Ovatodiolide suppresses yes-associated protein 1-modulated cancer stem cell phenotypes in highly malignant hepatocellular carcinoma and sensitizes cancer cells to chemotherapy in vitro

Accepted Manuscript Ovatodiolide suppresses yes-associated protein 1-modulated cancer stem cell phenotypes in highly malignant hepatocellular carcinoma and sensitizes cancer cells to chemotherapy in vitro

Hang-Lung Chang, Hsin-An Chen, Oluwaseun Adebayo Bamodu, Kwai-Fong Lee, Yew-Min Tzeng, Wei-Hwa Lee, Jo-Ting Tsai PII: DOI: Reference:

S0887-2333(18)30137-1 doi:10.1016/j.tiv.2018.04.010 TIV 4272

To appear in:

Toxicology in Vitro

Received date: Revised date: Accepted date:

13 October 2017 9 April 2018 22 April 2018

Please cite this article as: Hang-Lung Chang, Hsin-An Chen, Oluwaseun Adebayo Bamodu, Kwai-Fong Lee, Yew-Min Tzeng, Wei-Hwa Lee, Jo-Ting Tsai , Ovatodiolide suppresses yes-associated protein 1-modulated cancer stem cell phenotypes in highly malignant hepatocellular carcinoma and sensitizes cancer cells to chemotherapy in vitro. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Tiv(2018), doi:10.1016/j.tiv.2018.04.010

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ACCEPTED MANUSCRIPT Ovatodiolide Suppresses Yes-Associated Protein 1-Modulated Cancer Stem Cell Phenotypes in Highly Malignant Hepatocellular Carcinoma and Sensitizes Cancer Cells to Chemotherapy In Vitro Hang-Lung Chang, MD1#, Hsin-An Chen, MD2,3,4#, Oluwaseun Adebayo Bamodu, MD, PhD5,6, Kwai-Fong Lee, PhD 7, Yew-Min Tzeng, PhD8, 9, Wei-Hwa Lee, MD, PhD10*, Jo-

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Ting Tsai, MD, PhD 2,11,12* Department of General Surgery, En Chu Kong Hospital, New Taipei City, Taiwan

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Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University,

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Taipei, Taiwan.

Division of General Surgery, Department of Surgery, Taipei Medical University-Shuang Ho

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3

Hospital, New Taipei City, Taiwan.

Division of General Surgery, Department of Surgery, School of Medicine, College of

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4

Medicine, Taipei Medical University, Taipei, Taiwan.

Department of Hematology and Oncology, Cancer Center, Taipei Medical University -

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Shuang Ho Hospital, New Taipei City, Taiwan 6

Department of Medical Research & Education, Taipei Medical University - Shuang Ho

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Hospital, New Taipei City, Taiwan

Biobank management center, Tri-Service General Hospital, Taipei, Taiwan

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Center for General Education, National Taitung University, Taitung, Taiwan.

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Department of Appiled Chemistry, Chaoyang University of Technology, Taichung, Taiwan.

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Department of Pathology, Taipei Medical University-Shuang Ho Hospital, Taipei, Taiwan.

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Department of Radiology, School of Medicine, College of Medicine, Taipei Medical

University.

Department of Radiation Oncology, Taipei Medical University-Shuang Ho Hospital, New

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12

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10

Taipei City, Taiwan

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Contributed equally to this work

*Correspondence to: Wei-Hwa Lee; E-mail: [email protected] Jo-Ting Tsai; E-mail: [email protected]

ACCEPTED MANUSCRIPT Abstract The cancer stem cells (CSCs) theory recently became a focus of heightened attention in cancer biology, with the proposition that CSCs may constitute an important therapeutic target for effective anticancer therapy, because of their demonstrated role in tumor initiation, chemo-, and radio-resistance. Liver CSCs are a small subpopulation of poorly- or undifferentiated liver tumor cells, implicated in tumorigenesis, metastasis, resistance to

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therapy and disease relapse, enriched with and associated with the functional markers corresponding to the CSCs-enriched side population (SP), high aldehyde dehydrogenase (ALDH) activity, and enhanced formation of in vitro liver CSCs models, referred to herein as

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hepatospheres. In this study, we found YAP1 was significantly expressed in the SP cells, as

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well as in generated hepatospheres compared to non-SP or parental HCC cells, at transcript and/or protein levels. In addition, downregulation of YAP1 expression levels by small

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molecule inhibitor and siRNA transfection, in the HCC cell lines, PLC/PRF/5 and Mahlavu, were associated with marked loss of ability to form hepatospheres and increased sensitivity to sorafenib. Consistent with the above, we demonstrated that YAP1 expression positively

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correlated with that of Sox2, Oct4, c-Myc and GRP78, markers of stemness and drug resistance. This is suggestive of YAP1’s role as a modulator of cancer stemness, ER stress

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and chemoresistance. For the first time, we demonstrate that Ovatodiolide significantly attenuates YAP1 expression and subsequently suppressed YAP1-modulated CSCs

