Shuyuan Zhang1, Liem H Nguyen1, Kejin Zhou2, Ho-Chou Tu3, Alfica Sehgal3, Ibrahim Nassour1, Lin Li1, Purva Gopal4, Joshua Goodman1, Amit G Singal5, Adam Yopp6, Yu Zhang1, Daniel J Siegwart2, Hao Zhu7. 1. Children's Research Institute, Departments of Pediatrics and Internal Medicine, Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas. 2. Simmons Comprehensive Cancer Center, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas. 3. Alnylam Pharmaceuticals, Inc, Cambridge, Massachusetts. 4. Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas. 5. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas. 6. Department of Surgery, University of Texas Southwestern Medical Center, Dallas, Texas. 7. Children's Research Institute, Departments of Pediatrics and Internal Medicine, Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas. Electronic address: Hao.Zhu@utsouthwestern.edu.
Abstract
BACKGROUND & AIMS: Cytokinesis can fail during normal postnatal liver development, leading to polyploid hepatocytes. We investigated whether inhibiting cytokinesis in the liver slows tumor growth without compromising the health of normal hepatocytes. We inhibited cytokinesis in cancer cells by knocking down ANLN, a cytoskeletal scaffolding protein that regulates cytokinesis and might promote tumorigenesis, in mice with liver disease. METHODS: We analyzed clinical and gene expression data from The Cancer Genome Atlas, Oncomine, PrognoScan, and a hepatocellular carcinoma (HCC) tissue microarray. We knocked down ANLN with small interfering RNAs (siRNAs) in H2.35 liver cells and performed image analyses of cells undergoing cytokinesis. siRNAs were delivered to LAP-MYC mice, which develop hepatoblastoma, using lipid nanoparticles. H2.35 cells with knockdown of ANLN or control cells were injected into FRG mice, which develop chronic liver damage, and tumor growth was monitored. We also developed mice with inducible expression of transgenes encoding small hairpin RNAs (shRNAs) against Anln messenger RNA and studied liver tumorigenesis after administration of diethylnitrosamine and carbon tetrachloride. siRNAs against Anln messenger RNA were conjugated to N-acetylgalactosamine to reduce toxicity and increase hepatocyte tropism; their effects were studied in mouse models of liver cancer and regeneration. RESULTS: Levels of ANLN messenger RNA were increased in human HCC tissues compared to non-tumor liver tissues. siRNA knockdown of ANLN blocked cytokinesis in H2.35 liver cells. Administration of siRNA against ANLN increased survival times of LAP-MYC mice, compared to mice given a control siRNA. H2.35 liver cells with shRNA knockdown of ANLN formed tumors more slowly in FRG mice than control H2.35 cells. Mice with inducible expression of shRNAs against Anln mRNA developed fewer liver tumors after administration of diethylnitrosamine and carbon tetrachloride than control mice. Knockdown of ANLN did not affect liver regeneration after acute and chronic liver injuries. CONCLUSIONS: Knockdown of ANLN in liver cells blocks cytokinesis and inhibits development of liver tumors in mice. Agents that inhibit ANLN in the liver might be effective for prevention or treatment of HCC.
BACKGROUND & AIMS: Cytokinesis can fail during normal postnatal liver development, leading to polyploid hepatocytes. We investigated whether inhibiting cytokinesis in the liver slows tumor growth without compromising the health of normal hepatocytes. We inhibited cytokinesis in cancer cells by knocking down ANLN, a cytoskeletal scaffolding protein that regulates cytokinesis and might promote tumorigenesis, in mice with liver disease. METHODS: We analyzed clinical and gene expression data from The Cancer Genome Atlas, Oncomine, PrognoScan, and a hepatocellular carcinoma (HCC) tissue microarray. We knocked down ANLN with small interfering RNAs (siRNAs) in H2.35 liver cells and performed image analyses of cells undergoing cytokinesis. siRNAs were delivered to LAP-MYCmice, which develop hepatoblastoma, using lipid nanoparticles. H2.35 cells with knockdown of ANLN or control cells were injected into FRG mice, which develop chronic liver damage, and tumor growth was monitored. We also developed mice with inducible expression of transgenes encoding small hairpin RNAs (shRNAs) against Anln messenger RNA and studied liver tumorigenesis after administration of diethylnitrosamine and carbon tetrachloride. siRNAs against Anln messenger RNA were conjugated to N-acetylgalactosamine to reduce toxicity and increase hepatocyte tropism; their effects were studied in mouse models of liver cancer and regeneration. RESULTS: Levels of ANLN messenger RNA were increased in human HCC tissues compared to non-tumor liver tissues. siRNA knockdown of ANLN blocked cytokinesis in H2.35 liver cells. Administration of siRNA against ANLN increased survival times of LAP-MYCmice, compared to mice given a control siRNA. H2.35 liver cells with shRNA knockdown of ANLN formed tumors more slowly in FRG mice than control H2.35 cells. Mice with inducible expression of shRNAs against Anln mRNA developed fewer liver tumors after administration of diethylnitrosamine and carbon tetrachloride than control mice. Knockdown of ANLN did not affect liver regeneration after acute and chronic liver injuries. CONCLUSIONS: Knockdown of ANLN in liver cells blocks cytokinesis and inhibits development of liver tumors in mice. Agents that inhibit ANLN in the liver might be effective for prevention or treatment of HCC.
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