Phenethyl isothiocyanate inhibits CD133+/CD90+ liver cancer stem cells by modulation of microRNA‐214‐β‐catenin epigenome axis

Hepatocellular carcinoma (HCC) represents one of the most prevalent and lethal type of malignancies around the globe. Despite the advancement in medical research and therapeutics development, HCC still remains a taunting challenge in clinical settings. Recent studies indicate that the presence of cancer stem cells (CSCs) may be the underlying factor for treatment failure, distant metastasis, and disease recurrence. Elevated stemness gene expression has been correlated to disease stage and poorer prognosis in HCC patients. Initially, we established that β‐catenin is highly expressed in HCC clinical samples. We subsequently re‐validated the idea that CD133+/CD90+ subpopulation cells exhibited CSCs properties including elevated stemness expression (β‐catenin, Nanog, c‐Myc, and Twist1), increased self‐renewal capacity and metastatic potential. Using this cell model, we tested the potential anti‐CSCs effects of phenethyl isothiocynanate (PEITC), a phytochemical isolated from cruciferous vegetables. Treatment of PEITC led to a decreased percentage of CD133+/CD90+ cells in both Huh7 and Sk‐Hep1 cell lines. In addition, PEITC suppressed stemness gene expression, self‐renewal ability, and metastatic potential in HCC CSCs. Mechanistically, PEITC conveyed its anti‐CSCs effects via upregulating microRNA‐214, a negative regulator of β‐catenin. In conclusion, we provided evidence that PEITC could suppress HCC CSCs generation/maintenance. With further clinical testing, PEITC could be used either alone or in combination with currently available chemotherapeutic agents to achieve improved efficacy.


| INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the most prevalent types of liver cancer and the third leading cause of cancer-associated death. 1 The development of HCC is a complex series of events that are initiated from chronic hepatitis followed by cirrhosis, dysplasia, and finally malignant tumors with various genetic and epigenetic alterations. 2Despite the advance in the understanding of the molecular pathogenesis of HCC and the emergence of targeted therapeutic agents such as sorafenib, the incidence, and mortality of HCC have not been significantly reduced; more importantly, recurrence occurs in more than two-thirds of the patients who have received any forms of treatments.The current understanding of the precise factor(s) responsible for the aforementioned difficulties in treating HCC is limited due to the complexity of HCC etiology and development.
The emergence of cancer stem cell (CSC) theory entails tumor progression is driven by CSCs via their capacity for self-renewal, production of heterogeneous progenies, resistance to chemo-or radiation therapies, and ability to induce epithelial-to-mesenchymal transition (EMT). 3,4Of note, stem cell-associated signaling networks such as Wnt/β-catenin, TGF-β, Notch and Hedgehog are found to expedite tumorigenesis.In particular, the Wnt/β-catenin pathway is an evolutionarily conserved pathway that is critical in organ development, growth, survival, regeneration, and self-renewal. 5Aberrant Wnt/β-catenin activation is associated with a range of diseases and is frequently found in many cancers, especially in HCC. 6Based on these premises, agents which can target Wnt/β-catenin may also provide the means of eliminating CSCs.
Isothiocyanates from cruciferous vegetables, such as phenethyl isothiocynanate (PEITC), have received much attention due to their potent chemopreventive ability in different cancer types.PEITC mainly functions through modulating phases I and II drug-metabolizing enzymes as well as a wide spectrum of oncogenic pathways including NF-κB signaling. 7In addition, PEITC has been shown to suppress Sk-Hep1 metastasis via downregulating the expression of matrix metalloproteinases (MMPs) while upregulating tissue inhibitors of matrix metalloproteinase (TIMPs). 8Given the aforementioned anti-cancer properties of PEITC and the close correlation between EMT and the generation of CSCs, 9 PEITC may be a potential agent for suppressing the generation of CSCs.
In this study, we first isolate CD133+/CD90+ cells from human HCC cell lines Huh7 and Sk-Hep1 and demonstrated that these cells exhibited stem cell properties including increased stemness gene expression, enhanced self-renewal, and metastatic abilities.We then demonstrated that PEITC treatment decreased the abundance of CD133+/CD90+ cell population and major CSC properties in these cells.Mechanistically, we found that PEITC was able to modulate microRNA-214/β-catenin axis to achieve its anti-CSC effects.In short, we provided evidence for PEITC as a candidate agent to be used for improving treatment efficacy by targeting and eliminating CSCs in HCC patients in the future.

