Evaluation of Beta-Catenin Subcellular Localization and Water Channel Protein AQP1 Expression as Predictive Markers of Chemo-Resistance in Ovarian High-Grade Serous Carcinoma: Comparative Study between Preoperative Peritoneal Biopsies and Surgical Samples

Background. Mutations of the β-catenin gene (CTNNB1), leading to aberrant immunohistochemical expression of β-catenin, represent a key mechanism of WNT/β-catenin pathway alteration in ovarian cancer. Aquaporin 1 (AQP1), as component of transmembrane-water-channel family proteins, has been documented in different human tumors and, recently, also in ovarian carcinoma. Only few studies have investigated the pathogenetic and prognostic role of β-catenin and AQP1 in ovarian carcinoma. Methods. We evaluated the expression of β-catenin and AQP1 in the preoperative peritoneal biopsies of 32 patients with peritoneal carcinosis, in which a histological diagnosis of high grade serous ovarian carcinoma was made. Furthermore, we have investigated their potential association with chemotherapeutic response evaluated at the omental site, as well as with clinico-pathological parameters. Results. Sixteen cases showed an aberrant membranous and cytoplasmic β-catenin staining pattern. The remaining 16 cases showed a preserved β-catenin expression localized only in cell membranes; 20 cases showed positive membranous staining (AQP1+), while 12 cases were considered negative (AQP1–). In the AQP+ group, we detected a significant association of AQP1 expression with poor chemotherapy response in omental tissues complete response score (CRS) 1-2, while a CRS 3 was never observed in all positive cases. Conclusions. Our findings suggest that β-catenin and AQP1 are expressed in a sub-group of ovarian tumors and play important roles in carcinogenesis. Patients affected by high grade serous carcinoma could be categorized in two different predictive groups: as AQP+ and AQP–. AQP+ cases may represent a subset of poor responders who could be considered more eligible for cytoreductive surgery rather than for neoadjuvant chemotherapy.

AQP1 Is Up-Regulated by Hypoxia and Leads to Increased Cell Water Permeability, Motility, and Migration in Neuroblastoma

The water channel aquaporin 1 (AQP1) has been implicated in tumor progression and metastasis. It is hypothesized that AQP1 expression can facilitate the transmembrane water transport leading to changes in cell structure that promote migration. Its impact in neuroblastoma has not been addressed so far. The objectives of this study have been to determine whether AQP1 expression in neuroblastoma is dependent on hypoxia, to demonstrate whether AQP1 is functionally relevant for migration, and to further define AQP1-dependent properties of the migrating cells. This was determined by investigating the reaction of neuroblastoma cell lines, particularly SH-SY5Y, Kelly, SH-EP Tet-21/N and SK-N-BE(2)-M17 to hypoxia, quantitating the AQP1-related water permeability by stopped-flow spectroscopy, and studying the migration-related properties of the cells in a modified transwell assay. We find that AQP1 expression in neuroblastoma cells is up-regulated by hypoxic conditions, and that increased AQP1 expression enabled the cells to form a phenotype which is associated with migratory properties and increased cell agility. This suggests that the hypoxic tumor microenvironment is the trigger for some tumor cells to transition to a migratory phenotype. We demonstrate that migrating tumor cell express elevated AQP1 levels and a hypoxic biochemical phenotype. Our experiments strongly suggest that elevated AQP1 might be a key driver in transitioning stable tumor cells to migrating tumor cells in a hypoxic microenvironment.

AQP1-Driven Migration Is Independent of Other Known Adverse Factors but Requires a Hypoxic Undifferentiated Cell Profile in Neuroblastoma

