Hepatocellular Carcinoma, Steroid Hormones and Metalloproteases

Liver cancer is the fifth and seventh most frequently diagnosed cancer worldwide in men and women, respectively, but the second most frequent cause of cancer death in men [1]. In addition, hepatocellular carcinoma (HCC) represents the major histological subtype of primary liver cancers, accounting up to 90% of the total liver cancer burden worldwide [2]. The incidence of HCC has increased significantly over the past 10 years and is expected to increase because of the actual high prevalence of viral hepatitis C-seropositive individuals and also because of the known long latency period to HCC development from the initial hepatitis C virus (HCV) infection, which may take 2-3 decades [3]. Despite of the varied treatment options, the prognosis of HCC remains poor. Thus, estimated 5-year survival rates are in the range of 26% to 50%, and disease-free survival is 13% to 29% [4]. At present, systemic chemo‐ therapy is quite ineffective in HCC treatment, and is also known to express the multidrugresistance gene MDR-1 [5]. Therefore, is necessary to identify and characterize molecular abnormalities of clinical significance in HCC. Besides the heterogeneity of different HCC subtypes, these tumors may use different cellular pathways and oncogenic mechanisms at different development stages, and this is of essential importance to develop biologically-based clinical trials.


Introduction
Liver cancer is the fifth and seventh most frequently diagnosed cancer worldwide in men and women, respectively, but the second most frequent cause of cancer death in men [1]. In addition, hepatocellular carcinoma (HCC) represents the major histological subtype of primary liver cancers, accounting up to 90% of the total liver cancer burden worldwide [2]. The incidence of HCC has increased significantly over the past 10 years and is expected to increase because of the actual high prevalence of viral hepatitis C-seropositive individuals and also because of the known long latency period to HCC development from the initial hepatitis C virus (HCV) infection, which may take 2-3 decades [3]. Despite of the varied treatment options, the prognosis of HCC remains poor. Thus, estimated 5-year survival rates are in the range of 26% to 50%, and disease-free survival is 13% to 29% [4]. At present, systemic chemotherapy is quite ineffective in HCC treatment, and is also known to express the multidrugresistance gene MDR-1 [5]. Therefore, is necessary to identify and characterize molecular abnormalities of clinical significance in HCC. Besides the heterogeneity of different HCC subtypes, these tumors may use different cellular pathways and oncogenic mechanisms at different development stages, and this is of essential importance to develop biologically-based clinical trials. times higher incidence and mortality among men than women [9]. This male predominance is further supported by the fact that chronic liver disease progresses more rapidly to cirrhosis in males than in females, and therefore cirrhosis-derived HCC is largely seen in men and postmenopausal women disease [10].
The role of estrogens in modulating morphological and physiological features of liver became evident in early 1970s when a possible correlation between occurrence of hepatic neoplasms and use of oral contraceptives was suggested [11][12]. On the basis of these data, several in vitro and in vivo studies have explored the importance of sex hormones in HCC. Animal modelbased studies indicate that sex hormones play a key role in tumor progression, showing that ovarian estrogens protect against tumor progression, whereas androgens promote tumorigenesis [13][14][15]. It has been reported that the protective effect of estrogens against chemically induced liver tumors is mediated by prolactin (PRL) through liver prolactin receptors (PRLR) [16][17][18]. Nevertheless, the precise role of male and female sex hormones and their receptors in HCC remains still poorly understood. There is not enough information regarding the mechanism of estrogen and androgen in HCC.
Estrogen and androgen mediate their biological functions through binding to their specific receptors, the estrogen receptor (ER) and the androgen receptor (AR). Both ER and AR belong to the nuclear receptors family that, as transcription factors, regulate the expression level of several genes such as those involved in triggering immune responses, cell proliferation and apoptosis [19][20][21]. Therefore, sex hormones play a key role in normal physiology of organs other than these of the reproductive system.
