The previously authenticated cell lines were used in this study (Karagonlar et al., 2020). All cells were grown at 37°C, 5% CO2 in RPMI Medium 1640 supplemented with % 2–10 FBS, 1% NEAA, 2 mmol⋅L^–1 Glutamax, 1% pen/strep. Sorafenib and Regorafenib resistant cells were created by treating parental cell lines with increased doses of drugs starting with their IC50 values. For a period of 8–12 months, at each passage, the drug concentrations were increased between 0.2 and 0.5 μM according to cell viability and proliferation rate of the cells. MTT analysis was performed to confirm the resistance. Established cell lines are maintained under the following drug concentrations: Sorafenib: 7.2 μM and Regorafenib: 8.4 μM.

The plasmids used in TCF/LEF reporter assay were a gift from Dr. Hans Clevers at Hubrecht Institute. Transfections were performed as previously described (Karabicici et al., 2021) using Lipofectamine 2000. For TCF/LEF reporter assays, 24 h after transfection, cells were treated with Regorafenib (5 or 10 μM), Wnt3a/R-Spo1, IWR-1(10 μM) or a combination of these molecules. For TGF-β reporter assays, 24 h after transfection, cells were treated with 5 ng/mL TGF-β1 (Peprotech) (Cat#100-21) or 5 μM TGF-β type 1 receptor (ALK5) inhibitor (Selleckchem) (Cat: #SB525334) for an additional 24 h.

Immunofluorescence stainings were performed as previously described (Karabicici et al., 2021), Beta-catenin (BD, 610153)(1:50), p-Beta-catenin (S675)(4176)(1:50), Cytokeratin 19 (sc-6278)(1:50), E-Cadherin (sc8426)(1:50), EpCAM (2929S)(1:50), Vimentin (sc-373717)(1:250), alpha-SMA (ab21027)(1:50), p-Smad2 (S255)(ab188334)(1:50), t-Smad2 (ab40855)(1:50) and Phalloidin-iFluor 488 (ab176753) antibodies were used for the stainings. Cells were visualized using the Confocal LSM 800 microscope.

For the detection of apoptosis, cells were stained using AnnexinV Apoptosis Detection Kit (Biolegend; Cat #640922) as described in the manufacturer’s instructions. Annexin V and PI stainings were analyzed using BD LSR Fortessa flow cytometer.

Real-time q-PCR experiments were performed as previously described (Karabicici et al., 2021). using7500 Fast RT PCR System (Applied Biosystems). The relative gene expression was calculated by using the 2^–ΔΔCt method. The primers are given in Supplementary Table 1.

Flow cytometry experiments were performed as previously described (Karabicici et al., 2021). For all cell lines, 2 × 10⁶ cells were seeded into a 10 cm dish 1 day before the experiment. Cells were treated with Regorafenib, Wnt3a/R-spondin or their combination for 24 h and then were stained with EpCAM-FITC (1:50), CD133-APC (1:50) and CD24-APC (1:50) antibodies. Cells were analyzed using the BD LSR Fortessa flow cytometer.

3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay was performed as previously described (Karagonlar et al., 2020). Briefly, cells were seeded in 96-well plates (5 × 10³ cell/well) 1 day before the experiment, then treated with increasing concentrations of Sorafenib or Regorafenib (0, 2, 4, 8, 16, 32 μM) for 48 h.

For all colony formation assay cells were seeded six well plates at 500–1,000 cells per well. One day later cells treated with 5 μM Regorafenib or 5 μM Sorafenib. The cells were kept in culture for about 7–10 days so that they could form a colony of minimum 50 cells. During this time, fresh medium was added first after 3–4 days and then every 2 days.

At the end of the period, the medium on the cells was removed and cells were washed with 1 × PBS, then fixed with cold methanol for 20 min. After fixation, colonies were stained with crystal violet for 20 min and then washed by immersing them in a container kept under running water to remove excess dye. Plates were dried, then imaged using a camera. Colonies were analyzed with the Fiji cell counter tool of Image J and colony numbers were graphed using GraphPad Prism.

