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Imbalanced expression pattern of steroid receptor coactivator-1 and −3 in liver cancer compared with normal liver: An immunohistochemical study with tissue microarray

Steroids affect normal and pathological functions of the liver through receptors, which require coactivators for their transcriptional activation. Steroid receptor coactivator (SRC)-1 and SRC-3 have been demonstrated to be regulated in numerous cancers; however, their expression profiles in liver cancer including hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) remain unclear. Using tissue microarray immunohistochemistry, normal liver tissue and HCC tissue exhibited immunoreactivity of SRC-1, which were predominantly localized within extranuclear components; in CCC, they were detected within the cell nuclei; SRC-3 was also detected in the cell nuclei. Furthermore, no altered expression of SRC-1 and SRC-3 was observed in liver cancer compared with normal liver tissue; however, in CCC, the expression of SRC-3 was significantly increased compared with that detected in HCC. Importantly, although expression of SRC-1 and SRC-3 did not reveal any significant differences (30 vs. 40%) in normal liver tissue, HCC and CCC expression of SRC-1 was significantly decreased compared with that of SRC-3 (9.3 vs. 36%, and 6.7 vs. 67.7% for HCC and CCC, respectively). Further comparative analysis revealed that this discrepancy was detected in males with liver cancer, across all ages of HCC cases, younger CCC cases and all stages of liver cancer. The results suggested the presence of an imbalanced expression pattern of SRC-1 and SRC-3 from normal liver tissue to liver cancer (decreased SRC-1 and increased SRC-3), which may affect hepatic function and therefore promote liver carcinogenesis.


Introduction
Steroids, including androgens and estrogens (E 2 ), have been demonstrated to exert multiple effects not only on reproductive function, but also on numerous other organ systems, including the liver, in men and women (1). For example, previous human studies have demonstrated that female menopause has been associated with the increase in non-alcoholic fatty liver disease, hepatocellular carcinoma (HCC) and the progression of fibrosis (2). Furthermore, animal studies also revealed that bilateral ovariectomy increased the risk of HCC (3). There appears to be a sex difference in the survival of patients with HCC: HCC is a male-dominated cancer, with men 4-8-fold more likely to develop HCC than women; however, testosterone may act to protect against hepatic steatosis (4)(5)(6). Additionally, high levels of aromatase, an enzyme catalyzing the conversion of testosterone into E 2 , has been detected within the human liver, and aromatase overexpression has also been identified in hepatitis and HCC (7,8). Furthermore, aromatase gene-knockout mice exhibited hepatic glucose intolerance, which was able to be reversed by E 2 administration (9). Recent studies have identified that high circulating E 2 and low testosterone ratio may be associated with adverse clinical outcomes in men with advanced liver disease and patients with primary liver cancer (10,11).
The action of steroids is known to be mediated by their receptors. Androgen receptor (AR) has been detected in normal and cancerous liver tissue and cell lines; estrogen receptor (ER) α and decreased expression of ERβ have also been identified in HCC (5,12,13). These receptors have been demonstrated to regulate lipid and glucose metabolism in the liver. For example, ERs function to decrease lipogenesis, gluconeogenesis and fatty acid uptake, but enhance lipolysis, cholesterol secretion and glucose catabolism; AR functions to increase insulin receptor expression and glycogen synthesis, decrease glucose uptake and lipogenesis, and promote cholesterol storage (14). Additionally, studies have revealed that expression of ERα was associated with invasion and metastasis in HCC, and AR and ERα, but not ERβ, gene expression contributed to the prevalence of HCC in male rats (15,16).
Cholangiocellular carcinoma (CCC) is difficult to treat due to its chemo-resistance and its inability to be detected at an early stage of disease (17). It has been reported that, compared with males, females are more susceptible to several biliary tract diseases such as primary biliary cirrhosis, debilitating/symptomatic adult polycystic liver disease and autoimmune hepatitis (18). The significant increase of estrogen levels in the serum of patients with CCC has been reported (19), and has also been demonstrated to enhance the proliferation and invasiveness of CCC cells in vitro. Furthermore, the survival time of patients with CCC is associated with estrogen levels (20). Additionally, different levels of ERα and ERβ have been detected in CCC cells (18,21), ERα has been demonstrated to mediate estrogenic stimulation of interleukin-6 production and thus influence the pathology of CCC (18), the overexpression of ERβ has also been demonstrated to exhibit protective abilities against CCC (21).
Previous studies have demonstrated that steroid receptor coactivators (SRCs) are required for the transcriptional activation of target genes by a steroid receptor (22,23). Among SRCs, the p160 family members SRC-1 and SRC-3 have been investigated in a number of types of cancer including tissue and cell lines (24). For example, overexpression of SRC-1 and SRC-3 has been detected in breast cancer, non-small cell lung cancer, bone cancer and chondrosarcoma (25)(26)(27)(28)(29)(30)(31). Decreased expression of SRC-3 has been reported in astrocytic tumors and decreased expression of SRC-1 and SRC-3 has been identified in meningothelial tumors and neuroepithelial tumors (32,33). In liver tissue, an early study demonstrated that SRCs serve a role in the regulation of hepatic energy homeostasis (34), and further studies highlighted that SRC-1 was a critical mediator of glucose homeostasis as it functioned as the integrator of glucose and oxidized/reduced nicotinamide-adenine dinucleotide homeostasis (35,36). Thus, hepatic SRC-1 activity may have potential relevance for human metabolic pathogenesis (37). However, the expression profiles of SRC-1 and SRC-3 in HCC and CCC have not yet been reported. To address this question, the present study investigated the expression and significance of these two coactivators in HCC and CCC using tissue microarray (TMA) immunohistochemistry.

