ADP-ribosylation Factor-like Protein Research Articles

ARL13B promotes cell cycle through the sonic hedgehog signaling pathway to alleviate nerve damage during cerebral ischemia/reperfusion in rats

Cerebral ischemia/reperfusion (CIRI) is a leading cause of death worldwide. A small GTPase known as ADP-ribosylation factor-like protein 13B (ARL13B) is essential in several illnesses. The role of ARL13B in CIRI remains unknown, though. A middle cerebral artery occlusion/reperfusion (MCAO/R) in rats as well as an oxygen-glucose deprivation/reoxygenation (OGD/R) models in PC12 cells were constructed. The neuroprotective effects of ARL13B against MCAO/R were evaluated using neurological scores, TTC staining, rotarod testing, H&E staining, and Nissl staining. To detect the expression of proteins associated with the SHH pathway and apoptosis, western blotting and immunofluorescence were employed. Apoptosis was detected using TUNEL assays and flow cytometry. There was increased expression of ARL13B in cerebral ischemia/reperfusion models. However, ARL13B knockdown aggravated CIRI nerve injury by inhibiting the sonic hedgehog (SHH) pathway. In addition, the use of SHH pathway agonist (SAG) can increased ARL13B expression, reverse the effects of ARL13B knockdown exacerbating CIRI nerve injury. ARL13B alleviated cerebral infarction and pathological injury and played a protective role against MCAO/R. Furthermore, ARL13B significantly increased the expression of SHH pathway-related proteins and the anti-apoptotic protein BCL-2, while decreased the expression of pro-apoptotic protein BAX, thus reducing apoptosis. The results from the OGD/R model in PC12 cells were consistent with those obtained in vivo. Surprisingly, we demonstrated that ARL13B regulates the cell cycle to protect against CIRI nerve injury. Our findings indicate that ARL13B protects against CIRI by reducing apoptosis through SHH-dependent pathway activation, and suggest that ARL13B plays a crucial role in CIRI pathogenesis.

Proximity labeling of host factor ANXA3 in HCV infection reveals a novel LARP1 function in viral entry

Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.

Abstract 4683: Suppression of ARL6ip1 inhibits malignant phenotypes of human colorectal cancer cells in vivo and > in situ

Abstract Introduction: Conophylline is an alkaloid isolated from the leaves of the Thai plant, Ervatamia microphylla. We isolated conophylline as an inhibitor of K-Ras functions and an activator of pancreatic β-cell differentiation. It ameliorates various disease models in animals, including cancer, diabetes mellitus, NASH, and hepatic cirrhosis. On the other hand, its molecular target was determined to be ADP-ribosylation factor-like 6-interacting protein 1 (ARL6ip1) using a conophylline-biotin conjugate. ARL6ip1 is located in the endoplasmic reticulum (ER) membrane, and conophylline binds to the cytoplasmic portion of ARL6ip1. Known functions of ARL6ip1 include inhibition of apoptosis, inhibition of glutamate transporter, and modulation of the ER structure. Previously, we knocked out ARL6ip1 in human colon carcinoma cells by CRISPR-Cas9 and found that either deletion of ARL6ip1 or addition of conophylline inhibited migration, invasion and tumorigenicity in cultured cancer cells. In the present research we have studied the in vivo anticancer effect of conophylline and ARL6ip1 knockout, and the mechanism of anticancer activity in cultured cells. Materials and Methods: Conophylline was isolated from the leaves of Ervatamia microphylla. We employed HCT116 and DLD1 cell lines as human colorectal cancer cells. Cellular migration was measured by a wound-healing assay. The activity of Akt was measured by the phospho-Akt antibody. BALB/cSlc-nu/nu mice were used for the animal experiment. Results: Colon adenocarcinoma tissue microarray comparing tumor and adjacent normal tissues revealed that ARL6ip1 expression was significantly higher in cancer than in normal tissues. Intraperitoneal administration of conophylline and knockout of ARL6ip1 both inhibited the growth of HCT116 in mice. Neither influenced the bodyweight of animals. Conophylline and deletion of ARL6ip1 both inhibited the migration. Rescue of ARL6ip1 canceled the effect of ARL6ip1 deletion on migration in HCT116 and DLD1 cells. As the mechanism, either conophylline or ARL6ip1 knockout inhibited the Akt activity, which was canceled by the rescue of ARL6ip1 in both cell lines. Conclusion: ARL6ip1 was found to be highly expressed in the tumor tissue in colorectal cancer. CRISPR-Cas9 knockout of ARL6ip1 showed a similar anticancer activity as treatment with conophylline in the animal experiment. It is likely that the anticancer activity of conophylline would be mediated by Akt. Thus, ARL6ip1 would be a useful molecular target for the treatment of cancer. Citation Format: Yinzhi Lin, Sivasundaram Karnan, Hideaki Ito, Kazuo Umezawa. Suppression of ARL6ip1 inhibits malignant phenotypes of human colorectal cancer cells in vivo and > in situ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4683.

