β-catenin-driven hepatocellular carcinoma

Journal Pre-proofs Targeting Jak/Stat pathway as a therapeutic strategy against SP/CD44+ tumorigenic cells in Akt/β-catenin-driven hepatocellular carcinoma Tan Boon Toh, Jhin Jieh Lim, Lissa Hooi, Masturah Bte Mohd Abdul Rashid, Edward Kai-Hua Chow PII: DOI: Reference:

S0168-8278(19)30547-1 https://doi.org/10.1016/j.jhep.2019.08.035 JHEPAT 7480

To appear in:

Journal of Hepatology

Received Date: Revised Date: Accepted Date:

1 April 2019 20 August 2019 29 August 2019

Please cite this article as: Toh, T.B., Lim, J.J., Hooi, L., Rashid, M.B.M., Chow, E.K-H., Targeting Jak/Stat pathway as a therapeutic strategy against SP/CD44+ tumorigenic cells in Akt/β-catenin-driven hepatocellular carcinoma, Journal of Hepatology (2019), doi: https://doi.org/10.1016/j.jhep.2019.08.035

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© 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Targeting Jak/Stat pathway as a therapeutic strategy against SP/CD44+ tumorigenic cells in Akt/β-catenindriven hepatocellular carcinoma

Tan Boon Toh1,2,a,*, Jhin Jieh Lim2,a, Lissa Hooi2, Masturah Bte Mohd Abdul Rashid3, Edward Kai-Hua Chow2,3,*

1The

N.1 Institute for Health (N.1), National University of Singapore, Singapore

2Cancer

Science Institute of Singapore, National University of Singapore, Singapore

3Department

of Pharmacology, Yong Loo Lin School of Medicine, National University of

Singapore, Singapore *Corresponding

authors at: Center for Life Sciences, National University of Singapore, 28

Medical Drive, #05-COR, Singapore 117456 (Tan Boon Toh); Centre for Translational Medicine, National University of Singapore, 14 Medical Drive #12-01, Singapore 117599 (Edward Kai-Hua Chow) aThese

authors contributed equally to this work.

Graphical abstract Dysregulation and mutations of signaling pathways such as Akt/mTOR and Wnt/βcatenin are frequently observed in human liver cancer. Akt/β-catenin/Stat3 regulatory

axis plays an important role in governing cancer stem-like traits that contributes to liver tumorigenesis.

ga1

Highlights



Co-activation of both Akt/mTOR and Wnt/ β-catenin signaling pathways was found in 14.4% of HCC patients. More importantly, patients with co-activated pathways portend a poorer survival than patients who exhibited either Akt/mTOR or Wnt/β-catenin activation alone.



Akt/β-catenin tumors contained a subpopulation of cells with stem/progenitor-like characteristics identified through side population (SP) analysis and expression of the cancer stem-like marker CD44, which may contribute to tumor sustenance and drug resistance.



MDR1 as the main driver of SP phenotype in the Akt/β-catenin-driven HCC as identified through the high protein expression of MDR1 and the use specific drug transporter inhibitors.



Activated Stat3 play an important role in regulating the self-renewing and tumorigenic population of SP/CD44+ cells in Akt/β-catenin tumors and the use of Jak/Stat inhibitors to abrogate cell proliferation and consequent cancer cells with stemness properties.

Abstract Background & Aims Hepatocellular carcinoma (HCC) is the most common primary liver cancer, accounting for more than 780,000 deaths worldwide each year. Hepatic resection and liver

transplantation with adjuvant chemo- and radiotherapy are the mainstay of HCC treatment, but the 5-year survival rate remains poor because of frequent recurrence and intrahepatic metastasis. Currently, only Sorafenib and Lenvatinib are approved for firstline therapy against advanced, unresected HCC, but yield modest survival benefits. Thus, there is a need to identify new therapeutic targets to improve current HCC treatment modality.

Methods The HCC tumor model was generated by hydrodynamic transfection of Akt and βcatenin oncogenes. Cancer cells with stemness properties were characterized following isolation using side population (SP) and CD44 surface markers by flow cytometry. The effect of Jak/Stat inhibitors were analyzed in vitro by using tumorsphere culture and in

vivo using a xenograft mouse model.

Results Co-activation of both Wnt/β-catenin and Akt/mTOR pathways was found in 14.4% of our HCC patient cohort. More importantly, these patients showed poorer survival than those with either Wnt/β-catenin or Akt/mTOR pathway activation alone, demonstrating the

clinical relevance of our study. In addition, we observed that Akt/β-catenin tumors contained a subpopulation of cells with stem/progenitor-like characteristics identified through SP analysis and expression of the cancer stem cell-like marker CD44, which may contribute to tumor sustenance and drug resistance. Consequently, we identified small molecule inhibitors of Jak/Stat pathway that demonstrated efficacy in mitigating tumor proliferation and formation in Akt/β-catenin-driven HCC.

Conclusions Our study supports the use of a clinically relevant tumor model in the development of Jak/Stat-based drug therapies against Akt/β-catenin-driven HCC.

Lay Summary In this study, we identified a subpopulation of cancer cells with stemness properties in a mouse model of HCC that is critical for tumor maintenance and growth. Targeting Jak/Stat pathway may be a potential therapeutic intervention in HCC.

Keywords liver cancer

hepatocellular carcinoma Akt

β-catenin

Jak/Stat side population CD44

Introduction Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death worldwide with a very high mortality rate. [1] Hepatitis B and C viral infections, aflatoxin B1, and alcohol abuse are main risks factors for HCC development. [2] Despite treatment with advanced surgical resection, liver transplantation, or ablation, the 5-year survival rate for HCC patients remains poor due to frequent recurrence and intrahepatic metastasis. [3] Currently, the first-line FDA-approved drugs for the treatment of advanced HCC are Sorafenib and Lenvatinib, both of which are multi-kinase inhibitors of Raf, VEGF, PDGF and c-kit signaling with anti-proliferative and anti-angiogenic activity.[4-6] However, patients treated with these multi-kinase inhibitors showed marginal overall survival of 2 to 3 months improvement compared to placebo group, and in most instances with low partial treatment response rate due to drug resistance. [7] Therefore, there is a need to understand the biology of hepatocarcinogenesis and identify new therapeutic targets to improve current HCC treatment modality. Hepatocarcinogenesis is a multistep process involving multiple signaling cascades, contributing to a heterogeneous molecular profile. Recent deep molecular profiling efforts have identified several main mutations in HCC including the gene for β-catenin, CTNNB1 which occurs at a frequency of about 30% of

all HCC tumors. [8] In addition, dysregulated signaling pathways of the Wnt/β-catenin, Ras/Raf/MEK/ERK, and PI3K/Akt/mTOR axis have been frequently identified in HCC. [8] In particular, components of the Wnt/β-catenin pathway (e.g. APC, β-catenin and GSK3β) are dysregulated in more than 25% of HCC patients. Importantly, transcriptomic analysis of HCC clinical specimens identified specific pathway activation, in particular, the Wnt and Akt pathways that are associated with distinct clinical outcome. [9] Moreover, Wnt and Akt pathway activations were observed in more than fifty percent of the tumors studied, suggesting the important clinical implication in developing targeted therapies against these activated signaling modules. The existence of a subpopulation of cancer cells with stemness properties (CCSPs) has been identified in various solid tumors, including hepatic tumors.[10,11] Commonly used surface markers to identify selfrenewal and tumorigenic population of cells in HCC tumors include CD133, CD90, CD13, EpCAM and CD44.[11-14] However, no single marker has been shown to be specific and universal in identifying CCSPs due to the molecular heterogeneity of the tumors. Alternatively, the use of side population (SP) to prospectively isolate and characterize CCSPs based on their ability to efflux Hoechst 33342 is also frequently employed.[10,15-18] Activated Wnt/β-catenin pathway has been implicated in various aspects of both normal stem cell and CCSP biology, including in the liver.[19,20] Additionally, Akt has been previously identified in HCC to contribute to chemoresistance mechanisms in CCSPs. [21] To study the crosstalk between Akt and Wnt/β-catenin pathways in HCC, we utilized a hydrodynamic transfection mouse model of liver cancer that overexpresses the activated forms of AKT and CTNNB1.[22,23] We aimed to characterize and study the tumorspheres isolated from these Akt/β-catenin-driven tumors to screen for potential small molecule inhibitors with therapeutic implications in HCC.

