Arl4c is involved in tooth germ development through osteoblastic/ameloblastic differentiation.

Murine tooth germ development proceeds in continuous sequential steps with reciprocal interactions between the odontogenic epithelium and the adjacent mesenchyme, and several growth factor signaling pathways and their activation are required for tooth germ development. The expression of ADP-ribosylation factor (Arf)-like 4c (Arl4c) has been shown to induce cell proliferation, and is thereby involved in epithelial morphogenesis and tumorigenesis. In contrast, the other functions of Arl4c (in addition to cellular growth) are largely unknown. Although we recently demonstrated the involvement of the upregulated expression of Arl4c in the proliferation of ameloblastomas, which have the same origin as odontogenic epithelium, its effect on tooth germ development remains unclear. In the present study, single-cell RNA sequencing (scRNA-seq) analysis revealed that the expression of Arl4c, among 17 members of the Arf-family, was specifically detected in odontogenic epithelial cells, such as those of the stratum intermedium, stellate reticulum and outer enamel epithelium, of postnatal day 1 (P1) mouse molars. scRNA-seq analysis also demonstrated the higher expression of Arl4c in non-ameloblast and inner enamel epithelium, which include immature cells, of P7 mouse incisors. In the mouse tooth germ rudiment culture, treatment with SecinH3 (an inhibitor of the ARNO/Arf6 pathway) reduced the size, width and cusp height of the tooth germ and the thickness of the eosinophilic layer, which would involve the synthesis of dentin and enamel matrix organization. In addition, loss-of-function experiments using siRNAs and shRNA revealed that the expression of Arl4c was involved in cell proliferation and osteoblastic cytodifferentiation in odontogenic epithelial cells. Finally, RNA-seq analysis with a gene set enrichment analysis (GSEA) and Gene Ontology (GO) analysis showed that osteoblastic differentiation-related gene sets and/or GO terms were downregulated in shArl4c-expressing odontogenic epithelial cells. These results suggest that the Arl4c-ARNO/Arf6 pathway axis contributes to tooth germ development through osteoblastic/ameloblastic differentiation.

ARL4C is associated with epithelial-to-mesenchymal transition in colorectal cancer.

BACKGROUND: ADP-ribosylation factor-like protein 4 C (ARL4C) is a member of the ARF small GTP-binding protein subfamily. The ARL4C gene is highly expressed in colorectal cancer (CRC). ARL4C protein promotes cell motility, invasion, and proliferation. METHODS: We investigated the characteristics of ARL4C by comparing its expression at the invasion front and relationships with clinicopathological data using RNAscope, a highly sensitive RNA in situ method. RESULTS: In all cases, ARL4C expression was observed in cancer stromal cells and cancer cells. ARL4C expression in cancer cells was localized at the invasion front. In cancer stromal cells, ARL4C expression was significantly stronger in cases with high-grade tumor budding than in cases with low-grade tumor budding (P = 0.0002). Additionally, ARL4C expression was significantly increased in patients with high histological grade compared with those with low histological grade (P = 0.0227). Furthermore, ARL4C expression was significantly stronger in lesions with the epithelial-to-mesenchymal transition (EMT) phenotype compared with the non-EMT phenotype (P = 0.0289). In CRC cells, ARL4C expression was significantly stronger in cells that had the EMT phenotype compared with those with a non-EMT phenotype (P = 0.0366). ARL4C expression was significantly higher in cancer stromal cells than in CRC cells (P < 0.0001). CONCLUSION: Our analysis reinforces the possibility that ARL4C expression worsens the prognosis of patients with CRC. Further elucidation of the function of ARL4C is desired.

RAF1-MEK/ERK pathway-dependent ARL4C expression promotes ameloblastoma cell proliferation and osteoclast formation.