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phenotypes and associated disease progression, consistent with our previous finding in breast cancer. Taken together, our findings suggest that YAP1, highly expressed in malignant liver tumours, contributes to hepatocellular CSCs phenotype and is a molecular target of interest

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for CSCs targeted therapy in liver cancer patients. Keywords: Hepatocellular carcinoma, Yes-associated protein, YAP1, Ovatodiolide, Side-

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population, Cancer stemness, Cancer stem cells (CSCs), Chemoresistance

ACCEPTED MANUSCRIPT 1. Introduction Hepatocellular carcinoma (HCC) is one of the most common human cancers, and despite major advances in HCC diagnostic and therapeutic strategies, it remains the second most common cause of cancer-related death, with approximately 750,000 deaths in 2012 alone (Ferlay et al., 2014). Recently, targeted anti-cancer therapy has become the standard therapeutic strategy for malignant late stage hepatocellular carcinoma (HCC), and the first

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FDA-approved agent, sorafenib remains one of the most effective, if not the most effective anti-HCC systemic therapy, evidenced by improved patient prognosis (Zhu et al., 2012;

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Cheng et al., 2009). Recent studies of liver cancer biology have demonstrated that malignant HCC cells are associated with expression of stemness markers such as CD133, CD44,

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EpCAM, CD90 and keratin 19, and thus, suggest that the presence and activities of cancer stem cells in the hepatocarcinogenesis, growth, metastatic spread and progression of HCC

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(Zhu et al., 2010; Yamashita et al., 2013; Ma et al., 2010; Kim et al., 2011). In addition, these stemness markers are associated with the cancer stem cells (CSCs)-enriched side population

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(SP), high aldehyde dehydrogenase (ALDH) activity, and enhanced formation of in vitro liver CSC models, referred to herein as hepatospheres, all of which have been implicated in enhanced tumorigenicity in murine xenograft tumor models, and resistance to anti-cancer

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therapeutic agents (Castelli et al., 2017).

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Yes-associated protein 1 (YAP1) is a transcription co-activator and downstream target gene in the Hippo signalling pathway, with demonstrated role in embryogenesis-related control of organ size (Lian et al., 2010; Kango-Singh et al., 2009). YAP1 has also been

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implicated as a tumor-enhancing gene, evidenced by its expression-associated induction of epithelial-mesenchymal transition (EMT), promotion of cell proliferation, and suppression of

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apoptosis in different cancer types (Shao et al., 2014), driving of hepatocarcinogenesis in in vivo murine models (Tao et al., 2014), facilitating self-renewal and vascular mimicry of stemlike cells by regulating their Oct4 activity and Sox2 expression (Bora-Singhal et al., 2015; Zhu et al., 2016), subsequently resulting in resistance to chemotherapy and poor prognosis (Jeong et al., 2014). Remarkably, it has been reported that about half of all human HCC cases are associated with aberrant expression and nuclear translocation of the YAP oncogene (Zhao et al., 2014), thus, YAP1 phosphorylation and its cytoplasmic retention may represent an effective therapeutic strategy in HCC patients. Ovatodiolide, a macrocyclic diterpenoid, is a one of the principal bioactive components of Anisomeles indica (L.) Kuntze (Labiatae), which in traditional Chinese medicine is used as

ACCEPTED MANUSCRIPT an anti-inflammatory, anti-allergy, anti-viral and pain-modulatory therapeutic agent (Arisawa et al., 1986; Rao et al., 2013a, 2009b; Shahidul et al., 2000; Dharmasiri et al., 2003), as well as in the treatment of gastrointestinal and hepatic disorders (Wang et al., 2005; Rao et al., 2009b). More recently, the antiproliferative and pro-apoptotic effect of Ovatodiolide has also attracted much attention. In deed documented evidence indicate that Ovatodiolide via diverse mechanisms not only suppresses cellular proliferation in vitro, or induces apoptosis of cancer

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cells, but it also shows that this effect is replicated in vivo, with tumour growth inhibition, shrinkage of tumor size, reduction of distant tumor cell dissemination, in both blood and solid cancers (Hou et al., 2009; Lin et al., 2011; Bamodu et al., 2015; Ho et al., 2013).

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In this present study, we investigated the role of YAP 1 in the modulation of

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hepatocellular carcinoma stem cell phenotype and its associated aggressive tumor biology, as well as the CSC-inhibitory activity of Ovatodiolide in HCC as mono therapy and in

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combination with Food and Drug Administration (FDA)-approved advanced HCC chemotherapeutic agent, Sorafenib. To the best of our knowledge, our study is the first to examine the anticancer effect of Ovatodiolide in liver cancer, and its ability to inhibit YAP1

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expression and/or activity in the hepatic carcinoma cells.

