**4. Tumor "friendly" microenvironment attributed to altered glucose metabolism**

One would ask: what is the advantage for cancer cells to use energy-inefficient glycolysis under adequate oxygen supply. A potential advantage, as a result of Warburg effect, is high production of lactic acid due to enhanced glycolysis. Positive correlation between lactate serum levels and tumor burden in cancer patients has been well documented, implicating a role of acidic microenvironment in promoting tumor growth and development (McCarty and Whitaker, 2011).

First, accumulated evidence has suggested that acidic environment amplifies the capacity of invasion and metastasis of cancer cells. For instance, acid pretreatment of tumor cells enhance their ability to form metastases in tumor-transplanted mice (Rofstad et al., 2006). Consistently, it has been shown that increasing tumor pH via bicarbonate therapy significantly reduces the number and the size of metastases in a mouse model of breast cancer (Robey et al., 2009).

Second, the extracellular pH of solid tumors is significantly more acidic than that of normal tissues, thus impairing the uptake of weakly basic chemotherapeutic drugs (Raghunand et al., 1999). Several anticancer drugs such as doxorubicin, mitoxantrone and vincristine are weak bases that are protonated in slightly acid tumor microenvironments. The protonated forms of the drugs cannot easily diffuse across the plasma membrane and therefore their cellular uptake is suppressed. It has been demonstrated that the addition of sodium bicarbonate in the drinking water enhanced the anti-tumor effect of doxorubicin on xenotransplanted tumors presumably by enhancing the intracellular drug delivery through raising the pH of the extracellular milieu in mice (Raghunand et al., 1999). The reverse situation was also demonstrated in another study showing that glucose administration to mice led to a lower efficacy of doxorubicin on tumors presumably due to a decrease in the extracellular pH (Gerweck et al., 2006).

HIF-1 as their downstream effector. Consistently, among the HIF-1 regulated genes, most are those encoding glycolytic enzymes (Semenza, 2003). The loss of PTEN and concurrent increase of Akt and mTOR lead to the HIF-1 activation and the Warburg effect (Arsham et

Another oncogene, K-Ras, can alter glucose metabolism so as to provide tumor cells with a selective advantage. In cells with mutated K-Ras, GLUT1 is upregulated, leading to an augmented glucose uptake, glycolysis and lactate production. Interestingly, in these cells mitochondrial functions and oxidative phosphorylation are not compromised, which allows increased survival rate of the K-Ras mutant cells during glucose deprivation (Annibaldi and

Loss of p53 functions also leads to the Warburg effect. As described above, p53 represses transcription of the genes encoding GLUT1 and 4, and induces transcription of the TIGAR gene, which in turn lowers the intracellular level of PFK/FBPase (Bensaad et al., 2006). In addition, p53 inhibits the PI3K-Akt-mTOR pathways. This appears to be mediated by the transcriptional targets of p53 including PTEN, IGF-binding protein 3, tuberous sclerosis protein TSC-2, and the beta subunit of AMPK (Feng et al., 2007). Conceivably, loss of p53 functions and subsequent loss of expression of these target genes lead to a high HIF level

One would ask: what is the advantage for cancer cells to use energy-inefficient glycolysis under adequate oxygen supply. A potential advantage, as a result of Warburg effect, is high production of lactic acid due to enhanced glycolysis. Positive correlation between lactate serum levels and tumor burden in cancer patients has been well documented, implicating a role of acidic microenvironment in promoting tumor growth and development (McCarty

First, accumulated evidence has suggested that acidic environment amplifies the capacity of invasion and metastasis of cancer cells. For instance, acid pretreatment of tumor cells enhance their ability to form metastases in tumor-transplanted mice (Rofstad et al., 2006). Consistently, it has been shown that increasing tumor pH via bicarbonate therapy significantly reduces the number and the size of metastases in a mouse model of breast

Second, the extracellular pH of solid tumors is significantly more acidic than that of normal tissues, thus impairing the uptake of weakly basic chemotherapeutic drugs (Raghunand et al., 1999). Several anticancer drugs such as doxorubicin, mitoxantrone and vincristine are weak bases that are protonated in slightly acid tumor microenvironments. The protonated forms of the drugs cannot easily diffuse across the plasma membrane and therefore their cellular uptake is suppressed. It has been demonstrated that the addition of sodium bicarbonate in the drinking water enhanced the anti-tumor effect of doxorubicin on xenotransplanted tumors presumably by enhancing the intracellular drug delivery through raising the pH of the extracellular milieu in mice (Raghunand et al., 1999). The reverse situation was also demonstrated in another study showing that glucose administration to mice led to a lower efficacy of doxorubicin on tumors presumably due to a decrease in the

**4. Tumor "friendly" microenvironment attributed to altered glucose** 

al., 2002; Zundel et al., 2000).

and establishment of the Warburg effect.