Materials and methods
Tissue microarray. The two types of hepatic carcinoma and normal TMA used were purchased from US Biomax, Inc. (cat. no. BC03118; Rockville, MD, USA). The TMA contained 75 cases of malignant HCC (65 males and 10 females; mean age, 50.8 years), 15 cases of malignant CCC (8 males and 7 females; mean age, 48.5 years) and 10 cases of normal hepatic tissue (6 males and 4 females; mean age, 26.8 years). In total, 200 tissue cores featured on a single slide as 2 cores were punched from each case.
Immunohistochemistry. TMAs were deparaffinized in xylene, rehydrated with a gradient alcohol series and heat-mediated antigen retrieval (0.01 M sodium citrate buffer, pH 6.0) was performed according to our previous protocol (32,33 Image acquisition and data analysis. Images of immunohistochemical staining were captured using a digital camera (DP70; Leica, Germany) equipped with an Olympus microscope (BX60; Olympus Corporation, Tokyo, Japan; under x20 or x40 magnification). The strength of staining was scored in accordance with a four-point system (0-3) described in a previous study (32) by a pathologist double-blindly. A score of 3 indicated visible dark staining of >50% of cells; a score of 2 indicated either dark focal staining of <50% of cells or moderate staining of >50%; a score of 1 indicated either moderate focal staining of <50% of cells or pale staining in any proportion of cells not easily seen at low power; a score of 0 indicated no positive staining. A high level of expression was defined as a score of 2 or 3, and a low level of expression was defined as a score of 0 or 1. Pathologically, early-stage liver cancer was defined as stage I and II; advanced stage was defined as stage III and IIIb.
Statistical analysis. All data are expressed as n (%) and compared using a χ 2 test or Fisher's exact test with SPSS software (version 18.0; SPSS, Inc., Chicago, IL, USA). All P-values were two-tailed and P<0.05 was considered to indicate a statistically significant difference. Fig. 1 demonstrate the localization patterns of SRC-1-and SRC-3-immunoreactive materials. In normal liver and HCC tissue, SRC-1-immunoreactive materials were predominantly detected within the extra-nuclear component. However, in CCC, SRC-1 materials were predominantly detected within the cell nuclei. SRC-3-immunoreactive materials were primarily detected within the cell nuclei.

