Inhibition of glucosylceramide synthase eliminates the oncogenic function of p53 R273H mutant in the epithelial-mesenchymal transition and induced pluripotency of colon cancer cells

Abstract

Missense mutation of tumor suppressor p53, which exhibits oncogenic gain-of-function (GOF), not only promotes tumor progression, but also diminishes therapeutic efficacies of cancer treatments. However, it remains unclear how a p53 missense mutant contributes to induced pluripotency of cancer stem cells (CSCs) in tumors exposed to chemotherapeutic agents. More importantly, it may be possible to abrogate the GOF by restoring wild-type p53 activity, thereby overcoming the deleterious effects resulting from heterotetramer formation, which often compromises the efficacies of current approaches being used to reactivate p53 function. Herewith, we report that p53 R273H missense mutant urges cancer cells to spawn CSCs. SW48/TP53 cells, which heterozygously carry the p53 R273H hot-spot mutant (R273H/+, introduced by a CRISPR/Casp9 system), were subchronically exposed to doxorubicin in cell culture and in tumor-bearing mice. We found that p53-R273H (TP53-Dox) cells were drug-resistant and exhibited epithelial-mesenchymal transition (EMT) and increased numbers of CSCs (CD44v6+/CD133+), which resulted in enhanced wound healing and tumor formation. Inhibition of glucosylceramide synthase with d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) sensitized p53-R273H cancer cells and tumor xenografts to doxorubicin treatments. Intriguingly, PDMP treatments restored wild-type p53 expression in heterozygous R273H mutant cells and in tumors, decreasing CSCs and sensitizing cells and tumors to treatments. This study demonstrated that p53-R273H promotes EMT and induced pluripotency of CSCs in cancer cells exposed to doxorubicin, mainly through Zeb1 and β-catenin transcription factors. Our results further indicate that restoration of p53 through inhibition of ceramide glycosylation might be an effective treatment approach for targeting cancers heterozygously harboring TP53 missense mutations.

Keywords: cancer stem cells; epithelial-mesenchymal transition; glucosylceramide synthase; missense mutation; tumor suppressor p53.

Figure 1. p53 missense mutation and cancer cell response to doxorubicin

Cells were treated with doxorubicin in 5% FBS medium for 72 hr. A. Cell response to doxorubicin. MCF-12A (wt p53), SW48 (wt p53), COLO 320DM (mutant p53 R248W; COLO), WiDr (mutant p53 R273H), SW48/TP53 (mutant p53 R273H), SW48-Dox and TP53-Dox (mutant p53 R273H) cells were treated with doxorubicin for 72 hr. *, p<0.001, compared to wt p53 cells (MCF-12A, SW48). B. Doxorubicin induced EMT in cancer cells carrying a p53 R273H mutant allele. Cells (passages 8-10) of SW48, SW48/TP53, SW48-Dox and TP53-Dox were grown in multi-chamber slides with 10% FBS medium for 48 hr. Red, Alexa Fluor 555–vimentin (VIM); green, Alexa Fluor 488–E-cadherin (ECAD); blue, DAPI–nucleus. Scale bar represents 50 μm in photomicrographs (200x magnification). C. Western blotting for EMT marker assessment. Equal amounts of extracted detergent-soluble proteins (50 μg protein/lane) were resolved using 4-20% gradient SDS-PAGE and then immunoblotted with antibodies of E-cadherin (ECAD), vimentin (VIM) or GAPDH. Protein levels were represented by ratios (mean ± SD) of ECAD or VIM densities normalized against GAPDH from three blots. *, p<0.01 compared to SW48 cells; **, p<0.001 compared to SW48-Dox cells.

Figure 2. Effect of p53 R273H mutant on wound healing of colon cancer cells

A. Wound healing of cancer cells. Scale bar represents 50 μm in photomicrographs (×100 magnification). *, p<0.001 compared to TP53 cells; **, p<0.001 compared to SW48-Dox or TP53-Dox cells treated with vehicle. B. GCS activities analyzed by HPLC. Cer, NBD C6-ceramide. GlcCer, NBD C6-glucosylceramide. *, p<0.001 compared to Vehicle of TP53-Dox cells. C. Ceramides analyzed by ESI/MS/MS. Cer, ceramide. *, ≥twofold higher than vehicle.

Figure 3. p53 R273H mutant enhanced tumor sphere formation and CSCs among colon cancer cells

Cells of SW48-Dox and TP53-Dox lines were separately treated with 5 μM PDMP in 5% FBS medium. A. Tumor sphere formation. Scale bar represents 50 μm in photomicrographs (100x magnification). *, p>0.001 compared to SW48-Dox with vehicle; **, p<0.001 compared to TP53-Dox with vehicle. B. CSCs. Cells were incubated with Alexa-Fluor488 CD44v6 and APC-CD133 antibodies and analyzed by using flow cytometry. The detected CD44v6⁺/CD133⁺ cells (CSCs) are identified in the plots by enclosure with an ellipse (upper right), and compared with vehicle controls, as percentages of total cells in the adjacent bar graph.

