Chemically Modified Antisense Oligonucleotide Against ARL4C Inhibits Primary and Metastatic Liver Tumor Growth

The liver is an organ that develops primary and metastatic tumors. Hepatocellular carcinoma (HCC) is the fifth common type of cancer worldwide and the third leading cause of cancer death (1). The median survival is approximately 6 to 20 months after diagnosis for the intermediate and advanced stages, and the 5-year survival of patients with HCC is less than 30%. Even after apparently curative surgical resection, recurrent HCC develops in 80% of patients within 5 years because of intrahepatic metastasis. For unresectable HCC, different treatment modalities are available, including radiation, radioembolization, systemic chemotherapy, and molecularly targeted therapy. Oral sorafenib, which is a multityrosine kinase inhibitor, is currently the first-line treatment for advanced HCC but its effectiveness is limited (2). The liver is also a common site of metastasis from other solid tumors, especially tumors originating from the intra-abdominal organs, including the stomach, pancreas, colon, gallbladder, and ovary (3). Less than 10% to 15% of patients with liver-only solid tumor metastases are candidates for resection (4). For the majority of patients with primary and metastatic liver cancers who are not candidates for surgical resection, novel treatment approaches to control and potentially cure the liver tumors must be explored.

ADP-ribosylation factor (ARF)-like proteins (ARL) are one of the subgroups of the ARF family of proteins in the small GTP-binding protein superfamily (5). ARF family proteins are essential regulators of the actin remodeling and membrane-trafficking pathways, including those involved in secretion, endocytosis, and phagocytosis (6). However, the functions of most ARLs remain unclear (7). ARF-like 4c (ARL4C), a member of ARL family proteins, is a target protein that is expressed as a result of Wnt/β-catenin and EGF/RAS/MAPK signaling and plays an important role in both epithelial morphogenesis and tumorigenesis (8, 9).

Immunohistologic analysis has revealed that ARL4C is frequently overexpressed in colorectal cancer, lung cancer, and tongue cancer but little detected in nontumor regions of these tissues (9, 10). In HCT116 colorectal cancer cells and A549 lung adenocarcinoma cells, ARL4C expression is upregulated by Wnt/β-catenin or RAS/MAPK signaling (9). In addition, ARL4C is highly expressed in NCI-H520 lung squamous cell carcinoma cells because of DNA hypomethylation of the ARL4C gene (10). ARL4C is involved in the activation of Ras-related C3 botulinum toxin substrate (RAC), the inhibition of ras homolog family member (RHO), and the nuclear localization of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in these cancer cells. ARL4C depletion in cancer cells reduced migration, invasion, and proliferation both in vitro and in vivo. Furthermore, the direct injection of ARL4C siRNA into HCT116 xenograft tumors inhibited tumor growth in immunodeficient mice (9). Therefore, ARL4C may represent a novel molecular target for cancer therapy.

Oligonucleotide-based therapeutics has been recognized as a promising and potent systemic approach for the treatment of incurable diseases (11–13). There are several types of oligonucleotides, including antisense oligonucleotides (ASO), siRNAs, aptamers, and miRNAs. At present, some ASO therapies against cytomegalovirus retinitis, age-related macular degeneration of the retina, homozygous familial hypercholesterolemia, Duchene muscular dystrophy, and spinal muscular atrophy have received FDA approval (14). However, no oligonucleotide drugs have been indicated for cancer. Oligonucleotide therapeutics have problems with susceptibility to nuclease degradation, inefficient delivery to target cells following systemic administration, and potentially serious nonspecific side effects, which result in unsatisfactory in vivo gene silencing to warrant clinical trials. Therefore, chemical modification strategies have been developed to overcome these obstacles (15). In particular, a combination of a 2′,4′-bridged nucleic acid (also known as a locked nucleic acid, LNA) and phosphorothioate linkages exhibits high target RNA binding, resistance to nuclease degradation, and acceptable pharmacokinetics. Additional optimization of the bridged nucleic acids, in which the cyclic amide structure is introduced into the LNA (named amide-bridged nucleic acid, AmNA), has further improved these characteristics (16).