Variable expressions of ER and AR has been found in normal liver and HCC using different methods, such as immunohistochemistry, enzyme-immuno assays, or determining mRNA levels ( Table 1 and Table 2 give the details of these studies for ER and AR, respectively), which indicate a relationship between sex hormones and pathogenesis of HCC. The percentage of positivity for these receptors varied between the different studies [22]. These differences may be due to several methodological aspects, such as differences in the origin of the studied patients populations, sample types or technologies determining the receptor expression. In a recent study, we determined the expression of sex hormones receptors in 31 HCC patients by immunohistochemistry using tissue micro-arrays technology [23]. Our results demonstrate a wide variability in the immunohistochemical values for steroid receptors among HCCs: 67.7% of tumors stained positively for AR, 51.6% for ER and 83.8% for progesterone receptor (PgR), but, among the positive cases, immunostaining score values for each protein were largely variable.

Steroid receptors and hepatitis virus
It is of special interest the interaction between sex estrogen receptors and viral proteins in hepatitis B virus (HBV) and hepatitis C virus (HCV)-induced HCC. It has been reported that a HBV protein (HBx) interacts with ERα. HBx is a multifunctional protein involved in neoplastic transformation in cultured cells and in HCC induction in transgenic mice. Both HBx and vERα (

Endocrine therapy in hepatocellular carcinoma
Considering both the epidemiologic and the experimental data supporting the sex steroids influence in growth and progression from HCCs, different clinical studies analyzed if endocrine therapy could be interesting in advanced disease. Table 4 gives details of some of these studies. Trials using several agents, such as the anti-estrogen tamoxifen, megestrol acetate, progestin, the gonadotropin releasing hormone (GnRH) agonist leuprorelin and the antiandrogen flutamide have been uniformly disappointing [37][38][39][40]. At present, experts have concluded that hormonal manipulation should not be a part of the current management of patients with HCC [41][42]. However, it is possible that the rational design of an endocrine therapy requires a more complete understanding of the role of sex hormones in the tumorigenic process and how hormones and organ systems interact during this process [18], in addition to careful selection of the new studies patient populations, since patient stratification based on gender may uncover signals of activity of hormonal therapy in these settings. Notably, it has been shown that the anti-estrogens tamoxifen and raloxifene, behave differently in different tissues, producing estrogen-agonist activity in one tissue while behaving as an antagonist in another [43][44]. In addition, in vitro studies in the liver cell line Hep3B, indicate that raloxifene induces the insulin-like growth factor (IGF-I) gene transcription, whereas estradiol or tamoxifen inhibits it [45]. On the other hand, the lack of hormone efficacy in these clinical studies, in terms of tumor growth and survival, could be due to either a low expression of ER or AR in HCC or to mutated receptors expression. Therefore, further studies will be necessary to improve patient selection on the basis of gender, ER tumor expression and their functional status. With regard to this latter aspect, considering that PgR is an estrogen inducible protein, it led us to consider that this steroid receptor could be a possible marker of ER functional status in HCC, to select candidate patients to an anti-estrogenic therapy. In addition, we demonstrated that PgR expression have a prognostic implication in patients with resected HCCs, being a factor associated with better prognostic [23], which is a clinical finding previously demonstrated in breast cancer [46][47]. This finding is very important considering also our reported positive association between PgR expression and HCV infection [23], which represents an increasing aetiology of HCC in our patient's population [3].
