We previously showed that thiazolidinediones, commonly-used drugs for type 2 diabetes, were potent activators of the POX promoter through PPARgamma (Pandhare J, et al., J. Biol. Chem., 281:2044, 2006;Phang J. et al., PPAR Res. , 2008;2008: 542694). The coupling of POX to PPARgamma strongly suggests that POX is involved in regulation of bioenergetics and responses to nutrient stress, a finding which led us to consider the mTOR-AMPK signaling pathway. This pathway integrates signals from growth factors, nutrients, energy levels and cellular stress to regulate protein translation and cell growth. Mutations in this pathway have been associated with a number of neoplastic phenotypes. We tested the effects of rapamycin, an inhibitor of mTOR, LY 294002, an inhibitor of PI3-K/Akt, and 5-amino-4-carboxamide ribofuranoside (AICAR), a purine analog which activates AMP-directed protein kinase (AMPK). We found that these agents which block mTOR signaling at 3 different sites, all markedly activated POX activity. Additionally, rapamycin, by blocking mTOR, inhibited protein translation and cell growth and concomitantly increased cellular ATP levels, presumably to sustain a vegetative survival state. Interestingly, blockade of POX expression by POX siRNA or inhibiting POX catalytic activity with dehydroproline markedly inhibited the rapamycin-induced increase in cellular ATP. These studies suggest that proline can function as a stress substrate under the regulation of PPARgamma and the mTOR/AMPK signaling pathways (Pandhare J, et al., J. Cell. Biochem., 107:759, 2009;Phang JM less than I.et al., J. Nutr.138:2008S, 2008). Although we showed that POX expression generated ATP under conditions of nutrient stress, the biochemical source for the ATP required elucidation. Glucose is the main source for ATP in cultured cells;therefore we tested whether glycolysis was increased by POX overexpression. Surprisingly, glycolysis measured by the conversion of (5)-3H-glucose to 3H2O was not changed with POX overexpression. In contrast, the pentose phosphate shunt (PPS) was increased more than 5-fold when POX was induced. Furthermore, with limiting glucose (.05 mM), ATP levels progressively fell. However, when POX was induced, ATP levels were maintained in the presence or absence of added proline. Presumably, the cycling of endogenous proline could mediate the effect. These findings suggest that when glucose is limiting, POX promotes the metabolism of glucose through the PPS and the cycling of proline shuttles the NADPH generated from the shunt into a source of reducing potential for ATP generation (Pandhare J, et al., J. Cell. Biochem., 107:759, 2009). Another seminal finding is that POX-mediated signaling is involved in lipid metabolism and signaling initiated by oxidized low-density lipoprotein (oxLDL). Treatment of colorectal cancer cells with oxLDL markedly induced POX, and this induction was dependent on PPARgamma because 7-ketocholesterol, an oxidized metabolite contained in the oxLDL particle is a potent activator of PPARgamma. OxLDL can stimulate both autophagy and apoptosis. Interestingly, autophagy was partially blocked by knockdown of POX by siRNA, whereas oxLDL-activated apoptosis was not. Thus, POX appears to be involved in the autophagic activation by oxLDL. To directly assess this linkage, we used DLD-tet-off-POX cells, stable transfectants in which POX is induced when doxycycline is removed from the medium. We found that LC3-I is cleaved to LC3-II with POX expression, and LC3-II is incorporated into autophagosomes. Importantly the expression of beclin-1, a critical gene for autophagy, was induced by the expression of POX. Thus, the expression of POX , induced by oxLDL through PPARgamma, is a signaling mechanism for the activation of autophagy. This is of interest because we have proposed that POX initiates ecophagy, the consumption of substrates, e.g. collagen, in the microenvironment. Our studies suggested that ecophagy not only precedes but also may activate autophagy. With the aforementioned evidence that POX provides a source of ATP during nutrient stress, we also addressed the question of metabolic stress due to hypoxia. A variety of cultured cells subjected to hypoxia showed an increase in POX expression either monitored at the level of mRNA by real time PCR or by a luciferase assay for POX promoter acdtivity. POX mRNA and POX protein by Western blot increased as a function of hypoxia (5%, 0.5%, 0.05% oxygen) and duration of hypoxia. Interestingly, the increase in POX is not mediated by HIF-1alpha. Instead, the POX response was mediated by AMPK. Consistent with the dependence on AMPK, we found that ATP levels, which were decreased with hypoxia, decreased further with POX knockdown. Importantly, the decrease in cell proliferation due to hypoxia was accentuated by knockdown of POX with siRNA. Although POX was responsible, in large part, for the increase in ROS with hypoxia, it did not induce apoptosis as measured by PARP cleavage. Instead, Hypoxia induced autophagy and this autophagy was decreased by the knockdown of POX by siRNA. Taken together, these studies suggested that the response to hypoxia through the AMPK phosphorylation cascade involved the induction of POX which activated autophagy to maintain ATP and cell survival.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010744-05
Application #
8157426
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2010
Total Cost
$247,550
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Phang, James M; Liu, Wei; Hancock, Chad (2013) Bridging epigenetics and metabolism: role of non-essential amino acids. Epigenetics 8:231-6
Liu, Wei; Phang, James M (2012) Proline dehydrogenase (oxidase) in cancer. Biofactors 38:398-406
Phang, James Ming; Liu, Wei; Hancock, Chad et al. (2012) The proline regulatory axis and cancer. Front Oncol 2:60
Phang, James M; Liu, Wei (2012) Proline metabolism and cancer. Front Biosci 17:1835-45
Liu, Wei; Glunde, Kristine; Bhujwalla, Zaver M et al. (2012) Proline oxidase promotes tumor cell survival in hypoxic tumor microenvironments. Cancer Res 72:3677-86
Liu, Wei; Le, Anne; Hancock, Chad et al. (2012) Reprogramming of proline and glutamine metabolism contributes to the proliferative and metabolic responses regulated by oncogenic transcription factor c-MYC. Proc Natl Acad Sci U S A 109:8983-8
Liu, Wei; Phang, James M (2012) Proline dehydrogenase (oxidase), a mitochondrial tumor suppressor, and autophagy under the hypoxia microenvironment. Autophagy 8:1407-9
Liu, W; Zabirnyk, O; Wang, H et al. (2010) miR-23b targets proline oxidase, a novel tumor suppressor protein in renal cancer. Oncogene 29:4914-24
Phang, James M; Liu, Wei; Zabirnyk, Olga (2010) Proline metabolism and microenvironmental stress. Annu Rev Nutr 30:441-63
Tokar, Erik J; Qu, Wei; Liu, Jie et al. (2010) Arsenic-specific stem cell selection during malignant transformation. J Natl Cancer Inst 102:638-49

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