Suppression of tumorigenesis and metastasis of hepatocellular carcinoma by shRNA interference targeting on homeoprotein Six1

Abstract

We previously demonstrated that the overexpression of homeoprotein Six1 in hepatocellular carcinoma (HCC) patients is associated with venous infiltration, advanced pathologic tumor metastasis (pTNM) stage and poor overall survival rate (Ng et al. Br J Cancer 2006;95:1050-5). In this study, short hairpin RNA (shRNA) interference approach was used to suppress the expression of Six1 in a metastatic HCC cell line MHCC97L. Stable transfectant MHCC97L-shSix1 carrying Six1-specific shRNA plasmid was established to downregulate Six1 expression to about 40% when compared with MHCC97L-Control. In vitro functional assays demonstrated that the growth rate and proliferation ability of MHCC97L-shSix1 cells were markedly decreased. Moreover, significant decrease of cell motility and invasiveness were observed in MHCC97L-shSix1 cells. Data from in vivo xenograft tumorigenesis model demonstrated that the size of tumor in MHCC97L-shSix1 group was dramatically reduced. Experimental and spontaneous metastasis models indicated that targeting Six1 suppression noticeably reduced the pulmonary metastasis in MHCC97L-shSix1 group. To identify Six1-regulated targets, cDNA microarray was employed to compare the expression profiles of MHCC97L-Control and MHCC97L-shSix1 cells. Twenty-eight downregulated and 24 upregulated genes with known functions were identified in MHCC97L-shSix1. The functions of these target genes are involved in diverse biological activities. Our data suggest that Six1 may be involved in regulation of proliferation and invasiveness of HCC; thus targeting suppression of Six1 is a viable option for treating HCC patients.Hepatocellular carcinoma (HCC) is one of the most malignant tumors in the world, causing more than 600,000 deaths per year. 1 Surgical treatments in terms of hepatic resection and orthotopic liver transplantation are frontline treatments for HCC, but the long-term disease-free survival remains unsatisfactory. 2,3 Tumor recurrence and metastases are the major causes of death in HCC patients after surgical treatments, 4,5 indicating the necessity of developing new therapeutic strategies targeting at tumor recurrence and metastases in HCC. Up to now, the molecular mechanisms of HCC metastasis remain unclear; hence, identification and characterization of novel metastasis-associated genes are indispensable for development of effective treatment of HCC patients.Homeoprotein Six1 belongs to a subfamily of the Six family of homeodomain-containing transcription factors that shares a lysine within the DNA-binding helix of the homeodomain. 6,7 Six1, located at 14q23 of the chromosome, is involved in early developments of diverse organs such as the brain, ear, eye, muscle and kidney. 7-11 Alteration of Six1 expression takes place in human breast cancer, Wilms' cancer, cervical cancer, ovarian cancer and rhabdomyosarcoma, indicating its possible contributions in the tumorigenicity of different cancers. [12][13][14][15][16] Six1 can transform the adult mammary gland to hyperplasia and highly aggressive tumor as well as...

Identification of Transmembrane Protein 98 as a Novel Chemoresistance-Conferring Gene in Hepatocellular Carcinoma

“…MTT (Invitrogen) assay and colony formation assay were performed as previously described (18). Cells were treated with drugs for 72 hours and 2 weeks for MTT assay and colony formation assay, respectively.…”

“…cDNA microarray analysis cDNA microarray profiles of MHCC97L, MHCC97L/ CisR, and MHCC97L/DoxR were conducted by gene chip system Human U133 Plus 2.0 (Affymetrix Inc.), by Genome Research Centre, The University of Hong Kong (18). Microarray data (GEO accession number: GSE54175) were analyzed by GeneSpring Version 10 (Agilent Technologies).…”

“…Method of Western blot analysis was described in previous study (18). TMEM98 antibody was purchased from Sigma-Aldrich Corporation.…”

“…Xenograft ectopic liver tumor model in nude mice was adopted (18 curve was generated to analyze the sensitivity and 1-specificity of DDDCt(TMEM98) value to predict overall survival of patient with hepatocellular carcinoma after hepatectomy. Youden index was used to determine high deregulation (High group) and low deregulation (Low group) of patient with hepatocellular carcinoma.…”

SIX1 induces lymphangiogenesis and metastasis via upregulation of VEGF-C in mouse models of breast cancer

“…In addition, SIX1 overexpression correlates with poor prognosis in numerous tumor types, including breast cancer (10), suggesting that it plays an important role not only in tumorigenesis, but also in metastasis. Indeed, SIX1 is critical for metastasis in different models of cancer, including rhabdomyosarcoma (11), hepatocellular carcinoma (12), and breast cancer (10). Using an experimental metastasis model, we recently demonstrated that SIX1 plays a role in the later stages of metastasis through its ability to upregulate TGF-β signaling (10).…”