| Western blot
Western blotting was performed using standard methods.Cells were washed with PBS and then lysed in RIPA lysis buffer; cellular protein lysates were isolated using the Protein Extraction Kit (QIAGEN, USA) and quantified using the Bradford Protein Assay Kit (Thermo Fisher Scientific, USA).After preparing the whole cell lysate, the proteins are separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane, followed by blocking with Tris-buffered saline plus skimmed milk.These PVDF membranes were then probed with respective primary antibodies, followed by the secondary antibody.The commercial antibodies are shown in Table S1.Images were captured and analyzed using the UVP BioDoc-It system (Upland, CA).

| Real-time PCR
Total RNA was isolated from liver cancer cell lines using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.Reverse transcription reactions were performed with Transcriptor First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN).Real-time PCR (RT-PCR) was performed by a Rotor-Gene Q (QIAGEN).The reaction was subjected to 42-cycle amplification at 95 C for 20 s, at 60 C for 20 s, and at 72 C for 25 s.Relative mRNA expression of selected genes was normalized to HPRT and quantified using the ddCT method.The sequences of the RT-PCR primers are listed in Table S2.

| Colony formation assay
About 300 liver cancer cells were seeded in six-well plates and treated with PEITC alone or in combination.The cells were allowed to grow for another week, and they were then harvested, fixed, and counted.

| Migration and invasion assay
Cells were seeded in six-well plates (Corning, Corning, NY) with DMEM containing 10% FBS and cultured to 95%-100% confluence.A scratch along the median axis was made with a sterile yellow pipette tip across the cells.Cell migration pictures were captured immediately and at 48 h after the scratch under a microscope.Cells (2 Â 10 5 ) were seeded in 24-transwell chambers with an 8-μm pore membrane coated with Matrigel in the upper chamber of the transwell system containing serum-free DMEM.The lower chamber of the transwell chamber contained a medium with 20% FBS.After incubation at 37 C for 6 h, the noninvaded HCC cells on the upper side of the membrane were carefully removed with a cotton swab, whereas the invaded cells were stained with crystal violet dye, air dried, and photographed under a microscope.Images were analyzed with ImageJ software (https://imagej.nih.gov/ij/download.html,accessed on June 15, 2020).

| Flow cytometry
After the dissociation into a single-cell suspension from the HCC cell culture, the suspension was transferred into a 15-mL falcon tube.The cells were washed by 10 mL PBS (containing 10% FBS) and followed by centrifugation at 200g for 10 min.Supernatant was removed and then counted by hemocytometer.CD133 and CD90 staining were carried out using the CD133 and CD90 monoclonal antibody according to the manufacturer's instructions.For separation of HCC cells with CD133+ and CD90+ during FACS, we used the dilution concentration of antibody was 1:1000.CD133+ and CD90+ HCC cells were isolated via Florescence Associated Cell Sorting (FACS; BD Biosciences, Franklin Lakes, NJ).Using the sterile PBS washed three times to remove all serum in the cells after sorting, each washing should be centrifuged at 200g for 10 min.After the HCC cell classification, CD133+/CD90+enriched cells were tested for their stemness and differentiation potential in vitro.CD133+/CD90+ liver CSCs populations were sorted using a fluorescence-activated cell sorter (BD Biosciences, Franklin Lakes, NJ).The cells were measured using a FACS-Canto-II (Becton Dickinson) and data were analyzed using FlowJo software (Tree Star).