Neuroblastoma is a biologically very heterogeneous tumor with its clinical manifestation ranging from spontaneous regression to highly aggressive metastatic disease. Several adverse factors have been linked to oncogenesis, tumor progression and metastases of neuroblastoma including NMYC amplification, the neural adhesion molecule NCAM, as well as CXCR4 as a promoter of metastases. In this study, we investigate to what extent the expression of AQP1 in neuroblastoma correlates with changing cellular factors such as the hypoxic status, differentiation, expression of known adverse factors such as NMYC and NCAM, and CXCR4-related metastatic spread. Our results show that while AQP1 expression leads to an increased migratory behavior of neuroblastoma cells under hypoxic conditions, we find that hypoxia is associated with a reduction of NMYC in the same cells. A similar effect can be observed when using the tetracycline driven mechanism of SH-EP/Tet cells. When NMYC is not expressed, the expression of AQP1 is increased together with an increased expression of HIF-1α and HIF-2α. We furthermore show that when growing cells in different cell densities, they express AQP1, HIF-1α, HIF-2α, NMYC and NCAM to different degrees. AQP1 expression correlates with a hypoxic profile of these cells with increased HIF-1α and HIF-2α expression, as well as with NMYC and NCAM expression in two out of three neuroblastoma cell lines. When investigating cell properties of the cells that actually migrate, we find that the increased APQ1 expression in the migrated cells correlates with an increased NMYC and NCAM expression again in two out of three cell lines. Expression of the tumor cell homing marker CXCR4 varies between different tumor areas and between cell lines. While some migrated tumor cells highly express CXCR4, cells of other origin do not. In the initial phase of migration, we determined a dominant role of AQP1 expression of migrating cells in the scratch assay.

Aquaporin-1: a major modulator of aging-mediated platelet/endothelial dysfunction in atherothrombosis

Background Aging is associated with development of cardiovascular diseases, including atherothrombosis. Aquaporin-1 (AQP1) is a water channel that also transports hydrogen peroxide (H2O2). Regarding to the role of oxidative stress in atherothrombosis, we hypothesized that AQP1 modulates aging-associated platelet/endothelial dysfunction. Methods Human aortic endothelial cells (HAEC) from passages 5 (young) to 15 (senescent/old) were subjected to fluorescent immunocytochemistry to detect AQP1 protein expression and subcellular localization. The cells were also probed for AQP1 and the phospho/total proteins (AMPK, acetyl-coA-carboxylase (ACC), caveolin-1 and eNOS) for studying the signal transduction by immunoblotting. The endothelial cells were transfected with constructs containing H2O2 biosensor HyPer targeted to cell nucleus or cytosol followed by fluorescence imaging. The transcriptional levels of pro-inflammatory/pro-atherogenic vs. anti-inflammatory/atheroprotective genes in the cells were assessed by qRT-PCR. Human blood samples were taken and treated with or without AQP1 inhibitor (Bacopaside II, 10 μM) to examine the platelet adhesion and rolling velocity on vWF under high shear flow (100 dyn/cm2). Also, platelet aggregation in response to collagen (2 μM), ADP (1 μM) and TRAP (1 μM) were recorded. Results First, the senescence of HAEC was adjusted by a significant increase in β-galactosidase activity from passage 5 to 15. AQP1 immunofluorescence showed a remarkable increase in the young (P.5) compared to senescent (P.15) cells. Immunoblot analyses showed that aging leads to significant increases in AQP1 intensity and phosphorylation of caveolin-1 (Tyr14) and ACC (Ser79), along with decreases in phosphorylation of eNOS (Ser1177) and AMPK (Thr172) (p<0.01, n>6). Fluorescence imaging documented a robust H2O2 production in the senescent endothelial cell cytosol, but not nucleus, and activated TNF-α gene, whereas the transcription of hemoxygenase-1 gene enhanced in the young cells (p<0.01). AQP1 inhibition reduced platelet adhesion and thrombus formation, and elevated platelet rolling velocity on vWF under shear flow (p<0.01). Also, a decrease was found in platelet aggregation in response to AQP1 inhibition (p<0.05). Conclusion These studies, for the first time, demonstrate that aging induces AQP1 expression in endothelial cells and platelets, and modulates the dephosphorylation of AMPK/eNOS. These may lead to platelet/endothelial dysfunction and production of pro-coagulant/pro-inflammatory factors via ACC activation. Therefore, AQP1 inhibition could potentially be exploited as a therapeutic strategy for improving age-related atherothrombosis. Figure 1. Aging increases AQP1 expression in HAEC that leads to dephosphorylation of AMPK and eNOS. AQP inhibition also improves platelet function. *p<0.05, **p<0.01 compared to control. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation (SNSF)

