**4. Modification of PDH regulation is vital to malignant transformation**

If cancer metabolism is to be a therapeutic target it is vital to identify likely targets for intervention. In this section we will review some of the evidence that the PDKs regulating PDH activity are excellent potential candidates for such targets. Later we will return to the question of whether the other major dehydrogenase entry point for mitochondrial metabolism, KGDH, might also be an attractive therapeutic target.

48 Dehydrogenases

minority of tumors [49].

for such targets.

properties [40]. However, there is also reason to suspect that other features of the cancer cell version of the anabolic shift will not be shared with most normal, rapidly dividing cells. In particular, the poor vascularization of solid tumors reflects an environment rather different than rapidly dividing normal cells typically experience [41, 42]. Tumor cells are likely to

Of course, the existence of such metabolic adaptations to the extreme tumor microenvironment may still not be helpful therapeutically if these adaptations are idiosyncratic to each individual case of cancer. Fortunately, there is both empirical and theoretical reason to believe that these special adaptations will, in fact, be generally consistent and predictable, as we now discuss. It has long been recognized that solid tumors bear a striking resemblance to healing wounds [43-45]. Specifically, solid tumors resemble wounds that continue the early steps in wound healing, but never resolve as normal wound healing does. Thus, it is plausible to speculate that metabolic modifications in tumor cells usually or always involve the pathological, uncontrolled activation of regulatory pathways normally accommodating cells to the rigors of the wound environment. On this working hypothesis, tumor metabolism is expected to have features not shared with normal cells (except potentially in healing wounds) and to be potentially universal to most or all cancers. The possibility that tumor metabolism might have unique features is particularly important therapeutically. The dramatic increase in our knowledge about the genetic changes in cancer and our experience with genetically targeted therapies has produced some sobering potential insights as illustrated by the following examples. Specifically, in many or even most cancers, the "driver" mutations may represent loss-of-function in tumor suppressors rather than gain-of-function mutations [46-48]. Moreover, the fraction of tumor-producing changes that are epigenetic may be high. All of these features mean that targeting specific oncogene-targeted products, such as small molecules like gleevec, may be viable in only a

Finally, the levels of redundancy in the regulatory circuits mutated in cancers mean that targeting an individual gain-of-function driver, even when it is possible, may be

The upshot of these genetic considerations is that chemotherapy might be better focused on *properties* of the transformed state rather than on the genetic drivers producing that state. Moreover, non-redundant, indispensable properties of the transformed state are expected to represent the most propitious of such targets. Cancer metabolism may represent an environment in which such desirable targets exist. Below we will develop the argument that the dehydrogenases at key control points in metabolism are especially attractive candidates

**4. Modification of PDH regulation is vital to malignant transformation** 

If cancer metabolism is to be a therapeutic target it is vital to identify likely targets for intervention. In this section we will review some of the evidence that the PDKs regulating

incompletely successful and subject to evolved resistance.

have additional metabolic adaptations to this extreme environment.

PDKs have long been recognized as key regulators of PDH function (above). Moreover, in view of the central role of modification of carbohydrate metabolism in cancer (above), alteration of PDK regulation in malignancy is likely. Initially, analysis of clinical samples demonstrated that up-regulation of PDK levels was a frequent correlate of advanced malignancy [50] a pattern that has persisted in subsequent studies [51].

These and other observations stimulated systematic detailed study to great effect. Kim, et al., [52] and Papandreou, et al. [53] demonstrated that HIF-1, a global regulator of the hypoxic response, up-regulated PDK1, with the expected effect of down-regulating carbon flow through PDH. Moreover, PDK up-regulation correlated with increased resistance to programmed cell death, a hallmark of cancer as reviewed in [54]. In view of the consistent hypoxia in solid tumors as reviewed in [41, 42] and the frequent activation of HIF1 in tumors as reviewed in [55] these observations suggested that PDK up-regulation might be a central element of the cancer metabolic reconfiguration.

The next challenge was to ask if more direct evidence for the importance of PDK upregulation in cancer metabolism could be found. Was PDK up-regulation required for malignancy or marginal, epiphenomenal? Several observations argue strongly that PDK upregulation is essential to the malignant phenotype.

First, McFate, et al. [56] demonstrated that PDK1 up-regulation was essential for the malignant phenotype in head and neck squamous cell carcinoma cells (HNSC). Specifically, PDK1 expression contributed to induction of limited flux of carbon through mitochondrial metabolism and PKD1 over-expression further reduced this flux (as measured by glucose consumption and diversion of carbohydrate carbon to lactate formation). Confirming this relationship, PKD1 knockdown increased TCA processing of carbohydrate carbon. PDK1 knockdown reduced several indicators of malignant potential, including ability to resist cell death under hypoxia or to form colonies in soft agar in culture. Most importantly, PDK1 knockdown dramatically, reduced the rate of tumor growth in xenograft models.

Earlier work from this group indicated that accumulation of glycolytic intermediates might, itself, be sufficient to up-regulate HIF1 [57]. Thus, McFate [56] proposed a positive feedback loop central to the malignant state wherein PDK1 up-regulation by HIF1 enhanced production of glycolytic intermediates (by blocking pyruvate consumption) further upregulating HIF1 and so on.