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2.1.Reagents and Drugs

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2. Materials and Methods

Sorafenib were purchased from Santa Cruz Biotechnology (CAS 284461-73-0). Stock solution of 1 mM dissolved in PBS was stored at −20 °C away from light. Ovatodiolide

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(99.7% purity) was generously provided by Professor Yew-Min Tzeng (National Taitung University, Taitung, Taiwan). Ovatodiolide was dissolved in DMSO and further diluted in

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sterile culture medium immediately prior to use. Antibodies against YAP1, C-Myc, CD133, Sox2, Oct4 and GRP78 were purchased from Cell Signalling Technology (Beverly, MA, USA). Anti- β-actin antibody was purchased from Santa Cruz Biotechnology. Alexa Fluor 647 donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-rabbit IgG were purchased from Invitrogen (Grand Island, NY, USA).

2.2.Cells and cell culture Human liver cancer cell lines Mahlavu and PLC/PRF/5 purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-

ACCEPTED MANUSCRIPT streptomycin at 37oC, in 5% CO2 humidified incubator. Medium was changed, and cells passaged every 48h. Matrigel invasion assay revealed that Mahlavu and PLC/PRF/5 possess a high invasive potential with a mesenchymal phenotype could play some facilitatory role in invasion (Riou et al., 2006).

2.3.Side population (SP) analysis using flow cytometry Culture HCC cells were detached with Trypsin-EDTA (Invitrogen), centrifuged and pellet

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resuspended at 1 × 106 cells/mL in Hank’s balanced salt solution (HBSS) supplemented with 3% fetal calf serum (FCS) and 10mM HEPES, then incubated for 90 minutes at 37oC with 20

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μg/mL Hoechst 33342 (Sigma Chemical, St. Louis, MO), with or without 50 μM verapamil The cells were then

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(Sigma), for inhibition of verapamil-sensitive ABC transporter.

centrifuged at 300×g, 4oC for 5 minutes, resuspended in ice-cold HBSS, kept on ice for

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inhibition of Hoechst dye efflux and 1 μg/mL Propidium iodide (BD Pharmingen, San Diego, CA) was then added to for selection of viable cells. The viable cells were then filtered through a 40 μm cell strainer (BD Falcon) to obtain single cell suspension. This was followed

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by dual-wavelength analysis and purification of cells on a dual-laser FACS Vantage SE (BD). Hoechst 33342 was excited by 355nm UV light and detected using blue fluorescence of

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450/20 band-pass filter and red fluorescence of 675 nm edge filter long pass (EFLP). For separation of the emission wavelengths, we used a 610nm dichroic mirror short pass

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(DMSP). Propidium iodide was used to exclude dead cells from the analysis.

2.4. Immunohistochemical staining

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YAP1, C-Myc and GRP78 expressions in sections of formalin-fixed, paraffin-embedded (FFPE) tissues were evaluated by immunohistochemical staining. Briefly, sections of FFPE

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tissues were deparaffinised, rehydrated, subjected to antigen retrieval and then probed with primary antibodies for YAP1 (1:200, Cell Signaling Technology, MA, USA), c-Myc (1:100, Cell Signaling Technology), and GRP78 (1:100, Cell Signaling Technology). Slides were then washed, incubated in biotinylated universal antiserum and then in horseradish peroxidise-steptavidin conjugate. The slides were washed, 3, 3-diaminobenzidine hydrochloride chromogen was used for colour development, and then slide were rinsed again, counterstained with Haematoxylin and mounted for microscopy evaluation and photography.

2.5. Western blotting

ACCEPTED MANUSCRIPT Cultured HCC cells were trypsinized, collected and lysed. Protein lysates were then heated for 5 minutes, and then subjected to western blot analysis as previously described. Blots were blocked with 5% non-fat milk in TBST for 1 h, probed overnight at 4°C with specific antibodies against YAP 1 (1:1000), c-Myc (1:1000), CD133 (1:1000), GRP78 (1:1000), and β-actin (1:1000). After incubation with primary antibodies, the PVDF membranes were washed 3 times with TBST, and then incubated with horseradish peroxidise

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(HRP)-labelled secondary antibody for 1h at room temperature and washed again with TBST. Band detection was done using enhanced chemiluminescence (ECL) Western blotting

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reagents and the BioSpectrum Imaging System (UVP, Upland, CA).

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2.6. Tumorsphere formation assay

For evaluation of the HCC cells ability to form tumor spheres, Mahlavu and PLC/PRF/5

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cells were cultured in HEScGRO serum-free medium (Chemicon Technologies) which was supplemented with 20 ng/ml hEGF, NeuroCult NS-A and 10 ng/ml hFGF-b (STEMCELL

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Technologies). 1000 cells were seeded per 1ml medium in 12-well, non-treated plates. The generated spheroids (spherical, non-adherent cell-masses > 90 μm in diameter) were counted, then about 50 spheroids per group were measured using an ocular micrometer. For the

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secondary (F2) and tertiary (F3) spheroid generations, we mechanically dissociated the

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primary and secondary spheroids respectively, and processed exactly as for the primary assay. To estimate the percentage of spheroid-forming cells, we seeded one cell per well in

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96-well plates.

