We show here that attenuation of p32 expression increases glycolysis while reducing cell proliferation and tumorigenesis and that restored expression of p32 rescues the original phenotype of the cells. We also provide evidence that p32 sustains mitochondrial oxidative phosphorylation by playing a role in the synthesis of mitochondrial-DNA-encoded genes. These results show that the mitochondrial p32 protein is a critical regulator of tumor metabolism and important for tumor maintenance and malignancy. Moreover, that a protein overexpressed in cancer cells supports oxidative phosphorylation provides a novel perspective on the generally held belief that increased glycolysis under aerobic conditions (the Warburg effect) promotes tumor growth.
We provide several lines of evidence to show that p32 sustains OXPHOS and that its absence increases glycolysis, while impairing cell proliferation in vitro and tumor growth in vivo. The apparent molecular mechanism that our results provide for this metabolic effect is that p32 controls the synthesis of the proteins that the mitochondrial genome contributes to the oxidative electron transfer chain. This in itself is a novel finding. The way in which p32 specifically accomplishes this trigger mechanism will be a subject of further investigation; however, it is possible that a chaperone-like function of p32 is involved.
The expression of p32 has been tied to apoptosis and autophagy via the tumor suppressor ARF (30
), and autophagy consumes mitochondria (31
). However, as increasing p32 expression reportedly enhances autophagy by stabilizing the mitochondrial short form of ARF (56
) and as we show that knocking down p32 increases glycolysis, it is unlikely that the effect on glycolysis would be secondary to autophagy. Furthermore, the apoptosis-promoting activity of p32 through the long form of the ARF protein requires p53 activity (30
). As p53 is mutated in two of the cell lines we used, an involvement of apoptosis-related p32 activity in metabolic regulation is also unlikely. In fact, our in vitro
and in vivo
data present a protumorigenic function for p32 that is more consistent with the reported upregulation of p32 in tumors and that is likely linked to its role in metabolism. In support of this, our findings are consistent with earlier studies showing that depleting or reducing mitochondrial DNA levels with ethidium bromide produces cells with absent OXPHOS and a lower growth rate in vitro
). Inhibiting mitochondrial-DNA-encoded protein synthesis in tumor cells also produces effects similar to our p32 knockdown (5
). In addition, we obtained data showing that chemical inhibition of at least one OXPHOS complex (complex I) mimics the effect of p32 knockdown in vitro
and at least partially in vivo
. Collectively, we believe these findings couple p32 loss and the subsequent impaired OXPHOS with reduced tumorigenicity.
Our results on the metabolic effects of p32 are in agreement with studies on the role of p32 in yeast metabolism, which show that the p32 homologue is necessary for the maintenance of a normal level of OXPHOS (48
). As in yeast, p32 has a profound effect on tumor cell metabolism: knocking down p32 expression in cancer cells shifted the metabolism of the cells from OXPHOS to glycolysis, which was accompanied by an increase in lactate production and elevated glucose consumption. The p32 protein becomes extremely unstable when it is unable to localize to the mitochondria. This biological limitation thus rules out the possibility of clearly excluding the extramitochondrial functions of p32 from the regulation of tumor metabolism. Also, gene array analysis using control versus p32 knockdown cell lines did not reveal different gene signatures in the p32 knockdown cells that could readily explain the metabolic phenotypes we observed (data not shown). It is thus unlikely that extramitochondrial functions related to gene expression might have indirectly affected the mitochondrial metabolism of p32 knockdown cells.
The mammalian p32 protein may not be a general requirement for mammalian cells, as some normal cells show very low or undetectable expression of p32 (19
). Also, p32 overexpression is common in breast cancers but rare or nonexistent in some other malignant tissues, such as prostate carcinomas (19
The Warburg effect, evidenced by high aerobic glycolysis in tumors, is an almost universal feature of malignancy and is thought to provide a growth advantage to tumors (70
). Our results are not easily reconciled with the hypothesis that the glycolytic phenotype is advantageous to tumor growth. If the Warburg effect confers a growth advantage, why would tumor cells upregulate a protein that counteracts this effect and why do the highly glycolytic cells produced by knocking down p32 grow poorly and show impaired tumorigenicity?
It has been proposed that the change to glycolysis is an adaptation to hypoxic conditions encountered by premalignant lesions during their initial growth, which takes the newly added cells farther away from the blood supply than their normal counterpart cells (22
). However, cells transformed in vitro
also become glycolytic (2
), and some oncogenes, such as the c-Myc and Akt genes, enhance the expression of enzymes in the glycolytic pathway, promoting aerobic glycolysis (15
). Perhaps the glycolytic phenotype is a side product of malignant transformation that, depending on circumstances such as the positioning of a cell within a tumor, can confer a growth and/or survival advantage or conversely be disadvantageous. In this regard, a recent study suggests that tumors form a symbiotic environment. This environment consists of cells in hypoxic regions, which need the glycolytic pathway to produce ATP, and cells in the well-oxygenated regions, which use the lactic acid from the hypoxic cells in OXPHOS (64
). If this were the case, one would expect p32 expression to be the strongest outside the hypoxic regions within tumors. However, our results show that the opposite is true, i.e., that p32 expression is strongest in the hypoxic regions (19
), suggesting that p32 moderates glycolytic tendencies in these regions. However, we cannot exclude the possibility of a symbiotic relationship between cells that favor glycolysis and those that favor OXPHOS at the local microenvironment level.