Thalamic Neuron Resilience during Osmotic Demyelination Syndrome (ODS) Is Revealed by Primary Cilium Outgrowth and ADP-ribosylation factor-like protein 13B Labeling in Axon Initial Segment.

A murine osmotic demyelinating syndrome (ODS) model was developed through chronic hyponatremia, induced by desmopressin subcutaneous implants, followed by precipitous sodium restoration. The thalamic ventral posterolateral (VPL) and ventral posteromedial (VPM) relay nuclei were the most demyelinated regions where neuroglial damage could be evidenced without immune response. This report showed that following chronic hyponatremia, 12 h and 48 h time lapses after rebalancing osmolarity, amid the ODS-degraded outskirts, some resilient neuronal cell bodies built up primary cilium and axon hillock regions that extended into axon initial segments (AIS) where ADP-ribosylation factor-like protein 13B (ARL13B)-immunolabeled rod-like shape content was revealed. These AIS-labeled shaft lengths appeared proportional with the distance of neuronal cell bodies away from the ODS damaged epicenter and time lapses after correction of hyponatremia. Fine structure examination verified these neuron abundant transcriptions and translation regions marked by the ARL13B labeling associated with cell neurotubules and their complex cytoskeletal macromolecular architecture. This necessitated energetic transport to organize and restore those AIS away from the damaged ODS core demyelinated zone in the murine model. These labeled structures could substantiate how thalamic neuron resilience occurred as possible steps of a healing course out of ODS.

Increasing Ciliary ARL13B Expression Drives Active and Inhibitor-Resistant Smoothened and GLI into Glioma Primary Cilia.

ADP-ribosylation factor-like protein 13B (ARL13B), a regulatory GTPase and guanine exchange factor (GEF), enriches in primary cilia and promotes tumorigenesis in part by regulating Smoothened (SMO), GLI, and Sonic Hedgehog (SHH) signaling. Gliomas with increased ARL13B, SMO, and GLI2 expression are more aggressive, but the relationship to cilia is unclear. Previous studies have showed that increasing ARL13B in glioblastoma cells promoted ciliary SMO accumulation, independent of exogenous SHH addition. Here, we show that SMO accumulation is due to increased ciliary, but not extraciliary, ARL13B. Increasing ARL13B expression promotes the accumulation of both activated SMO and GLI2 in glioma cilia. ARL13B-driven increases in ciliary SMO and GLI2 are resistant to SMO inhibitors, GDC-0449, and cyclopamine. Surprisingly, ARL13B-induced changes in ciliary SMO/GLI2 did not correlate with canonical changes in downstream SHH pathway genes. However, glioma cell lines whose cilia overexpress WT but not guanine exchange factor-deficient ARL13B, display reduced INPP5e, a ciliary membrane component whose depletion may favor SMO/GLI2 enrichment. Glioma cells overexpressing ARL13B also display reduced ciliary intraflagellar transport 88 (IFT88), suggesting that altered retrograde transport could further promote SMO/GLI accumulation. Collectively, our data suggest that factors increasing ARL13B expression in glioma cells may promote both changes in ciliary membrane characteristics and IFT proteins, leading to the accumulation of drug-resistant SMO and GLI. The downstream targets and consequences of these ciliary changes require further investigation.

