Gremlin in the pathogenesis of hepatocellular carcinoma complicating chronic hepatitis C: an immunohistochemical and PCR study of human liver biopsies

The variability in the prognosis of individuals with hepatocellular carcinoma (HCC) suggests that HCC may comprise several distinct biological phenotypes. These phenotypes may result from activation of different oncogenic pathways during tumorigenesis and/or a different cell of origin [1]. Enhanced proliferation and maturation arrest of hepatic progenitor/stem cells have been considered the origin of HCCs [1]. However, the link between cirrhosis and stem cell proliferation has not been fully elucidated. This link is multifaceted and complex with interplay of multiple factors that enhance or inhibit the proliferative rate of hepatic progenitor/stem cells, which culminates in HCC development. The outcome depends on the balance between stimulatory and regulatory signals that reach these stem cells [2, 3]. The fate of hepatic progenitor/stem cells is determined by various growth factors, some of which are promoters of stem cell differentiation, and their exit from the stem cell niche as bone morphogenic proteins (BMPs), whereas other growth factors, such as basic fibroblast growth factor (b- FGF) and activin/nodal (the other arm of action of Transforming growth factor β (TGF-β) maintain their stemness and promote pluripotency [4]. During embryogenesis, BMP-7 mediates sprouting of the liver bud from the central foregut endoderm, which is essential for hepatocytic differentiation [4]. BMP-7 protein has been found in inflamed fibrotic livers [5] and its mRNA has been detected in stellate and Kupffer cells of the adult liver [6]. Several proteins antagonize BMP signaling, such as follistatin, noggin and members of the DAN family including cerberus and gremlin [7]. Gremlin is a protein secreted by fibroblasts and its mRNA is highly expressed under fibrotic conditions such as advanced diabetic nephropathy [8].

In this study, we presumed that gremlin, secreted by hepatic myofibroblasts in cases of fibrosis (cirrhosis), would antagonize the effects of BMP on stem cell differentiation and its anti-fibrogenic property. Antagonism of BMP-7 may arrest stem cell maturation and promote proliferation in an immature state, thereby possibly culminating in HCC development.

We investigated the role of cirrhosis and FGF-2 in the pathogenesis of HCC development complicating chronic HCV by detection of gremlin and BMP-7 mRNA and correlating their presence with hepatic progenitor cell numbers in cases of cirrhosis and HCC complicating chronic HCV in Egyptian patients with HCV type 4.

Patient ages ranged from 23 to 52 (mean ± standard deviation (SD) 36 ± 8.7), and 39 to 70 (mean ± SD 53.67 ± 7.4) for hepatitis, and cirrhosis and HCC cases, respectively (cirrhosis and HCC cases were obtained from the same segmentectomy specimens).

Immunohistochemical results

Positive staining of CK19 in cell membranes was observed in both hepatitis and cirrhosis groups. Positivity was observed in and around portal tracts and along the fibrous septa with few intra-acinar positive cells (Figure 1a, b and c). Values were in the range of 0–10 (× 2.63 ± 2.7) and 0–90 (× 18 ± 23.48) for hepatitis and cirrhosis cases, respectively. Cirrhosis cases (Figure 1b) showed higher levels of positivity (p = 0.004) than hepatitis cases (Figure 1). All HCC cases (Figure 1c) were negative for CK19 (Table 2).

CK 19	Hepatitis	Cirrhosis	HCC
	(n=35)	(n=35)	(n=35)
Range	0.0 – 10.0	0.0 – 90.0	0.0 – 0.0
Mean ± SD	2.63 ± 2.70	18.0 ± 23.48	0.0 ± 0.0
Median	2.0	9.0	0.0
Z 1 (p)		2.866* (0.004)	4.305* (<0.001)
Z 2 (p)			4.665* (<0.001)

Z ₁ Z for the Mann–Whitney test between hepatitis and other groups.

Z ₂ Z for the Mann–Whitney test between cirrhosis and HCC groups.

*Statistically significant.

Among hepatitis cases, positive correlations were observed between each of CK19 expression and each grade (Figure 1d) and stage (Figure 1e) (r = 0.63, p = 0.009 and r = 0.56, p = 0.025, respectively). Correlation with the stage was not found in cirrhosis cases (fixed stage 6 Hepatitis activity index (HAI) by definition). However, correlation with the grade was detected (r = 0.753, p = 0.001). Ductular reactions correlated with CK19 expression in hepatitis cases (r = 0.42, p = 0.012) (Figure 1f).

Hepatitis and cirrhosis groups showed positive FGF-2 expression as granular cytoplasmic staining in periportal (Figure 2a), perivenular (Figure 2b) and intermediate (Figure 2c) hepatocytes as well as bile ductular epithelial cells. In HCCs, malignant hepatocytes also expressed FGF-2 (Figure 2d). FGF-2 positivity was higher in HCCs than in hepatitic and cirrhotic tissues, but was not significant (Table 3).

