Daniela Mennerich

Antioxidant responses and cellular adjustments to oxidative stress

Redox biological reactions are now accepted to bear the Janus faceted feature of promoting both physiological signaling responses and pathophysiological cues. Endogenous antioxidant molecules participate in both scenarios. This review focuses on the role of crucial cellular nucleophiles, such as glutathione, and their capacity to interact with oxidants and to establish networks with other critical enzymes such as peroxiredoxins. We discuss the importance of the Nrf2-Keap1 pathway as an example of a transcriptional antioxidant response and we summarize transcriptional routes related to redox activation. As examples of pathophysiological cellular and tissular settings where antioxidant responses are major players we highlight endoplasmic reticulum stress and ischemia reperfusion. Topologically confined redox-mediated post-translational modifications of thiols are considered important molecular mechanisms mediating many antioxidant responses, whereas redox-sensitive microRNAs have emerged as key players in the posttranscriptional regulation of redox-mediated gene expression. Understanding such mechanisms may provide the basis for antioxidant-based therapeutic interventions in redox-related diseases.

Nutritional Countermeasures Targeting Reactive Oxygen Species in Cancer: From Mechanisms to Biomarkers and Clinical Evidence

Reactive oxygen species (ROS) exert various biological effects and contribute to signaling events during physiological and pathological processes. Enhanced levels of ROS are highly associated with different tumors, a Western lifestyle, and a nutritional regime. The supplementation of food with traditional antioxidants was shown to be protective against cancer in a number of studies both in vitro and in vivo. However, recent large-scale human trials in well-nourished populations did not confirm the beneficial role of antioxidants in cancer, whereas there is a well-established connection between longevity of several human populations and increased amount of antioxidants in their diets. Although our knowledge about ROS generators, ROS scavengers, and ROS signaling has improved, the knowledge about the direct link between nutrition, ROS levels, and cancer is limited. These limitations are partly due to lack of standardized reliable ROS measurement methods, easily usable biomarkers, knowledge of ROS action in cellular compartments, and individual genetic predispositions. The current review summarizes ROS formation due to nutrition with respect to macronutrients and antioxidant micronutrients in the context of cancer and discusses signaling mechanisms, used biomarkers, and its limitations along with large-scale human trials.

Hypoxia-Inducible Factors (HIFs) and Phosphorylation: Impact on Stability, Localization, and Transactivity

The hypoxia-inducible factor α-subunits (HIFα) are key transcription factors in the mammalian response to oxygen deficiency. The HIFα regulation in response to hypoxia occurs primarily on the level of protein stability due to posttranslational hydroxylation and proteasomal degradation. However, HIF α-subunits also respond to various growth factors, hormones, or cytokines under normoxia indicating involvement of different kinase pathways in their regulation. Because these proteins participate in angiogenesis, glycolysis, programmed cell death, cancer, and ischemia, HIFα regulating kinases are attractive therapeutic targets. Although numerous kinases were reported to regulate HIFα indirectly, direct phosphorylation of HIFα affects HIFα stability, nuclear localization, and transactivity. Herein, we review the role of phosphorylation-dependent HIFα regulation with emphasis on protein stability, subcellular localization, and transactivation.

PHD1 regulates p53-mediated colorectal cancer chemoresistance

Overcoming resistance to chemotherapy is a major challenge in colorectal cancer (CRC) treatment, especially since the underlying molecular mechanisms remain unclear. We show that silencing of the prolyl hydroxylase domain protein PHD1, but not PHD2 or PHD3, prevents p53 activation upon chemotherapy in different CRC cell lines, thereby inhibiting DNA repair and favoring cell death. Mechanistically, PHD1 activity reinforces p53 binding to p38α kinase in a hydroxylation‐dependent manner. Following p53–p38α interaction and chemotherapeutic damage, p53 can be phosphorylated at serine 15 and thus activated. Active p53 allows nucleotide excision repair by interacting with the DNA helicase XPB, thereby protecting from chemotherapy‐induced apoptosis. In accord with this observation, PHD1 knockdown greatly sensitizes CRC to 5‐FU in mice. We propose that PHD1 is part of the resistance machinery in CRC, supporting rational drug design of PHD1‐specific inhibitors and their use in combination with chemotherapy.

