Unfolded Protein Response Signaling Pathway Research Articles

Abstract PO4-08-01: Methylsterol monooxygenase 1 (MSMO1) modulates endoplasmic reticulum stress through cholesterol metabolic reprogramming to alter chemotherapy sensitivity in breast cancer

Abstract Background Endoplasmic reticulum stress (ER stress) is a process in which cells activate unfolded protein response and other signaling pathways in response to misfolded and unfolded protein aggregation and calcium ion balance disturbance in the endoplasmic reticulum cavity. Depending on the intensity of endoplasmic reticulum stress, it can not only induce chaperone expression to play a protective effect, but also induce apoptosis independently. Metabolic reprogramming is one of the characteristics of tumor, which refers to the metabolic changes that cells make in response to various stimuli. Here, we demonstrate that methylsterol monooxygenase 1 (MSMO1) regulates cellular cholesterol metabolism and influences breast cancer susceptibility to chemotherapy by regulating the relative intracellular T-MAS content. Methods RNA-seq was performed on core needle biopsy samples from 26 patients receiving neoadjuvant chemotherapy for breast cancer to screen for genes that may influence neoadjuvant chemotherapy sensitivity. Cell proliferation, CCK8 assay and apoptosis assay were performed for MSMO1 phenotype study. Transmission electron microscopy (TEM) was conducted to visualize the structure of endoplasmic reticulum. Western Blot was used to detect the activation level of the unfolded protein response pathway. The relative content of sterols in MSMO1 knockdown cells was detected by targeted lipid mass spectrometry. AO/PI staining of breast cancer organoids was performed to verify the effect of MSMO1 metabolic substrate T-MAS on chemotherapy drug sensitivity. Results RNA-seq of core needle biopsy samples from breast cancer patients receiving neoadjuvant chemotherapy suggested that MSMO1 was highly expressed in non-pCR patients, and clinical survival data suggested that MSMO1 was associated with poor prognosis in patients with all types of breast cancer, suggesting that MSMO1 might be related to chemotherapy sensitivity of breast cancer. Phenotypic experiments revealed that MSMO1 regulates chemotherapy sensitivity of breast cancer both in vivo and in vitro. Ectopic overexpression of MSMO1 promoted cancer cell resistance to chemotherapy. To uncover the underlying mechanisms, RNA-seq of MSMO1 knock-down cells were carried out and identified that MSMO1 was associated with ER stress. TEM and western blot assay indicated that downregulation of MSMO1 expression leads to endoplasmic reticulum swelling and activation of the unfolded protein response. Considering that MSMO1 is a cholesterol metabolizing enzyme, targeted lipids mass spectrometry was performed to detect the changes in the contents of various intermediate metabolites in the cholesterol pathway, which indicated that the relative content of T-MAS, a metabolic substrate of MSMO1, was significantly up-regulated in MSMO1 knockdown cells. Then, in order to verify whether this metabolite can independently regulate ER stress, breast cancer cells were given T-MAS externally, and TEM and Western Blot analysis indicated that T-MAS could induce ER stress and activate the unfolded protein response independently. Apoptosis assay and AO/PI staining of breast cancer organoids revealed that T-MAS can improve the sensitivity of breast cancer to chemotherapy drugs. Conclusion In summary, these results shed light on the role of MSMO1 in cholesterol metabolism reprogramming, which adjust the endoplasmic reticulum stress state of cells by changing the relative content of intracellular T-MAS, and thereby influencing the sensitivity of breast cancer chemotherapy. As an intermediate metabolite of cholesterol metabolism, T-MAS has potential clinical application value of chemotherapy sensitization. Citation Format: Hengyu Ren, Liren Wangxu, Yayun Chi, Jiong Wu. Methylsterol monooxygenase 1 (MSMO1) modulates endoplasmic reticulum stress through cholesterol metabolic reprogramming to alter chemotherapy sensitivity in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-08-01.