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2.7.Flow cytometry analysis of YAP1 Flow cytometry analysis of YAP1 was performed using the FACSAria (BD Biosciences, Franklin Lakes, NJ, USA). Cells were harvested and single-cell suspensions were prepared with the aid of StemPro Accutase (Life Technologies, Carlsbad, CA, USA). Spheroid cells were separated into single-cell suspensions with the aid of collagenase I (Sigma Aldrich) and adjusted to a concentration of 106cell/ml. To stain surface antigens, cells were incubated with antibodies against YAP1 for 30 min on ice. YAP1—FITC antibodies conjugated to APC (BD Biosciences) were used to determine liver cancer spheres cells.

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2.8.Real-time PCR analysis The comparative analysis of the mRNA expression levels of YAP1, GRP78, and c-Myc in hepatospheres versus parental PLC/PRF/5 or Mahlave cells were performed by real-time PCR, starting with the isolation of total RNA from the harvested HCC cells. 1 μg of total

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RNA was then reverse-transcribed to cDNA using the High-capacity cDNA reverse transcriptase kit (Applied Biosystems, Foster City, CA, USA). Then real-time PCR analysis

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of the cDNA was carried out on the iCycler iQ Real-Time detection system (Bio-Rad, Hercules, CA) for the expression of YAP1, GRP78, and c-Myc mRNA, with

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glyceraldehydes-3-phosphate-dehyhrogenase (GAPDH) serving as internal control. The specific primer pairs are as follows: GAPDH - 5′-GTGGTCTCCTCTGACTTCAAC-3′ and

5′-TCTCTTCCTCTTGT

GCTCTTG-3′

(anti-sense).

YAP1

-

5′-

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(sense)

CGCTCTTCAACGCCGTCA-3′ (sense) and 5′- AGTACTGGCCTGTCGG GAGT -3′ (antisense). GRP78 - 5′-GCCTGTATTTCTAGACCTGCC-3′ (sense) and 5′-TTCATCTTGCCAG

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CCAGTTG-3′ (anti-sense), and c-Myc - 5′-AAACACAAACTTGAACAGCTAC-3′ (sense) and 5ATTTGAGGCAGTTTAC ATTATGG-3′ (anti-sense). According to the manufacturer's

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instruction, SYBR-Green I was used as fluorescent dye for the detection of PCR products.

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The cycling conditions were 95°C for 3 min, followed by 40 cycles at 94°C for 0.5 min, 62°C for 0.5 min and 72°C for 1 min. We then normalized the expression of the YAP1, GRP78 and c-Myc mRNAs to that of GAPDH.

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2.9.Statistical analysis

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All experiments were performed in three separate times and in triplicates. Reported data reflects the mean ± standard deviation (SD). All statistical analyses were performed using paired student’s t test on the GraphPad Prism 5 software (GraphPad Software Inc., CA, USA). P-value < 0.05 was considered statistically significant.

3. Results 3.1

HCC-derived side population (SP) cells showed significantly enhanced ability to

form hepatospheres in vitro

ACCEPTED MANUSCRIPT 2.67% and 3.16% of PLC/PRF/5 and Mahlavu cells respectively were identified as side population (SP) cells using low Hoechst 33342 blue-red fluorescence light (Figure 1A). This subpopulation of cancer cells was reduced to 0.17% and 0.15% in PLC/PRF/5 and Mahlavu cells respectively in the presence of verapamil (Figure 1A). Detected SP cells were isolated and used for subsequent assays. To evaluate the cancer stem cell-like traits of the HCC cells in vitro, we assessed the ability of PLC/PRF/5 and Mahlavu-derived SP and non-SP (NSP)

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to form hepatospheres. As anticipated, the sorted SP and NSP cells exhibited divergent tumorsphere formation or growth patterns, with the SP cells displaying significantly increased ability to form hepatospheres. This is evidenced by the larger quantity and size of

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the SP cell-derived hepatospheres, compared to the fewer and smaller tumorspheres in their

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NSP counterparts (Figure 1B). This tendency was significant and noted in all repeated SP-

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NSP HCC tumorsphere formation assay (p < 0.001, Figure 1).

cancer stem cell-like phenotype To better understand

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3.2.YAP 1 expression links repression of ER-stress to the highly malignant HCC cells’

the cancer stem cell-like phenotype of HCC, using

immunohistochemical staining, RT-PCR and western blot assay, we probed the expression of

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selected genes in several HCC cell lines, including the highly malignant Mahlavu and

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PLC/PRF/5. RT-PCR showed that YAP 1 transcript is strongly overexpressed in Mahlavu and PLC/PRF/5, while moderate expression was noted in the less malignant Huh7 and HepG2 and weak expression in the Hep3B and J5 cells (Figure 1C and D). At protein level,

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YAP1 is significantly expressed in liver tumor tissues, compared to its non-expression in their non-tumor pair, and its expression profile positively correlates with that of GRP78, an

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anti-apoptotis marker for repressed endoplasmic reticulum (ER) stress, and c-Myc, a protooncogene and stemness marker (Figure 2A). In the cell lines, the co-expressed levels of YAP1, c-Myc, and GRP78 proteins were markedly enhanced in the Mahlavu and PLC/PRF/5-derived spheres, compared to their parental counterpart (Figure 2B). Similarly, on the transcript level, the expressions of YAP1, c-Myc and GRP78 mRNAs were significantly more in the hepatospheres generated from the PLC/PRF/5 and Mahlavu cell lines than in their parental cells (Figure 2C and D). These data do suggest the role of YAP1 as a probable indicator of repressed ER stress and marker of stemness, thus serving as a link between the two vital cellular events in HCC.

