Title : Phosphate analog PPF inhibits papillary thyroid cancer by disrupting phosphate balance and reprogramming tumor metabolism
Abstract:
Background: The global incidence of papillary thyroid cancer (PTC) has been increasing in recent years, placing greater demands on healthcare systems worldwide. While standard treatments such as surgery combined with radioactive iodine therapy and chemoradiotherapy are common, many patients still experience refractory or progressing disease due to the large number affected. Additionally, some patients are unable to tolerate surgery or the side effects of radiotherapy and chemotherapy, which can lead to treatment delays and poor outcomes. This highlights an urgent need for new, safe, and effective therapies for PTC. Our earlier research indicated that the inorganic phosphate (Pi) transporter XPR1 promotes PTC growth and progression, suggesting that disrupted phosphate regulation could be a key factor in the metabolic abnormalities and malignancy of thyroid cancer. Based on these findings, this study investigates an innovative strategy targeting tumor phosphate metabolism to develop metabolism-based treatments for PTC.
Methods: By examining the three-dimensional structure of the phosphate transporter XPR1, we used molecular dynamics simulations and structure-based screening to find small-molecule compounds that can specifically bind to the XPR1-mediated inorganic Pi transport channel. Importantly, this study is the first to suggest repurposing an FDA-approved phosphate analog, PPF, as a possible therapeutic for PTC. To thoroughly investigate the antitumor effects and mechanisms of the phosphate analog PPF, various experimental approaches were employed. These included CCK-8 cell viability tests, EdU proliferation assays, colony formation assays, wound healing tests, transwell invasion studies, transcriptomic sequencing, metabolomic profiling, and assessment of intracellular free Pi levels. Together, these methods aimed to uncover the molecular basis of PPF's effects and evaluate its potential for clinical use.
Results: CCK-8 assays demonstrated that treating TPC-1 cells with PPF at concentrations of 0.5 mM, 1 mM, 2 mM, 3 mM, and 4 mM for 48 hours significantly reduced cell viability (all p < 0.0001). Colony formation tests showed that 48-hour exposure to 1 mM and 2 mM PPF substantially decreased the clonogenic ability of TPC-1 cells (1 mM: p = 0.0042; 2 mM: p = 0.0002). Similarly, EdU incorporation assays indicated that 1 mM and 2 mM PPF treatments markedly inhibited DNA synthesis and cell proliferation (1 mM: p = 0.0072; 2 mM: p < 0.0001). Wound healing assays also revealed a significant reduction in cell migration after 48 hours of PPF treatment (1 mM: p = 0.0006; 2 mM: p < 0.0001). Transwell assays confirmed PPF's dose-dependent inhibition of TPC-1 cell invasion. Transcriptomic analysis showed that PPF caused significant alterations in genes involved in phosphate homeostasis, including SLC20A1, SLC25A3, and XPR1. Additionally, genes related to nucleoside, purine, and pyrimidine transport (SLC29A3 and SLC29A4) were notably downregulated, while those associated with ribosomal protein synthesis, such as RPS27L, exhibited significant dysregulation. Metabolomic profiling revealed that differentially expressed metabolites were mainly linked to nucleoside, purine, pyrimidine transport, amino acid metabolism, and aminoacyl-tRNA biosynthesis, consistent with transcriptomic findings. Furthermore, intracellular Pi measurements showed a significant decrease in phosphate levels in TPC-1 cells following PPF treatment (p = 0.0055).
Conclusions: PPF exerts antitumor effects by inhibiting proliferation, invasion, and metastasis in TPC-1 cells. Moreover, this study presents an innovative antitumor strategy that targets tumor metabolism to treat PTC. More importantly, the findings indicate that phosphate analogs, such as PPF, can modulate intracellular Pi levels and alter Pi-dependent metabolic pathways, particularly those related to nucleotide and amino acid synthesis. These metabolic shifts interfere with the production of essential cellular proteins, resulting in antitumor effects of PPF.