Because ASOs tend to be delivered to the liver and the kidney (17), we investigated whether ARL4C is involved in the proliferation of liver cancer cells and if AmNA–ASO therapeutics against ARL4C could represent a novel therapy for the primary and metastatic liver tumors.

This study involved 128 patients with HCC (stage I to IVA) with ages ranging from 21 to 81 years (median, 69 years), who underwent surgical resection at Kobe University Hospital between January 2010 and December 2012, and 115 patients with colorectal cancer with ages ranging from 28 to 91 years (median, 66 years), who underwent surgical resection at Osaka University Hospital between June 2007 and February 2017. The HCC cases did not include patients with distant metastasis. The colorectal cancer cases included stage 0 to IIIC (102 cases) and stages IVA and IVB (13 cases). Tumors were staged according to the Union for International Cancer Control (UICC) TNM staging system. Resected specimens were macroscopically examined to determine the location and size of tumors. Histologic specimens were fixed in 10% (v/v) formalin and paraffin embedded. The specimens were sectioned at a 4-μm thickness and stained with hematoxylin and eosin (H&E) or immunoperoxidase for independent evaluation.

The protocol for this study was approved by the ethical review board of the Graduate School of Medicine, Kobe University, Japan (No. 180048) and the Graduate School of Medicine, Osaka University, Japan (No. 13032) under Declaration of Helsinki, and written informed consent was obtained from all patients. The study was performed in accordance with the Committee guidelines and regulations.

Immunohistochemical studies were performed as previously described (9) with modification. When the total area of a tumor lesion showed >20% ARL4C staining, the results were defined as ARL4C-positive. Three investigators assessed the sections independently in a blinded fashion.

HCT116 cells were provided by Dr. T. Kobayashi (Hiroshima University, Hiroshima, Japan) in November 2003. A549 cells were provided by Dr. Y. Shintani (Osaka University, Suita, Japan) in January 2014. HCC cells (HLE, HLF, HuH-7, and PLC) were purchased from the Japanese Collection of Research Bioresources (Osaka, Japan) in June 2015. HepG2 cells were purchased from the ATCC in July 2017. Mycoplasma testing and cell line authentication were not conducted. HLE, HLF, HuH-7, PLC, HCT116, and A549 cells were maintained in DMEM supplemented with 10% FBS. HepG2 cells were grown in E-MEM (Wako; Catalog No. 051-07615) supplemented with GlutaMAX I (Life Technologies; Catalog No. 35050-061), MEM Non-Essential Amino Acids (Life Technologies; Catalog No. 11140-050), and 10% FBS. HLE cells stably expressing GFP (HLE/GFP) or ARL4C (HLE/ARL4C-GFP) and HCT116 stably expressing luciferase (HCT/Luc) were generated using lentivirus as described previously (8, 9).

Anti-ARL4C and Anti-PIK3CD antibodies were purchased from Abcam and Santa Cruz Biotechnology, respectively. Anti-phospho AKT (S473), anti-AKT, anti-Ki-67, and anti-YAP/TAZ antibodies were obtained from Cell Signaling Technology. PD184161 (a selective MEK1/2 inhibitor; ref. 18) and VivoGlo luciferin were from Sigma-Aldrich and Promega, respectively. SecinH3 [an inhibitor of ARF nucleotide-binding site opener (ARNO); ref. 19] and NSC23766 (a RAC inhibitor; ref. 20) were from TOCRIS Bioscience and Merck Millipore, respectively.

Phosphorothioate 15-mer or 19-mer ASOs containing AmNA monomers or 6-carboxyfluorescein (FAM)-labeled ASO were synthesized by GeneDesign based on the previously described method (16, 21). The sequences of the ASOs are listed in Supplementary Table S1.

