Upregulation of TRIB2 by Wnt/β‑catenin activation in BRAFV600E papillary thyroid carcinoma cells confers resistance to BRAF inhibitor vemurafenib
Nianxue Wang1 · Jing Wen2 · Wei Ren1 · Yuting Wu3 · Chaonan Deng3
Received: 18 November 2020 / Accepted: 25 March 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021
Purpose The BRAFV600E mutation is an oncogenic driver associated with aggressive tumor behaviors and increased mortal- ity among patients with papillary thyroid cancer (PTC). Although the BRAF inhibitor vemurafenib gave promising results in BRAFV600E-mutant PTC, resistance development remains a major clinical challenge. This study aimed to explore the mechanisms underlying drug resistance in PTC.
Methods Two vemurafenib-resistant PTC cell lines (KTC1 and BCPAP) were established by continuous treatment with vemurafenib for 5 months. The knockdown and upregulation of Tribbles homolog 2 (TRIB2) in PTC cells were achieved by the transfection with short hairpin RNA against TRIB2 or recombinant lentiviral vector carrying TRIB2, respectively. The β-catenin inhibitor, ICG-001, was used for the inhibition of the Wnt/β-catenin signaling in PTC cells.
Results Vemurafenib-resistant PTC cells showed higher TRIB2 expression, upregulated ERK and AKT activation, enhanced invasive capacity, and increased epithelial-mesenchymal transition compared to the drug-sensitive groups. TRIB2 knock- down repressed the activation of ERK and AKT, inhibited invasion and EMT, and induced apoptosis of PTC cells. TRIB2 deficiency also enhanced the sensitivity of both PTC cells to vemurafenib. Vemurafenib-resistant PTC cells showed elevated expression of β-catenin in both cytoplasm and nucleus. The pre-incubation of cells with β-catenin inhibitor significantly inhibited TRIB2 expression, suppressed EMT, and repressed the activation of ERK and AKT in vemurafenib-resistant cells.
Conclusion Our study showed that the upregulation of TRIB2 by the Wnt/β-catenin activation confers resistance to vemu- rafenib in PTC with BRAFV600 mutation. These findings support the potential use of TRIB2 as a therapeutic target for resist- ant PTC.
Tribbles homolog 2 · Wnt/β-catenin · BRAFV600E mutation · Papillary thyroid cancer · Vemurafenib
Thyroid cancer is one of the most common endocrine gland malignancies worldwide with an incidence increasing from 7.1 to 17.6 per 100,000 over the past decade . The four major histological categories of thyroid cancer are papillary, follicular, anaplastic, and medullary, representing approxi- mately 85%, 2–5%, 1%, and 3–5% of all thyroid cancer cases, respectively . Although papillary thyroid cancer (PTC) generally has a good overall prognosis, the cause metastasis remain poor . Therefore, it is of vital impor- tance to develop novel therapeutic strategies for patients with advanced PTC.
The BRAFV600E mutation is an oncogenic driver that occurs in nearly 45% of PTC . It is significantly associ- ated with aggressive tumor behaviors and increased cancer- related mortality among PTC patients . The mutation of BRAFV600E contributes to tumorigenesis, metastasis, and recurrence of PTC by constitutively activating the mitogen- activated protein kinase (MAPK) pathway, which results in the phosphorylation of downstream extracellular signal- regulated kinase 1/2 (ERK1/2) . Vemurafenib is a BRAF inhibitor that has been approved for the treatment of mela- noma patients harboring mutant BRAFV600E . Although clinical trials suggested that vemurafenib could also repre- sent a useful therapeutic option for PTC patients, the devel- opment of drug resistance after initial response remains a major clinical challenge, even in melanoma where vemu- rafenib has been approved . A recent study identified the hyperactivation of the Wnt/β-catenin signaling as a funda- mental cause of BRAF inhibitor resistance in colorectal cancer patients, providing a potential therapeutic strategy for BRAFV600E−mutant cancers .
Tribbles homolog 2 (TRIB2), a member of the tribbles family, is a scaffold protein involved in fundamental cel- lular processes in human cancers . It has been reported to promote tumorigenesis in acute myeloid leukemia, lung carcinoma, colorectal cancer, and melanoma via regulat- ing different signaling pathways [11–14]. Moreover, previ- ous evidence revealed that high TRIB2 expression induces chemoresistance in melanoma by activating the protein kinase B (AKT) pathway . However, the regulatory effects of TRIB2 in PTC remains unknown.
