Embryonic Lethal Abnormal Vision Research Articles

Knock out of ELAVL1 affects steroid synthesis in adrenocortical carcinoma cell line NCI-H295R and leads to a less malignant phenotype

Introduction: Adrenocortical carcinoma (ACC) is a rare malignancy of the adrenal gland. With a 5-year-survival rate below 50% and no curative treatment options in advanced stages new therapeutic options in ACC are needed. Analyzing the ACC cohort of the cancer genome atlas we found a significant negative correlation of the expression level of a gene called Embryonic Lethal Abnormal Vision Like 1 (ELAVL1) and ACC patient survival. ELAVL1 has been described to be unfavorable in a lot of different tumor entities, but its function in ACC progression is still unknown. Methods: To evaluate the function of ELAVL1 in ACC progression we performed a CRISPR/Cas9 mediated knock out (KO) of the ELAVL1 gene in the ACC cell line NCI-H295R. The KO was confirmed on DNA level via Sanger sequencing and on protein level via immunofluorescence and western blot. Transcriptome changes were analyzed by next generation RNA sequencing (NGS). To characterize changes in cell function we performed soft agar colony forming assays and evaluated changes in steroid production via mass spectrometry. Results: Via CRISPR/Cas9 we knocked out ELAVL1 in NCI-H295R cells. NGS data showed 6926 significantly deregulated genes in ELAVL1-KO cells compared to control cells. There was an upregulation of 3468 genes, while 3458 genes showed a lower expression in ELAVL1 KO cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed a significant upregulation of 29 genes associated with aldosterone synthesis and a significant downregulation of 87 genes associated with pathways in cancer. Analyzing cell function via soft agar colony forming assays an impaired colony forming ability in ELAVL1 KO cells was seen. Furthermore, we could prove significant changes in steroid production. ELAVL1 KO led to an inhibition of androgen and glucocorticoid synthesis and an enhancement in mineralocorticoid synthesis. These changes could not only be proven by measuring steroid concentrations in cell culture supernatants, but also by NGS showing a downregulation of the genes CYP17A1 and CYP11B1 and an upregulation of CYP21A2 and CYP11B2. Conclusion: Our study showed that ELAVL1 affects the regulation of around 7000 genes in the ACC cell line NCI-H295R. Especially the inhibition of glucocorticoid synthesis shows that ELAVL1 may be a new target for therapeutic approaches in ACC treatment since hypercortisolism is one of the main symptoms in ACC and a negative prognostic factor for patient survival. The authors have nothing to disclose. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

ELAVL1 ameliorates sevoflurane induced neurotoxicity by inhibiting NLRP3 inflammasome activation

Purpose: To investigate the role of embryonic lethal-abnormal vision like protein 1 (ELAVL1) in sevoflurane-induced neurotoxicity.
 Methods: A cell model was established in HT22 cells by exposing them to 3 % sevoflurane for 12 h. The HT22 cells were transfected with siRNA ELAVL1 (si-ELAVL1) or siRNA negative control (si-NC) to knockdown ELAVL1 expression. Enzyme-linked immunosorbent assay (ELISA) was performed to assess the levels of proinflammatory factors in HT22 cell culture supernatants. Cell apoptosis was analyzed using flow cytometry while apoptosis-related proteins and NLRP3 inflammasome pathway proteins were determined by western blot.
 Results: ELAVL1 was upregulated in sevoflurane-exposed HT22 cells. Sevoflurane exposure also resulted in inflammation, apoptosis and activation of NLRP3 inflammasome of HT22 cells. Importantly, knockdown of ELAVL1 inhibited inflammation and apoptosis in HT22 cells caused by sevoflurane through the inhibition of IL-6, IL-1β and TNF-α production and bys regulating Bax and Bcl-2 protein expression. Furthermore, knockdown of ELAVL1 suppressed NLRP3 inflammasome activity as reflected by the inhibition of the expression of caspase 1, ASC, IL-1β and IL-18. 
 Conclusion: The findings of this study suggest that the knockdown of ELAVL1 alleviates the inflammation and apoptosis of HT22 cells induced by sevoflurane which impeded the activation of NLRP3 inflammasome, suggesting that ELAVL1 would make a good therapeutic target for sevoflurane-caused neurotoxicity.

Analysis of RNA Interference Targeted Against Human Antigen R (HuR) to Reduce Vascular Endothelial Growth Factor (VEGF) Protein Expression in Human Retinal Pigment Epithelial Cells.

