Loss of USF2 promotes proliferation, migration and mitophagy in a redox-dependent manner.

The upstream stimulatory factor 2 (USF2) is a transcription factor implicated in several cellular processes and among them, tumor development seems to stand out. However, the data with respect to the role of USF2 in tumor development are conflicting suggesting that it acts either as tumor promoter or suppressor. Here we show that absence of USF2 promotes proliferation and migration. Thereby, we reveal a previously unknown function of USF2 in mitochondrial homeostasis. Mechanistically, we demonstrate that deficiency of USF2 promotes survival by inducing mitophagy in a ROS-sensitive manner by activating both ERK1/2 and AKT. Altogether, this study supports USF2's function as tumor suppressor and highlights its novel role for mitochondrial function and energy homeostasis thereby linking USF2 to conditions such as insulin resistance, type-2 diabetes mellitus, and the metabolic syndrome.

Cyclin-Dependent Kinase 5 (CDK5)-Mediated Phosphorylation of Upstream Stimulatory Factor 2 (USF2) Contributes to Carcinogenesis.

The transcription factor USF2 is supposed to have an important role in tumor development. However, the regulatory mechanisms contributing to the function of USF2 are largely unknown. Cyclin-dependent kinase 5 (CDK5) seems to be of importance since high levels of CDK5 were found in different cancers associated with high USF2 expression. Here, we identified USF2 as a phosphorylation target of CDK5. USF2 is phosphorylated by CDK5 at two serine residues, serine 155 and serine 222. Further, phosphorylation of USF2 at these residues was shown to stabilize the protein and to regulate cellular growth and migration. Altogether, these results delineate the importance of the CDK5-USF2 interplay in cancer cells.

The Circadian Clock Protein CRY1 Is a Negative Regulator of HIF-1α.

The circadian clock and the hypoxia-signaling pathway are regulated by an integrated interplay of positive and negative feedback limbs that incorporate energy homeostasis and carcinogenesis. We show that the negative circadian regulator CRY1 is also a negative regulator of hypoxia-inducible factor (HIF). Mechanistically, CRY1 interacts with the basic-helix-loop-helix domain of HIF-1α via its tail region. Subsequently, CRY1 reduces HIF-1α half-life and binding of HIFs to target gene promoters. This appeared to be CRY1 specific because genetic disruption of CRY1, but not CRY2, affected the hypoxia response. Furthermore, CRY1 deficiency could induce cellular HIF levels, proliferation, and migration, which could be reversed by CRISPR/Cas9- or short hairpin RNA-mediated HIF knockout. Altogether, our study provides a mechanistic explanation for genetic association studies linking a disruption of the circadian clock with hypoxia-associated processes such as carcinogenesis.

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 MESSAGE: As(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.

PAI-1 modulates cell migration in a LRP1-dependent manner via ß-catenin and ERK1/2.

Plasminogen activator inhibitor-1 (PAI-1) is the major and most specific acting urokinase (uPA) and tissue plasminogen activator (tPA) inhibitor. Apart from its function in the fibrinolytic system, PAI-1 was also found to contribute to processes like tissue remodelling, angiogenesis, and tumour progression. However, the role of PAI-1 in those processes remains largely controversial with respect to the influence of PAI-1 on cell signalling pathways. Although PAI-1 does not possess its own cellular receptor, it can be bound to low-density lipoprotein receptor-related protein 1 (LRP1) which was proposed to modulate the ß-catenin pathway. Therefore, we used wild-type mouse embryonic fibroblasts (MEFs), and MEFs deficient of LRP1 to study PAI-1 as modulator of the ß-catenin pathway. We found that PAI-1 influences MEF proliferation and motility in a LRP1-dependent manner and that ß-catenin is important for that response. In addition, expression of ß-catenin and ß-catenin-dependent transcriptional activity were induced by PAI-1 in wild type MEFs, but not in LRP1-deficient cells. Moreover, PAI-1-induced ERK1/2 activation was more prominent in the LRP1-deficient cells and interestingly knockdown of ß-catenin abolished this effect. Together, the data of the current study show that PAI-1 can promote cell migration via LRP1-dependent activation of the ß-catenin and ERK1/2 MAPK pathway which may be important in stage-specific treatment of human diseases associated with high PAI-1 levels.

GSK3ß-dependent phosphorylation alters DNA binding, transactivity and half-life of the transcription factor USF2.

The upstream stimulatory factor 2 (USF2) is a regulator of important cellular processes and is supposed to have also a role during tumor development. However, the knowledge about the mechanisms that control the function of USF2 is limited. The data of the current study show that USF2 function is regulated by phosphorylation and identified GSK3ß as an USF2-phosphorylating kinase. The phosphorylation sites within USF2 could be mapped to serine 155 and threonine 230. In silico analyses of the 3-dimensional structure revealed that phosphorylation of USF2 by GSK3ß converts it to a more open conformation which may influence transactivity, DNA binding and target gene expression. Indeed, experiments with GSK-3ß-deficient cells revealed that USF2 transactivity, DNA binding and target gene expression were reduced upon lack of GSK3ß. Further, experiments with USF2 variants mimicking GSK3ß phosphorylated USF2 in GSK3ß-deficient cells showed that phosphorylation of USF2 by GSK3ß did not affect cell proliferation but increased cell migration. Together, this study reports a new mechanism by which USF2 may contribute to cancerogenesis.