Regulation of the poly(A) Polymerase Star-PAP by a Nuclear Phosphoinositide Signalosome.

Star-PAP is a noncanonical poly(A) polymerase that controls gene expression. Star-PAP was previously reported to bind the phosphatidylinositol 4-phosphate 5-kinase PIPKI⍺ and its product phosphatidylinositol 4,5-bisphosphate, which regulate Star-PAP poly(A) polymerase activity and expression of specific genes. Recent studies have revealed a nuclear PI signaling pathway in which the PI transfer proteins PITP⍺/ß, PI kinases and phosphatases bind p53 to sequentially modify protein-linked phosphatidylinositol phosphates and regulate its function. Here we demonstrate that multiple phosphoinositides, including phosphatidylinositol 4-monophosphate and phosphatidylinositol 3,4,5-trisphosphate are also coupled to Star-PAP in response to stress. This is initiated by PITP⍺/ß binding to Star-PAP, while the Star-PAP-linked phosphoinositides are modified by PI4KII⍺, PIPKI⍺, IPMK, and PTEN recruited to Star- PAP. The phosphoinositide coupling enhances the association of the small heat shock proteins HSP27/⍺B-crystallin with Star-PAP. Knockdown of the PITPs, kinases, or HSP27 reduce the expression of Star-PAP targets. Our results demonstrate that the PITPs generate Star-PAP-PIPn complexes that are then modified by PI kinases/phosphatases and small heat shock proteins that regulate the linked phosphoinositide phosphorylation and Star-PAP activity in response to stress.

Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer.

The Hippo pathway effectors Yes-associated protein 1 (YAP) and its homolog TAZ are transcriptional coactivators that control gene expression by binding to TEA domain (TEAD) family transcription factors. The YAP/TAZ-TEAD complex is a key regulator of cancer-specific transcriptional programs, which promote tumor progression in diverse types of cancer, including breast cancer. Despite intensive efforts, the YAP/TAZ-TEAD complex in cancer has remained largely undruggable due to an incomplete mechanistic understanding. Here, we report that nuclear phosphoinositides function as cofactors that mediate the binding of YAP/TAZ to TEADs. The enzymatic products of phosphoinositide kinases PIPKIα and IPMK, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (P(I3,4,5)P3), bridge the binding of YAP/TAZ to TEAD. Inhibiting these kinases or the association of YAP/TAZ with PI(4,5)P2 and PI(3,4,5)P3 attenuates YAP/TAZ interaction with the TEADs, the expression of YAP/TAZ target genes, and breast cancer cell motility. Although we could not conclusively exclude the possibility that other enzymatic products of IPMK such as inositol phosphates play a role in the mechanism, our results point to a previously unrecognized role of nuclear phosphoinositide signaling in control of YAP/TAZ activity and implicate this pathway as a potential therapeutic target in YAP/TAZ-driven breast cancer.

A p85 isoform switch enhances PI3K activation on endosomes by a MAP4- and PI3P-dependent mechanism.

Phosphatidylinositol 3-kinase α (PI3Kα) is a heterodimer of p110α catalytic and p85 adaptor subunits that is activated by agonist-stimulated receptor tyrosine kinases. Although p85α recruits p110α to activated receptors on membranes, p85α loss, which occurs commonly in cancer, paradoxically promotes agonist-stimulated PI3K/Akt signaling. p110α localizes to microtubules via microtubule-associated protein 4 (MAP4), facilitating its interaction with activated receptor kinases on endosomes to initiate PI3K/Akt signaling. Here, we demonstrate that in response to agonist stimulation and p85α knockdown, the residual p110α, coupled predominantly to p85ß, exhibits enhanced recruitment with receptor tyrosine kinases to endosomes. Moreover, the p110α C2 domain binds PI3-phosphate, and this interaction is also required to recruit p110α to endosomes and for PI3K/Akt signaling. Stable knockdown of p85α, which mimics the reduced p85α levels observed in cancer, enhances cell growth and tumorsphere formation, and these effects are abrogated by MAP4 or p85ß knockdown, underscoring their role in the tumor-promoting activity of p85α loss.

Environmental pollutants and phosphoinositide signaling in autoimmunity.

