Paracrine effects of CCN3 from non-cancerous hepatic cells increase signaling and progression of hepatocellular carcinoma

Background The liver microenvironment plays a key role in the progression and metastasis of hepatocellular carcinoma (HCC). Gene expression profiling of non-cancerous hepatic tissues obtained from patients with metastatic HCC exhibit a unique immune response signature, including upregulation of CCN3. However, the role of CCN3 secreted from non-cancerous hepatic tissues in the progression of HCC remains unclear. Methods Using tissue microarrays, we examined CCN3 in non-cancerous hepatic tissues of patients with HCC and correlated expression with clinical and pathological features. In addition, CCN3 localization and mechanisms of HCC progression were investigated in tissues and cell lines. Finally, correlations between CCN3 and cirrhosis were explored in patients. Results CCN3 was primarily localized to hepatic cells of non-cancerous hepatic tissues and was associated with vascular invasion and poor prognosis in patients with HCC. CCN3 expression in non-cancerous hepatic tissues also correlated with the degree of liver fibrosis. Compared with conditioned media from wild-type LO2 cells, conditioned media from hepatic cell line LO2 activated by LX2 (aLO2-CM) induced CCN3 expression and HCC cell proliferation and metastasis. Further, aLO2-CM activated MAPK signaling and epithelial-mesenchymal transition in HCC cells. Finally, CCN3 was inversely related to cirrhosis in the prognosis of HCC and negatively regulated hepatic stellate cells (HSCs) in vitro with downregulation of α-SMA, TGF-β, and collagens. Conclusions CCN3 was secreted from hepatic cells activated by HSCs and increased MAPK signaling, EMT, proliferation and metastasis of HCC cells. CCN3 was also inversely related to cirrhosis, regulating HSCs through a negative feedback loop.


Background
Hepatocellular carcinoma (HCC) represents an extremely poor prognostic cancer, and more than 50% of the total number of HCC patients was occurred in China [1]. Most patients with HCC have a history of cirrhosis. As a primary risk factor for developing HCC, cirrhosis treatment is a potential strategy for preventing and treating HCC [2].
The tumor microenvironment plays a key role in HCC progression and metastasis [3]. HCC develops from chronically damaged hepatic tissue, which is surrounded by non-cancerous tissue containing stromal cells and cytokines. HCC exhibits a unique stromal signature associated with metastasis and recurrence [4]. Budhu et al. [5] previously explored the unique expression profiles of non-cancerous hepatic tissues obtained from patients with venous metastatic HCC. In the current study, we performed cDNA microarray-based gene expression profiling and found significant upregulation of CCN3, a matricellular protein encoded by the nephroblastoma overexpressed (NOV) gene in humans. The CCN family is a small, six-member family of cysteine-rich regulatory proteins in humans. CCN proteins function as core regulatory factors in the inflammatory microenvironment and are closely linked with HCC progression [6]. We previously reported that high CCN3 protein expression in tumor tissue is associated with low overall survival (OS) rates in patients with HCC [7]. However, CCN3 expression levels in non-cancerous hepatic tissues and the role of CCN3 in HCC progression remain unclear.
The goal of the present study was to explore expression and functional effects of CCN3 in non-cancerous hepatic tissues in correlation with HCC progression. We analyzed the localization and role of CCN3 in HCC. We found that CCN3 was secreted from hepatic cells and explored signaling pathways related to HCC malignant progression. Finally, we confirmed the antagonistic role of CCN3 on hepatic stellate cells (HSCs).