In the current study, we established vemurafenib-resistant PTC cell lines and investigated the effects of TRIB2 on the ERK, AKT, and Wnt/β-catenin pathways in these cells. Our findings demonstrated that the upregulation of TRIB2 by Wnt/β-catenin activation confers resistance to vemurafenib in PTC with BRAFV600 mutation.
Materials and methods
Immortalized human PTC cell lines (KTC1 and BCPAP) with a passage number of p20 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The KTC1 cell line, harboring the heterozygous BRAFV600E mutation, was originally derived from the pleu- ral effusion of a male patient with metastatic and recurrent PTC . The BCPAP cell line, harboring the homozygous BRAFV600E mutation, was established from the primary tumor of a female patient with poorly differentiated PTC . All cells were cultured in DMEM/F12 medium (Invit- rogen, Carlsbad, USA) containing 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, USA) and maintained in a humidified atmosphere of 5% CO2 at 37 °C.
Short hairpin RNA (shRNA) against TRIB2 (shTRIB2#1 and shTRIB2#2), scrambled control shRNA sequence (shNC), recombinant lentiviral vector carrying TRIB2 (TRIB2), and empty control vector (Vector) were designed and synthesized by GenePharma (Shanghai, China). KTC1 and BCPAP cells were transfected with designated shRNAs or lentiviral vectors for 48 h using the Lipofectamine 2000 Transfection Reagent (Invitrogen). Transfection efficiency was evaluated by western blot.
Vemurafenib exposure and ICG‑001 treatment
Vemurafenib was purchased from Selleck Chemicals (Houston, USA) and dissolved in DMSO (Sigma-Aldrich) to achieve a stock concentration of 2 mM. Then 2 mM vemurafenib was diluted to 2 µM in DMEM/F12 supple- mented with 0.2% FBS. The vehicle used in this study was 2% DMSO diluted in DMEM/F12 containing 0.2% FBS. Vemurafenib-resistant cell lines were selected by long-term exposure of 2 µM vemurafenib for 20 weeks (20 cycles of therapy). One cycle was defined as follows: treatment of cells with 2 µM vemurafenib in the presence of culture medium containing 0.2% FBS until 90% of the cells were dead and then recover them in culture medium containing 10% FBS without vemurafenib. The total passage number of these cells following the long-term vemurafenib treat- ment was passage 40 from their original line derivation. The control cells were treated with the same volume of DMSO vehicle following the same protocol.
Wnt/β-catenin inhibitor ICG-001 was purchased from Selleck Chemicals and diluted in DMSO to achieve a stock concentration of 5 mM. Cells subjected to ICG-001 treat- ment were continuously incubated with 5 µM ICG-001 for 4 days prior to vemurafenib exposure . The control groups received the same volume of vehicle.
Cell viability assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine the 50% maximal inhibitory concentration (IC50) of vemurafenib for cell proliferation as previously described . Following cell transfection and/or long-term exposure to vemurafenib, KTC1 and BCPAP cells (1 × 103 cells/well) were plated in 96-well plates and treated with various doses of vemurafenib for 48 h. Cell viability was measured using the MTT assay kit (Roche, Mannheim, Germany) and the dose–response curves were plotted using the Prism software (version 6.0, GraphPad, La Jolla, USA).
Following cell transfection and/or vemurafenib treatment, KTC1 and BCPAP cells (3 × 104 cells) were suspended in 200 µL culture medium and transferred to the upper chamber of the Transwell plate (Corning Life Sciences, Tewksbury, USA). Then the Transwell insert was placed on a 24-well plate filled with 600 µL DMEM/F12 containing 20% FBS. After 24-h incubation at 37 °C, cells on the upper side of the filter membrane were wiped off by cotton swabs. The Transwell chambers were fixed with 4% paraformalde- hyde, stained with 0.2% crystal violet, and then observed under a light microscope (400×magnification). The number of invading cells per well was counted from six randomly selected fields.
KTC1 and BCPAP cells were transfected with designated shRNAs or lentiviral vectors followed by long-term expo- sure to vemurafenib or vehicle. Then cells were trypsinized, centrifuged, and resuspended with 500 μL 1 × binding buffer, and incubated with 5 μL V-FITC and 5 μL propidium iodide from the Apoptosis Detection Kit (Biovision Research Prod- ucts, Mountain View, USA) in the dark for 5 min. Flow cytometry analysis was performed to determine the apopto- sis rate using a Navios flow cytometer with Kaluza analysis software (Beckman Coulter, Brea, USA).