The high rate of glycolytic metabolism in tumors was initially believed to result from impaired ability of cancer cells to carry out oxidative phosphorylation. However, this view has been challenged thorough the years and needs to be reappraised. In fact, defects in oxidative metabolism were not found in several highly proliferative tumor cell lines (47
), and transformation of mesenchymal stem cells was shown to increase cell dependency on oxidative phosphorylation (20
). Furthermore, that mitochondrial OXPHOS function might still be advantageous in highly glycolytic tumor cells is suggested by the facts that recycling of lactate via oxidative metabolism appears to be critical for tumorigenesis (64
) and that oncogenes known to promote the use of the glycolytic pathway by tumors can also upregulate genes important for mitochondrial physiology and increase mitochondrial metabolism (13
). More recently, it has been shown that mitochondrial STAT3 contributes to Ras-dependent malignant transformation via augmenting electron transport chain activity (26
). Altogether, these findings suggest that oncogene-driven glycolytic metabolism should be balanced at least in part by concomitant changes to mitochondria. In view of this, one could hypothesize that the overexpression of proteins such as p32 is required to counteract the otherwise detrimental activity of an oncogene. It is noteworthy that c-Myc changes are common in breast cancers (41
), which exhibit high glycolytic activity (29
). We and others found that breast cancers and some other adenocarcinomas upregulate p32 while others, notably prostate cancer, do not (19
). Interestingly, a majority of prostate cancers, in contrast to many other malignancies, are not highly glycolytic (43
). Thus, p32 may counteract the proglycolytic functions of c-Myc, while allowing its tumor-promoting effects to remain intact. In support of this model, database analysis shows that the p32 gene is a direct c-Myc target gene (Myc target database [77
] at www.myc-cancer-gene.org
In addition to increasing the expression of numerous enzymes in the glycolytic cascade, c-Myc has recently been shown to promote the use of glutamine (21
). Glutaminolysis is important not only for energy production but also for replenishing, through a process termed anapleurosis, the TCA cycle intermediates that are necessary precursors for the anabolic processes required for cancer cell growth (11
). Recently, coordination between glucose and glutamine utilization pathways has been described, and it has been suggested that this represents a metabolic checkpoint in highly glycolytic tumor cells (34
). In view of this, it is possible to speculate that p32 has a role in mediating glutamine metabolism downstream of Myc.
Tumor cells depend on the expression of the oncogenes that contributed to their transformation. This is the case even if several oncogenes with redundant activities have become mutated in the same tumor cell; eliminating just one of the oncogenes induces cell death. This phenomenon is referred to as “oncogene addiction” (71
). Perhaps a similar situation exists in tumor cell energy metabolism. Whether the Warburg effect is advantageous or not, a tumor cell cannot handle a drastic change in either direction from the metabolic balance that has developed during the tumorigenesis process. Existing data supporting this concept by Fantin et al. (16
) showed that suppressing one of the glycolytic enzymes, lactate dehydrogenase A, reduced tumor growth, whereas our results show that enhancing glycolysis produces the same effect. Moreover, it has been reported that the expression of the glycolytic enzyme PKM2 is required for the shift in cellular metabolism to aerobic glycolysis and for tumor growth (9
). Tumor maintenance and malignancy of the cancer cell lines used in this study depend on mitochondrial oxidative phosphorylation via p32, despite the fact that these cells express PKM2. Finally, while we were able to restore p32 expression to the original level in p32 knockdown cells using forced expression of p32, we had difficulties deriving MDA-MB-435 cell lines overexpressing p32. Interestingly, in MDA-MB-435 cells the basal level of p32 expression was the highest among the various tumor cell lines we tested (19
). This result suggests that shifting the balance too far toward OXPHOS is also detrimental, even under well-oxygenated cell culture conditions.
The expression of p32 is upregulated in tumors, particularly in breast cancers, and cell surface-expressed p32 is a tumor marker (19
). Peptides and antibodies that bind to p32 specifically home to tumors and have been used to deliver nanoparticles and nanoparticle-embedded drugs to tumors, particularly to the regions in tumors that are poorly served by blood vessels (19
). Moreover, a p32-binding peptide has an inherent antitumor activity (38
). The present results show that reducing p32 expression in tumor cells suppresses tumor growth. Thus, p32 is not only an important regulator of tumor metabolism but also a promising molecular target in tumor diagnosis and therapy.