Widmann, 2010).

**metabolism** 

and Whitaker, 2011).

cancer (Robey et al., 2009).

extracellular pH (Gerweck et al., 2006).

Third, acidic microenvironment inhibits anti-tumor immune response. For instance, lactic acid suppressed the proliferation and cytokine production of human cytotoxic T lymphocytes (CTLs) up to 95% and led to a 50% decrease in cytotoxic activity (Fischer et al., 2007). Activated lymphocytes themselves use glycolysis, which relies on the efficient secretion of lactic acid. Export of lactic acid from lymphocytes depends on a gradient between intracellular and extracellular lactic acid concentration. High extracelullar acidity would diminish this gradient and block the secretion of lactic acid from lymphocytes. The accumulation of intracellular lactic acid eventually disturbs the glycolysis process hence affecting the activity of lymphocytes. Acidification similarly inhibits the activity of other immune cells such as dendritic cells.

### **5. Coupled biological and metabolic processes and the logic of a mammalian metabolic cycle**

Glycolytic enzymes have multiple cellular functions. For instance, GAPDH has been implicated in numerous non-glycolytic functions (Colell et al., 2007; McKnight, 2003). In 2003, we published a paper that describes the isolation and characterization of OCA-S, which is a transcription cofactor complex that directly stimulates the transcription of the histone H2B gene in an S-phase-specific manner (Zheng et al., 2003). Surprisingly, a key component of the OCA-S complex represents a nuclear form of GAPDH, which regulates H2B transcription in a redox dependent manner. LDH was later shown to be an essential OCA-S component as well and can exercise the enzyme activity to reverse in vitro inhibition of H2B transcription by converting NADH to NAD+ in the presence of substrate pyruvate (Dai et al., 2008). Conceivably, the participation of these glycolytic enzymes in such a cell cycle event would subject cell cycle regulation to altered glucose metabolism in cancer cells, providing yet another mechanistic explanation of cancer growth and development. The "moonlighting" participation of the glycolytic enzymes in a cellular process would in theory impose a dynamic modulation of the redox status in the cellular compartment where this process is executed and subsequently affect the functions of other redox-sensitive proteins in the same intracellular compartment. Thus, in addition to histone expression, our study has suggested that other cellular processes including cell cycle regulation, DNA replication and damage repair are potentially all coupled through the redox signals (Yu et al., 2009). The coupling of these cellular processes is apparently crucial for the maintenance of chromatin integrity during cell cycle, and thus altered glucose metabolism in cancer cells potentially would disrupt the coupling of these processes and make cancer genomes more error-prone.

It is well delineated that in yeast, quite a few biological and metabolic processes are known to be compartmentalized in time, termed the yeast metabolic cycle (YMC) that is in sync with the cell cycle progression (Tu et al., 2005). The YMC has oxidative, reductive/building and reductive/charging phases, and the S-phase of yeast cell is synchronized with the most reductive stage of YMC. We subsequently found that the oxidative and reductive phases in mammalian cells are also synchronized with the cell cycle, and our study demonstrated that the free NAD+/NADH ratio fluctuated in a defined manner during cell cycle(Yu et al., 2009). At G1 phase, the intracellular NAD+/NADH ratio is high, suggesting that G1 cells maintain an oxidative status. Upon entering S phase, the ratio becomes lower, corresponding to a reductive status. When the cells exit S phase and enter G2 phase, the NAD+/NADH ratio becomes higher again. This oxidative status appears to be maintained until cells enter the next S phase. This phenomenon has been dubbed mammalian metabolic cycle (MMC) for its similarity with YMC. The fluctuating NAD+/NADH ratios in a mammalian cell cycle must reflect overall oscillatory cellular metabolism; whether and how glucose metabolism is synchronized with the cell cycle remains to be explored. Nonetheless, this synchronization must have been very precisely regulated. Conceivably, if glucose metabolism is altered, the cell cycle must be coordinately modulated, and vice versa. Therefore, cancer cells may have acquired the growth and proliferative advantage over normal cells through alteration in glucose metabolism.
