Sphingosine kinase 1 mediates head & neck squamous cell carcinoma invasion through sphingosine 1-phosphate receptor 1
Cancer Cell International
Sphingosine kinase 1 mediates head & neck squamous cell carcinoma invasion through sphingosine 1-phosphate receptor 1
Paulette M Tamashiro 1
Hideki Furuya 1
Yoshiko Shimizu 0 1
Toshihiko Kawamori 0 1
0 Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa , Honolulu, HI 96818 , USA
1 Cancer Biology Program, University of Hawaii Cancer Center , 701 Ilalo Street, Honolulu, HI 96813 , USA
Background: Head and neck squamous cell carcinoma (HNSCC) is characterized by aggressive loco-regional invasion. Sphingosine kinase1 (SphK1), an enzyme in sphingolipid metabolism, is emerging as a key player in HNSCC pathology. The observation that SphK1 is overexpressed in all HNSCC stages and is associated with depth of tumor invasion, metastasis and clinical failure underscores the importance of SphK1 in HNSCC pathology. Still, the mechanisms underlying SphK1 regulation of invasion have not been delineated. Therefore, we sought to mechanistically describe how SphK1 regulates invasion in HNSCC. Methods: Invasion assays were used to measure invasive ability of SphK1 overexpressing human tongue squamous cell carcinoma (SCC-25 cells). Western blotting, quantitative qPCR, ELISA and zymography were used to measure the effect of SphK1 and sphingosine 1-phoshate receptor 1 (S1P1) on invasion measures, MMP-2/9, E-cadherin, EGFR, IL-6/STAT3, in SCC-25 cells. Results: SphK1 expression is elevated in cells with an invasive phenotype as compared to non-invasive phenotype. We show SphK1 overexpression increased EGF-induced EGFR/ERK and AKT activity, increased matrix metalloproteinase (MMP)-2/9 mRNA and reduced E-cadherin. SphK1 overexpression also increased IL-6 concentration and EGF-induced STAT3 phosphorylation, exemplifying that SphK1 modulates IL-6/STAT3 signaling. Notably, we show that S1P1 knockdown reduced IL-6/STAT3 signaling, representing another pathway by which SphK1/S1P regulates invasion. Conclusions: Taken together, our data suggest that SphK1 sits at the hub of multiple key signaling cascades, all which have been implicated in the regulation of invasiveness, making SphK1 an attractive target for the development of HNSCC therapies.
Sphingosine kinase1; Invasion; HNSCC; EGFR; STAT3; Sphingosine 1-phosphate; Sphingosine 1-phosphate receptor 1; IL-6; SCC-25 cells
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Background
Recurrence rates for advanced-stage head and neck
squamous cell carcinoma (HNSCC) is greater than 50%
[1] and the 5-year survival rate for HNSCC has not
drastically improved over the last 30 years [2]. The bleak
survival rate is due to late presentation, the subsequent
delay of detection of lesions, invasion into loco-regional
lymph nodes, a high rate of metastasis [2] and the limited
availability of effective therapies.
Sphingolipids play a crucial role in cancer
pathogenesis by modulating cell signal transduction pathways to
influence biological outcomes such as cell senescence,
differentiation, apoptosis, migration, and proliferation
[3-5]. Sphingosine kinase-1 (SphK1) is an important
enzyme in sphingolipid metabolism which regulates
tumor growth in HNSCC. For example, SphK1 knockdown
results in lower cell proliferation and smaller HNSCC
tumors [6], and SphK1 inhibition increases radiation
sensitivity [7].
In addition to its well-documented role in cell
proliferation, SphK1 also regulates invasion. SphK1 overexpression
is positively associated with invasion, invasive morphology
and cell diameter in esophageal squamous carcinoma cells
(ESCC) [8]. In addition, immunodeficient mice
subcutaneously injected with ESCC cells overexpressing SphK1
exhibited 6-fold greater lung metastasis compared to
parent cells [8]. In clinical HNSCC samples, human
SphK1 expression was significantly higher compared to
normal mucosa, and this was positively associated with
depth of tumor invasion, metastasis, and clinical failure
[9]. Furthermore, SphK1 negative staining was associated
with a 6.5-year survival post-surgery, while SphK1 positive
staining was associated with only a 2-year survival period
post-surgery [9].
It is not known whether SphK1 is directly involved in
activation of epidermal growth factor receptor (EGFR) in
HNSCC. However, it is known that SphK1 expression
correlates with genes downstream of the EGFR pathway
(i.e., amphiregulin, integrin5, epiregulin) in ESCC as
demonstrated with microarray analyses [8]. Also, ESCC
cells overexpressing SphK1 had greater phosphorylation
of EGF, while cells transfected with siRNA against SphK1
showed reduced EGFR phosphorylation [8]. Another
study showed treatment with EGFR alone increased
invasion (~4) in carcinoma of the epiglottis (PCI-37A)
and inhibition of EGFR inhibited invasion by 47-fold
[10]. In addition, EGFR was also shown to mediate
invasion in conjunction with signal transducer and
activator of transcription 3 (STAT3) in HNSCC [11].
Together these studies suggest that a relationship may
exist between SphK1, EGFR and STAT3 to affect invasive
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