Carnitine o-octanoyltransferase is a p53 target that promotes oxidative metabolism and cell survival following nutrient starvation.

Whereas it is known that p53 broadly regulates cell metabolism, the specific activities that mediate this regulation remain partially understood. Here, we identified carnitine o-octanoyltransferase (CROT) as a p53 transactivation target that is upregulated by cellular stresses in a p53-dependent manner. CROT is a peroxisomal enzyme catalyzing very long-chain fatty acids conversion to medium chain fatty acids that can be absorbed by mitochondria during ß-oxidation. p53 induces CROT transcription through binding to consensus response elements in the 5'-UTR of CROT mRNA. Overexpression of WT but not enzymatically inactive mutant CROT promotes mitochondrial oxidative respiration, while downregulation of CROT inhibits mitochondrial oxidative respiration. Nutrient depletion induces p53-dependent CROT expression that facilitates cell growth and survival; in contrast, cells deficient in CROT have blunted cell growth and reduced survival during nutrient depletion. Together, these data are consistent with a model where p53-regulated CROT expression allows cells to be more efficiently utilizing stored very long-chain fatty acids to survive nutrient depletion stresses.

A role of cytoplasmic p53 in the regulation of metabolism shown by bat-mimicking p53 NLS mutant mice.

The transcription factor p53 suppresses tumorigenesis via a wide-ranging, concerted set of functions. Although several studies have identified cytoplasmic, transcription-independent functions of p53, the biological relevance of these activities has not been fully elucidated, particularly in vivo. Here, we generated a mouse model with a p53K316P mutation, which mimics a naturally occurring p53 nuclear localization signal (NLS) change observed in bat species. We find that the p53K316P mutation increases cytoplasmic localization of p53 and promotes a pleiotropic metabolic phenotype that includes increased adiposity, increased de novo lipogenesis, and decreased lactate generation. Mechanistic studies show that, independent of its transactivation function, p53K316P interacts with lactate dehydrogenase B (LDHB) and alters the composition and enzymatic activities of LDH complex favoring pyruvate generation and hindering lactate production. Overall, the study identifies a role for cytoplasmic p53 in the regulation of metabolism that favors energy generation and storage.

Genomics analysis of Drosophila sechellia response to Morinda citrifolia fruit diet.

Drosophila sechellia is an island endemic host specialist that has evolved to consume the toxic fruit of Morinda citrifolia, also known as noni fruit. Recent studies by our group and others have examined genome-wide gene expression responses of fruit flies to individual highly abundant compounds found in noni responsible for the fruit's unique chemistry and toxicity. In order to relate these reductionist experiments to the gene expression responses to feeding on noni fruit itself, we fed rotten noni fruit to adult female D. sechellia and performed RNA-sequencing. Combining the reductionist and more wholistic approaches, we have identified candidate genes that may contribute to each individual compound and those that play a more general role in response to the fruit as a whole. Using the compound specific and general responses, we used transcription factor prediction analyses to identify the regulatory networks and specific regulators involved in the responses to each compound and the fruit itself. The identified genes and regulators represent the possible genetic mechanisms and biochemical pathways that contribute to toxin resistance and noni specialization in D. sechellia.

MDMX is essential for the regulation of p53 protein levels in the absence of a functional MDM2 C-terminal tail.

BACKGROUND: MDM2 is an E3 ubiquitin ligase that is able to ubiquitinate p53, targeting it for proteasomal degradation. Its homologue MDMX does not have innate E3 activity, but is able to dimerize with MDM2. Although mouse models have demonstrated both MDM2 and MDMX are individually essential for p53 regulation, the significance of MDM2-MDMX heterodimerization is only partially understood and sometimes controversial. MDM2C462A mice, where the C462A mutation abolishes MDM2 E3 ligase activity as well as its ability to dimerize with MDMX, die during embryogenesis. In contrast, the MDM2Y487A mice, where the Y487A mutation at MDM2 C-terminus significantly reduces its E3 ligase activity without disrupting MDM2-MDMX binding, survive normally even though p53 is expressed to high levels. This indicates that the MDM2-MDMX heterodimerization plays a critical role in the regulation of p53. However, it remains unclear whether MDMX is essential for the regulation of p53 protein levels in the context of an endogenous MDM2 C-terminal tail mutation. RESULTS: Here, we studied the significance of MDM2-MDMX binding in an MDM2 E3 ligase deficient context using the MDM2Y487A mouse embryonic fibroblast (MEF) cells. Surprisingly, down-regulation of MDMX in MDM2Y487A MEFs resulted in a significant increase of p53 protein levels. Conversely, ectopic overexpression of MDMX reduced p53 protein levels in MDM2Y487A MEFs. Mutations of the RING domain of MDMX prevented MDMX-MDM2 binding, and ablated MDMX-mediated suppression of p53 protein expression. Additionally, DNA damage treatment and nuclear sequestration of MDMX inhibited MDMX activity to suppress p53 protein expression. CONCLUSIONS: These results suggest that MDMX plays a key role in suppressing p53 protein expression in the absence of normal MDM2 E3 ligase activity. We found that the ability of MDMX to suppress p53 levels requires MDM2 binding and its cytoplasmic localization, and this ability is abrogated by DNA damage. Hence, MDMX is essential for the regulation of p53 protein levels in the context of an MDM2 C-terminal mutation that disrupts its E3 ligase activity but not MDMX binding. Our study is the first to examine the role of MDMX in the regulation of p53 in the context of endogenous MDM2 C-terminal mutant MEF cells.

