ARL3 is downregulated and acts as a prognostic biomarker in glioma

Glioma is the most common primary intracranial tumor in adults and is notorious for its malignancy and unfavorable prognosis [1]. Despite standard treatment regimens, including surgery followed by radiation and chemotherapy, the prognosis of glioma patients is still dismal [2, 3]. Glioblastoma multiforme (GBM) is the most aggressive type, with a median survival between 14 and 18 months after diagnosis and an estimated 5-year survival rate of 5.1% [4]. Intratumoral heterogeneity of GBM is a key factor for the unsatisfactory therapeutic effect [5]. Heterogeneity in glioma could be affected by the tumor microenvironment, which provides a particular niche for glioma stem cells (GSCs) to promote glioma initiation, invasion, and therapeutic resistance [6]. Recently, several new therapeutic strategies, including oncogenic signal transduction inhibition/targeted therapy, antiangiogenesis, and immunotherapy, have attracted substantial attention and shed new light on the treatment of glioma [7‐9].

ADP-ribosylation factor-like 3 (ARL3) is a kind of small GTP-binding protein in the ADP-ribosylation factor (ARF) family belonging to the RAS superfamily,  which is involved in multiple biological processes and tumor occurrence and progression [10‐12]. ARF members regulate several essential cellular functions, such as membrane trafficking, cytoskeleton organization, and cell adhesion and migration, which are significantly relevant to tumor invasion and metastasis [13‐16]. Current evidence has indicated that ARF proteins are involved in cancer progression through three different mechanisms: cell–cell adhesion, integrin trafficking and actin cytoskeleton rearrangement [14]. As a member of the ARF family, ARL3 has been reported to regulate cell morphology and cytokinesis through microtubule-based processes [17]. ARL3 interacts with dynactin and dynein to regulate microtubule mediated retrograde transport [18]. ARL3 can act as an allosteric release factor for farnesylated cargo and a regulator of trafficking of lipid-modified proteins [19, 20]. In addition, ARL3 influences ciliogenesis and is involved in ciliary function affecting kidney and photoreceptor development in mice [21, 22]. However, the specific functions of ARL3 in tumors remain unknown.

In this study, we investigated ARL3 expression and its roles in glioma prognosis using clinical samples and data from The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA) and Repository for Molecular Brain Neoplasia Data (REMBRANDT) databases. Furthermore, a nomogram was constructed by applying the identified factors to predict 3- or 5-year survival for glioma patients. In addition, we explored the biological functions and pathways affected by ARL3 in glioblastoma, which may provide novel insights into glioma treatment.

Presently, treatment of glioblastoma is still an enormous challenge due to its aggressiveness and high rate of recurrence [48]. Although biotechnology and several innovative approaches have been adopted, no progress has been made in progression-free survival (PFS) and overall survival (OS) in GBM patients [49]. Intratumor heterogeneity is one of the most important hallmarks of GBM, which gives rise to therapeutic resistance and tumor recurrence [50]. It is thus urgent to precisely evaluate the prognosis of GBM patients and apply personalized treatment strategies.

For a more accurate prognostic prediction, nomograms have been developed, and these nomograms show better performance than conventional staging systems in some cancers [51, 52]. In this study, we identified ARL3 as a prognostic marker for glioma, and constructed a nomogram and risk classification system. The nomogram included five parameters that are readily available from clinical records and tissue specimens. As reported previously, age is an independent prognostic factor, and older ages are associated with poorer prognosis [53]. For sex, males have a higher incidence of GBM than females [54]. IDH mutation is an early event in gliomagenesis and is implicated in glioma progression [55]. Wild-type IDH and higher WHO grade (III or IV) have been proven to be associated with adverse outcomes [56]. These results are consistent with our findings. In the validation cohorts, the C-indexes, areas under the ROC curve (all above 0.85) and highly fitted calibration plots demonstrated that the nomogram performed well in predicting 3- or 5-year survival for patients with glioma. However, there are some limitations: first, the sample size used in the nomogram was small; second, the primary and validation cohorts were collected from datasets, and they did not contain details of intervention, such as extent of glioma resection, radiotherapy and chemotherapy. In future studies, we may incorporate detailed clinical records and apply the nomogram into clinical practice.