HLE, HCT116, and A549 cells were transfected with ASOs at 0.5 to 50 nmol/L and siRNAs at 10 to 20 nmol/L using Lipofectamine 3000 (Invitrogen) or RNAiMAX (Invitrogen) in antibiotics-free medium, respectively. The transfected cells were then used for experiments conducted at 36 to 48 hours after transfection.

A HLE cell (1 × 10⁷ cells) pellet was suspended in 100 μL of Matrigel matrix high concentration (BD Biosciences) and directly injected into the liver of anesthetized 8-week-old male BALB/cAJcl-nu/nu mice (nude mice; CLEA Japan). ASO (50 μg/body, approximately 2.5 mg/kg; 100 μg/body, approximately 5 mg/kg) was administered subcutaneously twice per week from day 0. The mice were euthanized 29 days after transplantation. Tumor weights were collected and subjected to histologic analyses.

HCT116/Luc cells (2.5 × 10⁵ cells) resuspended in 100 μL of PBS were injected into the spleens of anesthetized 8-week-old male nude mice. Nineteen days after implantation, ASO (50 μg/body) was administered subcutaneously twice per week. The mice were euthanized 47 days after transplantation, and histologic analysis was performed. Tumor sizes were measured once per week using the IVIS imaging system (Xenogen Corp.). For the in vivo imaging, 100 μL of VivoGlo luciferin (30 mg/mL) was administrated intraperitoneally and then bioluminescence imaging was recorded 10 minutes after administration of luciferin. All protocols used for animal experiments in this study were approved by the Animal Research Committee of Osaka University, Japan (No. 21-048-1).

The data of ARL4C mRNA expression in liver HCC and colorectal cancer were obtained from the Gene Expression Profiling Interactive Analysis (GEPIA) online database (http://gepia.cancer-pku.cn/). Tumors and normal samples in the GEPIA database were derived from TCGA and Genotype-Tissue Expression (GTEx) projects. Differential analysis was performed using one-way ANOVA. P values below 0.05 were considered statistically significant. The correlations of overall survival rates with ARL4C mRNA expression in the colorectal cancer of the TCGA dataset were analyzed using OncoLnc (http://www.oncolnc.org) and visualized using GraphPad Prism (GraphPad Software). High and low ARL4C expression groups were classified by the median value of ARL4C expression of patients with colorectal cancer in the dataset. Coexpression analysis using the TCGA dataset was performed using the “R2: Genomics Analysis Platform (http://r2.amc.nl)” and visualized using GraphPad Prism. P values and r values were calculated using GraphPad Prism.

Potential off-target genes for the ARL4C ASOs [hARL4C ASO-1316-AmNA [15-mers] (ARL4C ASO-1316) and hARL4C ASO-3223-AmNA [19-mers] (ARL4C ASO-3223)] were identified using basic local alignment search tools (BLAST+ programs) with the human genomic plus transcript (Human G+T) database (Build 2.7.1) and Megablast algorithm.

Each experiment was performed at least three times, and the results are presented as the mean ± SD. The cumulative probabilities of recurrence-free survival were determined using the Kaplan–Meier method; the log-rank test was used to assess statistical significance. The Student t test was used to determine the statistical significance in other experiments; P values <0.05 were considered statistically significant.

Cell migration assays and quantitative real-time PCR analyses were performed as previously described (9). The primers and siRNAs used in these experiments are listed in Supplementary Tables S2 and S3, respectively. The cell proliferation assay was performed using the CyQUANT NF Cell Proliferation Assay Kit (ThermoFisher Scientific) according to the manufacturer's instructions. Fluorescence was measured using a fluorescence microplate reader (Synergy HTX Multi-Mode Microplate Reader; BioTek).

