Abstract
Background/Aim: The role of androgen receptor (AR) in hepatocellular carcinoma (HCC) development is controversial. Therefore, the translational value of targeting AR in HCC is unknown. Sorafenib, a multiple kinase inhibitor, is the standard therapy for patients with unresectable HCC. This study investigated sorafenib effect on AR in experimental models of HCC. Material and Methods: AR cDNA was introduced into HCC cells and in vitro cell growth and in vivo tumor growth were measured. Sphere cells, as well as epithelial cell adhesion molecule-positive (EpCAM+) and CD133+ cells were isolated from HCC cells with/without AR expression to observe in vitro/in vivo effects. Liver specific AR knockout in mouse models of spontaneous HCC (carcinogen-induced and hepatitis B virus-related HCC) was also implemented to examine gene expression. HCC cells/tumors were treated with sorafenib in order to determine effects on tumor growth and related gene expression. Result: AR cDNA increased transactivation function, increased colony/sphere-forming activities, and enhanced tumorigenicity in HCC cells compared to their parental cells. Expression of the stemness marker EpCAM was also dramatically increased. In carcinogen-and HBV-induced HCC models, EpCAM+ cells were significantly reduced in AR-knockout mice compared to wild-type HCCs. In addition, AR reduced sorafenib-related signals, e.g. extracellular-regulated kinase, AKT serine/threonine kinase 1, and p38 mitogen-activated protein kinase, compared to that in parental cells. Regarding sorafenib cytotoxicity, AR-expressing cells were vulnerable to treatment. Moreover, the half maximal-inhibitory concentration (IC50) was drastically lowered in AR+/EpCAM+ compared to AR−/EpCAM− sphere cells. Strikingly, the IC50. in AR+/CD133+ vs. AR−/CD133+ cells were similar. Moreover, sorafenib robustly suppressed tumor growth in implanted AR+/EpCAM+ cells but not AR−/EpCAM− ones. Finally, bioinformatics analyses revealed EpCAM to be a prognostic biomarker in Asian and non-alcohol-consuming patients with HCC, suggesting suitability of a sorafenib regimen for such patients. Conclusion: AR+/EpCAM+ may be a marker of responsiveness to sorafenib for patients with HCC. Prospective surveys associating AR/EpCAM expression with therapy outcomes are essential.
Epidemiological research has revealed a number of factors involved in the carcinogenesis of hepatocellular carcinoma (HCC), including hepatitis B or C virus infection, alcohol consumption, genotoxic toxin ingestion (e.g. aflatoxin B1 ingestion), metabolic syndrome (or non-alcoholic steatotic hepatitis), and male sex, among others (1-3). Men are predominant in HCC populations, with one factor partially responsible for this predominance being the relatively high levels of androgen and the expression/genomic polymorphism of androgen receptor (AR) in men (4-7). However, studies have revealed somewhat paradoxical roles of androgen/AR in the progression and therapy responses of HCC (8, 9). Specifically, they appear to promote the initiation of HCC while also suppressing its progression (9-11). That said, while these biological roles of AR in HCC have been demonstrated, their translational value remains unknown.
Clinical strategies for combating HCC include combined adjuvant chemotherapy, the surgical blockade of blood flow in the liver portal veins, and hepatectomy (12). Surgical treatment remains the most effective treatment in terms of prolonging survival and managing HCC but only a low percentage (~30%) of patients meet the criteria for such treatment (12, 13). Moreover, the rate of recurrence in patients post-hepatic surgery unfortunately remains quite high (14-16). In fact, the rates of HCC recurrence within 3 years are quite high both in patients treated with hepatectomy (10-50%) and patients treated with liver transplantation (20-60%). Worse still, there was no available effective adjuvant therapy (17) for patients with unresectable HCC prior to 2008(12, 18).