Gene Expression Profiling to Unfolded Proteins Response as a Risk Modulator of Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease. Despite new methods of diagnostics and treatment as well as extensive biological and immunosuppressive treatment, the etiology of RA is not fully understood. Moreover, the problem of diagnosis and treatment of RA patients is still current and affects a large group of patients. It is suggested that endoplasmic reticulum (ER)-related features may impair adaptation to chronic stress, inferring the risk of rheumatoid arthritis. The main goal in this study was evaluation of changes in mRNA translation to determine chronic ER stress conditions in rheumatoid arthritis patients. The study group consist of 86 individuals including a total of 56 rheumatoid arthritis patients and 30 healthy controls. The expression level of mRNA form blood samples of RA patients as well as controls of the unfolded protein response (UPR)-associated genes (p-eIF2, BCL-2, PERK, ATF4, and BAX) were investigated using real-time qPCR. GAPDH expression was used as a standard control. Considering the median, the expression levels of PERK, BCL-2, p-eIF2, ATF4, and BAX were found to be significantly increased in the blood of RA patients compared with the control group. The p-value for the PERK gene was 0.0000000036, the p-value for the BCL-2 gene was 0.000000014, the p-value for the p-eIF2 gene was 0.006948, the p-value for the ATF4 gene was 0.0000056, and the p-value for the BAX gene was 0.00019, respectively. Thus, it can be concluded that the targeting of the components of the PERK-dependent UPR signaling pathway via small-molecule PERK inhibitors may contribute to the development of novel, innovative treatment strategies against rheumatoid arthritis.

Regulation of cardiac fibroblast cell death by unfolded protein response signaling.

The endoplasmic reticulum (ER) is a tightly regulated organelle that requires specific environmental properties to efficiently carry out its function as a major site of protein synthesis and folding. Embedded in the ER membrane, ER stress sensors inositol-requiring enzyme 1 (IRE1), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) serve as a sensitive quality control system collectively known as the unfolded protein response (UPR). In response to an accumulation of misfolded proteins, the UPR signals for protective mechanisms to cope with the cellular stress. Under prolonged unstable conditions and an inability to regain homeostasis, the UPR can shift from its original adaptive response to mechanisms leading to UPR-induced apoptosis. These UPR signaling pathways have been implicated as an important feature in the development of cardiac fibrosis, but identifying effective treatments has been difficult. Therefore, the apoptotic mechanisms of UPR signaling in cardiac fibroblasts (CFs) are important to our understanding of chronic fibrosis in the heart. Here, we summarize the maladaptive side of the UPR, activated downstream pathways associated with cell death, and agents that have been used to modify UPR-induced apoptosis in CFs.

FBXO5-mediated RNF183 degradation prevents endoplasmic reticulum stress-induced apoptosis and promotes colon cancer progression

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), and prolonged ER stress leads to cell apoptosis. Despite increasing research in this area, the underlying molecular mechanisms remain unclear. Here, we discover that ER stress upregulates the UPR signaling pathway while downregulating E2F target gene expression and inhibiting the G2/M phase transition. Prolonged ER stress decreases the mRNA levels of E2F2, which specifically regulates the expression of F-Box Protein 5(FBXO5), an F-box protein that functions as an inhibitor of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase complex. Depletion of FBXO5 results in increased ER stress-induced apoptosis and decreased expression of proteins related to PERK/IRE1α/ATF6 signaling. Overexpression of FBXO5 wild-type (not its ΔF-box mutant) alleviates apoptosis and the expression of the C/EBP Homologous Protein (CHOP)/ATF. Mechanistically, we find that FBXO5 directly binds to and promotes the ubiquitin-dependent degradation of RNF183, which acts as a ubiquitin E3 ligase in regulating ER stress-induced apoptosis. Reversal of the apoptosis defects caused by FBXO5 deficiency in colorectal cancer cells can be achieved by knocking down RNF183 in FBXO5-deficient cells. Functionally, we observed significant upregulation of FBXO5 in colon cancer tissues, and its silencing suppresses tumor occurrence in vivo. Therefore, our study highlights the critical role of the FBXO5/RNF183 axis in ER stress regulation and identifies a potential therapeutic target for colon cancer treatment.

Overproduction of mycotoxin biosynthetic enzymes triggers Fusarium toxisome-shaped structure formation via endoplasmic reticulum remodeling.