Patients and follow up
Clinicopathological analysis was performed on 374 HCC samples. Immunohistochemical analysis of tissue microarrays included 186 non-cancerous hepatic tissues obtained from HCC patients. Another 98 HCC samples were evaluated for CCN3 mRNA levels. Post-surgical follow up occurred until December 2013, with a median follow-up period of 63 months (range, 0-110 months). Curative resection was defined as complete resection of tumor nodules with clean post-surgical margins. Histopathological diagnosis was performed according to WHO criteria. The level of cirrhosis of liver tissue refers to the general observation of pathological specimens and the improved standard of METAVIR scoring system. The severity of cirrhosis was classified into four degree by histopathological evaluation of the liver tissue: No fibrosis, mild (portal fibrosis with few septa), moderate (numerous septa without cirrhosis), and severe (cirrhosis). The clinicopathological characteristics of patients are provided in Tables 1 and 2 Conditioned Media (CM) form HSC LX2 was used for treating hepatic cells LO2 for 3 days. Then LO2 cells were activated by LX2 and referred to as activated LO2 (aLO2). When we silenced endogenous CCN3 in aLO2 using lentiviral transfection, the CM from HSC LX2 was were continuously applied until the aLO2-CCN3-sh1 was successful constructed. We silenced endogenous CCN3 in aLO2 using lentiviral transfection and named these cells aLO2-CCN3-sh1. The aLO2 and aLO2-CCN3-sh1 cell lines were plated in T 75 flasks (1 × 10 6 cells). Media was replaced with 6 ml of fresh DMEM containing 2% FBS the next day. After 24 h, CM were centrifuged at 1000×g, and the supernatants were collected and designated aLO2-CM or aLO2-CCN3sh1-CM.

Migration and invasion assays
Migration of HCC cells was evaluated in Boyden chambers containing membranes with 8.0-μm pore size in 24-well plates (Corning, Tewksbury, MA, USA). Hep3B cells were seeded into the upper chamber of each well in serum-free DMEM (6 × 10 4 cells per well). DMEM containing 2% FBS or aLO2-CM was added to the lower chamber of each well. After 24 h, cells on the underside of the membrane were stained with Giemsa (Sigma Chemical Company, St. Louis, MO, USA), counted, and photographed at 200× magnification. Cell invasion assays were performed similarly, except that 80 μL Matrigel (BD Biosciences, San Jose, CA, USA) was added to each well 6 h before cells were seeded onto the membrane.

Statistical analyses
Time to recurrence rates (TRR) and OS of patients in different groups were compared by Kaplan-Meier analysis. Statistical analyses shown in Tables 1 and 2 were performed with Fisher's exact or Chi-squared tests. Quantitative data were evaluated by t-tests. IBM SPSS® 20.0 for Windows was used to perform statistical analyses, with statistical significance defined as p < 0.05.

Results
High expression of CCN3 localizes primarily to hepatic cells in non-cancerous hepatic tissues and correlates with metastasis and poor prognosis in patients with HCC Budhu et al. [5] previously examined gene expression profiles in non-cancerous hepatic tissues from patients with HCC and intrahepatic venous metastasis. In the cDNA microarray-based gene expression profiles, we found that CCN3 was significantly upregulated in HCC patients with intrahepatic venous metastasis than patients without intrahepatic venous metastasis (0.27 ± 0.39 vs. -0.46 ± 0.39 p = 0.0014, Fig. 1a). We examined mRNA expression in 98 non-cancerous hepatic tissues from patients with HCC. CCN3 mRNA levels were upregulated in HCC patients with venous metastasis than patients without intrahepatic venous metastasis (0.0064 ± 0.004 vs. 0.0037 ± 0.0037, p = 0.0175, Fig. 1b). To confirm the clinical role of CCN3, we examined another 186 non-cancerous hepatic tissues by immunohistochemistry. CCN3 staining was primarily localized to hepatic cells (Fig. 1c, Fig. 2a). Patients with HCC and high expression of CCN3 in non-cancerous hepatic tissues exhibited significantly lower OS (p = 0.013) and higher TRR (p = 0.021) (Fig.  1d). These data demonstrate that high expression of CCN3 in non-cancerous hepatic tissues correlates with metastasis and poor prognosis in patients with HCC.