Ameloblastoma is an odontogenic neoplasm characterized by slow intraosseous growth with progressive jaw resorption. Recent reports have revealed that ameloblastoma harbours an oncogenic BRAFV600E mutation with mitogen-activated protein kinase (MAPK) pathway activation and described cases of ameloblastoma harbouring a BRAFV600E mutation in which patients were successfully treated with a BRAF inhibitor. Therefore, the MAPK pathway may be involved in the development of ameloblastoma; however, the precise mechanism by which it induces ameloblastoma is unclear. The expression of ADP-ribosylation factor (ARF)-like 4c (ARL4C), induced by a combination of the EGF-MAPK pathway and Wnt/ß-catenin signalling, has been shown to induce epithelial morphogenesis. It was also reported that the overexpression of ARL4C, due to alterations in the EGF/RAS-MAPK pathway and Wnt/ß-catenin signalling, promotes tumourigenesis. However, the roles of ARL4C in ameloblastoma are unknown. We investigated the involvement of ARL4C in the development of ameloblastoma. In immunohistochemical analyses of tissue specimens obtained from 38 ameloblastoma patients, ARL4C was hardly detected in non-tumour regions but tumours frequently showed strong expression of ARL4C, along with the expression of both BRAFV600E and RAF1 (also known as C-RAF). Loss-of-function experiments using inhibitors or siRNAs revealed that ARL4C elevation depended on the RAF1-MEK/ERK pathway in ameloblastoma cells. It was also shown that the RAF1-ARL4C and BRAFV600E-MEK/ERK pathways promoted cell proliferation independently. ARL4C-depleted tumour cells (generated by knockdown or knockout) exhibited decreased proliferation and migration capabilities. Finally, when ameloblastoma cells were co-cultured with mouse bone marrow cells and primary osteoblasts, ameloblastoma cells induced osteoclast formation. ARL4C elevation in ameloblastoma further promoted its formation capabilities through the increased RANKL expression of mouse bone marrow cells and/or primary osteoblasts. These results suggest that the RAF1-MEK/ERK-ARL4C axis, which may function in cooperation with the BRAFV600E-MEK/ERK pathway, promotes ameloblastoma development. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The role of Wnt, ARL4C, and Sema3A in developmental process and disease pathogenesis.

Various types of tumors, including malignant and benign ones, occur in the oral cavity. These arise from the mucosal epithelium, odontogenic epithelium, and salivary gland. To date, few major driver events in oral tumors have been identified. Accordingly, molecular targets in anti-tumor therapy for oral tumors are lacking. We focused on elucidating the function of aberrantly activated signal transduction related to oral tumor formation, especially in oral squamous cell carcinoma, ameloblastoma, and adenoid cystic carcinoma, which are raised as common oral tumors. Wnt/ß-catenin-dependent pathway is involved in the developmental process, organ homeostasis and disease pathogenesis through regulating various cellular functions by enhancing transcriptional activity. Recently, we identified ADP-ribosylation factor (ARF)-like 4c (ARL4C) and Semaphorin 3A (Sema3A), the expression of which is regulated by Wnt/ß-catenin-dependent pathway, and characterized their functions in the developmental process and tumor formation. This review highlights the recent advances in understanding the roles of Wnt/ß-catenin-dependent pathway, ARL4C and Sema3A, as determined by pathological and experimental studies.

CRL5-dependent regulation of the small GTPases ARL4C and ARF6 controls hippocampal morphogenesis.

The small GTPase ARL4C participates in the regulation of cell migration, cytoskeletal rearrangements, and vesicular trafficking in epithelial cells. The ARL4C signaling cascade starts by the recruitment of the ARF-GEF cytohesins to the plasma membrane, which, in turn, bind and activate the small GTPase ARF6. However, the role of ARL4C-cytohesin-ARF6 signaling during hippocampal development remains elusive. Here, we report that the E3 ubiquitin ligase Cullin 5/RBX2 (CRL5) controls the stability of ARL4C and its signaling effectors to regulate hippocampal morphogenesis. Both RBX2 knockout and Cullin 5 knockdown cause hippocampal pyramidal neuron mislocalization and development of multiple apical dendrites. We used quantitative mass spectrometry to show that ARL4C, Cytohesin-1/3, and ARF6 accumulate in the RBX2 mutant telencephalon. Furthermore, we show that depletion of ARL4C rescues the phenotypes caused by Cullin 5 knockdown, whereas depletion of CYTH1 or ARF6 exacerbates overmigration. Finally, we show that ARL4C, CYTH1, and ARF6 are necessary for the dendritic outgrowth of pyramidal neurons to the superficial strata of the hippocampus. Overall, we identified CRL5 as a key regulator of hippocampal development and uncovered ARL4C, CYTH1, and ARF6 as CRL5-regulated signaling effectors that control pyramidal neuron migration and dendritogenesis.