Metastasis-Specific Gene Expression in Autochthonous and Allograft Mouse Mammary Tumor Models: Stratification and Identification of Targetable Signatures.

Breast cancer metastasis is a leading cause of cancer-related death of women in the United States. A hurdle in advancing metastasis-targeted intervention is the phenotypic heterogeneity between primary and secondary lesions. To identify metastasis-specific gene expression profiles we performed RNA-sequencing of breast cancer mouse models; analyzing metastases from models of various drivers and routes. We contrasted the models and identified common, targetable signatures. Allograft models exhibited more mesenchymal-like gene expression than genetically engineered mouse models (GEMM), and primary culturing of GEMM-derived metastatic tissue induced mesenchymal-like gene expression. In addition, metastasis-specific transcriptomes differed between tail vein and orthotopic injection of the same cell line. Gene expression common to models of spontaneous metastasis included sildenafil response and nicotine degradation pathways. Strikingly, in vivo sildenafil treatment significantly reduced metastasis by 54%, while nicotine significantly increased metastasis by 46%. These data suggest that (i) actionable metastasis-specific pathways can be readily identified, (ii) already available drugs may have great potential to alleviate metastatic incidence, and (iii) metastasis may be influenced greatly by lifestyle choices such as the choice to consume nicotine products. In summary, while mouse models of breast cancer metastasis vary in ways that must not be ignored, there are shared features that can be identified and potentially targeted therapeutically. IMPLICATIONS: The data we present here exposes critical variances between preclinical models of metastatic breast cancer and identifies targetable pathways integral to metastatic spread. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/9/1278/F1.large.jpg.

The genomic landscape of metastasis in treatment-naïve breast cancer models.

Metastasis remains the principle cause of mortality for breast cancer and presents a critical challenge because secondary lesions are often refractory to conventional treatments. While specific genetic alterations are tightly linked to primary tumor development and progression, the role of genetic alteration in the metastatic process is not well-understood. The theory of tumor evolution postulated by Peter Nowell in 1976 has yet to be proven in the context of metastasis. Therefore, in order to investigate how somatic evolution contributes to breast cancer metastasis, we performed exome, whole genome, and RNA sequencing of matched metastatic and primary tumors from pre-clinical mouse models of breast cancer. Here we show that in a treatment-naïve setting, recurrent single nucleotide variants and copy number variation, but not gene fusion events, play key metastasis-driving roles in breast cancer. For instance, we identified recurrent mutations in Kras, a known driver of colorectal and lung tumorigenesis that has not been previously implicated in breast cancer metastasis. However, in a set of in vivo proof-of-concept experiments we show that the Kras G12D mutation is sufficient to significantly promote metastasis using three syngeneic allograft models. The work herein confirms the existence of metastasis-driving mutations and presents a novel framework to identify actionable metastasis-targeted therapies.

Ferristatin II promotes degradation of transferrin receptor-1 in vitro and in vivo.

Previous studies have shown that the small molecule iron transport inhibitor ferristatin (NSC30611) acts by down-regulating transferrin receptor-1 (TfR1) via receptor degradation. In this investigation, we show that another small molecule, ferristatin II (NSC8679), acts in a similar manner to degrade the receptor through a nystatin-sensitive lipid raft pathway. Structural domains of the receptor necessary for interactions with the clathrin pathway do not appear to be necessary for ferristatin II induced degradation of TfR1. While TfR1 constitutively traffics through clathrin-mediated endocytosis, with or without ligand, the presence of Tf blocked ferristatin II induced degradation of TfR1. This effect of Tf was lost in a ligand binding receptor mutant G647A TfR1, suggesting that Tf binding to its receptor interferes with the drug's activity. Rats treated with ferristatin II have lower TfR1 in liver. These effects are associated with reduced intestinal (59)Fe uptake, lower serum iron and transferrin saturation, but no change in liver non-heme iron stores. The observed hypoferremia promoted by degradation of TfR1 by ferristatin II appears to be due to induced hepcidin gene expression.