Materials and Methods Hydrodynamic transfection mouse model To generate Akt/β-catenin hepatic tumor mouse model, 20 µg of two plasmids encoding for oncogenic forms of myr-Akt (myristoylated Akt) and

Δ90Nβ-catenin (first 90 amino acids deleted) and transposon were mixed with 2 µg of plasmids encoding the Sleeping Beauty transposase in 2.5 mL of phosphate-buffered saline (PBS) and injected into the lateral tail vein of 6- to 8-week old male (n=5) and female (n= 40) wild-type FVB/N mice. pT3myr-AKT-HA tagged (Addgene plasmid # 31789) and pT3-N90-betacatenin-Myc tagged (Addgene plasmid # 31785) were gifts from Xin Chen.[24,25] In all experiments, age- and gender-matched mice (female) were used, unless otherwise specified . All mice were maintained in an enriched environment on a 12 hours light-dark cycle and fed with Teklad global 18% protein rodent diet. All Akt/β-catenin tumorspheres were harvested from female Akt/β-catenin-hydrodynamically transfected FVB/N mice. All animal experimentations were performed according to guidelines and protocols (R18-1508) approved by the National University of Singapore Institutional Animal Care and Use Committee (IACUC). Tumorsphere isolation, allograft transplantation and in vivo tumor studies Liver tumor tissues from female Akt/β-catenin tumor mice were harvested and diced into 1–2 mm pieces before treating with 1 mg/ml collagenase/dispase (Roche, IN) for 5 minutes at 37°C. The tumor suspension was then filtered through a sterile medical gauze and centrifuged for 5 minutes at 1000 rpm, 4°C. This was repeated by passing

cells through a 70 and 40 µm cell strainer. Subsequently, tumor cells were resuspended in 1X red blood cell lysis buffer and incubated on ice for 3 minutes, followed by washing and centrifuging at 1000 rpm for 5 minutes. Thereafter, cells were resuspended in serum-free media supplemented with defined growth factors, [26] and seeded into cell culture flasks for generation of tumorspheres. For in vivo tumor xenograft study, five female FVB/N mice per group were injected with 2 x 106 live tumorsphere cells pre-treated with DMSO, AZ960, or TG101209 and monitored for tumor growth. Tumor volume was calculated according to the formula V =0.5 ×A2 ×B, where A is the smallest superficial diameter and B is the largest superficial diameter. For in vivo primary tumor study, female FVB/N mice were hydrodynamically injected with oncogenic forms of Akt (myristoylated Akt) and Δ90Nβ-catenin to generate Akt/β-catenin hepatic tumor. Mice were subsequently randomized into 3 groups (n=4)—Vehicle, AZ960 (10 mg/kg), and TG101209 (100 mg/kg) 6 weeks post injection. Mice were then administered with their respective treatments (AZ960 by intraperitoneal injection and TG101209 by oral gavage) for 4 consecutive weeks (alternate days at 3 times a week) before all the mice were euthanized. Total liver weight to body weight ratios were recorded as a measurement of tumor burden. Minimum sample size of mice (n=4) per treatment group was

determined by power analyses based on an expected minimum 2x difference with a 0.5 SD, a power of 80%, and a statistical significance of <0.05. Immunoblot analysis Cells were lysed in buffer containing 0.5% sodium deoxycholate, 1% NP-40 detergent, 0.1% SDS, 0.15M NaCl, 10mM Tris-HCl pH7.4, protease and phosphatase inhibitor cocktail tablets (Roche). Equal amounts of protein lysate were resolved by SDS-PAGE and electrotransferred onto PVDF membranes. Membranes were processed according to standard procedure and proteins were detected using the ImageQuant LAS 500 imaging system. Antibodies used for immunoblot analyses included active-β-catenin (Millipore), β-catenin (BD Transduction), MDR1 (Santa Cruz Biotechnology), phospho-Akt (Ser473), Akt, phospho-p70 S6 Kinase (Thr389), p70-S6 Kinase, cyclin D1, c-Jun, phospho-Stat3 (Tyr705), Stat3, Caspase 3, PARP, and ABCG2 (Cell Signaling), and β-actin (Sigma). Additional details on the antibodies are included in Supplementary Table S2. Proteins were detected using the ImageQuant LAS 500 imaging system. Side population analysis Tumor cell suspensions were labeled with Hoechst 33342 dye (Sigma) using the methods described by Goodell et al. with slight modifications.[18,27] Briefly, cells (1 × 106 cells/mL) were incubated in pre-warmed DMEM/2% FBS containing

freshly added Hoechst 33342 (5 μg/mL) either alone or in the presence of 100 μM verapamil (Sigma) for 90 minutes at 37°C with intermittent mixing. After incubation, cells were spun down at 4°C and resuspended in HBSS+ (HBSS with 2% FBS and 10 mM HEPES buffer) and stained with FITC-conjugated anti-CD44 antibody (BioLegend) for 30 minutes on ice. Cells were then washed with HBSS+, collected by centrifuging for 5 minutes and resuspended in HBSS+ containing 7AAD (5 µL/test, STEM CELLTM Technologies), which allows for discrimination of dead versus live cells. Subsequently, cells were filtered into FACS tubes and analysed using FACSAria (BD Bioscience). Hoechst 33342 was excited with the UV laser at 350 nm and its fluorescence was measured with 405/BP30 (Hoechst Blue) and 570/BP20 (Hoechst Red) optical filters. Side population (SP) gating was defined based on fraction of cells that were removed by verapamil treatment and the percentages of SP population that were positively-stained for CD44 (SP/CD44+) and non-SP that were negative for CD44 (NSP/CD44-) were recorded. Colony formation assay, limiting dilution assay and allograft tumor initiation assay SP and CD44-positive (SP/CD44+) and non-SP and CD44-negative (NSP/CD44-) tumorspheres were isolated by flow cytometry, and 30 cells were seeded per well in a 96-well cell culture plate, in growth factor-supplemented serum-free DMEM media (Biowest). The number of colonies formed was counted every 4 days, up to

