproline content, and weight loss. Moreover, during the inflammatory phase of the model (day 7), but not the fibrotic phase (day 23), bleomycin-treated mice presented with an enhanced leukocyte recruitment as assessed by 18 F-FBEM-labeled leukocyte PET/CT. Autoradiographic analysis of lung sections and CD45 immunostaining confirm the higher and early recruitment of leukocytes in bleomycin-treated mice, compared with control mice. Conclusion: 18 F-FDG-and 18 F-FBEM-labeled leukocyte PET/CT enable monitoring of metabolic activity and leukocyte recruitment in a mouse model of pulmonary fibrosis. Implications for preclinical evaluation of antifibrotic therapy are expected.