FGF 2	Hepatitis	Cirrhosis	HCC
	(n=35)	(n=35)	(n=35)
Range	0.060.0 – 16.0.0	0. 0.0 – 16.00 – 16.0	0.0 – 70.0
Mean ± SD	5.06 ± 4.30± 4.30	6.13 ± 5.0.0	14.67 ± 18.67
Median	4.544.50	4.04.0	9.0
Z1 (p)		.50.557(0.578)	1.308 (0.191)
Z2 (P)			0.937 (0.349)

Z ₁ Z for the Mann–Whitney test between HCV and other groups.

Z ₂ Z for Mann Whitney test between cirrhosis and HCC.

* Statistically significant at p ≤ 0.05.

FGF-2 correlated with each of grade (r = 0.603, p = 0.013) (Figure 3a, Table 4), stage (r = 0.721, p = 0.002) (Figure 3b, Table 4) and ductular reaction (r = 0.684, p = 0.004) (Figure 3c, Table 4) in hepatitis cases. In hepatitis and cirrhosis cases, positive correlations were detected between FGF-2 and CK19 positivity (r = 0.703, p = 0.002 (Figure 3d) and r = 0.536, p = 0.048, respectively) (Table 4).

FGF-2	Grade	Stage	Ductular reaction	CK19
HCV r	0.603 *	0.721 *	0.684 *	0.703 *
patients p	0.013	0.002	0.004	0.002
Cirrhotic r	0.446	0.388	0.483	0.536*
patients p	0.096	0.153	0.080	0.048

Rho spearman’s correlation coefficient.

*p < 0.05.

Gremlin expression and its correlations

Gremlin mRNA (198 bp) was expressed in 24/35 (68.7%) hepatitis cases (Figure 4a), 35/35 (100%) cirrhosis cases (Figure 4b), and 35/35 (100%) HCC cases (Figure 4c) (Table 5, Figure 4d). Gremlin mRNA levels were higher in HCCs and cirrhosis cases than in hepatitis cases (p = 0.009) (Table 5, Figure 4d).

Positive cases (%)	Hepatitis	Cirrhosis	HCC	MCp
	n (%)	n (%)	n (%)
Gremlin mRNA	24 (68.7%)	35 (100%)	35 (100%)	0.009*
BMP 7 mRNA	16 (43.8%)	27 (78.6%)	25 (73.3%)	0.115

n actual numbers of positive cases.

MCp p value for the Monte Carlo test.

*Statistically significant at p ≤ 0.05.

In hepatitis cases, gremlin expression correlated with the stage (r = 0.699, p = 0.003) (Figure 4e) and CK19 positivity (r = 0.223, p = 0.04) (Table 6), but not with the stage in the cirrhosis group due to the fixed stage (stage 6 HAI by definition). Gremlin expression did not correlate with the number of FGF-2 positive cells (r = 0.548, p = 0.191) or BMP-7 mRNA level (r = 0.051, p = 0.851) in the hepatitis group. However, it did positively correlate with BMP-7 levels in the cirrhosis group (r = 0.602, p = 0.023) (Table 6).

Gremlin	Grade	Stage	Ductular reaction	CK 19	BMP 7
HCV r	0.136	0.699 *	0.276	0.223	0.051
p	0.615	0.003	0.301	0.04	0.851
Cirrhosis
r	0.526		0.994		0.602
p	0.044	NP	0.001	NP	0.023

Rho (r) Spearman coefficient * Statistically significant at p ≤ 0.05.

NP Not performed because gremlin was expressed in 100% of cases and all 35 cases of this group received a score of 3 for CK19 positivity. Therefore, a correlation was not feasible.

Note: a correlation was not performed with the stage (fixed stage 6 by definition).

BMP-7 expression and its correlations

No significant differences in the level of BMP-7 mRNA expression between the three study groups were detected (Figure 5a–d and i, Table 5). However, BMP-7 mRNA correlated significantly with the levels of ductular reactions (r = 0.576, p = 0.031) in the cirrhosis group (Figure 5ii) (Table 7).

BMP 7	Grade	Stage	Ductular reaction	CK 19	FGF 2	Gremlin
HCV
r	−0.311	0.217	0.076	0.264	0.136	0.051
p	0.241	0.418	0.780	0.324	0.608	0.851
Cirrhosis
r	0.284	0.134	0.576 *	0.065	0.196	0.602*
p	0.326	0.635	0.031	0.825	0.501	0.023

Rho (r): Spearman coefficient.

* Statistically significant at p ≤ 0.05.

The current study aimed to identify a possible link between fibrosis/cirrhosis and HCC development in chronic HCV type 4 cases. Under normal conditions, FGFs, acting via BMP-7, stimulate maturation of stem cells into functional mature hepatocytes or cholangiocytes. However, under fibrotic conditions, gremlin is produced by fibrotic tissue [7, 8] and inhibits BMP function [7, 8]. This production of gremlin may cause maturation arrest of stem cells and their proliferation in an immature state predisposes HCC development.