| Microarray data set processing
To examine the expression of our interest genes, we utilize public microarray profiling that was reported by Woo et al. to determine expression profile in HCC, cholangiocarcinoma (CCA), and combination of both cancers using microarray platform. 11The data set was deposited in Gene Expression Omnibus (GEO) under accession number GSE15765.Limma package was used to assess differential expression genes among those cancer groups.The boxplot was then generated to visualize different expressions of interest gene in HCC, CCA, and combined HCC + CCA group.

| Statistical analysis
All assays were performed at least thrice in triplicate.Values are expressed as mean ± SD.Comparisons between groups were estimated using Student's t test for cell line experiments or the Mann-Whitney U test for clinical data, Spearman's rank correlation between variables, and the Kruskal-Wallis test for comparison of three or more groups were conducted.The Kaplan-Meier method was used for survival analysis, and the difference between survival curves was tested with a log-rank test.Univariate and multivariate analyses were performed using a Cox proportional hazards regression model.All statistical analyses were performed using IBM SPSS Statistics for Windows, version 20 (IBM, Armonk, NY).Statistical significance was indicated if P < .05.

| Isolation and characterization of CD133+/CD90+ HCC cells with increased β-catenin signaling
Using online Public microarray database search, we found that β-catenin mRNA is upregulated in different HCC samples.In Woo Liver database, 11 three major categories were shown, CCA, HCC, and combined CCA and HCC.The mRNA expression level of β-catenin (CTNNB1) was found to be significantly higher respectively in HCC (n = 70) than other two categories as described in the boxplot (Figure 1A).This finding provided us the foundation for exploring the role of β-catenin in the generation/maintenance of HCC CSCs.
Next, we utilized flow cytometry and CD markers to identify and isolate potential HCC CSCs from two HCC cell lines, Huh7 and Sk-Hep1.3][14] A distinct subpopulation of CD133+/CD90+ cells were identified in both Huh7 and Sk-Hep1 cell lines (6.61% and 5.48%, respectively, Figure 1B).Subsequently, we then examined the stemness gene expression profiles of these isolated CD133+/ CD90+ cells.Comparatively, CD133+/CD90+ Huh7 and Sk-Hep1 cells contained a significantly more abundant amount of c-Myc, Nanog, and β-catenin mRNA transcripts (stemness genes) as well as Twist1 (an EMT inducer).For instance, in CD133+/CD90+ Huh7 and Sk-Hep1 cells, β-catenin level was approximately 1.9-fold and 1.79-fold higher as compared to their parental counterparts (Figure 1C).Western blotting result also confirmed increased activation of epithelial-mesenchymal transition (EMT) in CD133+/CD90+ HCC cell lines as observed by upregulation of EMT regulators such as Twist1, Snail, Slug, Zeb1, and Zeb2 in accordance with EMT downstream marker such as overexpression of N-cadherin and vimentin while E-cadherin was downregulated (Figure 1D).Moreover, upregulation of ABC drug efflux transporter as chemoresistance marker was also observed in CD133+/CD90+ subset of HCC cells along with overexpression of c-MYC, Nanog, and β-catenin (Figure 1E).
Therefore, this study reaffirmed well-recognized findings concerning the association between β-catenin signaling, EMT, and chemoresistance associated with CD133+/ CD90+ of liver CSCs.