Quantitative real‑time PCR (qRT‑PCR)
Total RNA was extracted from KTC1 and BCPAP cells using the TRIzol Plus RNA Purification Kit (Invitrogen) and reversely transcribed to cDNA using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Target genes were amplified using the 7300 Real-Time PCR System (Thermo Fisher Scientific). The expressions of TRIB2 was normal- ized to GADPH. The primers used in this experiment were as follows: TRIB2 forward, 5′-CCCGACTGTTCTACC AGATT-3′, TRIB2 reverse, 5′-AAGCGTCTTCCAAAC TCTCC-3′; GAPDH forward: 5′-ATCACTGCCACCCAG AAGAC-3′, GAPDH reverse: 5′-TTTCTAGACGGCAGG TCAGG-3′.
Total protein was extracted from all groups of cells by RIPA buffer containing protease inhibitors and phosphatase inhibitors (Pierce, Rockford, USA). The cytoplasmic and nuclear fractions were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Fisher Scientific, Waltham, USA) following the manufacturer’s instructions. The protein content was quantified by BCA assay (Pierce, Rockford, USA). An equal amount of protein was separated on 12% SDS-PAGE under reducing condi- tions and transferred to 0.2-μm PVDF membranes. After blocking, the membranes were incubated with the follow- ing diluted primary antibodies at 4 °C overnight: ERK (1:1000, Cat No. 4695, Cell Signaling Technology, Dan- vers, USA), phosphorylated ERK (pERK, 1:1000, Cat No. 9101, Cell Signaling Technology), AKT (1:1000, Cat No. 9272, Cell Signaling Technology), phosphorylated AKT (pAKT, 1:1000, Cat No. 9271, Cell Signaling Technology), E-cadherin (1:2000, Cat no. 40772, Abcam, Cambridge, UK), N-cadherin (1:1000, Cat No. 76057, Abcam), snail (1:1000, Cat No. 216347, Abcam), TRIB2 (1:1000, Cat No.117981, Abcam), cleaved-caspase-3 (1:1000, Cat No. 49822, Abcam), β-catenin (1:2000, Cat No. 32572, Abcam), his- tone 3 (1:2000, Cat No. 176842, Abcam), β-actin (1:2000, Cat No. 32572, Abcam), followed by the incubation with a goat anti-rabbit secondary antibody (1:2000, Cat no. 6721, Abcam) for 50 min. The protein bands were visualized and quantified using the Alphalmager™ 2000 Imaging System (Alpha Innotech, San Leandro, USA). Histone 3 and β-actin were used as internal controls.
All experiments in this study were performed in triplicate and repeated three times. Data were analyzed by software SPSS (version 24.0) and are shown as mean ± standard deviation. One-way analysis of variance (ANOVA) was performed to compare the differences among groups. A p value of less than 0.05 was considered statistically signifi- cant. *p < 0.05, **p < 0.01, ***p < 0.001.
Long‑term exposure to vemurafenib induces the activation of ERK and AKT in PTC cells
To understand the long-term impacts of vemurafenib expo- sure to PTC cells, we continuously treated KTC1 cells (heterozygous BRAFV600E) and BCPAP cells (homozy- gous BRAFV600E) with 2 µM vemurafenib or vehicle for 20 weeks. Then cell proliferation assay was performed to determine the IC50 of vemurafenib for these cell lines. In vehicle-treated KTC1 and BCPAP cells (KTC1-S and BCPAP-S), the IC50 values of vemurafenib for cell viabil- ity were 2.536 μM and 3.425 μM, respectively. In vemu- rafenib-exposed groups (KTC1-R and BCPAP-R), the IC50 values increased to 8.707 μM and 9.858 μM, respectively, suggesting that KTC1 and BCPAP cells acquired resist- ance to vemurafenib after 20 weeks of exposure (Fig. 1a). Furthermore, long-term exposure to vemurafenib effec- tively induced the activation of ERK and AKT in both KTC1 and BCPAP cells, as shown by significantly increased the ratios of pERK/ERK and pAKT/AKT in KTC1-S and BCPAP-S groups relative to vehicle-treated cells (Fig. 1b). The analysis of the epithelial-mesenchymal transition (EMT) markers revealed that E-cadherin was markedly downregulated, while N-cadherin and snail were