Long Non-Coding RNA ZSCAN16-AS1 Promotes the Malignant Progression of Melanoma Through Regulating the miR-503-5p/ARL2 Axis.

LncRNA zinc finger and SCAN domain containing 16 antisense RNA 1 (ZSCAN16-AS1), a newly identified lncRNA, has been proven to accelerate hepatocellular carcinoma progression. However, the function and molecular mechanism of ZSCAN16-AS1 in melanoma are still unknown. The level of ZSCAN16-AS1 in melanoma tissues was detected and reported in The Cancer Genome Atlas (TCGA) and GEO#GSE15605. CCK-8, Transwell and flow cytometry assays were used to explore the role of ZSCAN16-AS1 in melanoma cells. Luciferase reporter assays and RNA pull-down assays were used to verify the molecular mechanism of ZSCAN16-AS1. Here, we found that ZSCAN16-AS1 expression was increased in melanoma. We confirmed that ZSCAN16-AS1 promotes the growth and metastasis of melanoma. ZSCAN16-AS1 exerts its pro-tumour role through sponging of miR-503-5p to liberate ADP-ribosylation factor-like protein 2 (ARL2) mRNA transcripts. These results demonstrated the role and molecular mechanism of ZSCAN16-AS1 in the occurrence and development of melanoma. Therefore, ZSCAN16-AS1 may be used as a specific biomarkerin the diagnosis and treatment of melanoma patients.

ARL6IP1 mediates small-molecule-induced alleviation of Alzheimer pathology through FXR1-dependent BACE1 translation initiation

Exploring the potential lead compounds for Alzheimer's disease (AD) remains one of the challenging tasks. Here, we report that the plant extract conophylline (CNP) impeded amyloidogenesis by preferentially inhibiting BACE1 translation via the 5' untranslated region (5'UTR) and rescued cognitive decline in an animal model of APP/PS1 mice. ADP-ribosylation factor-like protein 6-interacting protein 1 (ARL6IP1) was then found to mediate the effect of CNP on BACE1 translation, amyloidogenesis, glial activation, and cognitive function. Through analysis of the 5'UTR-targetd RNA-binding proteins by RNA pulldown combined with LC-MS/MS, we found that FMR1 autosomal homolog 1 (FXR1) interacted with ARL6IP1 and mediated CNP-induced reduction of BACE1 by regulating the 5'UTR activity. Without altering the protein levels of ARL6IP1 and FXR1, CNP treatment promoted ARL6IP1 interaction with FXR1 and inhibited FXR1 binding to the 5'UTR both invitro and invivo. Collectively, CNP exhibited a therapeutic potential for AD via ARL6IP1. Through pharmacological manipulation, we uncovered a dynamic interaction between FXR1 and the 5'UTR in translational control of BACE1, adding to the understanding of the pathophysiology of AD.

Abstract 3968: Conophylline-target ARL6ip1 regulates Ran-mediated cellular migration and invasion in human colorectal cancer cells