Mycotoxin deoxynivalenol (DON) produced by the Fusarium graminearum complex is highly toxic to animal and human health. During DON synthesis, the endoplasmic reticulum (ER) of F. graminearum is intensively reorganized, from thin reticular structure to thickened spherical and crescent structure, which was referred to as "DON toxisome". However, the underlying mechanism of how the ER is reorganized into toxisome remains unknown. In this study, we discovered that overproduction of ER-localized DON biosynthetic enzyme Tri4 or Tri1, or intrinsic ER-resident membrane proteins FgHmr1 and FgCnx was sufficient to induce toxisome-shaped structure (TSS) formation under non-toxin-inducing conditions. Moreover, heterologous overexpression of Tri1 and Tri4 proteins in non-DON-producing fungi F. oxysporum f. sp. lycopersici and F. fujikuroi also led to TSS formation. In addition, we found that the high osmolarity glycerol (HOG), but not the unfolded protein response (UPR) signaling pathway was involved in the assembly of ER into TSS. By using toxisome as a biomarker, we screened and identified a novel chemical which exhibited high inhibitory activity against toxisome formation and DON biosynthesis, and inhibited Fusarium growth species-specifically. Taken together, this study demonstrated that the essence of ER remodeling into toxisome structure is a response to the overproduction of ER-localized DON biosynthetic enzymes, providing a novel pathway for management of mycotoxin contamination.

Unfolded Protein Response Signaling in Hepatic Stem Cell Activation in Liver Fibrosis.

Frequent exposure to various external and internal adverse forces (stresses) disrupts cell protein homeostasis through endoplasmic reticulum (ER) capacity saturation. This process leads to the unfolded protein response (UPR), which aims to re-establish/maintain optimal cellular equilibrium. This complex mechanism is involved in the pathogenesis of various disorders, such as metabolic syndrome, fibrotic diseases, neurodegeneration, and cancer, by altering cellular metabolic changes integral to activating the hepatic stellate cells (HSCs). The development of hepatic fibrosis is one of the consequences of UPR activation. Therefore, novel therapies that target the UPR pathway effectively and specifically are being studied. This article covers the involvement of the UPR signaling pathway in cellular damage in liver fibrosis. Investigating the pathogenic pathways related to the ER/UPR stress axis that contribute to liver fibrosis can help to guide future drug therapy approaches.

HIV-1 replication requires optimal activation of the unfolded protein response.

Several human diseases including viral infections activate the unfolded protein response (UPR) due to abnormal accumulation of unfolded/misfolded proteins. However, UPR modulation and its functional relevance in HIV-1 infection lack comprehensive elucidation. This study reveals that HIV-1 activates IRE1, PERK, and ATF6 signaling pathways of UPR. The knockdown of PERK and ATF6 reduces HIV-1 long terminal repeat (LTR)-driven gene expression, whereas the endoplasmic reticulum (ER) chaperone HSPA5 prevents proteasomal degradation of HIV-1 p24 through its chaperone activity. Interestingly, overstimulation of UPR by a chemical inducer leads to anti-HIV activity through an enhanced type-1 interferon response. Also, treatment with a chemical ER stress inhibitor reduces HIV-1 replication. These findings suggest that an optimal UPR activation is crucial for effective viral replication, as either overstimulating UPR or inhibiting ER stress leads to viral suppression.

Lipotoxicity, ER Stress, and Cardiovascular Disease: Current Understanding and Future Directions.

The endoplasmic reticulum (ER) is a sub-cellular organelle that is responsible for the correct folding of proteins, lipid biosynthesis, calcium storage, and various post-translational modifications. In the disturbance of ER functioning, unfolded or misfolded proteins accumulate inside the ER lumen and initiate downstream signaling called unfolded protein response (UPR). The UPR signaling pathway is involved in lipolysis, triacylglycerol synthesis, lipogenesis, the mevalonate pathway, and low-density lipoprotein receptor recycling. ER stress also affects lipid metabolism by changing the levels of enzymes that are involved in the synthesis or modifications of lipids and causing lipotoxicity. Lipid metabolism and cardiac diseases are in close association as the deregulation of lipid metabolism leads to the development of various cardiovascular diseases (CVDs). Several studies have suggested that lipotoxicity is one of the important factors for cardiovascular disorders. In this review, we will discuss how ER stress affects lipid metabolism and their interplay in the development of cardiovascular disorders. Further, the current therapeutics available to target ER stress and lipid metabolism in various CVDs will be summarized.

Fundamental and Applicative Aspects of the Unfolded Protein Response in Yeasts.