VEGF-A or vascular endothelial growth factor-A is an important factor in enabling neovascularization and angiogenesis. VEGF-A is regulated transcriptionally as well as post transcriptionally. Human antigen R (HuR) belonging to the embryonic lethal abnormal vision (ELAV) family is a key regulator promoting stabilization of VEGF-A mRNA. In this research we investigate, whether HuRtargeted RNA interference would enable the reduction of the VEGF-A protein in human retinal pigment epithelial cells (ARPE-19) in-vitro, in normoxic conditions. Three siRNA molecules with sequences complementary to three regions of the HuR mRNA were designed. The three designed siRNA molecules were individually transfected in ARPE-19 cells using Lipofectamine™2000 reagent. Post-transfection (24h, 48h, 72h), downregulation of HuR mRNA was estimated by real-time polymerase reaction, while HuR protein and VEGF-A protein levels were semi-quantitatively determined by western blotting techniques. VEGF-A protein levels were additionally quantified using ELISA techniques. All experiments were done in triplicate. The designed siRNA could successfully downregulate HuR mRNA with concomitant decreases in HuR and VEGF-A protein. The study reveals that HuR downregulation can prominently downregulate VEGF-A, making the protein a target for therapy against pathological angiogenesis conditions such as diabetic retinopathy.

CERS6 antisense RNA 1 promotes colon cancer via upregulating mitochondrial calcium uniporter.

Colon cancer (CC) belongs to a common cancer of digestive system. Long non-coding RNAs (lncRNAs) are dysregulated in numerous cancers and affect their development. The function of lncRNA CERS6 antisense RNA 1 (CERS6-AS1) in CC remains unclear. CERS6-AS1 expression in colon adenocarcinoma tissues and CC cell lines was assessed by The Cancer Genome Atlas database and quantitative real-time polymerase chain reaction analysis. The function of CERS6-AS1 in CC was analysed by 5-ethynyl-2'-deoxyuridine, colony formation, flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labelling, wound healing, Transwell and immunofluorescence assays. Mechanistic analyses including RNA pull down, RNA-binding protein immunoprecipitation and luciferase reporter assay revealed the interaction between RNAs. CERS6-AS1 expression was aberrantly upregulated in colon adenocarcinoma tissues and CC cell lines. CERS6-AS1 knockdown inhibited CC cell malignant phenotypes and in vivo tumour growth. CERS6-AS1 served as the competing endogenous RNA of microRNA-16-5p in CC, and microRNA-16-5p inhibition partly rescued the effects of CERS6-AS1 depletion on CC development. Mitochondrial calcium uniporter was targeted by microRNA-16-5p. Mitochondrial calcium uniporter upregulation completely remedied the influence of CERS6-AS1 silencing in CC progression. Moreover, CERS6-AS1 enhanced the stability of mitochondrial calcium uniporter messenger RNA via recruiting RNA-binding protein embryonic lethal abnormal vision like 1. CERS6-AS1 promotes the development of CC via upregulating mitochondrial calcium uniporter expression.

Genomic organization, evolution and functional characterization of embryonic lethal abnormal vision like protein 1 (ELAVL1) in miiuy croaker (Miichthys miiuy)

Embryonic lethal vision-like protein 1 (ELAVL1), an AU-rich elements (AREs) binding protein involved in the regulation of inflammatory transcript stability, which has not been reported in fish. In this study, we identified the ELAVL1 gene in Miichthys miiuy (mmiELAVL1), and then analyzed its structure and evolution, furthermore described its expression pattern in miiuy croaker. The results showed that mmiELAVL1 and other vertebrate ELAVL1 genes all have three highly conserved RNA recognition motif (RRM) protein domains, and the structure and protein structure are evolutionarily conserved, indicating that their functions may also conservative. In healthy miiuy croaker, mmiELAVL1 was commonly expressed in the tested tissues, and mmiELAVL1 is mainly localized in the nucleus of kidney cells. In addition, mmiELAVL1 responds to poly(I:C) and SCRV stimulation and promotes antiviral genes, indicating its active role in immune process. In summary, this study will facilitate future studies on the role and underlying mechanisms of ELAVL1 in fish immune responses.

