Ekaterina Bobrovnikova-Marjon

The GCN2-ATF4 pathway is critical for tumour cell survival and proliferation in response to nutrient deprivation

The transcription factor ATF4 regulates the expression of genes involved in amino acid metabolism, redox homeostasis and ER stress responses, and it is overexpressed in human solid tumours, suggesting that it has an important function in tumour progression. Here, we report that inhibition of ATF4 expression blocked proliferation and survival of transformed cells, despite an initial activation of cytoprotective macroautophagy. Knockdown of ATF4 significantly reduced the levels of asparagine synthetase (ASNS) and overexpression of ASNS or supplementation of asparagine in trans, reversed the proliferation block and increased survival in ATF4 knockdown cells. Both amino acid and glucose deprivation, stresses found in solid tumours, activated the upstream eukaryotic initiation factor 2α (eIF2α) kinase GCN2 to upregulate ATF4 target genes involved in amino acid synthesis and transport. GCN2 activation/overexpression and increased phospho‐eIF2α were observed in human and mouse tumours compared with normal tissues and abrogation of ATF4 or GCN2 expression significantly inhibited tumour growth in vivo. We conclude that the GCN2‐eIF2α‐ATF4 pathway is critical for maintaining metabolic homeostasis in tumour cells, making it a novel and attractive target for anti‐tumour approaches.

PERK-dependent regulation of lipogenesis during mouse mammary gland development and adipocyte differentiation

The role of the endoplasmic reticulum stress-regulated kinase, PERK, in mammary gland function was assessed through generation of a targeted deletion in mammary epithelium. Characterization revealed that PERK is required for functional maturation of milk-secreting mammary epithelial cells. PERK-dependent signaling contributes to lipogenic differentiation in mammary epithelium, and perk deletion inhibits the sustained expression of lipogenic enzymes FAS, ACL, and SCD1. As a result, mammary tissue has reduced lipid content and the milk produced has altered lipid composition, resulting in attenuated pup growth. Consistent with PERK-dependent regulation of the lipogenic pathway, loss of PERK inhibits expression of FAS, ACL, and SCD1 in immortalized murine embryonic fibroblasts when cultured under conditions favoring adipocyte differentiation. These findings implicate PERK as a physiologically relevant regulator of the lipogenic pathway.

PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage

To proliferate and expand in an environment with limited nutrients, cancer cells co-opt cellular regulatory pathways that facilitate adaptation and thereby maintain tumor growth and survival potential. The endoplasmic reticulum (ER) is uniquely positioned to sense nutrient deprivation stress and subsequently engage signaling pathways that promote adaptive strategies. As such, components of the ER stress-signaling pathway represent potential antineoplastic targets. However, recent investigations into the role of the ER resident protein kinase, RNA-dependent protein kinase (PKR)-like ER kinase (PERK) have paradoxically suggested both pro- and anti-tumorigenic properties. We have used animal models of mammary carcinoma to interrogate the contribution of PERK in the neoplastic process. The ablation of PERK in tumor cells resulted in impaired regeneration of intracellular antioxidants and accumulation of reactive oxygen species triggering oxidative DNA damage. Ultimately, PERK deficiency impeded progression through the cell cycle because of the activation of the DNA damage checkpoint. Our data reveal that PERK-dependent signaling is used during both tumor initiation and expansion to maintain redox homeostasis, thereby facilitating tumor growth.

miR-211 Is a Prosurvival MicroRNA that Regulates chop Expression in a PERK-Dependent Manner

MicroRNAs typically function at the level of posttranscriptional gene silencing within the cytoplasm; however, increasing evidence suggests that they may also function in nuclear, Argonaut-containing complexes, to directly repress target gene transcription. We have investigated the role of microRNAs in mediating endoplasmic reticulum (ER) stress responses. ER stress triggers the activation of three signaling molecules: Ire-1α/β, PERK, and ATF6, whose function is to facilitate adaption to the ensuing stress. We demonstrate that PERK induces miR-211, which in turn attenuates stress-dependent expression of the proapoptotic transcription factor chop/gadd153. MiR-211 directly targets the proximal chop/gadd153 promoter, where it increases histone methylation and represses chop expression. Maximal chop accumulation ultimately correlates with miR-211 downregulation. Our data suggest a model in which PERK-dependent miR-211 induction prevents premature chop accumulation and thereby provides a window of opportunity for the cell to re-establish homeostasis prior to apoptotic commitment.

Ribosomal Stress Couples the Unfolded Protein Response to p53-dependent Cell Cycle Arrest

Protein misfolding in the endoplasmic reticulum (ER) triggers a signaling pathway termed the unfolded protein response path-way (UPR). UPR signaling is transduced through the transmembrane ER effectors PKR-like ER kinase (PERK), inositol requiring kinase-1 (IRE-1), and activating transcription factor 6 (ATF6). PERK activation triggers phosphorylation of eIF2α leading to repression of protein synthesis, thereby relieving ER protein load and directly inhibiting cyclin D1 translation thereby contributing to cell cycle arrest. However, PERK-/- murine embryonic fibroblasts have an attenuated G1/S arrest that is not attributable to cyclin D1 loss, suggesting a cyclin D1-independent mechanism. Here we show that the UPR triggers p53 accumulation and activation. UPR induction promotes enhanced interaction between the ribosome proteins (rpL5, rpL11, and rpL23) and Hdm2 in a PERK-dependent manner. Interaction with ribosomal proteins results in inhibition of Hdm2-mediated ubiquitination and degradation of p53. Our data demonstrate that ribosomal subunit:Hdm2 association couples the unfolded protein response to p53-dependent cell cycle arrest.