Sorafenib (Nexavar), an anti-angiogenesis multiple kinase blocker, is recognized as the safest and most durably effective molecular targeting adjuvant therapy to have passed a phase III clinical trial (19, 20). The molecular signaling inhibition by sorafenib targets the serine–threonine kinases RAF proto-oncogene serine/threonine-protein kinase and serine/threonine-protein kinase BRAF, as well as the receptor tyrosine kinase activity of vascular endothelial growth factor receptors 1, 2 and 3, and platelet-derived growth factor receptor β (20-22). Sorafenib has been reported to prolong patient survival by up to 10 months (20); however, clinical trials conducted in various areas have reported conflicting results (23, 24), with the overall survival benefit ranging from 1-6 months depending on the trial area (25, 26). As such, researchers are eager to find ways to improve sorafenib efficacy, whether by identifying suitable recipients by distinguishing biomarkers or by boosting the sensitivity to sorafenib of those receiving the treatment.
Stem cells are defined as undifferentiated cells with replicative potential which can differentiate into multiple cell types. Adult stem cells have been identified in tissues with fast turnover, e.g. gastrointestinal tract, skin, and bone marrow tissues. During acute liver injury, the fast growth of liver parenchymal cells does not involve stem cell activation (27). However, during chronic liver damage, e.g. fibrosis, hepatitis, cirrhosis, and cancer, the resulting oncogenic and inflammatory signals do wake up quiescent stem cells (28). Moreover, the full spectrum of cell stages from carcinoma stemness to differentiation can be identified by detecting the expression of surface markers on cells. A range of previous studies have reported a variety of surface markers in HCC that reflect a variety of clinical scenarios. For example, the CD133, CD13, CD90, CD24, CD44, epithelial cell adhesion molecule (EpCAM), oval cell markler 6, and aldehyde dehydrogenase gene families have variously been identified as markers of carcinogenesis, progression, or therapeutic response of HCC in patients (29-31).
In the current study, we examined the role AR plays in HCC cellular function, including stemness marker expression, and the responsiveness to sorafenib treatment.
Materials and Methods
Bioinformatics analysis using Kaplan–Meier Plotter RNA sequencing dataset-based progression analyzer in patients with HCC. In order to determine the associations between gene expression and HCC cancer progression, the Kaplan–Meier Plotter RNA sequencing dataset-based progression analyzer was used to analyze overall survival (OS) (http://kmplot.com/analysis/index.php?p=service&cancer=liverrnaseq) (32). OS was assessed in HCC cohorts and stratified by median classifier expression. The analyzed HCC cohorts included all the investigated patients (non-classified; n=364), Caucasians (n=184), Asians (n=158), males (n=250), females (n=121), those with hepatitis virus infection (n=153), those without hepatitis virus infection (n=169), and patients who did not consume alcohol (n=206). The input genes and classifiers used were AR (#367), EpCAM (#4072), and CD133 (PROM1; #8842).
Production of HCC and HBV-HCC wild-type vs. hepatocyte ARKO mice (6, 9). The Guidelines for the Care and Use of Laboratory Animals (Ministry of Sciences and Technology, Taiwan) were followed in performing animal experiments, which were approved by the China Medical University Committee of Laboratory Animal Welfare. Liver-specific ARKO (LARKO; albumin-Cre recombinase transgene-driven AR deletion) and HBVtg-LARKO (albumin-Cre recombinase transgene-driven AR deletion in HBVtg mouse) mice, as well as the spontaneous HCC mouse model, were generated using previously described protocols (6, 7, 9).
Cell lines. Human HCC cell lines (HCC, Huh7, and Tong) and the human embryonic kidney cell line (HEK293T) were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher, Taichung, Taiwan, ROC) with 10% fetal bovine serum (FBS; Thermo Fisher) and 1% penicillin/streptomycin (Thermo Fisher) at 37°C in a humidified atmosphere of 5% CO2. The cells were then cultured in fresh DMEM/10% charcoal-dextran-stripped FBS for 24 h before the experiments. Sorafenib was purchased from LC laboratories (Boston, MA, USA).
