**6. Targeting glycolysis for cancer treatment**

The aberrant metabolic pathways underlying the Warburg effect are being considered as novel targets for cancer therapy. Several strategies have been employed to target glucose metabolic pathways for cancer treatment.

**First,** inhibitors of glycolytic enzymes or glycolytic pathways are being searched to identify therapeutic agents that can inhibit cancer growth and development.

A number of small molecules have been reported to target glycolysis although none to date has been shown to have specific molecular targets. For example, 3-bromopyruvate, a highly active alkylating agent, was reported to target HK-2 and/or GAPDH (Dang et al., 2009). 2 deoxyglucose (2-DG) can be phosphorylated by HK-2, which in turn inhibits HK-2 (Ralser et al., 2008). It is also shown to be a GLUT inhibitor.

Many efforts have been made to identify specific inhibitors. Lonidamine has been described as a specific inhibitor of mitochondria-bound HK (Floridi et al., 1981) and has been tested in multiple clinical trials including a phase II study in combination with diazepam for the treatment of glioblastoma patients (Porporato et al., 2011). Unfortunately, none of these clinical trials have successfully shown its therapeutic benefit in terms of time-to-progression and overall survival (Oudard et al., 2003); one study showed its severe hepatic adverse effects.

Drugs that target more specific metabolic control points of glycolysis in cancer cells, such as PKM2 or LDH-A, warrant investigation as potential cancer therapies. gossypol/AT-101, a natural product and a non-specific LDH inhibitor that has more preferential inhibitory activity on malarial and spermoctye LDHs, has already been tested in human clinical trials for its anticancer effect(Porporato et al., 2011). A selective competitive inhibitor of LDH5, 3-dihydroxy-6 methyl-7-(phenylmethyl)-4-propylnaphthalene-1-carboxylicacid (FX11), has been identified through screening a library of compounds derived from gossypol (Yu et al., 2001). FX11 has been shown to suppress in vivo xenograft tumor growth of human B lymphoid tumor and pancreatic cancer cells (Le et al. 2010), providing a strong rationale for clinical development of therapeutic agents targeting LDH-A. Recently, *N*-Hydroxy-2-carboxy-substituted indole compounds have been identified as LDH5-specific inhibitors (Granchi et al., 2011).

Several clinical trials with the PKM2 inhibitor TLN-232/CAP-232, a seven amino-acid peptide, have been initiated. Encouraging preliminary results demonstrated that it is safe, well tolerated, and may offer disease control (Porporato et al., 2011). New small molecule inhibitors of PKM2 were also screened. Among them, the most potent one resulted in decreased glycolysis and increased cell death in respond to loss of growth factor signaling, supporting the feasibility and viability of targeting glucose metabolism as a novel strategy to treating human cancers (Vander Heiden et al., 2010a).

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

The aberrant metabolic pathways underlying the Warburg effect are being considered as novel targets for cancer therapy. Several strategies have been employed to target glucose

**First,** inhibitors of glycolytic enzymes or glycolytic pathways are being searched to identify

A number of small molecules have been reported to target glycolysis although none to date has been shown to have specific molecular targets. For example, 3-bromopyruvate, a highly active alkylating agent, was reported to target HK-2 and/or GAPDH (Dang et al., 2009). 2 deoxyglucose (2-DG) can be phosphorylated by HK-2, which in turn inhibits HK-2 (Ralser et

Many efforts have been made to identify specific inhibitors. Lonidamine has been described as a specific inhibitor of mitochondria-bound HK (Floridi et al., 1981) and has been tested in multiple clinical trials including a phase II study in combination with diazepam for the treatment of glioblastoma patients (Porporato et al., 2011). Unfortunately, none of these clinical trials have successfully shown its therapeutic benefit in terms of time-to-progression and overall survival (Oudard et al., 2003); one study showed its severe hepatic adverse effects.

Drugs that target more specific metabolic control points of glycolysis in cancer cells, such as PKM2 or LDH-A, warrant investigation as potential cancer therapies. gossypol/AT-101, a natural product and a non-specific LDH inhibitor that has more preferential inhibitory activity on malarial and spermoctye LDHs, has already been tested in human clinical trials for its anticancer effect(Porporato et al., 2011). A selective competitive inhibitor of LDH5, 3-dihydroxy-6 methyl-7-(phenylmethyl)-4-propylnaphthalene-1-carboxylicacid (FX11), has been identified through screening a library of compounds derived from gossypol (Yu et al., 2001). FX11 has been shown to suppress in vivo xenograft tumor growth of human B lymphoid tumor and pancreatic cancer cells (Le et al. 2010), providing a strong rationale for clinical development of therapeutic agents targeting LDH-A. Recently, *N*-Hydroxy-2-carboxy-substituted indole

Several clinical trials with the PKM2 inhibitor TLN-232/CAP-232, a seven amino-acid peptide, have been initiated. Encouraging preliminary results demonstrated that it is safe, well tolerated, and may offer disease control (Porporato et al., 2011). New small molecule inhibitors of PKM2 were also screened. Among them, the most potent one resulted in decreased glycolysis and increased cell death in respond to loss of growth factor signaling, supporting the feasibility and viability of targeting glucose metabolism as a novel strategy

compounds have been identified as LDH5-specific inhibitors (Granchi et al., 2011).

to treating human cancers (Vander Heiden et al., 2010a).

normal cells through alteration in glucose metabolism.