Upon the dysfunction or functional shortage of the endoplasmic reticulum (ER), namely, ER stress, eukaryotic cells commonly provoke a protective gene expression program called the unfolded protein response (UPR). The molecular mechanism of UPR has been uncovered through frontier genetic studies using Saccharomyces cerevisiae as a model organism. Ire1 is an ER-located transmembrane protein that directly senses ER stress and is activated as an RNase. During ER stress, Ire1 promotes the splicing of HAC1 mRNA, which is then translated into a transcription factor that induces the expression of various genes, including those encoding ER-located molecular chaperones and protein modification enzymes. While this mainstream intracellular UPR signaling pathway was elucidated in the 1990s, new intriguing insights have been gained up to now. For instance, various additional factors allow UPR evocation strictly in response to ER stress. The UPR machineries in other yeasts and fungi, including pathogenic species, are another important research topic. Moreover, industrially beneficial yeast strains carrying an enforced and enlarged ER have been produced through the artificial and constitutive induction of the UPR. In this article, we review canonical and up-to-date insights concerning the yeast UPR, mainly from the viewpoint of the functions and regulation of Ire1 and HAC1.

The Unfolded Protein Response: A Double-Edged Sword for Brain Health.

Efficient brain function requires as much as 20% of the total oxygen intake to support normal neuronal cell function. This level of oxygen usage, however, leads to the generation of free radicals, and thus can lead to oxidative stress and potentially to age-related cognitive decay and even neurodegenerative diseases. The regulation of this system requires a complex monitoring network to maintain proper oxygen homeostasis. Furthermore, the high content of mitochondria in the brain has elevated glucose demands, and thus requires a normal redox balance. Maintaining this is mediated by adaptive stress response pathways that permit cells to survive oxidative stress and to minimize cellular damage. These stress pathways rely on the proper function of the endoplasmic reticulum (ER) and the activation of the unfolded protein response (UPR), a cellular pathway responsible for normal ER function and cell survival. Interestingly, the UPR has two opposing signaling pathways, one that promotes cell survival and one that induces apoptosis. In this narrative review, we discuss the opposing roles of the UPR signaling pathways and how a better understanding of these stress pathways could potentially allow for the development of effective strategies to prevent age-related cognitive decay as well as treat neurodegenerative diseases.

AMPK Mediates Early Activation of the Unfolded Protein Response through a Positive Feedback Loop in Palmitate-Treated Muscle Cells.

Activation of the unfolded protein response (UPR) is closely related to the pathogenesis of many metabolic disorders. Accumulating evidence also shows that UPR and metabolic signaling pathways are interdependent. The AMP-activated protein kinase (AMPK) signal pathway controls the energy balance of eukaryotes. The aim of this study was therefore to investigate the possible interaction between AMPK signaling and UPR in muscle cells exposed to saturated fatty acids, as well as the potential mechanism. The saturated fatty acid palmitate was used to induce UPR in C2C12 myotubes. Compound C or knockdown of AMPKα with short hairpin RNA (shRNA) were used to inhibit the AMPK signaling pathway in palmitate-treated muscle cells. AMPK signaling in myotubes was activated using 5-amino-1-β-D-ribofuranosylimidazole-4-carboxamide (AICAR) or ex229. C2C12 myotubes were pre-treated with taurourdodeoxycholic acid (TUDCA) to inhibit UPR before adding palmitate. Real-time PCR and Western blotting were performed to evaluate the expression of UPR markers and activation of AMPK. Palmitate treatment induced UPR in C2C12 myotubes while activating AMPK signaling. Inhibition of the AMPK pathway with compound C or AMPK shRNA reduced palmitate-induced activation of UPR, while inhibition of UPR with TUDCA reduced palmitate-induced AMPK activation. This indicates a positive feedback loop between UPR and AMPK. Furthermore, activation of the AMPK pathway with AICAR or ex229 caused a dose-dependent upregulation of UPR markers, including activating transcription factor 4 (ATF4), binding immunoglobulin protein (BIP), and growth arrest and DNA damage-inducible 34 (GADD34) protein. These results provide the first evidence that AMPK signaling is involved in the early activation of UPR caused by saturated fatty acids in skeletal muscle. Furthermore, they indicate that physiological or pharmacological activation of the AMPK pathway (e.g., by exercise or phenformin, respectively) can promote muscle health and function, thereby improving the quality of life in individuals with metabolic disorders due to a high-fat diet or obesity.