Bolwig Organ and Its Role in the Photoperiodic Response of Sarcophaga similis Larvae

Flesh-fly Sarcophaga similis larvae exhibit a photoperiodic response, in which short days induce pupal diapause for seasonal adaptation. Although the spectral sensitivity of photoperiodic photoreception is known, the photoreceptor organ remains unclear. We morphologically identified the Bolwig organ, a larval-photoreceptor identified in several other fly species, and examined the effects of its removal on the photoperiodic response in S. similis. Backfill-staining and embryonic-lethal-abnormal-vision (ELAV) immunohistochemical-staining identified ~34 and 38 cells, respectively, in a spherical body at the ocular depression of the cephalopharyngeal skeleton, suggesting that the spherical body is the Bolwig organ in S. similis. Forward-fill and immunohistochemistry revealed that Bolwig-organ neurons terminate in the vicinity of the dendritic fibres of pigment-dispersing factor-immunoreactive and potential circadian-clock neurons in the brain. After surgical removal of the Bolwig-organ regions, diapause incidence was not significantly different between short and long days, and was similar to that in the insects with an intact organ, under constant darkness. However, diapause incidence was not significantly different between the control and Bolwig-organ-removed insects for each photoperiod. These results suggest that the Bolwig organ contributes partially to photoperiodic photoreception, and that other photoreceptors may also be involved.

Role of Cisplatin in Inducing Acute Kidney Injury and Pyroptosis in Mice via the Exosome miR-122/ELAVL1 Regulatory Axis

Although cisplatin is an effective chemotherapy drug for the treatment of various cancers, its clinical use is limited due to its side effects, especially nephrotoxicity. Unfortunately, acute kidney injury (AKI) caused by cisplatin remains one of the main challenges in effective cancer treatment. Evidence increasingly suggests that renal inflammation and pyroptotic inflammatory cell death of renal tubular epithelial cells (RTECs) mainly determine the progression and outcome of cisplatin-induced AKI. However, it is not clear how cisplatin regulates the pyroptosis of RTECs cells in AKI. The current study aimed to determine the regulation mechanism of AKI induced by cisplatin. We used cisplatin to induce AKI in vivo. We performed H&E staining of mouse kidney tissue sections and evaluated serological indicators of kidney injury (including blood urea nitrogen (BUN), serum creatinine, and tumor necrosis factor-alpha (TNF-α)). We used immunohistochemistry and western blot to detect the important substrate protein gasdermin D (GSDMD) and key target caspase-1 of pyroptosis, respectively. Cisplatin induced mouse AKI and RTECs pyroptosis. HK2 cell-derived exosomes treated with cisplatin influenced pyroptosis of the surrounding HK2 cells. Cisplatin-treated HK2 cells exosome-derived miR-122 regulated pyroptosis in the surrounding cells. Exosome-derived miR-122 affected cisplatin-induced AKI and HK2 cells pyroptosis by regulating the expression of embryonic lethal abnormal vision (ELAVL1). These results suggest that exosome miR-122 inhibited pyroptosis and AKI by targeting ELAVL1 under cisplatin treatment, and this offers a potential target for the treatment of AKI.

LINC00460 Promotes Cutaneous Squamous Cell Carcinoma Progression Through Stabilizing ELAVL1 Protein

Long intergenic noncoding ribonucleic acid (lncRNA) 460 is reportedly associated with carcinogenesis and progression in various types of cancer. However, the mechanisms underlying its action in cutaneous squamous cell carcinoma (CSCC) remain unclear. LINC00460 mRNA expression was analysed using data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Cell growth, migration, and invasion were evaluated using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU), transwell migration and invasion assays after inducing LINC00460 knockdown. A xenograft tumour model was used to determine the effects of LINC00460 on tumour growth and metastasis in vivo. To examine the interaction between LINC00460 and ELAVL1, RNA pulldown and RNA immunoprecipitation assays were performed. LINC00460 was found to be significantly upregulated in CSCC tissues and cell lines. Functionally, LINC00460 knockdown inhibited cell proliferation, migration, and invasion in vitro. Consistent with this, when LINC00460 expression decreased, CSCC tumorigenesis and metastasis in vivo were inhibited. Mechanistically, LINC00460 binds to embryonic lethal abnormal vision like RNA binding protein 1 (ELAVL1) and enhances its stability by inhibiting the β-transducin repeats-containing protein (β-TrCP)-mediated ubiquitination of ELAVL1. Moreover, the effect of LINC00460 silencing on the proliferation, migration, and invasion of CSCC cells could be reversed by overexpressing ELAVL1. Our findings demonstrated that LINC00460 plays a critical role in regulating ELAVL1 function. This highlights the potential targets for the clinical diagnosis and treatment of CSCC.

