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Abstract

Many stressful conditions on cells, including hypoxia, nutrition deprivation, inflammation, infections, and changes in cell microenvironment, challenge the folding capacity of the cell and promote endoplasmic reticulum (ER) stress. The unfolded protein response (UPR), a homeostatic signaling network, buffers and orchestrates the recovery of ER function, and when it fails, ER stress results in apoptosis. Tumor cells face endogenous and exogenous challenges when proliferating rapidly while maintaining metabolic homeostasis required for growth. The simultaneous tumorigenic and pro-apoptotic effects of ER stress in cancer were widely studied. This risky balancing act also endows cancer cells with selective vulnerabilities that could be harnessed to therapeutic advantage. Here, it is observed that rapid cell proliferation and protein synthesis occur in response to endogenous and exogenous cellular stress at low concentration, such as Sorafenib, Regorafenib, hypoxia, glucose, but except low glutamine cell culture in HCC cancer. Furthermore, it is confirmed that cancer cells increase extracellular glutamine uptake via the activation of glutamine classic transporter, SLC1A5 in adaptation to ER stress. The absorbed glutamine is used for nucleotide and protein synthesis, but not for ATP and NAPDH production. Notably, it is defined that glutamine is also used for the supply of asparagine de novo synthesis by up-regulating glutamine-dependent asparagine synthetase, which plays the key role in coordinating nucleotide and protein synthesis in resistance to ER stress induced apoptosis. Most solid tumors express glutamine-dependent ASNS and synthesize asparagine de novo. The current data demonstrate that ASNS, the transcriptional target of ATF4, is activated to synthesize intrinsic asparagine in response to ER stress. ATF4 knockout or ASNS knockout decreases the expression of the downstream targets mTOR, 4EBP1 and eIF4E at protein level. Therefore, it is defined that the key axis ATF4/ASNS/eIF4F complex activation is required to keep cellular metabolism homeostasis in response to cellular stress. Present data also suggest that intracellular depletion of asparagine via ASNS knockout induces apoptosis even in the face of an abundant supply of glutamine. This confirms that the influence of glutamine on cellular homeostasis may be in part mediated by glutamine-dependent asparagine synthesis via ASNS. Like glutamine, asparagine is shown to up-regulate the protein synthesis via the eIF4F complex in resistance to cellular stress. The eIF4F complex knockdown inhibits the protein synthesis and converts cells from adaptability into apoptosis when facing ER stress, which cannot be rescued at presence of glutamine or by adding exogenous asparagine. The present results support the hypothesis that glutamine-dependent de novo synthetic asparagine has a key role in keeping cellular adaptability to extrinsic and intrinsic stress in cancer, suggesting that targeting of amino acids has the possibility for additional treatments of tumors. Here, V-9302, an antagonist designed to abrogate all facets of glutamine signaling and metabolism downstream of SCL1A5-mediated import, is applied as an additional treatment and sensitizes HCC cells to ER stress and the treatments of Sorafenib and Regorafenib. Likewise, L- asparaginase—Spectrila is applied here to deplete asparagine and alert cells to sensitivity to ER stress and the treatments of Sorafenib and Regorafenib. Based on the findings above it can be suggested that combination treatment with V-9302, or Spectrila could be a therapeutic supplementation for Sorafenib and Regorafenib in HCC. Targeting of glutamine and asparagine metabolism may represent a new promising class of targeted oncological therapy.