Fig. 1 Effect of vemurafenib exposure on the activation of ERK and AKT in PTC cells. Human PTC cell lines KTC1 and BCPAP were continuously exposed to 2 µM vemurafenib for 20 weeks to acquire resistance to vemurafenib (KTC1-R and BCPAP-R). The control groups were treated with the same volume of DMSO (KTC1-S and BCPAP-S). a The IC50 values of vemurafenib for different groups of cells were determined by MTT assay, in which cells were treated with significantly upregulated in vemurafenib-resistant cells when compared to the vehicle control groups (Fig. 1c). The long-term treatment with vemurafenib also increased the invasive capacity of both KTC1 and BCPAP cells (Fig. 1d). The above results indicated that long-term vemurafenib exposure induced the activation of ERK and AKT, and cell migration/invasion in PTC cells. various doses of vemurafenib for 48 h followed by the assessment of cell viability. b The protein expressions of pERK, ERK, pAKT, and AKT were detected by Western blot. c The protein levels of E-cad- herin, N-cadherin, and snail were measured by Western blot. d Tran- swell assay was performed to evaluate the invasive capacity of PTC cells following vemurafenib exposure. The number of invading cells per well was calculated
TRIB2 is upregulated in PTC cells treated with vemurafenib
To explore the role of TRIB2 in vemurafenib resistance, we compared the mRNA and protein levels of TRIB2 between vemurafenib-resistant and -sensitive cell lines. Cells exposed to vemurafenib for 20 weeks showed significantly upregulated mRNA expression of TRIB2 compared with the vehicle control groups (Fig. 2a). Consistently, the protein levels of TRIB2 in the KTC1-R and BCPAP-R groups were also significantly higher than those in vemurafenib-sensitive cells (Fig. 2b). These findings suggested the involvement of TRIB2 in vemurafenib resistance in PTC.
The knockdown of TRIB2 reduces the resistance of PTC cells to vemurafenib
To investigate the correlation between TRIB2 expression and vemurafenib resistance in PTC cells, we transfected KTC1 and BCPAP cells with shRNAs (shTRIB2#1 and shTRIB2#2) against TRIB2 (or scrambled control sequence) or recombinant lentiviral vector carrying TRIB2 (or empty control vector). The shRNA (shTRIB2#2) with better inhibi- tory effect on TRIB2 expression and more robust regula- tory effect in PTC cells was used for this study, and termed shTRIB2 thereafter. The preliminary results are shown in
Fig. 2 Expression of TRIB2 in PTC cells following vemurafenib treatment. KTC1 and BCPAP cells were treated with 2 µM vemu- rafenib or the same volume of DMSO for 20 weeks. The (a) mRNA and (b) protein expressions of TRIB2 were measured by qRT-PCR and Western blot, respectively
Supplementary Fig. 1. The transfection of shTRIB2 sig- nificantly decreased the level of TRIB2 in both KTC1 and BCPAP cells, while the delivery of TRIB2 vector greatly upregulated TRIB2 in these cell lines (Fig. 3a). The IC50 values of vemurafenib for TRIB2-deficient cells were lower compared to those with normal TRIB2 expression, whereas the IC50 values for cells overexpressing TRIB2 were higher than those for cells transfected with empty vector (Fig. 3b). TRIB2 knockdown significantly suppressed the phospho- rylation of ERK and AKT in KTC1 and BCPAP cells, but the upregulation of TRIB2 successfully increased the ratios of pERK/ERK and pAKT/AKT in these cell lines (Fig. 3c). The level of TRIB2 also affected the expressions of EMT markers in PTC cells. The downregulation of TRIB2 sig- nificantly elevated the level of E-cadherin, but reduced the expressions of N-cadherin and snail compared to the scram- bled control group. The delivery of TRIB2 vector, however, exerted opposite effects on the expression of EMT-related proteins (Fig. 3d). The transfection with shRNA against TRIB2 almost completely inhibited the invasion of both KTC1 and BCPAP cells, while cells overexpressing TRIB2 exhibited significantly improved invasive capacity com- pared to the groups with normal TRIB2 expression (Fig. 3e). We further depleted TRIB1 in both vemurafenib-resistant PTC cell lines. TRIB2 deficiency enhanced the sensitivity of PTC cells to vemurafenib following 20 weeks of expo- sure, as shown by decreased IC50 values of vemurafenib for shTRIB2-transfected cells relative to the control groups (Fig. 3f). To determine whether TRIB2 would affect apop- totic cell death in vemurafenib resistance, we transfected KTC1 and BCPAP cells with shRNA against TRIB2 or vec- tor carrying TRIB2 prior to long-term vemurafenib expo- sure (2 µM). The apoptosis rate and expression of cleaved caspase-3 protein in PTC cells with insufficient TRIB2 were significantly higher than those in the control groups, whereas cells overexpressing TRIB2 had significantly lower apop- totic level and protein expression of cleaved caspase-3 com- pared to the groups transfected with empty vector (Fig. 4a, b). Taken together, these data suggested that TRIB2 knock- down reduced the resistance of PTC cells to vemurafenib.