Aquaporin 1 promotes sensitivity of anthracycline chemotherapy in breast cancer by inhibiting β-catenin degradation to enhance TopoIIα activity

Anthracyclines are a class of conventional and commonly used frontline chemotherapy drugs to treat breast cancer. However, the anthracycline-based regimens can only reduce breast cancer mortality by 20–30%. Furthermore, there is no appropriate biomarker for predicting responses to this kind of chemotherapy currently. Here we report our findings that may fill this gap by showing the AQP1 (Aquaporin1) protein as a potential response predictor in the anthracycline chemotherapy. We showed that breast cancer patients with a high level of AQP1 expression who underwent the anthracycline treatment had a better clinical outcome relative to those with a low level of AQP1 expression. In the exploration of the underlying mechanisms, we found that the AQP1 and glycogen synthase kinase-3β (GSK3β) competitively interacted with the 12 armadillo repeats of β-catenin, followed by the inhibition of the β-catenin degradation that led to β-catenin’s accumulation in the cytoplasm and nuclear translocation. The nuclear β-catenin interacted with TopoIIα and enhanced TopoIIα’s activity, which resulted in a high sensitivity of breast cancer cells to anthracyclines. We also found, the miR-320a-3p can attenuate the anthracycline’s chemosensitivity by inhibiting the AQP1 expression. Taken together, our findings suggest the efficacy of AQP1 as a response predictor in the anthracycline chemotherapy. The application of our study includes, but is not limited to, facilitating screening of the most appropriate breast cancer patients (who have a high AQP1 expression) for better anthracycline chemotherapy and improved prognosis purposes.

Aquaporin 1 (AQP1) Expression in Healthy Dog Tears

Aquaporins (AQPs) are a family of thirteen membrane proteins that play an essential role in the transport of fluids across the cell plasma membrane. Recently, the expression of AQPs in different ocular tissues and their involvement in the pathophysiology of eye diseases, have garnered attention. Considering that literature on AQP expression in the lacrimal glands and their secretion is scarce, we aimed to characterise AQP1 expression in the tears of healthy dogs using two tear collection methods (Schirmer tear strips (STS) and ophthalmic sponges (OS)). Fifteen healthy dogs, free of ophthalmic diseases, were included in the study. Tear collection was performed by using STS in one eye and OS in the other. After the extraction of proteins from the tears, the expression of AQP1 was analysed by Western blotting. AQP1 was expressed as a band of 28 kDa. In addition, differences were observed in the expression of AQP1 and in the correlation between tear volume and protein concentration, in tears collected by the two different methods. Our results suggest that AQP1 has a specific role in tear secretion; further research is required to assess its particular role in the function of the ocular surface in eye physiology and pathology.

Aquaporin 1, 3, and 5 Patterns in Salivary Gland Mucoepidermoid Carcinoma: Expression in Surgical Specimens and an In Vitro Pilot Study

Salivary gland aquaporins (AQPs) are essential for the control of saliva production and maintenance of glandular structure. However, little is known of their role in salivary gland neoplasia. Salivary gland tumors comprise a heterogeneous group of lesions, featuring variable histological characteristics and diverse clinical behaviors. Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy. The aim of this study was to evaluate the expression of AQP1, AQP3, and AQP5 in 24 MEC samples by immunohistochemistry. AQP1 expression was observed in vascular endothelium throughout the tumor stroma. AQP3 was expressed in epidermoid and mucosal cells and AQP5 was expressed in mucosal cells of MEC. These proteins were expressed in the human MEC cell line UH-HMC-3A. Cellular ultrastructural aspects were analyzed by electron microscopy to certificate the tumor cell phenotype. In summary, our results show that, despite the fact that these molecules are important for salivary gland physiology, they may not play a distinct role in tumorigenesis in MEC. Additionally, the in vitro model may offer new possibilities to further investigate mechanisms of these molecules in tumor biology and their real significance in prognosis and possible target therapies.