28 days of growth. For limiting dilution assay, cells were seeded at densities of 5, 10, 25 and 50 cells per well in 96-well plates, and colony formation was observed every 5 days, up to 20 days of growth. For allograft tumor initiation, sorted cells were resuspended in supplemented serum-free media with Growth Factor Reduced Matrigel (BD Bioscience) in a 1:1 ratio, and injected into the right and left flanks of FVB/N mice. SP/CD44+ allograft tumors that formed were subsequently harvested and serially passaged in mice for up to 3 generations (P0 – P2), as well as subjected to in vitro assays as described above. Immunohistochemistry Tissues were fixed in 4% paraformaldehyde (PFA) immediately after isolation and embedded in paraffin. Tissue sections of 4 μm thickness were stained with hematoxylin and eosin (H&E) as well as the following antibodies: β-catenin (BD Transduction), Ki-67 (Millipore), cyclin D1 (Cell Signaling), c-Jun (Cell Signaling), phospho-Akt (Ser473) (Cell Signaling), and phospho-p70 S6 Kinase (Abcam). Fluorescent immunohistochemistry and tissue microarray (TMA) analysis Two sets of tissue microarrays of human liver cancer patients (OutdoBiotech and Biomax) comprising a total of 118 matched normal adjacent and tumor tissues were subjected to multiplex fluorescent immunohistochemistry based on OpalTM protocol (PerkinElmer). Briefly, tissues were serially stained with β-catenin primary antibody (BD Transduction) for 1 hour followed by HRP-conjugated

secondary antibody and tyramide signal amplification (TSA) by fluorescein isothiocyanate (FITC) (PerkinElmer). Tissues were subsequently stained with pAkt primary antibody (Cell Signaling) followed by CD44 (BD Transduction), and the same TSA system was applied for both, using Cyanine 3 (Cy3) and Cyanine 5 (Cy5) as the respective fluorescent stains. DAPI was used as the nuclear counterstain. Image acquisition and signal quantification analysis of the tissues were performed using the Vectra® multispectral imaging system and inForm® software (PerkinElmer). Positive expression was determined by comparing fluorescence intensities of tumor tissues with the respective normal adjacent tissues (NAT). Low and high expression were determined using the first and third quartile mean intensities respectively as thresholds. Viability and sphere assays Tumorspheres were dissociated into single cells with AccutaseTM (Millipore) and seeded into 96-well plates, at 200 cells/µl, with DMEM/F12 medium supplemented with defined growth factors. Cells were allowed to recover over 2 days before drug treatment. Cell viability was assessed using alamarBlue® (Serotec) where cells were incubated with 10% culture volume of alamarBlue® for approximately 16 hours before reading at absorbance 570 and 600 nm. Dose response curves were generated using GraphPad Prism (GraphPad Software) and IC50 values were computed.

Quantitative RT-PCR RNA was isolated using TRI Reagent® (Sigma-Aldrich) and reverse transcribed into cDNA using the iScript Reverse Transcription Supermix (Bio-Rad Laboratories) according to the manufacturers’ instructions. For quantitative realtime PCR, each sample was done in triplicate and subjected to 40 amplification cycles of PCR (Applied Biosystems Prism 7500 sequence detection system). Primers used were as follows: EPCAM (forward primer, 5’TTGCTCCAAACTGGCGTCTA-3’; reverse primer, 5’ACGTGTCTCCGTGTCCTTGT-3’), CD44 (forward primer, 5’TCCGAATTAGCTGGACACTC-3’; reverse primer, 5’CCACACCTTCTCCTACTATTGAC-3’), CD90 (forward primer, 5’ATCCAGCATGAGTTCAGCCT-3’; reverse primer, 5’ATCCTTGGTGGTGAAGTTGG-3’), CD133 (forward primer, 5’GAAAAGTTGCTCTGCGAACC-3’; reverse primer, 5’TCTCAAGCTGAAAAGCAGCA-3’), MDR1 (forward primer, 5’CCCATCATTGCGATAGCTGG-3’; reverse primer, 5’TCCAACATATTCGGCTTTAGGC-3’), ABCG2 (forward primer, 5’GAACTCCAGAGCCGTTAGGAC-3’; reverse primer, 5’CAGAATAGCATTAAGGCCAGGTT-3’), G6PC (forward primer, 5’CAGGACTGGTTCATCCTT-3’; reverse primer, 5’GTTGCTGTAGTAGTCGGT-3’), TAT (forward primer, 5’-

ACCTTCAATCCCATCCGA-3’; reverse primer, 5’-TCCCGACTGGATAGGTA3’), NANOG (forward primer, 5’- TTCCTCTGAAGACCTGCCTCT-3’; reverse primer, 5’- ATCTGCTGGAGGCTGAGGTA-3’), POU5F1 (forward primer, 5’TGGACACCTGGCTTCAGACTT-3’; reverse primer, 5’ATCCCTCCGCAGAACTCGTAT-3’) and GAPDH (forward primer, 5’TGTGAACGGATTTGGCCGTAT-3’; reverse primer, 5’ACAAGCTTCCCATTCTCGGC-3’). The level of gene expression was determined using GAPDH as the normalizer gene and expressed as mean ± SEM of the triplicate PCR reactions. Cell cycle analysis Cells were first treated with AZ960, TG101209 or DMSO for 48 hours, then harvested and washed with cold phosphate-buffered saline (PBS), followed by overnight fixation with ice-cold 70% ethanol. Cells were then incubated with RNase A (100 μg/ml) and propidium iodide (PI, 50 μg/ml) for 10 mins. Cell cycle distribution was assessed by DNA content using flow cytometry (BD LSRII, BD Biosciences), and analysis was done using FlowJo software. Drug treatment using small molecule inhibitor Tumorspheres were dissociated with AccutaseTM (Millipore) and seeded into 96well plates at a density of 5,000 cells per well. Cells were allowed to adhere and recover for 24 hours prior to drug treatment. Thereafter, cells were subjected to 1

μM of each drug from the Stem cell Inhibitor library (SelleckChem). The drug treatment was executed by the liquid handling system, MiniJanus (Perkin Elmer). The drugs were diluted in media to a concentration of 20 μM before dispensing the drugs into master plates containing the respective media to a final concentration of 1 μM by the MiniJanus system. Thereafter, the library of drugs at 1 μM were added to the plates containing the respective cells and incubated for 24 hours. Statistical analysis All experimental data were performed in at least triplicates (unless otherwise stated), the results averaged, and the standard deviation (SD) or standard error of the mean (SEM) calculated. Unpaired two-tailed student's t test was used for statistical comparison of two different independent groups. Kaplan-Meier survival curves were compared using log-rank (Mantel-Cox) test. Pearson correlation coefficient was computed to assess the relationship between the expression levels of two proteins. p < 0.05 was accepted as statistically significant. Results Tumorspheres derived from Akt/β-catenin-driven tumors demonstrate cancer cells with stemness properties To assess the potential role of the cooperation of Wnt/β-catenin and Akt/mTOR signaling during hepatogenesis, we used a previously reported hepatic tumor mouse model, Akt/β-catenin, using sleeping beauty (SB)