Immunoblotting assay. Protein extraction and the immunoblot assay were performed as previously described (33). In brief, HCC 36 cells (treated with/without 5 μM sorafenib for 24-h were washed with 1xPBS and lysed in RIPA buffer (100 mM Tris, 5 mM EDTA, 5% NP40; pH8.0) with protease inhibitors (1 mM phenyl-methyl sulphonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin). Total proteins were resolved with and then transferred to membranes. Non-specific binding was blocked by 5% non-fat milk. After incubation with primary antibodies against AR, (N-20; Santa Cruz, Taichung, Taiwan, ROC), GAPDH (Santa Cruz), β-actin (Santa Cruz); E-cadherin, vimentin, p38 mitogen-activated protein kinase (MAPK), phospho-p38, AKT serine/threonine kinase 1 (AKT), phosphor-AKT, extracellular signal-regulated kinase (ERK), phosphor-ERK: Cell Signaling, (Taichung, Taiwan, ROC), secondary antibodies (1:3000; HRP-goat-anti-mouse and HRP-goat-anti-rabbit) were applied for 1 h at room temperature. Signals were enhanced using an ECL chemiluminescence kit (Millipore, Taichung, Taiwan, ROC) and detected by ChemiDoc TM XRS+ (BioRad Laboratories Inc., Taichung, Taiwan, ROC).
Transfection and lentivirus infection procedures. The lentiviral production and infection procedures were carried out as reported previously with minor modifications(34). In brief, cells were transfected with the following lentivirus plasmids: psPAX2 packaging plasmid, pMD2G envelope plasmid (Addgene, Taipei, Taiwan, ROC), pWPI-vector ctrl, and pWPI-hAR (Addgene). Lentiviral plasmids were co-transfected with psPAX2 and pMD2G into HEK293T cells at a ratio of 3: 2: 4 by lipofectamine 2000 (Invitrogen, Taipei, Taiwan, ROC) according to the manufacturer's instructions. After 6 h, the medium was replaced with fresh DMEM/10% FBS, and the cells were maintained at 37°C in a humidified incubator in an atmosphere with 5% CO2 for 48 h. Viral-containing medium was collected by centrifugation and filtered through a 0.45 μm filter. Polybrene (Sigma-Aldrich, Taichung, Taiwan, ROC; 0.8 mg/ml) was then added to culture medium and added onto 106 cells. After 16 h of infection, the medium containing virus was replaced with fresh DMEM/10% FBS, and the cells were maintained at 37°C in a humidified incubator in an atmosphere with 5% CO2 for 48 h. The infected cells were then collected and analyzed.
Total RNA isolation, cDNA synthesis, and quantitative real-time polymerase chain reaction (PCR). RNA extraction from HCC cells was reported previously (35) and modified. In brief, cells reached 80-90% confluence in 100-mm dishes and were then lysed with 1 ml Trizol (Invitrogen). Phenol/chloroform was then added for phase separation, and RNA-rich layers were separated by centrifugation. Soluble RNA was precipitated with 2-propanol. The salt was washed away with 75% ethanol and RNA was dissolved in RNase-free water. First-strand cDNA synthesis using 5 μg of total RNA was then reverse transcribed by PCR by PrimeScript™ RT reagent kit (TAKARA Bio Inc., Kyoto, Japan). cDNA was synthesized followed the manufacturer's instructions. A real-time detection system (Bio-Rad Laboratories, Inc.) and the KAPA™ SYBR FAST One-Step qRT-PCR Kit (Kapa Biosystems, Inc., Wilmington, MA, USA) were used according to the manufacturers' instructions. Relative gene expression was determined by normalizing the expression level of the stem marker genes to the expression level of housekeeping gene β-actin (ACTB). Threshold value (Ct) dynamics were used (2−ΔΔCt) for quantitation of gene expression.
