BIG3 Inhibits the Estrogen-Dependent Nuclear Translocation of PHB2 via Multiple Karyopherin-Alpha Proteins in Breast Cancer Cells

We recently reported that brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) binds Prohibitin 2 (PHB2) in cytoplasm, thereby causing a loss of function of the PHB2 tumor suppressor in the nuclei of breast cancer cells. However, little is known regarding the mechanism by which BIG3 inhibits the nuclear translocation of PHB2 into breast cancer cells. Here, we report that BIG3 blocks the estrogen (E2)-dependent nuclear import of PHB2 via the karyopherin alpha (KPNA) family in breast cancer cells. We found that overexpressed PHB2 interacted with KPNA1, KPNA5, and KPNA6, thereby leading to the E2-dependent translocation of PHB2 into the nuclei of breast cancer cells. More importantly, knockdown of each endogenous KPNA by siRNA caused a significant inhibition of E2-dependent translocation of PHB2 in BIG3-depleted breast cancer cells, thereby enhancing activation of estrogen receptor alpha (ERα). These data indicated that BIG3 may block the KPNAs (KPNA1, KPNA5, and KPNA6) binding region(s) of PHB2, thereby leading to inhibition of KPNAs-mediated PHB2 nuclear translocation in the presence of E2 in breast cancer cells. Understanding this regulation of PHB2 nuclear import may provide therapeutic strategies for controlling E2/ERα signals in breast cancer cells.     

Epidermal Growth Factor Induces Internalization But Not Degradation of the Epidermal Growth Factor Receptor in a Human Breast Cancer Cell Line

Abstract

Metabolism of the epidermal growth factor (EGF) receptor was studied in the MDA-MB-231 human breast cancer cell line. As in normal fibroblasts the EGF receptor from MDA-MB-231 cells was synthesized from a M_r =160,000 precursor and tunicamycin treatment of cells resulted in accumulation of a M_r =130,000 polypeptide. Unlike normal fibroblasts in which a M_r =170,000 mature form of the EGF receptor was found, MDA-MB-231 cells contained a M_r =172,000 mature form. Addition of EGF to MDA-MB-231 cells led to rapid internalization of EGF receptors, however, internalization did not affect receptor half-life and receptors did not recycle to the cell surface. EGF receptors could be visualized by immunofluorescence and remained sequestered in intracellular membranous structures following internalization. EGF was degraded slowly by MDA-MB-231 cells relative to degradation of EGF by normal cells. A high endogenous level of in vivo phosphorylation of threonine 654 of the EGF receptor was found in MDA-MB-231 cells and treatment of cells with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) further stimulated phosphorylation of this residue. EGF induced receptor internalization resulted in dephosphorylation of threonine 654. The significance of these unusual properties of EGF receptor metabolism in MDA-MB-231 cells is discussed.     

Clinicopathologic Characteristics of Oestrogen Receptor-Positive/Progesterone Receptor-Negative/Her2-Negative Breast Cancer According to a Novel Definition of Negative Progesterone Receptor Status: A Large Population-Based Study from China

PurposeA lack of progesterone receptor (PgR) expression in oestrogen receptor-positive (ER+) tumours is associated with worse survival. PgR status is usually defined as positive or negative using 1% positive nuclei as a cut-off point. In this study, we aimed to assess the clinicopathologic characteristics of ER+/PgR-/HER2- tumours by comparing them with ER+/PgR+/HER2- tumours using a PgR cut-off point of 20% as a divisive criterion.MethodsWe analysed 1,522 patients with primary breast cancer who had undergone surgery at the Cancer Center of Fudan University between 2012 and 2014. Age, grade, tumour size, lymph node status and lymphovascular invasion were assessed. Multinomial logistic regression, linear regression and chi-square test models were applied to assess associations between ER, PR and clinical features.ResultsER+/PgR-/HER2- tumours showed poorer clinicopathologic characteristics relative to ER+/PgR+/HER2- tumours using a PgR threshold of 20% instead of 1%. The clinicopathologic characteristics did not differ between tumours with purely negative PgR expression and tumours with a PgR percentage ranging from 1% to 19%. The prognostic significance of PR expression appeared more pronounced in patients under a high Ki-67 status than those under a low Ki-67 status.ConclusionsBased on these findings, we propose the use of a novel threshold of 20% to define PgR status. Nevertheless, the impact of this new criterion on patient management and clinical treatment requires additional study.     

Identification of a Novel Cell Death Receptor Mediating IGFBP-3-induced Anti-tumor Effects in Breast and Prostate Cancer