**6. Targeting glycolysis for cancer treatment** 

al., 2008). It is also shown to be a GLUT inhibitor.

therapeutic agents that can inhibit cancer growth and development.

metabolic pathways for cancer treatment.

Conceivably, ENOA, as a tumor-associated antigen with an ability of eliciting both B cell and T cell immune response (Capello et al., 2011), is an ideal target of cancer vaccine and immunotherapy. Comparing to small molecule inhibitors, immunotherapy offers superior target specificity and may be used as an alternative approach to target other glycolysis enzymes.

**Second**, inhibition of glycolytic enzyme or glycolysis pathways serves as a strategy to enhance the sensitivity of tumor cells to conventional cytotoxic chemotherapy agents.

Inhibition of LDH-A has been shown to re-sensitize Taxol-resistant cancer cells to Taxol (Zhou et al.). 2-DG is another example. The safety of using it as an anti-cancer agent has been questioned notably because of brain toxicity (Tennant et al., 2010). However, it has a proven efficacy in sensitizing human osteosarcoma and non-small cell lung cancers to adriamycin and paclitaxel (Maschek et al., 2004). Recently, a Phase I clinical study for prostate cancer has defined a maximum tolerance dose of 45mg/kg for Phase II trials (Stein et al., 2010). It will be interesting to test whether 2-DG can enhance the efficacy of chemotherapy agents even if it cannot offer anti-cancer activity by itself at this dose level.

As aforementioned, chemosensitivity is enhanced by counteracting the acidification of tumor's microenvironment. Inhibitors of glycolytic enzymes may impose an alkalizing effect in tumor's microenvironment particularly at the tumor tissue level and thus may have a more specific and powerful role in enhancing the sensitivity of tumor cells to basic chemotherapy drugs. Major targets for counteracting the acidification of tumor's microenvironment include carbonic anhydrasesis (CA)-9 and -12, sodium–proton exchanger 1 (NHE1), sodium bicarbonate cotransporter (NBC), vacuolar ATPase (V-ATPase), sodium– potassium (NaK) ATPase, and MCT4 (Porporato et al., 2011). Indisulam is a leading compound for CA9 inhibition. It is a sulfonamide derivative and shown to inhibit CA9 at nanomolar concentrations (Abbate et al., 2004; Supuran, 2008; Owa et al., 2002). It has been tested in multiple clinical trials for the treatment of melanoma, lung, pancreatic and metastatic breast cancers and has not been found in the completed clinical trials to have antitumor efficacy as a single agent (Talbot et al., 2007). Girentuximab, a specific antibody targeting CA9, is now being tested in Phase III clinical trials for the treatment of clear-cell renal cell carcinoma (Reichert, 2011). Several inhibitors of membrane-bound V-ATPase have been reported to have antitumor activity in preclinical studies (Perez-Sayans et al., 2009). Other enzymes involved in the cellular export of protons are also studied as targets of anticancer therapeutic development. However, future studies should emphasize on combining anti-acidification therapies with cytotoxic chemotherapy or immunotherapy to achieve the effective anti-cancer treatment.

**Third,** combination of inhibitors of glucose metabolic enzymes with inhibitors of oncogenic pathways may result in synergistic anti-tumor effects.

Inhibitors of oncogenic pathways have been extensively tested for cancer therapy, with only moderate success in a few types of human cancers. Among them, inhibitors of the Ras pathway are essentially not effective. As K-ras mutated cancer cells have an enhanced survival when glucose is deprived, a combinatorial treatment with both glycolysis inhibitors and Ras pathway inhibitors may target Ras-mutated cancer cells more effectively and more specifically. Supporting this hypothesis, the hexokinase inhibitor 3-bromopyruvate was demonstrated to be highly toxic specifically to cancer cells with K-ras mutation, but not to cancer cells with wild-type K-ras (Yun et al., 2009). BAY87-2243, which is a small molecule inhibitor of HIF-1 activity and of HIF-1α stability, and EZN-2968, which is an antisense oligonucleotide targeting HIF-1α, had been tested in clinical trials (Greenberger et al., 2008). Metformin is an AMPK-activating drug and is currently used for type-2 diabetes treatment. Epidemiological studies have shown reduced incidence of cancer in diabetic patients treated with metformin (Evans et al., 2005; Jalving et al., 2010; Libby et al., 2009). It is highly intriguing to test whether this clinically safe and known glucose metabolism modulating drug can enhance anti-cancer activity of cytotoxic chemotherapy and/or further lower the cancer recurrence following adjuvant chemotherapy. Similarly, other combination treatments with inhibitors of both glucose metabolisms and oncogenic pathways such as Akt, mTOR, etc. also warrant investigation.

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