ARL4C expression was analyzed in primary HCC using immunohistochemistry. Tumors in which greater than 20% of the tumor lesions were stained with the anti-ARL4C antibody were considered ARL4C-positive. Among the 128 HCC samples, ARL4C was detected and positive in 33 tumors (25.8%) but only minimally detected in matched nontumor regions (Fig. 1A). ARL4C staining was observed throughout the cytoplasm of the ARL4C -positive tumor cells only. The dataset GEPIA, which combined TCGA and GTEx, revealed that although the ARL4C mRNA levels in HCC are not statistically higher than those in normal liver, there is trend to higher expression of ARL4C mRNA in HCC compared with normal liver (Supplementary Fig. S1).

Clinicopathologic examination revealed that ARL4C positivity is associated with vascular invasion (P = 0.02; Supplementary Table S4). Univariate analysis demonstrated that sex, tumor number > 2, poorly differentiated type, lymph node metastasis, vascular invasion, stage IIIA to IVA, and ARL4C expression were associated with inferior relapse-free survival (Supplementary Table S5). Multivariate analysis showed that tumor number >2 is an independent prognostic factor (P = 0.0003). In addition, ARL4C positivity tended to be independently associated with poor prognosis (P = 0.066; Table 1). Furthermore, relapse-free survival was significantly decreased in patients with ARL4C-positive tumors compared with patients with ARL4C-negative tumors (P = 0.033; Fig. 1B). Taken together, these results suggest that ARL4C expression is correlated with the aggressiveness of HCC.

In a previous colorectal study (9), the detailed relationship between ARL4C expression and clinicopathologic parameters was not examined. In this study, 24.5% of the 102 stage 0 to IIIC patient with colorectal cancer tumors were ARL4C-positive (i.e., >20% of the tumor cells within the tumor lesions were positive; Supplementary Table S4). The positivity was associated with tumor invasion, vascular invasion, and stage IIA to IIIC (Supplementary Table S4). Univariate analysis demonstrated that a tumor invasion depth beyond the submucosa, vascular invasion, lymphatic invasion, lymph node metastasis, stage IIA to IIIC, and ARL4C expression are associated with inferior relapse-free survival (Supplementary Table S5). Multivariate analysis showed that ARL4C expression is an independent prognostic factor (P = 0.0004; Table 1). Relapse-free survival was significantly decreased in patients positive for ARL4C compared with patients negative for ARL4C (P < 0.0001; Fig. 1C). These results were consistent with two public databases: the GEPIA dataset showed that ARL4C mRNA is expressed at higher levels in colorectal cancer than in normal colon tissue (Supplementary Fig. 1A); the TCGA dataset indicated that the ARL4C high expression groups has a decreased overall survival rate compared with the ARL4C low expression groups (P = 0.003; Fig. 1D).

Colorectal cancer liver metastasis tumors (24 cases) were examined for ARL4C expression by IHC (Fig. 1E). The 24 cases included 11 cases of recurrent liver metastasis after colorectal cancer resection (metachronous liver metastasis) and 13 cases of colorectal cancer with liver metastasis (synchronous liver metastasis). For the metachronous liver metastasis cases, ARL4C was expressed in 11 (100%) and 9 (81.8%) cases of colon and liver tumor lesions, respectively (Fig. 1E). For the synchronous liver metastasis cases, ARL4C was positive in 11 (84.6%) and 10 (76.9%) cases of primary and metastatic tumor lesions, respectively (Fig. 1E). In all cases, ARL4C was minimally detected in nontumor regions. Therefore, ARL4C expression in colorectal cancer was associated with poor prognosis and the expression frequency increased in colorectal cancer cases with liver metastasis.

