Functional Consequences and Regulation of Hypoxia-Inducible Factor-2alpha in Human Breast Cancer

摘要

Breast cancer is one of the most common cancers worldwide and the most common cancers in women. As in other types of cancer, breast cancer is a multi-factorial disease, consisting of various internal and external factors influencing the development and progression of the disease. Hypoxia and estrogen signalling have been established to be involved in the development of breast cancer. Hypoxia plays a crucial role in tumour development and metastasis. The key regulator in cellular hypoxic adaptation is hypoxia-inducible factor (HIF), which consists of the constitutively expressed alpha-subunit and the oxygen-sensitive alpha-subunit. HIF-alpha can be classified into three different isoforms: HIF-1alpha, HIF-2alpha and HIF-3alpha, among which HIF-2alpha is one of the less well characterised isoforms. Although HIF-2alpha has long been reported, the functional consequences and regulation of HIF-2alpha are not well understood. Wnt-1-inducible signalling protein 2 (WISP-2) is one of the few known genes to be selectively induced by HIF-2alpha. Clinically, patients expressing both WISP-2 and AREG have been reported to possess significant overall survival. Four hypoxia response elements (HREs) were discovered within the WISP-2 promoter, in which two HREs were necessary for WISP-2 regulation by HIF-2alpha. Moreover, the two active HREs are located within microsatellite (MS) regions which have been established to play an essential role in tumour progression. A negative correlation between WISP-2 expression and tumour macrophage numbers was observed, supporting the better prognosis of patients expressing WISP-2, as lower macrophage infiltration means lower cancer aggressiveness. Furthermore, proliferation, anchorage-independent colony formation, and scratch recovery assays suggest the reduction in the tumourigenic properties of MCF-7 breast cancer cells, in line with the tumour-suppressor like characteristic of WISP-2 and its regulation by HIF-2alpha. Besides hypoxia, the involvement of estrogen (E2) in the development and progression of breast cancer is well established. In this study, treatment of E2 on estrogen receptor (ER)-positive breast cancer cell lines resulted in HIF-2alpha mRNA and protein downregulation. The involvement of ER was confirmed with the absence of HIF-2alpha downregulation upon treatment of E2 on ER-negative breast cancer cell lines. Moreover, the role of ER alpha in the downregulation was further established by utilising both pharmacological and siRNA approaches. An investigation of 690 samples from breast cancer patients revealed the association of HIF-2alpha tumour levels with a better prognosis in the triple-positive patients, which was validated with less pronounced HIF-2alpha downregulation on a triple-positive breast cancer cell line, BT474. To explore the potential molecular mechanism, trichostatin A (TSA), a histone deacetylate (HDAC) inhibitor, was added together with E2. The blunting of HIF-2alpha downregulation in the presence of TSA indicated the transcriptional origin of HIF-2alpha downregulation. In silico analysis revealed the presence of four bound estrogen response elements (EREs) within the first intron of EPAS1, in which one ERE was activated upon treatment with E2. In conclusion, this thesis reports functional consequences of HIF-2alpha in human breast cancer by its regulation of WISP-2. Furthermore, the novel regulation of HIF-2alpha by estrogen signalling was described, which might partially explain the association of high HIF-2alpha in triple-positive breast cancer patients with better prognosis.