Triggering of Endoplasmic Reticulum Stress by Tannic Acid Inhibits the Proliferation and Migration of Colorectal Cancer Cells.

Due to the pivotal role of endoplasmic reticulum (ER) stress in cancers, interfering with its function can cause the accumulation of unfolded proteins, which ultimately leads to the activation of the unfolded protein response (UPR) signaling pathway and apoptosis. Therefore, the use of plant compounds such as tannic acid with UPR-inducing properties can be proposed as a possible treatment method for cancer. In this study, we investigated the effect of tannic acid on cell migration, colony formation, growth, and UPR-induced apoptosis in the SW48 colorectal cancer cell line. The MTT assay was performed to investigate the cytotoxic effect of tannic acid. We performed the qPCR method to elucidate the effect of tannic acid on the expression of Bim, MMP-9, Bcl-xL, cyclin D1, CHOP, and ATF4 genes. We also used the colony formation and migration experiments to investigate the effect of this compound on the colony formation and migration ability of tumor cells. Finally, we used Hoechst staining to measure cell apoptosis. Tannic acid inhibited the cell survival, clonogenic, and migration of colon cancer cells. This compound increased the expression of ER stress-mediated UPR genes, ATF4 and CHOP. Moreover; tannic acid increased the expression of pro-apoptotic proteins like Bim, while at the same time causing a sharp decline in the expression of anti-apoptotic protein Bcl-xL. A decline in MMP-9 expression confirmed the anti-metastatic role of this compound. Taken together, tannic acid can induce apoptosis via ER stress-mediated UPR pathway, and has a suppressive effect on cell viability, growth, migration, colony formation, and metastasis, suggesting it may be a potential drug in colorectal cancer treatment.

The Unfolded Protein Response and Its Implications for Novel Therapeutic Strategies in Inflammatory Bowel Disease.

The endoplasmic reticulum (ER) is a multifunctional organelle playing a vital role in maintaining cell homeostasis, and disruptions to its functions can have detrimental effects on cells. Dysregulated ER stress and the unfolded protein response (UPR) have been linked to various human diseases. For example, ER stress and the activation of the UPR signaling pathways in intestinal epithelial cells can either exacerbate or alleviate the severity of inflammatory bowel disease (IBD), contingent on the degree and conditions of activation. Our recent studies have shown that EPICERTIN, a recombinant variant of the cholera toxin B subunit containing an ER retention motif, can induce a protective UPR in colon epithelial cells, subsequently promoting epithelial restitution and mucosal healing in IBD models. These findings support the idea that compounds modulating UPR may be promising pharmaceutical candidates for the treatment of the disease. In this review, we summarize our current understanding of the ER stress and UPR in IBD, focusing on their roles in maintaining cell homeostasis, dysregulation, and disease pathogenesis. Additionally, we discuss therapeutic strategies that promote the cytoprotection of colon epithelial cells and reduce inflammation via pharmacological manipulation of the UPR.

Inhibition of IRE1 RNase activity modulates tumor cell progression and enhances the response to chemotherapy in colorectal cancer.

Drug resistance is one of the clinical challenges that limits the effectiveness of chemotherapy. Recent reports suggest that the unfolded protein response (UPR) and endoplasmic reticulum stress-adaptation signalling pathway, along with increased activation of its inositol-requiring enzyme 1α (IRE1α) arm, may be contributors to the pathogenesis of colorectal cancer (CRC). Here, we aimed to target the IRE1α/XBP1 pathway in order to sensitise CRC cells to the effects of chemotherapy.The CT26 colorectal cell line was treated with tunicamycin, and then was exposed to different concentrations of 5-fluorouracil (5-FU), either alone and/or in combination with the IRE1α inhibitor, 4µ8C. An MTT assay, flow cytometry and RT-PCR were performed to determine cell growth, apoptosis and IRE1α activity, respectively. In vivo BALB/c syngeneic colorectal mice received chemotherapeutic drugs. Treatment responses, tumour sizes and cytotoxicity were assessed via a range of pathological tests.4µ8C was found to inhibit the growth of CRC, at a concentration of 10µg/ml, without detectable cytotoxic effects and also significantly enhanced the cytotoxic potential of 5-FU, in CRC cells. In vivo experiments revealed that 4µ8C, at a concentration of 50µM/kg prevented tumour growth without any cytotoxic or metastatic effects. Interestingly, the combination of 4µ8C with 5-FU remarkably enhanced drug responses, up to 40-60% and also lead to significantly greater inhibition of tumour growth, in comparison to monotherapy, in CRC mice.Targeting the IRE1α/XBP1 axis of the UPR could enhance the effectiveness of chemotherapy in both in vitro and in vivo models of CRC.