Second, Lu, et al. [58] found a similar role for HIF1-dependent up-regulation of PDK3 in malignant metabolic state. In this case also, reduction in mitochondrial carbohydrate metabolism with the corresponding up-regulation of oxygen-independent cytosolic glycolysis was driven by elevated PDK3 expression. Of particular importance here, the resistance of tumor cells to killing by chemotherapeutic agents paclitaxel and cisplatin was strongly improved by PDK3 up-regulation, while PDK3 knockdown sensitized to these agents. Finally, Lu, et al. [58] observed that both PDK1 and PDK3 collaborated to produce this drug resistance.

The Pyruvate Dehydrogenase Complex in Cancer:

Implications for the Transformed State and Cancer Chemotherapy 51

significantly inhibits tumor growth in mouse xenograft models. Specifically, DCA administered ad libitum in drinking water at 75mg/l produced both inhibition of xenograft tumor growth and modest, but significant reduction in tumor volume in larger tumors.

The clinical follow up on these preclinical results has been generally weak. This largely results from economic factors. DCA is a cheap, generic drug and no corporate actors have an incentive to develop the agent. To date clinical data consist of several anecdotal case reports [66,67] suggesting possible efficacy, including in a poorly differentiated metastatic carcinoma. In addition, in view of its low cost and wide availability, DCA is being prescribed off-label or self- administered. Anecdotal reports of efficacy from this use group can be found in patient/physician webshops (see, for example, www.thedcasite.com). It will be of considerable interest to see if sufficient evidence can be accumulated to assess the

An apparently common side effect of DCA use is peripheral neuropathy. Though this is generally limited and reversible, this may not always be the case. A patient report describes relatively severe encephalopathy probably induced by DCA doses within the range currently being experimented with by patients and their physicians [68]. These problems emphasize the importance for individual patients of using DCA only under professional

Finally, a series of small molecule PDK inhibitors have been developed, initially for the treatment of diabetes as reviewed in [8]. These compounds are far more potent than DCA on a mole basis and it will be of interest to see if they have significant anti-cancer activity. To date, no clinical trial data are available on any possible anti-cancer activities of these agents. Moreover, the design strategy for these compounds renders them relatively selective for individual PDK homodimers. We do not currently know which homo- or heterodimer(s)

**6. Thioctoid lipoate analogs exaggerate cancer-specific, PDK-mediated repression of PDH to shut down tumor cell mitochondria and induce cell** 

The PDH complex includes a dense cloud of lipoate residues making up the functional catalytic co-factor domain of the E2 activity (above). Acyl groups and reducing potential can be back-transferred to this lipoate cloud from the surrounding mitochondrial matrix pool as well as being generated by the conventional PDH forward reaction (above). Moreover, these acyl groups and reducing potential can apparently be rapidly shuttled between these residues before ultimately being transferred to CoA and NAD+[28]. Finally, the oxidation/acylation state of the elements of the lipoate cloud strongly effect the activity of the PDK regulatory kinases, with acylation/reduction stimulating PDK activity (above). Thus, each PDK homo- or heterodimer docked at any point on the PDH complex is apparently continuously exposed to the result of an ongoing, real-time poll of the

might be the most propitious anti-cancer target(s) for this class of agents.

Subsequent reports from other groups corroborate these preclinical findings [64, 65].

clinical potential of this agent.

medical supervision.

**death** 

Collectively, these results clearly indicate that PDK regulation of PDH activity is central to the malignant phenotype. Moreover, there is apparently no metabolic bypass of the requirement for properly managed mitochondrial metabolism. Mitochondria are not turned off in hypoxic tumor cells. Rather, they are redeployed to provide the limited carbon traffic they can sustain to the provision of anabolic intermediates (above). There appears to be no other way to provide some of these biosynthetic resources (citrate for lipid biosynthesis, for example). Further, mitochondria are central controllers of Ca++ signaling [see 59 and references therein] and of cell death control as reviewed in [60,61]. Thus, their continued properly controlled function is likely to be essential to cancer cell homeostasis and survival. These results and considerations are consistent with the hypothesis that this re-regulation of PDH cannot be dispensed with. Without this metabolic pattern cancer cells are likely to be unable to survive and prosper in the hypoxic environment crucial to malignant progression. Thus, PDK re-regulation is a candidate for the kind of essential, non-redundant therapeutic target needed if we are to have fundamentally new clinical options. We will argue below that this target can be therapeutically exploited in several different ways.

Before turning to assessment of how we might attack this potential target it is important to emphasize a particular implication of PDK re-regulation in cancer cells. Because of the possibility of PDK heterodimer formation (above), tumor-specific quantitative changes in individual PDK expression may be substantially amplified in their regulatory effects. For example, if PDK1 and PDK3 were both up-regulated 10 fold in the *in vivo* tumor environment, this could represent a 100-fold increase in the levels of the hypothetical PDK1/PDK3 heterodimer. Such very large effects might represent targets that are essentially qualitatively unique to tumor cells.
