Original Article
Dabrafenib stimulates autophagy in thyroid carcinoma cells via HMGB-1
2025 Volume 50 Issue 6 Pages 273-281
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2025 Volume 50 Issue 6 Pages 273-281
Autophagy has been implicated in the pathophysiology of thyroid cancer and in determining the response of cancer cells to anticancer therapy. Dabrafenib, a BRAF inhibitor, has demonstrated efficacy and safety in several types of cancers. However, it is unknown whether Dabrafenib exerts a protective effect on autophagy in thyroid carcinoma cells. In the current study, our findings demonstrate that treatment with Dabrafenib reduced cell viability and promoted LDH release in SW579 thyroid carcinoma cells. Dabrafenib was then shown to promote autophagy by increasing the level of Beclin1 and the LC3-II/LC3-I ratio while reducing the level of p62. Additionally, exposure to Dabrafenib upregulated the expression of HMGB-1 at both mRNA and protein levels. Interestingly, silencing of HMGB-1 abrogated Dabrafenib-induced autophagy, suggesting that the effects of Dabrafenib are mediated by HMGB-1. Further study revealed that Dabrafenib activated the JAK1/STAT1 signaling pathway and that blockage of the JAK1/STAT1 signaling pathway with its inhibitor Pyridone 6 ameliorated Dabrafenib-induced HMGB-1 upregulation and autophagy, implicating the involvement of the JAK1/STAT1 signaling pathway in this process. Collectively, these findings demonstrate that Dabrafenib induces autophagy in thyroid carcinoma cells via the JAK1/STAT1/HMGB-1 axis. Notably, this effect occurs independently of BRAF V600E mutation status, suggesting a novel therapeutic mechanism.
Thyroid cancer (TC), the most prevalent malignant tumor of the endocrine system, has emerged as a growing public health concern due to its alarming global surge in incidence over the past decade (Chen et al., 2023). This rise can be attributed to advancements in diagnostic technologies, which have enabled the detection of TC at an earlier stage than before (Chen et al., 2023). As a result, the annual increase in TC diagnoses is now higher than that of other cancers (Chen et al., 2023). In the female population, the incidence of TC is three times higher than in males (Shobab et al., 2022). Data from China indicates that TC has become the fastest-growing malignant tumor, with an average annual increase of 14.73% in men aged ≥ 25 years and an annual increase of 18.98% in women (Liu et al., 2019). TC can lead to symptoms such as thyroid dysfunction, neck masses, hoarseness, and even metastasis to other organs, severely threatening patients’ lives (Boucai et al., 2024). The current treatment bottlenecks for TC primarily encompass challenges such as residual cancer cells post-surgery, resistance to radioactive iodine therapy, and metastatic disease management (Nabhan et al., 2021). Traditional treatment methods such as surgical removal, radioactive iodine therapy, and hormone therapy still have certain limitations, therefore further research is needed to explore more effective treatment strategies, improve treatment outcomes, and reduce the mortality rate of patients. While Dabrafenib is approved for BRAF V600E-mutant cancers, its effects in BRAF-wildtype tumors remain poorly understood.
The latest research has demonstrated that autophagy significantly contributes to the pathogenesis of TC (Holm et al., 2022). Autophagy is a process of self-degradation and recycling that occurs within cells, whereby damaged proteins, organelles, and cellular waste are enclosed in vesicles and delivered to lysosomes for degradation and recycling (Debnath et al., 2023). In TC, autophagy has been demonstrated to inhibit cancer cell growth and migration. By promoting the process of autophagy, the survival rate of TC cells was reduced, and their proliferation and metastatic abilities were inhibited (Qu et al., 2022). Furthermore, autophagy is found to promote cancer cell apoptosis, thereby further inhibiting cancer cell growth (Fu et al., 2023). Therefore, autophagy plays a critical role in the treatment of TC as a promising target.