Dysregulated mTORC1 renders cells critically dependent on desaturated lipids for survival under tumor-like stress

Solid tumors exhibit heterogeneous microenvironments, often characterized by limiting concentrations of oxygen (O2), glucose, and other nutrients. How oncogenic mutations alter stress response pathways, metabolism, and cell survival in the face of these challenges is incompletely understood. Here we report that constitutive mammalian target of rapamycin complex 1 (mTORC1) activity renders hypoxic cells dependent on exogenous desaturated lipids, as levels of de novo synthesized unsaturated fatty acids are reduced under low O2. Specifically, we demonstrate that hypoxic Tsc2(-/-) (tuberous sclerosis complex 2(-/-)) cells deprived of serum lipids exhibit a magnified unfolded protein response (UPR) but fail to appropriately expand their endoplasmic reticulum (ER), leading to inositol-requiring protein-1 (IRE1)-dependent cell death that can be reversed by the addition of unsaturated lipids. UPR activation and apoptosis were also detected in Tsc2-deficient kidney tumors. Importantly, we observed this phenotype in multiple human cancer cell lines and suggest that cells committed to unregulated growth within ischemic tumor microenvironments are unable to balance lipid and protein synthesis due to a critical limitation in desaturated lipids.

PERK-dependent regulation of IAP translation during ER stress

Exposure of cells to endoplasmic reticulum (ER) stress leads to activation of phosphatidylinositol 3-kinase (PI3K)–Akt signaling pathway and transcriptional induction of the inhibitor of apoptosis family of proteins. One of the proximal effectors of the ER stress response, the PKR-like ER kinase (PERK), leads to cellular adaptation to stress by multiple mechanisms, including attenuation of protein synthesis and transcriptional induction of pro-survival genes. Although PERK activity leads to cellular adaptation to ER stress, we now demonstrate that PERK activity also inhibits the ER stress-induced apoptotic program through the induction of cellular inhibitor of apoptosis (cIAP1 and cIAP2) proteins. This induction of IAPs occurs through both transcriptional and translational responses that are PERK dependent. Reintroduction of cIAP1 or cIAP2 expression into PERK-/- murine embryonic fibroblasts during ER stress delays the early onset of ER stress-induced caspase activation and apoptosis observed in these cells. Furthermore, we demonstrate that the activation of the PI3K–Akt pathway by ER stress is dependent on PERK, suggesting additional ways in which PERK activity protects cells from ER stress-induced apoptosis.

Regulation of autophagy during ECM detachment is linked to a selective inhibition of mTORC1 by PERK

Adhesion to the extracellular matrix (ECM) is critical for epithelial tissue homeostasis and function. ECM detachment induces metabolic stress and programmed cell death via anoikis. ECM-detached mammary epithelial cells are able to rapidly activate autophagy allowing for survival and an opportunity for re-attachment. However, the mechanisms controlling detachment-induced autophagy remain unclear. Here we uncover that the kinase PERK rapidly promotes autophagy in ECM-detached cells by activating AMP-activated protein kinase (AMPK), resulting in downstream inhibition of mTORC1-p70S6K signaling. LKB1 and TSC2, but not TSC1, are required for PERK-mediated inhibition of mammalian target of rapamycinin MCF10A cells and mouse embryo fibroblast cells. Importantly, this pathway shows fast kinetics, is transcription-independent and is exclusively activated during ECM detachment, but not by canonical endoplasmic reticulum stressors. Moreover, enforced PERK or AMPK activation upregulates autophagy and causes luminal filling during acinar morphogenesis by perpetuating a population of surviving autophagic luminal cells that resist anoikis. Hence, we identify a novel pathway in which suspension-activated PERK promotes the activation of LKB1, AMPK and TSC2, leading to the rapid induction of detachment-induced autophagy. We propose that increased autophagy, secondary to persistent PERK and LKB1-AMPK signaling, can robustly protect cells from anoikis and promote luminal filling during early carcinoma progression.

PERK Utilizes Intrinsic Lipid Kinase Activity To Generate Phosphatidic Acid, Mediate Akt Activation, and Promote Adipocyte Differentiation

ABSTRACT The endoplasmic reticulum (ER) resident PKR-like kinase (PERK) is necessary for Akt activation in response to ER stress. We demonstrate that PERK harbors intrinsic lipid kinase, favoring diacylglycerol (DAG) as a substrate and generating phosphatidic acid (PA). This activity of PERK correlates with activation of mTOR and phosphorylation of Akt on Ser473. PERK lipid kinase activity is regulated in a phosphatidylinositol 3-kinase (PI3K) p85α-dependent manner. Moreover, PERK activity is essential during adipocyte differentiation. Because PA and Akt regulate many cellular functions, including cellular survival, proliferation, migratory responses, and metabolic adaptation, our findings suggest that PERK has a more extensive role in insulin signaling, insulin resistance, obesity, and tumorigenesis than previously thought.