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-ß1 signalling. Correspondingly, TGF-ß1 treatment dramatically increases ARL4C expression and ARL4C knockdown inhibits the phosphorylation level of Smads, downstream factors of TGF-ß1. Meanwhile, the coexpression of ARL4C and TGF-ß1 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-ß1. More importantly, we uncover the great promise of ARL4C-targeted therapy in improving the efficacy of TGF-ß1 inhibitors for GC patients.

Single-marker and haplotype-based genome-wide association studies for the number of teats in two heavy pig breeds.

The number of teats is a reproductive-related trait of great economic relevance as it affects the mothering ability of the sows and thus the number of properly weaned piglets. Moreover, genetic improvement of this trait is fundamental to parallelly help the selection for increased litter size. We present the results of single-marker and haplotypes-based genome-wide association studies for the number of teats in two large cohorts of heavy pig breeds (Italian Large White and Italian Landrace) including 3990 animals genotyped with the 70K GGP Porcine BeadChip and other 1927 animals genotyped with the Illumina PorcineSNP60 BeadChip. In the Italian Large White population, genome scans identified three genome regions (SSC7, SSC10, and SSC12) that confirmed the involvement of the VRTN gene (as we previously reported) and highlighted additional loci known to affect teat counts, including the FRMD4A and HOXB1 gene regions. A different picture emerged in the Italian Landrace population, with a total of 12 genome regions in eight chromosomes (SSC3, SSC6, SSC8, SSC11, SSC13, SSC14, SSC15, and SSC16) mainly detected via the haplotype-based genome scan. The most relevant QTL was close to the ARL4C gene on SSC15. Markers in the VRTN gene region were not significant in the Italian Landrace breed. The use of both single-marker and haplotype-based genome-wide association analyses can be helpful to exploit and dissect the genome of the pigs of different populations. Overall, the obtained results supported the polygenic nature of the investigated trait and better elucidated its genetic architecture in Italian heavy pigs.

ARL4C is associated with initiation and progression of lung adenocarcinoma and represents a therapeutic target.

Lung adenocarcinoma is the most common histological type of lung cancer and is classified into adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and invasive adenocarcinoma (IA). Atypical adenomatous hyperplasia (AAH) lesions are possible precursors to adenocarcinoma. However, the mechanism underlying the stepwise continuum of lung adenocarcinoma is unclear. In this study, the involvement of ADP-ribosylation factor (ARF)-like (ARL) 4C (ARL4C), a member of the small GTP-binding protein family, in the progression of lung adenocarcinoma and the possibility of ARL4C as a molecular target for lung cancer therapy were explored. ARL4C was frequently expressed in AAH and ARL4C expression in immortalized human small airway epithelial cells promoted cell proliferation and suppressed cell death. In addition, ARL4C was expressed with increased frequency in AIS, MIA and IA in a stage-dependent manner, and the expression was correlated with histologic grade, fluorine-18 fluorodeoxyglucose uptake and poor prognosis. An anti-sense oligonucleotide (ASO) against ARL4C (ARL4C ASO-1316) inhibited RAS-related C3 botulinum toxin substrate activity and nuclear import of Yes-associated protein and transcriptional coactivator with PDZ-binding motif, and suppressed in vitro proliferation and migration of lung cancer cells with KRAS or epidermal growth factor receptor (EGFR) mutations. In addition, transbronchial administration of ARL4C ASO-1316 suppressed orthotopic tumor formation induced by these cancer cells. Thus, ARL4C is involved in the initiation of the premalignant stage and is associated with the stepwise continuum of lung adenocarcinoma. ARL4C ASO-1316 would be useful for lung adenocarcinoma patients expressing ARL4C regardless of the KRAS or EGFR mutation.

Macrophage Lxrα reduces atherosclerosis in Ldlr-/- mice independent of Arl7 transactivation.