BRAF is a protein kinase involved in cell growth and proliferation. In some patients with malignant melanoma, the BRAF gene may mutate, leading to abnormal activation of the BRAF protein and thereby promoting the abnormal proliferation and metastasis of malignant melanoma cells (Zaman et al., 2019). Dabrafenib selectively binds to the BRAF protein, inhibiting its activity and thereby suppressing the proliferation and spread of malignant melanoma cells (Bouffet et al., 2023). Compared to chemotherapy drugs, dabrafenib offers greater precision by effectively inhibiting the growth of malignant melanoma cells while minimizing damage to normal cells, thereby reducing the side effects associated with treatment (Menzies et al., 2012). Patients receiving dabrafenib treatment often experience significant shrinkage or cessation of melanoma growth, effectively extending their survival and improving their quality of life (Long et al., 2017). Several clinical studies have demonstrated the growth-inhibiting effect of dabrafenib on TC (Busaidy et al., 2022; Lorimer et al., 2023; Subbiah et al., 2022). However, its potential mechanism of action in treating TC remains to be fully elucidated. Herein, the mechanism of dabrafenib in treating TC was explored by investigating its effects on autophagy in TC cells, providing a solid theoretical basis for the wide clinical application of dabrafenib in treating TC.
SW579 TC cells were purchased from ATCC (USA) and cultured in DMEM supplemented with 10% FBS. SW579 cells do not harbor the BRAF V600E mutation (CCLE database). The culture conditions were set at 5% CO2 and 37°C. To knock down HMGB-1 in SW579 cells, the cells were transduced with lentivirus containing a shRNA targeting HMGB-1 (sequence: 5’-CCATCACAGTGTTGTTAATGT-3’; GenePharma, Shanghai, China) at an MOI of 5 with 8 μg/mL polybrene. After 24 hr, the medium was replaced with fresh DMEM containing 10% FBS and 2 μg/mL puromycin for 7 days to select stable knockdown clones. Knockdown efficiency was confirmed by Western blot analysis.
CCK-8 assaySW579 cells were treated for 48 hr. Then, 10 μL of CCK - 8 reagents (Dojindo, Japan) was added, and the cell plate was incubated in a CO2 incubator for 2 more hours. Absorbance at 450 nm was measured with an ELISA reader (PerkinElmer, USA) to calculate cell viability.
LDH releaseSW579 cells were digested with pancreatic enzymes, collected into a centrifuge tube, and centrifuged at 400 g for 5 min. The supernatant was discarded, and the cells were resuspended in PBS-diluted LDH reagent and incubated for 1 hr. After another centrifugation at 400 g for 5 min, 120 μL of the supernatant was transferred to a new 96-well plate. Subsequently, 1× INT solution and LDH detection reagent were added. The plate was then incubated at 25°C in the dark for 30 min. Finally, the absorbance was measured at 490 nm using an ELISA reader (PerkinElmer, USA).
Western blot analysisProteins were extracted from SW579 cells using RIPA lysis buffer supplemented with proteinase inhibitors, and the protein concentration was determined using a BCA protein assay kit (BIO-RAD, USA). Equal amounts of protein lysates (20 μg per lane) were separated by 10% SDS-PAGE gel and then transferred to nitrocellulose membranes via electroblotting. The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 for 2 hr, followed by overnight incubation at 4°C with primary antibodies against LC3-II/I (1:1000), p62 (1:500), Beclin1 (1:2000), HMGB-1 (1:1000), p-JAK1 (1:1000), p-STAT1 (1:1000), and β-actin (1:5000, Abcam, USA). Subsequently, the membranes were incubated with secondary antibodies (1:4000, Abcam, USA) for 1 hr, and immunocomplexes were detected using an ECL kit (BIO-RAD, USA). Band intensity was quantified using Quantity One software (BIO-RAD, USA).
RT-PCR assayTotal RNA was isolated using a TRIzol reagent, and reverse transcription was performed with a reverse transcription kit (BIO-RAD, USA). Quantitative PCR (QPCR) was conducted in a final reaction volume of 20 μL using SYBR Green I Supermix (BIO-RAD, USA). Primer sequences for HMGB-1 were forward: 5’-TATGGCAAAAGCGGACAAGG-3’ and reverse: 5’-CTTCGCAACATCACCAATGGA-3’ (amplicon size: 196 bp), while GAPDH primers were forward: 5’-GGAGCGAGATCCCTCCAAAAT-3’ and reverse: 5’-GGCTGTTGTCATACTTCTCATGG-3’ (amplicon size: 197 bp). All detections were run in triplicate on an iCycler IQ multicolor real-time PCR detection system (BIO-RAD, USA) for a total of 40 cycles. PCR products were validated by 2% agarose gel electrophoresis to confirm amplicon specificity. Quantification of all genes was normalized to GAPDH, and relative expressions were calculated using the 2-ΔΔCt method.