Environmental pollution stands as one of the most critical challenges affecting human health, with an estimated mortality rate linked to pollution-induced non-communicable diseases projected to range from 20% to 25%. These pollutants not only disrupt immune responses but can also trigger immunotoxicity. Phosphoinositide signaling, a pivotal regulator of immune responses, plays a central role in the development of autoimmune diseases and exhibits high sensitivity to environmental stressors. Among these stressors, environmental pollutants have become increasingly prevalent in our society, contributing to the initiation and exacerbation of autoimmune conditions. In this review, we summarize the intricate interplay between phosphoinositide signaling and autoimmune diseases within the context of environmental pollutants and contaminants. We provide an up-to-date overview of stress-induced phosphoinositide signaling, discuss 14 selected examples categorized into three groups of environmental pollutants and their connections to immune diseases, and shed light on the associated phosphoinositide signaling pathways. Through these discussions, this review advances our understanding of how phosphoinositide signaling influences the coordinated immune response to environmental stressors at a biological level. Furthermore, it offers valuable insights into potential research directions and therapeutic targets aimed at mitigating the impact of environmental pollutants on the pathogenesis of autoimmune diseases. SYNOPSIS: Phosphoinositide signaling at the intersection of environmental pollutants and autoimmunity provides novel insights for managing autoimmune diseases aggravated by pollutants.

Heme biosynthesis regulates BCAA catabolism and thermogenesis in brown adipose tissue.

With age, people tend to accumulate body fat and reduce energy expenditure 1 . Brown (BAT) and beige adipose tissue dissipate heat and increase energy expenditure via the activity of the uncoupling protein UCP1 and other thermogenic futile cycles 2,3 . The activity of brown and beige depots inversely correlates with BMI and age 4-11 , suggesting that promoting thermogenesis may be an effective approach for combating age-related metabolic disease 12-15 . Heme is an enzyme cofactor and signaling molecule that we recently showed to regulate BAT function 16 . Here, we show that heme biosynthesis is the primary contributor to intracellular heme levels in brown adipocytes. Inhibition of heme biosynthesis leads to mitochondrial dysfunction and reduction in UCP1. Although supplementing heme can restore mitochondrial function in heme-synthesis-deficient cells, the downregulation of UCP1 persists due to the accumulation of the heme precursors, particularly propionyl-CoA, which is a product of branched-chain amino acids (BCAA) catabolism. Cold exposure promotes BCAA uptake in BAT, and defects in BCAA catabolism in this tissue hinder thermogenesis 17 . However, BCAAs' contribution to the TCA cycle in BAT and WAT never exceeds 2% of total TCA flux 18 . Our work offers a way to integrate current literature by describing heme biosynthesis as an important metabolic sink for BCAAs.

Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins.

Reactive oxygen species (ROS) are generated by aerobic metabolism, and their deleterious effects are buffered by the cellular antioxidant response, which prevents oxidative stress. The nuclear factor erythroid 2-related factor 2 (NRF2) is a master transcriptional regulator of the antioxidant response. Basal levels of NRF2 are kept low by ubiquitin-dependent degradation of NRF2 by E3 ligases, including the Kelch-like ECH-associated protein 1 (KEAP1). Here, we show that the stability and function of NRF2 is regulated by the type I phosphatidylinositol phosphate kinase g (PIPKIg), which binds NRF2 and transfers its product phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) to NRF2. PtdIns(4,5)P 2 binding recruits the small heat shock protein HSP27 to the complex. Silencing PIPKIg or HSP27 destabilizes NRF2, reduces expression of its target gene HO-1, and sensitizes cells to oxidative stress. These data demonstrate an unexpected role of phosphoinositides and HSP27 in regulating NRF2 and point to PIPKIg and HSP27 as drug targets to destabilize NRF2 in cancer. In brief: Phosphoinositides are coupled to NRF2 by PIPKIγ, and HSP27 is recruited and stabilizes NRF2, promoting stress-resistance.

Regulation of Cell Adhesion and Migration via Microtubule Cytoskeleton Organization, Cell Polarity, and Phosphoinositide Signaling.

The capacity for cancer cells to metastasize to distant organs depends on their ability to execute the carefully choreographed processes of cell adhesion and migration. As most human cancers are of epithelial origin (carcinoma), the transcriptional downregulation of adherent/tight junction proteins (e.g., E-cadherin, Claudin and Occludin) with the concomitant gain of adhesive and migratory phenotypes has been extensively studied. Most research and reviews on cell adhesion and migration focus on the actin cytoskeleton and its reorganization. However, metastasizing cancer cells undergo the extensive reorganization of their cytoskeletal system, specifically in originating/nucleation sites of microtubules and their orientation (e.g., from non-centrosomal to centrosomal microtubule organizing centers). The precise mechanisms by which the spatial and temporal reorganization of microtubules are linked functionally with the acquisition of an adhesive and migratory phenotype as epithelial cells reversibly transition into mesenchymal cells during metastasis remains poorly understood. In this Special Issue of "Molecular Mechanisms Underlying Cell Adhesion and Migration", we highlight cell adhesion and migration from the perspectives of microtubule cytoskeletal reorganization, cell polarity and phosphoinositide signaling.