We found that CK19 was expressed at higher levels in cirrhosis cases, compared with those of hepatitis cases. CK19 expression correlated with the grade and stage of the disease. These findings are consistent with data from Ravazoula et al [9] and Libbrecht et al [10]. The correlation between stem cell niche activation, manifested as significantly increased ductular reactions [11], and increased severity (grade/stage/fibrosis) of the disease, which were observed in the present study, confirms the notion that damage of the epithelial compartment of the liver by chronic disease causes replicative senescence of hepatocytes, which leads to reserve cell compartment activation [12]. Several studies using detailed immunophenotyping of HCCs show that a substantial number of HCCs (28–50%) express progenitor cell markers including CK7, CK19 and OV6 [13–15]. In the present study, although significantly higher numbers of CK19-positive cells were observed in cirrhosis cases, HCCs did not show CK19 positivity. This observation can be interpreted in two ways: first, expression of immune markers by stem cells is stage-specific and not lineage-specific [16]; and CK19 represents an early marker of stem cells, which is normally expressed in hepatoblasts [17]. In HCCs and chronic HCV type 4 cases, stem cells are probably at a different stage of maturation/differentiation than that required to express this particular marker. Alternatively, the negativity of our HCC cases for CK19 may be attributed to the clinical setting in which our study was conducted. Other studies have been performed using experimental models of carcinogenesis [18], or a different disease setting (etiologic factors other than HCV type 4) such as the studies by Durnez et al [15], Lyer et al [18] and others [16] where the etiology of liver disease was quite heterogeneous with only very few cases resulting from HCV infection. Our study was conducted exclusively with HCV type 4 induced HCCs; the main predisposing factor of HCC in Egypt [19]. The pathogenetic pathways activated in HCCs complicating HCV may be quite different from those occurring in other settings such as chemical carcinogenesis. In fact, even chemical carcinogenic regimens result in activation of different pathways. Lingala et al [20] show similar results using a different set of stem cell markers (CD44, CD90 and CD133) with few HCCs showing very limited expression of these markers, although they were found in cases of chronic HCV and dysplastic foci. Dunsford et al [21] used monoclonal antibodies to study sequential histopathologic changes that occurred during two regimens of chemical carcinogenesis in the rat. The Solt-Farber regimen caused prominent oval cell proliferation and multiple large dysplastic nodules, while continuous administration of diethylnitrosamine produced minimal oval cell proliferation and a few small nodules. These results demonstrate that different etiologies lead to HCC development via different pathogenetic pathways. Is it possible that different cancer stem cells are activated by different pathogenetic pathways, which results in a tumor (HCC) that is actually not a single entity, but biologically different tumors with a common morphology [22, 23].

Gremlin antagonizes BMP function under several disease conditions including idiopathic pulmonary fibrosis [24], proliferative vitreoretinopathy [25] and renal fibrosis [26]. It non-covalently binds to BMP family members to prevent interactions with their cognate receptors, thus altering the effective concentration of active signalling molecule [27]. There are also claims that gremlin possesses an oncogenic role. Data from Namkoong et al [28] and Sneddon et al [29] have shown that gremlin is over-expressed in various human malignancies including carcinomas of the cervix, lung, ovary, kidney, breast, colon and pancreas as well as sarcomas. The source of gremlin in highly cellular HCCs is Kupffer cells [30]. In the present study, all three groups of patients expressed gremlin mRNA that was significantly higher in cirrhosis cases than hepatitis cases. Furthermore, gremlin mRNA significantly correlated with the stage. This finding may support the theory that fibroblast-secreted gremlin blocks BMP-mediated stem cell maturation [31], with possible HCC development in cirrhosis patients.

We detected FGF-2 expression in all study groups, with a significant correlation noted between FGF-2 positivity and each grade and stage. FGF-2 is considered as an autocrine repair hormone secreted following injury to promote healing (fibrosis) [32, 33] and neoangiogenesis [34]. In our study, the distribution pattern of FGF-2 positivity in hepatitis cases is consistent with data from Xiaodong et al [35] who recently elucidated the localization of FGF-2 mRNA in these same regions in a mouse fibrotic liver model [35]. Furthermore, FGF-2 expression significantly correlated with CK19 expression, implying that promotion of fibrogenesis by FGF [36] leads to increased gremlin expression that blocks BMP-7-mediated stem cell differentiation, which leads to proliferation in an immature state, as shown by higher CK19 positivity. This explanation is further supported by our finding of a positive correlation between gremlin mRNA expression, FGF-2 and CK19 positivity in the hepatitis group. Thus, FGF-2 may have an oncogenic role in HCC development.

Gremlin participates in the pathogenesis of HCC complicating chronic hepatitis C type 4-related cirrhosis by antagonizing the maturation inducing function of BMP-7 rather than the actual production of BMP-7 in cirrhotic livers. This occurrence promotes the proliferation of stem cells in an immature state, which may contribute to HCC carcinogenesis.