Downregulation of low‐density lipoprotein receptor class A domain‐containing protein 4 (Ldlrad4) in the liver of rats treated with nongenotoxic hepatocarcinogen to induce transforming growth factor β signaling promoting cell proliferation and suppressing apoptosis in early hepatocarcinogenesis

• Published in: Journal of applied toxicology 2020-11, Vol.40 (11), p.1467-1479
• Main Authors: Ito, Yuko, Nakajima, Kota, Masubuchi, Yasunori, Kikuchi, Satomi, Okano, Hiromu, Saito, Fumiyo, Akahori, Yumi, Jin, Meilan, Yoshida, Toshinori, Shibutani, Makoto
• Format: Article
• Language: English
• Subjects: Activation; Adenoma; AKT protein; AKT2 protein; Apoptosis; Attenuation; Carcinogenesis; Carcinogens; Caspase-3; Caveolin; Cell proliferation; Density; Epidermal growth factor receptors; Glutathione; glutathione S‐transferase placental form (GST‐P); Growth factors; hepatocarcinogenesis; Liver; low‐density lipoprotein receptor class A domain‐containing protein 4 (LDLRAD4); Myc protein; nongenotoxic hepatocarcinogen; NOX4 protein; Placenta; Proliferating cell nuclear antigen; Proteins; PTEN protein; rat; Receptor density; Receptors; Rodents; Signal transduction; Signaling; Snail protein; transforming growth factor (TGF)‐β signaling; Transforming growth factor-b; Transforming growth factor-b1
• ISSN: 0260-437X; 1099-1263
• DOI: 10.1002/jat.3998

We previously found downregulation of low‐density lipoprotein receptor class A domain‐containing protein 4 (LDLRAD4), a negative regulator of transforming growth factor (TGF)‐β signaling, in glutathione S‐transferase placental form (GST‐P) expressing (+) pre‐neoplastic lesions produced by treatment with nongenotoxic hepatocarcinogens for up to 90 days in rats. Here, we investigated the relationship between LDLRAD4 downregulation and TGFβ signaling in nongenotoxic hepatocarcinogenesis. The transcripts of Tgfb and Hb‐egf increased after ≥28 days of treatment. After 84 or 90 days, Snai1 increased transcripts and the subpopulation of GST‐P+ foci downregulating LDLRAD4 co‐expressed TGFβ1, phosphorylated EGFR, or phosphorylated AKT2, and downregulated PTEN, showing higher incidences than those in GST‐P+ foci expressing LDLRAD4. The subpopulation of GST‐P+ foci downregulating LDLRAD4 also co‐expressed caveolin‐1 or TACE/ADAM17, suggesting that disruptive activation of TGFβ signaling through a loss of LDLRAD4 enhances EGFR and PTEN/AKT‐dependent pathways via caveolin‐1‐dependent activation of TACE/ADAM17 during nongenotoxic hepatocarcinogenesis. The numbers of c‐MYC+ cells and PCNA+ cells were higher in LDLRAD4‐downregulated GST‐P+ foci than in LDLRAD4‐expressing GST‐P+ foci, suggesting a preferential proliferation of pre‐neoplastic cells by LDLRAD4 downregulation. Nongenotoxic hepatocarcinogens markedly downregulated Nox4 after 28 days and later decreased cleaved caspase 3+ cells in LDLRAD4‐downregulated GST‐P+ foci, suggesting an attenuation of apoptosis by LDLRAD4 downregulation through activation of the EGFR pathway. At the late hepatocarcinogenesis stage in a two‐stage model, LDLRAD4 downregulation was higher in adenoma and carcinoma than in pre‐neoplastic cell foci, suggesting a role of LDLRAD4 downregulation in tumor development. Our results suggest that nongenotoxic hepatocarcinogens cause disruptive activation of TGFβ signaling through downregulating LDLRAD4 toward carcinogenesis in the rat liver. The present study investigated the relationship between LDLRAD4 downregulation and TGFβ signaling during nongenotoxic hepatocarcinogenesis in rats. Immunohistochemically, LDLRAD4‐downregulated GST‐P+ foci revealed a phenotype suggestive of disruptive activation of TGFβ signaling to enhance EGFR and PTEN/AKT‐dependent pathways via caveolin‐1‐dependent activation of TACE/ADAM17. The LDLRAD4‐downregulated foci also facilitated proliferation and attenuated apoptosis