BRAF V600E /p-ERK/p-DRP1(Ser616) Promotes Tumor Progression and Reprogramming of Glucose Metabolism in Papillary Thyroid Cancer

Abstract

Background:

Papillary thyroid cancer (PTC) with the BRAF^V600E mutation is associated with a poorer prognosis. BRAF inhibitors may demonstrate limited efficacy due to emerging drug resistance. The Warburg effect may have cancer therapeutic implications. It is not known if the BRAF^V600E mutation is associated with altered glucose metabolism in PTC.

Methods:

This study examined the effect of BRAF^V600E and dynamin-related protein 1 (DRP1) on various cellular processes in PTC cells, including cell proliferation, migration, invasion, mitochondrial fission, glucose metabolism, reactive oxygen species (ROS) generation, and apoptosis. We used RT-qPCR to assess the expression of key glycolytic enzymes in thyroid cancer tissues. Additionally, the regulatory interaction between BRAF^V600E and DRP1 was investigated through Western blot and immunohistochemical staining. We further evaluated the impact of DRP1 in PTC and the inhibitory effects of dabrafenib and 2-deoxy-d-glucose (2-DG) in vitro and in vivo.

Results:

We found that the BRAF^V600E mutation significantly augments aerobic glycolysis while suppressing oxidative phosphorylation in PTC. We identified the BRAF^V600E/p-ERK/p-DRP1(Ser616) signaling pathway as a critical mediator in PTC progression. First, the BRAF^V600E/p-ERK/p-DRP1(Ser616) signaling pathway enhances cell proliferation by upregulating hexokinase 2 expression and thereby increasing aerobic glycolysis. Second, it inhibits apoptosis by promoting mitochondrial fission and reducing ROS levels. Moreover, we demonstrated that the combination therapy of 2-DG and dabrafenib markedly impedes the progression of BRAF^V600E-positive PTC.

Conclusion:

The BRAF^V600E/p-ERK/p-DRP1(Ser616) signaling pathway plays a pivotal role in glucose metabolism reprogramming, contributing to the aggressiveness and progression of BRAF^V600E-positive PTC. Our findings suggest that a combined therapeutic approach using 2-DG and dabrafenib has the potential to improve the outcome of PTC patients with BRAF^V600E.

Introduction

Thyroid cancer (TC) is the most prevalent tumor in the endocrine system. Papillary TC (PTC) is the predominant histological subtype of TC. The BRAF^V600E mutation emerges as the most frequent oncogenic alteration, present in approximately 45–80% of PTC.^1,2 This mutation is strongly associated with adverse clinical outcomes, including increased recurrence rates, lymph node metastasis, advanced tumor stages, poorer prognosis, and increased cancer-related mortality.^3,4 Additionally, BRAF^V600E leads to increased resistance to postoperative I¹³¹ therapy.⁵ However, BRAF inhibitors (BRAFi) such as vemurafenib and dabrafenib have shown limited effectiveness due to the emergence of resistance and severe side effects.^6,7 Therefore, it is crucial to identify novel therapeutic targets for the treatment of BRAF^V600E-mutated PTC.

The evolving understanding of tumor biology has acknowledged metabolic reprogramming as a novel cancer hallmark.^8,9 The Warburg effect, a phenomenon where tumor cells predominantly rely on glycolysis for energy even under aerobic conditions, is a well-reported example of this metabolic shift.¹⁰ Therapeutics targeting the Warburg effect have shown effectiveness in halting the progression of various cancers.^11
–13 Notably, the BRAF^V600E has been related to augmented aerobic glycolysis in cancers such as melanoma, colorectal, and bladder cancer.^14
–16 Previous research suggests that BRAF^V600E-mutated differentiated TCs exhibit significantly higher 18F-FDG uptake compared with wild-type BRAF (BRAF^WT) tumors.¹⁷ However, the precise relationship between BRAF^V600E and glucose metabolism in PTC remains unknown.

Decreased mitochondrial respiration and increased aerobic glycolysis have been reported in various cancers.^18,19 Central to the regulation of cellular metabolism, and thereby playing a pivotal role in cancer development and progression, is the balance between mitochondrial fission and fusion as well as overall mitochondrial function.^20,21 Emerging evidence underscores that mitochondrial fission facilitates cell proliferation and metastasis.²² Conversely, mitochondrial fusion exhibits tumor-suppressive effects in various cancers.^23,24 Notably, BRAF^V600E has been identified to augment mitochondrial fission. A previous study demonstrated that inhibiting the MAPK pathway can suppress mitochondrial fission and biogenesis in melanoma, suggesting a potential therapeutic target for cancers with the BRAF^V600E mutation.²⁵

In light of these findings, it becomes imperative to delineate the relationship between BRAF^V600E, glucose metabolism, and mitochondrial dynamics, especially given the challenging therapeutic effects and poor prognosis associated with BRAF^V600E-mutated PTC. Our study identifies a strong association between BRAF^V600E and increased aerobic glycolysis, as well as decreased oxidative phosphorylation (OXPHOS) in PTC. We have systematically investigated the role and underlying mechanisms of BRAF^V600E in the regulation of mitochondrial dynamics and metabolic reprogramming in PTC cells. Our findings reveal that a combination therapy of 2-deoxy-d-glucose (2-DG) and dabrafenib markedly inhibits the progression of PTC.