Lactoferrin Alleviates Lipopolysaccharide-Induced Infantile Intestinal Immune Barrier Damage by Regulating an ELAVL1-Related Signaling Pathway.

As the most important intestinal mucosal barrier of the main body, the innate immune barrier in intestinal tract plays especially pivotal roles in the overall health conditions of infants and young children; therefore, how to strengthen the innate immune barrier is pivotal. A variety of bioactivities of lactoferrin (LF) has been widely proved, including alleviating enteritis and inhibiting colon cancer; however, the effects of LF on intestinal immune barrier in infants and young children are still unclear, and the specific mechanism on how LF inhibits infantile enteritis by regulating immune signaling pathways is unrevealed. In the present study, we firstly performed pharmacokinetic analyses of LF in mice intestinal tissues, stomach tissues and blood, through different administration methods, to confirm the metabolic method of LF in mammals. Then we constructed in Vitro and in Vivo infantile intestinal immune barrier damage models utilizing lipopolysaccharide (LPS), and evaluated the effects of LF in alleviating LPS-induced intestinal immune barrier damage. Next, the related immune molecular mechanism on how LF exerted protective effects was investigated, through RNA-seq analyses of the mouse primary intestinal epithelial cells, and the specific genes were analyzed and screened out. Finally, the genes and their related immune pathway were validated in mRNA and protein levels; the portions of special immune cells (CD4+ T cells and CD8+ T cells) were also detected to further support our experimental results. Pharmacokinetic analyses demonstrated that the integrity of LF could reach mice stomach and intestine after oral gavage within 12 h, and the proper administration of LF should be the oral route. LF was proven to down-regulate the expression levels of inflammatory cytokines in both the primary intestinal epithelial cells and mice blood, especially LF without iron (Apo-LF), indicating LF alleviated infantile intestinal immune barrier damage induced by LPS. And through RNA-seq analyses of the mouse primary intestinal epithelial cells treated with LPS and LF, embryonic lethal abnormal vision Drosophila 1 (ELAVL1) was selected as one of the key genes, then the ELAVL1/PI3K/NF-κB pathway regulated by LF was verified to participate in the protection of infantile intestinal immune barrier damage in our study. Additionally, the ratio of blood CD4+/CD8+ T cells was significantly higher in the LF-treated mice than in the control mice, indicating that LF distinctly reinforced the overall immunity of infantile mice, further validating the strengthening bioactivity of LF on infantile intestinal immune barrier. In summary, LF was proven to alleviate LPS-induced intestinal immune barrier damage in young mice through regulating ELAVL1-related immune signaling pathways, which would expand current knowledge of the functions of bioactive proteins in foods within different research layers, as well as benefit preclinical and clinical researches in a long run.

Study on the correlation of high expression level of HuD with seborrheic keratosis and skin aging.

Currently, most studies of ELAV (Embryonic Lethal, Abnormal Vision, Drosophila)-like protein 4 (Hu antigen D, HuD) focus on nervous system-related diseases; the role of HuD in the occurrence of skin aging and seborrheic keratosis (SK) has not been reported. To explore the role of HuD in the occurrence of SK and skin aging and its related clinical significance. The expression levels of HuD in the skin and blood of healthy people at different ages, SK lesions, and perilesional skin of SK patients were detected by both immunohistochemistry and Western blotting. The mRNA expression levels of HuD in the skin and blood of healthy peoples at different ages were detected by quantitative real-time reverse transcription-polymerase chain reaction. The expression level of HuD was compared with the skin of healthy people, SK lesion, and perilesional skin of SK patients of the same age. The immunohistochemistry and Western blotting showed that the expression levels of HuD in SK lesions were higher than those in healthy skin and perilesional skin. The immunohistochemical staining intensity, protein and mRNA expression levels of HuD in the skin and blood of healthy people were correlated with age, which gradually increased with increasing age. HuD is highly expressed in SK lesion and aged skin, indicating that a higher HuD expression level is correlated with occurrence of SK and aging skin; however, its mechanism needs to be further studied.