Monodansylcadaverine (MDC) stainingSW579 cells were collected and washed with 1× wash buffer, then adjusted to a concentration of 1×106 cells/mL. The cell suspension was transferred to new EP tubes, and MDC staining solution was added, gently mixed, and incubated for 30-40 min in the dark. Cells were washed 2-3 times with 1× wash buffer and collected. Antifade mounting medium was added, and the formation of autophagosomes in cells was observed under a fluorescence microscope (Zeiss, Germany). The number of autophagosomes was counted.
Statistical analysisThe data were statistically analyzed using SPSS 23.0 software, and the results are presented as mean ± standard deviation (SD). Student’s t-test was used for two-group comparisons, while analysis of variance (ANOVA) was employed for comparisons among three or more groups, followed by pairwise comparisons using the Student-Newman-Keuls (SNK-Q) test. P values less than 0.05 were considered statistically significant.
The molecular structure of Dabrafenib is shown in Fig. 1A. To determine the concentration-dependent effects of Dabrafenib, SW579 cells were treated with various concentrations of Dabrafenib (10, 25, 50, 100, and 500 nM) for 1 day. The cell viability was maintained at 100% with 10 nM Dabrafenib but was significantly repressed to 81.9%, 52.6%, 32.1%, 28.7%, and 22.3% by 10, 25, 50, 100, and 500 nM Dabrafenib, respectively (Fig. 1B). Moreover, there was a sharp increase in LDH release as the concentration of Dabrafenib increased from 10 to 500 nM (Fig. 1C). Based on these findings, 25, 50, and 100 nM Dabrafenib were selected for subsequent assays.
Cytotoxicity of Dabrafenib in SW579 cells. (A) Molecular structure of Dabrafenib; (B) Cells were treated with 10, 25, 50, 100, and 500 nM Dabrafenib for 24 hr. Cell viability was assessed using the CCK-8 assay; (C) LDH levels in thyroid carcinoma cells (*P < 0.05, **P < 0.01, ***P < 0.005 compared to the control group). Data are presented as mean ± SD (n=3).
The study further investigated the impact of Dabrafenib on autophagy in these cells. It was observed that the levels of LC3-II/I and Beclin1 were significantly increased, while p62 levels were notably reduced in SW579 cells treated with 25, 50, and 100 nM Dabrafenib (Fig. 2A-D). These findings suggest a promoting effect of Dabrafenib on autophagy in SW579 cells.
Dabrafenib-induced changes in autophagy biomarkers in SW579 cells. Cells were treated with 25, 50, and 100 nM Dabrafenib for 24 hr. (A) Representative western blot analysis of LC3-II/I, p62, and Beclin1; (B) Quantification of LC3-II/I; (C) Quantification of p62; (D) Quantification of Beclin1 (*P < 0.05, **P < 0.01, ***P < 0.005 compared to the control group). Data are presented as mean ± SD (n=3).
Autophagy was characterized by an increased number of autophagosomes (Cao et al., 2021), as assessed by MDC staining. The number of MDC-positive staining in SW579 cells was significantly increased following treatment with 25, 50, and 100 nM Dabrafenib (Fig. 3A). The presence of 3-MA reduces the number of autophagosomes induced by Dabrafenib, indicating that the observed effect was mediated by autophagy (Fig. 3B). These results indicate a facilitating effect of Dabrafenib on the formation of autophagosomes in these cells.
Dabrafenib-induced increase in autophagosomes in SW579 cells. Cells were treated with 100 nM Dabrafenib in the presence of the autophagy inhibitor 3-MA (5 mM) for 24 hr. (A) Representative images of monodansylcadaverine (MDC) staining. Scale bar: 50 µm. (B) Quantification of MDC staining (**P < 0.01 compared to the control group, ## P < 0.01 compared to the Dabrafenib group). Data are presented as mean ± SD (n=3).
HMGB-1 is an inflammatory protein that has recently been identified as highly associated with the development of autophagy (Singh et al., 2022). In SW579 cells, HMGB-1 was found to be significantly upregulated in response to 25, 50, and 100 nM Dabrafenib (Fig. 4A-B). Notably, Dabrafenib treatment increased cytoplasmic HMGB-1 levels while reducing nuclear HMGB-1 expression (Fig. S1). Collectively, these findings demonstrate that Dabrafenib modulates HMGB-1 expression and localization to promote autophagy in TC cells.
Dabrafenib increased the expression of HMGB-1 in SW579 cells. Cells were treated with 25, 50, and 100 nM Dabrafenib for 24 hr. (A) mRNA levels of HMGB-1; (B) protein levels of HMGB-1 (*P < 0.05, **P < 0.01, ***P < 0.005 compared to the control group). Data are presented as mean ± SD (n=3).
