Molecular Basis of Human Diseases and Targeted Therapy Based on Small-Molecule Inhibitors of ER Stress-Induced Signaling Pathways.

The Endoplasmic Reticulum (ER) provides a conserved protein quality control system and plays a fundamental role in cell growth and homeostasis. Disturbances in the ER homeostasis may originate especially from hypoxia, glucose deficiency, presence of mutant proteins, that directly impair protein folding capacity and after deposition of unfolded and misfolded proteins within ER lumen trigger ER stress conditions. This subsequently activates the Unfolded Protein Response (UPR) branches, which have a dual pro-adaptive or pro-apoptotic role depending on the severity and time of duration of ER stress conditions. This review is the first to offer a detailed overview on molecular mechanisms of all major ER stress-dependent signaling branches, that are activated through three specific ER transmembrane receptors of impaired protein folding: Protein kinase RNA (PKR)-like ER kinase (PERK), Inositol-requiring enzyme-1 (IRE1) and Activating transcription factor 6 (ATF6). Molecular crosstalk among ER transmembrane receptors-dependent pathways determines a final UPR response, but the recent data reported that especially PERK over-activation has a significant impact on the development and progression of a wide spectrum of disease entities. Based on these findings, small-molecules, highly specific PERK inhibitors may provide effective, groundbreaking treatment strategy against human diseases. However, after foregoing in vitro cellular and in vivo animal models conducted examination, supplementary investigations of PERK inhibitors are required for their further clinical use. Future research may answer the question of how to minimize toxicity and side effects of characterized small-molecule PERK inhibitors, that may be used, as breakthrough drugs, alone or in combination with currently known models of therapy.

The Role of the PERK/eIF2α/ATF4/CHOP Signaling Pathway in Tumor Progression During Endoplasmic Reticulum Stress.

Hypoxia is a major hallmark of the tumor microenvironment that is strictly associated with rapid cancer progression and induction of metastasis. Hypoxia inhibits disulfide bond formation and impairs protein folding in the Endoplasmic Reticulum (ER). The stress in the ER induces the activation of Unfolded Protein Response (UPR) pathways via the induction of protein kinase RNA-like endoplasmic reticulum kinase (PERK). As a result, the level of phosphorylated Eukaryotic Initiation Factor 2 alpha (eIF2α) is markedly elevated, resulting in the promotion of a pro-adaptive signaling pathway by the inhibition of global protein synthesis and selective translation of Activating Transcription Factor 4 (ATF4). On the contrary, during conditions of prolonged ER stress, pro-adaptive responses fail and apoptotic cell death ensues. Interestingly, similar to the activity of the mitochondria, the ER may also directly activate the apoptotic pathway through ER stress-mediated leakage of calcium into the cytoplasm that leads to the activation of death effectors. Apoptotic cell death also ensues by ATF4-CHOP- mediated induction of several pro-apoptotic genes and suppression of the synthesis of anti-apoptotic Bcl-2 proteins. Advancing molecular insight into the transition of tumor cells from adaptation to apoptosis under hypoxia-induced ER stress may provide answers on how to overcome the limitations of current anti-tumor therapies. Targeting components of the UPR pathways may provide more effective elimination of tumor cells and as a result, contribute to the development of more promising anti-tumor therapeutic agents.

Tumor progression and the different faces of the PERK kinase.

The serine/threonine endoplasmic reticulum (ER) kinase, protein kinase R (PKR)-like ER kinase (PERK), is a pro-adaptive protein kinase whose activity is regulated indirectly by protein misfolding within the ER. As the oxidative folding environment in the ER is sensitive to a variety of cellular stresses, many of which occur during neoplastic transformation and in the tumor microenvironment, there has been considerable interest in defining whether PERK positively contributes to tumor progression and whether it represents a significant therapeutic target. Herein, we review the current knowledge of PERK-dependent signaling pathways, the contribution of downstream substrates including recently characterized new PERK substrates transcription factors Forkhead box O protein and diacyglycerol a lipid signaling second messenger, and efforts to develop small molecule PERK inhibitors.

BCR-ABL1 kinase inhibits uracil DNA glycosylase UNG2 to enhance oxidative DNA damage and stimulate genomic instability.

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia in chronic phase (CML-CP). Unfortunately, 25% of TKI-naive patients and 50-90% of patients developing TKI-resistance carry CML clones expressing TKI-resistant BCR-ABL1 kinase mutants. We reported that CML-CP leukemia stem and progenitor cell populations accumulate high amounts of reactive oxygen species, which may result in accumulation of uracil derivatives in genomic DNA. Unfaithful and/or inefficient repair of these lesions generates TKI-resistant point mutations in BCR-ABL1 kinase. Using an array of specific substrates and inhibitors/blocking antibodies we found that uracil DNA glycosylase UNG2 were inhibited in BCR-ABL1-transformed cell lines and CD34(+) CML cells. The inhibitory effect was not accompanied by downregulation of nuclear expression and/or chromatin association of UNG2. The effect was BCR-ABL1 kinase-specific because several other fusion tyrosine kinases did not reduce UNG2 activity. Using UNG2-specific inhibitor UGI, we found that reduction of UNG2 activity increased the number of uracil derivatives in genomic DNA detected by modified comet assay and facilitated accumulation of ouabain-resistant point mutations in reporter gene Na(+)/K(+)ATPase. In conclusion, we postulate that BCR-ABL1 kinase-mediated inhibition of UNG2 contributes to accumulation of point mutations responsible for TKI resistance causing the disease relapse, and perhaps also other point mutations facilitating malignant progression of CML.

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.