Antitumor effect of the novel sphingosine kinase 2 inhibitor ABC294640 is enhanced by inhibition of autophagy and by sorafenib in human cholangiocarcinoma cells

Abstract

Sphingosine kinase 2 (Sphk2) has an oncogenic role in cancer. A recently developed first-in-class Sphk2 specific inhibitor ABC294640 displays antitumor activity in many cancer models. However, the role of Sphk2 and the antitumor activity of its inhibitor ABC294640 are not known in cholangiocarcinoma. We investigated the potential of targeting Sphk2 for the treatment of cholangiocarcinoma. We found that Sphk2 is overexpressed in five established human cholangiocarcinoma cell lines (WITT, HuCCT1, EGI-1, OZ and HuH28) and a new patient-derived cholangiocarcinoma cell line (LIV27) compared to H69 normal cholangiocytes. Inhibition of Sphk2 by ABC294640 inhibited proliferation and induced caspase-dependent apoptosis. Furthermore, we found that ABC294640 inhibited STAT3 phosphorylation, one of the key signaling pathways regulating cholangiocarcinoma cell proliferation and survival. ABC294640 also induced autophagy. Inhibition of autophagy by bafilomycin A1 or chloroquine potentiated ABC294640-induced cytotoxicity and apoptosis. In addition, ABC294640 in combination with sorafenib synergistically inhibited cell proliferation of cholangiocarcinoma cells. Strong decreases in STAT3 phosphorylation were observed in WITT and HuCCT1 cells exposed to the ABC294640 and sorafenib combination. These findings provide novel evidence that Sphk2 may be a rational therapeutic target in cholangiocarcinoma. Combinations of ABC294640 with sorafenib and/or autophagy inhibitors may provide novel strategies for the treatment of cholangiocarcinoma.

Keywords: ABC294640; autophagy; cholangiocarcinoma; sorafenib; sphingosine kinase 2.

Conflict of interest statement

CONFLICTS OF INTEREST

Charles D. Smith is the Chief Executive Officer and President of Apogee Biotechnology Corporation. Apogee produced clinical-grade ABC294640 for this study. None of the other authors have formal relationships with the company or other conflicts of interest.

Figures

Figure 1. Sphk2 is overexpressed in cholangiocarcinoma cells and promotes cell proliferation

(A) Publicly available microarray dataset GSE32225 was downloaded and the Sphk2 expression levels between human normal biliary epithelial cells (n = 6) or intrahepatic cholangiocarcinoma (iCCA) (n = 149) were compared. (B) Sphk2 expression level of normal human biliary epithelial cells (n = 6) or two subclasses of iCCA (inflammation class, n = 57; proliferation class, n = 92) were compared. (C) mRNA expression of Sphk2 in human cholangiocarcinoma cell lines and the H69 normal human cholangiocyte cell line were analyzed by real-time PCR. 18S was used as the internal control.*P < 0.05; **P < 0.01; ***P < 0.001, compared with H69 cells. (D) Cells were treated with ABC294640 at different concentrations (20–100 μM) for 72 h and cell proliferation was determined by BrdU ELISA assay. (E) Plated HuCCT1 and EGI-1 cells were treated with ABC294640 at different concentrations (10–50 μM) for 7 days and colonies were stained with 0.5% crystal violet. The results are presented as mean ± SEM from at least three independent experiments.

Figure 2. ABC294640 induces apoptosis and inhibits STAT3 phosphorylation in cholangiocarcinoma cells

(A) Percentage apoptosis of cholangiocarcinoma cells treated with the indicated concentrations of ABC294640 (20–100 μM) for 72 h. Apoptosis was quantified by assessing characteristic nuclear changes of apoptosis by fluorescence microscopy after DAPI staining. The results are presented as mean ± SEM from at least three independent experiments. (B) WITT cells were treated with 20-60 μM ABC294640 for 72 h. Apoptosis was then measured by Annexin V-FITC/PI labeling followed by flow cytometry. (C) Western immunoblots for PARP and caspases 9, 8 and 3 performed on cell lysates from the same treatment as the flow cytometry assay. (D) Western immunoblots for PARP and caspases 9, 8 and 3 performed on WITT cell lysates prepared after 72 h treatment with ABC294640 in the presence or absence of the pan-caspase inhibitor, Z-VAD-FMK. (E) Cholangiocarcinoma cells were treated with 20–60 μM ABC294640 for 48 h. STAT3 Y705 phosphorylation was determined by immunoblotting.