Luciferase reporter assay. The luciferase reporter assay was performed as previously described (35). In brief, androgen response element-driven luciferase reporter plasmid (pGL3-ARE) and thymidine kinase promoter-driven renilla luciferase plasmid (pRL-TK) were co-transfected transiently into HCC cells. The medium was replaced with fresh medium and 10% charcoal-dextran treated-FBS after 6-h of transfection. After that, cells were treated with or without 5α-dihydrotestosterone (DHT) (10 nM) for 24 h. Then cells were washed with 1xPBS and incubated in the presence of 100 μl cell lysis reagent (GeneLab Life Science Let., Taipei, Taiwan, ROC) for 30 min at room temperature. The cell lysates were then placed in a plastic tube and centrifuged at 10,000 × g for 5 min. Supernatant (5 μl) was then mixed with 50 μl luciferase assay reagent. Luciferase activity was measured immediately using a luminescence microplate reader and data are presented as relative luminescence units.
In vitro cytotoxic, colony formation assay, sphere formation assays, and sorafenib treatment. Cell viability assay, colony-formation assay, and sphere-formation assay were performed following published work (36, 37). WST-1 reagent (water-soluble tetrazolium salt; Roche, Basel, Switzerland) was used to assess cell viability. Firstly, 4×103 cells/100 μl/well seeded onto 96-well plates with DMEM/10% FBS. The cells were then treated with sorafenib (0, 5, 10, 20 μM) for 48 h. Next, 10 μl of WST-1 solution was added to each well, and plates were incubated for 1 h. Sorafenib cytotoxicity was quantified by colormetry using an enzyme-linked immunosorbent assay plate reader (Coulter Paradigm™ Detection Platform; Beckman, Taipei, Taiwan, ROC) at an absorbance of 450 nm and 690 nm to generate an optically densitometric value proportional to the abundance of live cells in the wells. For the colony-forming assay, 1.5×103 cells/well were seeded onto 6-well plates and incubated for 8 days. One milliliter of 4% formaldehyde solution was added to fix the cells, and the cells were allowed to incubate at room temperature for 1 h. Crystal violet staining was then performed. After 1 h, excess crystal violet dye was washed away, and the colonies were photographed and counted. For the sphere-forming assay, cells were collected and washed to remove serum and then suspended in serum-free DMEM supplemented with 20 ng/ml human recombinant epidermal growth factor (hEGF), 10 ng/ml human recombinant basic fibroblast growth factor (hrbFGF), 5 μg/ml insulin, and 0.4% bovine serum albumin (Sigma). The cells were subsequently cultured in ultra-low attachment 6-well plates (Corning Inc., Corning, NY, USA) at a density of less than 5,000 cells/well for 14 days. Spheres were observed under a microscope, and were photographed.
Immunohistochemistry and quantitation of staining score. The HCC histological studies from mouse models were performed as described in previous study (11) with modifications. For histologicaI inspection, tissue sections (2 μM) were started with hematoxylin and eosin or with EpCAM-specific antibodies, using ABC kit (Vector Laboratories; Taichung, Taiwan, ROC) to enhance the staining signals. The staining scores of EpCAM in label-blinded slides were determined by two co-authors inspecting independently. The scoring for immunohistochemistry was compared between genotypes (wild-type vs. LARKO).
Stem cell implantation for tumorigenicity, and xenograft tumor model for sorafenib growth suppression. The xenograft tumorigenesis method was carried out as reported in previous work with modifications (35). Six-week-old male athymic nude mice (CB17/Icr-Prkdcscid/Cr1Nar1) were purchased from the National Laboratory Animal Center (Taipei, Taiwan, ROC). For the effect of AR expression on tumorigenicity in mice, HCC36 cells (102 or 105 cells/implant site) were implanted into both flanks of athymic nude mice to observe tumor formation. For evaluation of sorafenib therapeutic efficacy, HCC36 parental and AR-overexpressing cells (5×106/site) were subcutaneously implanted into both flanks in each mouse. As the tumor volume reached 300 mm3, the mice were randomized into two groups (placebo vs. 30 mg/kg sorafenib). All treatments were given intra-peritoneally three times a week for 4 consecutive weeks. Tumor length and width as determined by caliper and mouse body weight were measured twice weekly. When the mice were sacrificed, the tumor volume (TV) was calculated as follows: TV=(tumor width2 × tumor length)/2.