The mTOR Signaling Pathway Interacts with the ER Stress Response and the Unfolded Protein Response in Cancer.

The mTOR complex 1 (mTORC1) coordinates several important environmental and intracellular cues to control a variety of biological processes, such as cell growth, survival, autophagy, and metabolism, in response to energy levels, growth signals, and nutrients. The endoplasmic reticulum (ER) is a crucial intracellular organelle that is essential for numerous cellular functions, including the synthesis, folding, and modification of newly synthesized proteins, stress responsiveness, and maintainence of cellular homeostasis. mTOR-mediated upregulation of protein synthesis induces the accumulation of misfolded or unfolded proteins in the ER lumen, which induces ER stress, leading to activation of the unfolded protein response (UPR) pathway. Reciprocally, ER stress regulates the PI3K/AKT/mTOR signaling pathway. Therefore, under pathologic conditions, the cross-talk between the mTOR and UPR signaling pathways during cellular stress can critically affect cancer cell fate and may be involved in the pathogenesis and therapeutic outcome of cancer. Here, we discuss accumulating evidence showing the mechanism of action, interconnections, and molecular links between mTOR signaling and ER stress in tumorigenesis and highlights potential therapeutic implications for numerous cancers.

Abstract 3528: ALKBH5 promotes cancer growth by regulating ER homeostasis via UPR, autophagy, and mitochondrial function

Abstract Background: N6-methyladenosine (m6A) RNA methylation is a dynamic reversible epitranscriptomic modification that includes methyl transferases (writes m6A), demethylases (erases m6A) and reader proteins, which proofreads m6A marks of a specific site of transcripts. Recent studies have suggested that RNA methylation affects several fundamental cellular and molecular functions including mRNA splicing, stability, export, stem cell fate, circadian rhythms, DNA repair and cell survival. The objective of this study is to establish the mechanisms by which RNA demethylase ALKBH5 (AlkB homolog 5) facilitates tumor growth and progression. Methods: To establish the significance of RNA methylation in children’s cancers, we performed siRNA screen targeting m6A writers, erasers, and readers in osteosarcoma (OS). We used several OS cell lines including 143B, MG63, SaOS2, U2OS and multiple patient derived OS cell lines. Mechanistic studies were conducted using ALKBH5 KO and knockdown cells and by measuring the status of Autophagy and UPR associated proteins using Western blot analysis, confocal and electron microscopy. Results: Our results revealed that depletion of ALKBH5, a demethylase that erases the m6A mark from the target gene, altered the autophagy in OS cells. Interestingly, we found that level of LC3, which is the universal marker for autophagy, was significantly increased in OS cells. RNA seq analysis showed that depletion of ALKBH5 significantly altered several autophagy related genes in the OS cells. To better understand the molecular mechanism by which ALKBH5 regulates autophagy, we investigated the Endoplasmic Reticulum (ER) stress-induced Unfolded Protein Response (UPR) pathway, which is a known activator of autophagy. We discovered that ER stress induced UPR signaling pathway is highly activated in ALKBH5 depleted cancer cells. Further, mechanistic studies suggested that ALKBH5 promoted ER homeostasis by controlling the expression of ER lipid raft associated 1 (ERLIN1), which binds to the activated inositol 1, 4, 5,-triphosphate receptor and facilitates its degradation via ERAD to maintain calcium flux between ER and mitochondria. Using functional studies and electron microscopy, we show that ALKBH5-ERLIN1-IP3R-dependent calcium signaling modulates the activity of AMP kinase, and consequently mitochondrial biogenesis. These findings thus reveal that ALKBH5 serves an important role in maintaining ER homeostasis and cellular fitness. Conclusion: These findings provide novel insight into how m6A may promote cancer cell growth by regulating the crosstalk among ER signaling, UPR, autophagy, and mitochondrial function. Our study is the first to show that RNA methylation plays an important role in osteosarcoma by regulating autophagy via UPR. Citation Format: Panneerdoss Subbarayalu, Daisy Medina, Pooja Yadav, Santosh Timilsina, Kunal Baxi, Ratna Vadlamudi, Yidong Chen, Manjeet Rao. ALKBH5 promotes cancer growth by regulating ER homeostasis via UPR, autophagy, and mitochondrial function. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3528.