The hanging drop method was used for the formation of spheroids. Briefly, 1 × 10³ cells/30 μl were prepared for all treated or transfected cell lines. Then 30 μl droplets were pipetted on the interior of a 10 cm plate lid. Then the lid was inverted and the plate was incubated for 3 days to start the formation of the spheroids. After 3 days of incubation, plate lids were inverted again and the spheroid medium was collected without disturbing the spheroids. Then the treatment medium was added to the spheroids. After 2 and 4 days in the hanging cell culture, cells were imaged under the stereo microscope using 5× zoom. Then spheroid areas were calculated using threshold calculating methods in the “Adjust” section in ImageJ. Results and statistical analyses were performed with GraphPad Prism.

Cells were seeded onto 6 cm dishes (1–1.5 × 10⁶ cells/plate) 1 day before treatments. TGFβ-R1 inhibitor (5 μM), Regorafenib (5 μM), Sorafenib (5 μM) or combined treatments were performed for 48 h for all conditions. After treatment, cell media were removed and washed with 1× PBS containing 0.1 mM NaF and 0.1 mM Na3VO4. Then cells were scraped on ice and collected into eppendorf tubes. Cell pellets were lysed using RIPA buffer (150 mM NaCl, %1 NP-40, %0.5 sodium deoxycholate, % 0.1 SDS, 50 mM Tris(pH:8),with 10 mM NaF, 10 mM Na3VO4, 1× phosphatase inhibitor (Pierce,Thermo,A32957,United States) and protease inhibitor (Pierce, Thermo 88666,United States) freshly added. Cell lysates were kept on ice for 30 min and were vortex for every 5 min. Then the cell lysates were sonicated (Diagenode SA Picoruptor 163007 E.C.) for 30 s and centrifuged at max speed for 20 min at 4°C. Supernatants were collected and protein concentrations were calculated using BCA Protein assay kit (Pierce, Thermo, 23225, United States). For all experiments 50 or 100°μg total protein was loaded to jels and incubated overnight with the following antibodies: Akt (p-Ser473)(4060S), Akt (9272),p-Smad3 (Ser423/425)(9520), p-Smad2 (S255)(ab188334),Smad2/3 (D7G7) (8685),Smad4 (sc-7966),p-B catenin (S675)(4176), Beta Catenin (BD, 610153), p-GSK-3α/β (Ser21/9) (9331), GSK-3α/β (sc-7291), p-p38 MAPK (Thr180/Tyr182) (D3F9) (4511),p38 MAPK (9212), p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4370), p44/42 MAPK (Erk1/2) (137F5) (4695), p-Stat3 (Tyr705) (D3A7) (9145),Stat3 (124H6) (9139). Blot images were taken using the Licor (CLX-1137 United States) detection system and analyzed using ImageJ “analyze”-“gel”-“plot lines” tools.

β-galactosidase assay was performed as previously described (Karabicici et al., 2021). Briefly, cells were seeded on the six well plates (7 × 10⁴) or 12 well plates (2 × 10⁴) 1 day before treatment. Then cells were treated with 5 ng/ml TGF-β1, 5 μM TGFβ -R1 inhibitor or transfected using shTGFβ -R1. 100 nM Doxorubicin treatment for 2 days was used as a positive control. After 6 days, the cell media were removed, cells were washed with 1 × PBS and then were stained with Biovision senescence detection kit as described in the manual. The cells were then imaged using a light microscope and results were analyzed using GraphPad Prism Software.

3–3.5 × 10⁵ cells were seeded on the 12 well plates 1 day before the experiment. Next day, a straight line was drawn using a yellow 200 μl pipette tip from top to down in the center of the cell monolayer. Residual cells were washed and cell media were replaced with control or treatment media. Cells were imaged at day 0, day 1, day 2, and day 3. From the images, wound area was calculated using Image J MRI wound healing tool with a range of 50–100 threshold parameter. Statistical analysis and wound closure percentages were calculated using GraphPad Prism software.

For the Sub G1 assay all mediums and cells were collected in 50 ml sterile tubes after the treatments (Regorafenib, Wnt3a/R CM (condition medium) or combination) for 48 h. Then the cell pellet was fixed with dropwise addition of cold %70 ethanol to the pellet while vortexing. Cells were kept on ice for 2 h then were centrifuged at 400 × g for 5 min. Then the pellet was washed two times with Phosphate-citrate buffer (192 parts of 0.2 M Na2HPO4 and eight parts of 0.1 M Citric acid; pH:7.8). To eliminate RNA, cells were treated with 50 μl 100 μg/ml Ribonuclease A solution for 15 min. After that 450 μl of 50 μg/ml PI was added and cells were incubated for an extra 15 min. Then cells were analyzed at the low flow rate under 400 events/seconds in BD LSR Fortessa flow cytometer and results were analyzed using FlowJo software (Becton Dickinson, Heidelberg, Germany).