To validate that Dabrafenib induces autophagy by upregulating HMGB-1, SW579 cells were transduced with lentiviral short hairpin RNA (shRNA) targeting HMGB-1 and then stimulated with 100 nM Dabrafenib. The knockdown of HMGB-1 in SW579 cells was confirmed by Western blotting assay (Fig. 5A). Furthermore, the significant increase in Beclin1 levels and the reduction in p62 levels observed in Dabrafenib-treated SW579 cells were notably reversed by silencing HMGB-1 (Fig. 5B-C). These results collectively confirm that HMGB-1 is a critical mediator of Dabrafenib-induced autophagy in TC cells.
HMGB-1 deficiency inhibited autophagy induced by Dabrafenib in SW579 cells. SW579 cells were transduced with HMGB-1 lentiviral-shRNA and then treated with 100 nM Dabrafenib. (A) Verification of shRNA-mediated knockdown of HMGB-1 by Western blot analysis; (B) Western blot results of Beclin1 and p62 levels were quantified (**P < 0.01 compared to the control group, ## P < 0.01 compared to the Dabrafenib group). Data are presented as mean ± SD (n=3).
HMGB-1 levels have been reported to be regulated by the JAK1/STAT1 signaling pathway (Xu et al., 2023). In this study, it was found that treatment with 25, 50, and 100 nM Dabrafenib significantly increased the levels of phosphorylated JAK1 (p-JAK1) and phosphorylated STAT1 (p-STAT1) in SW579 cells (Fig. 6). These results suggest that Dabrafenib activates the JAK1/STAT1 signaling pathway in SW579 cells.
Dabrafenib promoted activation of the JAK1/STAT1 signaling pathway in SW579 cells. SW579 cells were treated with 25, 50, and 100 nM Dabrafenib for 24 hr. The levels of p-JAK1 and p-STAT1 were measured by western blot analysis (*P < 0.05, **P < 0.01, ***P < 0.005 compared to the control group). Data are presented as mean ± SD (n=3).
To confirm that Dabrafenib induces autophagy and upregulates HMGB-1 in SW579 cells through activation of the JAK1/STAT1 signaling pathway, cells were treated with 100 nM Dabrafenib in the presence or absence of the JAK1/STAT1 inhibitor Pyridone 6 (30 nM) for 24 hr. Levels of HMGB-1 in SW579 cells were significantly increased by Dabrafenib, but this effect was notably suppressed by pyridone 6 (Fig. 7A). Additionally, the elevated Beclin1 levels and decreased p62 levels observed in Dabrafenib-treated SW579 cells were significantly reversed by pyridone 6 (Fig. 7B). Taken together, these findings confirm that JAK1/STAT1 signaling is essential for Dabrafenib-induced HMGB-1 upregulation and autophagy activation in TC cells.
Inhibition of the JAK1/STAT1 signaling pathway ameliorated Dabrafenib-induced increases in HMGB-1 and autophagy. SW579 cells were treated with 100 nM Dabrafenib in the presence or absence of the JAK1/STAT1 inhibitor Pyridone 6 (30 nM) for 24 hr. (A) mRNA levels of HMGB-1; (B) Western blot results of Beclin1 and p62 (**P < 0.01 compared to the control group, ## P < 0.01 compared to the Dabrafenib group). Data are presented as mean ± SD (n=3).
LC3 is a hallmark protein involved in the autophagy process, existing in two forms: the unmodified LC3-I and the phosphorylated LC3-II. LC3-I is commonly found in the early stages of autophagy. When autophagic activity intensifies, LC3-I undergoes modification to become LC3-II, which then associates with the autophagosome membrane and contributes to the formation of autophagic vesicles (Tanida et al., 2004; Tanida et al., 2008). p62 is a protein associated with autophagy that functions as an autophagy adaptor. It links aggregated waste proteins or damaged organelles in the cytoplasm and delivers them to autophagosomes for degradation. The level of p62 is inversely correlated with autophagic activity; that is, the accumulation of p62 increases when autophagic activity decreases (Lamark et al., 2017). Beclin1 is a crucial gene associated with autophagy and functions as an initiator of the process. Beclin1 can form complexes with other proteins, playing a role in the formation of autophagosomes and the regulation of autophagy. The level of Beclin1 is closely linked to autophagic activity; both overexpression and deficiency of Beclin1 can impact the autophagy process (Prerna and Dubey, 2022; Xu and Qin, 2019). In this study, similar to the effects of nitidine chloride on ovarian cancer cells reported by Lian (Lian et al., 2023), Dabrafenib significantly enhanced autophagy in SW579 cells. This was validated by increased LC3-II/I ratios and Beclin1 expression, as well as decreased p62 levels, accompanied by an increase in autophagosome production. These findings suggest that Dabrafenib may inhibit the growth of thyroid carcinoma (TC) cells by inducing autophagy.