transposon hydrodynamic transfection system. [23] Specifically, we co-inject the oncogenic forms of human AKT1 and constitutively active Δ90β-catenin into 6- to 8-week-old wild-type FVB/N mice. This procedure leads to the formation of high HCC tumor burden in both male and female FVB/N mice with 100% penetrance ( Supplementary Figure S1 A). We showed that FVB/N mice displayed perimoribund liver tumor burden after 90 days ( Figure 1 fig1 Ai) post hydrodynamic transfection with the oncogenes. The gross morphology of the tumors presented was multifocal with more than 100 nodules. Compared with the pT3-empty control, the histology of Akt/βcatenin tumors revealed distinct HCC-like morphological patterns ( Figure 1 Aii). HCC-like areas were composed of large cells with large nuclei, coarse chromatin, prominent nucleoli, and granular eosinophilic cytoplasm. In addition, the tumors had a high mitotic rate as measured by the Ki-67 staining ( Figure 1 Aiii). To confirm the presence of Wnt/β-catenin and Akt/mTOR pathway activation, we stained for β-catenin and phosphorylated Akt. β-catenin staining demonstrated both cytoplasmic and distinct nuclear staining, a key hallmark of active Wnt/β-catenin signaling ( Supplementary Figure S1 B). In addition, we also demonstrated that downstream targets of the Wnt/βcatenin (c-jun and cyclin D1) were also upregulated as compared to the

pT3-empty control group. Next, we performed an immunohistochemical (IHC) analysis with an antibody against phosphorylated Ser473 of Akt to recognize the active forms of all three Akt isoforms and verified the presence of high levels of phosphorylated Akt (p-Akt, Ser473) in Akt/βcatenin-driven tumor cells ( Supplementary Figure S1 B). High levels of phosphorylated p70S6K (p-p70S6K, Thr389), a downstream target of Akt, was also observed in these tumors ( Supplementary Figure S1 B). These tumors are also homogeneously immunoreactive for HA-tagged myr-Akt and Myc-tagged Δ90Nβ-catenin , indicating their origin from doublytransfected cells ( Supplementary Figure S1 C). Immunoblot analysis confirmed the activation of both Wnt/β-catenin and Akt/mTOR pathways in these tumors ( Figure 1 B). To demonstrate that both constitutively active Akt and mutant β-catenin are driving the tumorigenicity of Akt/β-catenin tumors, we isolated the primary tumors and subcutaneously injected the tissues into the allograft recipients. We showed that the allograft tumors formed were further enriched for the expression of nuclear β-catenin, p-Akt and their respective downstream targets ( Figure 1 B), suggesting that both Akt/mTOR and Wnt/β-catenin play a key role in driving the tumorigenesis of HCC.

Recent studies suggest that the heterogeneity and the maintenance of HCC tumors are attributed to a small subset of cancer cells with stemness properties (CCSPs). To identify and enrich for the CCSP population in the Akt/β-catenin tumors, we employed the use of side population (SP) analysis using Hoechst 33342 dye efflux and several commonly used HCC CCSP markers, in particular CD44, CD90 and EpCAM. We isolated and grew tumor spheroids (tumorspheres) derived from Akt/β-catenin tumors. These Akt/β-catenin tumorspheres could be grown and passaged in serumfree medium supplemented with defined growth factors ( Figure 1 Ci). We showed that tumorspheres derived from Akt/β-catenin tumors maintained a high expression of p-Akt and β-catenin in vitro ( Figure 1 Cii). More importantly, we are able to serially passage these tumorspheres in vitro and maintain these tumors subcutaneously in mouse allografts for more than 10 passages, demonstrating the tumorigenicity of these cells ( Figure 1 D). To determine the expression levels of commonly used HCC CCSP markers, we first performed quantitative real-time PCR and protein expression analysis on Akt/β-catenin primary tumors and their derived tumorspheres. We showed high mRNA expressions of CD44 and CD90, but not EPCAM and CD133 in both the primary tumors and the

tumorspheres as compared to their pT3-empty control counterparts ( Supplementary Figure S2 ). Flow cytometry analysis of these HCC CCSP markers demonstrated that only CD44, but not CD90 and EpCAM was consistently highly enriched in the Akt/β-catenin tumorspheres as compared to their primary tumors from which they were derived ( Figure 1 E). In addition, SP analysis with Hoechst 33342 demonstrated that Akt/βcatenin primary tumors and their derived tumorspheres contained SP fraction of 0.26% and 24.7% respectively ( Figure 1 F), further demonstrating that Akt/β-catenin tumorspheres have an enriched cellular fraction of SPlike tumorigenic cells. In addition, we also observed an increase of several key stemness markers such as POU5F1 and NANOG and the decrease in the expression of common hepatocyte differentiation markers such as tyrosine aminotransferase (TAT) and glucose-6-phosphatase (G6PC) in both the Akt/β-catenin tumors and tumorspheres ( Supplementary Figure S2 ). These data suggest that Akt/β-catenin tumorspheres derived from the Akt/β-catenin tumors are enriched with CCSP-like and tumor-propagating properties. Co-activation of Wnt/β-catenin and Akt/mTOR signaling portends poorer survival in HCC patients To demonstrate the clinical relevance of studying the co-expression of βcatenin and p-Akt (Ser473), we conducted multispectral fluorescent

imaging on tissue microarrays (TMAs) consisting of 118 HCC patients. Using the matched normal adjacent tissues (NAT) from each patient as a control, we showed that 23.7% and 21.2% of these patients expressed only β-catenin and p-Akt (Ser473) respectively. Co-activation of both β-catenin and p-Akt were observed in 14.4% of the cases ( Figure 2 fig2 A). Not surprisingly, our results showed that intracellular localization of β-catenin in HCC patient tumor tissues was heterogeneous, with 13% displaying membranous β-catenin staining, 18% with both membranous and cytoplasmic staining, 16% showing both nuclear and cytoplasmic staining, and 49% with membranous, nuclear and cytoplasmic staining ( Figure 2 B). In contrast, β-catenin expression in the NAT was either absent or present only on the membrane ( Figure 2 C). More importantly, Kaplan-Meier survival analysis demonstrated that patients with co-expression of both β-catenin and p-Akt (Ser473) have the worst overall survival with a median survival of 9 months as compared to the other groups individually ( Figure 2 D). Interestingly, when we stratified these 118 HCC patients according to their tumor stages, we observed that both high β-catenin and p-Akt coexpression can predict for poorer prognosis only amongst the stage 3 patients ( Figure 2 E). Specifically, p-Akt expression alone did not stratify for survival in stage 1 and stage 2 HCC patients. Log-rank (Mantel-Cox)

analysis of the Kaplan-Meier survival curves for stage 1 and stage 2 HCC patients based on p-Akt expression generated p-values of 0.3173 and 0.8891 respectively ( Figure 2 Ei). On the other hand, p-Akt expression in stage 3 HCC patients showed significant survival stratification with log-rank p-value of 0.0115. The prognostic value of p-Akt has been previously reported by Schmitz et al. where they showed increased p-Akt expression to be associated with overall poorer survival in HCC patients. [28] In addition, p‐Akt has been demonstrated as a risk factor for early disease recurrence and poor prognosis by Nakanishi and colleagues, suggesting the involvement of Akt activation in the progression of HCC. [29] Similarly, we observed that β-catenin expression did not stratify for HCC patient survival at clinical stages 1 and 2 with log-rank p-values of 0.3173 and 0.2872 respectively ( Figure 2 Eii). Kaplan–Meier survival curves of tumor stage stratified HCC patients expressing high β-catenin versus low βcatenin showed significant survival stratification for stage 3 HCC patients with a log-rank p-value of 0.0058. This is consistent with a previous study by Inagawa et al. where they showed that β-catenin expression correlated with poorer survival of HCC patients in only grade 3 but not grades 1 and 2 tumors, suggesting that β-catenin expression is an indicator of poorer prognosis in high grade HCC tumors. [30] This is also consistent with an