ACCEPTED MANUSCRIPT 3.3.YAP 1 is a critical regulator of HCC stemness Further, we investigated the role of YAP1 in liver cancer stemness, using the tumorsphere formation and FITC-cytometric assays, as well as silencing of YAP 1 by siRNA transfection. We demonstrated that YAP 1-enriched Mahlavu and PLC/PRF/5 side population (SP) cells showed significantly higher propensity for the formation of hepatospheres (Figure 3A), which are characteristically enriched with the stemness markers, including ALDH and

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CD133. The Mahlavu and PLC/PRF/5 -derived hepatospheres were then marked with FITCconjugated YAP1 antibodies and subjected to flow-cytometric analysis, which showed significantly enhanced YAP activity in the YAP 1-enriched hepatospheres (Figure 3B).

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Downregulation of YAP1 expression by siRNA resulted in severely suppressed hepatosphere

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formation and sphere size of the hitherto YAP1-enriched liver cancer cells (Figure 3C). Consistent with these findings, our western blot showed that the expression of stemness

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markers Sox2 and Oct4 were lower in the YAP1-silenced Mahlavu and PLC/PRF/5 cells, compared to their wild type counterpart (Figure 3D). These results indicate that YAP1

stemness markers, Sox2 and Oct4.

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modulates HCC stem-like properties by, in part, by modulating the expression of the

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3.4. Downregulation of YAP 1 increases the sensitivity of HCC cells to Sorafenib

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We further assessed the effects of suppressing YAP 1 expression on sensitivity of HCC cells to sorafenib. Using siRNA transfection, YAP1 expression in Mahlavu and PLC/PRF/5 cells was silenced (Figure 3D). 48 h after transfection with si-YAP1, the HCC cells were

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treated with various concentrations of sorafenib for 48 h and cell viability assessed by the SRB assay. As shown, YAP1-silenced Mahlavu cells had significantly lower number of

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viable cells in comparison to the vector-transfected control. Downregulation of YAP1 expression significantly increased the number of dead Mahlavu cells following sorafenib treatment, as well as sensitivity to same. A lower IC50 was noted in the YAP1-silenced cells (2.2 M) compared to the wild type (6.8 M) (Figure 4A). This finding was corroborated by results from colony formation assays which showed that the number of colonies formed per 100 Mahlavu cells was very significantly reduced when YAP1 expression was silenced in the cells before sorafenib treatment, compared to silencing YAP1 or treating with sorafenib alone (Figure 4B). Our results demonstrate that YAP1 expression may play a critical role in the resistance of HCC cells to conventional chemotherapy.

ACCEPTED MANUSCRIPT 3.5.

Ovatodiolide anticancer effect in HCC is mediated by efficient targeting of CSCs,

suppression of ALDH activity and downregulation of the expression of stemness markers

To investigate the effects of Ovatodiolide on HCC stemness and its associated highly malignant, as well as chemoresistant phenotype, isolated Mahlavu and PLC/PRF/5 SP cells were treated with ovatodiolide for 48h in surface treated dishes or for 5 days in ultra-low

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adhesion plates for generation of protein lysate and hepatosphere for western blot or spheroid formation assays respectively. The Mahlavu and PLC/PRF/5 SP cells formed significantly fewer tumorspheres when treated with ovatodiolide, compared to the untreated SP cells. This

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inhibitory effect was dose-dependent in nature (Figure 5A and B). The SP-derived

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hepatosphere sizes were also decreased by ovatodiolide treatment, as evidenced by marked reduction in number of large hepatospheres. As anticipated, and in correlation with earlier

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findings, flow cytometric analyses demonstrated a corresponding dose-dependent reduction in ALDH activity in the ovatodiolide-treated group (Figure 5C). In addition, western blot assay also revealed significant dose-dependent decrease of YAP 1 protein expression and

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corresponding downregulation of Sox2 and Oct4 after ovatodiolide treatment of the isolated Mahlavu and PLC/PRF/5 SP cells (Figure 5D).

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3.6. Ovatodiolide increased HCC cells’ sensitivity to chemotherapy Based on our earlier findings showing that ovatodiolide has significantly high anticancer effects in HCC, we further evaluated the effect of combining both agents on the

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chemosensitivity of HCC stem cell-like cells, using our SP-enriched Mahlavu-derived hepatospheres. Thus, we investigated the effects of ovatodiolide treatment on HCC cell sensitivity to sorafenib.