Regulation of Phosphoinositide Signaling by Scaffolds at Cytoplasmic Membranes.

Cytoplasmic phosphoinositides (PI) are critical regulators of the membrane-cytosol interface that control a myriad of cellular functions despite their low abundance among phospholipids. The metabolic cycle that generates different PI species is crucial to their regulatory role, controlling membrane dynamics, vesicular trafficking, signal transduction, and other key cellular events. The synthesis of phosphatidylinositol (3,4,5)-triphosphate (PI3,4,5P3) in the cytoplamic PI3K/Akt pathway is central to the life and death of a cell. This review will focus on the emerging evidence that scaffold proteins regulate the PI3K/Akt pathway in distinct membrane structures in response to diverse stimuli, challenging the belief that the plasma membrane is the predominant site for PI3k/Akt signaling. In addition, we will discuss how PIs regulate the recruitment of specific scaffolding complexes to membrane structures to coordinate vesicle formation, fusion, and reformation during autophagy as well as a novel lysosome repair pathway.

Lipid transfer proteins initiate nuclear phosphoinositide signaling.

The membrane-localized phosphatidylinositol (PI) 3-kinase (PI3K)/Akt pathway regulates cell growth and is aberrantly activated in cancer. Recent studies reveal a distinct nuclear PI3K/Akt pathway involving PI phosphate (PIP) kinases that bind the tumor suppressor protein p53 (wild-type and mutant) to generate nuclear p53-polyphosphoinositide (PIP n ) complexes that activate Akt. In the membrane pathway, PI transfer proteins (PITPs) transport PI, the precursor of PIP n s, to endomembranes to enable PIP n synthesis. In contrast, nuclear PIP n signaling relies on poorly characterized non-membranous PIP n pools. Here we show that PITPs accumulate in the non-membranous nucleoplasm in response to stress and are necessary to generate nuclear PIP n pools. Class I PITPα/ß bind p53 to form p53-PIP n complexes that activate nuclear Akt in response to stress, which inhibits apoptosis. These findings demonstrate an unexpected function for PITPα/ß in nuclear PIP n signaling by generating membrane-free, protein-linked PIP n pools that are modified by PIP kinases/phosphatases to regulate protein function. In brief: Phosphatidylinositol transfer proteins initiate the nuclear protein-associated PIP n network in membrane-free regions.

Breast cancer risk for women with diabetes and the impact of metformin: A meta-analysis.

BACKGROUND: Diabetes mellitus has been associated with increased breast cancer (BC) risk; however, the magnitude of this effect is uncertain. This study focused on BC risk for women with type 2 diabetes mellitus (T2DM). METHODS: Two separate meta-analyses were conducted (1) to estimate the relative risk (RR) of BC for women with T2DM and (2) to evaluate the risk of BC for women with T2DM associated with the use of metformin, a common diabetes treatment. In addition, subgroup analyses adjusting for obesity as measured by body mass index (BMI) and menopausal status were also performed. Studies were identified via PubMed/Scopus database and manual search through April 2021. RESULTS: A total of 30 and 15 studies were included in the first and second meta-analyses, respectively. The summary RR of BC for women with T2DM was 1.15 (95% confidence interval [CI], 1.09-1.21). The subgroup analyses adjusting BMI and adjusting BMI and menopause resulted in a summary RR of 1.22 (95% CI, 1.15-1.30) and 1.20 (95% CI, 1.05-1.36), respectively. For women with T2DM, the summary RR of BC was 0.82 (95% CI, 0.60-1.12) for metformin users compared with nonmetformin users. CONCLUSIONS: Women with T2DM were more likely to be diagnosed with BC and this association was strengthened by adjusting for BMI and menopausal status. No statistically significant reduction of BC risk was observed among metformin users. IMPACT: These two meta-analyses can inform decision-making for women with type 2 diabetes regarding their use of metformin and the use of screening mammography for early detection of breast cancer.

A p53-phosphoinositide signalosome regulates nuclear AKT activation.