MiR-29b-3p suppresses the malignant biological behaviors of AML cells via inhibiting NF-κB and JAK/STAT signaling pathways by targeting HuR

BackgroundHuR/ELAVL1 (embryonic lethal abnormal vision 1) was a downstream target of miR-29b in some cancer cells. HuR protein exerts important prognostic effects of involving in the pathogenesis and development of acute myeloid leukemia (AML). This study aims to investigate the role of miR-29b-3p in biological behaviors of AML cells by targeting HuR and the involvement of the NF-κB and JAK/STAT signaling pathways.MethodsThe expressions of HuR and miR-29b-3p in AML cells were determined using RT-qPCR and Western blot, and the association between them was analyzed using the Spearman method. Next, the target relationship between HuR and miR-29b-3p was predicted by biological information databases and verified by the dual-luciferase reporter gene assay. MTS, methyl cellulose, flow cytometry and transwell assay were employed to detect the cell proliferation, clone formation, cell cycle and apoptosis, invasion and migration respectively, the effect of miR-29b-3p targeted HuR on the biological behaviors of AML cells was explored after over- /down-expression of miR-29b-3p and rescue experiment. Then, immunofluorescence assay and western blot were employed to detect location expression and phosphorylation levels of NF-κB and JAK/STAT signaling pathways related molecules respectively.ResultsHuR was negatively correlated with miR-29b-3p, and was the downstream target of miR-29b-3p in AML cells. When miR-29b-3p was overexpressed in AML cells, HuR was down-regulated, accompanied by cell viability decreased, cell cycle arrest, apoptosis increased, invasion and migration weakened. Moreover, the opposite result appeared after miR-29b-3p was down-regulated. The rescue experiment showed that miR-29b-3p inhibitor could reverse the biological effect of HuR down-regulation in AML cells. Molecular pathway results showed that miR-29b-3p could inhibit p65 expression in nucleus and phosphorylation levels of p65, IκBα, STAT1, STAT3 and STAT5.ConclusionmiR-29b-3p can inhibit malignant biological behaviors of AML cells via the inactivation of the NF-κB and JAK/STAT signaling pathways by targeting HuR. miR-29b-3p and its target HuR can be used as a new potential molecular for AML treatment.

Abstract 4173: Improve anti-PD-1 immunotherapy response in immunologically cold tumors by harnessing RNA-binding protein HuR

Abstract Cancer immunotherapy, especially those targeting the immune checkpoint PD-1 and PD-L1 has emerged as a revolutionizing treatment modality. Immune checkpoint inhibitors (ICIs)-mediated anti-tumor response depends on T cell capability of recognizing and killing tumor cells. ‘Hot’ tumors, which are characterized by the T cell infiltration, show a better response rate to immune checkpoint blockade (ICB) treatment. In contrast, ‘Cold’ tumors have a particularly low objective response rate (ORR) to ICB. In consideration of the durable and impressive responses observed in ‘hot’ tumors, it will be valuable to make an endeavor to turn immunologically cold tumors into hot tumors. The RNA-binding protein HuR is a member of the embryonic lethal abnormal vision (ELAV) family that is overexpressed in a variety of cancers and promotes tumorigenesis by interacting with a subset of oncogenic mRNAs. In this study, we show the direct binding of HuR protein to PD-L1 mRNA in multiple types of cells by Ribonucleoprotein immunoprecipitation (RNP-IP) and further validated by RNA pulldown assay. We also show that PD-L1 mRNA decay is greatly expedited in HuR-knockout cells, which underlines the stabilization function of HuR on PD-L1 transcripts. Immunoblotting results demonstrate that PD-L1 protein expression level is decreased in HuR-knockout cells while up-regulated in HuR-proficient cells in comparison with control. These data together clarify HuR is engaged in PD-L1 post-transcription regulation. Based on this mechanism, we develop a combination strategy using HuR inhibitors and anti-PD1 antibodies to overcome the limitation by regulating the tumor immune microenvironment. KH-200 is a compound that disrupts the transportation of HuR from nuclear to cytoplasm. To test the combination strategy in vivo, we established the EMT6 syngeneic breast cancer model. After 4-week treatment, the combination group shows great effects in inhibiting tumor growth and prolonging survival. A similar combination effect was further observed in Lewis lung syngeneic lung cancer model. In conclusion, we preliminarily explored the role of HuR in tumor immune evasion. The method using HuR inhibitors as a combination in immunotherapy shows effectiveness in immunologically cold tumor treatment and may have reference significance for clinical cancer therapy. Citation Format: Qi Zhang, Yang Zhe, Xiaoqing Wu, Lan Lan, Lanjing Wei, Liang Xu. Improve anti-PD-1 immunotherapy response in immunologically cold tumors by harnessing RNA-binding protein HuR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4173.