To determine whether ARL4C could be a therapeutic target for the treatment of liver tumors, ARL4C ASOs were generated and their effects on cancer cells were examined. ARL4C sequences, which consisted of 15 nucleotides, were chosen based on the secondary structure of the ARL4C mRNA (22). Of the thousands of ARL4C ASO candidates, the sequences that might cause hepatotoxicity were excluded, and 38 ARL4C ASO sequences were selected by high-dimensional structure prediction of the ARL4C mRNA. All of the phosphodiester linkages in the ASOs used in this study were replaced by phosphorothioate linkages. Screening experiments using real-time PCR were performed to evaluate the knockdown efficiency of 38 ARL4C ASOs using A549 lung cancer cells, which were previously used to demonstrate reduced ARL4C protein levels in response to ARL4C siRNA treatment (Supplementary Fig. S2A; ref. 9). The screening was performed three times, and seven ARL4C ASOs, ASO-650, ASO-793, ASO-986, ASO-1065, ASO-1316, AS-1454, and ASO-3225 were selected for further evaluation.

Among the five HCC cell lines, HLE cells expressed the highest ARL4C mRNA levels compared with HLF, PLC, and HuH-7 cells, and HepG2 cells expressed little ARL4C mRNA (Supplementary Fig. S2B). Among the seven ARL4C ASOs, ASO-1316, ASO-1454, and ASO-3225 decreased ARL4C mRNA levels in both HLE and HCT116 cells without cytotoxicity (Fig. 2A). Transfection of control ASO did not significantly affect the expression of ARL4C mRNAs in HLE cells (Supplementary Fig. S2C). To optimize the effects of these three ARL4C ASOs, ARL4C ASO-1312, ASO-1450, and ASO-3223 were designed with 19 nucleotides, including the original 15 nucleotides. Among these six candidates, ARL4C ASO-1316 and ARL4C ASO-3223 (19-mer including the target sequence of ARL4C ASO-3225) were selected based on their strong downregulation of ARL4C mRNA expression in HLE cells at low doses (Fig. 2B; Supplementary Fig. S2D).

Four candidate off-target genes for ARL4C ASO-1316 and ASO-3223 were found by in silico analysis. These genes were neurolysin (NLN), sapiens F-box and leucine-rich repeat protein 19 (FBXL19), keratin-associated protein 15-1 (KRTAP15-1), and sialic acid binding Ig-like lectin 6 (SIGLEC6). KRTAP15-1 and SIGLEC6 were expressed at only low levels in HLE cells (Supplementary Table S6). ARL4C ASO-1316 and -3223 did not significantly affect the expression of NLN and FBXL9 mRNAs in HLE cells (Supplementary Fig. S2E). Thus, off-target effects resulting from treatment with ARL4C ASOs appear to be unlikely.

Both ARL4C ASO-1316 and ASO-3223 suppressed the migration of HLE cells, which was rescued by the overexpression of ARL4C, excluding off-target effects by the ARL4C ASOs (Fig. 2C and D). ARL4C ASOs also inhibited HLE cell proliferation (Fig. 2E) under the conditions that ASO transfection reagents did not affect cell proliferation (Supplementary Fig. S2F). Inhibition of cell migration by these ARL4C ASOs was also observed in HCT116 cells (Supplementary Fig. S2G; ref. 9). Taken together, these results suggest that ARL4C ASOs decreased both the cell proliferation and migration of ARL4C-expressing cancer cells in vitro.

ARL4C is expressed by Wnt/β-catenin and EGF/RAS signaling and can induce the nuclear localization of YAP/TAZ via cell relaxation by the appropriate inhibition of RHO signaling (8, 9). Treatment of HLE cells with PD184161, a selective MEK1/2 inhibitor, but not by knockdown of β-catenin (a CTNNB1 gene product), decreased the expression of ARL4C mRNA, suggesting that ARL4C is downstream of the MAPK pathway in HLE cells (Fig. 3A; Supplementary Fig. S3A). These findings were consistent with the correlation analysis data of the TCGA dataset, which showed that the mRNA expression levels of the target genes of the MAPK pathway (EGR1 and FOS), but not those of Wnt/β-catenin signaling pathway (AXIN2 and LGR5), are positively correlated with that of ARL4C in HCC (Supplementary Fig. S3B). Knockdown of ARL4C in A549 cells inhibited the nuclear localization of YAP, which did not occur in HLE cells (Supplementary Fig. S3C). Therefore, the genes that are expressed downstream of ARL4C and involved in the cell proliferation of HCC might be different from those in lung cancer cells.