Insulin-like growth factor-binding protein-3 (IGFBP-3), a major regulator of endocrine actions of IGFs, is a p53-regulated potent apoptotic factor and is significantly suppressed in a variety of cancers. Recent epidemiologic studies suggest that IGFBP-3 contributes to cancer risk protection in a variety of cancers, and a polymorphic variation of IGFBP-3 influences cancer risk, although other studies vary in their conclusions. Some antiproliferative actions of IGFBP-3 have been reported to be independent of IGFs, but the precise biochemical/molecular mechanisms of IGF-independent, antiproliferative actions of IGFBP-3 are largely unknown. Here we report a new cell death receptor, IGFBP-3R, that is a single-span membrane protein and binds specifically to IGFBP-3 but not other IGFBP species. Expression analysis of IGFBP-3 and IGFBP-3R indicates that the IGFBP-3/IGFBP-3R axis is impaired in breast and prostate cancer. We also provide evidence for anti-tumor effect of IGFBP-3R in vivo using prostate and breast cancer xenografts in athymic nude mice. Further in vitro studies demonstrate that IGFBP-3R mediates IGFBP-3-induced caspase-8-dependent apoptosis in various cancer cells. Knockdown of IGFBP-3R attenuated IGFBP-3-induced caspase activities and apoptosis, whereas overexpression of IGFBP-3R enhanced IGFBP-3 biological effects. IGFBP-3R physically interacts and activates caspase-8, and knockdown of caspase-8 expression or activity inhibited IGFBP-3/IGFBP-3R-induced apoptosis. Here, we propose that IGFBP-3R represents a novel cell death receptor and is essential for the IGFBP-3-induced apoptosis and tumor suppression. Thus, the IGFBP-3/IGFBP-3R axis may provide therapeutic and prognostic value for the treatment of cancer.     

Steroid Receptor Coactivator-3 (SRC-3/AIB1) as a Novel Therapeutic Target in Triple Negative Breast Cancer and Its Inhibition with a Phospho-Bufalin Prodrug

Triple negative breast cancer (TNBC) has the poorest prognosis of all types of breast cancer and currently lacks efficient targeted therapy. Chemotherapy is the traditional standard-of-care for TNBC, but is frequently accompanied by severe side effects. Despite the fact that high expression of steroid receptor coactivator 3 (SRC–3) is correlated with poor survival in estrogen receptor positive breast cancer patients, its role in TNBC has not been extensively investigated. Here, we show that high expression of SRC–3 correlates with both poor overall survival and post progression survival in TNBC patients, suggesting that SRC–3 can serve as a prognostic marker for TNBC. Furthermore, we demonstrated that bufalin, a SRC–3 small molecule inhibitor, when introduced even at nM concentrations, can significantly reduce TNBC cell viability and motility. However, because bufalin has minimal water solubility, its in vivo application is limited. Therefore, we developed a water soluble prodrug, 3-phospho-bufalin, to facilitate its in vivo administration. In addition, we demonstrated that 3-phospho-bufalin can effectively inhibit tumor growth in an orthotopic TNBC mouse model, suggesting its potential application as a targeted therapy for TNBC treatment.     

Identification of PDCL3 as a Novel Chaperone Protein Involved in the Generation of Functional VEGF Receptor 2

Angiogenesis, a hallmark step in tumor metastasis and ocular neovascularization, is driven primarily by the function of VEGF ligand on one of its receptors, VEGF receptor 2 (VEGFR-2). Central to the proliferation and ensuing angiogenesis of endothelial cells, the abundance of VEGFR-2 on the surface of endothelial cells is essential for VEGF to recognize and activate VEGFR-2. We have identified phosducin-like 3 (PDCL3, also known as PhLP2A), through a yeast two-hybrid system, as a novel protein involved in the stabilization of VEGFR-2 by serving as a chaperone. PDCL3 binds to the juxtamembrane domain of VEGFR-2 and controls the abundance of VEGFR-2 by inhibiting its ubiquitination and degradation. PDCL3 increases VEGF-induced tyrosine phosphorylation and is required for VEGFR-2-dependent endothelial capillary tube formation and proliferation. Taken together, our data provide strong evidence for the role of PDCL3 in angiogenesis and establishes the molecular mechanism by which it regulates VEGFR-2 expression and function.     

p21-activated Kinase 6 (PAK6) Inhibits Prostate Cancer Growth via Phosphorylation of Androgen Receptor and Tumorigenic E3 Ligase Murine Double Minute-2 (Mdm2)

The androgen receptor (AR) signaling pathway plays a crucial role in the development and growth of prostate malignancies. Regulation of AR homeostasis in prostate tumorigenesis has not yet been fully characterized. In this study, we demonstrate that p21-activated kinase 6 (PAK6) inhibits prostate tumorigenesis by regulating AR homeostasis. First, we demonstrated that in normal prostate epithelium, AR co-localizes with PAK6 in the cytoplasm and translocates into the nucleus in malignant prostate. Furthermore, AR phosphorylation at Ser-578 by PAK6 promotes AR-E3 ligase murine double minute-2 (Mdm2) association, causing AR degradation upon androgen stimuli. We also showed that PAK6 phosphorylates Mdm2 on Thr-158 and Ser-186, which is critical for AR ubiquitin-mediated degradation. Moreover, we found that Thr-158 collaborates with Ser-186 for AR-Mdm2 association and AR ubiquitin-mediated degradation as it facilitates PAK6-mediated AR homeostasis. PAK6 knockdown promotes prostate tumor growth in vivo. Interestingly, we found a strong inverse correlation between PAK6 and AR expression in the cytoplasm of prostate cancer cells. These observations indicate that PAK6 may be important for the maintenance of androgen-induced AR signaling homeostasis and in prostate malignancy, as well as being a possible new therapeutic target for AR-positive and hormone-sensitive prostate cancer.