The mTOR and PP2A Pathways Regulate PHD2 Phosphorylation to Fine-Tune HIF1α Levels and Colorectal Cancer Cell Survival under Hypoxia

Summary Oxygen-dependent HIF1α hydroxylation and degradation are strictly controlled by PHD2. In hypoxia, HIF1α partly escapes degradation because of low oxygen availability. Here, we show that PHD2 is phosphorylated on serine 125 (S125) by the mechanistic target of rapamycin (mTOR) downstream kinase P70S6K and that this phosphorylation increases its ability to degrade HIF1α. mTOR blockade in hypoxia by REDD1 restrains P70S6K and unleashes PP2A phosphatase activity. Through its regulatory subunit B55α, PP2A directly dephosphorylates PHD2 on S125, resulting in a further reduction of PHD2 activity that ultimately boosts HIF1α accumulation. These events promote autophagy-mediated cell survival in colorectal cancer (CRC) cells. B55α knockdown blocks neoplastic growth of CRC cells in vitro and in vivo in a PHD2-dependent manner. In patients, CRC tissue expresses higher levels of REDD1, B55α, and HIF1α but has lower phospho-S125 PHD2 compared with a healthy colon. Our data disclose a mechanism of PHD2 regulation that involves the mTOR and PP2A pathways and controls tumor growth.

The human mitochondrial DNA depletion syndrome gene MPV17 encodes a non-selective channel that modulates membrane potential

Background: MPV17 is a mitochondrial inner membrane protein with unknown function. Results: Recombinant human MPV17 shows highly regulated channel-forming activity; the mitochondrial membrane potential and the reactive oxygen species formation were elevated in embryonic fibroblasts from Mpv17−/− mice. Conclusion: MPV17 functions as a non-selective channel modulating the membrane potential to preserve mitochondrial homeostasis. Significance: Our data are important for understanding the role of MPV17 protein under physiological and pathological conditions. The human MPV17-related mitochondrial DNA depletion syndrome is an inherited autosomal recessive disease caused by mutations in the inner mitochondrial membrane protein MPV17. Although more than 30 MPV17 gene mutations were shown to be associated with mitochondrial DNA depletion syndrome, the function of MPV17 is still unknown. Mice deficient in Mpv17 show signs of premature aging. In the present study, we used electrophysiological measurements with recombinant MPV17 to reveal that this protein forms a non-selective channel with a pore diameter of 1.8 nm and located the channels selectivity filter. The channel was weakly cation-selective and showed several subconductance states. Voltage-dependent gating of the channel was regulated by redox conditions and pH and was affected also in mutants mimicking a phosphorylated state. Likewise, the mitochondrial membrane potential (Δψm) and the cellular production of reactive oxygen species were higher in embryonic fibroblasts from Mpv17−/− mice. However, despite the elevated Δψm, the Mpv17-deficient mitochondria showed signs of accelerated fission. Together, these observations uncover the role of MPV17 as a Δψm-modulating channel that apparently contributes to mitochondrial homeostasis under different conditions.

Differential transcriptional regulation of hypoxia-inducible factor-1α by arsenite under normoxia and hypoxia: involvement of Nrf2

Arsenite (As(III)) is widely distributed in nature and can be found in water, food, and air. There is significant evidence that exposure to As(III) is associated with human cancers originated from liver, lung, skin, bladder, kidney, and prostate. Hypoxia plays a role in tumor growth and aggressiveness; adaptation to it is, at least to a large extent, mediated by hypoxia-inducible factor-1α (HIF-1α). In the current study, we investigated As(III) effects on HIF-1α under normoxia and hypoxia in the hepatoma cell line HepG2. We found that As(III) increased HIF-1α protein levels under normoxia while the hypoxia-mediated induction of HIF1α was reduced. Thereby, the As(III) effects on HIF-1α were dependent on both, transcriptional regulation via the transcription factor Nrf2 mediated by NOX4, PI3K/Akt, and ERK1/2 as well as by modulation of HIF-1α protein stability. In line, the different effects of As(III) via participation of HIF-1α and Nrf2 were also seen in tube formation assays with endothelial cells where knockdown of Nrf2 and HIF-1α abolished As(III) effects. Overall, the present study shows that As(III) is a potent inducer of HIF-1α under normoxia but not under hypoxia which may explain, in part, its carcinogenic as well as anti-carcinogenic actions.Key messageAs(III) increased HIF-1α under normoxia but reduced its hypoxia-dependent induction.The As(III) effects on HIF-1α were dependent on ROS, NOX4, PI3K/Akt, and ERK1/2.The As(III) effects under normoxia involved transcriptional regulation via Nrf2.Knockdown of Nrf2 and HIF-1α abolished As(III) effects in tube formation assays.The data may partially explain As(III)’s carcinogenic and anti-carcinogenic actions.