Subcellular localization of SRCs in normal and cancerous liver tissue. Results presented in
Expression profiles of SRCs in normal and cancerous liver tissue. High levels of SRC-1 expression was detected in 30% (3/10) of the normal liver tissue, 9.3% (7/75) of HCC tissue and 6.7% (1/15) of CCC tissue. The χ 2 test indicated no statistically significant differences in association with the expression levels of SRC-1 between normal and HCC, normal and CCC, or HCC and CCC samples (Table I and Fig. 2A-C). High levels of SRC-3 expression was detected in 40% (4/10) of the normal liver tissue, 36% (27/75) of HCC tissue and 67.7% (10/15) of CCC tissue. The χ 2 test indicated no statistical significance in the expression levels of SRC-3 between normal and HCC or normal and CCC samples (Table I, Fig. 2D and E). However, expression of SRC-3 in CCC was significantly increased compared with HCC (67.7 vs. 6.7%; P=0.028) as indicated in Table I and Fig. 2F.
Comparison of the expression profile of SRCs in normal and cancerous liver tissue. Comparisons of SRC-1 and SRC-3 expression profiles in normal and liver cancer tissue were performed in order to determine any statistical significance. Normal liver tissue results identified that 30% (3/10) exhibited high levels of SRC-1 expression and 40% (4/10) exhibited high levels of SRC-3 expression. The difference in expression profiles was not statistically significant (P>0.05). In HCC, 9.3% (7/75) exhibited high levels of SRC-1 expression and 36% (27/75) exhibited high levels of SRC-3 expression. The HCC results indicated that SRC-3 expression was significantly increased compared with SRC-1 expression (P<0.01). In CCC results, 6.7% (1/15) exhibited high levels of SRC-1 expression, whereas SCR-3 expression was significantly increased in comparison (P= 0.02) at 67.7% (10/15). These results are presented in Table II and Fig. 2G-I.

Sex-specific analysis of SRCs in normal and cancerous liver tissue.
Male-specific analysis. The normal tissue group revealed that of the 6 cases, none exhibited high levels of SRC-1 and only 1 exhibited high levels of SRC-3; these results were not statistically significant (P>0.05). The HCC tissue group revealed that of 65 cases, 7.7% (5/65) exhibited high levels of SRC-1, whereas SCR-3 was significantly increased (P<0.01) at 38.5% (25/65). In CCC, of 8 cases none exhibited high levels of SRC-1, whereas 75% (6/8) exhibited high levels of SRC-3. SRC-3 expression was significantly increased compared with that of SRC-1 (P<0.01). These results are presented in Table III and Fig. 3.
Age-specific analysis of SRCs in normal and cancerous liver tissue. The mean age of normal cases was 26.8 years (n=10). A total of 4 normal cases were ≥26.8 years, none of which exhibited high levels of SRC-1 and 50% (2/4) exhibited high levels of SRC-3; these results were not statistically significant (P>0.05). A total of 6 normal cases were <26.8 years, 50% (3/6) exhibited high levels of SRC-1 and 33.3% (2/6) exhibited high levels of SRC-3; these results were not statistically significant (P>0.05). The mean age of HCC cases was 50.8 years (n=75). A total of 38 HCC cases were ≥50.8 years, of which 5.3% (2/38) exhibited high levels of SRC-1 and 36.8% (14/38) exhibited high levels of SRC-3. This was significantly increased compared with that of SRC-1 (P=0.02). The remaining 37 cases were <50.8 years, 13.5% (5/37) exhibited high levels of SRC-1 and 35.1% (13/37) exhibited high levels of SRC-3; this was also significantly increased compared with that of SRC-1 (P=0.030). The mean age of CCC cases was 48.5 years (n=15). A total of 7 CCC cases were ≥48.5 years,

Stage-specific analysis of SRCs in HCC and
In the early stage of CCC, no case exhibited high levels of SRC-1 and 40% (2/5) exhibited high levels of SRC-3; these results were not statistically significant (P>0.05). In the advanced stage of CCC, 10% (1/10) exhibited high levels of SRC-1 and 80% (8/10) indicated high levels of SRC-3; this was significantly increased compared with that of SRC-1 (P=0.007). These results are presented in Fig. 5C and D.