Anchorage-independent hepatosphere growth assay showed the

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sensitizing effect of 2.5M and 5M of ovatodiolide on the inhibition of Mahlavu cell growth by sorafenib concentrations ranging between 2.5M and 10M (Figure 6A). This augmenting effect was dose-dependent in nature. To understand the nature of the observed drug-drug augmentation, we used the Chou-Talalay drug combination isobologram method, which is based on the median-effect equation to derive the combination index (CI) and quantitatively define antagonism, additivity or synergism, based on CI >1, CI =1, CI <1, respectively (Chou et al., 2010). Drug combination isobologram analysis revealed existent synergism between sorafenib and ovatodiolide (Figure 6B and C). Collectively, these

ACCEPTED MANUSCRIPT findings indicate that ovatodiolide increases the sensitivity of hepatic tumor cells to sorafenib therapy in a synergistic manner. 4. Discussion

Aberrant expression of the Yes-associated protein 1 (YAP1), a potent transcription coactivator whose activity is initiated by interaction and formation of complex with the DNA-

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binding TEAD transcription factor, is not uncommon in cancers (Chan et al., 2011; Zhao et al., 2010), and is associated with poor prognosis in many of such cases including

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hepatocellular carcinomas (Xu et al., 2009). In general, the ectopic expression of the YAP1 gene regulates different embryogenic activities including organ size (Camargo et al., 2007),

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and modulates the acquisition of tumor-promoting properties and several oncogenic traits

(Overholtzer et al., 2006; Zhao et al., 2009).

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such as cell dedifferentiation, proliferation, migration, invasion and death evasion

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Liver CSCs, functionally defined by their ability to undergo dedifferentiation and selfrenewal, have been identified and implicated as critical causative agents in a vast array of malignancies, based on their expression of specific cell surface antigen and constitutively

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enhanced aldehyde dehydrogenase (ALDH) activity in malignancies (Liu et al., 2010).

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Similar to normal stem cells, these liver CSCs acquire a quiescent phenotype with a characteristic propensity for enhanced cellular longevity and self-renewal via modulation of pluripotent stemness factors and related cell cycle proteins, thus, rendering them as attractive

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therapeutic targets in the war against HCC. In this present study, we demonstrated that (i) malignant HCC cells are enriched with the YAP1 gene, (ii) YAP1 expression both at mRNA

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and protein levels, positively correlates with that of known pluripotent stemness factors, cMyc, Sox2 and Oct4, (iii) increased ectopic YAP1 expression enhances liver tumorsphere (in vitro representation of liver CSCs) formation and defines their CSCs-like phenotype (Figure 1 to 3). These findings in the light of our current knowledge of CSCs are consistent with those of Lian et al. which ascribed a regulatory role to YAP1 in the self-renewal and differentiation of stem cells (Lian et al., 2010). In their work, they demonstrated elevated YAP1expression and/or activity during induced pluripotent stem cell reprogramming with resultant maintenance of stem cell phenotypes, while conversely, loss of embryonic stem (ES) cell pluripotency and enhanced ES cell differentiation followed the silencing of YAP1 in the cells. Our findings where YAP1 expression, positively correlated with that of c-Myc

ACCEPTED MANUSCRIPT and GRP78 (Figure 2), is supported by the suppression of CSC phenotypes and Wnt inactivation, when GRP78 was silenced (van Lidth de Jeude et al., 2017). While validation studies are ongoing, we postulate that YAP1 by interacting with GRP78 on its nucleotidebinding domain enhances the expression of GRP78 protein, stabilizes YAP1 interaction with the stemness marker Oct4 or Sox2, consequently enhancing hepatosphere formation and tumorigenicity. This would be consistent with the documented roles of GRP78 which include

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maintenance of stemness, cellular survival under stress and, resistance to anticancer therapies (Hayashi et al., 2003). In addition, increased expression of GRP78 is associated with cancer initiating cells properties and its down-regulation suppressed self-renewal and tumorigenicity,

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and promotes apoptosis (Wu et al., 2010). It has been shown that gene co-expression provides

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a basis for predicting molecular interaction and functional similarity (Van Dam et al., 2017; Van Noort et al., 2003). It is very plausible that YAP1 serves as the repressed ER stress and

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marker of stemness in HCC tumorigenesis.

Like findings in ES cells, we demonstrate a rather interesting relationship between YAP1

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and c-Myc, Oct4 or Sox2 in the HCC parental and stem cells. Aberrant YAP1 expression positively modulates the expression of the stemness factors c-Myc, Oct4 and Sox2, while

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silencing YAP1 resulted in corresponding decrease or loss of the stemness factors (Figure 3). This further corroborates the assertion that YAP and Oct4 or Sox2 exist in a positive

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regulatory loop, and consequently the existence of high degree overlapping between YAP1 targets and those of the pluripotency stemness factors.

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Furthermore, to the best of our knowledge, our study demonstrates, for the first time, that (iv) silencing YAP1 sensitizes HCC cells to the anticancer activity of the FDA-approved

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sorafenib, (v) the novel anticancer phytocompound ovatodiolide significantly inhibits the self-renewal capacity of liver CSCs derived from HCC cell lines PLC/PRF/5 and Mahlavu, in vitro, and (vi) ovatodiolide enhances the sensitivity of HCC cells to sorafenib in a synergistic manner (Figure 4 to 6). These findings are relevant to practice in the HCC clinic today because HCC is noted to be highly resistant to contemporary chemotherapeutic agents, with a 0.25 response ratio to contemporary chemotherapy, and no significant increase in patient overall survival (Asghar et al., 2012); this may not be unconnected with its enrichment with YAP1 and related stemness factors, Sox2 and Oct4 as demonstrated in our study.