CCN3 expression correlates with level of cirrhosis, and HSCs induce expression of CCN3 in hepatic cells
In the 186 patients with HCC presented in the previous section, Cox regression analysis revealed a positive correlation between CCN3 expression in non-cancerous hepatic tissues and cirrhosis (p = 0.005), serum ALT (p = 0.023), and vascular invasion (p = 0.013) ( Table 1). CCN3 expression was higher in patients with cirrhosis (26.47 ± 9.81 vs. 18.73 ± 14.27 p = 0.0189, Fig. 2a, b), supporting a correlation between CCN3 levels in liver tissues and cirrhosis. Next, we evaluated the mRNA levels of CCN3 in 98 clinical samples. CCN3 was upregulated in non-cancerous hepatic tissues from patients with HCC and cirrhosis (0.0045 ± 0.0040 vs. 0.0022 ± 0.0023 p = 0.0149, Fig. 2c). Patients were then divided into four groups according to level of cirrhosis   Fig. 2d). Western blotting demonstrated that CCN3 expression was increased in hepatic LO2 cells activated by HSC LX2 compared with wild-type LO2 cells (Fig. 2e). ELISA further showed that the CCN3 concentration in CM from activated LO2 (aLO2-CM) was significantly increased compared with that of wild-type LO2 (427.31 ± 42.55 ng/L vs. 116.74 ± 33.56 ng/L, p = 0.0006, Fig. 2f).
These findings indicate that CCN3 expression in non-cancerous hepatic tissues correlates with the gradient level of cirrhosis, and that CCN3 is significantly increased in hepatic cells activated by HSCs.

CCN3 is inversely related to cirrhosis in the prognosis of HCC and reduces expression of α-SMA and TGF-β1 in HSCs
In the present study, we examined the role of cirrhosis in the prognosis of 186 patients with HCC. Patients with cirrhosis exhibited higher TRR (p = 0.045) and lower OS (p = 0.118, Fig. 4a) compared with those who did not have cirrhosis. Further analysis was performed for correlations between liver cirrhosis and clinicopathological features in 374 HCC patients. Cox regression analysis demonstrated a correlation between cirrhosis and HBsAg (p < 0.001), AFP (p = 0.026), and tumor dimension (p < 0.001) ( Table 2). We believe that cirrhosis alone is not a sufficient predictor of survival. To better understand the relationship between CCN3 and cirrhosis in the prognosis of HCC, we classified patients into two groups based on CCN3 expression level. Patients with high expression of CCN3 were further divided into two subgroups according to the presence or absence of cirrhosis. However, no difference in OS (p = 0.454) or TRR (p = 0.630) (Fig.  4b, a) was found between the two high CCN3 subgroups. By contrast, patients with low expression of CCN3 and cirrhosis exhibited a significantly lower OS (p = 0.016) and higher TRR (p = 0.005) than patients without cirrhosis (Fig. 4b, b). These data suggest that low expression of CCN3 may predict poor prognosis in patients with HCC and cirrhosis.
Patients were then divided into two groups based on the presence or absence of cirrhosis. Patients with cirrhosis were further divided into two subgroups according to CCN3 expression. There was no difference in OS (p = 0.454) or TRR (p = 0.630) between patients with high or low expression of CCN3 (Fig. 4c, a). However, in the subgroup without cirrhosis, patients with high expression of CCN3 exhibited significantly lower OS (p = 0.016) and higher TRR (p = 0.005) than patients with low expression of CCN3 (Fig. 4c, b). Thus, high expression of CCN3 may predict poor prognosis in patients with HCC who do not have cirrhosis.