Materials and Methods

Public database analysis

Gene set enrichment analysis (GSEA) was conducted to assess gene expression differences between PTCs with BRAF^WT and those with the BRAF^V600E mutation.

Clinical sample analysis

Eighty pairs of tumor and peritumor tissues were collected to extract RNA. These specimens were obtained from PTC patients who underwent primary surgery, including thyroidectomy and routine lymph node dissection (central and/or lateral lymph node dissection) at the Department of Head and Neck Surgery, Fudan University Shanghai Cancer Center (FUSCC), from March 2018 to June 2018, with follow-up until June 2022. No patients received thyroid surgery, radioiodine, or radiotherapy before the TC surgery. RT-qPCR was utilized to compare the expression levels of glycolysis-related enzymes in PTC versus peritumoral tissue. A total of 155 formalin-fixed, paraffin-embedded tissue PTC samples were collected to construct a tissue microarray (TMA). These samples were obtained from 97 patients who underwent primary PTC surgery at the Department of Head and Neck Surgery, FUSCC, between January 2010 and January 2015, with follow-up until June 2022. No patients received thyroid surgery, radioiodine, or radiotherapy before the TC surgery. The staging for T1/T2 PTC was based on the eighth edition of the AJCC staging system.²⁶ Locally advanced PTC (LA-PTC) was defined as PTC with invasion into muscles, blood vessels, nerves, esophagus, and trachea. BRAF status was ascertained by an amplification refractory mutation system based on an ultrasound-guided fine-needle biopsy. TMAs were stained by immunohistochemistry (IHC). The mean optical density (MOD) was employed for semiquantitative analysis of protein expression levels. The expression of phosphorylated dynamin-related protein 1 (DRP1) (Ser616) was compared among peritumoral tissue, T1/T2-stage PTC, and LA-PTC. Comparisons were also made between BRAF^WT PTC and BRAF^V600E PTC. Detailed immunohistochemical staining and evaluation methods are shown in Supplementary Data S1.

The study was approved by the Medical Ethics Committee of Fudan University Cancer Hospital (No. 050432-4-1911D), and patients provided informed consent to use their tissues in the study.

In vitro experiments

Three TC cell lines were used: TPC-1 (BRAF mutation negative), BCPAP (BRAF^V600E positive), and K1 (BRAF^V600E positive). DRP1 was knocked down by lentivirus–shRNA transfection. DRP1 was reintroduced by plasmid transfection. BRAF^V600E was overexpressed by lentivirus–plasmid transfection. BRAF^V600E was inhibited by dabrafenib. Cell viability, colony formation, migration, and invasion assays were conducted to assess the progression of PTC in vitro. Glycolysis was evaluated through the extracellular acidification rate (ECAR) and lactate production (LP) assays. OXPHOS was assessed via the oxygen consumption rate (OCR) assay. Mitochondrial morphology was analyzed using transmission electron microscopy (TEM). Cellular reactive oxygen species (ROS) production and apoptosis were assessed by test kits. The effects of dabrafenib, 2-DG, and their combined application were evaluated in vitro. Detailed experimental methods are presented in Supplementary Data S1.

In vivo experiments

Female BALB/C nude mice aged 4–5 weeks (purchased from Shanghai SLAC ANIMAL Co., Ltd.) were used to establish a subcutaneous xenograft model. Dead and nontumorous mice were excluded from the experiment. One researcher implanted tumor cells into the mice. The staff in the animal room who were unaware of the group allocation were in charge of feeding, cage allocation, and measurement. The research was approved by the Experimental Animal Care and Use Committee of Fudan University Cancer Hospital (no. FUSCC-IACUC-S2023-0506).

Knockdown of DRP1

Twelve mice were randomly divided into two groups using a random number generator in Excel software (version 2019; Microsoft, Redmond, WA): shNC and shDRP1. Each mouse was implanted with K1 cells on the right side. Tumor volumes were monitored weekly for 4 weeks. There was one mouse in each of the shNC group and the shDRP1 group, and no subcutaneous tumors formed.

