Steven P. Donald

The metabolism of proline, a stress substrate, modulates carcinogenic pathways.

The resurgence of interest in tumor metabolism has led investigators to emphasize the metabolism of proline as a “stress substrate” and to suggest this pathway as a potential anti-tumor target. Proline oxidase, a.k.a. proline dehydrogenase (POX/PRODH), catalyzes the first step in proline degradation and uses proline to generate ATP for survival or reactive oxygen species for programmed cell death. POX/PRODH is induced by p53 under genotoxic stress and initiates apoptosis by both mitochondrial and death receptor pathways. Furthermore, POX/PRODH is induced by PPARγ and its pharmacologic ligands, the thiazolidinediones. The anti-tumor effects of PPARγ may be critically dependent on POX/PRODH. In addition, it is upregulated by nutrient stress through the mTOR pathway to maintain ATP levels. We propose that proline is made available as a stress substrate by the degradation of collagen in the microenvironmental extracellular matrix by matrix metalloproteinases. In a manner analogous to autophagy, this proline-dependent process for bioenergetics from collagen in extracellular matrix can be designated “ecophagy”.

Proline oxidase functions as a mitochondrial tumor suppressor in human cancers.

Tumor metabolism and bioenergetics have become important topics for cancer research and are promising targets for anticancer therapy. Although glucose serves as the main source of energy, proline, an alternative substrate, is important, especially during nutrient stress. Proline oxidase (POX), catalyzing the first step in proline catabolism, is induced by p53 and can regulate cell survival as well as mediate programmed cell death. In a mouse xenograft tumor model, we found that POX greatly reduced tumor formation by causing G2 cell cycle arrest. Furthermore, immunohistochemical staining showed decreased POX expression in tumor tissues. Importantly, HIF-1alpha signaling was impaired with POX expression due to the increased production of alpha-ketoglutarate, a critical substrate for prolyl hydroxylation and degradation of HIF-1alpha. Combined with previous in vitro findings and reported clinical genetic associations, these new findings lead us to propose POX as a mitochondrial tumor suppressor and a potential target for cancer therapy.

Extracellular matrix and HIF‐1 signaling: The role of prolidase

Hypoxia‐inducible factor‐1 (HIF‐1) plays an important role in stress‐responsive gene expression. Although primarily sensitive to hypoxia, HIF‐1 signaling can be regulated by a number of stress factors including metabolic stress, growth factors and molecules present in the extracellular matrix (ECM). Degradation of ECM by metalloproteinases (MMP) is important for tumor progression, invasion and metastasis. ECM is predominantly collagen, and the imino acids (Pro and HyPro) comprise 25% of collagen residues. The final step in collagen degradation is catalyzed by prolidase, the obligate peptidase for imidodipeptides with Pro and HyPro in the carboxyl terminus. Defective wound healing in patients with inherited prolidase deficiency is associated with histologic features of angiopathy suggesting that prolidase may play a role in angiogenesis. Because HIF‐1α is central to angiogenesis, we considered that prolidase may modulate this pathway. To test this hypothesis, we made expression constructs of human prolidase and obtained stable transfectants in colorectal cancer cells (RKO). Overexpression of prolidase resulted in increased nuclear hypoxia inducible factor (HIF‐1α) levels and elevated expression of HIF‐1−dependent gene products, vascular endothelial growth factor (VEGF) and glucose transporter‐1 (Glut‐1). The activation of HIF‐1‐dependent transcription was shown by prolidase‐dependent activation of hypoxia response element (HRE)‐luciferase expression. We used an oxygen‐dependent degradation domain (ODD)‐luciferase reporter construct as a surrogate for HIF‐1α as an in situ prolyl‐hydroxylase assay. Since this reporter is degraded by VHL‐dependent mechanisms, the increased levels of luciferase observed with prolidase expression reflected the decreased HIF‐1α prolyl hydroxylase activity. Additionally, the differential expression of prolidase in 2 breast cancer cell lines showed prolidase‐dependent differences in HIF‐1α levels. These findings show that metabolism of imidodipeptides by prolidase plays a previously unrecognized role in angiogenic signaling.

Regulation and function of proline oxidase under nutrient stress

Under conditions of nutrient stress, cells switch to a survival mode catabolizing cellular and tissue constituents for energy. Proline metabolism is especially important in nutrient stress because proline is readily available from the breakdown of extracellular matrix (ECM), and the degradation of proline through the proline cycle initiated by proline oxidase (POX), a mitochondrial inner membrane enzyme, can generate ATP. This degradative pathway generates glutamate and α‐ketoglutarate, products that can play an anaplerotic role for the TCA cycle. In addition the proline cycle is in a metabolic interlock with the pentose phosphate pathway providing another bioenergetic mechanism. Herein we have investigated the role of proline metabolism in conditions of nutrient stress in the RKO colorectal cancer cell line. The induction of stress either by glucose withdrawal or by treatment with rapamycin, stimulated degradation of proline and increased POX catalytic activity. Under these conditions POX was responsible, at least in part, for maintenance of ATP levels. Activation of AMP‐activated protein kinase (AMPK), the cellular energy sensor, by 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR), also markedly upregulated POX and increased POX‐dependent ATP levels, further supporting its role during stress. Glucose deprivation increased intracellular proline levels, and expression of POX activated the pentose phosphate pathway. Together, these results suggest that the induction of proline cycle under conditions of nutrient stress may be a mechanism by which cells switch to a catabolic mode for maintaining cellular energy levels. J. Cell. Biochem. 107: 759–768, 2009.