earlier study by Kondo et al. which demonstrated β-catenin accumulation and mutations to be associated with late events in malignant hepatocarcinogenesis. [31] Collectively, these data indicate that both Wnt and Akt pathways are important for the progression into late tumor stages of the disease. Our study demonstrates the clinical relevance of studying co-activation of both Wnt/β-catenin and Akt/mTOR signaling in HCC patients. Side population in Akt/β-catenin-driven tumors is mediated through MDR1 transporter As previous studies have shown that both the ABCG2 and MDR1 drug transporter proteins are able to efflux Hoechst 33342 dye [32,33], we next determined which of these two transporters play a key role in mediating the SP phenotype of Akt/β-catenin tumorspheres. Interestingly, we showed that MDR1 but not ABCG2 was significantly overexpressed in these Akt/βcatenin tumorspheres as compared to the matched normal liver and the primary tumor ( Figure 3 fig3 A). Importantly, we also observed a similar high enrichment of MDR1 expression in the tumorspheres by immunoblot analysis ( Figure 3 B), further suggesting the role of CCSP-like cells in mediating the chemoresistance phenotype in these tumors. To verify that the side population is specifically mediated through the MDR1 transporter,

we used specific drug transporter inhibitors Fumitremorgin C and Ko143 (ABCG2 transporter inhibitors) or Tariquidar and Verapamil (MDR1 transporter inhibitors) and demonstrated that only Tariquidar and Verapamil treatment can inhibit the SP phenotype of these cells ( Figure 3 C). Collectively, our data suggests that Akt/β-catenin tumorspheres demonstrated SP phenotype primarily mediated through MDR1 drug transporter. SP/CD44+ population enriched for a highly tumorigenic population in Akt/βcatenin tumors and tumorspheres Our initial analysis of the CCSP markers in the Akt/β-catenin tumorspheres showed enriched CD44 expression and the SP phenotype. To highlight the importance of CCSP-like cells in maintaining the tumorigenicity of the tumors, we flow-sorted SP-positive cells with CD44 expression (SP/CD44+) and non-SP cells with no CD44 expression (NSP/CD44-) populations and performed long term colony formation assays over a period of 24 days to assess the stem cell frequency and self-renewal potential of these subfractions of tumorspheres in vitro at clonal densities. Specifically, flowsorted SP/CD44+ and NSP/CD44- cellular fractions were plated at clonal density to allow tumorspheres to arise from single cells as opposed to cellular aggregation. Our results demonstrated that the SP/CD44+ cells significantly formed more tumorspheres as compared to the NSP/CD44-

fraction, suggesting that SP/CD44+ subfraction is highly enriched for the tumorsphere-forming capacity ( Figure 4 fig4 A). The abilities to recapitulate tumor pathophysiology in an immunocompromised animal model and to serially transplant the tumor provide unequivocal evidence for the definition of CCSPs. [34] We flowsorted these two subpopulations of tumorspheres and investigated the tumor-formation ability of each fraction in vivo. Tumor formation analysis of Akt/β-catenin tumor cells revealed that SP/CD44+ subfraction were able to form tumors from 1000 cells (5/5 animals) ( Figure 4 B, Table 1 tbl1 ). This is in contrast with the NSP/CD44- fraction, in which no tumor formation was observed (0/5). More importantly, we could demonstrate the maintenance and high tumorigenicity of the SP/CD44+ fraction when we flow-sorted tumor cells from these tumors after serial passaging and performed colony formation assay ( Figure 4 C, Table 1 ). We observed that the SP/CD44+ population had higher tumor reformation capacity as compared to their NSP/CD44- counterparts, demonstrating that the self-renewal capacity resides mainly in the SP/CD44+ fraction of the tumorspheres. Subsequently, we also carried out a limiting dilution analysis on the Akt/βcatenin tumorspheres sorted based on their SP and CD44 profiles to

assess the CCSP frequency of these Akt/β-catenin tumorspheres. We showed significantly higher CCSP frequency in the SP/CD44+ fraction in contrast to the NSP/CD44- fraction from both Akt/β-catenin tumorspheres ( Figure 4 Di) and the Akt/β-catenin allograft tumors ( Figure 4 Dii). More importantly, when we analyzed the protein expression of the key components of Wnt/β-catenin and Akt/mTOR pathways, we found increased levels of Δ90β-catenin and p-Akt in the SP/CD44+ fraction as compared to the NSP/CD44- fraction as well as their associated downstream effector proteins, cyclin D1 and p-p70 S6K ( Figure 4 E), demonstrating that both pathways are crucial in driving the tumorigenic population marked by SP/CD44+. Furthermore, to determine if SP/CD44+ cells are found in human HCC, we analyzed six human HCC cell lines (Hep3B, Huh7, LM3, SNU387, SNU398, and SNU449) for the existence of SP/CD44+ CCSP population via flow cytometry. While all six HCC cell lines showed differential expression of SP and CD44+ expression, we observed that LM3 and SNU449 displayed relatively high SP/CD44+ CCSP population of 31.52 ± 2.885 % and 5.86 ± 3.417 % respectively ( Table 2 tbl2 , Supplementary Figure S3 ). Our observations are consistent with previous studies where high SP and CD44

expression has been identified in CCSP-like LM3 cells, suggesting the clinical relevance of studying SP/CD44+ CCSPs.[14,35] In addition, we also analyzed the TMA for CD44 expression in different tumor stages via multispectral fluorescent imaging. We observed that CD44 protein expression showed an increasing trend with tumor stage ( Supplementary Figure S4 A) and is positively correlated with β-catenin and pAkt expression in HCC patients with later stage tumors. Specifically, positive correlation was observed between CD44 expression and p-Akt for all stages of HCC tumors analyzed in the TMA, with higher Pearson correlation coefficient ‘r’ at later tumor stages of 2 and 3 ( Supplementary Figure S4 B). Interestingly, CD44 expression did not demonstrate positive

correlation with β-catenin in stage 1 tumors (r = -0.5709) and is only positively correlated for stage 2 and 3 HCC patients with pearson correlation coefficient r = 0.2027 and 0.1519 respectively ( Supplementary Figures S4 Bi – S4Biii). These data suggest that both Wnt/β-catenin and

Akt/mTOR pathways cooperate in maintaining the stemness properties (CD44 expression) of cancer cells and highlights the clinical relevance of studying SP/CD44+ CCSPs in human HCC. Jak/Stat inhibitors induced apoptosis and cell cycle arrest in Akt/β-catenin tumorspheres