Androgen receptor (AR) cDNA promotes hepatocellular carcinoma (HCC) cell growth and stemness. A: Western blotting showed that AR expression was significantly higher in HCC cells infected with AR cDNA (pWPI-hAR; AR) than in parental cells (pWPI vector; par). Actin served as an internal control. B: The androgen/AR axis was intact in HCC cells. 5α-Dihydrotestosterone (DHT) treatment (10 nM) turned on the AR transactivation function in Huh7, Tong, and HCC36 cells. An androgen response element (ARE)-luciferase assay was performed in DHT-treated HCC cells. AR transactivation activity was absent in control (EtOH) cells and vector control cells (par), whereas luciferase activity was robust in AR-overexpressing HCC cells. Thymidine kinase promoter-driven Renilla construct (pRL-TK) was co-transfected and measured as a transfection control. The relative ARE-luciferase activity (fold to EtOH/par) is presented as the transactivation activity of AR. C: AR cDNA facilitated cancer growth using colony-forming assay. The cells were seeded on plates and grown for 2 weeks allowing colonies to form. The absolute colony number was counted. D: AR cDNA facilitated cancer cell stemness, as demonstrated by sphere-forming assay. Cells were seeded on plates and grown for 2 weeks under unattached and low nutrition/growth factor conditions, allowing spheres to form. The absolute sphere number was then counted. All the data/values come from three reproducible experiments. Significantly different at *p<0.05, and ***p<0.001.
Statistical analysis. Statistical analyses were performed using Student's t-test. All experiments were repeated at least three times, and p-values less than 0.05 were considered to indicate statistical significance.
Results
AR promotes HCC cancer growth and stemness, and enriches EpCAM+ population. To assess the effects of AR on HCC cells, exogenous AR cDNA was stably introduced into three HCC cell lines (HCC36, Huh7, and Tong). AR expression was then detected using immunoblot assays (Figure 1A), which revealed the robust expression of AR in the AR-transfected cells. In order to evaluate whether AR exerts a complete transactivation function, ARE-driven luciferase reporter assay was performed. DHT, an AR ligand, was used to co-treat both parental control and AR-expressing cells to measure luminescence. Higher luminescence levels were detected in the AR-overexpressing HCC cells compared to the parental cells (Figure 1B). These data demonstrated the feasibility of using cell-based tools for examining AR functions in additional assays. We then tested the effects of AR on cancer cell colony-forming (Figure 1C) and sphere-forming (Figure 1D) capacity in the HCC cells, and found that AR also promotes cancer cell growth. To further test the effects of AR in terms of tumorigenicity, we subcutaneously implanted 102 and 105 cells/site into the flanks of nude mice. The tumor incidence at 3, 6, 9, 12, and 15 weeks was recorded, and it was found that only the AR-overexpressing HCC cells exhibited high levels of tumorigenicity Table II); tumors could not be found at the sites implanted with parental cells. These data suggested that AR expression facilitates HCC tumorigenesis.
Sorafenib the half maximal-inhibitory concentration (IC50) (mM) of parental and androgen receptor (AR)-overexpressing hepatocellular carcinoma cells.
In order to verify the effects of AR on tumorigenicity and stemness, EpCAM expression levels were examined (Figure 2A). We found that EpCAM expression was up-regulated under regular culture conditions with AR expression. However, EpCAM expression was more robustly up-regulated in sphere culture conditions. Furthermore, we also examined the EpCAM expression levels in HCC lesions from two spontaneous HCC mouse models, HBVtg-HCC and DEN-induced HCC, with liver-specific AR knockout (Figure 2B). EpCAM staining was detected in both wild-type and liver-specific AR-knockout lesions (Figure 2B); however, the staining score was significantly lower in AR-knockout lesions compared to wild-type lesions (Figure 2C and D).
