High-throughput transcriptomic analysis of anticancer drug activity is a suitable tool to identify novel target genes. However, confirmation that a particular drug-modulated gene specifically contributes to drug response requires detailed analysis similar to that performed for AQP3, a gene up-regulated by the 5-FU precursor and capecitabine catabolite, 5′-DFUR, in the breast cancer cell line MCF7
AQP3 is a broadly expressed aquaglyceroporin found in most epithelia, where it localizes to the basolateral membrane, as well as in several types of nonepithelial cells
. The extensive distribution pattern suggests that this water channel protein is a major player in barrier hydration and water and osmolyte homeostasis. AQP3 is a target of aldosterone in the collecting duct
 and under osmotic control in renal and keratocarcinoma cells, thus presumably contributing to cell volume adaptive regulatory processes
[23, 24]. While previous studies suggest that changes in cell size associated with cell division are facilitated by increased AQP1 abundance at the plasma membrane
, our results support a putative role of AQP3 in maintaining or promoting cell swelling induced by nucleoside-derived drugs. Interestingly, AQP3-related mRNA levels were not modified during cell cycle progression, suggesting that the role of the water channel in the increased cell volume is related to drug response. The nucleoside analogs 5′-DFUR and gemcitabine triggered G1/S cell cycle arrest, but not cisplatin. This DNA alkylating agent appeared to induce S/G2 arrest, which did not result in increased cell volume, in contrast to the effects of nucleoside-derived drugs.
Knockdown of AQP3 expression produced a partial but significant reversion of increased cell swelling associated with nucleoside-derived drug treatment, further supporting a role of AQP3 in this process. Nevertheless, the magnitude of cell volume reversion in MCF7 and HT29 (about 25%), even assuming that AQP3 expression is only partially blocked in siRNA-transfected cells, suggests that this water channel protein is not the only contributor to cell swelling associated with drug treatment. Interestingly, under similar conditions, suppression of AQP3 preserved cell growth inhibition to a better extent, and the magnitude of reversion of G1/S cell cycle arrest was significantly higher than reversion of cell swelling for 5′-DFUR and gemcitabine in MCF7 cells. Furthermore, in spite of achieving only a 20% of AQP3 mRNA knockdown in HT29, AQP3 suppression partially reverted cell cycle arrest and preserved cell growth inhibition in 5′-DFUR treated cells. Thus, it is possible that AQP3 plays roles other than those derived from its ability to mediate water transport. In fact, AQP3 plays a variety of roles in cell physiology associated with its ability to take up glycerol. AQP3-deficient mice show defective skin hydration and elasticity, which can be corrected by glycerol replacement
. Moreover, wound healing is significantly impaired in these animals, with low keratinocyte proliferation, a feature that can also be reversed in vivo by feeding mice with glycerol
. Interestingly, inhibition of AQP3 in keratinocyte cell cultures results in reduced water and glycerol permeability and impaired cell migration. The protein facilitates migration by functioning as a water channel, but is also implicated in epidermal cell proliferation as a glycerol transporter
. Consistent with this finding, mice lacking AQP3 expression not only display impaired epidermal cell proliferation but are also resistant to skin tumorigenesis
. This appears to be related to the ability of AQP3 to take up glycerol, a suitable energy substrate that supports cell growth. Nucleoside-derived drugs, particularly those used in antiviral therapy, may induce severe mitochondrial toxicity
[28, 29]. While this is not evident for nucleosides used in the treatment of solid tumors, recent evidence suggests that gemcitabine triggers moderate mitochondrial toxicity
 and blocks the activity of human mitochondrial DNA polymerase
. Nucleoside derivatives additionally compete with intracellular nucleotides and inhibit key enzymes of the nucleoside salvage pathways
[32, 33], consequently impairing the cellular energy metabolism. In this context, it is feasible to assume that AQP3 induced after exposure to these drugs plays a compensatory role as a provider of energy substrates.
AQP3 silencing also reversed the up-regulation of selective p53-dependent transcriptional targets, such as the death receptor, FAS, implicated in apoptosis, and the inhibitor of the cyclin-CDK2 and -CDK4 complexes, p21, implicated in the modulation of cell cycle progression at G1. It is not clear from these observations whether AQP3 contributes to apoptosis in addition to its reported effect on cell cycle arrest, which is significantly reversed upon silencing of the gene. Interestingly, AQP3 itself is transcriptionally regulated by p73, a member of the p53 family, which exhibits similar biochemical properties but is rarely mutated in cancer cells
. p73 interacts with the transcriptional coactivator, Yes-associated protein (YAP), leading to enhanced p73-dependent apoptosis in response to DNA damage. YAP is stabilized by the product of the p73/YAP target gene, PML, under negative control by the proto-oncogenic AKT/PKB kinase
. Interestingly, the anticancer drug, curcumin, down-regulates AQP3 expression in cancer ovarian cells via a mechanism that involves, at least partially, inhibition of the EGFR pathway and downstream AKT
. While AQP3 is a p73 target, its association with pro-apoptotic processes does not appear relevant, at least under the conditions used here. This hypothesis is based on evidence that AQP3 up-regulation is observed only at 5-FU concentrations triggering cell cycle arrest, but not at higher doses in which cells are committed to programmed cell death. Moreover, the decrease in cell growth associated with short treatment with low doses of 5-FU is significantly reversed by knockdown of AQP3 expression. These observations collectively support the view that induction of this aquaglyceroporin is related to cell cycle arrest rather than apoptosis.
Aquaporins, including AQP3, are overexpressed in different tumors
[12, 13, 15] and an oncogenic role was suggested for AQP5, although this protein is not an aquaglyceroporin
[13, 36]. To our knowledge, no correlation of basal or drug-induced AQP3 expression with drug chemoresistance has been reported to date. In view of the above findings, this issue deserves further investigation.