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Ⅲ-1-1. High expression level of COL6A3 in human HCC patients is associated with worsened outcomes
To explore the role of endotrophin in CLD, COL6A3 levels were assessed in HCC patients by analyzing the resources from public databases. It was revealed that mutations of COL6A3 in HCC patients resulted in poor survival compared to those without COL6A3 mutations, suggesting that COL6A3 is critically involved in HCC development (Figure III-1-1A). Moreover, we found higher level of COL6A3 in tumor regions, compared to that of paired nontumor regions in liver tissue specimens from HCC patients (Figure III-1-1B). Kaplan-Meier survival analysis revealed that COL6A3 levels in the neighboring nontumor liver tissue significantly discriminated overall survival of the HCC patients whereas COL6A3 levels in tumor tissues did not, revealing that high level of COL6A3 in nontumor regions was associated with poor outcomes in HCC patients (Figure III-1-1C, D). To further explore the prognostic relevance of endotrophin, gene expression profile of HCC patient, obtained from the GEO database, was analyzed.
It was found that there are positive correlations between COL6A3 levels and inflammation/fibrosis marker gene levels in tumor tissues from HCC patients and nontumor regions, as well (Figure III-1- 1E-R). Endotrophin levels were analyzed by IHC in liver tissue from HCC patients, revealing that endotrophin was abundantly expressed in tumors regions (12 out of 15, staining score greater than two) and, to a lesser extent, in paired nontumor liver tissues (5 out of 10, staining score greater than two).
However, endotrophin was barely observed in control liver tissues (1 out of 15, staining score greater than two) (Figure III-1-1S, T). Those results suggest that COL6A3/endotrophin in liver tissues could predict the outcome of the HCC patients, and thus act as promising prognostic marker for HCC.
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Figure III-1-1. COL6A3 is overexpressed and associated with poor prognosis in HCC.
(A) Kaplan-Meier survival analysis in HCC patients with (n = 18) and without (n = 343) COL6A3 gene alterations using the resources from the cBioPortal for Cancer Genomics. (B) COL6A3 levels in tumors and matched nontumor liver tissues as assessed by upper quantile normalized RNA-seq FPKM (FPKM- UQ) of HCC patient cohorts (n = 49) from the GDC portal and TCGA database. (C, D) Kaplan–Meier survival analysis in HCC patients from TCGA database. Subjects were grouped by (C) low (n = 12) or high (n = 38) COL6A3 levels in nontumor tissues or (D) low (n = 222) versus high (n = 149) COL6A3 levels in tumor tissues. (E-R) Correlations between COL6A3 and inflammation and fibrosis marker genes were assessed by linear regression analysis in tumor (E–K) and nontumor regions (L–R). (S) Immunostaining of endotrophin in liver tissues from control, patients-matched paired nontumor, and HCC tumors tissues (n = 10–15/group). Endotrophin staining scores were evaluated on the basis of intensity and positive cell proportions (Endotrophin = 0, no staining; endotrophin = 1, only partial staining in >10% of cells; endotrophin = 2, weak to moderate complete staining in >10% of cells;
endotrophin = 3, strong and complete staining in >10% of cells). (T) Representative images in liver tissues (Scale: 100 μm). Mean ± standard error of the mean (SEM). Statistical significance was evaluated by unpaired two-tailed Student's t-test or log-rank test (for survival probability). *p < 0.05;
**p < 0.01; ***p < 0.001.
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Ⅲ-1-2. Establishment of inducible and liver-specific endotrophin overexpression model
To investigate the direct effect of endotrophin in CLD, we established transgenic mouse line (Alb-ETP) that enables liver-specific endotrophin overexpression in dox inducible manner (Figure III-1-2A) [17].
We found that liver-specific endotrophin transgene expression was induced following 1week of Dox- chow feeding (Figure III-1-2B) and Col6a3 levels were found to be increased in the liver samples from Alb-ETP mice compared to that of control group (Figure III-1-2C). However, liver-specific endotrophin expression did not result in prominent changes in liver morphology and fibrotic or inflammatory gene expression profile was comparable between two genotypes (Figure III-1-2D-F).
