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. 2014 Nov;1841(11):1571-80.
doi: 10.1016/j.bbalip.2014.08.017. Epub 2014 Sep 6.

Ceramide Modulates pre-mRNA Splicing to Restore the Expression of Wild-Type Tumor Suppressor p53 in Deletion-Mutant Cancer Cells

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Free PMC article

Ceramide Modulates pre-mRNA Splicing to Restore the Expression of Wild-Type Tumor Suppressor p53 in Deletion-Mutant Cancer Cells

Gauri A Patwardhan et al. Biochim Biophys Acta. .
Free PMC article

Abstract

Mutants of tumor suppressor p53 not only lose the activity in genome stabilizing and in tumor suppression, but also exhibit oncogenic function in cancer cells. Most efforts in restoring p53 biological activity focus on either altering mutant-protein conformation or introducing an exogenous p53 gene into cells to eliminate p53-mutant cancer cells. Being different from these, we report that ceramide can restore the expression of wild-type p53 and induce p53-dependent apoptosis in deletion-mutant cancer cells. We show that endogenous long-carbon chain ceramide species (C16- to C24-ceramides) and exogenous C6-ceramide, rather than other sphingolipids, restore wild-type mRNA (intact exon-5), phosphorylated protein (Ser15 in exon-5) of p53, and p53-responsive proteins, including p21 and Bax, in ovarian cancer cells, which predominantly express a deleted exon-5 of p53 mutant before treatments. Consequently, the restored p53 sensitizes these p53-mutant cancer cells to DNA damage-induced growth arrest and apoptosis. Furthermore, we elucidate that ceramide activates protein phosphatase-1, and then the dephosphorylated serine/arginine-rich splicing-factor 1 (SRSF1) is translocated to the nucleus, thus promoting pre-mRNA splicing preferentially to wild-type p53 expression. These findings disclose an unrecognized mechanism that pre-mRNA splicing dysfunction can result in p53 deletion-mutants. Ceramide through SRSF1 restores wild-type p53 expression versus deletion-mutant and leads cancer cells to apoptosis. This suggests that heterozygous deletion-mutants of p53 can be restored in posttranscriptional level by using epigenetic approaches.

Keywords: Cancer; Ceramide; Pre-mRNA splicing; Restoration; SRSF1; p53 mutant.

Figures

Fig. 1
Fig. 1
Endogenous sphingolipids and p53 expression. A) Ceramide species. NCI/ADR-RES cells were treated with MBOs (100 nM, 48 h) and extracted lipids were analyzed by LC/MS. *, p < 0.001 compared to MBO-SC. B) Sphingolipids analyzed by ESI/MS/MS analysis. DHC16-Cer, C16-dihydroceramide; DHSph, dihydrosphingosine; Sph, sphingosine; Sph-1P, sphingosine 1 phosphate. C) Ceramide and p53 expression in NCI/ADR-RES cells. Cells were treated with agents, including MBO-asGCS (100 nM), FB1 (100 μM), C6-Cer (5 μM); C6-diH-Cer (5 μM), SMase (0.5 U/ml), and PDMP (10 μM) for 48 h. RT-PCR analyses were utilized to detect the pan p53 mRNA and deleted region in exon-5. Equal amounts of cellular protein (100 μg/lane) were used for Western analyses. pp53, phosphorylated p53; pp53/p53, the ratios of optical densities of pp53 bands normalized against total p53. *, p < 0.001 compared to cells treated with vehicle and exposed to Dox.
Fig. 2
Fig. 2
Cellular ceramide and wild-type p53. NCI/ADR-RES cells were treated with agents, including MBO-asGCS (100 nM), FB1 (100 μM), C6-Cer (5 μM), C6-diH-Cer (5 μM), SMase (0.5 U/ml), and PDMP (10 μM) for 48 h and exposed to Dox. Red, Alexa Fluor 555-Cer; green, Alexa Fluor 488-pp53; blue, DAPI-nucleus. Fluorescence micrographs (x 200 magnification) were captured by EVOS imaging system.
Fig. 3
Fig. 3
Restored expression of wild-type p53 induces cell death. NCI/ADR-RES cells were pretreated with M-asGCS (MBO-asGCS, 100 nM), C6-Cer (5 μM), C6-diH-Cer (5 μM), FB1 (100 μM), and PDMP (10 μM), and then treated with Dox (2.5 μM). A) Cell viability after 72 h of treatments. B) Apoptotic cells were analyzed by flow cytometry after 48 h of treatments. Active caspase 7 levels were detected by using Western blotting. *, p < 0.001 compared to vehicle (Dox alone); **, p < 0.001 compared to M-asGCS or C6-Cer, respectively.
Fig. 4
Fig. 4
PP1 and SRSF1 are associated with p53 restoration. A) PP1 activities. NCI/ADR-RES cells were pretreated with calyculin (Cal A, 5 nM) or okadaic acid (OA 10 nM), and treated with MBO-asGCS (M-asGCS, 100 nM) or C6-Cer (5 μM) in medium containing Dox. FLU, fluorescence units. *, p < 0.001 compared to vehicle; **, p < 0.001 compared to M-asGCS or C6-Cer. B) Western blotting for Nuclear SRSF1. NCI/ADR-RES cells were treated with MBO-asGCS (0–200 nM) for 6 days and then exposed to Dox in the last 48 h. Equal amounts of nuclear protein were used for immunoblotting. Nuclear SRSF1 levels are represented by SRSF1/tubulin, ratios of SRSF1 optical densities normalized against β-tubulin. C) RT-PCR analysis of wild-type p53 exon-5. D) Immunostaining of SRSF1 and pp53. SRSF1 and pp53 tagged with antibodies were further recognized by corresponding Alexa Fluor 488 or 555-conjugated goat IgG (×200, magnification).
Fig. 5
Fig. 5
PP1 and SRSF1 determine wild-type p53 expression. NCI/ADR-RES cells were pretreated with PP inhibitors (5 nM Cal A; 10 nM OA) and transfection of siSRSF1 (100 nM) or pSRSF1, and then treated with MBO-asGCS and C6-Cer followed by Dox exposure (2.5 μM, 48 h). A) RT-PCR analysis of p53 exon-5. B) Western blotting for wild-type p53. C) Effect of SRSF1 on p53 exon-5 mRNA, and D) p53 proteins. Total RNA and cellular protein were extracted from NCI/ADR-RES cells transfected with pEGFP-SRSF1 (pSRSF1) or pEFGP-C1 plasmids (mock) and then treated with MBO-asGCS (100 nM), C6-ceramide (5 μM) and Cal A (5 nM), respectively.
Fig. 6
Fig. 6
Restored p53 leads cancer cells to apoptosis. NCI/ADR-RES cells were pretreated with PP inhibitors (5 nM Cal A; 10 nM OA), and transfection of siSRSF1 (100 nM) or pSRSF1, and then treated with MBO-asGCS (100 nM) and Dox (2.5 μM). A) Cell viability. *, p < 0.001 compared to control (Dox alone); **, p < 0.001 compared to MBO-asGCS + OA or MBO-asGCS + siRNA-SC; #, p < 0.001 compared to Mock. B) Geimsa staining. Cells were treated with indicated conditions mentioned in Fig. 6A (×100 magnification). C) Apoptosis. *, p < 001 compared to control (Dox alone); **, p < 0.001 compared to MBO-asGCS + OA or MBO-asGCS + siRNA-SC; #, p < 0.001 compared to MBO-asGCS + mock or MBO-asGCS + siSRSF1.

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