Chloride Intracellular Channel 1 Research Articles

Salivary Chloride Intracellular Channel 1 (CLIC1) as a Hub of Gene-Gene Interactome of Periodontitis With Diabetes Mellitus.

Background and introduction Periodontal disease is one of the most prevalent chronic conditions that affects the oral cavity. Identifying and predicting biomarkers is essential for the prevention of high-morbidity oral diseases. The genomic interaction network identifies common hub genes involved in crucial protein formation in periodontal inflammation. Diabetes mellitus is a metabolic disorder that has a double-edged sword relationship with periodontitis. Chloride intracellular channel 1 (CLIC1) was identified as a hub gene linking the pathogenesis of periodontitis and diabetes mellitus using a bioinformatic tool. Therefore, this current study aimedto assess the concentration of the pro-inflammatory biomarker CLIC1 in saliva among individuals with periodontal health and those with periodontal disease linked to diabetes mellitus. Materials and methods Differentially expressed genes (DEGs) in periodontitis were identified using datasets retrieved from the Gene Expression Omnibus (GEO) database. DEGs were combined to build the network, and GeneMANIA was used to find and rank the interconnecting genes. CLIC1 was identified as the hub gene, and clinical validation was done using patient samples. The study involved 30 participants. Based on clinical and radiographic periodontal findings, they were split into three groups: healthy(group 1, n=10), with periodontitis but no diabetes mellitus (group 2, n=10), and with periodontitis anddiabetes mellitus(group 3, n=10). The collection of saliva samples, followed by quantifying these samples, was performed using an enzyme-linked immunosorbent assay (ELISA). Results Fromnetwork graph analysis, it was discovered that CLIC1 functions as a hub gene in the majority of toll-like receptor pathways.The mean concentration of CLIC1 in saliva increased consistently as the disease was observed in periodontitis patients and periodontitis patients with diabetes mellitus. Conclusion CLIC1 concentrations were positively correlated with periodontitis in individuals with diabetes. Therefore, CLIC1 could be a diagnostic biomarker for patients with periodontitis. However, large-scale studies are needed to confirm more positive associations.

Abstract LB_B11: Transmembrane chloride intracellular channel 1 (tmCLIC1) protein as akey regulator of colorectal cancers' aggressiveness

Abstract Colorectal cancer is the third leading cause of death in developed countries. Although the huge progresses reached in therapeutically approach, metastases remain the main issue treating colorectal cancer. Ion channels have a prominent role in the acquirement of aggressive phenotype, but most of them are involved in normal physiological processes. Among all the new potential biomarkers, Chloride Intracellular Channel 1(CLIC1) is a promising candidate.  As a matter of fact, in physiological homeostasis, CLIC1 is expressed by all cell types as a cytosolic monomer. CLIC1 is a metamorphic protein that coexist in both cytosolic and transmembrane form (tmCLIC1), the latter involved in several pathological states, including cancer. CLIC1 mRNA overexpression was identified in colorectal cancer cells and is related to a poor prognosis. In this scenario, the main aim of the present work is to understand the role of tmCLIC1 in tumorigenesis, invasion, and migration potential of colorectal cancer. We first investigated the level of expression of CLIC1 protein and the activity of its transmembrane form as an ion channel in several colorectal cancer cells at different stage of development. Our results have demonstrated that tmCLIC1 protein expression and activity is proportional to the level of aggressiveness of cells, and here its action is fundamental on cellular proliferation, migration, and invasion. The role of tmCLIC1 activity in colorectal cancer was confirmed also in in vivo models. Experiments performed in zebrafish and mice models have demonstrated that the silencing of CLIC1 results in the impairment of primary tumor development and a strong reduction of distal metastases formation. Moreover, many different metastatic genes are downregulated when CLIC1 protein is absent; in particular, genes related to oxidative stress conditions, typical of cancer cells. As a matter of fact, the peculiar activity of CLIC1 and its binding with glutathione makes this protein essential for redox balance of colorectal cancer cells. Altogether, all the results obtained might be considered a starting point to unveil a mechanism of action that could represent a new milestone in anticancer research. Citation Format: Francesca Cianci, Ivan Verduci, Carlotta Tacconi, Guido Rey, Luca Roz, Alessandro Fantin, Michele Mazzanti. Transmembrane chloride intracellular channel 1 (tmCLIC1) protein as akey regulator of colorectal cancers' aggressiveness [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B11.

