PKCε promotes oncogenic functions of ATF2 in the nucleus while blocking its apoptotic function at mitochondria.

The transcription factor ATF2 elicits oncogenic activities in melanoma and tumor suppressor activities in nonmalignant skin cancer. Here, we identify that ATF2 tumor suppressor function is determined by its ability to localize at the mitochondria, where it alters membrane permeability following genotoxic stress. The ability of ATF2 to reach the mitochondria is determined by PKCε, which directs ATF2 nuclear localization. Genotoxic stress attenuates PKCε effect on ATF2; enables ATF2 nuclear export and localization at the mitochondria, where it perturbs the HK1-VDAC1 complex; increases mitochondrial permeability; and promotes apoptosis. Significantly, high levels of PKCε, as seen in melanoma cells, block ATF2 nuclear export and function at the mitochondria, thereby attenuating apoptosis following exposure to genotoxic stress. In melanoma tumor samples, high PKCε levels associate with poor prognosis. Overall, our findings provide the framework for understanding how subcellular localization enables ATF2 oncogenic or tumor suppressor functions.

Lipid metabolism: new twists to the Yin and Yang of PKM2 in cancer.

While canonical and non-canonical functions of pyruvate kinase M2 (PKM2) are recognized to mediate often-opposing roles in cancer, its contribution to cellular and systemic fatty acid homeostasis remains poorly understood. A new study by Liu et al (2021) uncovers ER transmembrane protein TMEM33 as a novel target of PKM2, which is essential for regulation of cancer cell cholesterol metabolism. These findings highlight the diversity of tissue-specific functions of PKM2 and potential implications for cancer treatment.

Translation reprogramming is an evolutionarily conserved driver of phenotypic plasticity and therapeutic resistance in melanoma.

The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance. Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown. In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance. However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood. Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B. ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors. However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion. Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance. Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.

MAPK signaling regulates c-MYC for melanoma cell adaptation to asparagine restriction.

Amino acid restriction is among promising potential cancer treatment strategies. However, cancer cells employ a multitude of mechanisms to mount resistance to amino acid restriction, which impede the latter's clinical development. Here we show that MAPK signaling activation in asparagine-restricted melanoma cells impairs GSK3-ß-mediated c-MYC degradation. In turn, elevated c-MYC supports ATF4 translational induction by enhancing the expression of the amino acid transporter SLC7A5, increasing the uptake of essential amino acids, and the subsequent maintenance of mTORC1 activity in asparagine-restricted melanoma cells. Blocking the MAPK-c-MYC-SLC7A5 signaling axis cooperates with asparagine restriction to effectively suppress melanoma cell proliferation. This work reveals a previously unknown axis of cancer cell adaptation to asparagine restriction and informs mechanisms that may be targeted for enhanced therapeutic efficacy of asparagine limiting strategies.

Correction: Regulation of ATG4B Stability by RNF5 Limits Basal Levels of Autophagy and Influences Susceptibility to Bacterial Infection.

[This corrects the article DOI: 10.1371/journal.pgen.1003007.].

Targeting the Warburg effect via LDHA inhibition engages ATF4 signaling for cancer cell survival.

Nutrient restriction reprograms cellular signaling and metabolic network to shape cancer phenotype. Lactate dehydrogenase A (LDHA) has a key role in aerobic glycolysis (the Warburg effect) through regeneration of the electron acceptor NAD+ and is widely regarded as a desirable target for cancer therapeutics. However, the mechanisms of cellular response and adaptation to LDHA inhibition remain largely unknown. Here, we show that LDHA activity supports serine and aspartate biosynthesis. Surprisingly, however, LDHA inhibition fails to impact human melanoma cell proliferation, survival, or tumor growth. Reduced intracellular serine and aspartate following LDHA inhibition engage GCN2-ATF4 signaling to initiate an expansive pro-survival response. This includes the upregulation of glutamine transporter SLC1A5 and glutamine uptake, with concomitant build-up of essential amino acids, and mTORC1 activation, to ameliorate the effects of LDHA inhibition. Tumors with low LDHA expression and melanoma patients acquiring resistance to MAPK signaling inhibitors, which target the Warburg effect, exhibit altered metabolic gene expression reminiscent of the ATF4-mediated survival signaling. ATF4-controlled survival mechanisms conferring synthetic vulnerability to the approaches targeting the Warburg effect offer efficacious therapeutic strategies.