Drug experiments

Twelve mice were randomly divided into four groups using a random number generator: phosphate buffered saline (PBS), dabrafenib (30 mg/kg for each mouse), 2-DG (250 mg/kg for each mouse), and a combination of dabrafenib and 2-DG. Each mouse was implanted with BCPAP cells on both sides. Treatment began when tumor volumes reached ∼ 1500 mm³. Body weight and tumor volume were monitored weekly for 3 weeks.

Statistical analysis

Continuous variables were presented as mean ± standard error of the mean, and classified variable results were shown as frequency and percentage. Paired t and independent t tests were used to compare continuous variables between two groups. Pearson’s χ ² test and Fisher’s exact test were used for categorical variables. A p value below 0.05 was deemed statistically significant. Data analysis and visualization were performed using SPSS 22.0 and GraphPad Prism 6.0 software on the Windows platform.

Results

Clinical sample

In 80 pairs of samples for RNA extraction, 43 cases carried no BRAF mutation, and 37 cases carried the BRAF^V600E mutation. The median follow-up time was 49 months (interquartile range 2 months). Patients’ demographic and clinical characteristics are described in Table 1. In total, 155 TMA samples were subjected to IHC, including 45 PTC in the T1/T2 stage, 48 LA-PTC, and 62 normal thyroid tissues. Out of 97 patients, 30 cases carried no BRAF mutation, 33 cases carried the BRAF^V600E mutation, and the remaining BRAF status was unknown. The median follow-up duration was 111 months (interquartile range 27 months). Patients’ information is given in Table 2. A patient flow diagram is shown in Figure 1.

FIG. 1.

Patient flow diagram. IHC, immunohistochemistry; LA, locally advanced.

Table 1.

Demographic and Clinical Characteristics of Patients (80 Pairs of Tumors and Peritumors)

	BRAF^WT PTC	BRAF^V600E PTC	p Values
Total	43	37
Age			0.712
Median (years)	55	52
Range (years)	25–81	28–77
Sex			0.877
Female	26	23
Male	17	14
Tumor size			0.673
Average (cm)	1.4	1.3
Range (cm)	0.7–3.8	0.4–3.6
T stage			0.823
T1/T2	39	33
T3/T4	4	4
Multifocality			0.777
Yes	6	6
No	37	31
CLNM			0.960
Positive	23	20
Negative	20	17
LLNM			0.383
Positive	13	8
Negative	30	29
Recurrence			0.914
Yes	1	1
No	42	36
Stage			0.375
I	28	24
II	7	5
III	5	6
IV	3	2
Mortality due to PTC			0.351
Yes	1	0
No	42	37

CLNM, central lymph node metastasis; LLNM, lateral lymph node metastasis; PTC, papillary thyroid cancer.

Table 2.

Demographic and Clinical Characteristics of Patients (Tissue Microarray)

	T1/T2-PTC	LA-PTC	p Values	BRAF^WT PTC	BRAF^V600E PTC	p Values
Total	45	48		30	33
Age			0.702			0.340
Median (years)	58	56.5		58.5	58
Range (years)	19–74	21–77		23–76	21–77
Sex			0.904			0.516
Female	24	25		17	16
Male	21	23		13	17
Tumor size			0.002			0.003
Average (cm)	2.1	3.1		2.3	3.6
Range (cm)	0.5–3.8	0.4–6.5		0.4–5.5	0.4–6
T stage			<0.001			0.061
T1/T2	45	0		18	12
T3/T4	0	48		12	21
Multifocality			0.060			0.248
Yes	13	23		12	18
No	32	25		18	15
CLNM			0.270			0.547
Positive	24	31		15	19
Negative	21	17		15	14
LLNM			0.260			0.701
Positive	21	28		14	17
Negative	24	20		16	16
Recurrence			0.617			0.885
Yes	5	7		4	4
No	40	41		26	29
Stage			<0.001			0.056
I/II	45	20		24	19
III/IV	0	28		6	14
Mortality due to PTC			0.338			0.612
Yes	1	3		1	2
No	44	45		29	31

CLNM, central lymph node metastasis; LLNM, lateral lymph node metastasis; PTC, papillary thyroid cancer.