Abstract LB_B15: The antiproliferative activity of metformin in glioblastoma stem cells is mediated by the direct binding and functional inhibition of the membrane configuration of CLIC1 protein

Abstract Glioblastoma (GBM) is the deadliest brain tumor and represents a major clinical challenge. Even with the current standard of care and cutting-edge strategies, patients’ life expectancy remains invariably poor. Tumor heterogeneity and its extensive invasiveness make GBM difficult to be radically resected by neurosurgery and recurrence is common. There is scientific consensus that tumor relapse originates from progenitor/stem-like cells known as glioblastoma stem cells (GSCs). GSCs are endowed with distinct tumorigenic potential, with properties of self-renewal and multi-lineage differentiation that contribute to tumor mass heterogeneity. Thus, GSCs represent an appealing target for new therapies. In recent years, metformin's anti-cancer properties have been highlighted. Metformin administration to patients with type 2 diabetes (T2D) has been positively linked to a decrease in the risk of developing several cancers and in cancer-related mortality, including glioblastoma. Additionally, numerous studies on glioblastoma demonstrate that metformin’s anti-cancer properties are directed against GSCs. Despite this evidence, metformin’s mechanism of action on cancer cells has not been clarified, although it has been proposed to target the oxidative phosphorylation (OXPHOS) pathway. GSCs are able to shuttle between glycolysis and OXPHOS energetic pathways, and this metabolic plasticity could be the major reason for metformin’s low effectiveness in clinical trials so far. Chloride Intracellular Channel 1 (CLIC1) is a metamorphic protein that is predominantly cytoplasmic under physiological conditions. In response to persistent stress, it translocates to the plasma membrane, inducing chloride conductance. When GSCs are treated with metformin and agents that impair the activity of the transmembrane form of CLIC1 (tmCLIC1), they show a parallel and non-additive inhibition of proliferation. Our previous studies comprehensively demonstrated that tmCLIC1 contributes to the progression of GBM both in vitro and in vivo. In addition, its specific localization and enrichment at the GSC plasma membrane position tmCLIC1 as a prime pharmacological target for GBM treatment. Our investigation found that in vitro, metformin’s antiproliferative effect is exerted at a millimolar concentration range. However, in vivo, with metformin administered through drinking water, mice exhibit an increase in survival with brain levels of metformin quantified in the nanomolar range. It is possible that the constant fresh drug circulation, continuously reaching the brain during prolonged administration in vivo (up to 100 days) creates a drug steady state with persistent saturation and inhibition of all the active tmCLIC1 expressed by GBM cells. The need for lower concentrations in vivo than in vitro is also important to translate these studies into the clinic; the already-available extended-release formulation may provide the same sustained activity on GBM cells as the drug in mouse drinking water. Citation Format: Michele M Mazzanti, Francesca M Cianci. The antiproliferative activity of metformin in glioblastoma stem cells is mediated by the direct binding and functional inhibition of the membrane configuration of CLIC1 protein [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B15.

STEM-06. OSMR INTERACTOME MAPPING REVEALS THE SIGNIFICANCE OF A CHLORIDE CHANNEL IN CYTOKINE REGULATION AND BRAIN TUMOUR STEM CELL SELF-RENEWAL

Abstract Oncostatin M Receptor (OSMR) is a key regulator of brain tumour stem cell (BTSC) self-renewal and glioblastoma (GB) tumourigenesis. This receptor is found in a complex with Epidermal Growth Factor Receptor variant III (EGFRvIII) and orchestrates a positive feedback loop with STAT3. OSMR also regulates oxidative phosphorylation and participates in promoting the transition of cells towards mesenchymal-like glioma states. How OSMR drives GB tumorigenesis via multiple mechanisms remains largely unclear. Here, we map OSMR interactome in different GB subtypes using the Mammalian Membrane Two Hybrid High Throughput Screen (MaMTH-HTS). Analysis of these data followed by functional validation has uncovered distinct OSMR interactome in a context dependent manner. Follow up investigations, however, have led to the discovery of the Chloride Intracellular Channel 1 (CLIC1), essential in the regulation of OSMR cytokine signaling and GB progression independently of the genetic profile of the GB tumour. CLIC1 forms a complex with OSMR to promote BTSC self-renewal and knockout of CLIC1 or pharmacological inhibitors to CLIC1 impairs OSM mediated STAT3 phosphorylation. Our work suggests that CLIC1 and OSMR form a complex in BTSCs and functionally interact to activate STAT3.

Multi-omics analysis reveals CLIC1 as a therapeutic vulnerability of gliomas.

