Cell migration is a critical mechanism controlling tissue morphogenesis, epithelial wound healing and tumor metastasis. Migrating cells depend on orchestrated remodeling of the plasma membrane and the underlying actin cytoskeleton, which is regulated by the spectrin-adducin-based membrane skeleton. Expression of adducins is altered during tumorigenesis, however, their involvement in metastatic dissemination of tumor cells remains poorly characterized. This study investigated the roles of α-adducin (ADD1) and γ-adducin (ADD3) in regulating migration and invasion of nonsmall cell lung cancer (NSCLC) cells. ADD1 was mislocalized, whereas ADD3 was markedly downregulated in NSCLC cells with the invasive mesenchymal phenotype. CRISPR/Cas9mediated knockout of ADD1 and ADD3 in epithelial-type NSCLC and normal bronchial epithelial cells promoted their Boyden chamber migration and Matrigel invasion. Furthermore, overexpression of ADD1, but not ADD3, in mesenchymal-type NSCLC cells decreased cell migration and invasion. ADD 1-overexpressing NSCLC cells demonstrated increased adhesion to the extracellular matrix (ECM), accompanied by enhanced assembly of focal adhesions and hyperphosphorylation of Src and paxillin. The increased adhesiveness and decreased motility of ADD 1-overexpressing cells were reversed by siRNA-mediated knockdown of Src. By contrast, the accelerated migration of ADD1 and ADD3-depleted NSCLC cells was ECM adhesionindependent and was driven by the upregulated expression of pro-motile cadherin-11. Overall, our findings reveal a novel function of adducins as negative regulators of NSCLC cell migration and invasion, which could be essential for limiting lung cancer progression and metastasis.

Toxoplasma gondii is a prevalent protozoan parasite that can infect any nucleated cell but cannot replicate outside of its host cell. Toxoplasma is auxotrophic for several nutrients including arginine, tryptophan, and purines, which it must acquire from its host cell. The demands of parasite replication rapidly deplete the host cell of these essential nutrients, yet Toxoplasma successfully manages to proliferate until it lyses the host cell. In eukaryotic cells, nutrient starvation can induce the integrated stress response (ISR) through phosphorylation of an essential translation factor eIF2. Phosphorylation of eIF2 lowers global protein synthesis coincident with preferential translation of gene transcripts involved in stress adaptation, such as that encoding the transcription factor ATF4 (CREB2), which activates genes that modulate amino acid metabolism and uptake. Here, we discovered that the ISR is induced in host cells infected with Toxoplasma . Our results show that as Toxoplasma depletes host cell arginine, the host cell phosphorylates eIF2 via protein kinase GCN2 (EIF2AK4), leading to induced ATF4. Increased ATF4 then enhances expression of the cationic amino acid transporter CAT1 (SLC7A1), resulting in increased uptake of arginine in Toxoplasma -infected cells. Deletion of host GCN2, or its downstream effectors ATF4 and CAT1, lowers arginine levels in the host, impairing proliferation of the parasite. Our findings establish that Toxoplasma usurps the host cell ISR to help secure nutrients that it needs for parasite replication.

Toxoplasma gondii is an obligate intracellular parasite that persists in its vertebrate hosts in the form of dormant tissue cysts, which facilitate transmission through predation. The parasite must strike a balance that allows it to disseminate throughout its host without killing it, which requires the ability to properly counter host cell defenses. For example, oxidative stress encountered by Toxoplasma is suggested to impair parasite replication and dissemination. However, the strategies by which Toxoplasma mitigates oxidative stress are not yet clear. Among eukaryotes, environmental stresses induce the integrated stress response via phosphorylation of a translation initiation factor, eukaryotic initiation factor 2 (eIF2). Here, we show that the Toxoplasma eIF2 kinase TgIF2K-B is activated in response to oxidative stress and affords protection. Knockout of the TgIF2K-B gene, Δtgif2k-b, disrupted parasite responses to oxidative stresses and enhanced replication, diminishing the ability of the parasite to differentiate into tissue cysts. In addition, parasites lacking TgIF2K-B exhibited resistance to activated macrophages and showed greater virulence in an in vivo model of infection. Our results establish that TgIF2K-B is essential for Toxoplasma responses to oxidative stress, which are important for the parasite’s ability to establish persistent infection in its host. IMPORTANCE Toxoplasma gondii is a single-celled parasite that infects nucleated cells of warm-blooded vertebrates, including one-third of the human population. The parasites are not cleared by the immune response and persist in the host by converting into a latent tissue cyst form. Development of tissue cysts can be triggered by cellular stresses, which activate a family of TgIF2 kinases to phosphorylate the eukaryotic translation initiation factor TgIF2α. Here, we establish that the TgIF2 kinase TgIF2K-B is activated by oxidative stress and is critical for maintaining oxidative balance in the parasite. Depletion of TgIF2K-B alters gene expression, leading to accelerated growth and a diminished ability to convert into tissue cysts. This study establishes that TgIF2K-B is essential for the parasite’s oxidative stress response and its ability to persist in the host as a latent infection.

A stress adaptation pathway termed the integrated stress response has been suggested to be active in many cancers including prostate cancer (PCa). Here, we demonstrate that the eIF2 kinase GCN2 is required for sustained growth in androgen-sensitive and castration-resistant models of PCa both in vitro and in vivo, and is active in PCa patient samples. Using RNA-seq transcriptome analysis and a CRISPR-based phenotypic screen, GCN2 was shown to regulate expression of over 60 solute-carrier (SLC) genes, including those involved in amino acid transport and loss of GCN2 function reduces amino acid import and levels. Addition of essential amino acids or expression of 4F2 (SLC3A2) partially restored growth following loss of GCN2, suggesting that GCN2 targeting of SLC transporters is required for amino acid homeostasis needed to sustain tumor growth. A small molecule inhibitor of GCN2 showed robust in vivo efficacy in androgen-sensitive and castration-resistant mouse models of PCa, supporting its therapeutic potential for the treatment of PCa.

ABSTRACT Toxoplasma gondii is an intracellular parasite that reconfigures its host cell to promote pathogenesis. One consequence of Toxoplasma parasitism is increased migratory activity of host cells, which facilitates dissemination. Here, we show that Toxoplasma triggers the unfolded protein response (UPR) in host cells through calcium release from the endoplasmic reticulum (ER). We further identify a novel role for the host ER stress sensor protein IRE1 in Toxoplasma pathogenesis. Upon infection, Toxoplasma activates IRE1, engaging its noncanonical role in actin remodeling through the binding of filamin A. By inducing cytoskeletal remodeling via IRE1 oligomerization in host cells, Toxoplasma enhances host cell migration in vitro and dissemination of the parasite to host organs in vivo. Our study has identified novel mechanisms used by Toxoplasma to induce dissemination of infected cells, providing new insights into strategies for treatment of toxoplasmosis. IMPORTANCE Cells that are infected with the parasite Toxoplasma gondii exhibit heightened migratory activity, which facilitates dissemination of the infection throughout the body. In this report, we identify a new mechanism used by Toxoplasma to hijack its host cell and increase its mobility. We further show that the ability of Toxoplasma to increase host cell migration involves not the enzymatic activity of IRE1 but rather IRE1 engagement with actin cytoskeletal remodeling. Depletion of IRE1 from infected host cells reduces their migration in vitro and significantly hinders dissemination of Toxoplasma in vivo. Our findings reveal a new mechanism underlying host-pathogen interactions, demonstrating how host cells are co-opted to spread a persistent infection around the body.