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ASOs suppress ARL4C mRNA expression in vitro.

Fluorescence imaging confirms delivery of ASO into liver in tumor-free mouse.

ADP-ribosylation factor-like 4c (ARL4C) is identified as a small GTP-binding protein, which is expressed by Wnt and EGF signaling and plays an important role in tubulogenesis of cultured cells and the ureters. ARL4C is little expressed in adult tissues, but it is highly expressed in lung cancer and colorectal cancer and shown to represent a molecular target for cancer therapy based on siRNA experiments. This study revealed that ARL4C is highly expressed in primary hepatocellular carcinoma (HCC) tumors and colorectal cancer liver metastases, and that ARL4C expression is associated with poor prognosis for these cancers. Chemically modified antisense oligonucleotides (ASO) against ARL4C effectively reduced ARL4C expression in both HCC and colorectal cancer cells and inhibited proliferation and migration of these cancer cells in vitro. ARL4C ASOs decreased the PIK3CD mRNA levels and inhibited the activity of AKT in HCC cells, suggesting that the downstream signaling of ARL4C in HCC cells is different from that in lung and colon cancer cells. In addition, subcutaneous injection of ARL4C ASO was effective in reducing the growth of primary HCC and metastatic colorectal cancer in the liver of immunodeficient mice. ARL4C ASO accumulated in cancer cells more efficiently than the surrounding normal cells in the liver and decreased ARL4C expression in the tumor. These results suggest that ARL4C ASO represents a novel targeted nucleic acid medicine for the treatment of primary and metastatic liver cancers.

The sequences of ARL4C ASOs (Table S1), primers (Table S2) and siRNA (Table S3). Relationship between ARL4C expression and clinicopathological facrors of HCC and CRC cases (Table S4). Univariate analysis of relapse-free survival of HCC and CRC (Table S5). mRNA levels of candidate off-target genes for ARL4C ASO (Table S6).

YAP/TAZ is not a downstream target of ARL4C in HCC cells.

Wnt5a is a representative ligand that activates the β-catenin–independent pathway in Wnt signaling. It was reported that the expression of Wnt5a in human gastric cancer is associated with aggressiveness and poor prognosis and that knockdown of Wnt5a reduces the ability of gastric cancer cells to metastasize in nude mice. Therefore, Wnt5a and its signaling pathway might be important targets for the therapy of gastric cancer. The aim of this study was to examine whether an anti-Wnt5a antibody affects metastasis of gastric cancer cells. One anti-Wnt5a polyclonal antibody (pAb5a-5) inhibited migration and invasion activities in vitro of gastric cancer cells with a high expression level of Wnt5a. Previously, it was shown that Wnt5a induces the internalization of receptors, which is required for Wnt5a-dependent activation of Rac1. pAb5a-5 inhibited Wnt5a-dependent internalization of receptors, thereby suppressed Wnt5a-dependent activation of Rac1. Laminin γ2 is one of target genes of Wnt5a signaling and Rac1 was involved in its expression. pAb5a-5 also inhibited Wnt5a-dependent expression of laminin γ2. In an experimental liver metastasis assay, gastric cancer cells were introduced into the spleens of nude mice. Laminin γ2 was required for liver metastatic ability of gastric cancer cells in vivo. Furthermore, intraperitoneal injection of pAb5a-5 inhibited the metastatic ability of gastric cancer cells. These results suggest that an anti-Wnt5a antibody was capable of suppressing Wnt5a-dependent internalization of receptors, resulting in the prevention of metastasis of gastric cancer cells by inhibiting the activation of Rac1 and the expression of laminin γ2. Mol Cancer Ther; 11(2); 298–307. ©2011 AACR.

ARL4C mRNA levels in HCC and CRC.

PIK3CD is a downstream target of ARL4C in HCC cells.

ADP-ribosylation factor-like 4c (ARL4C) is identified as a small GTP-binding protein, which is expressed by Wnt and EGF signaling and plays an important role in tubulogenesis of cultured cells and the ureters. ARL4C is little expressed in adult tissues, but it is highly expressed in lung cancer and colorectal cancer and shown to represent a molecular target for cancer therapy based on siRNA experiments. This study revealed that ARL4C is highly expressed in primary hepatocellular carcinoma (HCC) tumors and colorectal cancer liver metastases, and that ARL4C expression is associated with poor prognosis for these cancers. Chemically modified antisense oligonucleotides (ASO) against ARL4C effectively reduced ARL4C expression in both HCC and colorectal cancer cells and inhibited proliferation and migration of these cancer cells in vitro. ARL4C ASOs decreased the PIK3CD mRNA levels and inhibited the activity of AKT in HCC cells, suggesting that the downstream signaling of ARL4C in HCC cells is different from that in lung and colon cancer cells. In addition, subcutaneous injection of ARL4C ASO was effective in reducing the growth of primary HCC and metastatic colorectal cancer in the liver of immunodeficient mice. ARL4C ASO accumulated in cancer cells more efficiently than the surrounding normal cells in the liver and decreased ARL4C expression in the tumor. These results suggest that ARL4C ASO represents a novel targeted nucleic acid medicine for the treatment of primary and metastatic liver cancers.