Research Article: Progressive Enrichment of Stemness Features and Tumor Stromal Alterations in Multistep Hepatocarcinogenesis

Date Published: January 23, 2017

Publisher: Public Library of Science

Author(s): Jeong Eun Yoo, Young-Joo Kim, Hyungjin Rhee, Haeryoung Kim, Ei Yong Ahn, Jin Sub Choi, Massimo Roncalli, Young Nyun Park, Taro Yamashita.


Cancer stem cells (CSCs), a subset of tumor cells, contribute to an aggressive biological behavior, which is also affected by the tumor stroma. Despite the role of CSCs and the tumor stroma in hepatocellular carcinoma (HCC), features of stemness have not yet been studied in relation to tumor stromal alterations in multistep hepatocarcinogenesis. We investigated the expression status of stemness markers and tumor stromal changes in B viral carcinogenesis, which is the main etiology of HCC in Asia. Stemness features of tumoral hepatocytes (EpCAM, K19, Oct3/4, c-KIT, c-MET, and CD133), and tumor stromal cells expressing α-smooth muscle actin (α-SMA), CD68, CD163, and IL-6 were analyzed in 36 low grade dysplastic nodules (DNs), 48 high grade DNs, 30 early HCCs (eHCCs), and 51 progressed HCCs (pHCCs) by immunohistochemistry or real-time PCR. Stemness features (EpCAM and K19 in particular) were progressively acquired during hepatocarcinogenesis in combination with enrichment of stromal cells (CAFs, TAMs, IL-6+ cells). Stemness features were seen sporadically in DNs, more consistent in eHCCs, and peaked in pHCCs. Likewise, stromal cells were discernable in DNs, showed up as consistent cell densities in eHCCs and peaked in pHCCs. The stemness features and tumor stromal alterations also peaked in less differentiated or larger HCCs. In conclusion, progression of B viral multistep hepatocarcinogenesis is characterized by an enrichment of stemness features of neoplastic hepatocytes and a parallel alteration of the tumor stroma. The modulation of neoplastic hepatocytes and stromal cells was at low levels in precancerous lesions (DNs), consistently increased in incipient cancer (eHCCs) and peaked in pHCCs. Thus, in B viral hepatocarcinogenesis, interactions between CSCs and the tumor stroma, although starting early, seem to play a major role in tumor progression.

Partial Text

Cancer stem cells (CSCs), a subset of tumor cells, exhibit the ability to self-renew and initiate (promote) tumor formation, and they also contribute to rapid tumor growth and chemoresistance [1]. Reportedly, hepatocellular carcinomas (HCCs) with stemness features, which express hepatic stem cell (HSC) markers, such as keratin19 (K19), CD133, or epithelial cell adhesion molecule (EpCAM), are associated with a higher incidence of vascular invasion and poorer prognosis, compared to HCCs lacking these markers [2, 3].

HCCs expressing stemness-related markers, such as K19, CD133, or EpCAM, are reportedly associated with a higher incidence of vascular invasion and a poorer prognosis, compared to HCCs that do not express these markers [2, 3]. However, whether CSCs (cancer stem cell features) are present in the early stages of human multistep hepatocarcinogenesis, including precancerous lesions, has yet to be clarified. In this study, we discovered a significant increase in the expression of stemness features with progression of B viral multistep hepatocarcinogenesis: cancer stemness features were seen sporadically in DNs, more consistent in eHCCs, and peaked in pHCCs. The expression levels of stemness features were also higher in less differentiated or larger HCCs. Taken together, these data suggest that the enrichment of stemness features takes place during B viral multistep hepatocarcinogenesis, with greater expansion in (more) progressed HCCs and thus in the more advanced phases of tumorigenesis. Recently, mature hepatocytes were found to exhibit unexpected plasticity upon direct de-differentiation to progenitor cells in culture [19], and complementary fate-tracing approaches that are able to label progenitor/biliary compartments and hepatocytes demonstrated that K19 and α-fetoprotein-positive cells within HCCs are hepatocyte-derived in murine hepatocarcinogenesis [20]. Similarly, K19 positivity was reported to be acquired with cancer progression in rat hepatocarcinogenesis [21]. Taken together, we speculate that CSCs of HCC are more likely (phenotypical features acquired from) to be derived from de-differentiated malignant hepatocytes that are selected and enriched during tumor progression. Although CSCs of HCC may possibly originate from pre-existing and transformed hepatic progenitor cells, this might be a minor pathway.