Combined BRAF and PIM1 inhibitory therapy for papillary thyroid carcinoma based on BRAFV600E regulation of PIM1: Synergistic effect and metabolic mechanisms

Abstract

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, and its incidence has increased rapidly in recent years. The BRAF inhibitor vemurafenib is effective against BRAFV600E-positive PTC; however, acquired resistance to single agent therapy frequently leads to tumor recurrence and metastasis, underscoring the need to develop tailored treatment strategies. We previously showed that the oncogenic kinase PIM1 was associated with the malignant phenotype and prognosis of PTC. In this study, we showed that sustained expression of the PIM1 protein in PTC was affected by the BRAFV600E mutation. Based on this regulatory mechanism, we tested the synergistic effects of inhibitors of BRAF (BRAFi) and PIM1 in BRAFV600E-positive PTC cell lines and xenograft tumors. LC-MS metabolomics analyses suggested that BRAFi/PIMi therapy acted by restricting the amounts of critical amino acids and nucleotides required by cancer cells as well as modulating DNA methylation. This study elucidates the role of BRAFV600E in the regulation of PIM1 in PTC and demonstrates the synergistic effect of a novel combination, BRAFi/PIMi, for the treatment of PTC. This discovery, along with the pathways that may be involved in the powerful efficacy of BRAFi/PIMi strategy from the perspective of cell metabolism, provides insight into the molecular basis of PTC progression and offers new perspectives for BRAF-resistant PTC treatment.

Keywords: BRAFV600E; Metabolomics; PIM1; Synergism; Thyroid carcinoma.

Fig. 1

The BRAFV600E oncoprotein regulates PIM1 expression and increases its stability in thyroid cancer (TC). A Mutations in the top 10 genes in PTC patients with high and low PIM1 expression. B Differential analysis of PIM1 expression in 470 thyroid papillary carcinoma tissues with BRAF WT and BRAFV600E mutation. TPM: reads per million mapped reads. C, D Expression of PIM1 in thyroid papillary carcinoma (PTC) and anaplastic cancer (ATC) with or without the BRAFV600E mutation. E, F Immunoblot of PIM1 protein from extracts of TPC-1 transfected with BRAFV600E or BRAF WT plasmid. G, H Expression level of PIM1 in TCs treated with 10 μM vemurafenib or DMSO for 48 h. I, J BRAFV600E PTC cells were treated with vemurafenib, MG-132, or a combination for 6 h. Lysates were harvested for western blotting. K, L Half-life analysis of PIM1 in PTC treated with vemurafenib. PTC cells carrying the BRAFV600E mutation in the control or vemurafenib group were treated with 100 μg/mL cycloheximide (CHX) and collected at the indicated times, followed by western blot analysis of PIM1 protein expression. Experiments were performed three times. (mean ± SD, N = 3) (*P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001, Student's t-test). M Quantitative data of the BRAF WT or V600E mutant PTC tissues and the corresponding analyses, which were performed by measuring the optical density (IOD) and all area with Image J and calculation of the average optical density values (IOD/area).

Fig. 2

Correlation between BRAF mutation and PIM1 level in PTC patients. D Representative images (100 ×, 400 ×) of HE and IHC staining of PIM1 protein level from PTC patients harboring BRAF WT (A and B) and BRAFV600E mutation (C and D).

Fig. 3

Synergistic effects of SGI-1776 and vemurafenib in BRAFV600E mutant PTC cells and a xenograft model. A-C BCPAP and KTC-1 cells were treated with different concentrations of vemurafenib and SGI-1776 alone or in combination for the indicated time points. The resulting cells were fixed and stained, and cell colony numbers were counted. (mean ± SD, N = 3) (*P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001, Student's t-test). D, E Combination effect curves and coefficient of drug in interaction (CI) of BCPAP and KTC-1 cells treated with vemurafenib and SGI-1776 alone or in combination for 48 h. F-I Subcutaneous xenografts generated by injecting BCPAP cells were treated with SGI-1776 (25 mg/kg), vemurafenib (20 mg/kg), or both drugs for 18 days. Tumor size and weight were monitored (mean ± SD, N = 5 mice per group) (*P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001, ANOVA test) (F, G and I). Change in body weight of nude mice in each group. (mean ± SD, N = 5 mice per group)(P > 0.05, ANOVA test) (H).

Fig. 4

Metabolomics analysis shows differences among control, vemurafenib, SGI-1776, and combination groups. A Score plots of QA-PCA models. Each point represents a sample; pink: control, red: QC, green: vemurafenib, blue: SGI-1776, purple: vemurafenib and SGI-1776. B Score plots of OPLS-DA models. Each point represents a sample, red: control, green: vemurafenib, blue: SGI-1776, purple: combination. C Permutation test results.

Fig. 5

Metabolite changes as a result of treatment with vemurafenib, SGI-1776 and combination. A Venn diagrams comparing the drug responsive metabolites identified in PTC in the indicated comparisons. B Differential expression of the metabolites in Table 1 between the vemurafenib, SGI-1776, and combination groups. *P < 0.5^⁎⁎P < 0.01^⁎⁎⁎P < 0.001^⁎⁎⁎⁎P < 0.0001 C-E Visualization of results from Joint pathway enrichment analyses (P < 0.05, impact > 0.1), using MetaboAnalyst, of the metabolites that were significantly altered in PTC cells treated with (C) vemurafenib, (D) SGI-1776, and (E) combination. The x-axis represents the pathway impact score, which is the relative degree to which the overall pathway is expected to be altered given the relative (positional) importance of altered metabolites in the pathway. The y-axis represents the -log₁₀-transformed P values for pathway enrichment. Nodes are colored according to P value and sized according to pathway impact score.