SIP/CacyBP promotes autophagy by regulating levels of BRUCE/Apollon, which stimulates LC3-I degradation.

BRUCE/Apollon is a membrane-associated inhibitor of apoptosis protein that is essential for viability and has ubiquitin-conjugating activity. On initiation of apoptosis, the ubiquitin ligase Nrdp1/RNF41 promotes proteasomal degradation of BRUCE. Here we demonstrate that BRUCE together with the proteasome activator PA28γ causes proteasomal degradation of LC3-I and thus inhibits autophagy. LC3-I on the phagophore membrane is conjugated to phosphatidylethanolamine to form LC3-II, which is required for the formation of autophagosomes and selective recruitment of substrates. SIP/CacyBP is a ubiquitination-related protein that is highly expressed in neurons and various tumors. Under normal conditions, SIP inhibits the ubiquitination and degradation of BRUCE, probably by blocking the binding of Nrdp1 to BRUCE. On DNA damage by topoisomerase inhibitors, Nrdp1 causes monoubiquitination of SIP and thus promotes apoptosis. However, on starvation, SIP together with Rab8 enhances the translocation of BRUCE into the recycling endosome, formation of autophagosomes, and degradation of BRUCE by optineurin-mediated autophagy. Accordingly, deletion of SIP in cultured cells reduces the autophagic degradation of damaged mitochondria and cytosolic protein aggregates. Thus, by stimulating proteasomal degradation of LC3-I, BRUCE also inhibits autophagy. Conversely, SIP promotes autophagy by blocking BRUCE-dependent degradation of LC3-I and by enhancing autophagosome formation and autophagic destruction of BRUCE. These actions of BRUCE and SIP represent mechanisms that link the regulation of autophagy and apoptosis under different conditions.

Targeting eIF4F translation initiation complex with SBI-756 sensitises B lymphoma cells to venetoclax.

BACKGROUND: The BCL2 inhibitor venetoclax has shown efficacy in several hematologic malignancies, with the greatest response rates in indolent blood cancers such as chronic lymphocytic leukaemia. There is a lower response rate to venetoclax monotherapy in diffuse large B-cell lymphoma (DLBCL). METHODS: We tested inhibitors of cap-dependent mRNA translation for the ability to sensitise DLBCL and mantle cell lymphoma (MCL) cells to apoptosis by venetoclax. We compared the mTOR kinase inhibitor (TOR-KI) MLN0128 with SBI-756, a compound targeting eukaryotic translation initiation factor 4G1 (eIF4G1), a scaffolding protein in the eIF4F complex. RESULTS: Treatment of DLBCL and MCL cells with SBI-756 synergised with venetoclax to induce apoptosis in vitro, and enhanced venetoclax efficacy in vivo. SBI-756 prevented eIF4E-eIF4G1 association and cap-dependent translation without affecting mTOR substrate phosphorylation. In TOR-KI-resistant DLBCL cells lacking eIF4E binding protein-1, SBI-756 still sensitised to venetoclax. SBI-756 selectively reduced translation of mRNAs encoding ribosomal proteins and translation factors, leading to a reduction in protein synthesis rates in sensitive cells. When normal lymphocytes were treated with SBI-756, only B cells had reduced viability, and this correlated with reduced protein synthesis. CONCLUSIONS: Our data highlight a novel combination for treatment of aggressive lymphomas, and establishes its efficacy and selectivity using preclinical models.

The mTORC1/4E-BP/eIF4E Axis Promotes Antibody Class Switching in B Lymphocytes.