In order to find therapeutic efficacy in mitigating tumor growth in CCSPs, we screened the effect of 81 small molecule inhibitors from a stem cell inhibitor library (SelleckChem) on the Akt/β-catenin tumorspheres cultured in serum-free media. After incubation for 24 hours with the compounds at 1 µM, viability of the cells were determined. From the screen, we identified 10 drug candidates that potently inhibited the viability of the Akt/β-catenin tumorspheres to less than 50% ( Figure 5 fig5 A). Interestingly, we observed that six out of the top ten hits were Jak/Stat pathway inhibitors. We showed that Akt/β-catenin tumor and tumorspheres contained enrichment of the activated Stat3 protein ( Figure 5 Bi). Moreover, activated Stat3 protein is significantly enriched in the SP/CD44+ fraction of the Akt/β-catenin tumorspheres as compared to the NSP/CD44- population ( Figure 5 Bii), suggesting that activated Stat3 may play a role in regulating the selfrenewing and tumorigenic population of SP/CD44+ cells in Akt/β-catenin tumors. [36] Moving forward, we used two Jak/Stat inhibitors (TG101209 and AZ960) identified in our initial screen to test their efficacy in the tumorspheres. Akt/β-catenin tumorspheres treated with TG101209 and AZ960 at their respective IC50 ( Figure 5 Ci) showed decreased p-Stat3 levels ( Figure 5 Cii). Interestingly, we also observed a similar reduction of active β-catenin and

p-Akt protein levels in the tumorspheres, suggesting the crosstalk of Jak/Stat pathway with Akt and β-catenin pathways. The reduction of these key signaling components of major survival pathways is accompanied by the increased expression of apoptotic markers (cleaved -p70S6K, -caspase 3, and -PARP) observed in the treated cells ( Figure 5 Cii). In addition, we explored the effects of Jak2 inhibition on cell-cycle distribution by flow cytometry. We showed that tumorspheres treated with AZ960 and TG101209 prominently induced accumulation of cells at G2/M phase of the cell cycle, with AZ960-treated cells having a higher apoptotic population than TG101209-treated cells ( Figure 5 D). More importantly, Jak/Stat inhibition reduced the CCSP phenotype of the Akt/β-catenin tumorspheres. Specifically, CD44-positive cells of AZ960- and TG101209-treated Akt/βcatenin tumorspheres significantly decreased to 39.6 ± 0.45% and 29.5 ± 2.31% from 63.9 ± 3.58% of DMSO-treated control cells ( Figure 5 Ei). In addition, AZ960 and TG101209 treatment significantly reduced the proportion of SP cells from 26.3 ± 2.54% to 11.6 ± 0.40% and 6.35 ± 0.53% respectively as compared to DMSO-treated control ( Figure 5 Eii). Taken together, our findings suggest that pharmacological inhibition of Jak/Stat activity by small molecule Jak/Stat inhibitors resulted in suppression of cell proliferation and consequent CCSP markers of the tumorspheres. Our

findings support the notion that Jak/Stat may be an effective therapeutic target for HCC treatment. Jak/Stat inhibitors delayed tumor formation and progression of Akt/βcatenin tumors To confirm the suppression of tumor growth upon Jak2 inhibitor treatment in vivo, we induced Akt/β-catenin tumor xenografts into mice subcutaneously by injecting 2 x 106 viable and singly dissociated Akt/βcatenin cells after 24 hours post treatment with either DMSO, AZ960 or TG101209. Tumor growth inhibition was then assessed by comparing tumor volumes of vehicle- and Jak/Stat inhibitors-treated mice. All of the DMSO-treated group formed tumors (5/5), whereas 2 out of 5 mice injected formed tumors for AZ960- and TG101209-treated groups. In addition, tumors that formed from AZ960- and TG101209-treated mice showed significant reduction in tumor volume compared with vehicle-treated mice ( Figure 6 fig6 Ai and Aii). Importantly, immunohistochemistry of tumor tissues from the treated groups showed significant reduction of p-Stat3 protein level as compared to the vehicle group ( Figure 6 Bi and Bii), suggesting that Jak2 inhibition-mediated reduction in tumorigenicity is due to decreased self-renewal and proliferation potential of the tumor cells.

In addition, we performed in vivo treatment on Akt/β-catenin tumors 6 weeks post hydrodynamic injection after the mice displayed moderate tumor burden. Mice were randomized into 3 different groups (vehicle, AZ960, and TG101209) to receive treatment for up to 4 weeks, at which all mice were then euthanized due to high liver tumor burden in the vehicle group ( Figure 6 Ci). We use liver weight to body weight ratio as a measurement of tumor burden in this model. Specifically, we showed that tumor burden was significantly reduced in AZ960-treated mice as compared to the vehicle control ( Figure 6 Cii). TG101209-treated mice did not show statistically significant lower tumor burden, but nevertheless displayed moderate reduction in tumor burden with a mean liver weight to body weight ratio of 1.56 fold reduction as compared to vehicle control. More importantly, we demonstrated that AZ960-treated mice displayed a significant reduction in p-Stat3 levels with concomitant reduction in Ki-67 proliferative index. ( Figure 6 Ciii, Supplementary Figure S5 ). These data suggest that the decrease in tumor burden might be contributed in part by the decrease in p-Stat3 and Ki-67 levels. More importantly, when we analyzed the SP/CD44+ subpopulation of cells by flow cytometry after in vivo drug treatment, we showed a significant reduction of SP/CD44+ cells in AZ960- and TG101209-treated groups as

compared to the vehicle. Specifically, SP/CD44+ cells of AZ960- and TG101209-treated Akt/β-catenin primary tumors significantly decreased to 0.116 ± 0.171% and 0.142 ± 0.283% respectively from 0.618 ± 0.242% of vehicle-treated group (Supplementary Table S1). These data suggest that Jak/Stat inhibition reduces the SP/CD44+ CCSPs in the Akt/β-catenin tumors. Collectively, our data suggest the importance of Jak/Stat signaling crosstalk with Akt and β-catenin signaling pathways and the feasibility of using Jak/Stat inhibitors in the suppression of Akt/β-catenin-driven tumors. Discussion HCC continues to remain a cancer that is difficult to treat and typically has a poor prognosis. Surgical resection and liver transplantation are often limited to a small proportion of HCC patients due to late presentation and more importantly, the resistance to conventional chemotherapy and high recurrence rate. The development of cancer recurrence is often attributed to the presence of cancer stem cells (CCSPs), a unique population of cancer cells that possess the ability to self-renew and proliferate extensively. Therefore, therapeutics targeting CCSPs is currently considered an important aspect for the effective treatment of liver cancers.

Our study provides insights into the tumorigenic population that can maintain liver tumor propagation and therapeutically targetable pathways that control these cells. Specific pathways that regulate stem cell maintenance are logical targets for anti-cancer stem cell directed therapies. Activation of Akt/β-catenin pathways leads to an enrichment of side population (SP) and CD44 expressing (SP/CD44+) HCC tumor cells. In addition, SP/CD44+ tumor cells demonstrated higher tumor-initiating properties, including self-renewal and tumorigenicity compared to NSP/CD44- counterparts. The potential for inhibition of the Akt and βcatenin pathways to target CCSPs is supported by the known involvement of these pathways in tumorigenesis and normal stem cell biology in HCC. [37] One of the most prevalent molecular alterations of HCC is the Wnt/βcatenin and Akt/mTOR pathways. Using comprehensive genomic sequencing, Wnt/β-catenin pathway-related molecular alterations have been shown to occur in approximately more than 60% of all HCC cases. [38] And interestingly, Wnt/β-catenin pathway alterations has been linked to CCSPs expressing surface markers such as CD133+, EpCAM, and Lgr5, demonstrating its importance in maintaining the CCSP phenotype.[20,39,40] Akt phosphorylation and its subsequent activation has been implicated in

