Telomere length, TERT and shelterin complex proteins in hepatocellular carcinomas expressing “stemness”-related markers

Abstract

Hepatocellular carcinomas (HCCs) expressing "stemness"-related markers have been associated with aggressive biological behavior and poor prognosis. We examined the relationship between "stemness"-related protein expression and telomere length, hTERT and shelterin complex protein expression and chromosomal instability. Quantitative fluorescent in situ hybridization for telomere length, immunohistochemistry for K19, EpCAM, CD133, c-kit, HepPar1, hTERT, TRF1, TRF2, POT1, RAP1 and TPP1, and TUNEL assay were performed in 137 HCCs, and array comparative genomic hybridization was performed with 24 HCCs. Telomeres were significantly longer in HCCs expressing "stemness"-related proteins (K19: p < 0.001, EpCAM: p = 0.002, CD133: p = 0.002). On analyzing different tumor cells within EpCAM-expressing HCCs, EpCAM-positive tumor cells showed longer telomeres (1.329 ± 0.246) compared to EpCAM-negative tumor cells (0.996 ± 0.381) within the same HCCs (p = 0.031). Telomeres were significantly longer in HCCs expressing hTERT (p = 0.048) and RAP1 proteins (p = 0.031). K19-expressing HCCs expressed hTERT (p = 0.002), TRF2 (p = 0.001) and TPP1 (p = 0.013) more frequently compared to K19-negative HCCs. EpCAM-positivity was associated with more frequent hTERT (p = 0.028), TPP1 (p = 0.017), TRF2 (p = 0.027) and POT1 (p = 0.004) expression. Copy number alterations were more frequent in K19 and EpCAM-expressing HCCs compared to HCCs without these markers (K19: p = 0.038, EpCAM: p = 0.009). HCCs with longer telomeres were associated with a shorter overall (p = 0.019) and disease-free survivals (p = 0.049), and decreased disease-free survivals were seen in TRF2-positive HCCs (p = 0.018). HCCs expressing "stemness"-related proteins are characterized by increased telomere length, increased expression of hTERT and shelterin complex proteins, and increased chromosomal instability compared to conventional HCCs. Longer telomeres and TRF2 expression in HCCs are associated with poor patient outcomes.