**3. Clinical implications of the reconfiguration of metabolism in cancer cells**

The hypothesis that metabolism may be altered in important ways in cancer is longstanding [30]. However, our understanding of cancer metabolism and its relationship to malignancy and clinical outcome has only recently begun to receive extensive attention. Though our knowledge about cancer metabolism remains quite incomplete, some important global insights have emerged.

First, fluxes of both glucose and glutamine, two major carbon sources, are substantially upregulated in many tumors as reviewed in [31-36]. More specifically, mutational changes altering signaling through the Akt pathway enhance glucose uptake and glycolytic metabolism [37]. Likewise, alterations of Myc expression substantially up-regulate glutamine utilization [3, 38, 39].

Second, evidence from multiple perspectives supports the view that one of the major functions of these tumor-specific changes is the redirection of metabolism toward provision of increased levels of anabolic substrates. For example, large fractions of glucose carbon are diverted into the pentose phosphate pathway in support of nucleotide synthesis and into amino acid biosynthesis. Likewise, a large portion of glutamine carbon is siphoned off of the TCA cycle as citrate to support lipid biosynthesis (including fatty acids and sterols) (Figure 1). We can call this the *anabolic shift* in tumor cells.

The therapeutic implications of the alterations in cancer metabolism depend heavily on how unique these changes are to tumor cells. There is good evidence that some elements of the anabolic shift are not unique to tumors cells, but may also be shared with rapidly dividing normal cells. For example, activated lymphocytes show some of these same metabolic 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 have additional metabolic adaptations to this extreme environment.

The Pyruvate Dehydrogenase Complex in Cancer:

Implications for the Transformed State and Cancer Chemotherapy 49

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

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

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

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 up-

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

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 up-

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

knockdown dramatically, reduced the rate of tumor growth in xenograft models.

metabolism, KGDH, might also be an attractive therapeutic target.

malignancy [50] a pattern that has persisted in subsequent studies [51].

central element of the cancer metabolic reconfiguration.

regulation is essential to the malignant phenotype.

regulating HIF1 and so on.

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 minority of tumors [49].

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 incompletely successful and subject to evolved resistance.

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 for such targets.