BRAF^V600E regulates malignant biological behaviors and glucose metabolism reprogramming in PTC

In vitro experiments demonstrated that BRAF^V600E accelerates malignant biological behaviors in PTC (Supplementary Fig. S1). GSEA indicated a notable downregulation in genes associated with respiratory electron transport and the citric acid cycle in BRAF^V600E-mutated PTC (Fig. 2A). The expression levels of key glycolytic enzymes, such as LDHA (p = 0.031), SCL2A1 (p = 0.004), and hexokinase 2 (HK2) (p < 0.001), were elevated in PTC compared with peritumor tissue (Fig. 2B). LDHA (p = 0.039) and HK2 (p = 0.014) levels were higher in BRAF^V600E-mutated PTC compared with BRAF^WT counterparts (Fig. 2C). BRAF^V600E was overexpressed in TPC-1 cells, while BCPAP and K1 cells were treated with dabrafenib to inhibit BRAF^V600E (Fig. 2D). We observed an increase in ECAR and LP (p < 0.001), along with a decrease in maximal OCR in TPC-1-BRAF^V600E cells (Fig. 2E). No significant changes were observed in ECAR, OCR, and LP in TPC-1 cells treated with dabrafenib (Fig. 2F). Dabrafenib treatment reduced ECAR in BCPAP (Fig. 2G) and K1 cells (Fig. 2H). LP significantly decreased in BCPAP when the concentration of dabrafenib reached 1000 nM (p < 0.001). LP significantly decreased in K1 when the concentration of dabrafenib reached 100 nM (p < 0.001). Conversely, OCR increased in dabrafenib-treated BCPAP (Fig. 2G) and K1 cells (Fig. 2H). These findings suggest that BRAF^V600E enhances glycolysis and suppresses OXPHOS in PTC.

FIG. 2.

The impact of BRAF^V600E on glucose metabolism reprogramming in PTC. (A) Gene set enrichment analysis of respiratory electron transport between PTC with BRAF^V600E and BRAF^WT. (B) The expression of LDHA, SCL2A1, and HK2 in PTC and peritumor. (C) The expression of LDHA, SCL2A1, and HK2 in BRAF^V600E PTC and BRAF^WT PTC. (D) The expression of BRAF V600E and p-ERK1/2 in BRAF^V600E-overexpressed TPC-1 and dabrafenib-treated BCPAP and K1. (E) ECAR, LP, and OCR alteration in BRAF^V600E-overexpressed TPC-1. (F) ECAR, LP, and OCR alteration in dabrafenib-treated TPC-1. (G) ECAR, LP, and OCR alteration in dabrafenib-treated BCPAP. (H) ECAR, LP, and OCR alteration in dabrafenib-treated K1. Each point represents five replicates in ECAR and OCR result. Each bar represents three replicates in LP result. ***p < 0.001, **p < 0.01, *p < 0.05. PTC, papillary thyroid cancer; ECAR, extracellular acidification rate; OCR, oxygen consumption rate; LP, lactate production.

BRAFV600E regulates mitochondrial fission, ROS production, and apoptosis in PTC

TEM analysis revealed an increase in the number of mitochondria per cell in BRAF^V600E-overexpressed TPC-1 and a decrease in BCPAP and K1 treated with 1000 nM dabrafenib. The length of mitochondria was shorter in BRAF^V600E-overexpressed TPC-1 and longer in dabrafenib-treated BCPAP and K1 (Fig. 3A). The results indicate that BRAF^V600E promotes mitochondrial fission in PTC.

FIG. 3.

The impact of BRAF^V600E on mitochondrial fission/fusion, ROS production, and apoptosis in PTC cells. (A) Representative TEM images; the length of mitochondria and the number of mitochondria per cell in BRAF^V600E-overexpressed TPC-1, dabrafenib-treated BCPAP and K1. (B) Representative FCA of JC-1 staining in BRAF^V600E-overexpressed TPC-1. (C) Representative FCA of JC-1 staining in dabrafenib-treated BCPAP. (D) Representative FCA of JC-1 staining in dabrafenib-treated K1. (E) Representative FCA of cellular ROS in BRAF^V600E-overexpressed TPC-1, dabrafenib-treated BCPAP and K1. (F) Apoptosis alteration in BRAF^V600E-overexpressed TPC-1. (G) Apoptosis alteration in dabrafenib-treated BCPAP. (H) Apoptosis alteration in dabrafenib-treated K1. Each bar represents three replicates in apoptosis assays. Each bar represents five randomly selected cells in number of mitochondria per cell and length of mitochondria. ROS, reactive oxygen species; PTC, papillary thyroid cancer; TEM, transmission electron microscopy; FCA, flow cytometric analysis.

JC-1 dye assays showed a reduced proportion of cells with high mitochondrial membrane potential in BRAF^V600E-overexpressed TPC-1 cells (Fig. 3B). In contrast, dabrafenib-treated BCPAP (Fig. 3C) and K1 (Fig. 3D) exhibited an increased proportion of cells with high mitochondrial membrane potential, suggesting impaired mitochondrial membrane stability caused by BRAF^V600E. Cellular ROS levels decreased in TPC-1 cells overexpressing BRAF^V600E and increased in BCPAP and K1 treated with dabrafenib (Fig. 3E). Apoptosis rates decreased in BRAF^V600E-overexpressed TPC-1 (Fig. 3F) cells and increased in dabrafenib-treated BCPAP (Fig. 3G) and K1 cells (Fig. 3H).