Beta‑catenin is upregulated in vemurafenib‑resistant PTC cells
To explore whether the Wnt/β-catenin signaling was involved in vemurafenib resistance in PTC, we first meas- ured the expression of β-catenin in whole cell lysates from vemurafenib-resistant and -sensitive cell lines. Cells exposed to vemurafenib for 20 weeks showed significantly elevated expression of β-catenin compared to the control vehicle groups (Fig. 5a). Then we separated the cytoplasmic and nuclear fractions of vemurafenib-resistant PTC cells and found that β-catenin was upregulated in both cytoplasm and
Fig. 3 Effect of TRIB2 knockdown on the resistance of PTC cells to vemurafenib. KTC1 and BCPAP cells were transfected with shRNA against TRIB2 (shTRIB2), scrambled control shRNA sequence (shNC), recombinant lentiviral vector carrying TRIB2 (TRIB2), or empty control vector (Vector). a The protein expression of TRIB2 was detected by Western blot. b The IC50 values of vemurafenib for cell proliferation were determined by MTT assay. c The protein lev- els of pERK, ERK, pAKT, and AKT were measured by Western blot. nucleus compared to the KTC1-S and BCPAP-S groups (Fig. 5b). These results indicated the activation of β-catenin in vemurafenib-resistant cells.
Beta‑catenin inhibitor reduces the resistance of PTC cells to vemurafenib
To examine whether the inhibition of β-catenin would affect the resistance of PTC cells to vemurafenib, we treated vemu- rafenib-sensitive and -resistant cells with 5 µM ICG-001, a β-catenin inhibitor, for 4 days. The treatment with ICG-001 decreased the IC50 values of vemurafenib for vemurafenib- resistant cells (Fig. 6a). Also, the pre-incubation with ICG- 001 significantly inhibited TRIB2 expression, restrained The protein expressions of E-cadherin, N-cadherin, and snail were assessed by Western blot. e The invasive capacity of transfected PTC cells was evaluated by Transwell assay. f KTC1 and BCPAP cells were transfected with shTRIB2 or scrambled control sequence fol- lowed by 20-week exposure to 2 µM vemurafenib. MTT assay was performed to determine the IC50 values of vemurafenib for cells with normal or downregulated TRIB2 expression following vemurafenib exposure EMT, and suppressed the activation of ERK and AKT in both KTC1-R and BCPAP-R cells (Fig. 6b). The Transwell assay revealed that the inhibition of β-catenin by ICG-001 significantly reduced the invasive capacity of vemurafenib- resistant PTC cells (Fig. 6c). Collectively, the above findings implied that the inhibition of β-catenin increased the sensi- tivity of PTC cells to vemurafenib via regulating TRIB2.
As the most common genetic alterations in PTC, the BRAFV600E mutation leads to constitutive activation of BRAF, ERK, and AKT, thus promoting tumor cell
Fig. 4 Effect of TRIB2 down- regulation on the apoptosis of PTC cells following vemu- rafenib exposure. KTC1 and BCPAP cells were transfected with shRNA against TRIB2, scrambled control sequence, vector carrying TRIB2, or empty control vector, followed by long-term exposure to 2 µM vemurafenib. a The apoptosis rate was measured by flow cytometry. b The expression level of cleaved caspase-3 was assessed by Western blot proliferation, inducing epithelial to mesenchymal transition, and eventually driving tumor progression . Although BRAF inhibitors gave encouraging results in BRAFV600E mutant PTC in clinical trials, efforts still need to be made to prevent, or at least reduce, acquired resistance in these patients . In this study, we showed that vemurafenib resistance induced the activation of the Wnt/β-catenin path- ways, resulting in TRIB2 upregulation and subsequent acti- vation of the ERK and AKT signaling.
