Endocrine Abstracts

Acquired resistance has limited the use of systemic molecular therapy in the management of hepatocellular carcinoma (HCC). Immunotherapy, alone and in combination with systemic molecular therapy, is now the first-line standard of care for HCC. The current in vitro study aims at investigating PER2 role in the HCC aggressiveness and drug resistance acquisition. Parental PLC/PRF/5, PLC/PRF/5 resistant to Everolimus (EveR) and to Sorafenib (SorR) cell lines were used. To evaluate PER2 involvement in aggressiveness and drug resistance, PER2 expression was genetically manipulated by using small interfering RNA (siRNA) to knockdown (KD) and CRISPR/Cas9 Plasmid to knockout (KO) PER2 gene in parental PLC/PRF/5. PER2 mRNA and protein expression were investigated by RT-qPCR and immunofluorescence (IF), respectively. The epithelial and mesenchymal transition (EMT) markers and TP53 expression was evaluated by IF and western blot, respectively. The role of PER2 on the onset of aggressiveness was evaluated through cell proliferation and cell migration assays. PLC/PRF/5 EveR, SorR, PER2KD and PER2KO significantly expressed lower mRNA PER2 levels, with a reduction of 72.7%, (P<0.01) in EveR, 59.6.% (P<0.05) in SorR, 91.5% (P<0.001) in PER2KD and 97.2% (P<0.001) in PER2KO compared to parental PLC/PRF/5. Remarkably, PER2 protein expression in PLC/PRF/5 EveR and SorR completely localized in cytoplasm contrary to parental ones. In PLC/PRF/5 EveR, PER2KD and PER2KO but not SorR, E-Cadherin was significantly decreased (24.9%, P=0.05; 33.8%, P=0.04; 55.5%, P=0.0002; +37.2%, P=0.05 respectively) while Vimentin protein expression was significantly increased in all cell models (+148.1%,+202.9%, +110.3%,+552.0%, P<0.0001 respectively). Interestingly, TP53 expression was reduced in PER2KO and completely absent in PLC/PRF/5 EveR and SorR. Significant cell proliferation inhibition of EVE10^-9M and SOR5×10^-6M in the parental PLC/PRF/5 (19.6%, P<0.0001 and 34.7%, P<0.05, respectively) was partially but not significantly reverted when PER2 was KD while it was completely reverted when PER2 was KO (P<0.01 vs EVE10^-9M and P<0.001 vs SOR5×10^-6M, respectively). Similarly, the significant inhibition of cell migration of EVE10^-9M and SOR5×10^-6M in the parental PLC/PRF/5 (20.7%, P<0.0001 and 8.6%, P<0.01, respectively) was reverted when PER2 was KD (P<0.0001 vs EVE10^-9M and vs SOR5×10^-6M, respectively), and when PER2 was KO (P<0.0001 vs EVE10^-9M and vs SOR5×10^-6M, respectively). These results suggest that the acquisition of aggressive phenotype is characterized by PER2 reduced expression and loss of nuclear translocation that in turn induces resistance to HCC systemic therapy. Further studies are mandatory to evaluate the impact of PER2 expression on immunotherapy efficacy in HCC improving patient’s management.