Small‑molecule PKR‑like endoplasmic reticulum kinase inhibitors as a novel targeted therapy for Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder in worldwide and remains a therapeutic challenge due to the low efficacy of current treatments. Numerous studies have demonstrated the pivotal role of endoplasmic reticulum (ER) stress in PD pathogenesis. ER stress, followed by activation of the protein kinase RNA‑like endoplasmic reticulum kinase (PERK)‑dependent branch of the unfolded protein response signaling pathway, ultimately leads to neural cell death and dopaminergic neurodegeneration in PD. Therefore, the present study evaluated the effectiveness of the small‑molecule PERK inhibitor LDN‑87357 in an invitro PD model using the human neuroblastoma SH‑SY5Y cell line. To assess the mRNA expression levels of the pro‑apoptotic ER stress markers, the TaqMan Gene Expression Assay was performed. Cytotoxicity was assessed using a colorimetric 2,3‑bis‑(2‑methoxy‑4‑nitro‑5‑sulfophenyl)‑ 2H‑tetrazolium‑5‑carboxanilide assay and apoptosis was assessed using a caspase‑3 assay. Moreover, cell cycle progression was evaluated using flow cytometry. The results indicated that LDN‑87357 treatment induced a significant decrease in ER stress markers gene expression in SH‑SY5Y cells exposed to ER stress. Furthermore, LDN‑87357 significantly increased viability, diminished apoptosis and restored the normal cell cycle distribution of SH‑SY5Y cells after ER stress induction. Therefore, the evaluation of small‑molecule PERK inhibitors, such as LDN‑87357, may lead to the development of novel therapeutic strategies against PD.

Effects of Spatial Expression of Activating Transcription Factor 4 on the Pathogenicity of Two Phenotypes of Bovine Viral Diarrhea Virus by Regulating the Endoplasmic Reticulum-Mediated Autophagy Process.

The endoplasmic reticulum (ER) stress response is a highly conserved stress-defense mechanism and activates the adaptive unfolded protein response (UPR) to mitigate imbalance. The ER stress-activated signaling pathways can also trigger autophagy to facilitate cellular repair. Bovine viral diarrhea virus (BVDV) utilizes the host cellular ER as the primary site of the life cycle. However, the interplay between cellular ER stress and BVDV replication remains unclear. This report reveals that cytopathic (cp) and noncytopathic (ncp) BVDV have distinct strategies to regulate UPR mechanisms and ER stress-mediated autophagy for their own benefit. Immunoblot analysis revealed that cp and ncp BVDV differentially regulated the abundance of ER chaperone GRP78 for viral replication, while the protein kinase RNA-like ER kinase (PERK)-eukaryotic translation initiation factor 2 subunit α (eIF2α)-activating transcription factor 4 (ATF4) pathway of the UPR was switched on at different stages of infection. Pretreatment with ER stress inducer promoted virion replication, but RNA interference (RNAi) knockdown of ATF4 in BVDV-infected cells significantly attenuated BVDV infectivity titers. More importantly, the effector ATF4 activated by cp BVDV infection translocated into the nucleus to mediate autophagy, but ATF4 was retained in the cytoplasm during ncp BVDV infection. In addition, we found that cp BVDV core protein was localized in the ER to induce ER stress-mediated autophagy. Overall, the potential therapeutic target ATF4 may contribute to the global eradication campaign of BVDV. IMPORTANCE The ER-tropic viruses hijack the host cellular ER as the replication platform of the life cycle, which can lead to strong ER stress. The UPR and related transcriptional cascades triggered by ER stress play a crucial role in viral replication and pathogenesis, but little is known about these underlying mechanisms. Here, we report that cytopathic and noncytopathic BVDV use different strategies to reprogram the cellular UPR and ER stress-mediated autophagy for their own advantage. The cytopathic BVDV unconventionally downregulated the expression level of GRP78, creating perfect conditions for self-replication via the UPR, and the noncytopathic BVDV retained ATF4 in the cytoplasm to provide an advantage for its persistent infection. Our findings provide new insights into exploring how BVDV and other ER-tropic viruses reprogram the UPR signaling pathway in the host cells for replication and reveal the attractive host target ATF4 for new antiviral agents.