Several data suggest that blocking AR may be interesting in HCC therapy. Studies in animal models suggest that increased hepatic AR expression correlated with accelerated tumor development [48], or even AR expression was associated with intrahepatic recurrence in HCC [49]. However, AR expression has also been reported to be associated to small tumor size, but not with a higher rate of recurrence [28]. More recent data indicate that suppression of androgen or AR signals also led to an increase in the number of infiltrating cells, such as macrophages as well as B or T-cells [50], which shows diverse and important roles in the promotion of HCC tumorigenicity [51]. Although there is few data on the effect of antiandrogens in HCC patients, it has been suggested that anti-androgen therapy may have some benefit in patients with androgen-positive tumors [52]. Likewise, recently it has been reported that a multicenter trial with anti-androgens in HCC male patients, has been interrupted because of digestive side effects [39]. Nevertheless, further studies will be necessary to assess whether AR status may be a useful marker to select more accurately candidate patients to further anti-androgenic therapies. Notably, it has been reported that expression of androgeninduced proteins, such as apolipoprotein D or Zinc-alpha2-glycoprotein, is associated with poor prognosis in HCC.
Apolipoprotein D (ApoD) is an androgen-induced protein increased in both prostate and breast cancer cells [53][54][55]. A report of Utsunomiya et al. showed that low ApoD expression correlated significantly with less-differentiated HCCs and therefore with a worse prognosis of patients [56]. However, we did not found any relationship between ApoD expression and the hormonal receptor status in HCC [23]. Nevertheless, further studies will be also necessary in order to assess the possible value of ApoD as a biological marker of androgen response and/ or other hormonal pathways in HCC. Thus, with regard to this latter aspect, although ApoD is an androgen-regulated protein, it can also been induced by other hormonal steroids or substances such as glucocorticoids, retinoic acid or 1,25-dihydroxyvitamin D 3 , that might be involved in the regulation of this ApoD in human tumors [54,[57][58]. This is relevant since it has been described that both retinoid and glucocorticoid receptors have been also detected in HCC [59][60], and that preliminary data show that retinoids are considered of clinical interest as cancer chemotherapeutic agents in advanced HCC [61].
Zinc-alpha2-glycoprotein (ZA2G) is also an androgen-induced protein increased in breast cancer [58,62]. Recently, ZA2G expression was found to be decreased in HCC tissues at both mRNA and protein levels. Moreover, low expression of ZA2G was notably more prevalent in patients with poor tumor differentiation, advanced liver cirrhosis, high serum alpha fetoprotein (AFP) level and shorter survival time. These results suggest that ZA2G may be a promising novel prognostic biomarker for HCC [63], and advocate for the need for validation studies.  Recent data indicates that estrogens attenuate tumor progression in HCC in vivo by reducing tumor cell invasion, arresting cell cycle progression, and promoting apoptosis, which is characterized by an increased expression of cleaved caspase-3, and by a decrease in the expression of Proliferating Cell Nuclear Antigen (PCNA), cyclin A, cyclin D1, Bcl-2, and matrix metalloproteases (MMP) 2 and 9 [64]. It is relevant the data indicating that steroid hormones, such as estrogens, may inhibit MMPs in several experimental models [65][66][67]. This may be of great importance due to MMPs are proteolytic enzymes which participate in the degradation of the stromal connective tissue and basement membrane component, therefore facilitating tumor invasion and metastasis. Here, we show a negative relationship between ER expression and MMP2, MMP-9 or MMP-11 expression in HCC ( Figure 1).

Metalloproteases and their inhibitors
Degradation of the stromal connective tissue and basement membrane components are key elements in tumor invasion and metastasis. Some components of the extracelluar matrix, particularly interstitial collagens, are very resistant to proteolytic attacks, being degraded only by matrix metalloproteases (MMPs) [68].
There are few available data referring to the integrated expression of these factors in relation with HCC. In this context, we have described new findings about MMPs and TIMPs expressions in HCC, together with an important expression by tumor stromal cells, as well as their clinical relevance. It is known that MMPs expression in neoplastic tissues is high due to regulation, in a paracrine manner, by growth factors and cytokines secreted by either tumor or stromal cells [106]. Nevertheless, high MMPs and TIMPs expression by HCCs tissues may be also due to the interplay between transformed cells and their microenvironment, particularly the surrounding extracellular matrix. In human livers, fibrogenesis underlies the development of HCCs in at least 90% of cases [107], and HCC is typically surrounded by a fibrous capsule at an early stage [108]. In addition, several studies have shown that some MMPs (MMP-1, -2, -9 and -13) and TIMPs (TIMP-1 and -2) are involved in the liver fibrosis processes [90,[109][110][111][112]. It has been demonstrated that extracellular matrix deposition induces overexpression of MMPs and TIMPs, such as MMP-2, secreted by human mesenchymal cells but also by hepatoma cells [87].