The tumour suppressor p53 and PI3K-AKT pathways have fundamental roles in the regulation of cell growth and apoptosis, and are frequently mutated in cancer. Here, we show that genotoxic stress induces nuclear AKT activation through a p53-dependent mechanism that is distinct from the canonical membrane-localized PI3K-AKT pathway. Following genotoxic stress, a nuclear PI3K binds p53 in the non-membranous nucleoplasm to generate a complex of p53 and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), which recruits AKT, PDK1 and mTORC2 to activate AKT and phosphorylate FOXO proteins, thereby inhibiting DNA damage-induced apoptosis. Wild-type p53 activates nuclear AKT in an on/off fashion following stress, whereas mutant p53 dose-dependently stimulates high basal AKT activity. The p53-PtdIns(3,4,5)P3 complex is dephosphorylated to p53-phosphatidylinositol 4,5-bisphosphate by PTEN to inhibit AKT activation. The nuclear p53-phosphoinositide signalosome is distinct from the canonical membrane-localized pathway and insensitive to PI3K inhibitors currently in the clinic, which underscores its therapeutic relevance.

Targeting the methionine addiction of cancer.

Methionine is the initiator amino acid for protein synthesis, the methyl source for most nucleotide, chromatin, and protein methylation, and the carbon backbone for various aspects of the cellular antioxidant response and nucleotide biosynthesis. Methionine is provided in the diet and serum methionine levels fluctuate based on dietary methionine content. Within the cell, methionine is recycled from homocysteine via the methionine cycle, which is linked to nutrient status via one-carbon metabolism. Unlike normal cells, many cancer cells, both in vitro and in vivo, show high methionine cycle activity and are dependent on exogenous methionine for continued growth. However, the molecular mechanisms underlying the methionine dependence of diverse malignancies are poorly understood. Methionine deprivation initiates widespread metabolic alterations in cancer cells that enable them to survive despite limited methionine availability, and these adaptive alterations can be specifically targeted to enhance the activity of methionine deprivation, a strategy we have termed "metabolic priming". Chemotherapy-resistant cell populations such as cancer stem cells, which drive treatment-resistance, are also sensitive to methionine deprivation, suggesting dietary methionine restriction may inhibit metastasis and recurrence. Several clinical trials in cancer are investigating methionine restriction in combination with other agents. This review will explore new insights into the mechanisms of methionine dependence in cancer and therapeutic efforts to translate these insights into enhanced clinical activity of methionine restriction in cancer.

Methionine restriction exposes a targetable redox vulnerability of triple-negative breast cancer cells by inducing thioredoxin reductase.

PURPOSE: Tumor cells are dependent on the glutathione and thioredoxin antioxidant pathways to survive oxidative stress. Since the essential amino acid methionine is converted to glutathione, we hypothesized that methionine restriction (MR) would deplete glutathione and render tumors dependent on the thioredoxin pathway and its rate-limiting enzyme thioredoxin reductase (TXNRD). METHODS: Triple (ER/PR/HER2)-negative breast cancer (TNBC) cells were treated with control or MR media and the effects on reactive oxygen species (ROS) and antioxidant signaling were examined. To determine the role of TXNRD in MR-induced cell death, TXNRD1 was inhibited by RNAi or the pan-TXNRD inhibitor auranofin, an antirheumatic agent. Metastatic and PDX TNBC mouse models were utilized to evaluate in vivo antitumor activity. RESULTS: MR rapidly and transiently increased ROS, depleted glutathione, and decreased the ratio of reduced glutathione/oxidized glutathione in TNBC cells. TXNRD1 mRNA and protein levels were induced by MR via a ROS-dependent mechanism mediated by the transcriptional regulators NRF2 and ATF4. MR dramatically sensitized TNBC cells to TXNRD1 silencing and the TXNRD inhibitor auranofin, as determined by crystal violet staining and caspase activity; these effects were suppressed by the antioxidant N-acetylcysteine. H-Ras-transformed MCF-10A cells, but not untransformed MCF-10A cells, were highly sensitive to the combination of auranofin and MR. Furthermore, dietary MR induced TXNRD1 expression in mammary tumors and enhanced the antitumor effects of auranofin in metastatic and PDX TNBC murine models. CONCLUSION: MR exposes a vulnerability of TNBC cells to the TXNRD inhibitor auranofin by increasing expression of its molecular target and creating a dependency on the thioredoxin pathway.

Assessing In Situ Phosphoinositide-Protein Interactions Through Fluorescence Proximity Ligation Assay in Cultured Cells.

