Sphingosine kinase 1 is overexpressed and promotes adrenocortical carcinoma progression

Abstract

Adrenocortical carcinoma (ACC) is a rare endocrine tumor with a very poor prognosis. Sphingosine kinase 1 (SphK1), an oncogenic kinase, has previously been found to be upregulated in various cancers. However, the role of the SphK1 in ACC has not been investigated. In this study, SphK1 mRNA and protein expression levels as well as clinicopathological significance were evaluated in ACC samples. In vitro siRNA knockdown of SphK1 in two ACC cell lines (H295R and SW13) was used to determine its effect on cellular proliferation and invasion. In addition, we further evaluated the effect of SphK1 antagonist fingolimod (FTY720) in ACC in vitro and in vivo, as a single agent or in combination with mitotane, and attempted to explore its anticarcinogenic mechanisms. Our results show a significant over-expression of SphK1 mRNA and protein expression in the carcinomas compared with adenomas (P < 0.01 for all comparisons). Functionally, knockdown of SphK1 gene expression in ACC cell lines significantly decreased cell proliferation and invasion. FTY720 could result in a decreased cell proliferation and induction of apoptosis, and the combination of mitotane and FTY720 resulted in a greater anti-proliferative effect over single agent treatment in SW13 cells. Furthermore, FTY720 could markedly inhibit tumor growth in ACC xenografts. SphK1 expression is functionally associated to cellular proliferation, apoptosis, invasion and mitotane sensitivity of ACC. Our data suggest that SphK1 might be a potential therapeutic target for the treatment of ACC.

Keywords: FTY720; adrenocortical carcinoma; prognosis; progression; sphingosine kinase 1.

Figure 1. Expression of Sphk1 is elevated in ACCs

(A) SphK1 mRNA expression analyzed by real-time RT-PCR in ACA and ACC tissues. SphK1 mRNA expression levels were normalized to GAPDH mRNA expression. SphK1 mRNA was determined to be at least three folds higher in the ACC tissues compared to ACA tissues ***P < 0.001; (B) Western blot analysis of SphK1 and sphingosine-1-phosphate Receptor 1 (S1P1R) protein in primary ACA and ACC tissues. Expression levels were normalized with β-actin; (C) Representative images from SphK1 immunohistochemistry in ACA and ACC tissues (20 × magnification). a) The expression of SphK1 was negative in ACA tissues; b) The expression of SphK1 was weakly positive staining in ACC with ENSAT 1; c) The expression of SphK1 was moderate positive staining in ACC with ENSAT 2; d) The expression of SphK1 was strong positive staining in ACC with ENSAT 3; (D) The association between SphK1 expression levels in tumors and overall survival in ACC patients using IHC score. Patients with high expression of SphK1 display a significant shorter overall survival after surgery. Kaplan-Meier method and log-rank test were used to evaluate overall survival and compare the differences between the two groups, P = 0.03.

Figure 2. Depletion of SphK1 abrogates the proliferation of ACC cells

(A) Knockdown of SphK1 in H295R and SW13 cell lines analyzed by real-time RT-PCR, SphK1 mRNA expression was normalized to GAPDH mRNA expression; (B) Knockdown of SphK1 in H295R and SW13 cell lines analyzed by western blot analysis, and β-actin was used as a loading control for western blot assays; (C) SphK1 enzymatic activity in SphK1-knocked down ACC cells was significantly decreased; (D) MTT assay was performed to evaluate the SphK1 on the proliferation of H295R at indicated time points; (E) MTT assay was performed to evaluate the SphK1 on the proliferation of SW13 at indicated time points.

Figure 3. Inhibition of SphK1 surpresses the invasion and proliferation of ACC cells

(A) Knockdown of SphK1 reduced invasion in H295R cell line; (B) Knockdown of SphK1 reduced invasion in SW13 cell line; (C) ACC cell lines were treated at the indicated concentrations for different time points, and the cell viability was analyzed by MTT assay. FTY720 induced cell death in H295R cells in a dose and time-dependent manner. (D) FTY720 induced cell death in SW13 cells in a dose and time-dependent manner; (E) FTY720 at a concentration of 10 μM significantly decreased SphK1 enzymatic activity in H295R cells; (F) FTY720 at a concentration of 10 μM significantly decreased SphK1 enzymatic activity in SW13 cells.

Figure 4. Effect of FTY720 on the activities of major signaling pathways, and effect of FTY720/mitotane combination on the proliferation of ACC cells

(A) The antibodies against PI3K, phospho-PI3K^Y607 (p-PI3K), AKT, phospho-AKT^S473 (p-AKT), ERK, phospho-ERK ^{pT202/pY204 + pT185/pY187} (p-ERK) were used to determine the effect of FTY720 on the activities of PI3K/Akt and MAPK signaling. Western blotting analysis revealed that FTY720 significantly reduced the levels of p-PI3K, p-AKT and p-ERK in a dose dependently manner in H295R cells; (B) Western blotting analysis revealed that FTY720 significantly reduced the levels of p-PI3K, p-AKT and p-ERK in a dose dependently manner in SW13 cells; (C) Cell viability after single or combined mitotane and FTY720 treatment. Cytotoxic response to mitotane and the combination of mitotane and FTY720 in H295R and SW13 cell lines.; (D) The combination of mitotane and FTY720 showed a significant synergistic anti-proliferative effect (CI = 0.90 ± 0.08) in SW13 cells that potentiated the cytotoxic effect observed by using FTY720 alone.

Figure 5. Induction of apoptosis by FTY720 in ACC cells

Flow cytometry results of annexin V-PI stained ACC cells after exposure to different concentration of FTY720 for 24 h. (A) An increase in apoptotic cells following treatment with FTY720 is shown in H295R cells; (B) An increase in apoptotic cells following treatment with FTY720 is shown in SW13 cells; (C) The combination of FTY720 and mitotane had a significant influence on the apoptotic rate in SW13 cells, but there is no effect of the combination of FTY720 and mitotane on the apoptosis of H295R; (D) The flow cytometry profile represents SW13 cells with annexin V-FITC staining on the x-axis and PI on the y-axis. Dual staining of cells with annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) FITC and PI enabled categorization of cells into 4 regions. Region Q1 shows dead cells, Q2 shows late apoptotic cells, Q3 shows early apoptotic cells, and Q4 shows living cells. Q2 and Q3 are collectively called apoptotic cells.

Figure 6. Inhibition of xenograft tumor growth by FTY720

(A) Time course of tumor growth, measured as tumor volume in each group at the indicated time of treatment with vehicle control or FTY720 (10 mg/kg; ip.). Data are presented as mean ± SD; (B) Representative picture of tumor growth in mice treated with the indicated concentration of FTY720 and vehicle control; (C) The bar graph represents mean of the tumor weight from FTY720-treated and control mice. Data are presented as mean ± SD. **, P < 0.01; (D) Compared with control group, the mean body weight was not significantly change in FTY720-treated group.