We next assessed the effect of chronic exposure of endotrophin on liver tissues by feeding the Dox- chow for 4 or 32 weeks. H&E staining revealed that no macroscopically apparent changes were observed in liver tissue from the both time point (Figure III-1-2G, H). Similarly, there were no apparent differences in inflammation, fibrosis, and vessel formations in liver samples from Alb-ETP compared to those of control group after four weeks of Dox-chow administration, as determined by the Mac2 staining (inflammation marker), αSMA and Masson's trichrome C staining (fibrosis marker) and endomucin staining (angiogenesis marker), respectively (Figure III-1-2I-L). Although fibrotic marker gene expression levels were upregulated in the liver tissue samples from 32 weeks of long-term Dox feeding, Masson's trichrome C staining did not reveal apparent changes in hepatic fibrosis between the genotypes (Figure III-1-2M, N). Furthermore, inflammation, cell proliferation, and apoptosis marker gene expression levels were not significantly changed by endotrophin overexpression in liver tissue (Figure III-1-2O, P), and also there were no significant differences in total body weight and liver to body weight ratio between the genotype (Figure III-1-2Q, R). Collectively, those results indicate that hepatic overexpression of endotrophin alone does not trigger liver pathogenesis, without any additional stress.
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Figure III-1-2. Liver specific inducible ETP transgenic mouse model.
(A) Graphical view of Alb-ETP mice model generation. Introduction of prolactin signal sequence at N- terminus of the endotrophin allowed efficient expression and secretion of endotrophin into extracellular space in TRE-endotrophin transgenic mice. In Alb-Cre and Rosa-rtTA and TRE-endotrophin crossed transgenic mice, TRE promoter is operated by rtTA, to provide hepatocyte-specific expression of endotrophin. The Cre recombinase driven by Albumin promoter removes stop sequence, and subsequently rtTA gene expression is enabled. Upon doxycycline (Dox) administration to the Alb-ETP
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mice model, endotrophin expression could be induced in liver tissue. (B) Endotrophin transgene expression was analyzed in various tissues from either littermate control or Alb-ETP mice, treated with Dox-diet (600mg/kg) for 7 days. (C) The mRNA levels for ETP transgene by using primer sets detecting C5 region of Col6a3. (D) H&E staining of liver tissues from either control or Alb-ETP mice treated with Dox-diet (600 mg/ kg) for five days (Scale bar: 50 μm). (E) Inflammation and (F) fibrosis marker gene expression levels in liver tissues from either control or Alb-ETP mice treated with Dox-diet (600 mg/ kg) for 7 days. (G, I-L) Both control and Alb-ETP groups were treated with 4 weeks of dox-diet (600 mg/kg), and histological analyses were performed in their liver tissues. (G) H&E staining, (I) Mac- 2 immunostaining, (J) Masson’s trichrome C staining, (K) α-SMA immunostaining, and (L) Endomucin immunostaining. (Scale bar: 50 μm). (H, M-R) Alb-ETP and control mice were treated with 32 weeks of dox-diet (600 mg/kg), and their liver tissues were analyzed. (H) H&E, and (M) Masson’s trichrome C staining revealed no differences between the two genotypes (Scale bar: 50 μm).
(N) Fibrosis, (O) inflammation, and (P) survival/apoptosis marker gene expression levels were analyzed by RT-qPCR. (Q) Total body weight or (R) liver to body weight ratio were determined and revealed no significant changes between the genotypes. Data are presented as the mean ± SEM. Statistical significances were analyzed by unpaired two-tailed Student’s t-test. (*p < 0.05; **p < 0.01; ***p < 0.001;) n.s., not significant.
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Ⅲ-1-3. Liver-specific endotrophin overexpression aggravates CCl4-driven liver damages
To assess the role of endotrophin under pathological condition, we established a CCl4-driven hepatic fibrosis model [18]. Wildtype C57BL/6J mice were administered with either vehicle or CCl4 two times in a week for one month. H&E and Trichrome C analysis of liver tissue revealed that most of the mice developed hepatic fibrosis, apoptosis, and inflammation (Figure III-1-3A, B), and similar results were found in mRNA levels (Figure III-1-3C–E). Importantly, both mRNA and protein levels of Col6a3, which encodes the precursor of endotrophin, were elevated in liver tissue from CCl4 injected mice (Figure III-1-4A, B). Thus, we investigated whether the endotrophin is directly involved in the pathological response in CLD. Alb-ETP and their littermate control mice were given liver injury, induced by one month of CCl4 administration, and we assessed the effect of endotrophin in early event of hepatic fibrosis development. Endotrophin levels were elevated in liver tissue form CCl4 treated mice, which was further increased in Alb-ETP mice (Figure III-1-4C). H&E staining revealed that liver tissue from Alb-ETP exhibited elevated number of ballooned hepatocytes, and irregular structures close to the portal veins compared to that of control mice. Furthermore, Alb-ETP mice developed several septa formation with piecemeal necrosis nearby the portal area while the control group had moderate fibrosis without septa formations within liver tissue (Figure III-1-4D). Hepatic apoptosis was elevated in liver tissues from Alb-ETP mice group compared to control, as assessed by cleaved caspase 3 staining (Figure III-1-4E). Likewise, fibrosis and inflammation were augmented in liver tissue samples from Alb-ETP group, as assessed by αSMA, Masson's trichrome C, and Mac2 staining (Figure III-1-4F-H).
