Impact of hypoxia inducible factors on estrogen receptor expression in breast cancer cells.

In women breast cancer is still the most commonly diagnosed cancer. This type of cancer is classified as a hormone-dependent tumor. Estrogen receptor (ER) expression and functional status contribute to breast cancer development and progression. Another important factor associated with cancer is hypoxia which is defined as the state of reduced oxygen availability in tissues. Intratumoral hypoxia results in the activation of the hypoxia inducible factors (HIFs). HIFs are heterodimeric transcription factors involved in the regulation of many cellular processes, such as angiogenesis, anaerobic metabolism, cell proliferation/survival, and promotion of metastasis. In this study we evaluated the interplay between hypoxia, HIF stabilization and the ER-α/ß-ratio in several ER-positive breast cancer cell lines. Hypoxia was shown to inhibit ER expression in ER-positive breast cancer cells. Further experiments using the hypoxia mimetic CoCl2 and HIF-1α knockdown cells indicated that the influence of hypoxia on breast cancer cells involves other pathways than the molecular oxygen sensing pathway. Moreover, we demonstrated that MCF-7 cells in long-term culture lost part of their ability to respond to hypoxic incubation. Understanding the relationships between HIF, ER-α and ER-ß expression holds the promise of the development of new therapeutic agents and may provide future advances in prognosis.

Prolyl-4-Hydroxylase 2 Potentially Contributes to Hepatocellular Carcinoma-Associated Erythrocytosis by Maintaining Hepatocyte Nuclear Factor-4α Expression.

BACKGROUND: Increased red blood cell count (Erythrocytosis) is an important paraneoplastic syndrome of hepatocellular carcinoma (HCC) and is a significant risk factor for lethal lung artery thromboembolism. HCC-associated erythrocytosis is partially caused by the ability of several HCC cells to produce erythropoietin (EPO). Prolyl-4-hydroxylase 2 (PHD2) is an enzyme encoded by the gene EGLN1. The best-known function of PHD2 is to mediate the oxygen-dependent degradation of the labile α-subunit of hypoxia-inducible factor (HIF). However, there is increasing evidence that PHD2 also regulates HIF-independent pathways by interacting with other substrates. METHODS: In the EPO-producing human HCC cell line HepG2, the expression of PHD2 gene was silenced with siRNA. EPO production was estimated using quantitative PCR and ELISA. RESULTS: In HepG2 cells, PHD2 suppresses the activity of TGF-ß1 pathway and consequently maintains the expression of hepatocyte nuclear factor-4α (HNF-4α), an important transcription factor promoting the EPO expression in hepatocytes. PHD2 knockdown caused a marked reduction of EPO production. HIF seemed not to be involved in this biology. CONCLUSION: Our findings show that PHD2 represents a potential contributing factor for HCC-associated erythrocytosis. Selective inhibition of PHD2 in HCC cells might be considered as a new way to manage erythrocytosis in HCC patients.

Distinguishing the impact of distinct obstructive sleep apnea syndrome (OSAS) and obesity related factors on human monocyte subsets.

Obstructive sleep apnea syndrome (OSAS) and obesity go hand in hand in the majority of patients and both are associated with a systemic inflammation, immune disturbance and comorbidities such as cardiovascular disease. However, the unambiguous impact of OSAS and obesity on the individual inflammatory microenvironment and the immunological consequences of human monocytes has not been distinguished yet. Therefore, aim of this study was to investigate the impact of OSAS and obesity related factors on the inflammatory microenvironment by performing flow cytometric whole blood measurements of CD14/CD16 monocyte subsets in normal weight OSAS patients, patients with obesity but without OSAS, and patients with OSAS and obesity, compared to healthy donors. Moreover, explicitly OSAS and obesity related plasma levels of inflammatory mediators adiponectin, leptin, lipocalin and metalloproteinase-9 were determined and the influence of different OSAS and obesity related factors on cytokine secretion and expression of different adhesion molecules by THP-1 monocytes was analysed. Our data revealed a significant redistribution of circulating classical and intermediate monocytes in all three patient cohorts, but differential effects in terms of monocytic adhesion molecules CD11a, CD11b, CD11c, CX3CR1, CD29, CD49d, and plasma cytokine levels. These data were reflected by differential effects of OSAS and obesity related factors leptin, TNFα and hypoxia on THP-1 cytokine secretion patterns and expression of adhesion molecules CD11b and CD49d. In summary, our data revealed differential effects of OSAS and obesity, which underlines the need for a customized therapeutic regimen with respect to the individual weighting of these overlapping diseases.

Selinexor decreases HIF-1α via inhibition of CRM1 in human osteosarcoma and hepatoma cells associated with an increased radiosensitivity.