Abstract Introduction: Conophylline is an alkaloid isolated from the leaves of Ervatamia microphylla. We isolated conophylline as an inhibitor of K-Ras functions. It is known to ameliorate various disease models in animals, including cancer, diabetes mellitus, NASH, and hepatic cirrhosis. On the other hand, its molecular target was determined to be ADP-ribosylation factor-like 6-interacting protein 1 (ARL6ip1) using a conophylline-biotin conjugate. ARL6ip1 is located in the endoplasmic reticulum (ER) membrane, and the conophylline-binding domain was determined by deletion mutation analysis. Known functions of ARL6ip1 include inhibition of apoptosis, inhibition of glutamate transporter, and modulation of the ER structure. However, whether ARL6ip1 is involved in the mechanisms of various biological activities of conophylline has not been proven, since knockdown of ARL6ip1 has often failed to change the cellular phenotypes. Therefore, in the present research, we knocked out ARL6ip1 in human colon carcinoma cells by CRISPR-Cas9 and studied the involvement and mechanism of ARL6ip1 in the anticancer activity of conophylline. Ran activity was measured by a pull-down assay with Ran-GTP antibody. Materials and Methods: Conophylline was isolated from the leaves of Ervatamia microphylla. We employed HCT116 and DLD1 cells as human colorectal cancer cells. Tumorigenicity was measured by soft agar colony formation. Cellular migration was measured by a wound healing assay and cell tracking analysis. Cellular invasion was measured by a Matrigel chamber assay. Results: Conophylline inhibited soft agar colony formation in HCT116 and DLD1 cells. It inhibited migration and invasion in HCT116 and DLD1 cells at nontoxic concentrations. Knockout of ARL6ip1 also decreased tumorigenicity, migration, and invasion in HCT116 and DLD1 cells. The mechanistic study was carried out with HCT116 cells. ARL6 and Ran are both G-proteins and are reported to interact with ARL6ip1. Although knockdown of ARL6 did not change the cell migratory activity, knockdown of Ran inhibited the migration. Conophylline inhibited the interaction of ARL6ip1 to Ran in the proximity ligation assay. Moreover, conophylline inhibited the Ran activity. Conclusion: CRISPR-Cas9 knockout of ARL6ip1 showed a similar anticancer activity as treatment with conophylline. It is likely that the anticancer activity of conophylline would be mediated by the ARL6ip1-Ran system. Thus, ARL6ip1 would be a useful molecular target for the treatment of cancer. Citation Format: Yinzhi Lin, Sivasundaram Karnan, Hideaki Ito, Kazuo Umezawa. Conophylline-target ARL6ip1 regulates Ran-mediated cellular migration and invasion in human colorectal cancer cells. [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 3968.

Investigating the prognosis gene profile of triple-negative breast cancer

Background: Current therapeutic strategies have poor effects in triple-negative breast cancer (TNBC) patients due to lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 expression. Identification of novel genes of TNBC prognosis aids in the development of effective treatment strategies. Aim: We aim at explore key genes related to TNBC recurrence. Methods: RNAseq and clinical characteristics data were derived from The Cancer Genome Atlas Breast Invasive Carcinoma project. Ninety-seven TNBC patients were included. We used DESeq2 and Cox regression to identify significant genes to TNBC recurrence. Pathway enrichment analysis and protein–protein interaction plot were conducted to understand the functions of target genes. Results: We discovered top nine important genes for TNBC recurrence. Lower mRNA expression of potassium voltage-gated channel subfamily Q member 5, H3 clustered histone 10, and ADP-ribosylation factor-like protein 17 and higher mRNA expression of synuclein beta, interleukin 6 (IL-6), casein kappa, RHOC, phosphodiesterase 8B, and laminin subunit alpha 3 (LAMA3) were associated with higher risk of recurrence. IL-6, LAMA3, and Ras homolog family member V (RHOV) genes out of nine candidate genes can make the best prediction of TNBC recurrence (area under receiver operating characteristic curve: 0.95, sensitivity: 0.89 and specificity: 0.97). The top three significant Gene Ontology (GO) pathways are nucleosome, ion gated channel activity, and epidermis development. Significant GO pathways can be categorized into four functions: cell–cell adhesion, cell transportation, cell proliferation, ion channel and transporter, and immune. Conclusion: We discovered that the gene set of IL6, LAMA3, and RHOV can accurately predict TNBC recurrence. These genes warrant further study to confirm their causal association with TNBC prognosis and possible treatment targets.

The inhibition of putrescine synthesis affects the in vitro shoot development of Cedrela fissilis Vell. (Meliaceae) by altering endogenous polyamine metabolism and the proteomic profile