early onset HCC and is associated with poorer prognosis. [29] Since constitutive activation of Akt/mTOR pathway is a major determinant of tumor growth and survival in various solid tumors, it serves as an attractive target for tumor inhibition. In addition, molecular classification of HCC identified six subgroups of which G1/G2 subgroup showed Akt activation. [41] Multiple lines of evidence suggest that Akt activation in CCSPs is a key mechanism by which CCSPs mediate chemoresistance, a common hallmark of CCSPs. Akt appears to be able to drive chemoresistance through several mechanisms, including upregulation of drug transporter proteins as well as pro-survival pathways, including the Bcl-2 pathway.[21,42] While activation of the PI3 kinase pathway, which includes activation of Akt, has been shown to increase ABCG2-driven SP+ glioma tumor-initiating CCSPs, [43] this study provides evidence that MDR1 and not ABCG2 drives the SP phenotype in Akt/β-catenin-driven tumors. Beyond upregulation of specific drug transporter and pro-survival proteins, there may be other unknown mechanisms of chemoresistance driven by Akt that remain to be discovered. Differences in these molecular mechanisms of chemoresistance are likely regulated by cancer type as well as co-activation of other signaling, such as β-catenin. Regardless, identifying therapeutic methods that can effectively treat CCSPs that are

commonly resistant to standard therapies is an attractive approach to improving treatment options against HCC. Interestingly, our results showed that Jak/Stat pathway inhibition may be effective in impairing growth of Akt/β-catenin-driven tumors, suggesting that Jak/Stat pathway is at the crossroad between both Akt/mTOR and Wnt/βcatenin pathways. Activated STAT3 is commonly observed across a wide range of HCC patients, but the mechanisms by which STAT3 is activated in HCC are largely unknown beyond hepatitis virus infection, NF-b activation and induction of IL-6.[44-46] Furthermore, it has been shown that uncontrolled activation of the Jak/Stat pathway is a dominant oncogenic event in human HCC tumorigenesis. [47] Recent studies by Yokogami et al. observed that using a mTOR inhibitor, rapamycin, can reduce the transcriptional activity of Stat3. [48] Furthermore, loss of TSC1/2 which activates mTORC1 can lead to increased Stat3 phosphorylation.[49,50] Activation of Wnt/β-catenin has also been shown to lead to upregulation and activation of Stat3 in embryonic stem cells. [51] In addition, Wnt3a ligand can also induce Stat3 activation and nuclear translocation. [52] In contrast, depletion of β-catenin in anaplastic large cell lymphoma led to significant reduction of total and active Stat3 levels. [53] More importantly, a recent study by Dhar et al. showed the induction of CD44 expression in

pericentral hepatocytes via recruitment of Stat3 to CD44 promoter in a diethylnitrosamine (DEN)-induced HCC mouse model, further supporting our observations that CD44 is one of the key tumor-initiating marker and the use of Jak/Stat inhibitors as a therapeutic strategy against HCC. [54] Stat3 regulates a multitude of cellular processes both in normal and neoplastic conditions and its aberrant activation leads to tumorigenesis by upregulating survival, angiogenesis, metastasis and immune evasion. There have been multiple studies implicating the targeting of aberrant Stat3 oncogenic pathway as a viable option for eradicating cancer stem -like cells at its root. For instance, the Jak/Stat3 pathway has been previously implicated in subset of CD133+ and ALDH+ cancer stem-like cells in non– small cell lung cancers (NSCLC) patient specimens. Shao and colleagues demonstrated the use of Jak/Stat inhibition in the downregulation of selfrenewal ability of NSCLC stem cells, suggesting the requirement of Stat3 pathway in the maintenance of NSCLC stem cells. [55] In addition, prostate cancer stem-like cells have also been demonstrated to constitutively activate Jak/Stat signaling as determined by their activated Stat3 expression in these cells. [56] Blocking Stat3 activity with IL-6 antibody or small molecule inhibitor LLL12 reduced colony forming ability and tumorigenicity, suggesting that p-Stat3 activity is required in these prostate

cancer stem-like cells. More recently, Dolatabadi et al. described a subset of cancer stem cells in myxoid liposarcoma with activated Jak/Stat signaling. [57] Pharmacological inhibition using Ruxolitinib, a Jak/Stat inhibitor, decreased p-Stat3 and the number of cancer stem cells via the SWI/SNF complex, which acts as a chromatin remodelling system known to regulate stem cell properties through Stat3. These studies support the findings that Jak/Stat signaling is activated in a subset of solid cancers and suggest the use of Jak/Stat inhibitors in mitigating cancer stem-like cells. In conclusion, we established the clinical relevance of studying Akt/βcatenin pathway activation and that these Akt/β-catenin tumors contained a subpopulation of tumor-initiating cells with stem/progenitor-like characteristics identified through side population analysis and expression of the cancer stemness marker CD44 that may contribute to tumor sustenance and drug resistance. More importantly, this study demonstrated that inhibition of the Jak/Stat pathway may be an alternative method to overcome drug resistance and effectively treat Akt/β-catenin-driven HCC tumors.

Declaration of Competing Interest

The authors have no potential conflicts of interest to disclose.

Financial support This study is supported by the National Research Foundation Cancer Science Institute of Singapore RCE Main Grant and Ministry of Education Academic Research Fund (MOE AcRF Tier 2 MOE2015-T2-2-126). Authors contributions T.B. Toh and E.K-H. Chow designed the experiments. T.B. Toh, J.J. Lim, M.B.M.A. Rashid and L. Hooi performed the experiments. T.B. Toh, J.J. Lim and E.K-H. Chow analyzed the data. T.B. Toh and J.J. Lim wrote the manuscript. T.B. Toh and E.K-H. Chow supervised the study.

Acknowledgements E. K-H. Chow gratefully acknowledges support from the National Research Foundation Cancer Science Institute of Singapore RCE Main Grant and Ministry of Education Academic Research Fund (MOE AcRF Tier 2 MOE2015-T2-2-126). This work is funded by the NCIS Yong Siew Yoon Research Grant through donations from the Yong Loo Lin Trust. We would like to thank Michelle Mok Meng Huang and Jessica Lee Kin-Mun from Fluorescence Activated Cell Sorting (FACS) Facility at the Cancer Science Institute of Singapore for technical support in flow cytometry.

Appendix A Supplementary data

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The following are the Supplementary data to this article: Supplementary data 1

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Figure 1. Akt/β-catenin-driven liver tumors display cancer stem cell phenotype.

(Ai) FVB/N mice injected with oncogenic forms of human AKT1 and constitutively active Δ90β-catenin displayed perimoribund liver tumor burden after 90 days compared to pT3-empty control. Scale bar = 1cm. (Aii) H&E and (Aiii) Ki-67

stainings of pT3-empty and Akt/β-catenin-driven livers. Scale bar = 50 µm. (B) Immunoblot analysis of Wnt/β-catenin and Akt/mTOR activation in pT3-empty, Akt/β-catenin and Akt/β-catenin-derived allograft tumors. β-actin was used as a loading control. (Ci) Akt/β-catenin tumorspheres grown under serum-free conditions. Scale bar = 50 µm. (Cii) Akt/β-catenin tumorspheres displayed high expression for both p-Akt and β-catenin in vitro. Scale bar = 50 µm. (D) Akt/βcatenin tumorspheres formed subcutaneous tumor allografts. Scale bar = 2 cm. (E) Flow cytometry analysis of CCSP markers for primary Akt/β-catenin-driven liver tumors and Akt/β-catenin tumorspheres. (F) Side population analysis of primary Akt/β-catenin-driven liver tumors and Akt/β-catenin tumorspheres. Verapamil was added as a control for the experiment. Figure 2. Co-activation of Akt and β-catenin pathways portends poorer survival in