Introduction: Despite advances in comprehending cancer biology, malignant gliomas remain incurable. The present work conducted a multi-omics analysis for investigating the significance of chloride intracellular channel 1 (CLIC1) in gliomas. Methods: Multi-omics data of glioma covering transcriptomics, genomics, DNA methylation and single-cell transcriptomics from multiple public cohorts were enrolled for analyzing CLIC1. In vitro experiments were conducted to measure apoptosis and cell mobility in U251 and U373 glioma cells following transfection of CLIC1 siRNAs. Results: Elevated CLIC1 expression was proven to stably and independently estimate worse survival outcomes. CLIC1 expression was higher in more advanced stage, wild-type IDH and unmethylated MGMT samples. Tumorigenic and anticancer immunity pathways were remarkably enriched in CLIC1-up-regulated tumors. Additionally, CLIC1 was positively linked with cancer-immunity cycle, stromal activation, DNA damage repair and cell cycle. Suppressing CLIC1 resulted in apoptosis and attenuated cell motility of glioma cells. More frequent genomic alterations were found in CLIC1-up-regulated tumors. CLIC1 expression presented a remarkably negative connection to DNA methylation. High CLIC1 expression samples were more sensitive to camptothecin, cisplatin, doxorubicin, erlotinib, paclitaxel, rapamycin, clofarabine, tanespimycin, methotrexate, everolimus, TAK-733, trametinib and AZD8330. Tumors with upregulated CLIC1 presented abundant immune cell infiltration, higher expression of immune-checkpoints and -modulators and similar transcriptome profiling, indicative of well response to immune-checkpoint blockade (ICB). Nevertheless, due to elevated TIDE score, tumors with CLIC1 upregulation appeared to be resistant to ICB. Single-cell analysis unveiled that CLIC1 was expressed ubiquitously in tumor cells and tumor microenvironment. Conclusions: Overall, CLIC1 was a promising treatment vulnerability in glioma.

OSMR INTERACTOME MAPPING REVEALS THE SIGNIFICANCE OF A CHLORIDE CHANNEL IN CYTOKINE REGULATION AND BRAIN TUMOUR STEM CELL SELF-RENEWAL

Abstract Oncostatin M Receptor (OSMR) is a key regulator of brain tumour stem cell (BTSC) self-renewal and glioblastoma (GB) tumourigenesis. This receptor is found in a complex with Epidermal Growth Factor Receptor variant III (EGFRvIII) and orchestrates a positive feedback loop with STAT3. OSMR also regulates oxidative phosphorylation and participates in promoting the transition of cells towards mesenchymal-like glioma states. How OSMR drives GB tumorigenesis via multiple mechanisms remains largely unclear. Here, we map OSMR interactome in different GB subtypes using the Mammalian Membrane Two Hybrid High Throughput Screen (MaMTH-HTS). Analysis of these data followed by functional validation has uncovered distinct OSMR interactome in a context dependent manner. Follow up investigations, however, have led to the discovery of the Chloride Intracellular Channel 1 (CLIC1), essential in the regulation of OSMR cytokine signaling and GB progression independently of the genetic profile of the GB tumour. CLIC1 forms a complex with OSMR to promote BTSC self-renewal and knockout of CLIC1 or pharmacological inhibitors to CLIC1 impairs OSM mediated STAT3 phosphorylation. Our work suggests that CLIC1 and OSMR forms a complex in BTSCs and functionally interact to activate STAT3.

Chloride Intracellular Channel Protein 1 Expression and Angiogenic Profile of Liver Metastasis of Digestive Origin

Chloride intracellular channel 1 (CLIC1) is involved in cell migration and metastasis. The histological growth patterns of liver metastasis are as follows: desmoplastic (d-HGP), replacement (r-HGP), pushing (p-HGP), and mixed. The aim of this study was to evaluate the relation between HGP, angiogenesis, and CLIC1 expression. Materials and Methods: A total of 40 cases of primary tumors and their LM: d-HGP (12 cases), r-HGP (13 cases), and p-HGP (15 cases), were evaluated through simple and double immunostaining. CLIC1 assessment was conducted as follows: scores of 0 (less than 10% of positive cells), 1 (10-30%), 2 (30-50%), or 3 (more than 50%) were assigned. Heterogeneous CLIC1 expression was found. CLIC1 in primary tumors correlated with grade G for all cases of LM with a p-HGP (p = 0.004). The CLIC1 score for LMs with an r-HGP correlated with grade G of the corresponding primary tumor (p = 0.027). CLIC1 and CD34+/Ki67+ vessels (p = 0.006) correlated in primary tumors. CLIC1 in primary tumors correlated with CD34+/Ki67+ vessels of LMs with a d HGP (p = 0.024). Conclusions: The CLIC1 score may have prognostic value, mainly for LMs with a p-HGP and r-HGP, and therapeutic value for LMs with a d-HGP.