The AR-enriched EpCAM+ population is vulnerable to sorafenib treatment in vitro and in vivo. Sorafenib is considered the standard treatment for the management of late-stage or unresectable HCC. We therefore tested the effects of AR on the downstream signaling, e.g. ERK, AKT, and p38MAPK signaling, targeted by sorafenib (Figure 3A). We found that sorafenib treatment resulted in the up-regulation of AKT and ERK phosphorylation, but not p38MAPK phosphorylation. This finding was consistent with the past literature regarding sorafenib resistance mechanisms (38, 39). Interestingly, in AR-expressing HCC cells, sorafenib-induced AKT and ERK phosphorylation was significantly reduced or abolished. Therefore, we were interested in determining whether AR expression results in increased sensitivity to the cytotoxic effects of sorafenib. The results showed that AR-expressing HCC cells exhibited increased vulnerability to sorafenib treatment (Figure 3B), with the IC50 value for sorafenib dropping by around two-to three-fold (Figure 3).
As cancer stem cells exhibit multiple biological characteristics, including therapy responsiveness, we tested the cytotoxic effects of sorafenib on the spheres of AR-overexpressing and vector control cells. Surprisingly, we found that AR overexpression led to even greater sensitivity to sorafenib treatment (Figure 4). After sorting out the EPCAM+ cells to compare the cytotoxic efficacy of sorafenib against those cells with its efficacy against the residuaI cells (EPCAM−), we found that the IC50 value for sorafenib was robustly reduced in AR-expressing cells (Table I). In contrast, when we used cell sorting to compare the cytotoxic efficacy levels of sorafenib against cells expressing CD133 antibody (CD133+) compared with CD133− cells, we did not observe such a difference in efficacy (Table II). To verify our in vitro observations regarding enhanced sensitivity to sorafenib due to AR in an animal model, we examined the tumor-suppressive effect of sorafenib in HCC36 tumor-bearing mice. As shown in Figure 4, the HCC36-AR implanted xenograft tumors almost vanished under low-dose (30 mg/kg, 3 times/week for four consecutive weeks) sorafenib treatment. However, the HCC36 tumors resulting from implanted parental cells grew larger under the same treatment regimen.
Parental (par) and AR-overexpressing (AR) HCC cells (102 and 105) were subcutaneously implanted into both flanks of each mouse (n=12). The tumor size and tumor incidence were determined at the indicated times. All the data come from three reproducible experiments.
AR+ is a favorable therapeutic marker for HCC prognosis. The online Kaplan–Meier Plotter transcriptome database is an excellent tool for determining associations between gene expressions and patient prognoses (32). It allows for retrospective analyses of the gene expressions of tumor lesions using either cDNA microarray or RNA sequencing technology, including determination of the associations with multiple clinical modalities, including demographic characteristics and therapeutic regimens. With the Kaplan–Meier Plotter we were able to associate AR/EpCAM expression and the prognoses of patients with HCC who received standard care, which enabled us in turn to identify populations that would potentially be better suited for treatment with sorafenib. Interestingly, we found the AR expression level to be a prognostically favorable biomarker in almost all clinical modalities (Table III) This finding was compatible with our findings that AR expression is beneficial against HCC progression (9, 11). AR enriches the EpCAM+ population and, as revealed in Figures 3 and 4, facilitated the cytotoxic efficacy of sorafenib. When we analyzed the association of EpCAM expression with patient prognoses, we found EpCAM to be an unfavorable marker for Asian patients (Figure 5A) and non-alcohol-consuming patients (Figure 5B). Due to the importance of EpCAM expression in these patients, sorafenib regimens might be beneficial to them.