Viral plasmids were produced in Hek293T cells as previously described (Karagonlar et al., 2020). 2 × 10⁵ cells/well were plated in six well plates 1 day before the experiment. Next day, cells were transfected with shPLKO.1 empty control plasmid or shTGFB-RI plasmid (TRCN221535, Broad Institute) targeting TGFB-RI transcript. Cells were then with 6 μg/ml puromycin (A1113803, Gibco) for 3 days. After that, puromycin was removed and cells were maintained in their standard growing media.

Nucleofection protocol was performed according to Lonza P3 Primary Cell 4D-Nucleofector^TM X Kit L. Briefly, 5 μg TCF/LEF 5 μg Renilla and shB-cateninC4 or empty pSUPER plasmids were mixed with 100 μl of nucleofection solution, which contains 5 × 10⁵ cells. Transfection was done using the CA-137 program. Cells were incubated for 10 min in RT after nucleofection and then were seeded in 12 well plates. After 48 h, nucleofection efficiency was measured by Dual-luciferase reporter assay.

Tg(7xTCF-Xla.Siam: nlsmCherry)^ia5(designated TCFsiam) zebrafish embryos were crossed with WT embryos. At 8 h postfertilization (hpf), embryos were dechorionated with Pronase enzyme and incubated with 5 μM Regorafenib containing E3 medium for 24 h and 48 h. At the end of the 24 and 48 h incubation times, embryos were fixed in 4% PFA in PBS overnight. mCherry probe synthesis and whole-mount in situ hybridizations were performed as described previously (Moro et al., 2012).

Untreated HuH7 cells (CTRL), HuH7 cells treated with 5 μM Regorafenib (Reg5), Regorafenib resistant cells (RRC), and Sorafenib resistant cells (SRC) were labeled with 2 mg/ml DiI (V2288, Molecular Probes) before injection. The cells were then resuspended to a final density of 40,000 cells/μl in 10%FBS in PBS and injected into the yolk of 2 days old dechorionated embryos (∼250 cells/embryo). The injected embryos were incubated at 34°C in E3 media until 5 dpi.

Statistical analyses were done using GraphPad Prism 7(GraphPad Software, Inc., California, United States) software. Two-tailed unpaired student t-test was used to determine statistical significance between 2 experimental groups. Differences between groups were considered as “> 0.05” (n.s.), “≤ 0.05” (^∗), “≤ 0.01” (^∗∗), “≤ 0.001” (^∗∗∗) “≤ 0.0001” (^****).

We first performed MTT analyses to determine IC50 values of drugs on hepatoblast-like (HuH-7, Hep3B, and HepG2), mesenchymal (SNU387, SNU449) cell lines and acquired drug resistant clones of HuH-7 (SRC and RRC). MTT analyses demonstrated that IC50 values of mesenchymal cell lines for regorafenib were significantly higher than hepatoblast-like cells (Figure 1A). Regorafenib treatment also significantly reduced migration, 3-D growth and colony formation of hepatoblast-like cells (Supplementary Figure 1). Of note, we showed that the sorafenib resistant clones also had acquired regorafenib resistance while regorafenib resistant clones had become sorafenib resistant (Figure 1A).

It has previously been shown that hepatoblast-like cell lines exhibit higher basal levels of Wnt/β-catenin pathway activity while mesenchymal-like Snu387 and Snu449 cell lines have repressed Wnt/β-catenin pathway activity (Yuzugullu et al., 2009). So, we investigated if the difference on drug resistance of HCC lines can be explained via the characteristics of Wnt/β-catenin signaling. We first analyzed the effect of sorafenib and regorafenib treatment on TCF/LEF reporter activity of HCC cell lines under basal culture conditions as well as with ligand induction. Here we demonstrated that sorafenib and regorafenib treatments alone significantly increase TCF/LEF reporter transcriptional activity of hepatoblast-like cell lines while in mesenchymal-like cell lines Snu387 and Snu449, drug treatments have no significant effect on TCF/LEF reporter activity (Figure 1B). Moreover, although treatment with canonical Wnt pathway ligands Wnt3a/R-Spo1 was able to induce TCF/LEF activity of both hepatoblast like and mesenchymal-like cell lines, additional enhancement of Wnt3a/R-Spo1 induced TCF/LEF reporter activity upon regorafenib treatment was only detected in hepatoblast-like HuH7, HepG2, and Hep3B cell lines (Figure 1C). Notably, regorafenib treatment alone or in combination with Wnt3a/R CM significantly increased TCF/LEF activity and β-catenin phosphorylation in HuH7 cells (Figure 1D).

