**6.2. AQP 3**

microenvironment leading to enhanced tumor angiogenesis and invasion [129]. AQP 4 also plays a role in glioblastoma cell migration and invasion through rearrangement of the actin cytoskeleton [130]. Furthermore, overexpression of AQP 5 in non-small lung cancer cells enhanced cell metastasis through c-Src activation and induction of the EMT process [122].

While AQPs have been shown to be involved in the delivery of water to the mammary glands which is critical for milk production and secretion during lactation [131], their expression in

Immunostaining indicates a predominantly membranous localization with some presence in the cytoplasm in large tumor cells (more pronounced at the tumor invasion front), but no expression was seen in smaller tumor cells. All of the AQP 1 positive invasive carcinomas are found to be of ductal type, ER−ve and HER2/neu −ve (triple −ve form), and its expression was significantly associated with poor clinical prognosis [132, 133]. A recent report suggested that the cytoplasmic expression of AQP 1 promotes breast cancer progression and was associated with a shorter survival rate especially in luminal subtype patients [134]. Its cytoplasmic expression was positively correlated with advanced pathological features of invasive ductal carcinoma and lymph node metastasis [134]. Another study reported that AQP 1 was highly expressed in blood vessels (mainly in CD31+ve endothelial cells) of human breast and endometrial carcinoma tissues, suggesting a role in tumor angiogenesis [135]. Using human umbilical vein endothelial cells (HUVECs), Zou et al. [135] showed that estrogen treatment significantly up-regulated AQP 1 expression in a time- and dose-dependent fashion, which was mediated through a functional estrogen response element motif in the promoter region of the AQP1 gene. Estrogen treatment significantly increased HUVEC proliferation, migration, invasion and tubule formation; all of these effects were inhibited by pretreatment of cells with AQP1-specific siRNA. These data suggest an important role of AQP 1 in cell invasion in part through regulating actin stress fiber formation through colocalization with the ezrin/radixin/ moesin protein complex [135]. Qin et al. [134] showed that overexpression of AQP 1 in MCF-7 and MDA-MB-231 cells significantly enhanced (by approximately 2 fold) cell proliferation and invasion. Epidermal growth factor (EGF) stimulation induced AQP 1 redistribution from the cytoplasm to the cell membrane, further supporting a role in promoting cell invasion. In the mouse mammary tumor virus-driven polyoma middle T oncogene (MMTV-PyVT) model (which spontaneously develops a well-differentiated luminal-type breast carcinoma with lung metastasis), AQP 1 deficiency significantly reduced the breast tumor mass (by 46%) and volume (by 50%), vessel density and the number of lung metastases compared to the control group [136]. This effect was in part due to decreased expression of vascular endothelial growth factor receptor-2 (VEGFR2) and increased levels of hypoxia inducible factor-1α (HIF-1α) in the

**6. Role of AQPs in the pathogenesis of breast cancer**

breast tumors is modified and correlates with tumor grade.

**6.1. AQP 1**

66 Tumor Metastasis

AQP 1 knockout mice [136].

AQP 3 overexpression in early breast cancer patients was shown to be associated with worse prognosis in patients with HER2-overexpressing phenotype after curative surgery [137]. Its expression was correlated with advanced stage, large tumor size and lymphatic and vascular invasion, highlighting its role in angiogenesis and invasion. In addition, Huang et al. [138] showed higher AQP 3 protein expression in breast cancer tissues (mainly in the cell membrane and the cytoplasm) of premenopausal compared to postmenopausal patients, and was associated with higher histopathological grade and lymph node metastasis in ER+ve breast cancer patients. Estrogen stimulation significantly up-regulated AQP 3 expression in ER+ve breast cancer cells (MCF-7 and T47D) by activating the estrogen response elements (EREs) in the promoter region of the AQP 3 gene. siRNA mediated knockdown of AQP 3 in ER+ve breast cancer cells significantly reduced estrogen-induced cell migration (by 30–70%) and invasion (by 43–71%). Overexpression of AQP 3 in T47D cells significantly enhanced cell migration and invasion. The role of AQP 3 in cell invasion was suggested to be in part through mediating actin cytoskeleton rearrangement (by the formation of filopodia and stress fibers required for invasion) and EMT induction (evident by reduced expression of the epithelial marker Ecadherin, and increased levels of the mesenchymal markers N-cadherin and snail-1) [138]. Using breast cancer cell lines MDA-MB-231 and Bcap-37, Cao et al. [139] showed that fibroblast growth factor-2 (FGF-2) significantly increased AQP 3 expression, and lentivirus-mediated shRNA inhibition of AQP3 expression significantly reduced FGF-2 induced cell migration by approximately 50%. This effect was mediated through AQP-3-induced activation of Akt and ERK1/2. A recent report showed that AQP 3 expression in the triple negative breast cancer cell lines MDA-MB-231 and DU4475 (as well as in HUVEC) was required for the transport of extracellular hydrogen peroxide into the cells in response to CXCL-12 stimulation to induce directional cell migration [140]. AQP 3 silencing in these cells was associated with impaired CXCL-12 induced directional migration due to impaired F-actin polymerization, PTEN and PTP1B oxidation, Akt phosphorylation, and the accumulation of the intracellular hydrogen peroxide at the reading edge of migrating cells was needed for polarity sensing. Furthermore, the role of AQP3 in invasion was tested by the injection of fluorescently labeled breast cancer cells into severe combined immunodeficient (SCID) mice. Lung metastasis was significantly reduced in AQP-3-deficient breast cancer cells, whereas its overexpression significantly increased the number of cells migrating to the lungs [140]. In addition, the expression of AQP 3 was also increased in MCF-7 cells by treatment with the chemotherapeutic agent 5′-deoxy-5 fluorouridine (5′-DFUR) [141], which was required for the 5′-DFUR-induced cell cycle arrest (through its action on G1/S phase transition and up-regulation of p21 and FAS).

#### **6.3. AQP 4**

The role of this AQP is not well studied in breast cancer, however, one report showed that AQP 4 expression (at both mRNA and protein level) was significantly higher in normal compared to cancer tissue [133], and was mainly expressed in the cell membrane and the cytoplasmic compartments.
