Abstract
Background/Aim: Expression of sterol regulatory element-binding protein 1 (SREBP1) is upregulated in thyroid cancer and associated with shorter disease-specific survival. The molecular regulatory mechanisms governing SREBP1 over-expression in thyroid cancer are still unclear. Materials and Methods: Thyroid cancer cell lines BHT-101 (with the BRAF V600E mutation) and FTC-131 (wild-type for BRAF) were treated with specific inhibitors. The expression of SREBP1 was determined at the mRNA level using quantitative real-time PCR and at the protein level using immunoblotting. Results: Lenvatinib and a MEK inhibitor, selumetinib, suppressed SREBP1 expression in BHT-101 but not FTC-133 cells. Olitigaltin, a galectin-3 inhibitor, decreased SREBP1 expression in a time- and dose-dependent manner in both cells. MK2206, an allosteric AKT inhibitor, did not change SREBP1 expression in either cell line. Conclusion: The galectin-3 inhibitor attenuates SREBP1 expression in thyroid cancer cells, likely independent of AKT phosphorylation. Lenvatinib and selumetinib decreases SREBP1 expression in the BRAF-mutant cell line BHT-101.
Alterations in lipid metabolism occur in multiple types of cancer (1). Activation of de novo lipogenesis and cholesterogenesis provides cancer cells bulk availability for glycerophospholipids, which are important for redox homeostasis and membrane composition. Dysregulation of lipid metabolism not only sustains tumor growth but also promotes cell invasion and angiogenesis. Furthermore, changes in fatty acid metabolism are implicated in acquired treatment resistance (2). Although the majority of thyroid cancers are well-differentiated, with excellent outcomes, metabolomics and lipidomics analyses indicate differential metabolite content in malignant thyroid tissue, particularly regarding cholesterol, choline, and choline-containing lipids (3). The diagnostic and prognostic significance of aberrant lipid metabolism in thyroid cancer remains to be further defined.
Cholesterol and fatty acid biosynthesis is transcriptionally regulated by sterol regulatory element-binding proteins (SREBPs). The SREBP family includes three transcription factors: SREBF1a and SREBF1c, which are derived from the SREBF1 gene by alternative splicing, and SREBF2, which is encoded by the SREBF2 gene (4). Recently, we demonstrated that SREBP1 expression is significantly upregulated in invasive thyroid cancer and that higher tumoral SREBP1 expression was associated with shorter disease-specific survival in patients with differentiated thyroid cancer (5). However, the molecular regulatory mechanisms governing SREBP1 over-expression in thyroid cancer are still unclear. In the present study, we aimed to investigate whether different inhibitors might be able to modulate SREBP1 expression at the transcriptional and translational levels.
Materials and Methods
Cell cultures. The human thyroid cancer cell line BHT-101 was obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Braunschweig, Germany (6). The human thyroid cancer cell line FTC-133 was purchased from the European Collection of Authenticated Cell Cultures (ECACC), Salisbury, UK (7). The BHT-101, but not the FTC-133 cell line, carries the BRAF c.1799T>A (p.Val600Glu) mutation (8, 9). Genotyping of the cell lines in our laboratory was verified by Sanger Sequencing at Mission Biotech, Taipei, Taiwan. BHT-101 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA), whereas FTC-133 cells were cultured in DMEM/F-12 Medium (Gibco) supplemented with 10% FBS. Cells were maintained at 37°C in a humidified incubator with 5% CO2. Cell lines were routinely checked for mycoplasma contamination using the EZ-PCR Mycoplasma Detection Kit (Biological Industries, Sartorius AG, Kibbutz Beit-HaEmek, Israel).
Reagents. Lenvatinib (catalog number S1164), selumetinib (catalog number S1008), and MK2206 (catalog number S1078) were purchased from Selleck Chemicals, Houston, TX, USA. Olitigaltin (TD139, catalog number B2266) was purchased from BioVision, Milpitas, CA, USA. Dimethyl sulphoxide (DMSO) was used as the standard stock solution and vehicle control.