Discussion
HCC is a deadly cancer and is usually accompanied by cirrhosis due to chronic inflammation and necrotic hepatic tissue [10]. Numerous studies indicate that HCC progression is greatly influenced by the tumor microenvironment [11,12]. The cirrhosis microenvironment supports the malignant progression of HCC [13,14]. Understanding the role of cirrhosis in HCC progression may help identify new therapies for a more personalized medical approach [15,16].
HSCs are a key cell type in cirrhotic tissue and play an important role in hepatic fibrosis [17]. Activated HSCs promote HCC progression primarily through paracrine effects. We previously showed that HSCs interact with hepatoma cells via secretion of cytokines, which modulate the malignant changes of HCC [18,19]. In this study, we confirmed that patients with cirrhosis have a higher TRR and lower OS, although the differences were not significant. Therefore, identification of a molecular marker in HCC with a cirrhosis microenvironment may improve understanding of the relationship between cirrhosis and HCC. Budhu et al. [5] demonstrated that the NOV gene, which encodes the secreted protein CCN3 in humans, is significantly upregulated in non-cancerous hepatic tissues from patients with HCC and intrahepatic venous metastases. In the current study, tissue microarrays showed that CCN3 localized primarily to the hepatic cells of non-cancerous hepatic tissues. CCN3 was significantly upregulated in HCC patients with vascular invasion and poor prognosis. Tissue microarrays also demonstrated that increased expression of CCN3 in hepatic cells correlated with the level of cirrhosis. In vitro analyses showed paracrine effects of CCN3 from hepatic cells; these effects were significantly increased in hepatic cells activated by HSCs. Therefore, CCN3 paracrine effects from hepatic cells may be closely related to cirrhosis and may play an important role in HCC progression.
The CCN family includes six protein members. Similar to other family members, CCN3 is composed Fig. 4 CCN3 is inversely related to cirrhosis in the prognosis of HCC and inhibits HSC proliferation. Patients with cirrhosis exhibit lower OS and higher TRR (a). There was no difference in OS or TRR when patients with high CCN3 expression were stratified according to presence or absence of cirrhosis (b, a). There was a significant difference in OS and TRR when patients with low CCN3 expression were stratified according to presence or absence of cirrhosis (b, b). There was no difference in OS or TRR when patients with cirrhosis were stratified according to CCN3 expression (c, a). There was a significant difference in OS and TRR when patients without cirrhosis were stratified according to CCN3 expression (c, b). aLO2-CM and CCN3 significantly inhibit proliferation of LX2 (d). The proportion of LX2 cells in S phase is decreased after treatment with aLO2-CM or CCN3 (e). LX2 cells treated with aLO2-CM or CCN3 exhibit reduced expression of TGF-β1, α-SMA, Collagen I, and Collagen III; this trend is reversed in aLO2-CCN3sh1-CM (f). LX2 cells treated with aLO2-CM or CCN3 exhibit significantly decreased concentrations of TGF-β1 (g) of the following four functional modules, IGFBP, VWC, TSP1, and CT. CCN3 functions as a localized multitasking signal integrator in the microenvironment, binding with multiple receptors involved in the regulation of cell proliferation, chemotaxis, angiogenesis, and adhesion [20]. CCN3 was overexpressed in metastatic melanoma compared with the primary tumor and was associated with higher metastatic potential of melanoma cells [21]. Previously, our group showed that CCN3 is a potential therapeutic target that may affect upregulation of Ostopontin (OPN)and coagulation factors, enhancing the stemness and blood coagulation microenvironment of HCC tissue [7]. We also found that CCN3 orchestrated the stroma-derived resistance to chemotherapy in HCC. In the present study, we demonstrated that treatment of HCC cell line Hep3B with aLO2-CM, which has high CCN3 levels, induced migration and proliferation with upregulation of p-RAF, p-MEK, p-ERK, and Vimentin and downregulation of E-cadherin. These results indicate that hepatic cells promote the malignant progression of HCC via MAPK signaling and EMT.
The CCN family of proteins also participate in the assembly of the extracellular matrix [22]. In the present study, we examined the expression of CCN3 in hepatic tissue microarrays. High expression of CCN3 was localized to hepatic cells and associated with more severe cirrhosis. In addition, CCN3 was significantly increased in hepatic cell line LO2 after treatment with LX2-CM. Andrew et al. [23] reported that CCN3 is a potential anti-fibrotic treatment. We further showed that CCN3 was inversely related to cirrhosis in the prognosis of HCC and inhibited expression of α-SMA and TGF-β1 in HSCs. In patients without cirrhosis, high CCN3 expression correlated with lower OS and higher TRR compared with those who had low CCN3 expression. In patients with low CCN3 expression, patients with cirrhosis had significantly worse prognosis than patients without cirrhosis. We also concluded that CCN3 was inversely related to cirrhosis in the prognosis of HCC and acted in a negative feedback loop in HSCs (Fig. 5).
Although cirrhosis promoting tumors progression is very clearly, while cirrhosis was insufficient factor alone to predict prognosis of patients with HCC, cause of many cytokines such as CCN3 may influence the relationship between cirrhosis and HCC progression. Maybe that is why anti-fibrosis is not an very effect way to anti-HCC. Further studies will focus on the relationship between HCC and cirrhosis to identify a comprehensive treatment strategy.

Conclusions
From our experimental results and our review of the literature, we propose the following conclusions. (1) HCC progression is profoundly influenced by the tumor microenvironment. (2) Cirrhosis and CCN3 in hepatic cells are associated with a malignant phenotype and