**4. Concluding remarks**

20 Cancer Prevention – From Mechanisms to Translational Benefits

CAIX has been used as an endogenous marker to delineate hypoxic regions in solid tumors and is a prognostic marker of aggressive cancers. Inhibition of CAIX has been shown to suppress tumor growth in xenograft models (Chiche et al., 2009). CAIX small molecule inhibitors and antibodies have been developed and evaluated as potential anti-cancer therapeutics. A CAIX monoclonal antibody exhibited anti-tumor activities in the mouse xenograft model of colorectal cancer (Zatovicova et al., 2010). Moreover, CAIX inhibitors were shown to increase the therapeutic effects of tumor radiation (Dubois et al., 2011).

Monocarboxylate transporters (MCT) facilitate the efflux of lactate and protons from cells. The up-regulation of MCTs has been observed in a variety of tumors such as breast, colorectal, ovarian, prostate, and central nervous system carcinomas, and associated with cancer progression and poor prognosis in some instances (Fang et al., 2006; Froberg et al., 2001; Pertega-Gomes et al., 2011; Pinheiro et al., 2010; Pinheiro et al., 2008). MCT1 and MCT4 were related to the invasiveness of human lung cancer cells and drug resistance of ovarian cancer cells (Chen et al., 2010; Izumi et al., 2011). Inhibition of MCT1 could retard the growth of cancer cells in culture and animal models (Fang et al., 2006; Sonveaux et al., 2008).

Vacuolar-type H+-ATPases (V-ATPases) are multi-subunit, complex enzymes that transport protons from the cytoplasm to vacuolar lumens or to extracellular space (Izumi et al., 2003). These proton pumps are important for maintaining intracellular and extracellular pH homeostasis. V-ATPases overexpression has been detected in many types of tumors such as oral squamous cell cancer and melanoma (Nishisho et al., 2011; Perez-Sayans et al., 2010). Studies showed that V-ATPases increased cancer metastasis and drug resistance (Nishisho et al., 2011; You et al., 2009). Inhibition of V-ATPases by small molecule inhibitors or small interfering RNA suppressed tumor growth and metastasis, induced tumor cell apoptosis, and overcame chemoresistance in several cancer models (De Milito et al., 2007; Lu et al.,

Sodium/hydrogen exchangers (NHE) regulate the pH homeostasis of cells by extruding intracellular H+ in exchange of extracellular Na+ at a 1:1 ratio. Nine NHE isoforms have been identified in mammalian cells (De Vito, 2006). NHEs were found to regulate cytoskeletal structures and tumor cell migration and invasion (Paradiso et al., 2004). Treatment with NHE1 inhibitors sensitized the paclitaxel-induced apoptosis of human breast cancer cells (Reshkin et al., 2003). Moreover, increased activity of NHE was observed in doxorubicinresistant human colon cancer cells and the treatment with the NHE inhibitor 5-(N-ethyl-Nisopropyl)-amiloride (EIPA) sensitized the resistant cells to doxorubicin (Miraglia et al., 2005). Inhibition of NHEs has also been shown to reduce the proliferation and VEGF

Proton-sensing G protein-coupled receptors (GPCRs), including GPR4, TDAG8 (GPR65), OGR1 (GPR68), and G2A (GPR132), can be activated by acidic extracellular pH to transduce multiple downstream signaling pathways such as the Gs/cAMP, Gq/phospholipase C/Ca2+, and G13/Rho pathways (Ludwig et al., 2003; Murakami et al., 2004; Radu et al., 2005; Tobo et al., 2007; Wang et al., 2004; Yang et al., 2007). Different from the proton transporters, the proton-sensing GPCRs do not directly transport protons but, instead, perceive acidic extracellular pH to trigger signal transduction. Potential roles of the proton-sensing GPCRs in cancer biology have been emerging, with differential roles for each family member in a cell context-dependent manner. Activation of GPR4 by acidic pH has been shown to inhibit

MCTs may, therefore, represent potential targets for cancer treatment.

production of leukemia cells (He et al., 2007; Turturro et al., 2007).

2005; Nishisho et al., 2011; You et al., 2009).

Cancer cells do not exist in isolation; instead, they closely interact with blood vessels, inflammatory cells, and fibroblasts in a unique tumor microenvironment characterized by hypoxia and acidosis. The interaction between cancer cells and the tumor microenvironment plays a pivotal role in cancer progression and somatic evolution, which follows very similar principles of Darwinian selection. It is increasingly recognized that in addition to killing cancer cells, targeting the components of the tumor microenvironment can help develop more effective approaches for cancer prevention and therapy. For instance, antiangiogenesis therapy, combined with conventional chemotherapy, has shown significant clinical benefits in multiple cancer types (Ferrara and Kerbel, 2005; Kerbel, 2008). Furthermore, a number of agents targeting inflammation, cancer cell metabolism, and hypoxia and acidosis pathways have been developed and added to the arsenal for cancer treatment, detection, diagnosis, prognosis and chemoprevention.

While significant progress has been made to understand the tumor-microenvironment interaction, considerable knowledge gaps still remain. This aspect is exemplified by the lessons learned from anti-angiogenesis therapy. Whereas angiogenesis inhibitors have offered therapeutic benefits in cancer patients, some unexpected adverse effects deserve a close attention. In certain experimental settings, anti-angiogenesis therapy has been shown to promote tumor invasion and metastasis (De Bock et al., 2011; Ebos and Kerbel, 2011; Ebos et al., 2009). The underlying cause is largely attributed to the anti-angiogenesis therapyinduced hypoxia, which is known to stimulate cancer cell metastasis. These observations illustrate that cancer cells constantly evolve and adapt to the changing tumor microenvironment during therapeutic interventions and/or tumor development. In

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### **5. Acknowledgment**

We thank Dr. Adam Asch for reading the manuscript. The research in the authors' laboratory has been supported by the American Heart Association, Brody Brothers Endowment Fund, Golfers against Cancer, and North Carolina Biotechnology Center (to L.V.Y). We apologize to these whose work could not be cited due to the space limitation of this manuscript.