During an adaptive immune response, activated mature B cells give rise to Ab-secreting plasma cells to fight infection. B cells undergo Ab class switching to produce different classes of Abs with varying effector functions. The mammalian/mechanistic target of rapamycin (mTOR) signaling pathway is activated during this process, and disrupting mTOR complex 1 (mTORC1) in B cells impairs class switching by a poorly understood mechanism. In particular, it is unclear which mTORC1 downstream substrates control this process. In this study, we used an in vitro murine model in which the mTORC1 inhibitor rapamycin, when added after a B cell has committed to divide, suppresses class switching while preserving proliferation. Investigation of mTORC1 substrates revealed a role for eukaryotic translation initiation factor 4E (eIF4E) and eIF4E-binding proteins in class switching. Mechanistically, we show that genetic or pharmacological disruption of eIF4E binding to eIF4G reduced cap-dependent translation, which specifically affected the expression of activation-induced cytidine deaminase protein but not Aicda mRNA. This translational impairment decreased Ab class switching independently of proliferation. These results uncover a previously undescribed role for mTORC1 and the eIF4E-binding proteins/eIF4E axis in activation-induced cytidine deaminase protein expression and Ab class switching in mouse B cells, suggesting that cap-dependent translation regulates key steps in B cell differentiation.

Translational homeostasis via the mRNA cap-binding protein, eIF4E.

Translational control of gene expression plays a key role in many biological processes. Consequently, the activity of the translation apparatus is under tight homeostatic control. eIF4E, the mRNA 5' cap-binding protein, facilitates cap-dependent translation and is a major target for translational control. eIF4E activity is controlled by a family of repressor proteins, termed 4E-binding proteins (4E-BPs). Here, we describe the surprising finding that despite the importance of eIF4E for translation, a drastic knockdown of eIF4E caused only minor reduction in translation. This conundrum can be explained by the finding that 4E-BP1 is degraded in eIF4E-knockdown cells. Hypophosphorylated 4E-BP1, which binds to eIF4E, is degraded, whereas hyperphosphorylated 4E-BP1 is refractory to degradation. We identified the KLHL25-CUL3 complex as the E3 ubiquitin ligase, which targets hypophosphorylated 4E-BP1. Thus, the activity of eIF4E is under homeostatic control via the regulation of the levels of its repressor protein 4E-BP1 through ubiquitination.

UPR, autophagy, and mitochondria crosstalk underlies the ER stress response.

Cellular stress, induced by external or internal cues, activates several well-orchestrated processes aimed at either restoring cellular homeostasis or committing to cell death. Those processes include the unfolded protein response (UPR), autophagy, hypoxia, and mitochondrial function, which are part of the global endoplasmic reticulum (ER) stress (ERS) response. When one of the ERS elements is impaired, as often occurs under pathological conditions, overall cellular homeostasis may be perturbed. Further, activation of the UPR could trigger changes in mitochondrial function or autophagy, which could modulate the UPR, exemplifying crosstalk processes. Among the numerous factors that control the magnitude or duration of these processes are ubiquitin ligases, which govern overall cellular stress outcomes. Here we summarize crosstalk among the fundamental processes governing ERS responses.

Fine-tuning of Drp1/Fis1 availability by AKAP121/Siah2 regulates mitochondrial adaptation to hypoxia.

Defining the mechanisms underlying the control of mitochondrial fusion and fission is critical to understanding cellular adaptation to diverse physiological conditions. Here we demonstrate that hypoxia induces fission of mitochondrial membranes, dependent on availability of the mitochondrial scaffolding protein AKAP121. AKAP121 controls mitochondria dynamics through PKA-dependent inhibitory phosphorylation of Drp1 and PKA-independent inhibition of Drp1-Fis1 interaction. Reduced availability of AKAP121 by the ubiquitin ligase Siah2 relieves Drp1 inhibition by PKA and increases its interaction with Fis1, resulting in mitochondrial fission. High AKAP121 levels, seen in cells lacking Siah2, attenuate fission and reduce apoptosis of cardiomyocytes under simulated ischemia. Infarct size and degree of cell death were reduced in Siah2(-/-) mice subjected to myocardial infarction. Inhibition of Siah2 or Drp1 in hatching C. elegans reduces their life span. Through modulating Fis1/Drp1 complex availability, our studies identify Siah2 as a key regulator of hypoxia-induced mitochondrial fission and its physiological significance in ischemic injury and nematode life span.