Discussion
It is well known that liver disease is a major health concern worldwide (38). Statistics published in 2014 estimated that the number of new liver cancer cases in the USA totaled 33,190 (24,600 male cases), and the estimated mortality rate was 23,000 (15,870 male cases). Therefore, these statistics indicate that liver cancer is the fifth leading cause of male (15,870) and the ninth leading cause of female (7,130) cancer mortality in the USA in 2014 (39). Similar results were also reported in China, where liver cancer was ranked within the top five causes of cancer-associated mortality with clear male predominance (310,600 vs. 111,500) (40). However, the molecular mechanisms underlying the occurrence and disease progression, as well as sex differences observed in liver cancers are poorly understood. Therefore, in the Table II. Comparison of the expression of SRC-1 and SRC-3 in normal, HCC and CCC.
Furthermore, results indicated that expression of SRC-1 did not demonstrate any significant differences among normal, HCC and CCC liver tissue; similar phenomena for SRC-3 were also detected; however, significantly increased levels of SRC-3 were detected in CCC tissue when compared with those in HCC tissue. These tissue results indicated that there was an unchanged SRC-1 but an evident overexpression of SRC-3 in HCC and CCC when compared with that detected in the normal liver tissue. Some previous studies have also reported different changes of SRC-1 or SRC-3 in specific cancers compared to normal tissue. For example, overexpression of SRC-1 is present in breast and ovarian cancer cell lines as well as primary breast cancer, prostate cancer and endometrial carcinoma (42-44) when compared with normal tissue. Additionally, overexpression of SRC-3 was Figure 4. Age-specific comparison of SRC-1 and SRC-3 in normal, HCC and CCC liver tissue. In the older or equal to mean age cases, significantly decreased expression of SRC-1 was detected in the HCC compared to SRC-3, whereas in the normal and CCC liver tissue, no significant difference was detected. Additionally, in the cases, significantly decreased expression of SRC-1 was detected in HCC and CCC compared with SRC-3.     (29,(45)(46)(47)(48)(49)(50). However, previous studies also demonstrated a decrease in SRC-1 protein in endometrial carcinoma, a decrease in SRC-3 but unchanged SRC-1 in the high-grade astrocytic tissue and a decrease in   (53) reported SRC-1 overexpression in 62.5% of HCC tissues using western blot analysis. In the present study, levels of SRC-1 and SRC-3 remain unchanged in liver cancer including HCC and CCC when compared with that in normal tissue. The reasons for these differences are unclear; however, the relatively small normal sample size (10 cases) in the present study may be a factor, and thus further examinations are required to confirm these results.
Owing to a lack of significant differences regarding the levels of SRC-1 and SRC-3 between normal and cancerous liver tissue, focus was placed on the increased expression profiles of SRC-3, and comparisons were made between the levels of SCR-1 and SRC-3 in normal and cancerous liver tissue. A key point of interest was the absence of statistical significance regarding the different levels of SRC-1 and SRC-3; however, in liver cancer, including HCC and CCC, significantly decreased expression of SRC-1 was detected when compared with that of SRC-3. Further sex-, age-and stage-specific analysis revealed that significantly decreased expression of SRC-1 was detected in the following groups: HCC cases (male), CCC cases (male), HCC (all ages), CCC cases (below mean age) and liver cancer stages (all stages). However, levels of SRC-1 and SRC-3 did not exhibit any significant differences in the following categories: Normal cases (male), all cases (female), normal cases (all ages) and CCC cases (above or equal to mean age). The decreased SRC-1/SRC-3 ratio in liver cancer but not normal liver tissue may be due to the slight decrease in SRC-1 expression and increase in SRC-3 expression observed. This indicates an imbalanced expression between these two coactivators, and may contribute to the occurrence and progression of liver cancer. Additionally, the loss-of-balance expression pattern of SRC-1/SRC-3, detected in males caused by their distinct expression profiles (decreased SRC-1; increased SRC-3), was positively associated with previous reports demonstrating a high occurrence and mortality in males with liver cancer (39,40). A similar imbalanced expression profile was also detected in other tumors including high-grade astrocytoma, as a decrease in SRC-3 but unchanged SRC-1 was reported (32).
In summary, although no significant changes in SRC-1 and SRC-3 were identified in liver cancer tissue when compared with that detected in the normal liver tissue, it was noted that there was a significantly decreased SRC-1/SRC-3 ratio in liver cancer, compared with that detected in normal liver tissue. The significance of this decreased ratio is currently unclear. Louet et al (35) reported that SRC-1 is a key coordinator of the hepatic gluconeogenic program and a critical mediator of liver glucose homeostasis; Ma et al (54) reported that SRC-3 serves a crucial role in regulating hepatic lipid metabolism (35,54). Thus, the imbalanced expression of SRC-1 and SRC-3 in the liver tissue may induce abnormal hepatic metabolism and finally induce tumorigenesis. However, further studies are urgently required to explore the precise roles of these two coactivators in both the normal liver and liver disease.

Funding
The present study was supported by the National Science Foundation of China (grant nos. 81571059 and 81270525).

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Author's contributions
SL, HZ and YY conducted the experiments. ML photographed the pictures. DG conducted the pathological scoring. SL and HZ prepared the draft of this manuscript. JZ and XZ conceived this study and finalized this manuscript.

Ethics approval and consent to participate
Not applicable since the tissue microarray used in the present study is commercial available.

Pateint consent for publication
Not applicable since the tissue microarray used in the present study is commercial available.