BACKGROUND: Liver X receptor alpha (Lxrα) is a sterol-regulated transcription factor that limits atherogenesis by regulating cholesterol homeostasis and inflammation in macrophages. Transcriptional profiling identified the reverse cholesterol transport protein Arf-like 7 (Arl7, Arl4c) as a Lxrα target gene. We hypothesized that the LXR response element (LXRE) sequence on the murine macrophage Arl7 promoter may play a critical role in Lxrα's atherosuppressive effects. METHODS: Employing low density lipoprotein receptor-deficient mice with macrophage-specific Lxrα overexpression (Ldlr-/- MΦ-Lxrα), we constructed a novel in vivo Ldlr-/- MΦ-Lxrα Arl7MutLXRE model possessing macrophage-specific mutations within the Arl7 promoter LXRE sequences (Arl7MutLXRE) using the CRISPR/spCas9 genome editing technique. In vitro and in vivo transplantation studies were conducted using bone marrow-derived macrophages (BMDMs) and peritoneal macrophages (PMs). RESULTS: Ldlr-/-, Ldlr-/- MΦ-Lxrα, and Ldlr-/- MΦ-Lxrα Arl7MutLXRE mice on a 60% high-fat diet displayed no significant differences in body weight, fat mass, glucose homeostasis, or lipid metabolism. Macrophage Lxrα promoted Arl7 expression, enhanced cholesterol efflux, and reduced foam cell formation in an Arl7 LXRE-dependent manner. In contrast, Lxrα reduced macrophage activation, inflammatory cytokine expression, and efferocytosis independent of Arl7 LXRE. Western diet-fed Ldlr-/- mice reconstituted with transgenic BMDMs revealed that macrophage Lxrα reduced atherosclerotic plaque formation independent of Arl7 LXRE. CONCLUSION: Lxrα's anti-atherosclerotic effects in Ldlr-/- mice are not primarily attributable to Lxrα's influence on Arl7 expression. This evidence suggests that Lxrα's effects on plaque inflammation may be more critical to in vivo atherogenesis than its effects on macrophage cholesterol efflux and foam cell development.

ARL4C stabilized by AKT/mTOR pathway promotes the invasion of PTEN-deficient primary human glioblastoma.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) deficiency in primary human glioblastoma (GBM) is associated with increased invasiveness and poor prognosis with unknown mechanisms. Therefore, how loss of PTEN promotes GBM progression remains to be elucidated. Herein, we identified that ADP-ribosylation factor like-4C (ARL4C) was highly expressed in PTEN-deficient human GBM cells and tissues. Mechanistically, loss of PTEN stabilized ARL4C protein due to AKT/mTOR pathway-mediated inhibition of ARL4C ubiquitination. Functionally, ARL4C enhanced the progression of GBM cells in vitro and in vivo. Moreover, microarray profiling and GST pull-down assay identified that ARL4C accelerated tumor progression via RAC1-mediated filopodium formation. Importantly, targeting PTEN potently inhibited GBM tumor progression in vitro and in vivo, whereas overexpression of ARL4C reversed the tumor progression impaired by PTEN overexpression. Clinically, analyses with patients' specimens validated a negative correlation between PTEN and ARL4C expression. Elevated ARL4C expression but PTEN deficiency in tumor was associated with poorer disease-free survival and overall survival of GBM patients. Taken together, ARL4C is critical for PTEN-deficient GBM progression and acts as a novel prognostic biomarker and a potential therapeutic candidate. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A risk stratification model based on four novel biomarkers predicts prognosis for patients with renal cell carcinoma.

BACKGROUND: Accurate prediction of the prognosis of RCC using a single biomarker is challenging due to the genetic heterogeneity of the disease. However, it is essential to develop an accurate system to allow better patient selection for optimal treatment strategies. ARL4C, ECT2, SOD2, and STEAP3 are novel molecular biomarkers identified in earlier studies as survival-related genes by comprehensive analyses of 43 primary RCC tissues and RCC cell lines. METHODS: To develop a prognostic model based on these multiple biomarkers, the expression of four biomarkers ARL4C, ECT2, SOD2, and STEAP3 in primary RCC tissue were semi-quantitatively investigated by immunohistochemical analysis in an independent cohort of 97 patients who underwent nephrectomy, and the clinical significance of these biomarkers were analyzed by survival analysis using Kaplan-Meier curves. The prognostic model was constructed by calculation of the contribution score to prognosis of each biomarker on Cox regression analysis, and its prognostic performance was validated. RESULTS: Patients whose tumors had high expression of the individual biomarkers had shorter cancer-specific survival (CSS) from the time of primary nephrectomy. The prognostic model based on four biomarkers segregated the patients into a high- and low-risk scored group according to defined cut-off value. This approach was more robust in predicting CSS compared to each single biomarker alone in the total of 97 patients with RCC. Especially in the 36 metastatic RCC patients, our prognostic model could more accurately predict early events within 2 years of diagnosis of metastasis. In addition, high risk-scored patients with particular strong SOD2 expression had a much worse prognosis in 25 patients with metastatic RCC who were treated with molecular targeting agents. CONCLUSIONS: Our findings indicate that a prognostic model based on four novel biomarkers provides valuable data for prediction of clinical prognosis and useful information for considering the follow-up conditions and therapeutic strategies for patients with primary and metastatic RCC.

