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. 2016 Sep 13;7(37):60181-60192.
doi: 10.18632/oncotarget.11112.

Suppression of c-Myc and RRM2 expression in pancreatic cancer cells by the sphingosine kinase-2 inhibitor ABC294640

Clayton S Lewis  1 , Christina Voelkel-Johnson  2 , Charles D Smith  3
Affiliations

Suppression of c-Myc and RRM2 expression in pancreatic cancer cells by the sphingosine kinase-2 inhibitor ABC294640

Clayton S Lewis et al. Oncotarget. .

Abstract

Pancreatic cancer remains extremely difficult to treat, with the average lifespan following diagnosis being only 3-6 months, resulting in a death to incidence ratio of 0.94. A major reason for this high mortality rate is resistance to the main chemotherapeutic agent used to treat this disease, gemcitabine. Alterations in nucleoside and gemcitabine metabolism, specifically over-expression of ribonucleotide reductase, have been implicated as a major mechanism of resistance to this drug. Here, we show that inhibition of sphingosine kinase-2 by the specific inhibitor ABC294640 is synergistically cytotoxic with gemcitabine toward three human pancreatic cancer cell lines. Treatment with ABC294640 results in decreased expression of both RRM2 and MYC in all three cell lines. Additionally, expression of c-Myc protein and phosphorylation of Rb at S780 both decrease in a dose-dependent manner in response to ABC294640, while acetylation of H3-K9 and p21 levels increase. Pretreatment with the protein phosphatase 1 inhibitor okadaic acid or the ceramide synthase inhibitor fumonisin B1 fails to prevent the effects of ABC294640 on Rb phosphorylation. These data indicate a role for sphingosine kinase-2 in E2F and c-Myc mediated transcription through alteration of histone acetylation and p21 expression. These effects of ABC294640 suggest that it may be an effective agent for pancreatic cancer, particularly in combination with gemcitabine.

Keywords: c-Myc; gemcitabine; pancreatic cancer; ribonucleotide reductase; sphingosine kinase-2.

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Conflict of interest statement

CONFLICTS OF INTEREST

Charles Smith is employed by and holds equity in Apogee Biotechnology Corporation.

Figures

Figure 1
Figure 1. Cytotoxicity of ABC294640 and gemcitabine alone and in combination
Panel A. BxPC3 (●), MiaPaCa-2 (■) or Panc-1 (▲) pancreatic cell lines were exposed to gemcitabine and/or ABC294640 at the indicated concentrations for 96-hours. Cell survival was then quantified using the SRB assay. Panel B. Combination index values of the combination of ABC294640 and gemcitabine in three pancreatic cell lines were calculated using Calcusyn. All experiments were repeated three times, and values represent mean ± SEM.
Figure 2
Figure 2. Effects of ABC294640 on expression of c-Myc and RRM2
Panels A and C. BxPC-3, MiaPaCa-2 and Panc-1 cells were treated with the indicated concentration of ABC294640 for 24 hr. Cells were then harvested and analyzed for protein expression levels of c-Myc and GAPDH (Panel A) or RRM2 and GAPDH (Panel C). Western blots are representative of three independent experiments. Panels B and D. BxPC-3, MiaPaCa-2 and Panc-1 cells were treated with 30 μM ABC294640 for 24 hr. Cells were then harvested and analyzed for mRNA expression levels of MYC (Panel B) and RRM2 (Panel D) (p < 0.05*,0.005**, or 0.0001***). qRT-PCR was conducted in two independent experiments. Panel E. Untreated BxPC-3, MiaPaCa-2 and Panc-1 cell lysates were analyzed for expression of c-Myc by western blotting.
Figure 3
Figure 3. Effects of ABC294640 on proteasomal degradation of c-Myc
The indicated pancreatic cancer cells were incubated with DMSO (control) or MG-132 for 2 hr and then treated for 24 hr with 30 μM (BxPC-3) or 40 μM (MiaPaCa-2 and Panc-1) ABC294640. Cells were then harvested and analyzed for protein expression levels of c-Myc and GAPDH. Western blots are representative of two independent experiments.
Figure 4
Figure 4. Effects of ABC294640 on phosphorylation of Rb
Panel A. BxPC-3, MiaPaCa-2 and Panc-1 cells were treated with the indicated concentration of ABC294640 for 24 hr. Cells were then harvested and analyzed for protein expression levels of total Rb and pRb (S780) by western blotting. Panel B. The ratio of pRb S780 to Rb in BxPC-3, MiaPaCa-2, and Panc-1 following treatment with the indicated concentration of ABC294640 for 24 hr. Panel C. BxPC-3 cells were treated with 100 μM fumonisin B1 and/or 30 μM ABC294640 for 24 hr. Cells were then harvested and analyzed for protein expression levels of total Rb, pRb (S780), c-Myc and GAPDH by western blotting. Panel D. BxPC-3 cells were treated with the indicated concentration of OA and/or 30 μM ABC294640 for 24 hr. Cells were then harvested and analyzed for protein expression levels of pRb (S780), c-Myc and GPDH by western blotting. Western blots are representative of three independent experiments, the ratios in Panel B are derived from the blots in Panel A.
Figure 5
Figure 5. Effects of ABC294640 on H3K9ac, p21, and cell cycle
Panel A. BxPC-3, MiaPaCa-2 and Panc-1 cells were treated with the indicated concentration of ABC294640 for 24 hr. Cells were then harvested and analyzed for protein expression levels of H3-K9ac, p21 and GAPDH by western blotting. Western blots shown are representative of three independent experiments. Panel B. BxPC-3, MiaPaCa-2 and Panc-1 cells were treated with the indicated concentration of ABC294640 for 24 hr. Cells were then harvested and analyzed by flow cytometry, and the percentage of cells in G1 is indicated (p < 0.05*,0.005**, or 0.0005***). These are the combined results of three independent experiments.
Figure 6
Figure 6. Postulated mechanism of action of ABC294640
ABC294640 acts by inhibiting SphK2 in the nucleus leading to HDAC inhibition because of loss of nuclear S1P and/or elevation of nuclear sphingosine levels. This results in an increase in p21 which binds CDK/Cyclin-D1 complexes preventing the phosphorylation of Rb. The resulting hypophosphorylated Rb binds E2F preventing its transcriptional activity. It should also be noted that c-Myc inhibits the transcription of p21 and enhances the transcription of CDKs, Cyclin-D1, and E2F1, 2 and 3. The overall result is a decrease in proliferative signaling thereby resulting in inhibition of cancer growth.
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