Cooperative antiproliferative effect of coordinated ectopic expression of DLC1 tumor suppressor protein and silencing of MYC oncogene expression in liver cancer cells: Therapeutic implications

Abstract

Abstract. Human hepatocellular carcinoma (HCC) is one of the most common types of cancer and has a very poor prognosis; thus, the development of effective therapies for the treatment of advanced HCC is of high clinical priority. In the present study, the anti-oncogenic effect of combined knockdown of c-Myc expression and ectopic restoration of deleted in liver cancer 1 (DLC1) expression was investigated in human liver cancer cells. Expression of c-Myc in human HCC cells was knocked down by stable transfection with a Myc-specific short hairpin (sh) RNA vector. DLC1 expression in Huh7 cells was restored by adenovirus transduction, and the effects of DLC1 expression and c-Myc knockdown on Ras homolog gene family, member A (RhoA) levels, cell proliferation, soft agar colony formation and cell invasion were measured. Downregulation of c-Myc or re-expression of DLC1 led to a marked reduction in RhoA levels, which was associated with decreases in cell proliferation, soft agar colony formation and invasiveness; this inhibitory effect was augmented with a combination of DLC1 transduction and c-Myc suppression. To determine whether liver cell-specific delivery of DLC1 was able to enhance the inhibitory effect of c-Myc knockdown on tumor growth in vivo, DLC1 vector DNA complexed with galactosylated polyethylene glycol-linear polyethyleneimine was administered by tail vein injection to mice bearing subcutaneous xenografts of Huh7 cells transfected with shMyc or control shRNA. A cooperative inhibitory effect of DLC1 expression and c-Myc knockdown on the growth of Huh7-derived tumors was observed, suggesting that targeted liver cell delivery of DLC1 and c-Myc shRNA may serve as a possible gene therapy modality for the treatment of human HCC. IntroductionHuman hepatocellular carcinoma (HCC) is one of the most common and lethal types of cancer, and its incidence is increasing worldwide (1,2). As numerous cases of HCC are diagnosed at later stages when the prognosis is poor, the development of effective therapy for advanced and metastatic HCC is a high clinical priority (1,2). HCC is a heterogeneous disorder with a complex pattern of genetic alterations, and increasing our knowledge of the specific genes and signaling pathways involved in the pathogenesis of HCC is critical for identifying novel clinical tools for diagnosis, classification and targeted therapy of the disease (3,4).Despite the heterogeneity of genomic alterations in HCC, certain chromosomes or chromosomal sites are more frequently deleted or amplified, resulting in deregulation of crucial genes that may ultimately trigger malignant transformation of healthy hepatocytes (5). Chromosome 8 exhibits a highly recurrent pattern of DNA copy number loss on the short arm (8p21-p22) and gain on the long arm (8q22-q24) (5,6), and these genetic alterations are associated with HCC subclasses with a less favorable outcome (7). The 8p and 8q sites correspond with the loci of the tumor suppressor gene deleted in liver cancer 1 (DLC1) and the c-Myc proto-oncogene, respectively (8...

ENOblock, a unique small molecule inhibitor of the non-glycolytic functions of enolase, alleviates the symptoms of type 2 diabetes

“…This phenomena has been observed for the small molecule, GAPDS, which increases the nuclear localization of the glycolysis enzyme, GAPDH, to regulate gene expression 33 . Suppression of the known enolase-binding gene, c-Myc, was not observed after ENOblock treatment in Huh7 hepatocytes, although this may be due to the cell line used in this study (Huh 7), which is a cancer line with dysregulated c-Myc regulation 34 . In support of this, expression of the known enolase-binding genes, c-Myc, Erbb2 and Cox-2, were all down regulated in the liver of ENOblock treated mice ( Fig.…”

A tumor suppressor DLC1: The functions and signal pathways

“…In vivo, DLC1 is a critical regulator of proliferation in endothelial cells of angiosarcoma, an endothelial cell-derived invasive malignancy (Sanchez-Martin et al, 2018). Ectopic restoration of the DLC1 expression inhibits the growth of Huh7 and NPC cells, and reduces tumorigenicity potential in nude mice (Yang, Zhou, Tone, Durkin, & Popescu, 2016). Furthermore, the colony formation rate indicates the independent viability of cells.…”