ACCEPTED MANUSCRIPT In the context of (iv) to (vi), the scientific rationale for this aspect of study comes from previous published works performed in our lab by Bamodu O.A. and co-workers, which demonstrated that ovatodiolide sensitizes aggressive breast cancer cells to the systemic chemotherapeutic antibiotic doxorubicin, eliminates the cancer stem cell-like phonotype of triple negative breast cancer (TNBC) and reduces doxorubicin-associated toxicity (Bamodu et al., 2015) as well as by Lee C.M and colleagues showing the efficacy of the phytochemical

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Pterostilbene as a potent anti-CSC agent in the highly malignant HCC (Lee et al., 2013). Recently, concurrent treatment of advanced, highly malignant and/or unresectable HCC

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with sorafenib and conventional transarterial chemoembolization (TACE) showed some efficacy with acceptable level of safety (Park et al., 2012). Thus, suggesting that overcoming

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chemoresistance, a major challenge of current anti-HCC therapy could depend on a combination of targeted therapies to potentiate chemosensitivity. As alluded above, in our

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study, we further demonstrated the significant role of YAP1 in modulating the sensitivity of HCC parental and stem cells to sorafenib, with significant increase in cytotoxicity and

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decrease in IC50 of sorafenib in the siYAP1-infected Mahlavu cells compared to their wildtype counterparts (3.09 + 0.96 folds, p< 0.01). This increased sorafenib cytotoxicity because of YAP1 silencing, translated into marked reduction in the number of cancer cell colonies

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formed per 100 HCC cells (Figure 4). This validated our initial hypothesis that the aberrant

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expression of YAP1 in HCC cells may confer sorafenib resistance while suppression of its endogenous expression attenuated this effect. We suggest that this is partially attributed to YAP1-induced enhanced ALDH activity (Abdullah et al., 2013). Therefore, targeting YAP1

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in HCC could be used in combination with sorafenib to enhance chemosensitivity to the later. Recently, Bao and his team from a cell-based assay to screen stimulators of the Hippo-

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YAP1 pathway discovered that dobutamine has an inhibitory effect on the nuclear translocation of YAP1 and consequently impair YAP1-dependent gene transcription (Bao et al., 2011). Their findings flared our interest and informed our search for existent synergistic HCC antitumor effects in our array of therapeutic agents. Our study also revealed that YAP1dependent sorafenib resistance which was strongly associated with increased ALDH activity and expression of pluripotency factors Sox2 and Oct4, was abrogated by treatment with ovatodiolide, as evidenced by severely impaired ability to form hepatospheres, significantly suppressed ALDH activity and self-renewal capability of the HCC CSCs and marked downregulation of YAP1, Sox2 and Oct4 in a dose-dependent manner after incubation with

ACCEPTED MANUSCRIPT ovatodiolide alone (Figure 5 and Supplementary Figure 1) or in combination with sorafenib (Figure 6), when compared with untreated control group. Summarily, our results indicate that the novel putative anti-CSC agent, ovatodiolide abrogates YAP1-conferred HCC cell resistance to sorafenib which is in the least partially mediated by activation of Oct4/Sox2 signalling and upregulation of Sox2 and Oct4 protein and mRNA expression, as well as potentiates the anticancer effect of sorafenib.

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In conclusion, herein we provide corroborative evidence that aberrant expression of YAP1 plays a critical role in the induction of HCC cells chemoresistance to sorafenib, and

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that molecularly or therapeutically targeting YAP1 via genetic engineering or use of pharmacological agents respectively, could sensitize the cells to sorafenib-induced death,

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ovatodiolide as a potent sensitizer of sorafenib.

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Thus, projecting YAP1 as a therapeutic target in HCC in combination with sorafenib, and

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Authors’ contributions

HLC: Experimental design, Collation and/or assembly of data, Data analysis and interpretation, Manuscript writing.

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HAC: Conception and design of study, Data analysis and interpretation, Manuscript writing.

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OAB: Conception and design of study, Data analysis and interpretation, Manuscript writing and revision

KFL: Collection and/or assembly of data.

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YMT: Collection and/or assembly of data. WHL: Data analysis and interpretation, Manuscript writing, Final approval of manuscript JTT: Conception and design, Data analysis and interpretation, Manuscript writing, Final

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approval of manuscript. All authors approve the final submitted version. Acknowledgements

This work was supported by National Science Council of Taiwan: Yew-Min Tzeng (MOST103-2113-M-324-001-MY2; MOST-103-2811-M-324-001), and Wei-Hwa Lee (MOST 1052320-B-038-054). This study was also supported by grants from Taipei Medical University (102TMU-SHH-02) to Wei-Hwa Lee and grants from Taipei Medical University (105TMUSHH-15 and 106-FRP-04) to Jo-Ting Tsai. Conflict of interest statement

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The authors declare that there are no potential conflicts of interest.