HCC patients. (A) Percentage distribution of HCC patients analyzed (n=118) with no positive stainings, staining for only β-catenin or p-Akt, and co-staining for both

β-catenin and p-Akt. (B) Percentage distribution of β-catenin-positive patients with membranous (M), cytoplasmic (C) and nuclear (N) localization. (C) Fluorescent immunohistochemistry (FIHC) images of β-catenin and p-Akt expression in primary tumor tissue sections from representative HCC patients using Vectra® automated quantitative pathology imaging system. Green fluorescence indicates β-catenin expression (Yellow arrowheads indicate βcatenin nuclear localization); Red indicates p-Akt; Blue indicates DAPI staining. Scale bar = 50 µm. (D) Kaplan–Meier survival curves and median survival of HCC patients expressing β-catenin (n=28), p-Akt (n=25) and co-staining for βcatenin and p-Akt (n=17). Statistical analyses were performed using Log-rank (Mantel-Cox) test to compare β-catenin and p-Akt co-staining with the other groups. (E)(i) Kaplan–Meier survival curves of tumor stage stratified HCC patients expressing high p-Akt vs. low p-Akt. (ii) Kaplan–Meier survival curves of tumor stage stratified HCC patients expressing high β-catenin vs. low β-catenin.

Log-rank (Mantel-Cox) test was used to compare low and high p-Akt or β-catenin expression within each stage (n=3 for each group in Stage 1; n=12 for Stage 2; n=12 for Stage 3). Figure 3. Side population of Akt/β-catenin tumorspheres is mediated via MDR1

drug transporter. (A) mRNA expression of drug transporters MDR1 and ABCG2 in pT3-empty, Akt/β-catenin tumors and Akt/β-catenin tumorspheres. Data represented as mean ± SEM from 3 independent experiments. **, indicates p<0.01; ***, indicates p<0.001 (two-tailed Student's t test) (B) Immunoblot analysis of drug transporters ABCG2 and MDR1 in pT3-empty, Akt/β-catenin tumors and Akt/β-catenin tumorspheres. β-actin was used as a loading control. (C) Side population analysis of Akt/β-catenin tumorspheres with ABCG2-specific (Fumitremorgin C and Ko143) and MDR1-specific (Verapamil and Tariquidar) drug transporter inhibitors.

Figure 4. Cancer cells with stemness properties are highly enriched in SP/CD44+

subpopulation of Akt/β-catenin-driven tumors. (A) Long term colony formation assay over 24 days showed significant reduction in tumorsphere-forming abilities of NSP/CD44- as compared to SP/CD44+ subpopulation. Data shown as mean ± SD (n=10). ***, indicates p<0.001 (two-tailed Student's t test). (B) Representative image of mouse seeded in hindquarters with 1000 flow-sorted NSP/CD44- cells on the left and 1000 flow-sorted SP/CD44+ cells on the right. (C) Long term colony formation assay over 24 days showed significant reduction in tumorsphere-forming abilities of Akt/β-catenin allograft tumor-derived NSP/CD44as compared to SP/CD44+ subpopulation. Data shown as mean ± SD (n=10). ***, indicates p<0.001 (two-tailed Student's t test). (D) Limiting dilution assays demonstrating higher CCSP frequency of SP/CD44+ cells as compared to NSP/CD44- fraction for both (i) Akt/β-catenin tumorspheres and (ii) Akt/β-catenin allograft tumors. (E) Immunoblot analysis of Wnt/β-catenin and Akt/mTOR

activation in flow sorted NSP/CD44- and SP/CD44+ fractions of Akt/β-catenin tumorspheres. β-actin was used as a loading control. Figure 5. Jak/Stat inhibitors induced apoptosis and cell cycle arrest in Akt/β-

catenin tumorspheres. (A) Cell viability assay of 20 top-ranked inhibitors from the Stem cell inhibitor library screened at 1 µM. (B) Immunoblot analysis of Stat3 activation status in (i) Akt/β-catenin tumorspheres and (ii) flow-sorted SP/CD44+ and NSP/CD44- populations. β-actin was used as a loading control. (C)(i) Dose response curves of AZ960 and TG101209 on Akt/β-catenin tumorspheres. (ii) Immunoblot analysis of Stat3, Wnt/β-catenin, Akt/mTOR and apoptosis pathways in DMSO-, AZ960-, and TG101209-treated Akt/β-catenin tumorspheres. (D) Histogram plots of cell cycle analysis of Akt/β-catenin tumorspheres treated with (i) DMSO, (ii) AZ960, or (iii) TG101209. (iv) Corresponding quantification of different phases of cell cycle was carried out and presented with error bars (n=3). (E)(i) CD44-positive and (ii) SP cells of Akt/β-catenin tumorspheres treated with

vehicle, AZ960, or TG101209 at their respective IC50. Data represented as mean ± SD from 3 independent experiments. Figure 6. Jak/Stat inhibitors mitigate Akt/β-catenin tumor formation. (A)(i) Jak/Stat

inhibitors (AZ960 and TG101209) delay Akt/β-catenin tumor formation as compared to DMSO control. (ii) In vivo tumor volume of AZ960 and TG101209treated Akt/β-catenin tumors. Data shown as mean ± SD (n=5); *, p < 0.05 as compared to DMSO control. (B)(i) Representative images of immunohistochemical stainings of p-Stat3 and Ki67 in DMSO-, AZ960-, and TG101209-treated tumors. (ii) Quantification of IHC stainings of p-Stat3 in DMSO-, AZ960-, and TG101209-treated tumors. Data shown as mean ± SD (n=3) *, p < 0.05; ***, p < 0.001. (C)(i) Gross images, H&E (Scale bar = 200 µm) , and immunohistochemical stainings of p-Stat3 and Ki-67 in vehicle-, AZ960-, and TG101209-treated Akt/β-catenin tumors. Scale bar = 50 µm. (ii) Liver weight to body weight ratios of vehicle-, AZ960-, and TG101209-treated Akt/β-catenin

tumors. Data represented as mean ± SD (n=4). *, p < 0.05. (iii) IHC quantification of p-Stat3 protein levels in vehicle-, AZ960-, and TG101209-treated Akt/β-catenin tumors. Data presented as mean ± SD (n=4). *, p < 0.05. All statistical analyses were performed using two-tailed Student's t test.

Table 1. Tumor incidence and CCSP markers analysis in mice injected with

SP/CD44+ and NSP/CD44- fractions.

Group

Cell

Tumor

Percentage population (%)

Number

Incidence SP

SP/CD44+ 1000

CD44+

5/5 (100%) 5.73 ± 5.09 41.07 ±

SP/CD44+ 2.9 ± 1.47

18.23 NSP/CD44- 1000

0/5 (0%)

-

-

-

Table 2. Side population (SP) and CD44 expression in human HCC cell lines.

HCC line Hep3B Huh7 LM3 SNU387 SNU398

SP 0.0187 ± 0.007 0.0064 ± 0.002 31.53 ± 2.899 0.182 ± 0.138 0.028 ± 0.018

CD44+ 0.0147 ± 0.017 11.23 ± 0.702 99.6 ± 0.671 99.7 ± 0.1 0

SP/CD44+ 31.52 ± 2.885 -

SNU449

5.932 ± 3.485

99.36 ± 0.578

5.86 ± 3.417