The phosphatidylinositol-3-kinase catalytic subunit δ isoform (the PIK3CD gene product) is expressed in many cancers, including HCC (23–26). TCGA dataset analysis revealed that the PIK3CD mRNA expression level, but not that of PIK3CA and PIK3CB, is positively correlated with ARL4C expression in HCC (Fig. 3B; Supplementary Fig. S4A). ARF6 and RAC can act downstream of ARL4C in colon and lung cancer cells (9). Treatment of HLE cells with SecinH3 (an inhibitor of ARNO) or NSC23766 (a RAC inhibitor) decreased the PIK3CD mRNA level (Fig. 3C). Overexpression of ARL4C increased the PIK3CD mRNA level in HepG2 cells, and the increment was abrogated by SecinH3 or NSC23766 in HepG2 cells (Fig. 3D), suggesting that ARL4C upregulates PIK3CD expression through ARF6 and RAC. Consistent with these findings, TCGA dataset analysis showed that the mRNA expression levels of ARF6 and RAC1, but not ARF1, are positively correlated with that of PIK3CD in HCC (Supplementary Fig. S4B). PIK3CD knockdown inhibited proliferation and AKT activity in HLE cells (Fig. 3E and F; Supplementary Fig. S4C) under the conditions that siRNA transfection reagents did not affect cell proliferation (Supplementary Fig. S4D). ARL4C ASO-1316 and ASO-3223 decreased PIK3CD mRNA levels and inhibited AKT activity in HLE cells (Fig. 3G and H). In contrast, PIK3CD knockdown did not inhibit HCT116 cell proliferation (Supplementary Fig. S4C and S4E), and ARL4C ASOs did not affect PIK3CD mRNA levels in HCT116 cells (Supplementary Fig. S4F). These results suggest that PIK3CD is a downstream target of ARL4C in HCC but not colorectal cancer cells and that ARL4C ASOs inhibit cell proliferation by the suppression of PIK3CD expression in HCC.

The effects of subcutaneous injection of ARL4C ASO-1316 and ASO-3223 on liver tumors were examined in two mouse cancer models: a primary liver tumor model in which HLE cells were transplanted directly in the liver and a metastatic tumor model in which HCT116 cells were transplanted in the spleen and then metastasized to the liver.

HLE cells were injected into the liver, and mice were subcutaneously injected with control ASO and two ARL4C ASOs (Fig. 4A). ARL4C ASO-1316 suppressed HLE tumor formation and reduced tumor weight in a dose-dependent manner compared with control ASO (Fig. 4A). ARL4C ASO-1316 also inhibited ARL4C and PIK3CD expression in the liver tumors and decreased the number of Ki-67-positive cells (Fig. 4B). ARL4C ASO-1316 did not induce any histological damages or cell death in the nontumor regions of the liver (Supplementary Fig. S5A). In contrast, ARL4C ASO-3223 (50 μg/body) did not significantly inhibit HLE tumor formation (Fig. 4A), the expression of ARL4C and PIK3CD, or the numbers of Ki-67-positive cells (Fig. 4B) despite being effective in vitro (see Fig. 2).

Subcutaneous injection of 6-FAM-labeled ARL4C ASO-1316 into nontumor bearing mice showed that the ASO is specifically delivered to the liver compared with other organs, although its incorporation was found to be weak by the assessment of the fluorescence intensity (Supplementary Fig. S5B). However, when 6-FAM-labeled ARL4C ASO-1316 was injected into HLE tumor-bearing mice, the ASO highly accumulated in tumor lesions of the liver (Fig. 4C). Confocal imaging revealed that the ASO accumulated in nuclear punctate sites of tumor cells or hepatocytes (Fig. 4D; Supplementary Fig. S5C).