Label-free LC-MS/MS proteomics analyses reveal CLIC1 as a predictive biomarker for bladder cancer staging and prognosis.

Bladder cancer (BC) is a significant carcinoma of the urinary system that has a high incidence of morbidity and death owing to the challenges in accurately identifying people with early-stage BC and the lack of effective treatment options for those with advanced BC. Thus, there is a need to define new markers of prognosis and prediction. In this study, we have performed a comprehensive proteomics experiment by label-free quantitative proteomics to compare the proteome changes in the serum of normal people and bladder cancer patients-the successful quantification of 2064 Quantifiable proteins in total. A quantitative analysis was conducted to determine the extent of changes in protein species' relative intensity and reproducibility. There were 43 upregulated proteins and 36 downregulated proteins discovered in non-muscle invasive bladder cancer and normal individuals. Sixty-four of these proteins were elevated, and 51 were downregulated in muscle-invasive and non-muscle-invasive bladder cancer, respectively. Functional roles of differentially expressed proteins were annotated using Gene Ontology (GO) and Clusters of Orthologous Groups of Proteins (COG). To analyze the functions and pathways enriched by differentially expressed proteins, GO enrichment analysis, protein domain analysis, and KEGG pathway analysis were performed. The proteome differences were examined and visualized using radar plots, heat maps, bubble plots, and Venn diagrams. As a result of combining the Venn diagram with protein-protein interactions (PPIs), Chloride intracellular channel 1 (CLIC1) was identified as the primary protein. Using the Gene Set Cancer Analysis (GSCA) website, the influence of CLIC1 on immune infiltration was analyzed. A negative correlation between CD8 naive and CLIC1 levels was found. For validation, immunohistochemical (IHC), qPCR, and western blotting (WB) were performed.Further, we found that CLIC1 was associated with a poor prognosis of bladder cancer in survival analysis. Our research screened CLIC1 as a tumor-promoting protein in bladder cancer for the first time using serum mass spectrometry. And CLIC1 associated with tumor stage, and immune infiltrate. The prognostic biomarker and therapeutic target CLIC1 may be new for bladder cancer patients.

CLIC1 plasma concentration is associated with lymph node metastases in oral squamous cell carcinoma.

Previous studies have shown that the chloride intracellular channel 1 (CLIC1) protein is overexpressed in oral squamous cell carcinoma (OSCC) and nasopharyngeal carcinoma. Patients with these diseases had significantly higher CLIC1 plasma levels than healthy controls. To determine the plasma concentration of CLIC1 in patients with OSCC and laryngeal squamous cell carcinoma (LSCC). We collected blood samples from patients diagnosed with OSCC (n = 13) and LSCC (n = 7), as well as from healthy controls (n = 8). The blood samples were centrifuged to obtain plasma and stored at -80°C. The CLIC1 plasma concentration was determined using enzyme-linked immunosorbent assay (ELISA). The mean CLIC1 plasma concentration was higher in the OSCC group than in the LSCC and control groups. Patients with OSCC and nodal metastases had significantly higher CLIC1 plasma concentration levels than nonmetastatic patients (p < 0.0001; Tukey's multiple comparisons test) and controls (p = 0.0004). The CLIC1 concentration correlated significantly with the presence of nodal spread (p = 0.0003; Spearman's r = 0.8613) and overall TNM staging (p = 0.0167; Spearman's r = 0.6620). No differences in CLIC1 plasma levels were observed between the LSCC and control groups. The CLIC1 plasma concentration was not associated with age, sex, tumor stage, or tumor grade. There were no differences in CLIC1 plasma concentration between healthy controls and patients with LSCC. However, our findings suggest that the presence of this protein in plasma may be associated with lymphatic metastasis in patients with OSCC. More research is needed to confirm this possible association.

Comprehensive analysis of clinical prognosis and CLIC1 immune invasion in lung adenocarcinoma.