Harness RNA‐binding protein HuR to boost Anti‐PD‐1 Immunotherapy in immunologically cold tumors

Cancer immunotherapy, especially those targeting the immune checkpoint PD‐1 and PD‐L1 has emerged as a revolutionizing treatment modality. Immune checkpoint inhibitors (ICIs)‐mediated anti‐tumor response depends on T cell capability of recognizing and killing tumor cells. ‘Hot’ tumors, which are characterized by the T cell infiltration, show a better response rate to immune checkpoint blockade (ICB) treatment. In contrast, ‘Cold’ tumors have a particularly low objective response rate (ORR) to ICB. In consideration of the durable and impressive responses observed in ‘hot’ tumors, it will be valuable to make an endeavor to turn immunologically cold tumors into hot tumors. The RNA‐binding protein HuR is a member of the embryonic lethal abnormal vision (ELAV) family that is overexpressed in a variety of cancers and promotes tumorigenesis by interacting with a subset of oncogenic mRNAs.In this study, we show the direct binding of HuR protein to PD‐L1 mRNA in multiple types of cells by Ribonucleoprotein immunoprecipitation (RNP‐IP) and further validated by RNA pulldown assay. We also show that PD‐L1 mRNA decay is greatly expedited in HuR‐knockout cells, which underlines the stabilization function of HuR on PD‐L1 transcripts. Immunoblotting results demonstrate that PD‐L1 protein expression level is decreased in HuR‐knockout cells while up‐regulated in HuR‐proficient cells in comparison with control. These data together clarify HuR is engaged in PD‐L1 post‐transcription regulation.Based on this mechanism, we develop a combination strategy using HuR inhibitors and anti‐PD1 antibody to overcome the limitation by regulating the tumor immune microenvironment.KH‐200 is a compound that disrupts the transportation of HuR from nuclear to cytoplasm. To test the combination strategy in vivo, we established the EMT6 syngeneic breast cancer model. After 4‐week treatment, the combination group shows great effects in inhibiting tumor growth and prolonging survival. The similar combination effect was further observed in Lewis lung syngeneic lung cancer model.In conclusion, we preliminarily explored the role of HuR in tumor immune evasion. The method using HuR inhibitors as a combination in immunotherapy shows an effective in immunologically cold tumor treatment and may have reference significance for clinical cancer therapy.

Glial regenerative response in the imaginal discs of Drosophila melanogaster.

Glial regenerative response in the imaginal discs of Drosophila melanogaster.

Glucose deprivation enhances resistance to paclitaxel via ELAVL2/4-mediated modification of glycolysis in ovarian cancer cells.

The dysregulation of glycolysis regardless of oxygen availability is one of the major characteristics of cancer cells. While the drug resistance of ovarian cancer cells has been extensively studied, the molecular mechanism of anticancer drug resistance under low-glucose conditions remains unknown. In this study, we investigated the pathway mediating drug resistance under low-glucose conditions by examining the relationship between embryonic lethal abnormal vision Drosophila homolog-like (ELAVL) protein and glycolysis-related enzymes. Ovarian cancer cells resistant to 2.5 nM paclitaxel were exposed to low-glucose media for 2 weeks, and the expression levels of ELAVL2, ELAVL4, glycolytic enzymes, and drug resistance-related proteins were elevated to levels comparable to those in cells resistant to 100 nM paclitaxel. Gene silencing of ELAVL2/4 using small interfering RNA prevented the upregulation of glycolysis-related enzymes, reduced lactate production, and sensitized 2.5 nM paclitaxel-resistant ovarian cancer cells to anticancer agents under hypoglycemic conditions. Furthermore, pharmacological inhibition of glycolytic enzymes with 2-deoxyglucose, a specific inhibitor of glycolysis, triggered caspase-dependent apoptosis, reduced lactate generation, and blocked the expression of drug resistance-related proteins under low-glucose conditions. These results suggest that the level of ELAVL2/4 is responsible for the development of chemoresistance through activation of the glycolysis pathway under glucose deprivation conditions.

