Proline oxidase functions as a mitochondrial tumor suppressor in human cancers - PubMed (original) (raw)

Proline oxidase functions as a mitochondrial tumor suppressor in human cancers

Yongmin Liu et al. Cancer Res. 2009.

Abstract

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.

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Figures

Fig. 1

in vivo tumor growth inhibition. DLD-1 Tet-off POX cells and vector control cells were injected into Balb/C nude mice. (A). Numbers of mice which developed tumors after injection of cells when mice were given water with or without DOX. The left panel shows mice injected with POX cells; the right panel shows mice injected with vector cells. For those mice injected with POX cells in the (−) DOX group that didn’t develop tumors, half (8 mice) was switched to receive water with DOX. The numbers of mice developed tumors were recorded. H&E staining and immunohistochemical staining for POX are shown to demonstrate the induced expression and location of POX. (B) TUNEL assay showed relatively high apoptosis induction in POX-expressing cells. The right panel shows quantitation of at least 3 slide preparations. Mean ± SD are shown. (C). Immunohistochemical staining shows the reduced BrdU incorporation by POX. Right panel shows quantification from at least 3 slides. Mean ± SD are shown. (D). Soft agar assay showed reduced clonogenic ability of DLD-1 cells by POX. Right panel is a quantitation of clones per well and represent data ± of at least 4 determinations. The asterisks indicated statistically significant difference (* p < 0.05, ** p < 0.01). Magnification for C-E was×40 or×100.

Fig. 2

POX caused G2 cell cycle arrest. (A). POX was dose-dependently repressed by increasing DOX concentrations in DLD-1 Tet-Off POX cells. Cell growth assay showed increasing DOX concentrations released DLD-1 cells from POX-mediated inhibition of growth. (B). Flow cytometry assay showed that increasing POX expression with decreasing DOX concentration induced G2 cell cycle arrest. (C). Western blot showed the induction of Geminin, by increasing POX expression with decreasing DOX. (D). The effects of POX expression with decreasing DOX concentrations on several important regulators of G2 phase determined by Western blots.

Fig. 3

The involvement of GADD family proteins. The DLD-1 Tet-off POX cells were cultured in medium with 20 ng/ml [(+) DOX], or 100 and 1000 dilution (0.2 ng/ml, 0.02 ng/ml) for 1 or 3 days. The cells were harvested and RNA and cell lysates were prepared and then semi-quantitative RT-PCR (A & B) and western blots (C) for GADD members were performed. Actin was used as control.

Fig. 4

The reduced expression of POX in human tumor tissues. 92 pairs of human cancer and normal tissues from same patient, including 36 pairs from gastrointestinal system and 6 pairs from kidney were immunohistochemically stained for POX. (A). The representative images were shown from tissues of colon, stomach, liver, pancreas and kidney. Magnification,×100. (B). Z-test was used to do statistical analysis (Null hypothesis of proportion of decrease is equal to 50 %). DT: digestive tract tissues.

Fig. 5

The affect of POX on HIF signaling and TCA cycle intermediates. (A). The inhibition of HIF-1α and its downstream gene VEGF was demonstrated by Western blots in both normoxic and hypoxic conditions (1 % of O2). The relative average densitometric data of four Western blots to HIF-1α in normoxic condition were also shown (B). To determine the effect of ROS/superoxide on HIF signaling, MnSOD was introduced by an adenovirus vector, which did not reverse the POX effect on the expression of HIF-1α. (C). DLD-POX cells were plated with doxycycline. After 18 h, fresh medium with or without doxycycline was substituted for the plating medium. Levels of α-KG were determined by high-pressure liquid chromatography. Levels of succinate, fumerate, and lactate were determined by GC/MS. Data are expressed as nmol/106 cells and represent the mean ± SD of at least 3 determinations. (D) The effect of DMOG on HIF-1α levels was demonstrated by Western blots. The asterisks indicated statistically significant difference (**p < 0.01. HIF-1α* indicated longer exposure).

Fig. 6

Proposed mechanisms by which POX inhibit HIF signaling. The interconversion between proline and P5C, catalyzed by POX and P5CR, respectively, forms the proline cycle. When POX expression is high, P5C, glutamate and α-KG are sequentially produced, forming an important link between proline and the TCA cycle. As an important substrate of prolyl hydroxylase, α-KG increases the hydroxylation of HIF-1α, which will be degradated through ubiquitinal and proteosomal degradation systems. This is probably the principal mechanism by which POX expression decreases HIF signaling. On the other hand, the TCA cycle is affected, as demonstrated by decreased cellular levels of fumarate, succinate, and lactate. The decreased levels of these TCA cycle intermediates may also contribute to impair HIF signaling. In fact, inhibition of HIF could be another mechanism, along with those previously identified, by which POX exerts its tumor suppressing role.

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Proline oxidase functions as a mitochondrial tumor suppressor in human cancers - PubMed (original) (raw)