BRAF^V600E/p-ERK/p-DRP1(Ser616) signaling pathway in PTC

Western blot (WB) analysis revealed that both p-ERK and p-DRP1(Ser616) were upregulated in BRAF^V600E-overexpressed TPC-1 and HEK-293 cells and downregulated in dabrafenib-treated BCPAP and K1 cells (Fig. 4A). Moreover, Figure 4B shows that the MOD of p-DRP1(Ser616) was significantly elevated in PTC with BRAF^V600E (mean: 0.192 [CI 0.190–0.193]) compared with PTC with BRAF^WT (mean: 0.185 [CI 0.183–0.187]). These results suggest the presence of a BRAF^V600E/p-ERK/p-DRP1(Ser616) signaling pathway in PTC.

FIG. 4.

BRAF^V600E/p-ERK/p-DRP1(Ser616) signaling pathway in PTC. (A) Expression of BRAF V600E, and p-DRP1(Ser616) in BRAF^V600E-overexpressed TPC-1 and HEK-293, dabrafenib-treated BCPAP and K1. (B) Representative images of the immunohistochemical staining of tissue microarray; MOD of p-DRP1(S616) analysis in peritumor, BRAF^WT PTC, BRAF^V600E -PTC. ***p < 0.001, **p < 0.01, *p < 0.05. DRP1, dynamin-related protein 1; PTC, papillary thyroid cancer; MOD, mean optical density.

DRP1 promotes the progression of BRAF^V600E-muted PTC

DRP1 was knocked down in BCPAP, K1, and TPC-1 cells (Fig. 5A). As shown in Figure 5B, the colonies of BCPAP and K1 cells were significantly reduced after DRP1 knockdown (p < 0.001) but were rescued after the reintroduction of DRP1, while the colonies of TPC-1 cells were slightly reduced (p = 0.035) and increased after the reintroduction of DRP1 (p = 0.266). In Figure 5C, the cell viability of BCPAP and K1 decreased and was rescued after the reintroduction of DRP1, while the cell viability of TPC-1 changed little. In Figure 5D, the cell migration of BCPAP was significantly weakened (p < 0.001) and was rescued after the reintroduction of DRP1; the cell migration of K1 was slightly weakened and increased after the reintroduction of DRP1 (p = 0.002). TPC-1 did not show significant changes. In Figure 5E, the cell invasion of BCPAP (p < 0.001) and K1 (p = 0.011) was reduced and rescued after the reintroduction of DRP1, while TPC-1 showed less change. Figure 5F shows that with DRP1 knockdown, the volume of subcutaneous xenograft tumors in mice was significantly smaller than that in the negative control group (p = 0.008). Additionally, the MOD of p-DRP1(Ser616) in LA-PTC (mean: 0.189 [CI 0.188–0.191]) was higher than in T1/T2-PTC (mean: 0.186 [CI 0.184–0.187]), and both were elevated compared with peritumoral tissue (mean: 0.181 [CI 0.180–0.183]) (Fig. 5G). These findings support the role of the BRAF^V600E/p-ERK/p-DRP1(Ser616) pathway in promoting PTC progression.

FIG. 5.

The impact of DRP1 on the malignant behavior of PTC. (A) WB assay for protein expression of DRP1 and p-DRP1(s616) in cells with shDRP1, reintroduction of DRP1 in shDRP1, and their negative control. (B) Colony formation assay results in cells with shDRP1, reintroduction of DRP1 in shDRP1, and their negative control. (C) Cell viability assay results in cells with shDRP1, reintroduction of DRP1 in shDRP1, and their negative control. (D) Migration assay results in cells with shDRP1, reintroduction of DRP1 in shDRP1, and their negative control. (E) Invasion assays result in cells with shDRP1, reintroduction of DRP1 in shDRP1, and their negative control. (F) The subcutaneous tumor growth of K1-shDRP1 and K1-shNC cells in nude mice. (G) Representative images of IHC staining; MOD of p-DRP1(s616) analysis in peritumor, T1/T2-PTC, LA-PTC. ***p < 0.001, **p < 0.01, *p < 0.05. PTC, papillary thyroid cancer; DRP1, dynamin-related protein 1; WB, Western blot; IHC, immunohistochemistry; MOD, mean optical density; LA, locally advanced.