Those results suggest that hepatic apoptosis is likely to be the leading event, mostly affected by endotrophin at an early process of CLD. In addition, endotrophin could promote primary liver damage to deteriorate disease progressions, such as inflammation, fibrosis, and hepatic apoptosis whereas endotrophin alone is not sufficient to trigger advanced progression.
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Figure III-1-3. CCl4-induced liver injury develops inflammation, apoptosis, and fibrosis.
Either CCl4 or vehicle were administered to wildtype male mice twice in a week for one month, and their liver samples were analyzed. (A) H&E and (B) Masson’s trichrome C staining. (Scale bar: 200 μm). (C) Inflammation, (D) apoptosis, and (E) fibrosis marker gene expression levels were analyzed by qPCR Data represented the mean ± S.E.M. Statistical significance was evaluated by unpaired two-tailed Student’s t-test. *p < 0.05; **p < 0.01
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Figure III-1-4. Endotrophin facilitates CCl4-driven chronic liver damages in vivo.
(A, B) Either CCl4 or vehicle were administered to wildtype male mice twice in a week for 4 weeks.
(A) Gene expression levels of Col6a1, Col6a2, and Col6a3 in liver tissues were analyzed by qPCR and (B) Protein levels were assessed by western blot analysis. (C–H) Alb-ETP and control mice were treated with 5 days of dox-diet (600 mg/kg) and subsequently administered with CCl4 every five days for 4 weeks. Mice were dissected three days after final CCl4 treatment. (C) Endotrophin staining, (D) H&E staining (scale: 50 μm), (E) CC3 staining (scale: 20 μm), (F) αSMA staining, (G) trichrome C staining, and (H) Mac-2 staining (scale: 50 μm). Staining positive areas were quantified and are presented as the mean ± SEM. Statistical significance was evaluated by unpaired two-tailed Student's t-test. *p < 0.05;
**p < 0.01.
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Ⅲ-1-4. Endotrophin exacerbates cancer progression in DEN-induced HCC model
As the HCC is predominantly developed under CLD settings, we next tested DEN-induced liver cancer model [18, 19]. Most of the mice exhibited tumor development in liver tissues, where the Alb-ETP mice had increased tumor size and number compared to control (Figure III-1-5A). H&E staining revealed that both genotypes had severe liver damages, with more tumor regions being appeared in Alb-ETP group compared to control mice (Figure III-1-5B). Cancer incidence was slightly increased in Alb-ETP mice and they had significantly increased number of primary tumor nodules in liver tissues (Figure III- 1-5C-D). Immunostaining of liver tissue showed that Alb-ETP mice had elevated proportion of Ki-67 positive cells, especially near tumor lesions, compared to control mice (Figure III-1-5E). Consistently, Alb-ETP mice had increased level of AFP, a liver cancer marker, in serum (Figure III-1-5F). At the same time, hepatic apoptosis was also assessed by immunostaining of CC3, revealing that Alb-ETP mice had augmented apoptotic cells, particularly in non-tumor region of the liver compared to the control mice (Figure III-1-5G). Similar results were found in Ki-67 combined with TUNEL staining assay (Figure III-1-5H). In a transcript level, Bad and Bax were significantly elevated in Alb-ETP mice while Bcl2 and Birc5 levels were not changed between the genotypes (Figure III-1-5I). Several inflammation and fibrosis marker gene expressions were increased in liver tissues from Alb-ETP mice compared to those of controls (Figure III-1-5J, K). Immunostaining of αSMA in liver tissue also showed that Alb-ETP mice had elevated αSMA positive area compared to that of control mice (Figure III-1-5L). Notably, mRNA levels of the inflammation and fibrosis marker gene were positively correlated with Col6a3 level in nontumor regions of liver tissues from wildtype mice (Figure III-1-6A- G), but not in tumor region (Figure III-1-6H-N). Those results suggest that endotrophin overexpression facilitate hepatic apoptosis, inflammation, and fibrosis mostly in nontumor regions, where the tumor- prone niche is established within the hepatic tumor microenvironment. Furthermore, we validated the role of endotrophin in a bile duct ligation (BDL) induced hepatic injury model, which is an experimental obstructive cholestasis model displaying cholestasis and inflammation and severe fibrosis initiating from the periportal area. Similarly, Alb-ETP mice had exacerbated hepatic fibrosis, apoptosis and enlarged periportal area compared to control mice (Figure III-1-7A-E). As the JNK1 signaling pathway is involved in hepatic apoptosis and liver pathology [20-24], we assessed the JNK activity in DEN- injured liver tissues from the both mice groups. Alb-ETP mice had elevated JNK1 phosphorylation and cleaved caspase 3 level compared to control mice (Figure III-1-5M). Altogether, those in vivo results suggest that endotrophin plays a crucial role in the liver pathogenesis, acting as a necessary but not sufficient factor of pathological conditions in liver disease, such as hepatic apoptosis, inflammation, and fibrosis.