Quantitative real-time polymerase chain reaction (qRT-PCR). Following treatment with a specific inhibitor at 10 μM for 24 or 48 h, thyroid cancer cells were harvested. RNA was extracted with TRIzol reagent (Thermo Fisher Scientific) and reverse-transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) according to the manufacturers’ instructions. qRT-PCR reactions were performed on a 7500 Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific). The primers used in this study are listed in Table I. Cycling conditions were as follows: 1 cycle of 50°C for 2 min, an initial denaturation at 95°C for 10 min, 40 cycles of 95°C denaturation for 15 s, and 60°C annealing for 1 min. After PCR amplification, a melting curve was generated, and the expression of SREBF1a and SREBF1c was normalized against the expression of the housekeeping gene β-actin (10).
Forward and reverse primer sequences for quantitative real-time PCR.
Western blot analysis and antibodies. Following treatment with a specific inhibitor at indicated concentrations for 48 h, thyroid cancer cells were harvested. Proteins were extracted using the M-PER reagent (Thermo Fisher Scientific), and the amount of total protein was determined using a Pierce BCA Protein Assay (Thermo Fisher Scientific) (11). Protein lysate samples (30 μg/lane) were electrophoresed on 10% polyacrylamide gel and then transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked in 5% bovine serum albumin for 1 h and then incubated with primary antibodies at 4°C overnight against SREBP1 (1:500 dilution; catalog number NB600-582; Novus Biologicals, Centennial, CO, USA), phospho-ERK Thr202/Tyr204 (1:1,000; #4370; Cell Signaling Technology, Danvers, MA, USA), ERK (1:1,000; #9102; Cell Signaling), phospho-AKT Ser473 (1:1,000; #9271; Cell Signaling), AKT (1:1,000; #4691; Cell Signaling), or β-actin (1:2,500; A5441; Sigma-Aldrich, Merck KGaA, St. Louis, MO, USA), followed by incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies for 2 h at room temperature. Labeled proteins were visualized using the enhanced chemiluminescence system (Merck Millipore, Burlington, MA, USA).
Gene expression analysis. To explore the association of different gene expression levels in thyroid cancer, we used the “Correlation Analysis” module of the Gene Expression Profiling Interactive Analysis (GEPIA2) web server that analyzes gene expression from RNA sequencing data in The Cancer Genome Atlas (12). The correlation coefficient was determined by the Spearman method.
Statistical analysis. The level of significance was set to p=0.05. The data were compared using Student’s t-test and are presented as the mean±standard deviation from three independent experiments.
Results
Effects of lenvatinib on SREBP1 expression. To investigate the effects of lenvatinib on mRNA and protein expression of SREBP1, we first determined the mRNA levels in thyroid cancer cells following treatment with 10 μM lenvatinib for 24 or 48 h. The SREBF1 gene is located on chromosome 17p11.2, and alternative splicing results in different transcript variants including SREBF1a and SREBF1c. As shown in Figure 1, lenvatinib treatment decreased the expression levels of SREBF1c in BHT-101 cells after 48 h of treatment. Consistently, immunoblotting revealed a slight reduction in SREBP1 protein expression after lenvatinib treatment in BHT-101 but not FTC-133 cells. Furthermore, ERK and AKT phosphorylation decreased after lenvatinib treatment, in agreement with the anti-angiogenetic activity of lenvatinib (13).
Effects of lenvatinib, a multi-receptor tyrosine kinase inhibitor, on mRNA and protein expression of sterol regulatory element-binding protein 1 (SREBP1) in BHT-101 and FTC-133 thyroid cancer cells. **p<0.01 versus control.