Demography of Kaplan–Meier Plotter data for overall survival of patients with hepatocellular carcinoma in relation to the effect of high expression of androgen receptor (AR) and epithelial cell adhesion molecule (EpCAM) on overall survival (data were modified from http://kmplot.com/analysis/index.php?p=service&cancer=liver_rnaseq).
Finally, the overall study revealed a translational value insofar as it showed that AR promotes stemness, specifically, the expression of EpCAM, such that AR expression might be a new indicator for sorafenib regimen efficacy in HCC therapy.
Discussion
Translational significance of bimodal role of AR in HCC progression. While the majority of previous studies(6, 7, 40, 41) have stated that androgen/AR signaling plays a positive role in carcinogenesis, antiandrogen clinical trial have failed to support this (8). In addition, our team has collected multiple forms of evidence demonstrating that the expression of AR itself suppresses HCC metastasis in a liver-specific AR-knockout HCC mouse model (9), inhibits cell mobility through β1-integrin (10), and reduces post-surgery recurrence risk through the suppression of CD90+ population of circulating cancer cells (11). The bimodal role of AR, in which it promotes tumor growth while also suppressing metastasis, has been confirmed in the past decade(1, 10, 11). Therefore, researchers have been unsure about the translational significance of AR for HCC therapy, namely whether targeting AR would suppress or enhance the efficacy of such therapy. In the current study, we found that AR not only enriched the EpCAM+ HCC population but also enhanced the sensitivity of HCC cells to sorafenib. Considering that sorafenib is the approved standard therapy for unresectable HCC, we suggest the populations of patients regarded as suitable for sorafenib treatment might also be extended to Asian and non-alcohol-consuming patients. The expressions of AR and EpCAM might also serve as biomarkers for therapy-related decision making.
Targeting AR for sorafenib treatment. Since the bimodal role of AR has been verified, clarification of the underlying mechanism has been pursued from various perspectives. Cisplatin, an adjuvant chemotherapeutic for HCC, was suspected of exerting its anti-HCC effects through the down-regulation of AR and cytokine production, which would in turn cause it to enhance the cytotoxic capacity of infiltrating natural killer cells (42, 43). On the other hand, an AR-enhancing strategy involving the use of miR-367-3p combined with sorafenib provided a therapeutic benefit in a laboratory model (44). The underlying mechanism was the influence of mouse double minute 2 homolog (MDM2) stability related to AR ubiquitination and dephosphorylation of AKT and ERK (44). That was consistent, in turn, with our finding that AR increased sensitivity to sorafenib. In addition, one recent study discovered that AR/miR-520f-3p/SRY-box transcription factor 9 signaling enhanced the sensitivity of HCC cells to sorafenib under hypoxia (45). Interestingly, the data from that study showed that EpCAM was super-expressed under hypoxic conditions, which thus linked such conditions to AR expression. Although that study did not distinguish the effects of sorafenib on EpCAM+/AR+ cells, their data showed strong consistency with the data of the present study. Another previous study summarized the influence of AR in stem cells (46). As discussed, AR plays a suppressor role in the self-renewal of embryonic stem cells (47, 48) but it also promotes the differentiation of embryonic stem cells into cardiomyocytes. Another example relates to the prostate. AR promotes normal prostate stem/progenitor cells (49); however, it suppresses cancer stem cells of the prostate (50). There are other examples of the bimodal role of AR, e.g. its effects on bone marrow stem cells or hematopoietic stem cells (46). In this study, we examined the functions of AR in HCC stem cell populations. We found that AR enriched the EpCAM+ population in HCC. Furthermore, contrary to expectations, we found that this AR+EpCAM+ population is vulnerable to sorafenib treatment.
