Combating head and neck cancer metastases by targeting Src using multifunctional nanoparticle-based saracatinib

Abstract

Background: Inhibition of metastasis of head and neck squamous cell carcinoma (HNSCC) is one of the most important challenges in cancer treatment. Src, a non-receptor tyrosine kinase, has been implicated as a key promoter in tumor progression and metastasis of HNSCC. However, Src therapy for HNSCC is limited by lack of efficient in vivo delivery and underlying mechanisms remain elusive.

Methods: Src knockdown cells were achieved by lentiviral-mediated interference. Cell migration and invasion were examined by wound healing and Transwell assays. Protein levels were determined by Western blot and/or immunohistochemistry. The Src inhibitor saracatinib was loaded into self-assembling nanoparticles by the solvent evaporation method. An experimental metastasis mouse model was generated to investigate the drug efficacy in metastasis.

Results: Blockade of Src kinase activity by saracatinib effectively suppressed invasion and metastasis of HNSCC. Mechanistic assessment of the drug effects in HNSCC cells showed that saracatinib induced suppression of Src-dependent invasion/metastasis through downregulating the expression levels of Vimentin and Snail proteins. In tests in mice, saracatinib loaded into the novel multifunctional nanoparticles exhibited superior effects on suppression of HNSCC metastasis compared with the free drug, which is mainly attributed to highly specific and efficient tumor-targeted drug delivery system.

Conclusions: These findings and advances are of great importance to the development of Src-targeted nanomedicine as a more effective therapy for metastatic HNSCC.

Keywords: HNSCC; Metastasis; Nanoparticles; Saracatinib; Src.

Fig. 1

Saracatinib effectively inhibits Src phospho-activation and migration in HNSCC cells. a The effects of shRNAs against Src on the expression of Src protein. b The effects of Src knockdown on cell migration within 24 h. c The effects of saracatinib and dasatinib on the phosphorylation levels of Src. d The effects of saracatinib and dasatinib on cell migration within 24 h. b, d The representative images and quantitative data were shown in the left and right panels, respectively. *p < 0.05; **p < 0.01

Fig. 2

Saracatinib suppresses Src-dependent Vimentin/Snail signaling in HNSCC cells. a The effects of saracatinib and dasatinib on the expression of EMT-related proteins. b The effects of saracatinib and dasatinib on the expression of cell tight junction-related proteins. c The effects of Src knockdown on the expression of Vimentin, E-cadherin, and Snail proteins. d Quantification of relative protein levels of Vimentin and Snail among the different treatments from three independent experiments

Fig. 3

The synthesis and working principle of Nano-sar. a Schematic representation of the self-assemble Nano-sar and its disassembly upon CTSB digestion. b Schematic illustration of the working principle of Nano-sar for targeting tumor cells. c Solid-phase synthesis of peptide for Ac-K(Boc)GFLG-OH as a CTSB-cleavable linker. d Chemical structure of linear-dendritic polymeric drug carrier and saracatinib. e Saracatinib-loaded formulation to form nanoscale assembly as characterized by DLS. f The drug release profile at various conditions determined by HPLC

Fig. 4

Nano-sar inhibits the Src signaling pathways, migration, and invasion in HNSCC cells. a The effects of saracatinib (Sar) and Nano-sar on Src phospho-activation and the downstream pathways. b The effects of Sar and Nano-sar on colony formation of HNSCC cells within 2 weeks. In this assay, colonies with more than 50 cells were scored and counted under the microscope. c, d The effects of Sar and Nano-sar on migration and invasion of HNSCC cells within 24 h. In b–d, the representative images and quantitative data were shown in the left and right panels, respectively. e The effects of Sar and Nano-sar on 3D invasion in Matrigel within 72 h. *p < 0.05; **p < 0.01

Fig. 5

Nano-sar has superior effects on suppression of head and neck tumor metastasis than the free drug in mice. a–c Tumor growth curve, tumor weight, and body weight for mice treated with phosphate-buffered saline vehicle control, saracatinib (Sar), or Nano-sar (n = 5/group). d Tumor progression and metastasis monitored by examining bioluminescence in Xenogen IVIS-200 In Vivo imaging system. e The number of nodules on the lung surface. f Histology examination (HE staining) of the lung sections for mice treated with saline vehicle control, Sar, or Nano-sar. g Histology examination (HE staining) of tissues taken from major organs after therapy.*p < 0.05; **p < 0.01

Fig. 6

Nano-sar suppresses Src activation and the downstream pathways more efficiently in head and neck tumors than the free drug in mice. a–c The effects of saracatinib (Sar) or Nano-sar on Src signaling pathways in head and neck tumors. The representative IHC images were shown in the left panel and quantification of IHC staining with Image pro-Plus6.0 was shown in the right panel. *p < 0.05; **p < 0.01