Knockdown of ARL4C inhibits osteogenic differentiation of human adipose-derived stem cells through disruption of the Wnt signaling pathway.

ADP-ribosylation factor-like 4C (ARL4C) has been shown to play an important role in cholesterol secretion, microtubule dynamics, and cell morphological changes. However, its role in osteogenesis has not been explored. In this study, we found that ARL4C is downregulated during the osteogenic differentiation of human adipose derived stem cells (hASCs). Knockdown of ARL4C suppresses osteogenesis of hASCs in vitro and in vivo. We demonstrate that ARL4C knockdown likely attenuates osteogenesis of hASCs through inhibition of the Wnt signaling pathway. These results provide new insights into the mechanisms of osteogenic differentiation and provide a potential molecular target for bone tissue engineering.

Induction of expression of aryl hydrocarbon receptor-dependent genes in human HepaRG cell line modified by shRNA and treated with ß-naphthoflavone.

The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. In this study, the effects of administration of ß-naphthoflavone (BNF), a potent AhR ligand, on the expression of AhR-dependent genes were examined by microarray and qPCR analysis in both, differentiated and undifferentiated HepaRG cell lines. To prove that BNF-induced changes of investigated genes were indeed AhR-dependent, we knock down the expression of AhR by stable transfection of HepaRG cells with shRNA. Regardless of genetical identity, our results clearly demonstrate different expression profiles of AhR-dependent genes between differentiated and undifferentiated HepaRG cells. Genes involved in metabolism of xenobiotics constitute only minute fraction of all genes regulated by AhR in HepaRG cells. Participation of AhR in induction of expression of genes associated with regulation of apoptosis or involved in cell proliferation as well as AhR-dependent inhibition of genes connected to cell adhesion could support suggestion of involvement of AhR not only in initiation but also in progression of carcinogenesis. Among the AhR-dependent genes known to be involved in metabolism of xenobiotics, cytochromes P4501A1 and 1B1 belong to the most inducible by BNF. On the contrary, expression of GSTA1 and GSTA2 was significantly inhibited after BNF treatment of HepaRG cells. Among the AhR-dependent genes that are not involved in metabolism of xenobiotics SERPINB2, STC2, ARL4C, and TIPARP belong to the most inducible by BNF. Our results imply involvement of Ah receptor in regulation of CYP19A1, the gene-encoding aromatase, and an enzyme responsible for a key step in the biosynthesis of estrogens.

Single-Cell Multi-Dimensional data analysis reveals the role of ARL4C in driving rheumatoid arthritis progression and Macrophage polarization dynamics.

Rheumatoid arthritis (RA) is an enduring autoimmune inflammatory condition distinguished by continual joint inflammation, hyperplasia of the synovium, erosion of bone, and deterioration of cartilage.Fibroblast-like synoviocytes (FLSs) exhibiting "tumor-like" traits are central to this mechanism.ADP-ribosylation factor-like 4c (ARL4C) functions as a Ras-like small GTP-binding protein, significantly impacting tumor migration, invasion, and proliferation.However, it remains uncertain if ARL4C participates in the stimulation of RA FLSs exhibiting "tumor-like" features, thereby fostering the advancement of RA. In our investigation, we unveiled, for the inaugural instance, via the amalgamated scrutiny of single-cell RNA sequencing (scRNA-seq) and Bulk RNA sequencing (Bulk-seq) datasets, that activated fibroblast-like synoviocytes (FLSs) showcase high expression of ARL4C, and the ARL4C protein expression in FLSs derived from RA patients significantly surpasses that observed in individuals with osteoarthritis (OA) and traumatic injury (trauma).Silencing of the ARL4C gene markedly impeded the proliferation of RA FLSs by hindered the transition of cells from the G0/G1 phase to the S phase, and intensified cell apoptosis and diminished the migratory and invasive capabilities. Co-culture of ARL4C gene-silenced RA FLSs with monocytes/macrophages significantly inhibited the polarization of monocytes/macrophages toward M1 and the repolarization of M2 to M1.Furthermore, intra-articular injection of shARL4C significantly alleviated synovial inflammation and cartilage erosion in collagen-induced arthritis (CIA) rats. In conclusion, our discoveries propose that ARL4C assumes a central role in the synovial inflammation, cartilage degradation, and bone erosion associated with RA by triggering the PI3K/AKT and MAPK signaling pathways within RA FLSs.ARL4C holds promise as a prospective target for the development of pharmaceutical agents targeting FLSs, with the aim of addressing RA.