Effects of olitigaltin on SREBP1 expression. Galectin-3 is frequently over-expressed in thyroid cancer (14). Recently, we showed that galectin-3 inhibitors counteracted anoikis resistance and decreased the migratory and invasive abilities in thyroid cancer cells (15). Interestingly, olitigaltin (10 μM) reduced the expression of SREBF1a and SREBF1c transcripts in a time-dependent fashion in both BHT-101 and FTC-133 cells (Figure 2). A dose-dependent decrease in SREBP1 protein expression was also observed in both cell lines. Consistent with our previous findings (15), ERK phosphorylation increased whereas AKT phosphorylation decreased after olitigaltin treatment.
Effects of olitigaltin, a galectin-3 inhibitor, on mRNA and protein expression of sterol regulatory element-binding protein 1 (SREBP1) in BHT-101 and FTC-133 thyroid cancer cells. *p<0.05, **p<0.01 versus controls.
Effects of selumetinib on SREBP1 expression. The expression of SREBP1 decreased in BHT-101 cells following treatment with either lenvatinib or olitigaltin. However, lenvatinib decreased and olitigaltin increased ERK phosphorylation. To further characterize the role of the mitogen-activated protein kinase (MAPK) pathway in the regulation of SREBP1 expression, thyroid cancer cells were treated with a MEK inhibitor, selumetinib. Of interest, mRNA and protein expression of SREBP1 decreased after selumetinib treatment in BHT-101 but not FTC-133 cells (Figure 3). Selumetinib effectively abolished ERK phosphorylation in both cell lines, but the expression of ERK phosphorylation was remarkably higher in BHT-101 cells than in FTC-133 cells. These findings suggest that SREBP1 expression is susceptible to MAPK inhibition only in BRAF-mutant thyroid cancer cells.
Effects of selumetinib, a MEK inhibitor, on mRNA and protein expression of sterol regulatory element-binding protein 1 (SREBP1) in BHT-101 and FTC-133 thyroid cancer cells. *p<0.05, **p<0.01, ***p<0.001 versus controls.
Effects of MK2206 on SREBP1 expression. Both lenvatinib and olitigaltin decreased AKT phosphorylation. To gain a better understanding of the role of AKT in the regulation of SREBP1 expression, thyroid cancer cells were treated with a pan-AKT inhibitor, MK2206. As shown in Figure 4, the AKT inhibitor did not change either the mRNA or protein expression of SREBP1. These results suggest that attenuation of SREBP1 expression by the galectin-3 inhibitor is probably independent of AKT phosphorylation.
Effects of MK2206, an allosteric AKT inhibitor, on mRNA and protein expression of sterol regulatory element-binding protein 1 (SREBP1) in BHT-101 and FTC-133 thyroid cancer cells.
Positive correlation between SREBP1 and galectin-3 expression. Considering that the galectin-3 inhibitor reduced SREBP1 expression irrespective of BRAF mutation status, we hypothesized that there was an association between SREBP1 and galectin-3 expression. As shown in Figure 5, the expression of SREBP1 and galectin-3 in thyroid cancer samples from The Cancer Genome Atlas showed a strong positive correlation (p<0.001). The correlation did not exist in matched normal thyroid samples (p=0.26).
Correlation between mRNA expression of SREBF1 and LGALS3 in papillary thyroid cancer using RNA sequencing data from The Cancer Genome Atlas.
Discussion
Most well-differentiated thyroid cancers can be successfully treated with surgery with or without radioactive iodine therapy. However, for advanced thyroid cancer refractory to standard treatment, multikinase and selective inhibitors have improved progression-free survival in clinical trials and real-world studies (16). Lenvatinib is an antiangiogenic tyrosine kinase inhibitor that targets VEGFR1-3 and other prooncogenic receptor tyrosine kinases, including FGFR1-4, PDGFR, KIT, and RET. Lenvatinib is currently the preferred first-line systemic treatment in patients with radioiodine-refractory thyroid cancer (17). In this regard, we examined the effect of lenvatinib on SREBP1 expression in thyroid cancer cells. Intriguingly, we found that lenvatinib attenuated SREBP1 expression exclusively in BHT-101 cells. Similar results were observed for selumetinib treatment in the present study. Our data suggest that MAPK inhibition may attenuate SREBP1 expression in BRAF-mutant thyroid cancer cells.