Another novel finding of this study is that ARL3 is involved in the glioma immune microenvironment and angiogenesis. Angiogenesis has been reported to contribute to glioma growth, invasion and metastasis, and increased tumor microvascular density (MVD) indicates poor prognosis [57]. Several proangiogenic factors secreted by tumor cells, stromal cells and inflammatory cells in the tumor microenvironment promote angiogenesis [45, 58]. The proangiogenic factors include VEGF family proteins (including VEGFA, VEGFB, VEGFC and VEGFD) and placental growth factors, and serve as treatment targets [57]. Although bevacizumab, an anti-VEGFA antibody, has shown efficacy by prolonging PFS in clinical trials for glioblastoma, it fails to affect overall survival [59]. The reason for the lack of benefits on OS is ascribed to the use of the drug for an unselected patient population; thus, it is necessary to identify biomarkers to predict the response of antiangiogenic agents [59, 60]. Using bioinformatics analyses, our study revealed that ARL3 was closely correlated with angiogenesis. More importantly, we found that ARL3 was highly expressed in proliferating microvascular area of GBM and negatively correlated with proangiogenic genes, such as VEGFA. Overall, these results suggested that ARL3 negatively regulates angiogenesis and represents a potential target for antiangiogenic therapy in GBM.

In addition, angiogenesis plays an important role in the immune composition of the tumor microenvironment [61]. A recent study revealed that antiangiogenesis therapy increases the abundance of mature DCs and enhances CD8 T cell immunity against glioma [9]. Here, we also observed that ARL3 influences the infiltration of immune cells in the glioblastoma microenvironment. Samples with low ARL3 expression tended to harbor a higher proportion of dendritic cells (DCs), macrophages, NK cells, CD4 T cells and a lower proportion of CD8 T cells. Infiltrating immune cells are important components of the tumor microenvironment and are associated with tumor behavior and patient outcomes. Glioma cells release multiple cytokines, interleukins and growth factors that promote the infiltration of various cells, including astrocytes, pericytes, endothelial cells, circulating progenitor cells, and immune cells such as microglia, peripheral macrophages, myeloid-derived suppressor cells, leukocytes, CD4 T cells, and Tregs into the tumor [62]. Glioma not only recruits immune cells, but also modifies them to evade immune surveillance. In a mouse glioma model, it has been found that DCs downregulate costimulatory molecules (CD40, B7.1, B7.2) and are unable to stimulate T cells [47]. Another study observed that glioma cells induce abnormal Nrf2 expression in DCs to suppress their maturation and T cell activation, in turn leading to immune escape [63]. Moreover, glioma cells actively recruit glioma-associated microglia/macrophages (GAMs) and induce M2 polarization [64]. M2-type GAMs produce numerous cytokines, interleukins, and growth factors that generate an immunosuppressive microenvironment and promote glioma cell growth, invasion and angiogenesis [62, 65]. Furthermore, infiltration by M2-polarized macrophages indicates an unfavorable prognosis in high-grade gliomas and confers an aggressive glioma subtype [66]. These changes give rise to a supportive environment, enrich for extracellular substrates, and maintain glioma growth or progression [62]. Since it is well established that tumor-infiltrating immune cells play a key role in tumor development, the mechanism by which ARL3 influences infiltrating immune cells in the glioma microenvironment should be investigated in future studies.

As a member of the ARF family, ARL3 is involved in regulating ciliary functions and lipid-modified proteins transport [20, 67]. Notably, ARL3 is a newly identified binding partner of STAT3 and enhances the phosphorylation and nuclear accumulation of STAT3 [26]. According to a recent study, ARL3 induces autophagy in HEK293T cells [68]. In the present study, we discovered that ARL3 expression was decreased in glioma samples and was associated with tumor grade. Furthermore, low expression of ARL3 was related to adverse outcomes and radiation and chemotherapy resistance in glioma. A nomogram with ARL3 was constructed and proven to accurately predict 3- or 5-year survival for glioma patients. Regarding biological function, we proved that ARL3 negatively regulates angiogenesis and influences immune cell infiltration into the glioma microenvironment. These findings complement the biological functions of ARL3 and may provide new options for the management of glioma.

In conclusion, our study demonstrated that ARL3 expression was decreased in glioma samples and was associated with tumor grade. Furthermore, low ARL3 expression was related to adverse outcomes and radiation and chemotherapy resistance in glioma. We also constructed a nomogram with ARL3 to predict 3- or 5-survival for glioma patients. Regarding biological functions, we demonstrated that ARL3 was involved in angiogenesis and immune cell infiltration in the GBM immune microenvironment. Taken together, these results suggest a potential role of ARL3 as a prognostic marker and therapeutic target for glioma.