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Figure III-1-5. Endotrophin facilitates DEN-induced HCC development in vivo.
Alb-ETP and control mice were treated with a single injection of DEN (25 mg/kg) at postnatal day 14 and they were fed dox-diet (200 mg/kg). After 32 weeks of the injection, mice were dissected and analyzed for tumor progression. (A) Representative liver images from control and Alb-ETP. White arrows indicate tumors. (B) H&E staining of liver tissues. (T, tumor lesions, scale: 50 μm). (C) Tumor incidence rate, and (D) quantification of tumor number. (E) Ki-67 staining (NT, nontumor; T, tumor, scale: 50 μm) in liver tissues and their quantification of positive cell number. (F) Quantification of serum AFP levels. (G) CC3 immunofluorescence staining and their quantification of positive cell number
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(scale: 20 μm). (H) TUNEL, Ki-67 and DAPI co-staining in liver tissues (scale: 20 μm). (I) Apoptosis, (J) inflammation, and (K) fibrosis marker gene levels were analyzed by qPCR. (L) αSMA staining, (scale: 50 μm). (M) Western blot analysis of JNK1 phosphorylation and cleaved caspase 3 in liver tissues from the both genotypes. Mean ± SEM. Statistical significance was evaluated by unpaired two- tailed Student's t-test, and Mann–Whitney U-test for (D). *p < 0.05; **p < 0.01; ***p < 0.001.
Figure III-1-6. Inflammation and fibrosis marker genes are positively correlated with Col6a3 in nontumor region of liver tissues from HCC mice model.
(A-N) Linear regression analysis of Col6a3 with inflammation and fibrosis marker genes in (A-G) nontumor or (H-N) tumor regions of liver tissues from DEN-induced HCC mice model. A single intraperitoneal injection of DEN (25 mg/kg) was given to 2-week-old C57BL/6 wildtype male mice and they were dissected at 9 months of age. Tumor and non-tumor regions of DEN-induced HCC liver tissues were separately subjected for this analysis. Statistical significance was evaluated as **p<0.01,
***p<0.001 by linear regression (n=18).
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Figure III-1-7. Endotrophin promotes hepatic inflammation, fibrosis, and apoptosis upon bile- duct ligation (BDL) induced cholestasis mice model.
BDL surgery was performed in both Alb-ETP and control mice after 1 week of dox-diet treatment, and their liver tissues were analyzed 10 days after BDL surgery. (A) H&E staining (scale bar: 100 μm), (B) trichrome C staining (scale bar: 50 μm), and (C) cleaved caspase 3 staining (scale bar: 20 μm) revealed elevated inflammatory cell infiltrations, fibrotic regions, and apoptosis in liver tissue from Alb-ETP compared to control mice, respectively. (D) Cytoketatin7 (CK7) and (E) Cd133 staining (scale bar: 50 μm) revealed elevated population of progenitors and cholangiocyte in Alb-ETP compared to control mice, suggesting that endotrophin augments fibrosis in damaged liver tissues.