BRAF^V600E/p-ERK/p-DRP1(Ser616) pathway regulates mitochondrial fission and glucose metabolism reprogramming in PTC

Knockdown of DRP1 in BCPAP and K1 cells resulted in an increased average mitochondrial length and a decreased number of mitochondria per cell (Fig. 6A). Furthermore, with DRP1 knockdown, BCPAP, K1, and BRAF^V600E-overexpressed TPC-1 exhibited a higher proportion of cells with high mitochondrial membrane potential (Fig. 6B). A reduction in ECAR and LP was observed in BCPAP-shDRP1 (Fig. 6C), K1-shDRP1 (Fig. 6D), and BRAF^V600E-overexpressed TPC-1 with DRP1 knockdown (Fig. 6E). This implies that the BRAF^V600E/p-ERK/p-DRP1(Ser616) pathway promotes glycolysis. Conversely, OCR was upregulated in BCPAP-shDRP1 (Fig. 6C), K1-shDRP1 (Fig. 6D), and BRAF^V600E-overexpressed TPC-1 with DRP1 knockdown (Fig. 6E), indicating that the pathway inhibits OXPHOS. WB analysis and RT-qPCR showed an upregulation of HK2 with BRAF^V600E overexpression in TPC-1 and a reduction following DRP1 knockdown in BCPAP and K1 cells (Fig. 6F).

FIG. 6.

Regulation of mitochondrial fission/fusion and glucose metabolism reprogramming by BRAF^V600E/p-ERK/p-DRP1(Ser616) pathway in PTC. (A) Representative TEM images; the length of mitochondria, and the number of mitochondria per cell in BCPAP-shDRP1, K1-shDRP1. (B) FCA of JC-1 staining in BCPAP-shDRP1, K1-shDRP1, and BRAF^V600E-TPC1-shDRP1. (C) ECAR, LP, and OCR alteration in BCPAP-shDRP1. (D) ECAR, LP and OCR alteration in K1-shDRP1. (E) ECAR, LP, and OCR alteration in BRAF^V600E-TPC1-shDRP1. (F) RT-qPCR and WB assays for mRNA and protein expression levels of HK2 in PTC cells with BRAF^V600E overexpression, BRAF^V600E overexpression, and DRP1 knockdown. ***p < 0.001, **p < 0.01, *p < 0.05. DRP1, dynamin-related protein 1; PTC, papillary thyroid cancer; TEM, transmission electron microscopy; FCA, flow cytometric analysis; ECAR, extracellular acidification rate; OCR, oxygen consumption rate; LP, lactate production; WB, Western blot.

BRAF^V600E/p-ERK/p-DRP1(Ser616) signaling pathway regulates ROS and apoptosis in PTC

Knockdown of DRP1 led to an increase of cellular ROS in BRAF^V600E-overexpressed TPC-1, BCPAP, and K1 (Fig. 7A). Increased apoptosis occurred following DRP1 knockdown in BRAF^V600E-overexpressed TPC-1, BCPAP, and K1 (Fig. 7B, C, and D). These findings indicate that the BRAF^V600E/p-ERK/p-DRP1(Ser616) signaling pathway plays a regulatory role in both ROS production and apoptosis in PTC.

FIG. 7.

Regulation of ROS production and apoptosis by BRAF^V600E/p-ERK/p-DRP1(Ser616) pathway in PTC and drug experiments. (A) Representative FCA of cellular ROS in BRAF^V600E-TPC1-shDRP1, BCPAP-shDRP1, and K1-shDRP1. (B) Apoptosis alteration in BRAF^V600E-TPC1-shDRP1. (C) Apoptosis alteration in BCPAP-shDRP1. (D) Apoptosis alteration in K1-shDRP1. (E) Colony formation assay results in BCPAP, K1, and TPC-1 treated with different concentration of 2-DG. (F) Cell viability assay results in BCPAP, K1, and TPC-1 treated with DMSO, 1000 nM dabrafenib, 5 mM 2-DG, and 1000 nM dabrafenib + 5 mM 2-DG, respectively. (G) Comparison of colony formation between 2-DG alone and 2-DG combined with 1000 nM dabrafenib in BCPAP, K1, and TPC-1. (H) Dissected tumors from mice treated with PBS, dabrafenib, 2-DG, and dabrafenib combined with 2-DG respectively; the subcutaneous tumor volume curve of BCPAP cells; the weight curve of mice. Each bar represents three replicates in apoptosis assays. ***p < 0.001, **p < 0.01, *p < 0.05. ROS, reactive oxygen species; DRP1, dynamin-related protein 1; PTC, papillary thyroid cancer; FCA, flow cytometric analysis; 2-DG, 2-deoxy-d-glucose.