| CD133+/CD90+ HCC cells exhibit stem cell properties and enhanced β-catenin transcriptional activity
After establishing that CD133+/CD90+ cells express increased level of stemness genes, we further demonstrated that these cells contained stem cell properties.First, CD133+/CD90+ cells appeared to form a higher number of tumor spheres in serum-deprived culture medium in an anchorage-independent manner.For example, CD133+/CD90+ Sk-Hep1 cells formed approximately twice as many tumor spheres than their parental counterparts (Figure 2A).The increased sphere formation in those subsets indicated potentiation of self-renewal capability among CD133+/CD90+-enriched CSCs.In addition, colony-forming ability was also examined and compared between the parental and CD133+/CD90+ HCC cells.Similarly, CD133+/CD90+ Huh7 and Sk-Hep1 cells formed significantly more colonies than parental cells (Figure 2B).Another important feature of CSC is the enhanced metastatic potential.A marked increase in the migratory ability was observed in both CD133+/CD90+ HCC cell lines.CD133+/CD90+ Huh7 cells were estimated 1.5-fold more mobile than their parental cells 1.4-fold in Sk-Hep1 cells (upper panel, Figure 2C).The increased migratory ability was accompanied by an elevated invasive ability.As measured by matrigel-coated Boyden chamber assay, Huh7 CD133+/CD90+ HCC cells were approximately 2.1-fold more invasive than their parental cells while 1.9-fold in Sk-Hep1 cells (lower panel, Figure 2C).Given that β-catenin is elevated in HCC samples (our public microarray database search results) and aberrant β-catenin activity has also been observed in the malignancy and associated with the generation of CSCs in a variety of different cancer types, [15][16][17] we assessed the transcriptional activity of β-catenin in both Huh7 and Sk-Hep1 tumor spheres, using a luciferase-based reporter system.As expected, tumor spheres from both cell lines exhibited a marked increase in β-catenin transcriptional activity.For instance, Huh7 tumor spheres showed and F I G U R E 3 PEITC suppresses stemness of CD133+/CD90+ HCCs.(A) PEITC dose-dependently decreased the percentage of CD133+/ CD90+ cells in both Huh7 and Sk-Hep1 HCC cell lines.Cells were treated with increasing concentrations of PEITC (0, 5, and 10 μM) for 24 h and examined using the changes in the number of cells with CD133+/CD90+ phenotype.(B) PEITC inhibited the self-renewal ability of HCC CSCs.Both Huh7 and Sk-Hep1 cells were treated with PEITC (0, 5, and 10 μM).(C) The mRNA genes expression was evaluated after PEITC treatment.Gene expression of stemness markers including β-catenin, c-Myc, and Nanog; drug resistance marker ABCG2 and EMT marker Twist1.(D) Western blot showed the protein expression of several drug-resistant markers ABCG2, MDR1, ABCC5, and ABCC4 along with stemness marker β-catenin.CSC, cancer stem cell; EMT, epithelial-to-mesenchymal transition; HCC, hepatocellular carcinoma; PEITC, phenethyl isothiocynanate.*p < .05;**p < .012.3-fold increase in β-catenin activity while 1.9-fold in Sk-Hep1 spheres (Figure 2D).

| PEITC suppressed CSC properties in HCC in vitro
Previous studies suggested that phytochemicals isolated from cruciferous vegetables, such as phenethyl isothiocyanate (PEITC), are effective in chemoprevention and tumor suppression. 18,19Thus, we intended to examine if PEITC could also pose inhibitory effects on HCC CSCs.First, we treated Huh7 and Sk-Hep1 cells with different concentrations of PEITC and analyzed CD133/CD90 profiles of these two cell lines.PEITC treatment led to a dose-dependent decrease in the number of CD133+/ CD90+ cells (Figure 3A).For instance, PEITC (at 10 μM) treatment resulted in a 13% and 16% decrease in CD133+/CD90+ Huh7 and Sk-Hep1 cells, respectively.Subsequently, we demonstrated that PEITC reduced the self-renewal ability of HCC tumor spheres in a dosedependent manner (Figure 3B).PEITC effectively suppressed the tumor sphere-forming ability at 10 μM where approximately 59% and 44% reduction in sphere formation was observed in Huh7 and Sk-Hep1 cells, respectively.Next, we analyzed the stemness profiles of PEITC- treated HCC CSCs (CD133+/CD90+) using real-time PCR technique.PEITC-treated HCC CSCs exhibited a significantly reduced transcript level of β-catenin, c-Myc, Nanog, and Twist1 (Figure 3C).For instance, PEITC treatment reduced the mRNA level of β-catenin by approximately 47% and 39% in Huh7 and Sk-Hep1 CSCs, respectively.Additionally, the expression levels ABCG2, MDR1, ABCC5, and ABCC4, which are family member of ABC multidrug transporter suggested for drug resistance in CSCs and stem cells, were also reduced in the presence of PEITC (Figure 3D).Thus, this result presented anti-CSC effect following PEITC treatment in HCC cell lines mainly through downregulation of β-catenin, causing apoptosis of CD133+/CD90+ CSCs and reduction of drug-efflux transporters.