Giardia duodenalis-induced G0/G1 intestinal epithelial cell cycle arrest and apoptosis involve activation of endoplasmic reticulum stress in vitro.

Giardia duodenalis is a zoonotic intestinal protozoan parasite that may cause host diarrhea and chronic gastroenteritis, resulting in great economic losses annually and representing a significant public health burden across the world. However, thus far, our knowledge on the pathogenesis of Giardia and the related host cell responses is still extensively limited. The aim of this study is to assess the role of endoplasmic reticulum (ER) stress in regulating G0/G1 cell cycle arrest and apoptosis during in vitro infection of intestinal epithelial cells (IECs) with Giardia. The results showed that the mRNA levels of ER chaperone proteins and ER-associated degradation genes were increased and the expression levels of the main unfolded protein response (UPR)-related proteins (GRP78, p-PERK, ATF4, CHOP, p-IRE1, XBP1s and ATF6) were increased upon Giardia exposure. In addition, cell cycle arrest was determined to be induced by UPR signaling pathways (IRE1, PERK and ATF6) through upregulation of p21 and p27 levels and promotion of E2F1-RB complex formation. Upregulation of p21 and p27 expression was shown to be related to Ufd1-Skp2 signaling. Therefore, the cell cycle arrest was induced by ER stress when infected with Giardia. Furthermore, the apoptosis of the host cell was also assessed after exposure to Giardia. The results indicated that apoptosis would be promoted by UPR signaling (PERK and ATF6), but would be suppressed by the hyperphosphorylation of AKT and hypophosphorylation of JNK that were modulated by IRE1 pathway. Taken together, both of the cell cycle arrest and apoptosis of IECs induced by Giardia exposure involved the activation of the UPR signaling. The findings of this study will deepen our understanding of the pathogenesis of Giardia and the associated regulatory network.

Cycloartane triterpenoid from Euphorbia macrostegia modulates ER stress signaling pathways to induce apoptosis in MDA-MB231 and MCF-7 breast cancer cell lines.

Unfolded protein response (UPR) is involved in breast cancer (BC) progression and drug resistance. Many natural products (NPs) could modulate UPR and used for therapeutic purposes. Herein, we aimed to investigate the molecular mechanism of Cycloart-23E-ene-3β, 25-diol (Cycloart-E25), cytotoxicity, as a NP extracted from Euphorbia macrostegia and focused on endoplasmic-reticulum stress (ERS) and UPR signaling pathways. Reactive oxygen species (ROS) were probed by DCFDA fluorescence dye. Apoptosis was assayed by annexin V/propidium iodide (PI), immunoblotting of anti- and proapoptotic, Bcl-2 and Bax proteins, and mitochondrial transmembrane potential (ΔΨm) changes. Thioflavin T (ThT) staining and immunoblotting of UPR signaling components (CHOP, PERK, ATF6, BiP, and XBP1) were recruited for the assessment of ERS. Our results indicated that Cycloart-E25 noticeably increases ROS levels in both MB-231 MDA-MB-231 and MCF-7 cell lines, p>0.05. Flow cytometry assessments revealed an increase in the cell population undergoing apoptosis. Also, the Bax/Bcl-2 ratio increased in a dose-dependent manner following Cycloart-E25 treatment, significantly, p>0.05. Mitochondrial involvement could be deduced by significant decreases in ΔΨm, p>0.05. Cycloart-E25 potently induces protein aggregation and upregulated CHOP, PERK, ATF6, BiP, and XBP1 factors in both MDA-MB-231 MB-231 and MCF-7 cell lines, indicating the involvement of ERS in Cycloart-E25-mediated apoptosis. In conclusion, Cycloart-E25 increased the accumulation of misfolded proteins and upregulated UPR components. Therefore, induction of ERS may be involved in the trigger of apoptosis in BC cell lines. Cycloart-E25 induced apoptosis in breast cancer cell lines through ERS. More assessments are needed to confirm its in vivo anti-tumoral effects.