Abstract 1560: Targeting RNA-binding protein HuR to improve anti-PD-1 immunotherapy response in breast cancer

Abstract Breast cancer has been considered as a less immune-sensitive disease. Patients with metastatic breast cancer have a particularly low objective response rate (ORR) to anti-PD-1/PD-L1 therapy (3%~24%). It will be valuable to improve the treatment efficacy of anti-PD-1 immunotherapy in breast cancer. Recent studies reported that exosomal PD-L1 derived from tumor cells mediated immune surveillance evasion by interacting with the PD-1 receptor on T cells and contributed to the low ORR in anti-PD-1/PD-L1 immunotherapy.The RNA-binding protein Hu antigen R (HuR) is a member of the embryonic lethal abnormal vision (ELAV) family that is overexpressed in a variety of cancers and promotes tumorigenesis by interacting with a subset of oncogenic mRNAs, including PD-L1 mRNA. HuR also plays an important role in the exosome secretion process by regulating RAB27B mRNA. In this study, we aim to identify HuR biological functions in the cancer-immune system and evaluate the combination strategy using a HuR inhibitor with an anti-PD-1 antibody in breast cancer. We show here the direct binding of HuR protein to PD-L1 mRNA in human breast cancer cell line MDA-MB-231 by Ribonucleoprotein immunoprecipitation (RNP-IP). We also show that PD-L1 protein expression level is decreased in HuR knockout clones and in exosomes derived from HuR knockout clones. HuR knockout downregulates exosome secretion with decreased size in MDA-MB-231 cells. KH-3 is a small molecule identified from several chemical libraries that disrupts the binding of HuR and its target mRNAs.To test the combination strategy using a HuR inhibitor and anti-PD-1 antibody in vivo, we established the EMT6 orthotopic mouse breast cancer model. A combination treatment with HuR inhibitor KH-3 and anti-PD-1 antibody inhibits tumor growth and prolongs survival in this breast cancer model. In conclusion, we preliminarily explored the role of HuR in the cancer-immune system, we showed that HuR may be a promising therapeutic target to overcome immune evasion and improve immunotherapy response in breast cancer. Citation Format: Qi Zhang, Xiaoqing Wu, Lan Lan, Lanjing Wei, Liang Xu. Targeting RNA-binding protein HuR to improve anti-PD-1 immunotherapy response in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1560.

HuR Protein in Hepatocellular Carcinoma: Implications in Development, Prognosis and Treatment.

Hu-antigen R (HuR) is a post-transcriptional regulator that belongs to the embryonic lethal abnormal vision Drosophila-like family (ELAV). HuR regulates the stability, translation, subcellular localization, and degradation of several target mRNAs, which are implicated in carcinogenesis and could affect therapeutic options. HuR protein is consistently highly expressed in hepatocellular carcinoma (HCC) compared to the adjacent normal liver tissue and is involved in the post-transcriptional regulation of various genes implicated in liver malignant transformation. Additionally, HuR protein seems to be a putative prognosticator in HCC, predicting worse survival. This review summarizes the recent evidence regarding the role of HuR in primary liver tumors, as presented in clinical studies, in vitro experiments and in vivo animal models. In conclusion, our review supports the consistent role of HuR protein in the development, prognosis, and treatment of HCC. Additional studies are expected to expand current information and exploit its putative employment as a future candidate for more personalized treatment in these tumors.

High-resolution epitope mapping of anti-Hu and anti-Yo autoimmunity by programmable phage display.

Paraneoplastic neurological disorders are immune-mediated diseases understood to manifest as part of a misdirected anti-tumor immune response. Paraneoplastic neurological disorder-associated autoantibodies can assist with diagnosis and enhance our understanding of tumor-associated immune processes. We designed a comprehensive library of 49-amino-acid overlapping peptides spanning the entire human proteome, including all splicing isoforms and computationally predicted coding regions. Using this library, we optimized a phage immunoprecipitation and sequencing protocol with multiple rounds of enrichment to create high-resolution epitope profiles in serum and cerebrospinal fluid (CSF) samples from patients suffering from two common paraneoplastic neurological disorders, the anti-Yo (n = 36 patients) and anti-Hu (n = 44 patients) syndromes. All (100%) anti-Yo patient samples yielded enrichment of peptides from the canonical anti-Yo (CDR2 and CDR2L) antigens, while 38% of anti-Hu patients enriched peptides deriving from the nELAVL (neuronal embryonic lethal abnormal vision like) family of proteins, the anti-Hu autoantigenic target. Among the anti-Hu patient samples that were positive for nELAVL, we noted a restricted region of immunoreactivity. To achieve single amino acid resolution, we designed a novel deep mutational scanning phage library encoding all possible single-point mutants targeting the reactive nELAVL region. This analysis revealed a distinct preference for the degenerate motif, RLDxLL, shared by ELAVL2, 3 and 4. Lastly, phage immunoprecipitation sequencing identified several known autoantigens in these same patient samples, including peptides deriving from the cancer-associated antigens ZIC and SOX families of transcription factors. Overall, this optimized phage immunoprecipitation sequencing library and protocol yielded the high-resolution epitope mapping of the autoantigens targeted in anti-Yo and anti-Hu encephalitis patients to date. The results presented here further demonstrate the utility and high-resolution capability of phage immunoprecipitation sequencing for both basic science and clinical applications and for better understanding the antigenic targets and triggers of paraneoplastic neurological disorders.

