We previously elucidated the pleotropic role of solute carrier family A1 member 5 (SLC1A5) as the primary transporter of glutamine (Gln), a modulator of cell growth and oxidative stress in non-small cell lung cancer (NSCLC). The aim of our study was to evaluate SLC1A5 as a potential new therapeutic target and candidate biomarker predictive of survival and response to therapy. SLC1A5 targeting was examined in a panel of NSCLC and human bronchial cell lines by RNA interference and by a small molecular inhibitor, gamma-L-glutamyl-p-nitroanilide (GPNA). The effects of targeting SLC1A5 on cell growth, Gln uptake, ATP level, autophagy and cell death were examined. Inactivation of SLC1A5 genetically or pharmacologically decreased Gln consumption, inhibited cell growth, induced autophagy and apoptosis in a subgroup of NSCLC cell lines that overexpress SLC1A5. Targeting SLC1A5 function decreased tumor growth in NSCLC xenografts. A multivariate Cox proportional hazards analysis indicates that patients with increased SLC1A5 mRNA expression have significantly shorter overall survival (p =0.01, HR =1.24, 95% CI: 1.05–1.46), adjusted for age, gender, smoking history and disease stage. In an immunohistochemistry study on 207 NSCLC patients, SLC1A5 protein expression remained highly significant prognostic value in both univariate (p < 0.0001, HR =1.45, 95% CI: 1.15–1.50) and multivariate analyses (p =0.04, HR =1.22, 95% CI: 1.01–1.31). These results position SLC1A5 as a new candidate prognostic biomarker for selective targeting of Gln-dependent NSCLC.

Alveolata comprises diverse taxa of single-celled eukaryotes, many renowned for their ability to live inside animal cells. Notable examples are apicomplexan parasites and dinoflagellate symbionts, the latter of which power coral reef ecosystems. Although functionally distinct, they evolved from a common, free-living ancestor and must evade their hosts’ immune response for persistence. Both the initial cellular events that gave rise to this intracellular lifestyle and the role of host immune modulation in coral-dinoflagellate endosymbiosis are poorly understood. Here, we use a comparative approach in the cnidarian endosymbiosis model Aiptasia, which re-establishes endosymbiosis with free-living dinoflagellates every generation. We find that uptake of microalgae is largely indiscriminate, but non-symbiotic microalgae are expelled by vomocytosis, while symbionts induce host-cell innate immune suppression and form a LAMP1-positive niche. We demonstrate that exogenous immune stimulation results in symbiont expulsion and conversely, inhibition of canonical toll-like receptor (TLR) signaling enhances infection of host animals. Our findings indicate that symbiosis establishment is dictated by local innate immune suppression, to circumvent expulsion and promote niche formation. This work provides insight into the evolution of the cellular immune response and key steps involved in mediating endosymbiotic interactions.

To coordinate development and growth with nutrient availability, animals must sense nutrients and acquire food from the environment once energy is depleted. A notable exception are reef-building corals that form a stable symbiosis with intracellular photosynthetic dinoflagellates (family Symbiodiniaceae (LaJeunesse et al., 2018)). Symbionts reside in 'symbiosomes' and transfer key nutrients to support nutrition and growth of their coral host in nutrient-poor environments (Muscatine, 1990;Yellowlees et al., 2008). To date, it is unclear how symbiont-provided nutrients are sensed to adapt host physiology to this endosymbiotic lifestyle. Here we use the symbiosis model Exaiptasia pallida (hereafter Aiptasia) to address this. Aiptasia larvae, similar to their coral relatives, are naturally non-symbiotic and phagocytose symbionts anew each generation into their endodermal cells (Bucher et al., 2016;Grawunder et al., 2015;Hambleton et al., 2014).Using cell-specific transcriptomics, we find that symbiosis establishment results in downregulation of various catabolic pathways, including autophagy in host cells. This metabolic switch is likely triggered by the highly-conserved mTORC1 (mechanistic target of rapamycin complex 1) signaling cascade, shown to integrate lysosomal nutrient abundance with animal development (Perera and Zoncu, 2016). Specifically, symbiosomes are LAMP1-positive and recruit mTORC1 kinase. In symbiotic anemones, mTORC1 signaling is elevated when compared to non-symbiotic animals, resembling a feeding response. Moreover, symbiosis establishment enhances lipid content and cell proliferation in Aiptasia larvae. Challenging the prevailing belief that symbiosomes are early arrested phagosomes (Mohamed et al., 2016), we propose a model in which symbiosomes functionally resemble lysosomes as core nutrient sensing and signaling hubs that have co-opted the evolutionary ancient mTORC1 pathway to promote growth in endosymbiotic cnidarians.

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Emergence of the symbiotic lifestyle fostered the immense diversity of all ecosystems on Earth, but symbiosis plays a particularly remarkable role in marine ecosystems. Photosynthetic dinoflagellate endosymbionts power reef ecosystems by transferring vital nutrients to their coral hosts. The mechanisms driving this symbiosis, specifically those which allow hosts to discriminate between beneficial symbionts and pathogens, are not well understood. Here, we uncover that host immune suppression is key for dinoflagellate endosymbionts to avoid elimination by the host using a comparative, model systems approach. Unexpectedly, we find that the clearance of nonsymbiotic microalgae occurs by non-lytic expulsion (vomocytosis) and not intracellular digestion, the canonical mechanism used by professional immune cells to destroy foreign invaders. We provide evidence that suppression of TLR signalling by targeting the conserved MyD88 adapter protein has been co-opted for this endosymbiotic lifestyle, suggesting that this is an evolutionarily ancient mechanism exploited to facilitate symbiotic associations ranging from coral endosymbiosis to the microbiome of vertebrate guts. Main Text SummarySymbiotic interactions appear in all domains of life and are key drivers of adaption and evolutionary diversification. A prime example is the endosymbiosis between corals and eukaryotic, photosynthetic dinoflagellates, which the hosts take up from the environment by phagocytosis.Once intracellular, endosymbionts transfer vital nutrients to the corals, a process fundamental for survival in nutrient-poor environments (Muscatine, 1990;Yellowlees et al., 2008). A central question is how endosymbionts circumvent the hosts' defensive strategies to persist intracellularly and conversely, how hosts prevent invasion by non-symbiotic microorganisms. Here, we use a comparative approach in the anemone model Exaiptasia pallida (commonly Aiptasia) (Hambleton et al., 2019;Tolleter et al., 2013;Weis et al., 2008) to address this. Using RNA-seq, we show that symbiont phagocytosis leads to a broad scale, cell-specific transcriptional suppression of host innate immunity, but phagocytosis of non-symbiotic microalgae does not. We show that targeting MyD88 to inhibit canonical TLR signalling promotes symbiosis establishment. Using live imaging and chemical perturbations, we demonstrate that non-symbiotic microalgae are cleared from the host by ERK5-dependent vomocytosis (non-lytic expulsion) (Alvarez and Casadevall, 2006;Gilbert et al., 2017;Ma et al., 2006), and that immune suppression is key for symbiont persistence and for establishment of an intracellular LAMP1 niche. We propose that expulsion is a fundamental mechanism of ancient innate immunity used to eliminate foreign cells; a process that is subverted by symbionts in the endosymbiotic association that forms the foundation of coral reef ecosystems. University) Postdoctoral Program, and a PhD scholarship within the Graduate School "Evolutionary Novelty & Adaptation by the Baden-Württemberg Landesgraduier...