Stepwise activation of p63 and the MEK/ERK pathway induces the expression of ARL4C to promote oral squamous cell carcinoma cell proliferation.

Carcinogenesis is a multistep process wherein cells accumulate multiple genetic alterations and progress to a more malignant phenotype. It has been proposed that sequential accumulation of gene abnormalities in specific genes drives the transition from non-tumorous epithelia through a preneoplastic lesion/benign tumor to cancer. Histologically, oral squamous cell carcinoma (OSCC) progresses in multiple ordered steps that begin with mucosal epithelial cell hyperplasia, which is followed by dysplasia, carcinoma in situ and invasive carcinoma. It is therefore hypothesized that genetic alteration-mediated multistep carcinogenesis would be involved in the development of OSCC; however, the detailed molecular mechanisms are unknown. We clarified the comprehensive gene expression patterns and carried out an enrichment analysis using DNA microarray data from a pathological specimen of OSCC (including a non-tumor region, carcinoma in situ lesion and invasive carcinoma lesion). The expression of numerous genes and signal activation were altered in the development of OSCC. Among these, the p63 expression was increased and the MEK/ERK-MAPK pathway was activated in carcinoma in situ lesion and in invasive carcinoma lesion. Immunohistochemical analyses revealed that p63 was initially upregulated in carcinoma in situ and ERK was sequentially activated in invasive carcinoma lesions in OSCC specimens. ADP-ribosylation factor (ARF)-like 4c (ARL4C), the expression of which is reportedly induced by p63 and/or the MEK/ERK-MAPK pathway in OSCC cells, has been shown to promote tumorigenesis. Immunohistochemically, in OSCC specimens, ARL4C was more frequently detected in tumor lesions, especially in invasive carcinoma lesions, than in carcinoma in situ lesions. Additionally, ARL4C and phosphorylated ERK were frequently merged in invasive carcinoma lesions. Loss-of-function experiments using inhibitors and siRNAs revealed that p63 and MEK/ERK-MAPK cooperatively induce the expression of ARL4C and cell growth in OSCC cells. These results suggest that the stepwise activation of p63 and MEK/ERK-MAPK contributes to OSCC tumor cell growth through regulation of ARL4C expression.

The role of ARL4C in predicting prognosis and immunotherapy drug susceptibility in pan-cancer analysis.

Background: ARLs, which are a class of small GTP-binding proteins, play a crucial role in facilitating tumor tumorigenesis and development. ARL4C, a vital member of the ARLs family, has been implicated in the progression of tumors, metastatic dissemination, and development of resistance to therapeutic drugs. Nevertheless, the precise functional mechanisms of ARL4C concerning tumor prognosis and immunotherapy drug susceptibility remain elusive. Methods: By combining the GTEx and TCGA databases, the presence of ARL4C was examined in 33 various types of cancer. Immunohistochemistry and immunofluorescence staining techniques were utilized to confirm the expression of ARL4C in particular tumor tissues. Furthermore, the ESTIMATE algorithm and TIMER2.0 database were utilized to analyze the tumor microenvironment and immune infiltration associated with ARL4C. The TISCH platform facilitated the utilization of single-cell RNA-seq datasets for further analysis. ARL4C-related immune escape was investigated using the TISMO tool. Lastly, drug sensitivity analysis was conducted to assess the sensitivity of different types of tumors to compounds based on the varying levels of ARL4C expression. Results: The study found that ARL4C was highly expressed in 23 different types of cancer. Moreover, the presence of high ARL4C expression was found to be associated with a poor prognosis in BLCA, COAD, KIRP, LGG, and UCEC. Notably, ARL4C was also expressed in immune cells, and its high expression was found to be correlated with cancer immune activation. Most importantly, the drug sensitivity analysis revealed a positive correlation between ARL4C expression and the heightened sensitivity of tumors to Staurosporine, Midostaurin, and Nelarabine. Conclusion: The findings from our study indicate that the expression level of ARL4C may exert an influence on cancer development, prognosis, and susceptibility to immunotherapy drugs. In addition, the involvement of ARL4C in the tumor immune microenvironment has expanded the concept of ARL4C-targeted immunotherapy.