Agent experiments targeting BRAF^V600E and glycolysis

In vitro experiments demonstrated the efficacy of 2-DG in inhibiting cell proliferation in BCPAP, K1, and TPC-1 cells (Fig. 7E). The inhibitory effect of 2-DG on TPC-1 is weaker than that of BCPAP and K1 when the concentration is lower than 2.5 mM. Notably, the combination of 2-DG and dabrafenib exhibited a more pronounced inhibitory effect on BCPAP and K1 compared with either drug used alone (Fig. 7F, G). In vivo experiments showed that the combination of 2-DG and dabrafenib more effectively suppressed tumor growth than either agent individually, and no significant differences in mouse body weight were observed among the different treatment groups (Fig. 7H).

Discussion

Despite notable advancements in treating BRAF^V600E TC following the introduction of BRAFi, the prognosis for PTC with the BRAF^V600E mutation is limited by drug resistance.²⁷ A previous study found that targeting ACYP1, which promotes glycolysis in hepatocellular carcinoma, can significantly reduce lenvatinib resistance and inhibit tumor progression.²⁸ Thus, drugs targeting glucose metabolism may be the key to overcoming resistance to tyrosine kinase inhibitors. Our study not only confirms that BRAF^V600E supports the Warburg effect in PTC through the BRAF^V600E/p-ERK/p-DRP1(Ser616) signaling pathway but also demonstrates its detailed role in regulating glucose metabolism and tumor growth. Furthermore, we propose a promising therapeutic strategy for BRAF^V600E-mutated PTC.

GSEA of the TCGA database revealed a significant downregulation of the mitochondrial OXPHOS electron transport chain pathway in patients with BRAF^V600E mutations. RT-qPCR analysis of 80 pairs of tissues showed increased expression of glycolysis-related enzymes in PTC, especially in samples with the BRAF^V600E compared with BRAF^WT. TEM image analysis and flow cytometric analysis indicated that BRAF^V600E promotes mitochondrial fission in PTC. Further investigations demonstrated an elevation in glycolysis and simultaneous inhibition of OXPHOS in PTC cells due to BRAF^V600E. Additionally, BRAF^V600E was found to reduce cellular ROS production and apoptosis in PTC cells.

DRP1 is a fission-mediating GTPase that localizes to mitochondria and initiates membrane scission upon phosphorylation at serine 616.^29,30 After discovering the effect of BRAF^V600E on mitochondrial morphology in PTC cells, we attempted to explore the regulation of BRAF^V600E on DRP1, which plays a crucial role in mitochondrial fission. WB analysis revealed a BRAF^V600E/p-ERK/p-DRP1(ser616) regulatory axis in PTC. This pathway also exists in HEK-293 cells, indicating its widespread presence. IHC results demonstrated that p-DRP1(Ser616) expression is elevated in PTC compared with peritumor tissue and is higher in BRAF^V600E-positive PTC than in PTC without BRAF mutation. Furthermore, its expression was higher in LA-PTC than in early-stage T1/T2 PTC, suggesting a possible correlation between p-DRP1(Ser616) expression and poor PTC prognosis. Cellular experiments revealed that the BRAF^V600E/p-ERK/p-DRP1(Ser616) pathway enhances mitochondrial fission, glycolysis, and the malignant behavior of PTC cells. Animal models demonstrated that DRP1 knockdown inhibited PTC growth. No significant changes in glucose metabolism and malignant behavior were observed in TPC-1 after knocking down DRP1, suggesting that DRP1 depends on BRAF^V600E to exert its function in PTC.

HK2, the initial rate-limiting enzyme in the glycolytic pathway, was found to be influenced by the BRAF^V600E/p-DRP1(Ser616) axis. This aligns with the previous study, which reported that DRP1 in Ras-driven pancreatic tumors enhances glycolysis through HK2 regulation.³¹ Aerobic glycolysis not only boosts ATP production in energy-deficient environments but also supplies intermediates for rapid cell proliferation and acidifies the tumor microenvironment. It has been observed that cells with higher glycolytic capability exhibit more aggressive malignant behaviors in tumors.^32,33 Therefore, our findings corroborate that the BRAF^V600E/p-ERK/p-DRP1(Ser616) axis enhances glycolysis to promote malignant behaviors in PTC cells with BRAF ^V600E by regulating HK2.

Moreover, mitochondria, the site of OXPHOS, rely on a stable mitochondrial inner membrane for efficient electron chain transfer. Thus, mitochondrial fragmentation and excessive fission can impair OXPHOS, which is a primary source of intracellular ROS.³⁴ ROS acts as a double-edged sword in tumorigenesis and progression. While it activates protumorigenic signals and enhances cell survival, it also triggers oxidative stress-induced tumor cell apoptosis.³⁵ Our study suggests that the BRAF^V600E/p-ERK/p-DRP1(Ser616) axis promotes mitochondrial fission, thereby weakening OXPHOS, reducing ROS production, and ultimately decreasing cell apoptosis in BRAF^V600E-positive PTC.