The BRAF mutation is the most frequent oncogenic driver for thyroid cancer (18). In a multicenter retrospective study, the presence of the BRAF V600E mutation was associated with increased cancer-related mortality among patients with papillary thyroid cancer (19). SREBP1-dependent lipogenesis may result in the acquisition of resistance to BRAF-targeted therapy (20). However, whether oncogenic BRAF signaling regulates SREBP1 expression and the corresponding lipogenic phenotype in thyroid cancer has not been well studied. A recent study revealed that the BRAF mutation in thyroid cancer was associated with the down-regulation of acetyl-CoA carboxylase and dysregulation of lipid synthesis (21). In the present study, our findings seem contrary to previous research in malignant melanoma cells that indicated an upregulation in gene expression of de novo fatty acid biosynthesis following MAPK inhibition (22).
In melanoma, it was proposed that AKT activation following MAPK inhibition might contribute to upregulation of de novo fatty acid biosynthesis (22). Previous studies have suggested that AKT activation induces SREBP1 synthesis and expression of enzymes involved in fatty acid and cholesterol synthesis (23, 24). However, we did not substantiate these results in thyroid cancer cell lines. Treatment with MK2206 modulated neither mRNA nor protein expression of SREBP1 in the present study. Additionally, lenvatinib decreased AKT phosphorylation but did not change SREBP1 expression. This unexpected finding suggests that regulation of SREBP1 expression might depend on different cellular contexts. Further investigation into possible mechanisms for this discrepancy is needed.
Galectin-3 plays a role in numerous cellular functions including proliferation, apoptosis, adhesion, and cell-cell as well as cell-matrix interactions. Over-expression of galectin-3 is correlated with neoplastic progression and metastatic spread in some cancers (25). In addition to its diagnostic application, galectin-3 inhibition could be exploited as a potential treatment for thyroid cancer. In this study, olitigaltin treatment consistently decreased SREBP1 expression. Moreover, bioinformatics analysis suggested a high correlation between SREBP1 and galectin-3 expression in papillary thyroid cancer. The link between galectin-3 and SREBP1 has been unclear thus far. In agreement with previous reports (15, 26), olitigaltin attenuated AKT phosphorylation. However, we found that the AKT inhibitor, MK2206, did not change SREBP1 expression in thyroid cancer cells. This discrepancy suggests that olitigaltin suppressed SREBP1 expression independent of AKT activation. In animal studies, galectin-3 inhibition ameliorated cardiac lipotoxicity and fatty liver induced by a high-fat diet (27, 28). How cellular galectin-3 regulates SREBP1 expression is certainly a fascinating subject for future studies.
In summary, we demonstrated for the first time that a galectin-3 inhibitor, olitigaltin, attenuates SREBP1 expression in thyroid cancer cells. Lenvatinib or the MEK inhibitor selumetinib decreases SREBP1 expression only in the BRAF-mutant cell line.
Acknowledgements
This work was supported by research grants from the Ministry of Science and Technology of Taiwan (MOST-110-2314-B-195-018-MY3) and MacKay Memorial Hospital (MMH-11116 and MMH-E-111-08).
Footnotes
Authors’ Contributions
Huang TS and Cheng SP conceived the study, designed and carried out the experiments, and wrote the manuscript. Lee JJ helped in the conception of the study and critiqued the manuscript. Huang SY assisted in carrying out the experiments and critiqued the manuscript.
Conflicts of Interest
The Authors declare no competing interests in relation to this study.
- Received March 6, 2022.
- Revision received March 23, 2022.
- Accepted March 24, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
