CCATT/enhancer binding protein alpha (CEBPA) is a transcription factor that can act as a tumor suppressor. Its expression is downregulated in a number of cancers including hepatocellular carcinoma (HCC). We have investigated the role of CEBPA in models of HCC using small activating RNAs (saRNA) to transcriptionally upregulate its expression.
Three liver tumor cell lines (HepG2, Hep3B, PLCPRF5) were transfected with 20nM CEBPA-saRNAs (CEBPA51). The transcriptional regulation of two key members of the CEBP family: CEBPA and CEBPB and their protein expression level were measured. The impact on cell growth was assessed by way of an SRB and WST-1 assay. To investigate the role of CEBPB in protecting cells from the activity of CEBPA, siRNAs were used to knock down CEBPB. Using i.v. delivery of CEBPA51 oligonucleotide, the impact on tumor growth was investigated in a DEN (N-nitrosodiethylamine) model of liver cancer. Rats were treated with DEN for 7 weeks, followed by a 2 week wash out and then treated with the CEBPA51 (3-4mg/kg) complexed with PAMAM-dendrimers or encapsulated in a nanoparticle formulation (SMARTCLES). The impact on CEBPA mRNA levels in the liver, tumor growth and liver functions (including, bilirubin, ALT and AST) were measured.
Transfection of CEBPA51 into HepG2, Hep3B or PLCPRF5 cells after 72hr led to a significant increase in both CEBPA mRNA (1.7-2.5 fold by qPCR) and protein expression measured by western blot in all 3 cell lines. A significant inhibition in cell growth compared to either PBS or control oligonucleotide was observed in HepG2 and Hep3B but not in PLCPRF5 cells measured by both SRB and WST-1 assays. The levels of CEBPB mRNA and protein, which may act as an antagonist of CEBPA, were found to be higher (1.4-2 fold) in PLCRF5 cells compared to HepG2 and Hep3B cells. Co-transfection of PLCPRF5 cells with siRNAs to CEBPB and CEBPA51 saRNA led to downregulation of CEBPB and senstized the PLCPRF5 cells to growth inhibition by CEBPA51. Administration of CEBPA51 complexed to either dendrimers or encapsulated in SMARTCLES nanoparticle at doses of 3-4mg/kg i.v. over 2 weeks led to a significant elevation of CEBPA mRNA in the liver. This was accompanied by a reduction (80-90%) in the size of DEN induced liver tumour nodules compared to a control oligonucleotide using both delivery vehicles. The antitumor effects following treatment with CEBPA51 using both delivery vehicles were also accompanied by reduction in markers of liver injury (bilirubin, ALT and AST).
These studies support an important role for CEBPA in suppressing progression of HCC. Activation of the CEBPα gene by saRNA leading to restoration of CEBPA levels in the liver represents a promising novel approach for inhibiting the growth of HCC whilst improving normal liver function. The SMARTCLES formulation, MTL-CEBPA, was chosen for clinical development and is currently in a Phase 1 trial in patients with liver cancer (NCT02716012).
Citation Format: Nagy Habib, Vikash Reebye, Xiaoyang Zhao, Jon Voutila, Robert Habib, Pål Sætrom, Hans Huber, Kai-Wen Huang, John J. Rossi, David C. Blakey. MTL-CEBPA activates the transcription factor CEBPalpha leading to inhibition of hepatocellular cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1508. doi:10.1158/1538-7445.AM2017-1508