ATF2, a paradigm of the multifaceted regulation of transcription factors in biology and disease.

Stringent transcriptional regulation is crucial for normal cellular biology and organismal development. Perturbations in the proper regulation of transcription factors can result in numerous pathologies, including cancer. Thus, understanding how transcription factors are regulated and how they are dysregulated in disease states is key to the therapeutic targeting of these factors and/or the pathways that they regulate. Activating transcription factor 2 (ATF2) has been studied in a number of developmental and pathological conditions. Recent findings have shed light on the transcriptional, post-transcriptional, and post-translational regulatory mechanisms that influence ATF2 function, and thus, the transcriptional programs coordinated by ATF2. Given our current knowledge of its multiple levels of regulation and function, ATF2 represents a paradigm for the mechanistic complexity that can regulate transcription factor function. Thus, increasing our understanding of the regulation and function of ATF2 will provide insights into fundamental regulatory mechanisms that influence how cells integrate extracellular and intracellular signals into a genomic response through transcription factors. Characterization of ATF2 dysfunction in the context of pathological conditions, particularly in cancer biology and response to therapy, will be important in understanding how pathways controlled by ATF2 or other transcription factors might be therapeutically exploited. In this review, we provide an overview of the currently known upstream regulators and downstream targets of ATF2.

Fine tuning of the UPR by the ubiquitin ligases Siah1/2.

The endoplasmic reticulum (ER) responds to changes in intracellular homeostasis through activation of the unfolded protein response (UPR). Yet, it is not known how UPR-signaling coordinates adaptation versus cell death. Previous studies suggested that signaling through PERK/ATF4 is required for cell death. We show that high levels of ER stress (i.e., ischemia-like conditions) induce transcription of the ubiquitin ligases Siah1/2 through the UPR transducers PERK/ATF4 and IRE1/sXBP1. In turn, Siah1/2 attenuates proline hydroxylation of ATF4, resulting in its stabilization, thereby augmenting ER stress output. Conversely, ATF4 activation is reduced upon Siah1/2 KD in cultured cells, which attenuates ER stress-induced cell death. Notably, Siah1a(+/-)::Siah2(-/-) mice subjected to neuronal ischemia exhibited smaller infarct volume and were protected from ischemia-induced death, compared with the wild type (WT) mice. In all, Siah1/2 constitutes an obligatory fine-tuning mechanism that predisposes cells to death under severe ER stress conditions.

Dysregulation of ubiquitin ligases in cancer.

Ubiquitin ligases (UBLs) are critical components of the ubiquitin proteasome system (UPS), which governs fundamental processes regulating normal cellular homeostasis, metabolism, and cell cycle in response to external stress signals and DNA damage. Among multiple steps of the UPS system required to regulate protein ubiquitination and stability, UBLs define specificity, as they recognize and interact with substrates in a temporally- and spatially-regulated manner. Such interactions are required for substrate modification by ubiquitin chains, which marks proteins for recognition and degradation by the proteasome or alters their subcellular localization or assembly into functional complexes. UBLs are often deregulated in cancer, altering substrate availability or activity in a manner that can promote cellular transformation. Such deregulation can occur at the epigenetic, genomic, or post-translational levels. Alterations in UBL can be used to predict their contributions, affecting tumor suppressors or oncogenes in select tumors. Better understanding of mechanisms underlying UBL expression and activities is expected to drive the development of next generation modulators that can serve as novel therapeutic modalities. This review summarizes our current understanding of UBL deregulation in cancer and highlights novel opportunities for therapeutic interventions.

ß-Catenin-independent activation of TCF1/LEF1 in human hematopoietic tumor cells through interaction with ATF2 transcription factors.

