Increased Expression of CCN2, Epithelial Membrane Antigen, and Fibroblast Activation Protein in Hepatocellular Carcinoma with Fibrous Stroma Showing Aggressive Behavior

Abstract

Tumor behavior is affected by the tumor microenvironment, composed of cancer-associated fibroblasts (CAFs). Meanwhile, hepatocellular carcinomas (HCC) with fibrous stroma reportedly exhibit aggressive behavior suggestive of tumor-stroma interaction. However, evidence of the crosstalk remains unclear. In this study, CCN2, epithelial membrane antigen (EMA), fibroblast activation protein (FAP), and keratin 19 (K19) expression was studied in 314 HCCs (cohort 1), 42 scirrhous HCCs (cohort 2), and 36 chronic hepatitis/cirrhosis specimens by immunohistochemistry. Clinicopathological parameters were analyzed according to the expressions of these markers. In tumor epithelial cells from cohort 1, CCN2 and EMA were expressed in 15.3% and 17.2%, respectively, and their expressions were more frequent in HCCs with fibrous stroma (≥5% of tumor area) than those without (P<0.05 for all); CCN2 expression was well correlated with K19 and EMA expression. In tumor stromal cells, FAP expression was found in 6.7%. In cohort 2, CCN2, EMA, and FAP expression was noted in 40.5%, 40.5%, and 66.7%, respectively, which was more frequent than that in cohort 1 (P<0.05 for all). Additionally, EMA expression was associated with the expression of K19, CCN2, and FAP (P<0.05 for all); EMA expressing tumor epithelial cells showed a topographic closeness to FAP-expressing CAFs. Analysis of disease-free survival revealed CCN2 expression to be a worse prognostic factor in both cohort 1 (P = 0.005) and cohort 2 (P = 0.023), as well as EMA as a worse prognostic factor in cohort 2 (P = 0.048). In conclusion, expression of CCN2, EMA, and FAP may be involved in the activation of CAFs in HCC, giving rise to aggressive behavior. Significant correlation between EMA-expressing tumor cells and FAP-expressing CAFs and their topographic closeness suggests possible cross-talk between tumor epithelial cells and stromal cells in the tumor microenvironment of HCC.

Fibroblast activation protein‐α, a stromal cell surface protease, shapes key features of cancer associated fibroblasts through proteome and degradome alterations

“…These finding corroborate stromal fibroblast expression of FAPα in colorectal cancers and are in line with previous reports, including several tumor entities (Scanlan et al., 1994; Cheng et al., 2002; Huber et al., 2003; Henry et al., 2007; Shan et al., 2012; Yang et al., 2013a; Kim et al., 2014a; Mhawech‐Fauceglia et al., 2015).…”

Tumour epithelial and stromal characteristics of hepatocellular carcinomas with abundant fibrous stroma: fibrolamellar versus scirrhous hepatocellular carcinoma

“…The tumour microenvironment has been receiving increasing attention, as there is accumulating evidence that the tumour stroma is an important component of epithelial neoplasms, affecting the biological behaviour of tumours and the tumorigenetic process. The tumour stroma is composed of CAFs, immune cells including tumour‐associated macrophages, extracellular matrix proteins and blood vessels, and there is increasing evidence for cross‐talk between tumour cells and the peritumoral stroma in various malignancies, including HCCs …”

“…We have demonstrated previously that S‐HCCs are more aggressive compared to typical HCCs, with increased expression of stemness‐related and epithelial–mesenchymal transition‐related markers and activation of the transforming growth factor (TGF)‐β signature . In addition, we also found that the presence of FAP‐positive CAFs in the fibrous stroma of S‐HCCs was associated with frequent vascular invasion, and that CCN2 (a fibrogenic cytokine which mediates fibrotic processes including desmoplastic reactions in cancer) was expressed more frequently in HCCs with fibrous stroma and correlated significantly with K19 expression …”

“…The tumour stroma is composed of CAFs, immune cells including tumour-associated macrophages, extracellular matrix proteins and blood vessels, and there is increasing evidence for cross-talk between tumour cells and the peritumoral stroma in various malignancies, including HCCs. [8][9][10] While HCC is characterized usually by absent or minimal amounts of fibrous stroma, unlike many other epithelial malignancies, there are less common subtypes of HCC that demonstrate abundant fibrous stroma, the most well-known ones being FL-HCC and S-HCC. Although both these subtypes demonstrate abundant intratumoral fibrous stroma, they show clinicopathological differences.…”

“…3 In addition, we also found that the presence of FAP-positive CAFs in the fibrous stroma of S-HCCs was associated with frequent vascular invasion, and that CCN2 (a fibrogenic cytokine which mediates fibrotic processes including desmoplastic reactions in cancer) was expressed more frequently in HCCs with fibrous stroma and correlated significantly with K19 expression. 8 In order to take a closer look into the tumour microenvironment of HCCs with abundant fibrous stroma, we studied two separate cohorts of S-HCCs and FL-HCCs, and analysed the expression status of FAP, CD68 and CD163 in the intratumoral stroma as markers of CAFs and tumour-infiltrating macrophages, respectively, and the expression of CCN2 in the tumour epithelial cells, in addition to stemnessrelated markers, K7, K19, EpCAM and CD56/NCAM. As there are a limited number of clinicopathological studies on FL-HCCs so far, especially regarding the tumour microenvironment of FL-HCC, we were interested to see whether there were any differences in the intratumoral fibrous stroma between the two tumours.…”