Cancer Metabolism: New Validated Targets for Drug Discovery

Recent studies in cancer metabolism directly implicate catabolic fibroblasts as a new rich source of i) energy and ii) biomass, for the growth and survival of anabolic cancer cells. Conversely, anabolic cancer cells upregulate oxidative mitochondrial metabolism, to take advantage of the abundant fibroblast fuel supply. This simple model of “metabolic-symbiosis” has now been independently validated in several different types of human cancers, including breast, ovarian, and prostate tumors. Biomarkers of metabolic-symbiosis are excellent predictors of tumor recurrence, metastasis, and drug resistance, as well as poor patient survival. New pre-clinical models of metabolic-symbiosis have been generated and they genetically validate that catabolic fibroblasts promote tumor growth and metastasis. Over 30 different stable lines of catabolic fibroblasts and >10 different lines of anabolic cancer cells have been created and are well-characterized. For example, catabolic fibroblasts harboring ATG16L1 increase tumor cell metastasis by >11.5-fold, despite the fact that genetically identical cancer cells were used. Taken together, these studies provide >40 novel validated targets, for new drug discovery and anti-cancer therapy. Since anabolic cancer cells amplify their capacity for oxidative mitochondrial metabolism, we should consider therapeutically targeting mitochondrial biogenesis and OXPHOS in epithelial cancer cells. As metabolic-symbiosis promotes drug-resistance and may represent the escape mechanism during anti-angiogenic therapy, new drugs targeting metabolic-symbiosis may also be effective in cancer patients with recurrent and advanced metastatic disease.

Catabolic fibroblasts also show a pro-inflammatory phenotype, due to oxidative stress and NFkB activation, which leads to cytokine production. This, in turn, attracts and serves to activate inflammatory cells (macrophages and neutrophils), which produce more ROS and hydrogen peroxide species. These findings link inflammation directly with energy transfer to anabolic cancer cells [2, 41,42,54,55], explaining how inflammation energetically promotes tumor initiation and cancer progression.
To stringently test the validity of these energy transfer mechanism(s), stable cell lines of constitutively catabolic fibroblasts were generated by genetically increasing glycolysis, ketogenesis, autophagy, mitophagy, oxidative stress, and/or senescence. This was accomplished by the recombinant over-expression or knock-down of key metabolic target genes in hTERT-immortalized fibroblasts. Similar results were obtained by the genetic manipulation of either growth factors or extracellular matrix proteins, indicating that these "signaling networks" also converge on catabolic metabolism in stromal fibroblasts.
As metabolic-symbiosis may represent the underlying basis of drug-resistance [31,32], and/or the escape mechanism [35, 43,44,47,48] during antiangiogenic therapy [53], new drugs that target metabolicsymbiosis may prove to be effective in patients with recurrent cancers and even for the treatment of advanced metastatic disease [25-27, 35, 43].
The existence of metabolic-symbiosis (a.k.a., twocompartment tumor metabolism) has also been directly validated in human breast cancer tissue sections, by employing mitochondrial activity staining in situ. Using this approach, it is clear that oxidative, mitochondrialrich cancer cell nests, are physically surrounded by glycolytic, mitochondrial-poor stromal fibroblasts ( Figure  2) [58]. Virtually identical results were also obtained with metabolic protein biomarkers in primary breast tumors and secondary lymph-node metastases (Figure 3), reflecting a common organizing principle, with the juxtaposition of oxidative and glycolytic energetic compartments [52,59]. As such, tumor architecture also "mirrors" these energybased tumor-stromal interactions.
Remarkably, new studies suggest that normal adjacent epithelial cells, and stromal adipocytes, can also serve as functional metabolic partners for anabolic cancer cells [22,40,56,60]. Therefore, cancer cells may be able to use many different cell types, in addition to fibroblasts, as partners to engage in metabolic-symbiosis [52,61].
Finally, oncogene-transformed epithelial cancer cells also show significant increases in mitochondrial mass, which is strictly dependent on oxidative stress [56]. Figure 4 shows that NAC treatment (N-acetyl-cysteine; an anti-oxidant) dramatically reduced mitochondrial staining, selectively in Ras-transformed cancer cells, but not in matched normal control epithelial cells. Thus, NAC selectively blocks mitochondrial biogenesis in Ras-   subjected to immuno-staining with TOMM20, which is a well-established mitochondrial marker. Note that Ras-transformed cells, treated with NAC, show a significant decrease in mitochondrial mass. DAPI (blue nuclear staining) is also shown. Reproduced, with permission, from [56]. Human breast cancer samples (from primary tumors or lymph node metastases) were immuno-stained with antibodies directed against MCT4 (shown in RED) and TOMM20 (shown in BROWN). MCT4 is a marker of cellular stress, such as ROS production, glycolysis, and mitochondrial dysfunction, which reflects catabolic metabolism in cancer-associated fibroblasts. Conversely, TOMM20 is a marker of mitochondrial mass that has been shown to correlate with oxidative mitochondrial metabolism. Note that two distinct metabolic compartments (oxidative vs. glycolytic) co-exist, side-by-side, in human primary tumors. Virtually identical results were obtained with metastatic breast cancer lesions, within lymph node tissue. Insets are also shown at higher magnification. Reproduced, with permission, from [52].

LYMPH NODE
transformed cells, illustrating how new drug discovery of more powerful anti-oxidants could be used therapeutically to "starve" cancer cells. Quantitation indicated that the mitochondrial marker TOMM20 was decreased by >5-fold during NAC-treatment [56]. As such, oxidative stress and ROS production may drive mitochondrial biogenesis in certain aggressive epithelial cancer cells.
In summary, Table 1 lists >40 validated therapeutic target(s), related to metabolic-symbiosis, that could be exploited for new drug discovery. Thus, we should consider metabolic-symbiosis as a novel conceptual framework or platform to design more effective anticancer therapies.