Recently we reported that immunostaining for MMPs and TIMPs was localized predominantly in tumor cells, but also in peritumor stromal cells in a significant percentage of cases. However, we found no stromal expression of MMPs or TIMPs in normal liver samples [113]. We also found a positive and noteworthy association between MMP-1 expression by fibroblasts or by mononuclear inflammatory cells (MICs) and a larger tumor size. In addition, our data showed that MMP-1 expression by stromal fibroblasts, as well as the expression of MMP-13, TIMP-1 and 2, by MICs, were significantly associated with a shorter overall survival (Figure 2) [113]. There are discrepancies between different studies regarding the prognostic significance of several of these factors in HCC, such as MMP-9 and MMP-2 expression in stromal compartments [113,93]. Nevertheless, it is worth considering the existence of possible differences in MMPs or TIMPs expression between different patient's populations. Thus, for example, in Asian countries HCC originates more frequently in healthy liver tissues, with less incidence of cirrhosis, than in Europe, whereas in the Mediterranean area as well as in the North American countries liver HCC only develops in chronically injured livers, where altered turn-over and increased deposition of ECM proteins has been described. In a such environment, upregulation of MMPs and TIMPs has been reported and it is possible for them to play an important role in the rearrangement of the liver tissue architecture an aspect that could influence the different expression pattern of MMPs and TIMPs in stromal HCC cells [110,114].
On the other hand, a decrease on TIMP-2 and -3 expression has been reported to be associated with invasion and metastases in HCC [115][116]. Certainly, if TIMPs inhibit MMPs in vivo, it should be expected that high levels of these inhibitors would prevent tumor progression and thus to be related with good outcome in cancer patients. However, TIMPs are multifunctional proteins that in addition to its MMP-inhibitory effect also shown distinct tumor-stimulatory functions, such as the induction of proliferation and the inhibition of apoptosis [84]. Thus, it is of note that TIMP-1 and -2 expressions by stromal cells were associated with shortened overall survival [113]. Accordingly, it has been reported that TIMP-1 overexpression leads to an increase of hepatoma cells migration [117], and also that it is associated with invasion and metastases in HCC [115,117]. The unsupervised hierarchical cluster analysis of MMPs/TIMPs expressions in HCCs led us to identify 2 well-defined clusters of cases ( Figure 3): one with low expression of MMPs and TIMPs (Group 1), and another with high expression of these factors (Group 2). This classification could have biological interest in order to identify patient candidates to new therapies based on enzymatic MMP inhibition. With regard to this, although there are no published data about use of MMP inhibitors in HCC, recently it has been reported that decreased MMP activity mediated by statins reduced progression and limits metastatic diffusion of established HCC [118]. Likewise, it was demonstrated that blocking the tumor-related glycoprotein HAb18G/ CD147 by gene silence in HCC cells or with HA18 monoclonal antibody, resulted in a suppressive effect on MMP secretion and cell invasion [119]. More recent studies have shown an antimetastatic effect of Norcantharidin on HCC by transcriptional inhibition of MMP-9 [120], as well as a metastasis inhibition of HCC cells due to down-regulation of Osteopontin via a mechanism involving MMP-2 and urinary plasminogen activator (uPA) [121].

Conclusion
In summary, we consider that expression analysis of steroid hormone receptors, MMPs and TIMPs, contributes to a better knowledge in the biological characterization of HCC and highlights the need for further studies exploring new therapeutic targets for this common tumor.