Vemurafenib is a small-molecule, competitive inhibi- tor that selectively binds to the ATP-binding domain of the mutant BRAFV600E, thereby inhibiting kinase activity . The inhibitory effect of vemurafenib on tumor cell viability and motility has been fairly well-defined . Decreased activation of ERK and AKT by vemurafenib has also been demonstrated in human melanoma cells harboring BRAFV600E mutation . In this study, vemurafenib-resist- ant PTC cells showed increased phosphorylation levels of ERK and AKT, and enhanced invasive capacity compared to the vehicle-treated groups. The EMT is a fundamental step during metastasis, by which tumor cells acquire mesen- chymal properties and exhibit enhanced migratory/invasive capacities . By analyzing the EMT markers, including E-cadherin (epithelial phenotype marker), N-cadherin (mes- enchymal marker), and snail (EMT transcription factor), we found that long-term exposure to vemurafenib resulted in the loss of E-cadherin and the upregulation of N-cadherin and snail in both KTC1 and BCPAP cells, suggesting increased EMT potential in vemurafenib-resistant PTC cell lines.
TRIB2 was first recognized as an oncogene in multiple human cancers via the mechanisms involving the MAPK signaling pathway . In recent years, increasing evidence
Fig. 5 Expression of beta- catenin in PTC cells resistant to vemurafenib. a Whole cell lysates were isolated from vemurafenib-resistant KTC1 and BCPAP cell lines for the detection of β-catenin expres- sion by Western blot. b The protein levels of β-catenin in the cytoplasmic and nuclear fractions of vemurafenib- resistant cells were determined by Western blot. Beta-actin was used as an internal control for the cytoplasmic fraction while histone 3 served as an internal control for the nuclear fraction suggests that the overexpression of TRIB2 confers resistance to chemotherapeutics in both primary and immortalized can- cer cells by activating the ERK and AKT signaling pathways [15, 26]. In the current study, we first found that TRIB2 was upregulated in PTC cells following the long-term exposure to vemurafenib. In TRIB2-deficient PTC cells, the IC50 val- ues of vemurafenib were lower compared to the groups with normal TRIB2 expression. Also, the knockdown of TRIB2 effectively repressed the activation of ERK and AKT, inhib- ited the invasion and EMT, and induced the apoptosis of PTC cells. Furthermore, TRIB2 deficiency enhanced the sensitivity of both KTC1 and BCPAP cells to vemurafenib, indicating that TRIB2 contributed to the resistance to BARF inhibitor in PTC.
Aberrant activation of the Wnt/β-catenin signaling pathway is commonly observed during PTC initiation and progression . It has been reported that the acti- vated Wnt/β-catenin signaling contributes to therapeutic resistance in cancer cells by augmenting the expression of resistance-related genes responsible for the extrusion of chemotherapeutic drugs out of cells, such as ATP- binding cassette B1 and CD44 . Our results showed that β-catenin was upregulated in both the cytoplasm and nucleus of PTC cells following vemurafenib exposure, implying the activation of β-catenin signaling in vemu- rafenib-resistant cells. TRIB2 was identified as a direct target of the Wnt/β-catenin signaling in liver cancer cells. Here, we found that the pre-incubation of the β-catenin inhibitor significantly inhibited TRIB2 expression, sup- pressed EMT, and repressed the activation of ERK and AKT in vemurafenib-resistant cells. From the above results, we concluded that the inhibition of β-catenin downregulated TRIB2 and increased the sensitivity of PTC cells to vemurafenib.
Taken together, our study describes a novel regula- tory mechanism underlying chemotherapeutic resistance in PTC and suggests that the upregulation of TRIB2 by the Wnt/β-catenin activation confers resistance to BRAF inhibitor vemurafenib. These findings support further investigation and potential use of TRIB2 as a therapeutic target for resistant PTC.
Fig. 6 Effect of beta-catenin inhibitor on the resistance of PTC cells to vemurafenib. KTC1 and BCPAP cells with or without vemurafenib resist- ance were treated with 5 µM ICG-001 for 4 days. a The IC50 values of vemurafenib for cell proliferation was deter- mined by MTT assay. b The protein expressions of TRIB2, E-cadherin, N-cadherin, snail, pERK, ERK, pAKT, and AKT were analyzed by Western blot. c The invasive capacity of dif- ferent groups of PTC cells was evaluated by Transwell assay
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