Background:Chloride intracellular channel 1 (CLIC1) plays an important role in the process of cell epithelial transport, and is also involved in tumor invasion and metastasis. Due to its aberrant expression in cancer, the mechanism of action of CLIC1 in cancer has been carefully studied. In this study, we tried to investigate the relationship between CLIC1 and lung adenocarcinoma (LUAD).Methods:The RNA-sequencing data and clinical information of CLIC1 in lung adenocarcinoma were collected from the the cancer genome altas (TCGA) database and analyzed with R software. Paired t test and Mann–Whitney U test were used to detect differences between LUAD tissue and adjacent normal tissue, and the pROC software package performed reactive oxygen species (ROC) curves to detect cutoff values for CLIC1. The expression of CLIC1 in normal human tissues was extracted from the human protein altas (HPA) database, and analyzed clinical proteomic tumor analysis consortium by using UALCAN programme. The relationship between CLIC1 and LUAD was explored by enrichment analysis using gene oncology and Kyoto encyclopedia of genes and genomes. The tumor immunity estimation resource (TIMER) and integrated repository portal for tumor-immune system interactions (TISIDB) databases were used to analyze the correlation between CLIC1 and LUAD immune cell infiltration. Survival analysis of CLIC1 in LUAD was assessed by the PrognoScan database.Results:Compared with normal tissues, both mRNA (messenger Ribose Nucleic Acid) and protein of CLIC1 were overexpressed in LUAD, which was associated with shorter overall survial (OS). In addition, CLIC1 expression was in connection with some clinical-pathological characteristics like tumor node metatasis stages and lymph node metastases. What’s more, CLIC1 may play a role in the immune infiltration of LUAD.Conclusion:In summary, CLIC1 is up-regulated in LUAD and is associated with tumor metastasis, tumor staging, and OS. It may be regarded as a novel marker for prognostic judgement in LUAD.

Human antimicrobial peptide LL-37 contributes to Alzheimer's disease progression.

As a prime mover in Alzheimer's disease (AD), microglial activation requires membrane translocation, integration, and activation of the metamorphic protein chloride intracellular channel 1 (CLIC1), which is primarily cytoplasmic under physiological conditions. However, the formation and activation mechanisms of functional CLIC1 are unknown. Here, we found that the human antimicrobial peptide (AMP) LL-37 promoted CLIC1 membrane translocation and integration. It also activates CLIC1 to cause microglial hyperactivation, neuroinflammation, and excitotoxicity. In mouse and monkey models, LL-37 caused significant pathological phenotypes linked to AD, including elevated amyloid-β, increased neurofibrillary tangles, enhanced neuronal death and brain atrophy, enlargement of lateral ventricles, and impairment of synaptic plasticity and cognition, while Clic1 knockout and blockade of LL-37-CLIC1 interactions inhibited these phenotypes. Given AD's association with infection and that overloading AMP may exacerbate AD, this study suggests that LL-37, which is up-regulated upon infection, may be a driving force behind AD by acting as an endogenous agonist of CLIC1.

Chloride Intracellular Channel 1 is a Potential Biomarker for Breast Cancer.

PurposeMultiple reports have demonstrated that highly expressed chloride intracellular channel 1 (CLIC1) exists in a range of malignant tumors and is involved in proliferation, invasion, and migration of cancer cells. There are few studies on CLIC1 and breast cancer (BC). The purpose of this research was to evaluate the expression level of CLIC1 in BC and its impact on prognosis of BC patients.Patients and MethodsDifferences in CLIC1 expression levels in 25 pairs of BC and corresponding paracancerous specimens were tested by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB). Immunohistochemistry (IHC) was performed to discuss the relevance between CLIC1 expression in BC tissue chips and clinicopathological parameters of BC patients. The effect of CLIC1 expression on patient prognosis was evaluated by Kaplan–Meier survival curve and Cox regression analysis. Receiver operating characteristic (ROC) curve assessed the diagnostic performance of CLIC1 for BC.ResultsThe experimental results of qRT-PCR and WB demonstrated that CLIC1 was highly expressed in BC tissues. IHC results showed that overexpression of CLIC1 was strictly correlated with tumor size, TNM classification, pathological grade, lymph node metastasis and Ki67. Patients with lower CLIC1 expression had longer overall survival (OS) and progression-free survival (PFS). Cox regression analysis and ROC curve confirmed that CLIC1 could independently influence the prognosis of BC patients and might have diagnostic efficiency.ConclusionOverexpressed CLIC1 is closely related to the progression of BC and the poor prognosis of the patients, suggesting that it may act as a potential biological diagnostic index for BC.