Polyamines —(PAs) and proteins have been demonstrated to be fundamental for in vitro shoot development of Cedrela fissilis. We evaluated the influence of 6-benzyladenine (BA) and putrescine (Put) on the growth of shoots, PA metabolism and proteomic profiles of C. fissilis. The longest shoots were obtained under 2.5 µM BA + 2.5 mM Put treatment. The inhibition of Put synthesis by d-arginine (d-arg) reduced the activities of the Put biosynthesis enzymes arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) and the endogenous contents of free Put, resulting in reduced shoot growth. The ODC activity was higher than that of ADC, being ODC the main enzyme in the synthesis of Put in C. fissilis. Inhibition of Put synthesis affected the proteomic profile, reducing the accumulation of the ubiquitin receptor RAD23c, peroxidase 15, ADP-ribosylation factor 1, ADP-ribosylation factor-like protein 8a, profilin-4, profilin-2, glucan endo-1,3-beta-glucosidase, and expansin-like B1 and increasing the accumulation of V-type proton ATPase catalytic subunit A and methionine gamma-lyase, highlighting the relevance of these proteins in promoting the length of shoots. Moreover, the transport protein SEC13 homolog B and the basic isoform glucan endo-1,3-beta-glucosidase, unique proteins in shoots treated with BA + Put, were related to the promotion of shoot growth. Our results show that the modulation of endogenous PAs and proteomic profiles is necessary to regulate in vitro morphogenesis in C. fissilis. Moreover, the ODC enzyme is highly involved in the synthesis of Put during in vitro shoot development and is described for the first time in this species.

ARL11 correlates with the immunosuppression and poor prognosis in breast cancer: A comprehensive bioinformatics analysis of ARL family members

ADP-ribosylation factor-like protein (ARL) family members (ARLs) may regulate the malignant phenotypes of cancer cells. However, relevant studies on ARLs in breast cancer (BC) are limited. In this research, the expression profiles, genetic variations, and prognostic values of ARLs in BC have been systematically analyzed for the first time using various databases. We find that ARLs are significantly dysregulated in BC according to the TCGA database, which may result from DNA methylation and copy number alteration. Prognostic analysis suggests that ARL11 is the most significant prognostic indicator for BC, and higher ARL11 predicts worse clinical outcomes for BC patients. Further functional enrichment analysis demonstrates that ARL11 enhances the immunosuppression in BC, and dysregulation of ARL11 is significantly associated with immune infiltration in various types of cancer. Our results demonstrate the potential of ARL11 as an immune therapeutic target for BC.

Autophagy promotes hepatic cystogenesis in polycystic liver disease by depletion of cholangiocyte ciliogenic proteins.

Polycystic liver disease (PLD) is characterized by defective cholangiocyte cilia that regulate progressive growth of hepatic cysts. Because formation of primary cilia is influenced by autophagy through degradation of proteins involved in ciliogenesis, we hypothesized that ciliary defects in PLD cholangiocytes (PLDCs) originate from autophagy-mediated depletion of ciliogenic proteins ADP-ribosylation factor-like protein 3 (ARL3) and ADP-ribosylation factor-like protein 13B (ARL13B) and ARL-dependent mislocation of a ciliary-localized bile acid receptor, Takeda G-protein-coupled receptor 5 (TGR5), the activation of which enhances hepatic cystogenesis (HCG). The aims here were to determine whether: (1) ciliogenesis is impaired in PLDC, is associated with increased autophagy, and involves autophagy-mediated depletion of ARL3 and ARL13B; (2) depletion of ARL3 and ARL13B in PLDC cilia impacts ciliary localization of TGR5; and (3) pharmacological inhibition of autophagy re-establishes cholangiocyte cilia and ciliary localization of ARL3, ARL3B, and TGR5 and reduces HCG. By using liver tissue from healthy persons and patients with PLD, in vitro and in vivo models of PLD, and in vitro models of ciliogenesis, we demonstrated that, in PLDCs: ciliogenesis is impaired; autophagy is enhanced; ARL3 and ARL13B are ubiquitinated by HDAC6, depleted in cilia, and present in autophagosomes; depletion of ARL3 and ARL13B impacts ciliary localization of TGR5; and pharmacological inhibition of autophagy with mefloquine and verteporfin re-establishes cholangiocyte cilia and ciliary localization of ARL3, ARL13B, and TGR5 and reduces HCG. The intersection between autophagy, defective cholangiocyte cilia, and enhanced HCG contributes to PLD progression and can be considered a target for therapeutic interventions.

The molecular appearance of native TRPM7 channel complexes identified by high-resolution proteomics.