ARL4C depletion suppresses the resistance of ovarian cancer to carboplatin by disrupting cholesterol transport and autophagy via notch-RBP-Jκ-H3K4Me3-OSBPL5.

Increasing studies indicate that cholesterol plays an important role in drug resistance. ARL4C is implicated in the export and import of cholesterol, therefore this study aimed to explore the effect of ARL4C on the resistance of ovarian cancer (OVC) to Carboplatin. This study collected OVC tissue samples from patients who are sensitive or resistant to carboplatin, and established Carboplatin-resistant OVC cell lines, OVCAR3(R) and SKOV3(R) using OVCAR3 and SKOV3. High throughput sequencing was conducted to find genes that regulated by ARL4C. Cholesterol esterification was performed to evaluate the transport of cholesterol from Lysosome (LY) to Endoplasmic reticulum (ER). The fluorescence of LC3-GFP-mRFP was used to evaluate the function of autophagy flux. As indicated by PCR, western blot and Immunohistochemistry, ARL4C was increased in the Carboplatin-resistant OVC tissues and cells. Knockdown of ARL4C attenuated the resistance of OVCAR3(R) and SKOV3(R) to Carboplatin. By suppressing Notch signal, ARL4C knockdown inhibited the transcriptional function of RBP-Jκ and RBP-Jκ-induced H3K4Me3, which collectively reduced OSBPL5 expression. OSBPL5 deficiency inhibited the transport of cholesterol from LYs to ER, which led to the accumulation of cholesterol in LYs and the dysfunction of autophagy. In summary, ARL4C knockdown attenuated the resistance of OVC to Carboplatin by disrupting cholesterol transport and autophagy. This study revealed a promising target to attenuate the resistance of OVC to Carboplatin and elucidated the potential mechanism.

Clear cell squamous cell carcinoma of the tongue exhibits characteristics as an undifferentiated squamous cell carcinoma.

Clear cell squamous cell carcinoma (CCSCC), where cells show abundant clear cytoplasm, -is a variant of squamous cell carcinoma (SCC) and a rare entity in the oral cavity. The characteristics of CCSCC, especially in immunohistochemical features, remain unclear. We characterized a case of CCSCC arising from the oral mucosal epithelium of tongue, where the clear cell lesion accounted for a predominant portion of the tumor. This CCSCC, which was partially surrounded by conventional SCC, exhibited cellular atypia immunohistopathologically and histopathologically with a high Ki-67 index, increased number of mitotic figures and enlarged nuclei. Intravascular invasion of the carcinoma cells was also observed. Furthermore, the CCSCC recurred and metastasized to the cervical lymph nodes and both lungs three months after resection. Immunohistochemical analyses demonstrated decreased expression of p40 (an isoform of SCC marker p63), ADP-ribosylation factor (ARF)-like 4c (ARL4C), yes-associated protein (YAP) and 5-methylcytosine (5mC) in the CCSCC lesion compared with the surrounding SCC lesion, where the expression of ARL4C was upregulated compared with non-tumor region and YAP showed nuclear translocation. In addition, siRNA loss-of-function experiments revealed that p63 expression was required for ARL4C expression and DNA methylation was induced by p63 and YAP/transcriptional co-activator with PDZ-binding motif (TAZ) signaling in oral SCC cell lines. These results suggest that CCSCC, in which several markers of SCC-associated intracellular signaling pathways are downregulated, together with evidence of altered epigenetic regulation, is characterized as an undifferentiated SCC variant.

Identifying a novel IRF3/circUHRF1/miR-1306-5p/ARL4C axis in pancreatic ductal adenocarcinoma progression.