Proximity ligation assay (PLA) is a well-established method for detecting in situ interactions between two epitopes with high resolution and specificity. Notably, PLA is not only a robust method for studying protein-protein interaction but also an efficient approach to characterize and validate protein posttranslational modifications (PTM) using one antibody against the core protein and one against the PTM residue. Therefore, it could be applied as a powerful approach to detect specific interactions of endogenous phosphoinositides and their binding proteins within cells. Importantly, we have specifically detected the PLA signal between PtdIns(4,5)P2 and its binding effector p53 in the nucleus. This cutting-edge method fully complements other conventional approaches for studying phosphoinositide-protein interactions and provides important localization signals and robust quantitation of the detected interactions. Here, we present the PLA fluorescence protocol for detecting in situ phosphoinositide-protein interactions in cultured cells and is semiquantitative for interactions that are regulated by cellular signaling.

Synthetic Lethal Metabolic Targeting of Androgen-Deprived Prostate Cancer Cells with Metformin.

The initiation of androgen-deprivation therapy (ADT) induces susceptibilities in prostate cancer cells that make them vulnerable to synergistic treatment and enhanced cell death. Senescence results in cell-cycle arrest, but cells remain viable. In this study, we investigated the mechanisms by which prostate cancer cells undergo senescence in response to ADT, and determined whether an FDA-approved antidiabetic drug metformin has a synergistic effect with ADT in prostate cancer both in vitro and in vivo Our results show that longer term exposure to ADT induced senescence associated with p16INK4a and/or p27kip2 induction. The activation of PI3K/AKT and inactivation of AMPK in senescent cells resulted in mTORC1 activation. In addition, the antiapoptotic protein XIAP expression was increased in response to ADT. The addition of metformin following ADT induced apoptosis, attenuated mTOR activation, reduced senescent cell number in vitro, and inhibited tumor growth in prostate cancer patient-derived xenograft models. This study suggests that combining ADT and metformin may be a feasible therapeutic approach to remove persistent prostate cancer cells after ADT.

Lysine oxidase exposes a dependency on the thioredoxin antioxidant pathway in triple-negative breast cancer cells.

PURPOSE: Transformed cells are vulnerable to depletion of certain amino acids. Lysine oxidase (LO) catalyzes the oxidative deamination of lysine, resulting in lysine depletion and hydrogen peroxide production. Although LO has broad antitumor activity in preclinical models, the cytotoxic mechanisms of LO are poorly understood. METHODS: Triple (ER/PR/HER2)-negative breast cancer (TNBC) cells were treated with control media, lysine-free media or control media supplemented with LO and examined for cell viability, caspase activation, induction of reactive oxygen species (ROS) and antioxidant signaling. To determine the role of nuclear factor erythroid 2-related factor 2 (NRF2) and thioredoxin reductase-1 (TXNRD1) in LO-induced cell death, NRF2 and TXNRD1 were individually silenced by RNAi. Additionally, the pan-TXNRD inhibitor auranofin was used in combination with LO. RESULTS: LO activates caspase-independent cell death that is suppressed by necroptosis and ferroptosis inhibitors, which are inactive against lysine depletion, pointing to fundamental differences between LO and lysine depletion. LO rapidly induces ROS with a return to baseline levels within 24 h that coincides temporally with induction of TXNRD activity, the rate-limiting enzyme in the thioredoxin antioxidant pathway. ROS induction is required for LO-mediated cell death and NRF2-dependent induction of TXNRD1. Silencing NRF2 or TXNRD1 enhances the cytotoxicity of LO. The pan-TXNRD inhibitor auranofin is synergistic with LO against transformed breast epithelial cells, but not untransformed cells, underscoring the tumor-selectivity of this strategy. CONCLUSIONS: LO exposes a redox vulnerability of TNBC cells to TXNRD inhibition by rendering tumor cells dependent on the thioredoxin antioxidant pathway for survival.

Ionizing Radiation-induced Proteomic Oxidation in Escherichia coli.

Recent work has begun to investigate the role of protein damage in cell death because of ionizing radiation (IR) exposure, but none have been performed on a proteome-wide basis, nor have they utilized MS (MS) to determine chemical identity of the amino acid side chain alteration. Here, we use Escherichia coli to perform the first MS analysis of IR-treated intact cells on a proteome scale. From quintuplicate IR-treated (1000 Gy) and untreated replicates, we successfully quantified 13,262 peptides mapping to 1938 unique proteins. Statistically significant, but low in magnitude (<2-fold), IR-induced changes in peptide abundance were observed in 12% of all peptides detected, although oxidative alterations were rare. Hydroxylation (+15.99 Da) was the most prevalent covalent adduct detected. In parallel with these studies on E. coli, identical experiments with the IR-resistant bacterium, Deinococcus radiodurans, revealed orders of magnitude less effect of IR on the proteome. In E. coli, the most significant target of IR by a wide margin was glyceraldehyde 3'-phosphate dehydrogenase (GAPDH), in which the thiol side chain of the catalytic Cys residue was oxidized to sulfonic acid. The same modification was detected in IR-treated human breast carcinoma cells. Sensitivity of GAPDH to reactive oxygen species (ROS) has been described previously in microbes and here, we present GAPDH as an immediate, primary target of IR-induced oxidation across all domains of life.