| Decreased miR-214 level is associated with increased β-catenin level in HCC CSCs
A recent report indicated an inverse relationship between the expression of miR-214 and β-catenin in the HCC. 20ased on this finding, we decided to examine whether miR-214 is involved in the generation/maintenance of HCC CSCs.First, we demonstrated that in CD133+/ CD90+ Huh7 and Sk-Hep1 CSCs, the level of miR-214 was significantly downregulated approximately by 2-fold (Figure 4A).More importantly, when miR-214 level was increased by ectopic miR-214 mimics, the self-renewal ability was markedly hampered, estimated by 1.6-fold and 1.5-fold in Huh7 and Sk-Hep1, respectively (Figure 4B).In addition, an increased miR-214 level resulted in the decreased percentage of CD133+/CD90+ cell population in both miR-214 mimic-treated Huh7 and Sk-Hep1 cells as compared to those control cells (Figure 4C).More importantly, PEITC-treated (10 μM) HCC cells demonstrated a comparable decrease in the percentage of CD133+/CD90+ cells (Figure 4C).More importantly, PEITC-treatment (10 μM) appeared to increase miR-214 level in CD133+/CD90+ HCC CSCs (upper panel, Figure 4D) and concomitantly decrease β-catenin level (lower panel, Figure 4D).This reverse relationship between miR-214 and β-catenin was in agreement with the findings by Xia et al. 20