HMGB-1 is a nuclear protein that is expressed in both the nucleus and cytoplasm of cells. It participates in various biological processes, such as DNA repair and transcriptional regulation, within these cellular compartments (Casciaro et al., 2021). Furthermore, HMGB-1 acts as a cytokine and plays a significant role in physiological processes such as inflammatory responses, immune responses, and tissue repair. It binds to various receptors, including RAGE and TLR4, within cells to initiate signal transduction pathways, thereby regulating inflammatory and immune responses (Kamaşak et al., 2020). Recent studies have demonstrated that HMGB-1 is involved in regulating the autophagy process. It has been reported that HMGB-1 levels are positively correlated with autophagy in bladder cancer patients and can be detected in circulation (Singh et al., 2022). Additionally, HMGB1 has been shown to induce autophagy in endometrial cells, leading to the degradation of cellular components and promoting the development of endometriosis (Huang et al., 2021). In this study, the enhanced autophagy observed in Dabrafenib-challenged SW579 cells was accompanied by a significant increase in HMGB-1 expression. This suggests that Dabrafenib may induce autophagy in TC cells by activating HMGB-1. Furthermore, silencing HMGB-1 notably abolished the autophagy induced by Dabrafenib in SW579 cells, providing further validation that the effect of Dabrafenib on autophagy in TC cells is mediated through HMGB-1 activation.
The JAK1/STAT1 axis is a pivotal signaling pathway that becomes activated upon specific receptor stimulation. When the receptor is engaged, JAK1 is activated and subsequently phosphorylates STAT1. The phosphorylated STAT1 forms dimers, translocates into the cell nucleus, and binds to specific DNA sequences, thereby regulating gene transcription (Li et al., 2022; Ott et al., 2023). The JAK1/STAT1 pathway plays a significant role in immune responses, cell proliferation, and apoptosis, among other biological processes. It is involved in regulating inflammatory responses, antiviral immune responses, and anti-tumor effects (Fung et al., 2022; Ma et al., 2018). Recently, it has been suggested that the activation of the JAK1/STAT1 pathway contributes to increased secretion of HMGB-1 (Xu et al., 2023). Moreover, the activation of the JAK1/STAT1 pathway is a crucial triggering factor for inducing cell autophagy (Cheng et al., 2023; Lu et al., 2023; Dong et al., 2015). In this study, the activation of autophagy and the elevated levels of HMGB-1 in Dabrafenib-stimulated SW579 cells were accompanied by the activation of JAK1/STAT1 signaling. This suggests that Dabrafenib might trigger autophagy activation and HMGB-1 production through the activation of JAK1/STAT1 signaling. Furthermore, the increase in HMGB-1 and autophagy in Dabrafenib-challenged SW579 cells was significantly abrogated by an inhibitor of JAK1/STAT1 signaling, further verifying that the influence of Dabrafenib on autophagy and HMGB-1 production in TC cells is triggered by JAK1/STAT1 activation. The lack of BRAF V600E mutation in SW579 cells prompts inquiries into Dabrafenib's mechanism of action. Recent studies suggest that Dabrafenib can inhibit other kinases (CRAF, SRC) or modulate signaling pathways in BRAF-wildtype cells (King et al., 2013). Our data support a model where Dabrafenib activates the JAK1/STAT1/HMGB-1 axis independently of BRAF, providing a rationale for its use in BRAF-mutation-negative TC. As we progress in our work, the autophagy-inducing function of Dabrafenib and the related mechanisms will be validated using a TC xenograft model.
Overall, Dabrafenib stimulated autophagy in TC cells by enhancing HMGB-1 production through JAK1/STAT1 activation. Our findings offer valuable insights into the treatment strategy of TC.
This study was funded by the Heilongjiang Provincial Health Commission (20220404110755).
Data availability statementData are available upon reasonable request to the corresponding author.
Conflict of interestThe authors declare that there is no conflict of interest.