For the metastatic liver tumor model, HCT116/Luc cells were injected into the spleen, and the luminescence signal was detected in the liver starting on day 19 (Fig. 5A). Control ASO and ARL4C ASO-1316 and ASO-3223 were subcutaneously injected beginning on day 19, and the effect of treatment on tumor formation was followed by biweekly bioluminescence measurements (Fig. 5A and B). By day 47, ARL4C ASO-1316 suppressed the growth of HCT116 liver metastasis (Fig. 5A). In the liver tumors of the ARL4C ASO-1316-treated mice, ARL4C expression and the number of Ki-67-positive cells were reduced (Fig. 5B and C). In contrast, ARL4C ASO-3223 did not exhibit any antitumor activity (Fig. 5).

In this study, we examined whether ARL4C is involved in the proliferation of cancer cells in the liver and if ARL4C ASO could represent a novel treatment for HCC and metastatic liver cancer. The key findings were: (i) ARL4C is a prognostic factor for HCC and colorectal cancer; (ii) ARL4C upregulated PIK3CD in HCC; and (iii) ARL4C ASO inhibited liver tumor formation in two independent mouse models.

ARL4C expression in HCC was correlated with vascular invasion and inversely associated with relapse-free survival. Consistent with these findings, cell biology studies revealed that ARL4C is involved in proliferation and migration of HCC cells. Thus, ARL4C expression may be associated with the aggressiveness of HCC. In colon, lung, and tongue cancer cells, ARL4C modulates actomyosin through the regulation of ARF6, RAC, and RHO, which results in the nuclear localization of YAP/TAZ and cellular proliferation (8–10). Thus, the ARNO–ARF6 pathway is a direct downstream signaling target of ARL4C to regulate YAP/TAZ-dependent cancer cell proliferation (5). However, ARL4C did not affect YAP/TAZ subcellular localization in HCC cells but was involved in the expression of PIK3CD, which is expressed in many cancers, including HCC, colorectal cancer, breast cancer, glioblastoma, and multiple myeloma (23–26). In these cancers, PIK3CD is a therapeutic target, and a selective PIK3CD inhibitor and microRNA-7, which targets PIK3CD, exhibit antitumor activity. The underlying mechanism by which ARL4C induces PIK3CD expression in HCC cells remains to be clarified although ARF6 and RAC are likely to be involved in this process. Thus, ARL4C may activate multiple downstream signaling pathways in a cancer cell-context manner.

ARL4C was expressed in 24.5% of colorectal cancer cases without distant metastasis. The frequency increased to 95% of the primary lesions in colorectal cancer with liver metastasis, and more than 80% of the metastatic liver lesions were positive for ARL4C in these cases. Because ARL4C can stimulate migration and invasion of colorectal cancer cells, it is thought that liver metastasis occurs with high frequency in colorectal cancer expressing ARL4C. Indeed, the relapse-free survival was poor in patients with colorectal cancer that were positive for ARL4C. These results were supported by public databases. Taken together with the observations that ARL4C expression in gastric cancer is associated with the depth of invasion, peritoneal dissemination, and poor prognosis (27), ARL4C expression may be a molecular marker for the prognosis of these cancers.