The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed membrane protein consisting of ion channel and protein kinase domains. TRPM7 plays a fundamental role in the cellular uptake of divalent cations such as Zn2+, Mg2+, and Ca2+, and thus shapes cellular excitability, plasticity, and metabolic activity. The molecular appearance and operation of TRPM7 channels in native tissues have remained unresolved. Here, we investigated the subunit composition of endogenous TRPM7 channels in rodent brain by multi-epitope affinity purification and high-resolution quantitative mass spectrometry (MS) analysis. We found that native TRPM7 channels are high-molecular-weight multi-protein complexes that contain the putative metal transporter proteins CNNM1-4 and a small G-protein ADP-ribosylation factor-like protein 15 (ARL15). Heterologous reconstitution experiments confirmed the formation of TRPM7/CNNM/ARL15 ternary complexes and indicated that complex formation effectively and specifically impacts TRPM7 activity. These results open up new avenues towards a mechanistic understanding of the cellular regulation and function of TRPM7 channels.

A Novel Deoxynivalenol-Activated Wheat Arl6ip4 Gene Encodes an Antifungal Peptide with Deoxynivalenol Affinity and Protects Plants against Fusarium Pathogens and Mycotoxins.

Deoxynivalenol (DON) is one of the most widespread trichothecene mycotoxins in contaminated cereal products. DON plays a vital role in the pathogenesis of Fusarium graminearum, but the molecular mechanisms of DON underlying Fusarium–wheat interactions are not yet well understood. In this study, a novel wheat ADP-ribosylation factor-like protein 6-interacting protein 4 gene, TaArl6ip4, was identified from DON-treated wheat suspension cells by suppression subtractive hybridization (SSH). The qRT-PCR result suggested that TaArl6ip4 expression is specifically activated by DON in both the Fusarium intermediate susceptible wheat cultivar Zhengmai9023 and the Fusarium resistant cultivar Sumai3. The transient expression results of the TaARL6IP4::GFP fusion protein indicate that TaArl6ip4 encodes a plasma membrane and nucleus-localized protein. Multiple sequence alignment using microscale thermophoresis showed that TaARL6IP4 comprises a conserved DON binding motif, 67HXXXG71, and exhibits DON affinity with a dissociation constant (KD) of 91 ± 2.6 µM. Moreover, TaARL6IP4 exhibited antifungal activity with IC50 values of 22 ± 1.5 µM and 25 ± 2.6 µM against Fusarium graminearum and Alternaria alternata, respectively. Furthermore, TaArl6ip4 interacted with the plasma membrane of Fusarium graminearum spores, resulting in membrane disruption and the leakage of cytoplasmic materials. The heterologous over-expression of TaArl6ip4 conferred greater DON tolerance and Fusarium resistance in Arabidopsis. Finally, we describe a novel DON-induced wheat gene, TaArl6ip4, exhibiting antifungal function and DON affinity that may play a key role in Fusarium–wheat interactions.

De novo purine biosynthesis is a major driver of chemoresistance in glioblastoma.

Glioblastoma is a primary brain cancer with a near 100% recurrence rate. Upon recurrence, the tumour is resistant to all conventional therapies, and because of this, 5-year survival is dismal. One of the major drivers of this high recurrence rate is the ability of glioblastoma cells to adapt to complex changes within the tumour microenvironment. To elucidate this adaptation's molecular mechanisms, specifically during temozolomide chemotherapy, we used chromatin immunoprecipitation followed by sequencing and gene expression analysis. We identified a molecular circuit in which the expression of ciliary protein ADP-ribosylation factor-like protein 13B (ARL13B) is epigenetically regulated to promote adaptation to chemotherapy. Immuno-precipitation combined with liquid chromatography-mass spectrometry binding partner analysis revealed that that ARL13B interacts with the purine biosynthetic enzyme inosine-5'-monophosphate dehydrogenase 2 (IMPDH2). Further, radioisotope tracing revealed that this interaction functions as a negative regulator for purine salvaging. Inhibition of the ARL13B-IMPDH2 interaction enhances temozolomide-induced DNA damage by forcing glioblastoma cells to rely on the purine salvage pathway. Targeting the ARLI3B-IMPDH2 circuit can be achieved using the Food and Drug Administration-approved drug, mycophenolate mofetil, which can block IMPDH2 activity and enhance the therapeutic efficacy of temozolomide. Our results suggest and support clinical evaluation of MMF in combination with temozolomide treatment in glioma patients.

