Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKa. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors.

The Chk2-p53-PUMA pathway is a major regulator of DNA-damage-induced apoptosis in response to double-strand breaks in vivo. Through analysis of 53BP1 complexes we have discovered a new ubiquitin protease, USP28, which regulates this pathway. Using a human cell line that faithfully recapitulated the Chk2-p53-PUMA pathway, we show that USP28 is required to stabilize Chk2 and 53BP1 in response to DNA damage. In this cell line, both USP28 and Chk2 are required for DNA-damage-induced apoptosis, and they accomplish this in part through regulation of the p53 induction of proapoptotic genes like PUMA. Our studies implicate DNA-damage-induced ubiquitination and deubiquitination as a major regulator of the DNA-damage response for Chk2, 53BP1, and a number of other proteins in the DNA-damage checkpoint pathway, including several mediators, such as Mdc1, Claspin, and TopBP1.

FKHRL1 (FOXO3a) and p53 are two potent stress-response regulators. Here we show that these two transcription factors exhibit ''crosstalk'' in vivo. In response to DNA damage, p53 activation led to FKHRL1 phosphorylation and subcellular localization change, which resulted in inhibition of FKHRL1 transcription activity. AKT was dispensable for p53-dependent suppression of FKHRL1. By contrast, serum-and glucocorticoid-inducible kinase 1 (SGK1) was significantly induced in a p53-dependent manner after DNA damage, and this induction was through extracellular signal-regulated kinase 1͞2-mediated posttranslational regulation. Furthermore, inhibition of SGK1 expression by a small interfering RNA knockdown experiment significantly decreased FKHRL1 phosphorylation in response to DNA damage. Taken together, our observations reveal previously unrecognized crosstalk between p53 and FKHRL1. Moreover, our findings suggest a new pathway for understanding aging and the age dependency of human diseases governed by these two transcription factors.H uman longevity depends on genome stability. Several mouse models have revealed that an age-related decrease of DNA repair or an increase in DNA damage plays a role in mammalian aging (1-3). The idea that cellular responses to stress may be important in aging is supported by studies of p53, the functions of which are critical for both the apoptotic and senescence responses to DNA damage, telomeric shortening, and oxidative stress (4-6).The mammalian FOXO family of forkhead transcription factors (including FKHR, FKHRL1, and AFX) have been proposed as antiaging factors based on evidence from their orthologues, DAF-16 in Caenorhabditis elegans and dFOXO in Drosophila melanogaster, which regulate longevity in response to reduced insulin͞insulin-like growth factor I (IGF-I) signaling or by overexpressing constitutively active FOXO (7,8). Growth factor signaling to FOXO family members through phosphatidylinositol 3-kinase (PI3K) and its downstream kinase, Akt, has been found to be evolutionarily conserved for FOXO phosphorylation, subcellular translocation, and inhibition of its transcriptional activity (9). However, the role of FOXO in response to DNA damage, as well as the signaling pathway upstream of FOXO, is, as yet, unclear. Overexpression of FKHRL1 can protect cells from oxidative stress-induced cell death, as described in refs. 10-12. Induction of a number of antioxidant enzymes and stress-related gene products has been proposed as a potential mechanism (9, 13-15). The fact that the precise biological consequences of FOXO activation are cell-typespecific and stress-type-dependent suggests that there might be crosstalk between FOXO and other stress regulators. How FOXO communicates and coordinates with other signaling pathways in response to genotoxic stress remains unknown.The opposing functions of p53 and FKHRL1 with regard to the aging process suggest that a regulatory mechanism might exist to integrate these two signaling pathways. We sought to elucidate the mechanism underlying thi...

Despite the prominent pro-apoptotic role of p53, this protein has also been shown to promote cell survival in response to metabolic stress. However, the specific mechanism by which p53 protects cells from metabolic stress-induced death is unknown. Earlier we reported that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific member of a family of mitochondria-associated enzymes that have a central role in fatty acid metabolism promotes cell survival and tumor growth. Unlike other members of the CPT family, the subcellular localization of CPT1C and its cellular function remains elusive. Here, we report that CPT1C is a novel p53-target gene with a bona fide p53-responsive element within the first intron. CPT1C is upregulated in vitro and in vivo in a p53-dependent manner. Interestingly, expression of CPT1C is induced by metabolic stress factors such as hypoxia and glucose deprivation in a p53 and AMP activated kinase-dependent manner. Furthermore, in a murine tumor model, depletion of Cpt1c leads to delayed tumor development and a striking increase in survival. Taken together, our results indicate that p53 protects cells from metabolic stress via induction of CPT1C and that CPT1C may have a crucial role in carcinogenesis. CPT1C may therefore represent an exciting new therapeutic target for the treatment of hypoxic and otherwise treatment-resistant tumors. Hypoxia is an important chronic stress on tumor cell growth and has been shown to correlate with poor disease-free and reduced overall survival in a variety of carcinomas and sarcomas. 1 To enhance survival in an altered environment such as hypoxia cancer cells undergo a so-called metabolic transformation. [2][3][4] The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells upregulate glycolysis to limit their energy consumption. However, there is increasing evidence that not only glucose metabolism, but also fatty acid oxidation (FAO) is involved in metabolic transformation. Although glucose seems to be the major energy source for tumor growth and survival, there is increasing evidence that alternative energy sources such as fatty acid metabolism are altered in cancer cells, even under hypoxic conditions. Indeed, fatty acid synthase has been found to be upregulated in many human cancers, 5 and inhibitors of the fatty acid synthase show antitumor activity. 6 As recently published, we identified carnitine palmitoyltransferase (CPT) 1C (CPT1C) as a potential novel p53-target gene. 7 By their restriction of fatty acid import into mitochondria, 4 the CPT 1 (CPT1) family of enzymes represent key regulatory factors of FAO. There are three tissue-specific isoforms of CPT1: CPT1A that is found in liver, CPT1B in muscle and CPT1C in brain and testes. Loss-of-function of CPT1C was generated in mouse embryonic stem cells (Cpt1c gt/gt ES cells). Importantly, Cpt1c gt/gt ES cells readily succumbed to cell death under hypoxic conditions, whereas control cells were resistant. ES cells deficient for CPT1C showed a spontaneous induction in...