Conditionally Replicative Adenovirus Controlled by the Stabilization System of AU-Rich Elements Containing mRNA.

AU-rich elements (AREs) are RNA elements that enhance the rapid decay of mRNAs, including those of genes required for cell growth and proliferation. HuR, a member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, is involved in the stabilization of ARE-mRNA. The level of HuR in the cytoplasm is up-regulated in most cancer cells, resulting in the stabilization of ARE-mRNA. We developed the adenoviruses AdARET and AdAREF, which include the ARE of TNF-α and c-fos genes in the 3′-untranslated regions of the E1A gene, respectively. The expression of the E1A protein was higher in cancer cells than in normal cells, and virus production and cytolytic activities were also higher in many types of cancer cells. The inhibition of ARE-mRNA stabilization resulted in a reduction in viral replication, demonstrating that the stabilization system was required for production of the virus. The growth of human tumors that formed in nude mice was inhibited by an intratumoral injection of AdARET and AdAREF. These results indicate that these viruses have potential as oncolytic adenoviruses in the vast majority of cancers in which ARE-mRNA is stabilized.

Deletion of Elavl1 in renal collecting duct cells is associated with a specific gene expression pattern resulting in major functional changes

IntroductionThe action of vasopressin and aldosterone on water and sodium reabsorption in the renal collecting duct includes the action of the RNA binding protein human antigen R also known as Embryonic Lethal Abnormal Vision Like 1 (Elavl1). Elavl1 modifies the protein expression of the epithelial sodium channel EnaC by stabilizing the mRNA. Under hypertonic conditions it down regulates the expression of the mineralocorticoid receptor (MR), the endogenous target of aldosterone. So far, the role of Elavl1 on the expression of other factors has not been studied, nor whether hypertonicity has an effect on Elavl1 mediated gene expression. Within this study we identified hundreds of genes that show differences in expression upon Elavl1 knock out.MethodsWe used the CRISPR‐Cas9 technology to delete Elavl1. First we cloned specific guideRNAs targeting Elavl1 and scrambled gRNA into the lentiV2‐vector and used them to produce virus particles in HEK293T‐cells. With those particles we transduced a mouse cortical collecting duct cell line (mpkccd). The successful induction of mutation in the Elavl1 locus was verified by Sanger‐Sequencing. Single cell clones were selected and the deletion on protein level was confirmed by immunofluorescence analysis. The Elavl1‐deficient cells were used for functional analysis like cell proliferation, migration and colony forming capacity. Using next generation sequencing we analyzed the gene expression profile comparing Elavl1 deficient to control cells and identified signaling pathways, which were deregulated by deletion of Elavl1.ResultsThe successful deletion of Elavl1 was functionally associated with significantly slower proliferation, migration and lower colony forming capacity. As expected, Elavl1 deficiencies lead to down regulation of the MR gene. Beside MR the expression of more than 440 genes is significantly (p<0,001) up and more than 2700 genes are down regulated. Classification by gene ontology terms showed that gene members of the ontology categories like “enzyme binding”, „rna binding”,„cell stress” and „cell death” are significantly enriched. KEGG pathway analysis showed that the differentially expressed genes are involved in metabolic and cancer associated pathways, such as „PI3K‐AKT‐Signaling pathway” or „HIF1A Signaling pathway”.DiscussionWithin this study we showed a comprehensive analysis of Elval1 function in renal collecting duct cells. Beside mayor physiological differences, Elavl1 deficiency is associated with massive changes in gene expression. Further analyses are needed to investigate its role for in vivo renal function.