BACKGROUND: The nuclear pore complexes (NPCs) are built of about 30 different nucleoporins and act as key regulators of molecular traffic between the cytoplasm and the nucleus for sizeable proteins (> 40 kDa) which must enter the nucleus. Various nuclear transport receptors are involved in import and export processes of proteins through the nuclear pores. The most prominent nuclear export receptor is chromosome region maintenance 1 (CRM1), also known as exportin 1 (XPO1). One of its cargo proteins is the prolyl hydroxylase 2 (PHD2) which is involved in the initiation of the degradation of hypoxia-inducible factors (HIFs) under normoxia. HIFs are proteins that regulate the cellular adaptation under hypoxic conditions. They are involved in many aspects of cell viability and play an important role in the hypoxic microenvironment of cancer. In cancer, CRM1 is often overexpressed thus being a putative target for the development of new cancer therapies. The newly FDA-approved pharmaceutical Selinexor (KPT-330) selectively inhibits nuclear export via CRM1 and is currently tested in additional Phase-III clinical trials. In this study, we investigated the effect of CRM1 inhibition on the subcellular localization of HIF-1α and radiosensitivity. METHODS: Human hepatoma cells Hep3B and human osteosarcoma cells U2OS were treated with Selinexor. Intranuclear concentration of HIF-1α protein was measured using immunoblot analysis. Furthermore, cells were irradiated with 2-8 Gy after treatment with Selinexor compared to untreated controls. RESULTS: Selinexor significantly reduced the intranuclear level of HIF-1α protein in human hepatoma cells Hep3B and human osteosarcoma cells U2OS. Moreover, we demonstrated by clonogenic survival assays that Selinexor leads to dose-dependent radiosensitization in Hep3B-hepatoma and U2OS-osteosarcoma cells. CONCLUSION: Targeting the HIF pathway by Selinexor might be an attractive tool to overcome hypoxia-induced radioresistance.

The Nuclear Export Inhibitor Selinexor Inhibits Hypoxia Signaling Pathways And 3D Spheroid Growth Of Cancer Cells.

PURPOSE: The nucleocytoplasmic transport of macromolecules is critical for both cell physiology and pathophysiology. Exportin 1 (XPO1), the major nuclear export receptor, is involved in the cellular adaptation to reduced oxygen availability by controlling the nuclear activity of the hypoxia-inducible factors (HIFs). Recently, a specific inhibitor of XPO1, selinexor (KPT-330), has been identified that inhibits nuclear export of cargo proteins by binding to the XPO1 cargo-binding pocket. PATIENTS AND METHODS: We used different cancer cell lines from human tissues and evaluated the physiological activity of selinexor on the hypoxia response pathway in two-dimensional (2D) monolayer cell cultures in quantitative real-time (qRT)-PCR experiments and luciferase reporter gene assays. A three-dimensional (3D) tumor spheroid culture model of MCF-7 breast cancer cells was established to analyze the effect of selinexor on 3D tumor spheroid structure, formation and viability. RESULTS: Selinexor treatment reduces HIF-transcriptional activity and expression of the HIF-1 target gene solute carrier family 2 member 1 (SLC2A1). Moreover, 3D tumor spheroid structure, formation and viability are inhibited in response to selinexor-induced nuclear export inhibition. CONCLUSION: Here, we demonstrate the effect of specific XPO1-inhibition on the hypoxic response on the molecular level in 2D and 3D culture models of MCF-7 cells.

Nuclear-cytoplasmatic shuttling of proteins in control of cellular oxygen sensing.

In order to pass through the nuclear pore complex, proteins larger than ∼40 kDa require specific nuclear transport receptors. Defects in nuclear-cytoplasmatic transport affect fundamental processes such as development, inflammation and oxygen sensing. The transcriptional response to O2 deficiency is controlled by hypoxia-inducible factors (HIFs). These are heterodimeric transcription factors of each ∼100-120 kDa proteins, consisting of one out of three different O2-labile α subunits (primarily HIF-1α) and a more constitutive 1ß subunit. In the presence of O2, the α subunits are hydroxylated by specific prolyl-4-hydroxylase domain proteins (PHD1, PHD2, and PHD3) and an asparaginyl hydroxylase (factor inhibiting HIF-1, FIH-1). The prolyl hydroxylation causes recognition by von Hippel-Lindau tumor suppressor protein (pVHL), ubiquitination, and proteasomal degradation. The activity of the oxygen sensing machinery depends on dynamic intracellular trafficking. Nuclear import of HIF-1α and HIF-1ß is mainly mediated by importins α and ß (α/ß). HIF-1α can shuttle between nucleus and cytoplasm, while HIF-1ß is permanently inside the nucleus. pVHL is localized to both compartments. Nuclear import of PHD1 relies on a nuclear localization signal (NLS) and uses the classical import pathway involving importin α/ß receptors. PHD2 shows an atypical NLS, and its nuclear import does not occur via the classical pathway. PHD2-mediated hydroxylation of HIF-1α occurs predominantly in the cell nucleus. Nuclear export of PHD2 involves a nuclear export signal (NES) in the N-terminus and depends on the export receptor chromosome region maintenance 1 (CRM1). Nuclear import of PHD3 is mediated by importin α/ß receptors and depends on a non-classical NLS. Specific modification of the nuclear translocation of the three PHD isoforms could provide a promising strategy for the development of new therapeutic substances to tackle major diseases.