The role of Wnt signaling in embryonic development and stem cell maintenance is well established and aberrations leading to the constitutive up-regulation of this pathway are frequent in several types of human cancers. Upon ligand-mediated activation, Wnt receptors promote the stabilization of ß-catenin, which translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to regulate the expression of Wnt target genes. When not bound to ß-catenin, the TCF/LEF proteins are believed to act as transcriptional repressors. Using a specific lentiviral reporter, we identified hematopoietic tumor cells displaying constitutive TCF/LEF transcriptional activation in the absence of ß-catenin stabilization. Suppression of TCF/LEF activity in these cells mediated by an inducible dominant-negative TCF4 (DN-TCF4) inhibited both cell growth and the expression of Wnt target genes. Further, expression of TCF1 and LEF1, but not TCF4, stimulated TCF/LEF reporter activity in certain human cell lines independently of ß-catenin. By a complementary approach in vivo, TCF1 mutants, which lacked the ability to bind to ß-catenin, induced Xenopus embryo axis duplication, a hallmark of Wnt activation, and the expression of the Wnt target gene Xnr3. Through generation of different TCF1-TCF4 fusion proteins, we identified three distinct TCF1 domains that participate in the ß-catenin-independent activity of this transcription factor. TCF1 and LEF1 physically interacted and functionally synergized with members of the activating transcription factor 2 (ATF2) family of transcription factors. Moreover, knockdown of ATF2 expression in lymphoma cells phenocopied the inhibitory effects of DN-TCF4 on the expression of target genes associated with the Wnt pathway and on cell growth. Together, our findings indicate that, through interaction with ATF2 factors, TCF1/LEF1 promote the growth of hematopoietic malignancies in the absence of ß-catenin stabilization, thus establishing a new mechanism for TCF1/LEF1 transcriptional activity distinct from that associated with canonical Wnt signaling.

Regulation of ATG4B stability by RNF5 limits basal levels of autophagy and influences susceptibility to bacterial infection.

Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with-and regulation of its ubiquitination and stability by-RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5(-/-) MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5(-/-) using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5(-/-) mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5(-/-) macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.

ATF2 - at the crossroad of nuclear and cytosolic functions.

An increasing number of transcription factors have been shown to elicit oncogenic and tumor suppressor activities, depending on the tissue and cell context. Activating transcription factor 2 (ATF2; also known as cAMP-dependent transcription factor ATF-2) has oncogenic activities in melanoma and tumor suppressor activities in non-malignant skin tumors and breast cancer. Recent work has shown that the opposing functions of ATF2 are associated with its subcellular localization. In the nucleus, ATF2 contributes to global transcription and the DNA damage response, in addition to specific transcriptional activities that are related to cell development, proliferation and death. ATF2 can also translocate to the cytosol, primarily following exposure to severe genotoxic stress, where it impairs mitochondrial membrane potential and promotes mitochondrial-based cell death. Notably, phosphorylation of ATF2 by the epsilon isoform of protein kinase C (PKCε) is the master switch that controls its subcellular localization and function. Here, we summarize our current understanding of the regulation and function of ATF2 in both subcellular compartments. This mechanism of control of a non-genetically modified transcription factor represents a novel paradigm for 'oncogene addiction'.

Ubiquitin-recognition protein Ufd1 couples the endoplasmic reticulum (ER) stress response to cell cycle control.

The ubiquitin-recognition protein Ufd1 facilitates clearance of misfolded proteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. Here we report that prolonged ER stress represses Ufd1 expression to trigger cell cycle delay, which contributes to ERAD. Remarkably, down-regulation of Ufd1 enhances ubiquitination and destabilization of Skp2 mediated by the anaphase-promoting complex or cyclosome bound to Cdh1 (APC/C(Cdh1)), resulting in accumulation of the cyclin-dependent kinase inhibitor p27 and a concomitant cell cycle delay during the G1 phase that enables more efficient clearance of misfolded proteins. Mechanistically, nuclear Ufd1 recruits the deubiquitinating enzyme USP13 to counteract APC/C(Cdh1)-mediated ubiquitination of Skp2. Our data identify a coordinated cell cycle response to prolonged ER stress through regulation of the Cdh1-Skp2-p27 axis by Ufd1 and USP13.