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Ⅲ-1-5. High levels of ETP in hepatocytes contributes to hepatocyte apoptosis partly through JNK activation in chronic liver injury
In order to investigate how endotrophin facilitate pathological process of CLD, we first examined the Col6a3 expressions in primary hepatic cells under stress condition by treating either CCl4 or vehicle to the wildtype mice. Their liver tissues were extracted and subsequently fractionated into hepatocytes and nonparenchymal cells (NPCs) (Figure III-1-8A). Both hepatocyte and NPCs exhibited elevated Col6a3 levels in response to acute CCl4 treatment (Figure III-1-8B), and hepatic apoptosis and inflammation marker gene expressions were also augmented by CCl4 treatment (Figure III-1-8C). To address the direct role of endotrophin in CCl4-induced hepatocyte damages, either CCl4 or vehicle were treated to the primary hepatocytes isolated from liver tissues from wildtype mice. Western blot analysis revealed that in the presence of endotrophin overexpression, CCl4 treatment resulted in elevated JNK phosphorylation and apoptotic marker, such as BAD and CC3. However, CCl4 treatment elicited limited response in the absence of endotrophin (Figure III-1-8D). Notably, Immunostaining for CC3 further showed that CCl4 induced hepatic apoptosis was further augmented by endotrophin treatment, which was blocked by JNK inhibitor treatment, SP600125 (Figure III-1-8E). Our ex vivo findings suggest that endotrophin is critically involved in hepatocyte apoptosis by acting as a signaling molecule activating the JNK pathway upon liver damage, at least in part. Moreover, we found that intraperitoneal administration of CCl4 led to robust phosphorylation of JNK1 in liver tissue from control group in vivo, which was further augmented in liver tissue from Alb-ETP. The CCl4 triggered JNK1 activation could be efficiently blocked by SP600125 treatment (Figure III-1-8F).
Dead cell bodies from damaged liver tissues release pro-inflammatory signals to activate hepatic stellate cell (HSC) and Kupffer cell in a paracrine manner, and subsequent activation of residing cell population further contribute to hepatic apoptosis, fibrosis, and inflammation [25]. As the endotrophin is ECM- driven molecules that affects nearby cells, we investigated whether the endotrophin inhibition could block the paracrine interactions between hepatocytes and NPCs in injured liver tissues. Either endotrophin neutralizing antibodies (10B6) or control IgG were treated to hepatocytes isolated from either CCl4 or vehicle treated wildtype mice, and their conditioned medium (CM) were collected after 24 h of incubation. Freshly isolated NPCs were then treated with the CM from each condition for another 24 h, and analyzed for inflammation, fibrosis, and apoptosis marker gene expression (Figure III-1-8G). We found that expression levels of inflammatory marker genes including Il6, Ccl2, and Tnf were significantly elevated by the hepatocyte CM from CCl4-injected mice (CCl4-CM) compared to that of control mice (Ctrl-CM). Notably, the effect of CCl4-CM was profoundly abolished by inhibition of endotrophin, where the CCl4-CM was collected from 10B6 treated hepatocyte (Figure III-1-8H).
Furthermore, several fibrosis marker genes, including Timp1, and Ctgf, were significantly elevated by
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CCl4-CM treatment compared to Ctrl-CM treatment, whereas the 10B6 suppressed the effect of CCl4- CM leading to downregulation of fibrosis marker gene expressions (Figure III-1-8I). In this experimental setting, CCl4-CM treatment did not result in significant changes in other fibrotic marker genes such as Des, Vim, Acta2, and Col6a3 (Figure 4I) as well as apoptotic marker genes such as Bad, and Bax (Figure III-1-8J), implying that inflammation is likely to drive the paracrine crosstalks between NPCs and hepatocytes at an early stage of liver damages, rather than fibrosis or apoptosis.
Notably, basal levels of fibrotic gene expression were significantly decreased by 10B6-contained CM, in either Ctrl or CCl4 groups (Figure III-1-8I). In addition to NPCs, the paracrine interactions were further validated in LX-2 HSCs and Raw264.7 macrophages. Similarly, CCl4-CM treatment in LX-2 cells led to increase in ACTA2, and CTGF levels, while silencing of COL6A3 blunted responses to CCl4-CM, indicating that COL6A3 plays a crucial role in the paracrine interactions in HSCs (Figure III-1-8K, L). JNK inhibitor, SP600125, diminished the inflammatory and fibrotic responses exerted by endotrophin in CCl4-CM in LX-2 and Raw264.7 cells (Figure III-1-8M, N). Taken together, those results suggest that chronic liver injury induces endotrophin overexpression in hepatocytes, which contributes to hepatocyte apoptosis and inflammation partly through JNK1 activation. Moreover, inhibition of endotrophin in damaged hepatocytes could efficiently suppress the paracrine interactions of hepatocyte with NPCs, resulting in diminished inflammatory response at an early stage of hepatic injury.