DDRE-14. DE-NOVO PURINE BIOSYNTHESIS IS A MAJOR DRIVER OF CHEMORESISTANCE IN GLIOBLASTOMA

Glioblastoma is a primary brain cancer with a near 100% recurrence rate. Upon recurrence, the tumor is resistant to all conventional therapies, and because of this, 5-year survival is dismal. One of the major drivers of this high recurrence rate is glioblastoma cells’ ability to adapt to complex changes within the tumor microenvironment. To elucidate this adaptation’s molecular mechanisms, specifically during chemotherapy temozolomide, we employed chromatin immunoprecipitation followed by sequencing and gene expression analysis. We identified a molecular circuit in which the expression of ciliary protein ADP-ribosylation factor-like protein 13B (ALR13B) is epigenetically regulated to promote adaptation to chemotherapy. Immuno-precipitation combined with Liquid Chromatography-Mass Spectrometry binding partner analysis revealed that that ARL13B interacts with the purine biosynthetic enzyme inosine-5’-monophosphate dehydrogenase 2 (IMPDH2). Further, radioisotope tracing revealed that this interaction function as a negative regulator for purine salvaging. Inhibition of ARL13B-IMPDH2 interaction enhances temozolomide-induced DNA damage by forcing glioblastoma cells to rely on the purine salvage pathway. Targeting the ARLI3B-IMPDH2 circuit can be achieved using a Food and Drug Administration-approved drug, Mycophenolate Mofetil, that can block the IMPDH2 activity and enhance the therapeutic efficacy of TMZ. Our results suggest and support clinical evaluation of MMF in combination with TMZ treatment in glioma patients.

ARL4C might serve as a prognostic factor and a novel therapeutic target for gastric cancer: bioinformatics analyses and biological experiments.

The ADP‐ribosylation factor‐like proteins (ARLs) have been proved to regulate the malignant phenotypes of several cancers. However, the exact role of ARLs in gastric cancer (GC) remains elusive. In this study, we systematically investigate the expression status, interactive relations, potential pathways, genetic variations and clinical values of ARLs in GC. We find that ARLs are significantly dysregulated in GC and involved in various cancer‐related pathways. Subsequently, machine learning models identify ARL4C as one of the two most significant clinical indicators among ARLs for GC. Furthermore, ARL4C silencing remarkably inhibits the growth and metastasis of GC cells both in vitro and in vivo. Moreover, enrichment analysis indicates that ARL4C is highly correlated with TGF‐β1 signalling. Correspondingly, TGF‐β1 treatment dramatically increases ARL4C expression and ARL4C knockdown inhibits the phosphorylation level of Smads, downstream factors of TGF‐β1. Meanwhile, the coexpression of ARL4C and TGF‐β1 worsens the prognosis of GC patients. Our work comprehensively demonstrates the crucial role of ARLs in the carcinogenesis of GC and the specific mechanisms underlying the GC‐promoting effects of TGF‐β1. More importantly, we uncover the great promise of ARL4C‐targeted therapy in improving the efficacy of TGF‐β1 inhibitors for GC patients.

LncRNA PVT1 promotes cervical cancer progression by sponging miR-503 to upregulate ARL2 expression.

Cervical cancer (CC) is a huge threat to the health of women worldwide. Long non-coding RNA plasmacytoma variant translocation 1 gene (PVT1) was proved to be associated with the development of diverse human cancers, including CC. Nevertheless, the exact mechanism of PVT1 in CC progression remains unclear. Levels of PVT1, microRNA-503 (miR-503), and ADP ribosylation factor-like protein 2 (ARL2) were measured by quantitative reverse transcription-polymerase chain reaction or western blot assay. 3-(4,5)-Dimethylthiazole-2-y1)-2,5-biphenyl tetrazolium bromide (MTT) and flow cytometry were used to examine cell viability and apoptosis, respectively. For migration and invasion detection, transwell assay was performed. The interaction between miR-503 and PVT1 or ARL2 was shown by dual luciferase reporter assay. A nude mouse model was constructed to clarify the role of PVT1 in vivo. PVT1 and ARL2 expressions were increased, whereas miR-503 expression was decreased in CC tissues and cells. PVT1 was a sponge of miR-503, and miR-503 targeted ARL2. PVT1 knockdown suppressed proliferation, migration, and invasion of CC cells, which could be largely reverted by miR-503 inhibitor. In addition, upregulated ARL2 could attenuate si-PVT1-mediated anti-proliferation and anti-metastasis effects on CC cells. Silenced PVT1 also inhibited CC tumor growth in vivo. PVT1 knockdown exerted tumor suppressor role in CC progression via the miR-503/ARL2 axis, at least in part.

