Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

EGF induces the translocation of EGF receptor (EGFR) from the cell surface to the nucleus where EGFR activates gene transcription through its binding to an AT-rich sequence (ATRS) of the target gene promoter. However, how EGFR, without a DNA-binding domain, can bind to the gene promoter is unclear. In the present study, we show that RNA helicase A (RHA) is an important mediator for EGFRinduced gene transactivation. EGF stimulates the interaction of EGFR with RHA in the nucleus of cancer cells. The EGFR/RHA complex then associates with the target gene promoter through binding of RHA to the ATRS of the target gene promoter to activate its transcription. Knockdown of RHA expression in cancer cells abrogates the binding of EGFR to the target gene promoter, thereby reducing EGF/EGFR-induced gene expression. In addition, interruption of EGFR-RHA interaction decreases the EGFR-induced promoter activity. Consistently, we observed a positive correlation of the nuclear expression of EGFR, RHA, and cyclin D1 in human breast cancer samples. These results indicate that RHA is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus.cyclin D1 | nuclear translocation | inducible nitric oxide synthase | transcription C ell surface EGF receptor (EGFR) has been shown to be localized in the nucleus (1-4). Nuclear EGFR has been demonstrated to contribute to cancer cell resistance to cetuximab and radiation treatment (5, 6) and to be negatively correlated with overall survival of patients with multiple cancer types (7-11). Moreover, nuclear EGFR interacts with signal transducer and activator of transcription 3 (STAT3), signal transducer and activator of transcription 5A (STAT5A), E2F1, DNA-dependent protein kinase (DNA-PK), and proliferating cell nuclear antigen (PCNA) and plays important roles in cell transformation, proliferation, and DNA repair and replication (12-16). Nuclear EGFR regulates gene expression by binding to an AT-rich sequence (ATRS) of the gene's promoter (13,16,17). Additionally, a recent unbiased protein-DNA interactome study indicates that EGFR is a DNAbinding protein (18). However, EGFR does not contain a DNAbinding domain, and evidence supporting direct binding of EGFR to the specific DNA sequence is lacking. Thus, identifying the DNA-binding partner for EGFR is crucial for understanding how EGFR regulates gene transcription in the nucleus.RNA helicase A (RHA), the human homolog of Drosophila maleless (MLE) that increases the transcription of male X-linked genes (19), is a multifunctional protein and is conserved in Drosophila and mammals (20)(21)(22). RHA belongs to the aspartateglutamate-alanine-aspartate (DEAD) box family of proteins and has the ability to bind to RNA and DNA (23,24). RHA regulates gene transcription by interacting with transcription factors (22) or by binding directly to the target gene promoter (25). Moreover, Drosophila MLE activates rox2 transcription by binding to an ATrich region of the gene promoter (26). Interestingly, this AT-rich region contains the previo...

Inhibitors against poly (ADP-ribose) polymerase (PARP) are promising targeted agents currently used to treat BRCA-mutant ovarian cancer and are in clinical trials for other cancer types, including BRCA-mutant breast cancer. To enhance the clinical response to PARP inhibitors (PARPi), understanding the mechanisms underlying PARP inhibitor sensitivity is urgently needed. Here, we show enhancer of zeste homolog 2 (EZH2), an enzyme which catalyzes H3 lysine trimethylation and associates with oncogenic function, contributes to PARPi sensitivity in breast cancer cells. Mechanistically, upon oxidative stress or alkylating DNA damage, PARP1 interacts with and attaches poly ADP-ribose (PAR) chains to EZH2. PARylation of EZH2 by PARP1 then induces PRC2 complex dissociation and EZH2 downregulation, which in turn reduces EZH2-mediated H3 tri-methylation. In contrast, inhibition of PARP by PARPi attenuates alkylating DNA damage-induced EZH2 downregulation, thereby promoting EZH2-mediated gene silencing and cancer stem cell property compared to PARPi-untreated cells. Moreover, the addition of an EZH2 inhibitor sensitizes the BRCA-mutant breast cells to PARPi. Thus, these results may provide a rationale for combining PARP and EZH2 inhibition as a therapeutic strategy for BRCA-mutated breast and ovarian cancers.