Pancreatic ductal adenocarcinoma (PDAC) is considered one most aggressive and lethal cancer types worldwide. While its underlying mechanisms are still poorly understood. CircRNAs play essential roles in various biological progression, including PDAC. Here, our results found that circUHRF1 was highly expressed in PDAC tumor tissues compared with normal tissues. Next, Cell or animal models were constructed, CCK-8, cell colony, EdU, flow cytometry assay, transwell migration, and Western blot assays were applied. CircUHRF1 knockdown influenced PDAC cell proliferation, apoptosis, migration and EMT level in vitro, and tumor growth in vivo. Subsequently, bioinformatics analysis, AGO2-RIP, RNA pull-down, and dual-luciferase reporter assays were used to explore the downstream targets in PDAC progression. Our findings suggest that circUHRF1 regulated ARL4C expression to promote PDAC progression through sponging miR-1306-5p. The role of miR-1306-5p in PDAC cellular progression has been elucidated, and the expression association between miR-1306-5p and circUHRF1 or ARL4C in PDAC tissues was analyzed. Furthermore, circUHRF1 expression in PDAC cells could be transcriptionally regulated by IRF3. Collectively, our study demonstrated the role of IRF3/circUHRF1/miR-1306-5p/ARL4C axis in PDAC progression. Our results suggest that circUHRF1 is one promising diagnosis or therapeutic target for PDAC management.Abbreviations : CircRNA; Circular RNAPDAC; pancreatic ductal adenocarcinomaUHRF1; Ubiquitin-like with PHD and RING finger domain 1ARL4C; ADP Ribosylation Factor Like GTPase 4CRIP; RNA immunoprecipitationEDU; 5-Ethynyl-2'-deoxyuridineEMT; epithelial to mesenchymal transitionAGO2; Argonaute RISC Catalytic Component 2CCK8; Cell counting Kit-8IRF3; Interferon Regulatory Factor 3.

Localization of KRAS downstream target ARL4C to invasive pseudopods accelerates pancreatic cancer cell invasion.

Pancreatic cancer has a high mortality rate due to metastasis. Whereas KRAS is mutated in most pancreatic cancer patients, controlling KRAS or its downstream effectors has not been succeeded clinically. ARL4C is a small G protein whose expression is induced by the Wnt and EGF-RAS pathways. In the present study, we found that ARL4C is frequently overexpressed in pancreatic cancer patients and showed that its localization to invasive pseudopods is required for cancer cell invasion. IQGAP1 was identified as a novel interacting protein for ARL4C. ARL4C recruited IQGAP1 and its downstream effector, MMP14, to invasive pseudopods. Specific localization of ARL4C, IQGAP1, and MMP14 was the active site of invasion, which induced degradation of the extracellular matrix. Moreover, subcutaneously injected antisense oligonucleotide against ARL4C into tumor-bearing mice suppressed metastasis of pancreatic cancer. These results suggest that ARL4C-IQGAP1-MMP14 signaling is activated at invasive pseudopods of pancreatic cancer cells.

Most cases of pancreatic cancer are detected in the later stages when they are difficult to treat and, as a result, survival is low. Over 90% of pancreatic cancers contain genetic changes that increase the activity of a protein called KRAS. This hyperactive KRAS drives cancer growth and progression. Attempts to treat pancreatic cancer using drugs that reduce the activity of KRAS have so far failed. The KRAS protein can accelerate growth in healthy cells as well as in cancer and it does this by activating various other proteins. Drugs that target some of these other proteins could be more effective at treating pancreatic cancer than the drugs that target KRAS. One of these potential targets is called ARL4C. ARL4C is active during fetal development, but it is often not present in adult tissues. Harada et al. investigated whether the protein is important in pancreatic cancer, and what other roles it has in the body, to better understand if it is a good target for cancer treatment. First, Harada et al. used cells grown in the lab to show that ARL4C contributes to the aggressive spread of human pancreatic cancers. Using mice, Harada et al. also showed that blocking the activity of ARL4C in pancreatic cancers helped to slow their progression. Harada et al.'s results suggest that ARL4C could be a good target for new drugs treating pancreatic cancers. Given that this protein does not seem to have important roles in the cells of adults, targeting it is unlikely to have major side effects. Further investigation of ARL4C in more human-like animal models will help to confirm these results.