SLC26A4 Mutation Promotes Cell Apoptosis by Inducing Pendrin Transfer, Reducing Cl- Transport, and Inhibiting PI3K/Akt/mTOR Pathway.

Objective Pendrin is encoded by SLC26A4, which is expressed in the apical membrane of inner ear epithelial cells and drives chloride reabsorption in the apical septum. In the inner ear, pendrin dysfunction and hypofunctional mutations lead to vestibular aqueduct (EVA) enlargement and sensory neural hearing loss. Mutations in SLC26A4 are a common reason of deafness. However, the underlying mechanisms of SLC26A4 mutants in hearing loss remain unknown. Methods In the present study, pEGFP-N1 carrying wild-type and mutant SLC26A4 (c.85G>A, c.2006A>T, and c.853G>A) were transfected into HEK-293T cells. GFP fluorescence and GFP levels were determined. SLC26A4 mRNA levels were examined by quantitative real-time polymerase chain reaction (qRT-PCR). Then, the expression of chloride intracellular channel 1 (CLIC1) and CLIC2 was measured by Immunofluorescence assay. Intracellular chloride concentration and apoptotic rate were analyzed by flow cytometry. The levels of membrane/cytoplasmic pendrin, apoptosis-associated proteins, and the PI3K/Akt/mTOR pathway members were determined by Western blot. Results Constructed SLC26A4 mutant 1 (c.85G>A), SLC26A4 mutant 2 (c.2006A>T), and SLC26A4 mutant 3 (c.853G>A). The wild-type and 3 mutations were stably expressed in HEK-293T. SLC26A4 mRNA expression was significantly increased after transfection with wild-type SLC26A4 and mutant SLC26A4 compared with the untransfected vector group (P < 0.01). Compared with the vector group, the expression levels of membrane pendrin, cytoplasmic pendrin, CLIC1, CLIC2, Bcl-2, p-PI3K, p-Akt, and p-mTOR were upregulated. Compared with the vector group, the chloride concentration, cell apoptotic rate, and the expression levels of caspase-3, caspase-9, and Bax were downregulated. Compared with the vector group, the above effects of SLC26A4 were reversed after the SLC26A4 mutant. Conclusion After SLC26A4 mutation, pendrin was transferred from the membrane, the chloride intracellular channel function was reduced, and the excessive accumulation of chloride in the cytoplasm induced cell apoptosis by inhibited PI3K/Akt/mTOR pathway phosphorylation.

Abstract 2407: Transmembrane chloride intracellular channel 1 (tmCLIC1) protein is a key regulator of colorectal cancers' metastasis

Abstract Colorectal cancer (CRC) is the third most common tumor worldwide and the fourth cause of cancer-related death. The presence of distal metastasis at the time of the first diagnosis represents a worst prognosis factor for patients’ long-term survival. Therefore, finding new targets to arrest the metastatic process is a main challenge for successful CRC therapy. Our laboratory focused its attention on the Chloride Intracellular Channel 1 (CLIC1), a metamorphic protein able to switch from a cytoplasmic to a transmembrane form associated to ion channel activity. Previous experiments demonstrate that tmCLIC1 functional activity is negligible in normal colon cells, while increases proportionally to CRC cells’ aggressiveness. Moreover, we also reveal that tmCLIC1 supports CRC cells abnormal proliferation rate. Here, we show that tmCLIC1 pharmacological inhibition or its stable downregulation led to a significative reduction of CRC cells migrative and invasive potential, in vitro. To further confirm our hypothesis, we analysed downregulation of metalloproteinase-7 (MMP-7) protein and p38 mitogen-activated protein kinase (MAPK) signalling, both involved in migration and metastatic processes. Moreover, we demonstrate in murine model that CLIC1 knock-down impairs tumor growth and limits cancer cells dissemination. We tried to investigate the alteration of metastatic pathways after CLIC1 silencing. We obtained that many different genes are downregulated when CLIC1 protein is absent; in particular, genes regulated by Reactive Species of Oxygen (ROS) levels. In this scenario, we tried to define the mechanism of action in which CLIC1 is involved. Altogether, all the results obtained in the present work could be considered a starting point to design a new therapeutic strategy to counteract against metastatic colorectal cancer. Citation Format: Francesca Cianci, Ivan Verduci, Valentina Carlini, Carlotta Tacconi, Matteo Ranucci, Alessandro Fantin, Tullio Florio, Michele Mazzanti. Transmembrane chloride intracellular channel 1 (tmCLIC1) protein is a key regulator of colorectal cancers' metastasis [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 2407.