SummaryIron superoxide dismutases (FeSODs; FSDs) are primary antioxidant enzymes in Arabidopsis thaliana chloroplasts. The stromal FSD1 conferred the only detectable FeSOD activity, whereas the thylakoid membrane-and nucleoid-co-localized FSD2 and FSD3 double mutant showed arrested chloroplast development.FeSOD requires cofactor Fe for its activity, but its mechanism of activation is unclear. We used reversed-phase high-performance liquid chromatography (HPLC), gel filtration chromatography, LC-MS/MS, protoplast transient expression and virus-induced gene silencing (VIGS) analyses to identify and characterize a factor involved in FeSOD activation.We identified the chloroplast-localized co-chaperonin CHAPERONIN 20 (CPN20) as a mediator of FeSOD activation by direct interaction. The relationship between CPN20 and FeSOD was confirmed by in vitro experiments showing that CPN20 alone could enhance FSD1, FSD2 and FSD3 activity. The in vivo results showed that CPN20-overexpressing mutants and mutants with defective co-chaperonin activity increased FSD1 activity, without changing the chaperonin CPN60 protein level, and VIGS-induced downregulation of CPN20 also led to decreased FeSOD activity.Our findings reveal that CPN20 can mediate FeSOD activation in chloroplasts, a role independent of its known function in the chaperonin system.

Summary Posttranslational modifications of histones play fundamental roles in many biological functions. Specifically, histone H4-K20 methylation is critical in DNA synthesis and repair. However, little is known about how these functions are regulated by the upstream stimuli. Here, we identify a tyrosine phosphorylation site at Y72 of histone H4, which facilitates recruitment of histone methyltransferases (HMTases), SET8 and SUV4-20H, to enhance its K20 methylation, thereby promoting DNA synthesis and repair. Phosphorylation-defective histone H4 mutant is deficient in K20 methylation, leading to reduced DNA synthesis, delayed cell cycle progression, and decreased DNA repair ability. Disrupting the interaction between epidermal growth factor receptor (EGFR) and histone H4 by Y72 peptide significantly reduced tumor growth. Furthermore, EGFR expression clinically correlates with histone H4-Y72 phosphorylation, H4-K20 mono-methylation, and the Ki-67 proliferation marker. These findings uncover a mechanism by which EGFR transduces signal to chromatin to regulate DNA synthesis and repair.

In 3 experiments, the authors examined the role of adenosine regulation in escape deficits produced by earlier exposure to inescapable shock in rats (learned helplessness). Adenosine analogs injected before escape testing mimicked the effect of earlier inescapable shock, with the magnitude of the deficit varying with dose and drug specificity for A2 adenosine receptors. Agonist-induced and stress-induced escape deficits were eliminated by pretest treatment with the centrally acting adenosine receptor antagonist theophylline but not the peripheral antagonist 8-[p-sulfophenyl]-theophylline. Finally, preexposure to an ineffective number of inescapable shocks interacted in synergy with an ineffective pretest injection of adenosine agonist to maximize deficits in escape performance. These data implicate energy regulation and a central compensatory action by adenosine in the aspects of helplessness related to conservation-withdrawal.

In 3 experiments, the authors examined the effect of methylxanthine and amphetamine stimulants on deficits in shuttle-escape responding produced by earlier exposure to inescapable electric shock in rats. Caffeine completely reversed escape deficits in inescapably shocked rats when injected just before shuttle-escape testing but failed to prevent a test deficit when injected before shock pretreatment. Dose-response curves indicated that, whereas caffeine and theophylline were equally effective at reversing escape deficits, amphetamine not only failed to improve performance in preshocked rats but retarded escape in restrained (no-shock) controls. This amphetamine-induced deficit was reversed by cotreatment with caffeine. These data are discussed in terms of the role of adenosine receptor activation in helplessness and conservation-withdrawal.

The present study determined whether individual differences in neophobia during an open-field pretest predict vulnerability to inescapable electric shock, as measured by 2 tests of learned helplessness in rats. Shuttle-escape latencies and saccharin finickiness increased across groups that had received increasing numbers of inescapable shocks 24 hr earlier. Dispersion in the test measure as well as the percentage of variance explained by pretest neophobia were greater when no or few shocks were delivered in the interpolated stress phase. Pretest neophobia was positively related to stress vulnerability in both tests under these conditions. Further increments in stressor severity overwhelmed even the most stress-resistant rats, thereby decreasing dispersion in the test measure and eliminating the predictive value of pretest neophobia. This pattern of outcomes was more robust for the shuttle-escape measure of helplessness.