A role of sphingosine kinase 1 in head and neck carcinogenesis

Abstract

It is important to identify novel and effective targets for cancer prevention and therapy against head and neck squamous cell carcinoma (HNSCC), one of the most lethal cancers. Accumulating evidence suggests that the bioactive sphingolipids, such as sphingosine-1-phosphate (S1P) and its generating enzyme, sphingosine kinase 1 (SphK1) play pivotal roles in several important biological functions including promoting tumor growth and carcinogenesis. However, roles of SphK1/S1P in HNSCC development and/or progression have not been defined previously. Therefore, in this study, we first analyzed the expression of SphK1 in human HNSCC tumor samples and normal head & neck tissues (n = 78 and 17, respectively) using immunohistochemistry. The data showed that SphK1 is overexpressed in all of the HNSCC tumors tested (stages I-IV). We next investigated whether SphK1 is necessary for HNSCC development. To define the role of SphK1/S1P in HNSCC development, we utilized 4-nitroquinoline-1-oxide (4-NQO)-induced HNSCC model in wild-type mice compared with SphK1(-/-) knockout (KO) mice. Remarkably, we found that the genetic loss of SphK1, which reduced S1P generation, significantly prevented 4-NQO-induced HNSCC carcinogenesis, with decreased tumor incidence, multiplicity, and volume when compared with controls. Moreover, our data indicated that prevention of 4-NQO-induced HNSCC development in SphK1(-/-) KO mice might be associated with decreased cell proliferation, increased levels of cleaved (active) caspase 3, and downregulation of phospho (active) AKT expression. Thus, these novel data suggest that SphK1/S1P signaling may play important roles in HNSCC carcinogenesis, and that targeting SphK1/S1P might provide a novel strategy for chemoprevention and treatment against HNSCC.

Fig. 1

Expression of SphK1 protein in human head & neck tissues using immunohistochemistry. Applied tissues include normal mucosa, normal mucosa adjacent to HNSCC, and HNSCC stages I ~ IV. A. Expression levels of SphK1 in all stages of HNSCC are significantly higher than both normal mucosa and normal mucosa adjacent HNSCC (*P<0.0001). B and C. Representative photos of immunohistochemistry of SphK1 in HNSCC (B. original magnification x20, bar=50μm ; C. original magnification x40, bar=20μm).

Fig. 2

4-NQO-treated HNSCC animal model. A. A protocol for HNSCC induced by 4-NQO in drinking water. The mice were treated with 4-NQO at 40 μg/ml in drinking water for 16 weeks starting at 6 weeks of age and then observed for another 8 weeks with normal drinking water. The tongues were taken out when the animals were sacrificed. B. Macroscopic findings of tongue tumors induced by 4-NQO in wild type (upper) and SphK1 KO (lower) mice. Wild type mice showed bigger, harder, more number of tumors on their tongues than SphK1 KO mice. Tongue tumors and normal tissues were fixed in 10% buffered formalin and embedded in paraffin blocks for histological evaluation with H&E staining and SphK1 immunohistochemistry. C. Microscopic findings of tongue tumors including dysplastic lesion (a) and SCC (b) induced by 4-NQO in wild type mice (H&E staining and SphK1 immunohistochemistry, original magnification x20). Bars indicate 0.1mm.

Fig. 3

Sphingolipid profile in blood samples from SphK1 KO and wild type mice. The mice were treated with 4-NQO at 40 μg/ml in drinking water for 16 weeks and observed for another 8 weeks with normal drinking water. Blood samples were collected via heart puncture after complete euthanasia. Levels of sphingolipids were analyzed by sphingolipid profiling using a tandem mass spectrometry with positive mode electrospray ionization in the Lipidomics Core Facility, MUSC. Levels of S1P (A), Sphingosine (B), total ceramide (C), and C₁₆-ceramide (D) in blood samples from SphK1 and wild type mice. Levels of S1P (**P<0.001), Sphingosine (**P<0.001), and C₁₆-ceramide (*P<0.05) are significantly decreased in blood from SphK1 KO mice as compared to those in wild type mice.

Fig. 4

Cell proliferation and apoptosis in HNSCC induced by 4-NQO in SphK1 KO and wild type mice. The mice were treated with 4-NQO at 40 μg/ml in drinking water for 16 weeks and observed for another 8 weeks with normal drinking water. All animals received BrdU at 50 mg/kg body weight ip injection 1 hour prior sacrifice. All tongue tumors and normal tongue tissues were fixed in 10% buffered formalin and embedded in paraffin blocks. A. Cell proliferation assay by BrdU immunohistochemistry and representative photos in SphK1 KO and wild type mice. Number of S-phase cells in tumors developed in SphK1 KO mice was significantly decreased as compared to that in wild type mice (*P<0.05). B. Apoptosis assay by cleaved caspase-3 immunohistochemistry and representative photos in SphK1 KO and wild type mice. Number of cleaved caspase-3 positive cells, apoptotic cells, in tumors developed in SphK1 KO mice was significantly increased as compared to that in wild type mice (**P<0.01). Original magnification x40 and bars indicate 0.02 mm.

Fig. 5

Phospho-Akt immunohistochemistry in HNSCC induced by 4-NQO in wild type and SphK1 KO mice (original magnification x20 and bars indicate 0.1 mm). The mice were treated with 4-NQO at 40 μg/ml in drinking water for 16 weeks and observed for another 8 weeks with normal drinking water. After complete autopsy, all tongue tumors and normal tissues were fixed in 10% buffered formalin and embedded in paraffin blocks. Tumor sections were stained with phospho-Akt (Ser⁴⁷³) antibody as described in “Materials and Methods.” Tumors developed in SphK1 KO mice showed slightly lower expression of phospho-Akt than those in wild type mice.