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ACCEPTED MANUSCRIPT Figure Legends

Figure 1 HCC is enriched in SP cells with enhanced ability to form tumorsphere. (A.) Detection of SP from HCC cells stained with Hoechst 33342 and analyzed. Indicated percentage represents SP cell proportion. (B.) Tumorsphere formation of SP and NSP cells derived from HCC cells. SP cell from Mahlavu and PLC/PRF/5 cells showed enhanced

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ability to form hepatosphere, evidenced by the larger number and size, compared to the NSP cells. (C and D) RT-PCR assay show the expression of YAP1 in selected group of liver cancer cell lines. Overexpression of YAP1 is seen in the highly malignant PLC/PRF/5 and

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Mahlavu, compared to the less malignant Huh7, Hep3B, HepG2 and J5 cells. GAPDH was

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used as internal control and quantitative graphical representation was relative to GAPDH expression.

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Figure 2. YAP1 expression is a putative link between repressed ER-stress and liver cancer stem cell-like phenotype

Immunohistochemical staining of paired tumor-non tumor liver tissues. Staining shows

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strong YAP1, c-Myc and GRP78 staining in the tumor part, while non-tumor tissue showed very weak or no staining for any of the probed proteins. (B) Western blot and (C and D) RT-

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PCR analyses of YAP1 expression in parental and generated hepatospheres show that YAP1 is overexpressed in PLC/PRF/5 and Mahlavu–derived spheres compared to their parental

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counterparts. Expression of YAP1 correlates with that of c-Myc and GRP78. -actin was

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loading control. * p<0.05

Figure 3. YAP 1 regulates HCC cancer stemness (A) Flow cytometric analysis of hepatospheres generated from side population (SP) enriched

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cancer stem cells and their non-side population (non-SP) counterpart show that the PLC/PRF/5 and Mahlavu hepatospheres generated from SP were more in quantity compared to their non-SP counterparts. (B) Mahlavu and PLC/PRF/5 -derived hepatospheres marked with FITC-conjugated YAP1 antibodies, showed significantly enhanced YAP1 activity in the YAP1-enriched hepatospheres, represented by the violet histogram. (C) YAP 1-positive cells formed bigger and many hepatospheres, while the YAP1-silenced HCC cells were unable to form spheres. (D) Western blot analysis of PLC/PRF/5 and Mahlavu hepatospheres showed that when YAP1 is knocked down, there is a corresponding downregulation of Sox2 and Oct4. GAPDH served as loading control. * p<0.05

ACCEPTED MANUSCRIPT Figure 4 Downregulation of YAP 1 increases the sensitivity of HCC cells to Sorafenib. Post- treatment cell viability assay show the effect of sorafenib on HCC Mahlavu cells. (A) IC50 of sorafenib in wild type and YAP1-silenced cells was 6.8 M and 2.2 M, respectively. (B) The number of colonies formed per 100 cells was very significantly reduced when YAP1 was silenced in the Mahlavu cells before sorafenib treatment, compared to silencing YAP1 or treating with sorafenib alone. * p<0.05, ** p<0.01, *** p<0.001

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Figure 5 Ovatodiolide shows more potent anticancer effect by targeting the CSCs. (A and B) Treatment of the HCC cells with a novel anti-CSC agent, ovatodiolide, significantly

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inhibited their ability to form tumorspheres in a dose-dependent manner. (C) Ovatodiolide

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treatment severely suppressed ALDH activity and thus, self-renewal ability in the HCC CSCs as demonstrated by the diminished immunofluorescence signal. (D) Ovatodiolide treatment led to a significantly decreased expression of YAP, Sox2 and Oct4 in the HCC PLC/PRF/5

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and Mahlavu cells. * p<0.05

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Figure 6 Ovatodiolide enhances HCC cells’ sensitivity to sorafenib. Anchorageindependent cell growth assay for assessing the sensitivity of Mahlavu cells to ovatodiolide, sorafenib or combination of the two drugs showed that (A) combination of ovatodiolide and

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sorafenib significantly reduced growth of the HCC cell line, Mahlavu. (B) Isobologram and

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(C) Drug CI table indicating synergism between sorafenib and ovatodiolide. CI, combination index; CI = 1, additivity; CI < 1, synergism; CI > 1, antagonism.

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Supplementary Figure 1. Ovatodiolide inhibits the HCC stem cells’ self-renewal. Graphical representation of the inhibitory effect of Ova on the CSCs self-renewal capacity of

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hepatospheres generated from Mahlavu cells. Cells were pre-treated with 5 μM of Ova before performing the tumorsphere formation assay spanning three generations of hepatospheres, as described in the Materials and Methods section. * p < 0.05, ** p < 0.01, *** p < 0.001.

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Highlights  Increased YAP1 expression in malignant HCC.  Ovatodiolide suppresses HCC stemness.  Ovatodiolide enhances sorafenib’s effect via suppressing YAP1