The first step of glycolysis is that glucose is phosphorylated by HK to form glucose 6-phosphate (G-6-P). 2-DG, a mimic of glucose, interacts with HK to generate 2-deoxy-d-glucose-6-phosphate, which accumulates in cells but cannot exert the effect of G-6-P, thus inhibiting glycolysis.³⁶ Despite high expectations for 2-DG as a potential anticancer agent, its clinical use has been restricted due to its toxicity at high doses. Currently, it is primarily studied as an adjunct to other anticancer drugs.³⁷ This study demonstrates that when combined with dabrafenib, 2-DG exhibits significant potential for inhibiting BRAF^V600E PTC cell growth both in vitro and in vivo, surpassing the effects of either drug used alone. For PTC cells without BRAF mutations, 2-DG also shows inhibitory effects, albeit requiring higher drug concentrations. The combined application of the two drugs enables 2-DG to effectively inhibit PTC at lower doses and mitigate its toxicity. Furthermore, this combination may address the challenges posed by the drug resistance of dabrafenib in BRAF^V600E PTC.

While this study provides valuable insights into the treatment of BRAF^V600E-positive PTC, it is not without its limitations. One of the critical gaps in our understanding is the detailed molecular mechanism underlying the increased expression of HK2 in PTC. Future studies should focus on unraveling these molecular pathways. The combination of 2-DG and dabrafenib shows promise in treating BRAF^V600E PTC. However, more comprehensive trials are needed to detect the efficacy and safety of this drug combination. Such trials will be crucial in translating these findings into clinical practice.

Conclusion

In summary, our findings confirm that the BRAF^V600E mutation promotes glucose metabolism reprogramming and mitochondrial fission in PTC through the BRAF^V600E/p-ERK/p-DRP1(Ser616) pathway. The dual inhibition of this pathway using 2-DG and dabrafenib presents a promising therapeutic approach for patients with BRAF^V600E PTC.

Footnotes

Authors’ Contributions

N.Q., B.M., and Q.J. proposed the concept and conducted the work. S.W. and Y.W. drafted and revised the review. J.W. and Y.W. assisted with data analysis and interpretation. S.D. and X.X. provided technical support. Y.W. collected samples. Y.W. and N.H. assisted in collecting follow-up information. S.W., JW., and Z.N. conducted basic experiments. All authors gave final approval of the version to be published.

Author Disclosure Statement

The authors declare no financial interests related to this study.

Funding Information

This study was supported by grants from the National Natural Science Foundation of China (82373315 to Ning Qu, 82203052 to Ben Ma).

Supplementary Material

Supplementary Data S1

Supplementary Figure S1

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BRAF V600E /p-ERK/p-DRP1(Ser616) Promotes Tumor Progression and Reprogramming of Glucose Metabolism in Papillary Thyroid Cancer

Abstract

Background:

Methods:

Results:

Conclusion:

Introduction

Materials and Methods

Public database analysis

Clinical sample analysis

In vitro experiments

In vivo experiments

Knockdown of DRP1

Drug experiments

Statistical analysis

Results

Clinical sample

BRAFV600E regulates malignant biological behaviors and glucose metabolism reprogramming in PTC

BRAFV600E regulates mitochondrial fission, ROS production, and apoptosis in PTC

BRAFV600E/p-ERK/p-DRP1(Ser616) signaling pathway in PTC

DRP1 promotes the progression of BRAFV600E-muted PTC

BRAFV600E/p-ERK/p-DRP1(Ser616) pathway regulates mitochondrial fission and glucose metabolism reprogramming in PTC

BRAFV600E/p-ERK/p-DRP1(Ser616) signaling pathway regulates ROS and apoptosis in PTC

Agent experiments targeting BRAFV600E and glycolysis

Discussion

Conclusion

Footnotes

Authors’ Contributions

Author Disclosure Statement

Funding Information

Supplementary Material

References

BRAF^V600E regulates malignant biological behaviors and glucose metabolism reprogramming in PTC

BRAF^V600E/p-ERK/p-DRP1(Ser616) signaling pathway in PTC

DRP1 promotes the progression of BRAF^V600E-muted PTC

BRAF^V600E/p-ERK/p-DRP1(Ser616) pathway regulates mitochondrial fission and glucose metabolism reprogramming in PTC

BRAF^V600E/p-ERK/p-DRP1(Ser616) signaling pathway regulates ROS and apoptosis in PTC

Agent experiments targeting BRAF^V600E and glycolysis