Figure 3. ABC294640 induces autophagy in cholangiocarcinoma cells

(A) WITT, HuCCT1, EGI-1, OZ, HuH28 and LIV27 cells were treated with 20–60 μM ABC294640 for 24 h and 48 h. Autophagy was assessed by the conversion of cytosolic LC3-I into membrane-bound LC3-II by immunoblotting. (B) WITT, HuCCT1 and EGI-1 cells were treated with 40 μM ABC294640 for 24 h. LC3 detected by immunostaining was analyzed and compared with controls. Quantification of puncta is shown (lower panel). The results are presented as mean ± SEM from at least three independent experiments.*P < 0.05; **P < 0.01; ***P < 0.001. (C) EGI-1 cells were treated with 60 μM ABC294640 for 24 h and subjected to transmission electron microscopy. Representative micrographs at low magnification (left) or high magnification (right) are shown. The arrows show autophagic vacuoles.

Figure 4. ABC294640 activates autophagic flux in cholangiocarcinoma cells

(A) Cells were treated with 40 μM ABC294640 (ABC) alone or in combination with 0.5 nM bafilomycin A1 (Baf) or 15 μM chloroquine (CQ) for 48 h and cell lysates were subjected to immunoblotting. Autophagic flux results in substantial accumulation of LC3-II. (B) OZ cells were treated with ABC294640 (20–60 μM) for 24 h and 48 h. Beclin-1 expression was measured by immunoblotting.

Figure 5. Targeting autophagy potentiates ABC294640-induced cytotoxicity and apoptosis in cholangiocarcinoma cells

(A) WITT, HuCCT1, EGI-1 and OZ cells were treated with 40 μM ABC294640 (ABC) alone or in combination with 0.5 nM bafilomycin A1 (Baf), or 15 μM chloroquine (CQ) for 48 h. Cell proliferation was then determined by the BrdU ELISA assay. (B) HuCCT1 and EGI-1 cells were treated with 20 μM ABC294640 alone or in combination with 5 μM CQ for 7 days. Cell proliferation was analyzed using the colony formation assay. (C) WITT, HuCCT1, EGI-1 and OZ cells were treated with 40 μM ABC294640 alone or in combination with 0.5 nM Baf, or 15 μM CQ for 48 h. Cell apoptosis was quantified by DAPI staining. The results are presented as mean ± SEM from at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6. ABC294640 in combination with sorafenib synergistically inhibits proliferation in cholangiocarcinoma cells

(A) HuCCT1 and WITT cells were treated with different fixed-ratio combinations of ABC294640 and sorafenib and proliferation was determined by BrdU ELISA assay. The results are presented as mean ± SEM from at least three independent experiments. The combination index (CI) was determined using the Chou-Talalay Method. CI < 1 indicates that the interaction between ABC294640 and sorafenib was synergistic. (B) HuCCT1 and WITT cells were treated with 40 μM ABC294640 (ABC) and/or 6 μM sorafenib (Sor) for 24 h and whole cell lysates were then subjected to immunoblotting.

Figure 7. Schematic representation of the effect of Sphk2 specific inhibitor ABC294640 on CCA

ABC294640 inhibits proliferation, induces apoptosis and autophagy in CCA cells. Autophagy inhibitors potentiates ABC294640-induced cytotoxicity and apoptosis. ABC294640 has a synergistic effect with sorafenib in inhibiting cell proliferation.