The Role Of AQP3 In The Pathogenesis Of ER-positive Breast Cancer

Breast cancer is the most common cancer in women worldwide. The majority of breast cancers are estrogen-dependent for tumor progression. Endocrine therapy to block the ER pathway is highly effective, but its usefulness is limited by common intrinsic and acquired resistance. Well understanding of the mechanisms of cell migration, invasion and proliferation in breast cancer is important for investigating possible anti-tumor therapies.Aquaporins (AQPs) are a class of small integral membrane proteins distributed widely in organisms. AQP3, a member of the aquaglyceroporin subgroup, is involved in many biological functions, including urinary concentrating, skin wound healing and alimentary tract repairing. Recent studies showed the over-expressed AQP3 in several kinds of tumors, including breast cancer. However, whether the high-level expression of AQP3 in breast cancer tissues had any clinical implications in breast cancer patients is poorly understood. On the other hand, the mechanisms of AQP3 up-regulation in breast canceralso remain unclear.This study aimed to investigate whether AQP3 had any clinical implication in estrogen-receptor (ER) positive breast cancer, and explore the regulatory mechanisms of AQP3 in estrogen-related breast cancer progression. Here we show that AQP3 is an important enforcer of migration and invasion in breast cancer. We, for the first time, reported that ER-positive breast cancer tissues obtained from premenopausal patients had higher AQP3 expression when compared to those obtained from postmenopausal patients. Estrogen directly up-regulates AQP3 by activating ERE in the promoter of the AQP3 gene. Upregulation of AQP3 by E2 increases the cell migration and invasion in ER-positive breast cancer cells through regulating the expression of EMT-related genes and influencing reorganization of actin cytoskeleton.Part I Expression of AQP3 and its clinical significance in ER-positive breast cancer tissuesObjective:The objective of this part is to explore the expression of AQP3 and its clinical significance in ER-positive breast cancer tissues.Methods:1. qRT-PCR was used to analyse the expression of AQP3 in human breast cancer tissues and their corresponding normal tissues derived from 20 patients.2. Using immunohistochemistry and immunoreactivity scoring system (IRS), we examined the expression level of AQP3 protein and its location in breast invasive ductal carcinoma samples obtained from 56 ER-positive breast cancer patients.Results:1. The expression of AQP3 higher in breast cancer tissues than corresponding normal tissues.2. Higher AQP3 expression level was associated with higher histopathological grade and more lymph node metastasis in the patients with ER-positive breast cancer.3. AQP3 expression level in ER-positive breast cancer was higher in the premenopausal patients than which in the postmenopausal patients.Conclusion:Higher expression of AQP3 in ER-positive breast cancer might indicate higher malignity.Part II Estrogen directly upregulated AQP3 by activating ERE in the promoter of the AQP3 geneObjective:The objective of this part is to investigate whether E2 could regulate the expression of AQP3 in ER-positive breast cancer and its mechanism.Methods:1. Breast cancer cells were treated with E2 and/or estrogen receptor antagonist (ICI182780), and then were examined by Realtime-PCR and Western-blot for the expression of AQP3.2. Putative EREs in promoter of AQP3 gene were predicted, using the Regulatory Sequence Analysis Tools (RSAT).3. ChIP analysis and luciferase reporter assay were used to test the function of the putative EREs.Results:1. The E2-induced upregulation of AQP3 expression in ER-positive breast cancer cells was dose-dependent, and could be blocked by the estrogen receptor antagonist.2. The promoter of AQP3 gene contains a functional ERE motif, which could mediate E2-induced upregulation of AQP3 expression in ER-positive breast cancer cells.Conclusion:E2 directly increased expression of AQP3 in ER-positive breast cancer cells.Part III Upregulated AQP3 increases cell migration and invasion in ER-positive breast cancer cells through regulating the expression of EMT-related genes and the reorganization of actin cytoskeletonObjective:The objective of this part is to explore the role of AQP3 in E2-induced cell migration and invasion in ER-positive breast cancer.Methods:1. MTT assays were performed to analyze the effect of AQP3 on the E2-induced proliferation in ER-positive cells.2. Wound-healing assays were used to examine the effect of AQP3 on the E2-induced migration in ER-positive cells.3. Transwell assays were used to examine the role of AQP3 on the E2-induced invasion in ER-positive cells.4. Rhodamine-phalloidin stain was used to identify the effect of AQP3 in regulating the organization of actin cytoskeleton.5. Realtime-PCR was used to explore the effect of AQP3 in regulating the expression of EMT-related genes.Results:1. E2 significantly promoted cell migration, invasion and proliferation in ER-positive cells. Knockdown of AQP3 significantly reduced E2-promoted cell migration and invasion, not proliferation.2. E2 induced a marked reorganization of actin cytoskeleton in ER-positive cells, which characterized by formation of filopodia and change of the arrangement of stress fibers. Knockdown of AQP3 significantly inhibit E2-regulated reorganization of actin cytoskeleton.3. Overexpression of AQP3 significantly increased cell migrationand invasion, not proliferation.4. Overexpression of AQP3 induced a marked reorganization of actin cytoskeletonin cells, and, regulated the expression of several EMT-related genes.Conclusion:Upregulation of AQP3 by E2 increases the cell migration and invasion in ER-positive breast cancer cells through regulating the expression of EMT-related genes and influencing reorganization of actin cytoskeleton.