Abstract 2315: Synergic antitumoral activity of TTFields stimulation and Chloride Intracellular Channel 1 (CLIC1) inhibition in human glioblastoma primary culture

Abstract Glioblastoma relapse inevitably occurs in the majority of patients after surgery. Chemo- and radio- therapy are in most cases ineffective, while Tumor Treating Fields (TTFields) therapy delays average tumor recurrence by a few months. It is widely accepted that tumor reappearance is a result of the proliferation of residual glioblastoma stem cell (GSC) subpopulation, which escaped surgery. Long-term TTFields stimulation in in vitro experiments mimic glioblastoma cells recrudescence. After the first three days of TTFields exposure, cell growth is totally inhibited. However, in prolonged experiments, despite the chronic presence of TTFields stimulation, glioblastoma cells reestablish the ability to proliferate, even if at low rate. At the end of twelve days, exponentially growing cells show an increase of stemness markers, a drop of the membrane resistance with a related increase of membrane current, and an average depolarized membrane potential. Twelve days of TTFields treatment resulted in progressive increase in GSCs in the tumor cell population from 10% to more than 30%. Our long-term goal is to improve TTFields antitumoral activity by linking brain stimulation with a pharmacological strategy targeting GSC proliferation. The objective of our study is to test the therapeutic potential of combining TTFields exposure with the inhibition of the protein transmembrane Chloride Intracellular Channel 1 (tmCLIC1), which is crucial for GSCs proliferation. tmCLIC1 activity increases under oxidative stress conditions. Preliminary experiments using the INOVITRO-LIVE system showed a cytoplasmic oxidation increase under acute TTFields stimulation. tmCLIC1 impairment by ion channel specific antagonists affected the G1/S checkpoint and hence, tumor cell proliferation. Targeting synergically different glioblastoma cell cycle checkpoints, G2/M with TTFields stimulation and G1/S with tmCLIC1 inhibition, would include the vast majority of glioblastoma cell population. Combination TTFields exposure and tmCLIC1 ion channel inhibition could represent an efficacious step forward in the treatment of glioblastoma. Citation Format: Michele Mazzanti, Stefania Castiglione, Matteo Ranucci, Gaetano Cannavale, Francesca Cianci, Ivan Verduci. Synergic antitumoral activity of TTFields stimulation and Chloride Intracellular Channel 1 (CLIC1) inhibition in human glioblastoma primary culture [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 2315.

CLIC1 Drives Angiogenesis in Hepatocellular Carcinoma by Modulating VEGFA.

Background: Chloride intracellular channel 1 (CLIC1) is upregulated in hepatocellular carcinoma (HCC). The present study aimed to investigate the role of CLIC1 in HCC angiogenesis. Materials and Methods: Immunohistochemistry (IHC) was used to test the expression of CLIC1 and CD34 in 67 pairs of HCC and paracarcinoma tissues. The prognosis data of the patients were used to analyze the clinical relevance of CLIC1. We built a coculture system of HCC cells and endothelial cells to explore the migration of endothelial cells. Conditioned media (CMs) from HCC cells was then collected to assess endothelial cell migration. Experiments were then conducted to confirm the relationship between CLIC1 and angiogenesis in a subcutaneous tumor model. Results: CLIC1 expression was higher in HCC tumor tissues than in paracarcinoma tissues. Patients with increased CLIC1 expression showed a higher microvascular density (MVD; P = .013). Kaplan-Meier curves indicated that patients with lower expression of CLIC1 had better overall survival (P < .001) and recurrence-free survival (P = .046). Vascular endothelial growth factor A (VEGFA) in CMs from CLIC1-knockdown cells was lower than in the control group, while VEGFA in CMs from CLIC1 overexpression cells was higher than in the control group. CMs from CLIC1 overexpression cell lines promote the in vitro migration of EA.hy926 cells. Meanwhile, adding Bevacizumab to CMs from CLIC1 overexpression cells significantly inhibited this migration. The growth of xenograft tumors derived from CLIC1-knockdown Huh7 cells was restrained compared with the control group (P < .001). IHC staining showed MVD was higher in tumors with CLIC1 overexpression. Conclusion: CLIC1 is a promising biomarker for predicting the prognosis of HCC patients, and expression of CLIC1 correlates with angiogenesis in HCC through regulating VEGFA