Moreover, to detect if regorafenib activates Wnt/β-catenin signaling in vivo, we utilized Tg(7xTCF-Xla.Siam) Wnt/β-catenin reporter fish (Moro et al., 2012). The transgenic fishes were crossed with WT fishes and at 8 hpf, embryos were treated with 5 μM regorafenib for 24 and 48 h. At indicated time points, Wnt/β-catenin reporter activity was detected by in situ hybridization. For both time points, TCF/LEF activity was greater in zebrafish treated with regorafenib supporting the activation of Wnt/β-catenin signaling by this drug in vivo (Figure 1E).

To further understand the regulation of the Wnt/β-catenin pathway by regorafenib, we treated HuH7 cells with 10 μM IWR-1 which stabilizes the destruction complex member Axin2 and thus silences the Wnt/β-catenin pathway. When combined with regorafenib, IWR-1 treatment increased Annexin V+/PI- cell population and augmented expression of cleaved PARP (Figures 2A–C and Supplementary Figure 2). Moreover, IWR-1 treatment significantly decreased basal and regorafenib-induced TCF/LEF reporter activity (Figure 2D) and β-catenin phosphorylation (Figure 2E) in the HuH7 cell line. On the other hand, when regorafenib was used in combination with Wnt3a/R-Spo1, regorafenib-induced cell death was greatly reduced (Figure 2F). We also detected a significant decrease in cleaved PARP levels in Wnt3a/R-Spo1treated cells (Figure 2G) and a decrease in sub-G1 cell population (Supplementary Figure 2). Taken together, these findings indicate that Wnt/β-catenin signaling activation prevents regorafenib-induced apoptosis while its inhibition can enhance cell death upon regorafenib treatment.

To evaluate the effect of regorafenib treatment on EMT/MET transition, which is one of the essential hallmarks of cancer progression and metastasis, we detected the expression of epithelial and mesenchymal markers in regorafenib treated cells. Importantly, E-CAD expression increased when cells were treated with 5 μM regorafenib while the expression of mesenchymal markers decreased upon regorafenib treatment except VIM (Figure 3A). There was a decrease in the actin stress fibers in regorafenib treated cells (Supplementary Figure 3). Moreover, regorafenib treatment also induced the expression of hepatic stem/progenitor markers LGR5, AXIN2, CCND1, EpCAM and CK19 (Figure 3B). Also, the expression of ANXA3 which promotes angiogenesis, drug resistance, and stemness in HCC (Tong et al., 2015, 2018), and the expression of KLF4 which is one of the Yamanaka factors that also regulates liver cancer stem cell plasticity (Karagonlar et al., 2020), increased upon regorafenib treatment (Figure 3B). EpCAM is a known Wnt/β-catenin signaling target gene and an important liver cancer stem cell marker (Yamashita et al., 2007; Terris et al., 2010). Regorafenib treatment increased membranous expression of EpCAM (Supplementary Figure 3). Strikingly, flow cytometry analysis showed that when cells were treated with Wnt3a/R-Spo1 in combination with regorafenib, the induction of EpCAM+ cell population was greatly enhanced (from 42.4 to 68.5%). On the other hand, CD133+ and CD24+ cell populations were reduced under combined treatment (Figure 3C). Importantly, when we knocked down β-catenin expression using a shβ-catenin plasmid (Figure 3D), the regorafenib induced increase in EpCAM was abolished. Although the mRNA expression of E-CAD also dropped, the protein level did not seem to be significantly altered (Figures 3E,F). Knock-down of β-catenin also affected the 3-D growth of cells and their colony forming ability. Moreover, knock-down of β-catenin rendered 3-D spheroids more sensitive to regorafenib (Figures 3G,H).