Circ_0084927 Facilitates Cervical Cancer Development via Sponging miR-142-3p and Upregulating ARL2.

BackgroundCervical cancer is a fatal burden for women. Circular RNAs (circRNAs) are important regulators in cancer development. Our study aimed to investigate the function and action mechanism of a novel circRNA, circ_0084927, in cervical cancer.MethodsThe expression of circ_0084927, miR-142-3p and ADP-ribosylation factor-like protein 2 (ARL2) mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). For functional analyses, cell proliferation was assessed using cell counting kit-8 (CCK-8) assay. Cell cycle distribution was monitored by flow cytometry assay. Cell migration and cell invasion were evaluated by transwell assay. The interaction between miR-142-3p and circ_0084927 or ARL2 was predicted by the bioinformatics analysis and validated by dual-luciferase reporter assay and RNA immunoprecipitation assay (RIP) assay. The expression of ARL2 at the protein level was detected by Western blot. Animal tumor formation assay was performed to monitor the tumorigenicity of circ_0084927 in vivo.ResultsThe expression of circ_0084927 and ARL2 was enhanced in cervical cancer tissues and cells, while the expression of miR-142-3p was opposite to them. Circ_0084927 knockdown significantly blocked cervical cancer cell proliferation, migration and invasion and induced cell cycle arrest. MiR-142-3p was targeted by circ_0084927, and miR-142-3p inhibition reversed the effects of circ_0084927 knockdown. Besides, miR-142-3p bound to ARL2, and the inhibitory effects of miR-142-3p restoration on cell proliferation, cycle, migration and invasion were counteracted by ARL2 overexpression. More importantly, circ_0084927 upregulated ARL2 expression by sponging miR-142-3p. Circ_0084927 knockdown retarded tumor growth in vivo by regulating miR-142-3p and ARL2.ConclusionCirc_0084927 accelerated the progression of cervical cancer partly by mediating the miR-142-3p/ARL2 axis.

Suppressed microRNA-195-5p expression in mycosis fungoides promotes tumor cell proliferation.

Several cancers, including mycosis fungoides (MF), have reported dysregulation of miR-195-5p. miR-195-5p plays a role in cell cycle regulation in several malignant diseases. This study aimed to investigate: (a) the expression level of miR-195-5p in lesional MF skin biopsies and (b) the potential regulatory roles of miR-195-5p in MF. Quantitative real-time polymerase chain reaction (RT-qPCR) was used to determine miR-195-5p expression in MF skin biopsies and cell lines. The effect of miR-195-5p and ADP-ribosylation factor-like protein 2 (ARL2) on cell cycle and apoptosis was measured by flow cytometry assays. Changes in ARL2 expression were determined by RT-qPCR and Western blotting (WB). We found lower expression levels of miR-195-5p in lesional skin from MF patients compared with non-lesional MF skin and skin from healthy volunteers. Additionally, miR-195-5p showed lower expression levels in the skin from patients with disease progression compared with patients with stable disease. In vitro studies showed that overexpression of miR-195-5p induced a cell cycle arrest in G0G1. Using microarray analysis, we identified several genes that were regulated after miR-195-5p overexpression. The most downregulated gene after miR-195-5p mimic transfection was ARL2. RT-qPCR and WB analyses confirmed downregulation of ARL2 following transfection with miR-195-5p mimic. Lastly, transfection with siRNA against ARL2 also induced a G0G1 arrest. Upregulation of miR-195-5p in MF inhibits cycle arrest by downregulation of ARL2. miR-195-5p may thus function as a tumor suppressor in MF and low miR-195-5p expression in lesional MF skin may promote disease progression.