Chloride Intracellular Channel 1 Expression Is Associated With Poor Prognosis of Lung Adenocarcinoma

Chloride intracellular channel 1 (CLIC1) is a member of the chloride channel protein family. The aim of this study was to clarify the role of CLIC1 in lung adenocarcinoma. The expression levels of CLIC1 in 74 patients with completely resected lung adenocarcinoma were analyzed by immunohistochemistry. Overall survival was assessed in relation to the expression level of CLIC1. Moreover, in the lung cancer cell lines A549 and PC9, CLIC1 expression was inhibited by small interfering RNA. The function of CLIC1 was analyzed in these cell lines. High expression of CLIC1 was associated with short overall survival compared to low expression (p=0.0327). Multivariate analysis revealed that CLIC1 expression was an independent prognostic factor. Knockdown of CLIC1 inhibited cell proliferation and migration through suppression of the p38 MAPK signaling pathway in A549 and PC9 cells. CLIC1 may be a useful prognostic factor in lung adenocarcinoma.

34P The interrelations between CLIC 1 expression and the histological growth pattern of liver metastases of digestive origin

Chloride intracellular channel 1 (CLIC1) is expressed by several malignancies, and it is involved in cell migration and metastasis. The histological growth pattern (HGP) such as desmoplastic (DGP), replacement (RGP), pushing (PGP) and mixed has been noticed in the liver metastases of colorectal, pancreatic, gastric, breast and uveal melanoma origin. The aim of this abstract is to determine the CLIC 1 immunoexpression variability amongst different liver metastasis HGP.

Transmembrane Chloride Intracellular Channel 1 (tmCLIC1) as a Potential Biomarker for Personalized Medicine.

Identification of potential pathological biomarkers has proved to be essential for understanding complex and fatal diseases, such as cancer and neurodegenerative diseases. Ion channels are involved in the maintenance of cellular homeostasis. Moreover, loss of function and aberrant expression of ion channels and transporters have been linked to various cancers, and to neurodegeneration. The Chloride Intracellular Channel 1 (CLIC1), CLIC1 is a metamorphic protein belonging to a partially unexplored protein superfamily, the CLICs. In homeostatic conditions, CLIC1 protein is expressed in cells as a cytosolic monomer. In pathological states, CLIC1 is specifically expressed as transmembrane chloride channel. In the following review, we trace the involvement of CLIC1 protein functions in physiological and in pathological conditions and assess its functionally active isoform as a potential target for future therapeutic strategies.

Abstract 2893: Transmembrane Chloride Intracellular Channel 1 (tmCLIC1) protein as a potential target for colorectal cancer metastasis

Abstract Colorectal cancer (CRC) is the third most common tumor worldwide and the fourth cause of cancer-related death. The presence of distal metastasis at the time of the first diagnosis represents a worst prognosis factor for patients' long-term survival. Therefore, finding new targets to arrest the metastatic process is a main challenge for successful CRC therapy. Our laboratory focused its attention on the Chloride Intracellular Channel 1 (CLIC1), a metamorphic protein able to switch from a cytoplasmic to a transmembrane form associated to ion channel activity. Previous experiments demonstrated that transmembrane CLIC1 (tmCLIC1) functional activity is negligible in normal colon cells, while increases proportionally to CRC cells' aggressiveness. Moreover, we also revealed that tmCLIC1 supports CRC cells abnormal proliferation rate. Here, we show that tmCLIC1 pharmacological inhibition or its stable knock-down by gene silencing led to a significative reduction of CRC cells migrative and invasive potential, in vitro. To further confirm our hypothesis, we assessed in CLIC1 knock-down cells the downregulation of metalloproteinase-7 (MMP-7) protein and p38 mitogen-activated protein kinase (MAPK) signaling, both involved in migration and metastatic processes. Moreover, by injecting subcutaneously transfected cell lines in nude mice, we demonstrate that CLIC1 knock-down impairs tumor growth and limits cancer cells dissemination to lung. Our results suggest a role for tmCLIC1 in promoting CRC metastasis, making this protein a promising pharmacological target to arrest or slow-down CRC metastasis development. Moreover, thanks to its functional expression exclusively associated with malignant progression, tmCLIC1 could allow to limit side effects on healthy cell population. Citation Format: Francesca Cianci, Valentina Carlini, Ivan Verduci, Matteo Ranucci, Michele Mazzanti. Transmembrane Chloride Intracellular Channel 1 (tmCLIC1) protein as a potential target for colorectal cancer metastasis [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 2893.