EMT is known to be a critical step in acquisition of drug resistance (Aiello and Kang, 2019; Derynck and Weinberg, 2019) and TGF-β signaling is a master regulator of EMT (Reichl et al., 2012; Papageorgis, 2015; Hao et al., 2019). Interestingly, we detected an increase in the expression of TGF-β1 upon regorafenib treatment, although the expression of TGFβ-R1 was decreased (Figure 4A). When we treated cells with both TGF-β1 and regorafenib, the increase in the expression of E-CAD and EpCAM upon regorafenib treatment was partly inhibited (Figure 4A). On the contrary, regorafenib induced expression of LGR5 and CK19 was further augmented by TGF-β1 treatment (Figure 4A). To analyze the effect of regorafenib treatment on TGF-β signaling, we utilized a reporter plasmid. While TGFβ-1 treatment significantly increased TGF-β signaling, regorafenib treatment suppressed TGFβ-1 induced activation (Figure 4B).

Confocal staining also confirmed that while regorafenib treatment induced the membranous expression of E-CAD, and p-β-CAT, upon TGF-β1 treatment regorafenib induced increase in their expression was abolished (Figure 4C). Consistent with the RNA expression data, Vimentin expression was also significantly induced by Regorafenib treatment in these cells. Moreover, upon TGF-β1 treatment, we saw a further increase in Vimentin expression and an increase in actin stress fibers consistent with the acquisition of a more-mesenchymal morphology (Deguchi and Sato, 2009; Figure 4C). Consistently, while regorafenib treatment decreased cell motility, TGF-β1 treatment alone increased the motility of cells. When two treatments combined, the inhibitory effect of regorafenib on cell motility was partly attenuated by TGF-β1 (Figure 4D).

To analyze acquired drug resistance, we created Sorafenib and Regorafenib resistant cells by treating HuH7 cells with increasing doses of drugs starting with their IC50 values in long-term culture. Over 8–12 months, Sorafenib resistant (SRC) and Regorafenib resistant (RRC) cell lines were established and thereafter maintained constantly with media containing 7.2 μM Sorafenib (SRC line) or 8.4 μM Regorafenib (RRC line). MTT analysis confirmed that these resistant lines exhibited significantly higher IC50 values for both Sorafenib and Regorafenib, similar to mesenchymal-like HCC cell lines (Figure 1A and Supplementary Figure 3). Consistently, when compared to the parental HuH7 cell line, SRC and RRC cell lines had upregulation of several mesenchymal markers such as SNAI1, ZEB2 and VIM and α-SMA (Figures 5A,C), supporting the acquisition of a mesenchymal phenotype. Interestingly, CDH1 expression was also increased in the RRC line. Although CDH1 is a well-defined epithelial marker, distant metastases of invasive cancers were shown to re-express CDH1 which contributes to the establishment of metastatic foci. Moreover, similar to acute regorafenib treated cells, LGR5, CK19, KLF4, ANXA3, and CCND1 gene expressions were also still significantly higher in SRC and RRC lines compared to parental cells (Figures 5B,C). Also, there was a significant upregulation in TGF-β1 expression in the SRC and RRC lines. Consistently, the expressions of total and p-SMAD2 (S255) were increased in SRC and RRC cell lines, while the expression of p-β-catenin was reduced (Figure 5C). Reporter assays also confirmed reduced β-catenin signaling and increased TGF-β signaling in the resistant cell lines (Figures 6A,B).

We also analyzed the expression of cancer stem cell surface markers via flow cytometry. We detected that EpCAM+ cell population decreased in both SRC and RRC lines. On the other hand, CD133 and CD24 expressing cell populations significantly increased in the resistant lines (Figure 5D). Moreover, although under 3-D growth conditions, resistant cell lines formed smaller spheroids, RRC spheroids were significantly more resistant to regorafenib treatment (Figure 7A). In addition, RRC cell line demonstrated significantly higher in vitro motility than parental and SRC cells in scratch assay (Figure 7B). Similarly, although basal colony forming capacity of SRC and RRC lines were lower than parental HuH7 cells, upon regorafenib treatment the colony forming ability of parental HuH7 cell line was greatly lost, while the colony forming abilities of SRC and RRC cells were not affected (Figure 7C).

