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Identification of keratin 19-positive cancer stem cells associating human hepatocellular carcinoma using CYFRA 21-1.

ABSTRACT: The current lack of an easily measurable surrogate marker of cancer stem cells (CSCs) prevents the clinical application of CSCs for hepatocellular carcinoma (HCC). We previously reported that keratin 19 (K19) is a novel HCC-CSC marker associated with transforming growth factor beta (TGFβ)/Smad signaling, and that K19⁺ HCC-CSCs could be a new therapeutic target of TGFβ receptor 1 inhibitor LY2157299. In this study, we examined whether K19⁺ HCC-CSCs can be tracked using cytokeratin 19 fragment CYFRA 21-1. In 147 HCC patients who underwent curative resection and evaluated K19 expression by immunohistochemistry, preoperative serum CYFRA 21-1 levels were significantly higher in K19⁺ patients than in K19^- patients (P < 0.01). Receiver operating characteristic analyses revealed that serum CYFRA 21-1 was the statistically significant and the most sensitive predictor of tumor K19 expression among preoperative laboratory test values (P < 0.001). In HCC cells encoding with a K19 promoter-driven enhanced green fluorescent protein, fluorescence-activated cell sorting (FACS)-isolated K19⁺ cells displayed significantly higher levels of supernatant CYFRA 21-1 than K19^- cells (P < 0.01). Gain/loss of K19 function experiments confirmed that CYFRA 21-1 levels were regulated by K19 function in HCC cells. Furthermore, CYFRA 21-1 levels reflected the treatment efficacy of LY2157299 in K19⁺ cells. In conclusion, CYFRA 21-1 can be used to predict K19 expression in HCC, and should thereby aid in the development of novel therapeutic strategies targeting K19⁺ HCC-CSCs.

SUBMITTER: Kawai T

PROVIDER: S-EPMC5673926 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectiveKeratin 19 (K19) expression is a potential predictor of poor prognosis in patients with hepatocellular carcinoma (HCC). To clarify the feature of K19-proficient HCC, we traced epigenetic footprints in cultured cells and clinical materials.Patients and methodsIn vitro, KRT19 promoter methylation was analyzed and 5-aza-2'-deoxycytidine with trichostatin A (TSA) treatment was performed. Among 564 surgically resected HCCs, the clinicopathological relevance of K19-proficent HCCs was performed in comparison with hepatocytic (HepPar-1 and arginase-1), epithelial-mesenchymal transition (E-cadherin and vimentin), biliary differentiation-associated (K7 and NOTCH-1) markers, and epigenetic markers (KRT19 promoter/long interspersed nucleotide element-1 [LINE-1] methylation status).ResultsKRT19 promoter methylation was clearly associated with K19 deficiency and 5-aza-2'-deoxycytidine with TSA treatment-stimulated K19 re-expression, implicating DNA methylation as a potential epigenetic process for K19 expression. After excluding HCCs with recurrence, TNM stage as IIIB or greater, preoperative therapy, transplantation, and combined hepatocellular cholangiocarcinoma, we assessed 125 of 564 HCC cases. In this cohort, K19 expression was found in 29 HCCs (23.2%) and corresponded with poor survival following surgery (p = 0.025) and extrahepatic recurrence-free survival (p = 0.017). Compared with K19-deficient HCCs, lower KRT19 promoter methylation level was observed in K19-proficient HCCs (p < 0.0001). Conversely, HCC with genome-wide LINE-1 hypermethylation was frequently observed in K19-proficient HCCs (p = 0.0079). Additionally, K19 proficiency was associated with K7 proficiency (p = 0.043), and reduced E-cadherin and HepPar-1 expression (p = 0.043 and p < 0.0001, respectively).ConclusionsK19-proficient HCC exhibited poor prognosis owing to extrahepatic recurrence, with molecular signatures differing from those in conventional cancer stem cells, providing novel insights of the heterogeneity underlying tumor development.

Project description:Human hepatocellular carcinomas (HCCs) expressing the biliary/hepatic progenitor cell marker keratin 19 (K19) have been linked with a poor prognosis and exhibit an increase in platelet-derived growth factor receptor ? (PDGFR?) and laminin beta 1 (LAMB1) expression. PDGFR? has been reported to induce de novo synthesis of LAMB1 protein in a Sjogren syndrome antigen B (La/SSB)-dependent manner in a murine metastasis model. However, the role of this cascade in human HCC remains unclear. This study focused on the functional role of the PDGFR?-La/SSB-LAMB1 pathway and its molecular link to K19 expression in human HCC. In surgical HCC specimens from a cohort of 136 patients, PDGFR? expression correlated with K19 expression, microvascular invasion and metastatic spread. In addition, PDGFR? expression in pre-operative needle biopsy specimens predicted poor overall survival during a 5-year follow-up period. Consecutive histological staining demonstrated that the signaling components of the PDGFR?-La/SSB-LAMB1 pathway were strongly expressed at the invasive front. K19-positive HCC cells displayed high levels of ?2?1 integrin (ITG) receptor, both in vitro and in vivo. In vitro activation of PDGFR? signaling triggered the translocation of nuclear La/SSB into the cytoplasm, enhanced the protein synthesis of LAMB1 by activating its internal ribosome entry site, which in turn led to increased secretion of laminin-111. This effect was abrogated by the PDGFR?-specific inhibitor crenolanib. Importantly LAMB1 stimulated ITG-dependent focal adhesion kinase/Src proto-oncogene non-receptor tyrosine kinase signaling. It also promoted the ITG-specific downstream target Rho-associated coiled-coil containing protein kinase 2, induced K19 expression in an autocrine manner, invadopodia formation and cell invasion. Finally, we showed that the knockdown of LAMB1 or K19 in subcutaneous xenograft mouse models resulted in significant loss of cells invading the surrounding stromal tissue and reduced HepG2 colonization into lung and liver after tail vein injection. The PDGFR?-LAMB1 pathway supports tumor progression at the invasive front of human HCC through K19 expression.

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Identification of keratin 19-positive cancer stem cells associating human hepatocellular carcinoma using CYFRA 21-1.

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