RESUMEN
BACKGROUND & AIMS: Levels of atonal homolog 8 (ATOH8) are reduced in 48% of hepatitis B virus-associated hepatocellular carcinoma cells (HCCs). ATOH8 downregulation is associated with loss of tumor differentiation, indicating an effect mediated by cancer stem cells. We investigated the effects of loss of ATOH8 in human hepatocellular carcinoma (HCC) cells and cell lines. METHODS: HCC and adjacent nontumor tissues were collected, from 2001 through 2012, from 242 patients undergoing hepatectomy at Sun Yat-Sen University Cancer Center in China; 83% of HCCs were associated with hepatitis B virus (HBV) infection. CD133(+) cells were isolated from tumor tissues by flow cytometry. Experiments were performed in HBV-positive and HBV-negative HCC cell lines, the immortalized liver cell line LO2, and 8 other HCC cell lines. ATOH8 was expressed from lentiviral vectors in PLC8024 and Huh7 cells; levels were knocked down with small interfering RNAs in QSG7701 cells. Cells carrying empty vectors were used as controls. Gene regulation by ATOH8 was assessed in mobility shift and luciferase reporter assays. Cells were analyzed in proliferation, foci formation, and colony formation assays. The tumorigenic and chemo-resistant potential of cells were investigated by assessing growth of xenograft tumors in immunocompromised mice. Metastatic features of cells were assessed in Matrigel invasion assays and wound healing analyses. RESULTS: Levels of ATOH8 mRNA were reduced by more than 4-fold, compared to nontumor tissues, in 118 of 242 HCC samples (48.8%). Patients with tumor reductions in ATOH8 had significantly shorter times of disease-free survival (mean, 41.4 months) than patients with normal tissue levels (mean, 52.6 months). ATOH8 expression was reduced in HepG2, Huh7, PLC8024 and CRL8064 HCC cells, as well as CD133(+) cells isolated from human HCC samples. Transgenic expression of ATOH8 in HCC cell lines significantly reduced proliferation and foci colony formation, as well as their invasive and migratory abilities. Transgenic expression of ATOH8 reduced the ability of HBV-positive PLC8024 cells to form tumors in mice, compared to control cells. Cells with ATOH8 knockdown formed xenograft tumors more rapidly, in more mice, than control cells. ATOH8 repressed transcription of stem-cell associated genes including OCT4, NANOG, and CD133. Knockdown of ATOH8 in CD133-negative QSG7701 cells caused them to express CD133; acquire self-renewal, differentiation, chemo-resistance properties; form more xenograft tumors in mice; and generate induced pluripotent stem cells (based on staining for alkaline phosphatase and their ability to form embryoid bodies and teratomas). Alternatively, expression of ATOH8 in PLC8024 and Huh7 cells significantly reduced the numbers of cells expressing CD133, and increased the chemo-sensitivity of Huh7 cells to 5-fluorouracil (5-FU) and cisplatin, in vitro and in mice. CONCLUSIONS: ATOH8 appears to be a tumor suppressor that induces stem-cell features and chemoresistance in HCC cells. Strategies to restore its levels and activities might be developed to treat patients with liver cancer.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma Hepatocelular/metabolismo , Desdiferenciación Celular , Neoplasias Hepáticas/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Antineoplásicos/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Supervivencia sin Enfermedad , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/metabolismo , Células Hep G2 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Estimación de Kaplan-Meier , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Ratones SCID , Invasividad Neoplásica , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Péptidos/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , Tratamiento con ARN de Interferencia , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Transfección , Proteínas Supresoras de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hepatocellular carcinoma (HCC), which accounts for 85-90% of primary liver cancer, is now the second leading cause of cancer-related mortality worldwide. Here we reported that Aldo-Keto Reductase family 7A isoform 3 (AKR7A3) is frequently down-regulated in HCC, associating with poor overall survival rate, elevated serum α-fetoprotein (AFP) and poor differentiation of HCC. The promoter region of AKR7A3 was detected to be hypermethylated. Loss of heterozygosity (LOH) was also detected in AKR7A3. Functional assays on both AKR7A3 overexpressed and knockdown cells, including foci formation, colony formation in soft agar, migration, invasion and tumor formation in nude mice, demonstrated the strong tumor suppressive functions of AKR7A3. In addition, treatment of chemotherapy drug cisplatin showed that AKR7A3 sensitizes tumor cells to apoptosis. Mechanistically, western blot analysis showed that overexpression of AKR7A3 inhibits the activation of ERK, c-Jun and NF-κB. In summary, we found that AKR7A3 functions as a tumor suppressor gene in HCC through attenuating c-Jun, ERK and NF-κB signaling pathways.