Because knockdown or knockout of ARL4C inhibits the proliferation of cancer cells (9, 10), ARL4C represents a potential novel target for cancer therapy. ARL4C is an intracellular protein that mediates a signal through protein–protein interactions (5). Therefore, one strategy for targeting ARL4C is the use of nucleic acids rather than low molecular compounds or antibodies. Oligonucleotide-based drugs are promising therapeutics in that they can be designed rapidly and rationally against virtually any genetic target (15). The delivery and potency of the drugs are derived from the chemical structure of the oligonucleotide whereas their target is decided by the nucleic acid sequence. We designed and synthesized chemically modified ASOs, which were flanked by AmNA with a phosphorothioate backbone. This type of ASO was shown to be stable in the blood and highly incorporated into liver cells in vivo (16). Although LNAs have been associated with liver toxicity (28), the AmNA-ASOs inhibit target mRNA expression in vivo and exhibit less toxicity to the liver (29, 30).

From the thousands of candidate ARL4C ASOs, two ARL4C ASOs, which showed the highest suppression of ARL4C expression and inhibition of HCC and colorectal cancer proliferation in vitro, were selected for in vivo testing using mouse models of primary HCC and colorectal cancer liver metastasis. ARL4C ASO-1316, but not ARL4C ASO-3223, decreased the expression of ARL4C and inhibited tumor growth in both models. The results demonstrate that the in vitro effects of ASOs do not always reflect the in vivo effects probably due to differences in incorporation efficiency and inhibition of mRNA synthesis in intact cells in vivo. The mechanisms of cellular uptake, trafficking, and distribution of ASOs and the proteins that bind and regulate ASOs remain to be clarified (13). Although it has been reported that ASO length may play a role in dystrophin exon skipping (31), at present it is necessary to test the in vivo effects of all synthesized ASOs that are effective in vitro.

ARL4C ASO-1316 specifically accumulated in the nuclei of liver tumor cells and was indeed effective for liver tumors at low doses (twice per week with approximately 2.5 mg/kg) when compared with doses shown in previous reports (e.g., 50 mg/kg Msi-2 ASO given daily for pancreatic cancer; 20 mg/kg Stat3 ASO given daily for lymphoangiogenesis; refs. 32, 33). Histologic damage to the nontumor regions of the liver was not observed following the administration of ARL4C ASO-1316. Because ARL4C-positive primary HCC is about 26% of total HCC cases that we examined, the therapy using ARL4C ASO for primary HCC may be limited. Nonetheless, it is expected that ARL4C ASO-1316 is appropriate for clinical use without side effects.

In conclusion, ARL4C is involved in liver cancer cell proliferation through PIK3CD expression and ARL4C ASO represents the novel treatment for primary and metastatic liver cancers.

No potential conflicts of interest were disclosed.

Conception and design: S. Matsumoto, A. Kikuchi

Development of methodology: S. Matsumoto, S. Obika, A. Kikuchi

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Harada, S. Matsumoto, S. Hirota, S. Fujii, Y. Kasahara, H. Gon, T. Yoshida, N. Haraguchi, T. Mizushima, T. Noda, H. Eguchi, E. Morii, T. Fukumoto

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Matsumoto, H. Kimura, Y. Kasahara, H. Gon, E. Morii, S. Obika

Writing, review, and/or revision of the manuscript: T. Harada, H. Kimura, T. Fukumoto, A. Kikuchi

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Hirota, T. Itoh, T. Noda, S. Nojima, A. Kikuchi

Study supervision: T. Mizushima, A. Kikuchi

The authors would like to thank to Dr. Sae Murakami at Division of Clinical and Translational Research Center, Kobe University Hospital, for helping statistical analyses. The authors also thank Drs. T. Kobayashi and A. Shintani for donating cells. This work was supported by Grants-in-Aid for Scientific Research to A. Kikuchi (2016-2020; No. 16H06374) and for Scientific Research on Innovative Areas “Integrated analysis and regulation of cellular diversity” to A. Kikuchi (2018-2019; No. 18H05101) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; by the Project Promoting Support for Drug Discovery to A. Kikuchi (2015-2018; No. DNW-15005) from the Japan Agency for Medical Research and Development, AMED, by Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University to A. Kikuchi; and by grants from the Yasuda Memorial Foundation to A. Kikuchi.

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