Androgen receptor (AR) enriches epithelial cell adhesion molecule-positive (EpCAM+) population in hepatocellular carcinoma (HCC) cells. A: AR cDNA-transfected HCC cells expressed higher levels of EpCAM mRNA than did the parental cells in regular adherent and sphere culture conditions. B: EpCAM expression was higher in wild-type tumor lesions than in the lesions of liver-specific AR-knockout littermates in two mouse models of spontaneous HCC. The left panels show HBVtg-HCC liver tumors, and the right panels show carcinogen (DEN)-induced HCC tumors. The upper panels show wild-type (WT) HCC, whereas the lower panels show liver-specific ARKO (LARKO) HCC. C: Quantitation of EpCAM immunohistochemistry staining shown in (B). Significantly different at *p<0.05, **p<0.01, ***p<0.001.
Androgen receptor (AR) reduces sorafenib downstream signaling and enhances sensitivity to sorafenib. A: The downstream signaling molecules affected by sorafenib, e.g. p38 mitogen-activated protein kinases (p38MAPK), protein kinase B (AKT), and extracellular signal-regulated kinase (ERK) and phosphorylation signaling was measured by western blot assay. Sorafenib treatment increased AKT and ERK phosphorylation, whereas AR expression reduced that effect of sorafenib. B: The cytoxicity of sorafenib was assayed in three HCC cell lines with (green line) or without (red line) AR expressions. The half maximal-inhibitory concentration (IC50) values for sorafenib are presented. All the data were from at least three reproducible experiments. Significantly different at *p<0.05, **p<0.01.
In conclusion, this study found that even though AR promotes cancer stemness, it enhances the efficacy of sorafenib treatment. In addition, AR+/EpCAM+ might be a good biomarker for making decisions regarding sorafenib regimens for Asian and non-alcohol-consuming patients with HCC. Future prospective studies are still needed, however, to determine the value of AR+/EPCAM+ as a biomarker for sorafenib interventions.
The Author are grateful to George W. Whipple Distinguished Professor Dr. Chawnshang Chang for providing AR flox transgene mice for this study.
Androgen receptor (AR) enriches epithelial cell adhesion molecule-positive (EpCAM+) population for increased sensitivity to sorafenib treatment. AR+/EpCAM+ and AR−/EpCAM− HCC36 sphere cells were implanted into both flanks of each mouse (n=5 for each group). Each tumor grew to 400 mm3, after which the low-dose treatment regimen (30 mg/kg/mouse, 3 times/week for 4 consecutive weeks) was started. The appearances of the mice with double-positive vs. double-negative tumors differed. The tumors had nearly vanished in the mice with AR+/EpCAM+ hepatocellular carcinoma implants, while the mice with AR−/EpCAM− tumors presented large tumors in spite of being treated with the sorafenib regimen.
Kaplan–Meier plots of overall survival of Asian patients with hepatocellular carcinoma according to expression of epithelial cell adhesion molecule (EpCAM) (A) and of Asian non-alcohol-consuming patients with hepatocellular carcinoma according to androgen receptor (AR) expression.
Acknowledgements
This work was partially supported by the Taiwan Ministry of Science and Technology (MOST108-2320-B-039-017; MOST107-2314-B-039-011; MOST108-2314-B-039-052; MOST108-2314-B-039-043-MY3); the National Health Research Institute (NHRI-EX109-10705BI); and China Medical University/Hospital (CMU107-S-05; CMU106-S-28; CMU107-TC-02; DMR-108-080; DMR-108-179).
Footnotes
↵* These Authors contributed equally to this study.
Authors' Contributions
HC Lai and WM Chung executed experiments and drafted the article. CM Chang, PY Liao, YT Su executed partial in vitro and in vivo experiments. CC Yeh and LB Jeng edited the article and provide translational suggestions. WL Ma and WC Chang initiated, supervised and supported the entire project. WC Chang approved the final article.
Conflicts of Interest
All the Authors declare no conflicts of interest in this work.
- Received January 20, 2020.
- Revision received February 11, 2020.
- Accepted February 14, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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