| DISCUSSION
The CSCs hypothesis entails that the initiation, growth, and progression of tumor are maintained by a subpopulation of cells that exhibit stem cell-like properties.Clinically, the existence of CSCs represents the major obstacles for managing cancer because CSCs have been shown to contribute to virtually every aspect of tumorigenesis including tumor initiation, growth, metastasis, drug resistance, and recurrence.However, our understanding of CSCs remains limited which leads to inability to develop effective agents to target and eradicate CSCs.
HCC CSCs have been identified and isolated by several cell surface markers such as c-kit, CD133, CD90, CD44, OV6, and EpCAM, 12,[21][22][23][24] or alternatively identified by the side population (SP) methodology. 25,26Here, we used dual cell surface markers CD133 and CD90 for isolating HCC CSCs and demonstrated that these CD133+/CD90+ cells exhibit key stem-like properties such as the expression of "stemness" genes.In this case, four major stemness genes including c-Myc, Nanog, β-catenin and Twist1, were found to be elevated in CD133+/CD90+ HCC CSCs as compared to the parental cells.In addition, EMT regulators such as Snail, Slug, Zeb1, and Zeb2, along with EMT activation markers such as N-cadherin and Vimentin were upregulated in CSCs subset.Our data are supported by a previous study which also isolated HCC CSCs using CD133 as a marker. 27Isolated CD133+/CD90+ Huh7 and Sk-Hep1 CSCs not only expressed a high level of stemness genes but also demonstrated increased self-renewal ability (as reflected by tumor sphere formation under serum-derived and anchorage-independent conditions).The increased selfrenewal ability of CD133+ HCC cells has been correlated with de-differentiated phenotype, advanced disease stage, shorter survival, and higher recurrence rates as compared to patients with low CD133 expression. 28More importantly, the elevated expression of c-Myc, Nanog, β-catenin, and Twist1 contributed to the more mesenchymal status in both Huh7 and Sk-Hep1 CSCs as compared to their non-CSCs counterparts.Recently, c-Myc and Nanog have been shown to participate in murine hepatoma progression, where c-Myc/Nanog/CD133+ CSCs were able to produce progenies with different lineages. 29qually important, the aberrant activation of β-catenin has also been correlated to tumor progression and CSCs generation.Our public microarray database search revealed that β-catenin expression is elevated in CCA, combined hepatocellular and CCA and HCC.Supporting studies demonstrated that β-catenin plays a critical for the regeneration of hepatocytes. 30,31In addition, within a growing tumor, β-catenin enhances hypoxia-induced EMT in HCC by increasing the EMT-associated activity of HIF-1α and preventing apoptosis. 17Our data were in agreement with these studies, where CD133+/CD90+ Huh7 and Sk-Hep1 CSCs demonstrated activated EMT markers and increased metastatic potential which was accompanied by increased β-catenin transcriptional activity.These findings indicated that β-catenin activity is essential in the generation of CSCs and promotion of metastasis.More importantly, β-catenin has been reported to upregulate Nanog by physically interacting with Oct-3/4.Thus, agents which can target and disrupt β-catenin activity may inhibit the generation/maintenance of CSCs via suppressing multiple stemness genes.
3][34] In addition, PEITC was shown to suppress cell migration and invasion in human colon and gastric cancer cells. 35,36In this study, we demonstrated a novel function of PEITC where it effectively inhibited the CSCs generation/maintenance.First, PEITC dose-dependent decreased the percentage of CD133+/CD90+ subpopulation cells in HCC cell lines.Second, PEITC inhibited the formation of tumor spheres (self-renewal ability).Finally, we demonstrated that PEITC treatment suppressed the expression of stemness genes including β-catenin, c-Myc, Nanog, and Twist1.A previous study demonstrated that another member of isothiocyanate family, sulforaphane, in combination with sorafenib suppressed pancreatic CSCs, 37 supporting our notion that isothiocyanate could be a potential anti-CSC agent.
An inverse expression relationship between β-catenin and miR-214 has been shown in human HCC cell lines and clinical specimens. 20Here, we showed that this inverse expression was also present in CD133+/CD90+ Huh7 and Sk-Hep1 CSCs.When the level of miR-214 was increased ectopically, the percentage of CD133+/CD90+ Huh7 and Sk-Hep1 cells decreased significantly; consistently, the self-renewal ability of both CSCs was concomitantly decreased, indicating that miR-214 acts as a suppressor of stemness.More importantly, we demonstrated that PEITC treatment could increase the level of miR-214 while decreasing the level of β-catenin in HCC CSCs.Pharmacokinetically, it was shown that plasma concentration of PEITC in rats could reach 9.2-42.1 μM after oral dose of 10 and 100 μmol/kg of PEITC, 38 indicating that micromolar concentrations of PEITC are achievable in vivo.Taken together, PEITC could be used as a potential anti-CSCs agent which targets and modulates the miR-214/β-catenin axis in HCC CSCs.
In summary, as shown in Figure 5, we first demonstrated that CD133+/CD90+ HCC cells exhibit CSC properties including high stemness gene expression (β-catenin, c-Myc, Nanog, and Twist1), increased selfrenewal and metastatic abilities.In addition, increased β-catenin expression was found in clinical samples of HCC and correlated to poor prognosis.Importantly, we found that phytochemical, PEITC was able to suppress the generation of CD133+/CD90+ cells within Huh7 and Sk-Hep1 cell lines.Equally important, PEITC treatment suppressed the self-renewal ability of CD133+/CD90+ CSCs via decreasing the stemness gene expression and increasing miR-214 expression.We believe that this is the first report describing PEITC as a potential anti-HCC CSCs agent via modulating β-catenin/miR-214 circuit.Since PEITC can be ingested orally and micromolar concentrations which are required to suppress HCC CSCs can be reached in vivo, PEITC could be considered as a combined treatment option with clinically available agents such as sorafenib and/or cisplatin for better HCC management.