Expression of prostaglandin endoperoxide H synthase-2 induced by nitric oxide in conditionally immortalized murine colonic epithelial cells

Increased expression of prostaglandin endoperoxide H synthase‐2 (PGHS‐2) has been implicated in pathological conditions such as inflammatory bowel diseases and colon cancer. Recently, it has been demonstrated that inducible nitric oxide syn‐thase (NOS II) expression and nitric oxide (NO) production are up‐regulated in these diseases as well. However, the apparent link between PGHS‐2 and NOS II has not been thoroughly investigated in nontransformed and nontumorigenic colonic epithelial cells. In the present study, we examined the concomitant expression of PGHS‐2 and NOS II as well as the production of prostaglandin E2 (PGE2) and NO in conditionallyimmortalized mouse colonic epithelial cells, namely YAMC (Apc+/+). We found that the induction of PGHS‐2 and generation of PGE2 in these cells by IFN‐γ and lipopolysaccharide (LPS) were greatly reduced by two selective NOS II inhibitors, L‐NIL and SMT. To ascertain the effect of NO on PGHS‐2 overexpression, we tested NO‐releasing compounds, NOR‐1 and SNAP, and found that they caused PGHS‐2 expression and PGE2 production. This effect was abolished by hemoglobin, a NO scavenger. Using electrophoretic mobility shift assays, we found that both NOR‐1 and SNAP caused β‐catenin/LEF‐1 DNA complex formation. Super‐shift by anti‐β‐catenin antibody confirmed the presence of β‐catenin in the complex. Cell fractionation studies indicated that NO donors caused an increase in free soluble cytoplasmic β‐catenin. This is further corroborated by the immunocytochemistry data showing the redistribution of β‐catenin from the predominantly membrane localization into the cytoplasm and nucleus after treatment with NO donors. To further explore the possible connection between PGHS‐2 expression and β‐catenin/LEF‐1 DNA complex formation, we studied IMCE (ApcMin/+) cells, a sister cell line of YAMC with similar genetic background but differing in Apc genotype and, consequently, their β‐catenin levels. We found that IMCE cells, in comparison with YAMC cells, had markedly higher β‐catenin/LEF‐1 DNA complex formation under both resting conditions as well as after induction with NO. In parallel fashion, IMCE cells expressed significantly higher levels of PGHS‐2 mRNA and protein, and generated more PGE2. Overall, this study suggests that NO may be involved in PGHS‐2 overexpression in conditionally immortalized mouse colonic epithelial cells. Although the molecular mechanism of the link is still under investigation, this effect of NO appears directly or indirectly to be a result of the increase in free soluble β‐catenin and the formation of nuclear β‐catenin/LEF‐1 DNA complex.—Mei, J. M., Hord, N. G., Winterstein, D. F., Donald, S. P., and Phang, J. M. Expression of prostaglandin endoperoxide H synthase‐2 induced by nitric oxide in conditionally immortalized murine colonic epithelial cells. FASEB J. 14, 1188–1201 (2000)

A novel function for hydroxyproline oxidase in apoptosis through generation of reactive oxygen species.

Proline and hydroxyproline are metabolized by distinct pathways. Proline is important for protein synthesis, as a source of glutamate, arginine, and tricarboxylic acid cycle intermediates, and for participating in a metabolic cycle that shuttles redox equivalents between mitochondria and cytosol. Hydroxyproline, in contrast, is not reutilized for protein synthesis. The first steps in the degradation of proline and hydroxyproline are catalyzed by proline oxidase (POX) and hydroxyproline oxidase (OH-POX), respectively. Because it is well documented that POX is induced by p53 and plays a role in apoptosis, we considered whether OH-POX also participates in the response to cytotoxic stress. In LoVo and RKO cells, which respond to adriamycin with a p53-mediated induction of POX and generation of reactive oxygen species, we found that adriamycin also induced OH-POX gene expression and markedly increased OH-POX catalytic activity, and this increase in activity was not observed in the cell lines HT29 and HCT15, which do not have a functional p53. We also observed an increase in reactive oxygen species generation and activation of caspase-9 with adriamycin in a hydroxyproline-dependent manner. Therefore, we hypothesize that OH-POX plays a role analogous to POX in growth regulation, ROS generation, and activation of the apoptotic cascade.