Methyl-Metabolite Depletion Elicits Adaptive Responses to Support Heterochromatin Stability and Epigenetic Persistence.

S-adenosylmethionine (SAM) is the methyl-donor substrate for DNA and histone methyltransferases that regulate epigenetic states and subsequent gene expression. This metabolism-epigenome link sensitizes chromatin methylation to altered SAM abundance, yet the mechanisms that allow organisms to adapt and protect epigenetic information during life-experienced fluctuations in SAM availability are unknown. We identified a robust response to SAM depletion that is highlighted by preferential cytoplasmic and nuclear mono-methylation of H3 Lys 9 (H3K9) at the expense of broad losses in histone di- and tri-methylation. Under SAM-depleted conditions, H3K9 mono-methylation preserves heterochromatin stability and supports global epigenetic persistence upon metabolic recovery. This unique chromatin response was robust across the mouse lifespan and correlated with improved metabolic health, supporting a significant role for epigenetic adaptation to SAM depletion in vivo. Together, these studies provide evidence for an adaptive response that enables epigenetic persistence to metabolic stress.

The nuclear phosphoinositide response to stress.

Accumulating evidence reveals that nuclear phosphoinositides (PIs) serve as central signaling hubs that control a multitude of nuclear processes by regulating the activity of nuclear proteins. In response to cellular stressors, PIs accumulate in the nucleus and multiple PI isomers are synthesized by the actions of PI-metabolizing enzymes, kinases, phosphatases and phospholipases. By directly interacting with effector proteins, phosphoinositide signals transduce changes in cellular functions. Here we describe nuclear phosphoinositide signaling in multiple sub-nuclear compartments and summarize the literature that demonstrates roles for specific kinases, phosphatases, and phospholipases in the orchestration of nuclear phosphoinositide signaling in response to cellular stress. Additionally, we discuss the specific PI-protein complexes through which these lipids execute their functions by regulating the configuration, stability, and transcription activity of their effector proteins. Overall, our review provides a detailed landscape of the current understanding of the nuclear PI-protein interactome and its role in shaping the coordinated response to cellular stress.

Methionine restriction activates the integrated stress response in triple-negative breast cancer cells by a GCN2- and PERK-independent mechanism.

Transformed cells are often selectively susceptible to depletion of the amino acid methionine, which induces growth arrest and/or apoptosis. In non-transformed cells, amino acid deficiency is sensed by two stress-activated kinases, general control nonderepressible 2 (GCN2) and protein kinase R-like endoplasmic reticulum kinase (PERK), which phosphorylate and inactivate elongation initiation factor 2 α (eIF2α), thereby suppressing global mRNA translation and inducing activated transcription factor (ATF4). ATF4 and its downstream transcriptional targets including Sestrin-2 constitute an adaptive integrated stress response. We postulated that methionine depletion activates the integrated stress response in breast cancer cells by a GCN2- and/or PERK-dependent mechanism and that selective disruption of one or both of these kinases would enhance the therapeutic activity of methionine restriction. Here we demonstrate that methionine restriction induces eIF2α phosphorylation and enhances ATF4 gene expression and protein levels of ATF4 and Sestrin-2 in triple (ER/PR/HER2)-negative breast cancer (TNBC) cells. However, knockdown of GCN2, PERK or both in TNBC cells did not prevent induction of ATF4 or Sestrin-2 by methionine restriction. In contrast, deletion of GCN2 in murine embryonic fibroblasts abrogated ATF4 and Sestrin-2 induction in response to methionine restriction. Moreover, knockdown of GCN2, PERK or both did not affect TNBC cell growth or apoptosis in response to methionine restriction. Overall, our findings point to a GCN2- and PERK-independent mechanism(s) by which methionine restriction activates the integrated stress response in TNBC cells. Elucidation of this pathway(s) could lead to strategies to enhance the therapeutic response of methionine restriction.