To evaluate our in vitro results, we also tested the migration ability of SRC and RRC lines in a zebrafish xenograft model. 5 μM regorafenib treated HuH7 cells as well as SRC and RRC lines were implanted into the yolk sac of 2 dpf zebrafish embryos. 5 days after injection, migration to the tail was quantified. In total, 106 fishes from the control group, 117 fishes from the 5 μM regorafenib treated group, 77 fishes from the SRC group, and 81 fishes from the RRC group were counted. We detected migrated cells in 27% of zebrafish injected with HuH7 cells, while only 15 % of zebrafish injected with HuH7 cells treated with 5 μM regorafenib had migrated cells suggesting that regorafenib treated cells have reduced migration ability. Interestingly SRC line did not exhibit significantly higher migration ability (around 32% of zebrafish injected with SRC line had migration). However, we detected migrated cells around 53% of zebrafish injected with RRC line suggesting RRCs have significantly enhanced in vivo migration ability (Figure 7D).

Wnt/β-catenin signaling increases upon acute regorafenib treatment. However, in SRC and RRC lines, we detected significantly reduced basal TCF/LEF activity (Figure 6A). Moreover Wnt3a/R-Spo-induced Wnt/β-catenin signaling was also diminished in SRC and RRC lines (Supplementary Figure 3). On the other hand, luciferase reporter assay using a reporter plasmid consisting of TGF-β responsive Smad-binding elements (pSBE4-Luc) demonstrated that basal TGF-β pathway activity is significantly higher in SRC (∼7.5-fold) and RRC (∼ 9-fold) lines compared to parental HuH7 cells (Figure 6B) consistent with significantly higher levels of TGF-β1 expression in these cells. We then utilized a TGFβ-R1 inhibitor that reduces TGF-β signaling in these cells (Figure 6C) and compared the various abilities of SRC and RRC cells with and without this inhibitor. Upon treatment of SRC and RRC spheroids with regorafenib and/or sorafenib and TGFβ-R1 inhibitor, cell death was augmented in the spheroids (Figure 6D and Supplementary Figure 4). Also compared to regorafenib alone, the combined treatment with TGFβ-R1 inhibitor and drugs significantly reduced colony formation (Figure 6E and Supplementary Figure 4) and in vitro migration of resistant cells (Figure 6F and Supplementary Figure 4). We also tested the effect of TGFβ-R1 inhibitor on the in vivo migration ability of RRC cells in the zebrafish model. In total, migration to the tail was counted in 143 fishes from the control group, in 139 fishes from the RRC group, and in 139 fishes from the RRC group treated with TGFβ-RI inhibitor (Figure 6G). We detected migrated cells in 21.6% of zebrafish injected with HuH7 cells, while 43% of zebrafish injected with the RRC group had migration again demonstrating the high migration ability of the RRC line. On the other hand, only 12.9% of zebrafish injected with the TGFβ-RI inhibitor treated RRCs had migrated suggesting that TGF-β pathway inhibition significantly prevents in vivo migration ability of regorafenib resistant cells.

Activation of the TGFβ pathway by TGF-β1 treatment is known to induce senescence in HCC cells (Senturk et al., 2010). In accordance with previous literature, we demonstrated that TGF-β1 treatment induces senescence in parental HuH7, SRC and RRC lines (Supplementary Figure 5). Interestingly, however, when we knocked down TGFβ-R1 in parental and resistant cells using a lentiviral plasmid, the majority of RRCs entered senescence while senescence was not detected in parental and SRC lines (Figure 6H). The induction of senescence by TGFβ-R1 knockdown in RRC line suggests that TGF-β pathway promotes growth and survival of regorafenib resistant cells. Phosphorylation of Smad2 at Ser255 via ERK was shown to serve as a STAT3 co-activator (Yoon et al., 2015). Western blot analysis demonstrated that in the RRC line, there are higher levels of phospho-STAT3, and phospho-SMAD2 (S255) (Figure 8). Importantly, regorafenib treatment reduced phosphorylations of SMAD2 and STAT3. However, this inhibition was even more significantly augmented when resistant cells were treated with regorafenib in combination with TGFβ-R1 inhibitor. Also, upon regorafenib treatment, phosphorylation of ERK1/2 was completely inhibited in HuH7 cells whereas in the resistant cell lines, the inhibition of pERK1/2 by regorafenib was not significant. However when regorafenib was combined with TGFβ-R1 inhibition, phosphorylation of ERK1/2 was completely inhibited even in SRC and RRC lines. On the other hand, upon regorafenib treatment phospho-β-catenin level increased in parental HuH7 and in the resistant lines. However when TGFβ-R1 inhibitor was applied in addition to regorafenib, the regorafenib induced increase in phospho-β-catenin was abolished while GSK3β phosphorylation increased (Figure 8).