Direct phosphorylation events involved in HIF-α regulation: the role of GSK-3β

Hypoxia-inducible factors (HIFs), consisting of α- and β-subunits, are critical regulators of the transcriptional response to hypoxia under both physiological and pathological conditions. To a large extent, the protein stability and the recruitment of coactivators to the C-terminal transactivation domain of the HIF α-subunits determine overall HIF activity. The regulation of HIF α-subunit protein stability and coactivator recruitment is mainly achieved by oxygen-dependent posttranslational hydroxylation of conserved proline and asparagine residues, respectively. Under hypoxia, the hydroxylation events are inhibited and HIF α-subunits stabilize, translocate to the nucleus, dimerize with the β-subunits, and trigger a transcriptional response. However, under normal oxygen conditions, HIF α-subunits can be activated by various growth and coagulation factors, hormones, cytokines, or stress factors implicating the involvement of different kinase pathways in their regulation, thereby making HIF-α-regulating kinases attractive therapeutic targets. From the kinases known to regulate HIF α-subunits, only a few phosphorylate HIF-α directly. Here, we review the direct phosphorylation of HIF-α with an emphasis on the role of glycogen synthase kinase-3β and the consequences for HIF-1α function.

Non-electron transfer chain mitochondrial defects differently regulate HIF-1α degradation and transcription

Mitochondria are the main consumers of molecular O2 in a cell as well as an abundant source of reactive oxygen species (ROS). Both, molecular oxygen and ROS are powerful regulators of the hypoxia-inducible factor-1α-subunit (HIF-α). While a number of mechanisms in the oxygen-dependent HIF-α regulation are quite well known, the view with respect to mitochondria is less clear. Several approaches using pharmacological or genetic tools targeting the mitochondrial electron transport chain (ETC) indicated that ROS, mainly formed at the Rieske cluster of complex III of the ETC, are drivers of HIF-1α activation. However, studies investigating non-ETC located mitochondrial defects and their effects on HIF-1α regulation are scarce, if at all existing. Thus, in the present study we examined three cell lines with non-ETC mitochondrial defects and focused on HIF-1α degradation and transcription, target gene expression, as well as ROS levels. We found that cells lacking the key enzyme 2-enoyl thioester reductase/mitochondrial enoyl-CoA reductase (MECR), and cells lacking manganese superoxide dismutase (MnSOD) showed a reduced induction of HIF-1α under long-term (20 h) hypoxia. By contrast, cells lacking the mitochondrial DNA depletion syndrome channel protein Mpv17 displayed enhanced levels of HIF-1α already under normoxic conditions. Further, we show that ROS do not exert a uniform pattern when mediating their effects on HIF-1α, although all mitochondrial defects in the used cell types increased ROS formation. Moreover, all defects caused a different HIF-1α regulation via promoting HIF-1α degradation as well as via changes in HIF-1α transcription. Thereby, MECR- and MnSOD-deficient cells showed a reduction in HIF-1α mRNA levels whereas the Mpv17 lacking cells displayed enhanced HIF-1α mRNA levels under normoxia and hypoxia. Altogether, our study shows for the first time that mitochondrial defects which are not related to the ETC and Krebs cycle contribute differently to HIF-1α regulation by affecting HIF-1α degradation and HIF-1α transcription where ROS play not a major role.

USP28 Deficiency Promotes Breast and Liver Carcinogenesis as well as Tumor Angiogenesis in a HIF-independent Manner

Recent studies suggest that the ubiquitin-specific protease USP28 plays an important role in cellular repair and tissue remodeling, which implies that it has a direct role in carcinogenesis. The carcinogenic potential of USP28 was investigated in a comprehensive manner using patients, animal models, and cell culture. The findings demonstrate that overexpression of USP28 correlates with a better survival in patients with invasive ductal breast carcinoma. Mouse xenograft experiments with USP28-deficient breast cancer cells also support this view. Furthermore, lack of USP28 promotes a more malignant state of breast cancer cells, indicated by an epithelial-to-mesenchymal (EMT) transition, elevated proliferation, migration, and angiogenesis as well as a decreased adhesion. In addition to breast cancer, lack of USP28 in mice promoted an earlier onset and a more severe tumor formation in a chemical-induced liver cancer model. Mechanistically, the angio- and carcinogenic processes driven by the lack of USP28 appeared to be independent of HIF-1α, p53, and 53BP1. Implications: The findings of this study are not limited to one particular type of cancer but are rather applicable for carcinogenesis in a more general manner. The obtained data support the view that USP28 is involved in tumor suppression and has the potential to be a prognostic marker. Mol Cancer Res; 16(6); 1000–12. ©2018 AACR.