Figure 4. p53 expression and p53-responsive proteins in R273H p53 mutant cells

Cells were treated with PDMP (5 μM, 6 days) followed by doxorubicin exposure (50 nM, 48 hr). Equal amounts of detergent-soluble proteins (50 μg/lane) were resolved by 4-20% SDS-PAGE and immunoblotted with corresponding primary and secondary antibodies, sequentially. A. Effects of doxorubicin-induction and PDMP on p53 and p53-responsive protein expression. B. p53 modulates iPSC factor expression in cancer cells exposed to Dox. PDMP inhibits ceramide glycosylation, thereby increasing cellular ceramide levels, which may favor wt p53 expression over that of the R273H missense mutant. C. Protein expression levels. Protein levels are presented here as ratios of their densities normalized against GAPDH from three Western blots. *, p<0.05 compared to parental (SW48, TP53) or Dox-induced sublines (SW48-Dox, TP53-Dox); **, p<0.05 compared to SW-48, or SW48-Dox and SW48-Dox PDMP. pp53, phosphorylated p53 (Ser15); β-Cat, β-catenin; TGF-β, transforming growth factor β. D. Immunostaining of ceramide and pp53. The scale bar represents 5 μm in photomicrographs (200x magnification).

Figure 5. Tumor responses to doxorubicin treatments

Cells of SW48 and SW48/TP53 (TP53) lines were subcutaneously inoculated into athymic nude mice. Low doses of doxorubicin (200 μg/kg and 300 μg/kg, per 6 days, i.p.) and PDMP (4.0 mg/kg, per 3 days, i.p.) were administered for 32 days (5 cases/group). A. SW48 tumor growth. B. TP53 tumor growth. *, p<0.01, compared to doxorubicin alone treatments. C. Tumor weight after treatments. *, p<0.01, compared to doxorubicin alone treatments. D. GCS activities in tumors after treatments. *, p<0.001, compared to SW48 tumors treated with Dox (200 μg/kg); **, p<0.001, compared to TP53 tumors treated with doxorubicin (200 μg/kg); #, p<0.001, compared to SW48 tumors treated with Dox and PDMP.

Figure 6. Inhibition of GCS restored p53 expression in tumors of mice during doxorubicin treatments

A. Western blotting. Equal amounts of detergent-soluble proteins (50 μg/lane) extracted were resolved using 4-20% gradient SDS-PAGE and then immunoblotted with corresponding antibodies. Protein levels in the bar graph are presented as mean±SD of their densities normalized against GAPDH from three blots. *, p<0.01 compared to SW48 tumors treated with Dox; **, p<0.01 compared to TP53 tumors treated with Dox; #, p<0.01 compared to SW48 tumors treated with Dox and PDMP. B. Immunostaining of pp53 and p21. The scale bar indicates 25 μm in photomicrographs (200x magnification). Red, Alexa Fluor 555-pp53; green, Alexa Fluor 488-p21; blue, DAPI-nucleus. The scale bar indicates 50 μm in photomicrographs (200x magnification).

Figure 7. Effects of R273H p53 mutant on iPSC in tumors exposed to doxorubicin

A. Flow cytometry analysis of colon CSCs (CD133+/CD44V6) from tumors in mice treated with doxorubicin (Dox, 200 μg/kg, per 6-days, i.p.) alone or combined with PDMP (4.0 mg/kg, per 3-days, i.p.), for 32 days. *, p<0.001 compared to SW48 tumors treated with Dox; **, p<0.001 compared to TP53 tumors treated with Dox. B. Western blotting of pluripotency regulators in tumors. Equal amounts of detergent-soluble proteins (50 μg/lane) extracted were resolved using 4-20% gradient SDS-PAGE and then immunoblotted with corresponding antibodies. Protein levels are represented as mean ± SD of their densities normalized against GAPDH from three blots. *, p<0.01 compared to Dox treatments (200 μg/kg); **, p<0.01 compared to SW48 tumors.

Figure 8. Ceramide restores wt p53 expression in cells carrying heterozygous R273H mutation

After 48 h pretreatments with PDMP (5 μM), PDMP plus FB1 (25 μM), MBO-asGCS (100 nM), siRNA-p53 (100 nM), or siRNA-SC (100 nM), TP53-Dox cells were cultured in medium containing doxorubicin (50 nM) with each of these agents for 48 h to induce DNA damage and 72 h for cell viability assays. For the Western blot studies, the cells pretreated under each of the various sets of conditions, the doxorubicin (50 nM) exposure was combined with continuation of the various pretreatment agents for an additional 48 h to induce DNA damage before protein extraction. A. Western blotting. Equal amounts of detergent-soluble proteins extracted (50 μg/lane) were resolved using 4-20% gradient SDS-PAGE and then immunoblotted with corresponding antibodies; representative blots are presented. GCS, glucosylceramide synthase; pp53, phosphorylated p53 (Ser15); β-cat, β-catenin; siRNA-SC, siRNA scrambled control. B. Ceramide affects wt p53 expression. Protein levels are represented as mean ± SD of their densities normalized against GAPDH from three settings of blots. *, p<0.001 compared to vehicle or siRNA-SC; **, p<0.001 compared to PDMP or MBO-asGCS treatment. C. Cell responses to doxorubicin. D. IC₅₀ values for doxorubicin. *, p<0.001 compared to siRNA-SC or vehicle. **, p<0.001 compared to PDMP or MBO-asGCS treatment.

Figure 9. Ceramide restores wild-type p53 expression in cells heterozygously carrying a p53 R273H mutant allele

When mutant-heterozygous (HZ) cancer cells are exposed to a sub-lethal dose of doxorubicin (Dox), DNA damage induces overexpression of p53 R273H, and the presence of this missense mutant protein upregulates Zeb1 and β-catenin (β-Cat) stem-like factors. The consequent gain-of-function (GOF) presents as epithelial-mesenchymal transition (EMT) and induced pluripotency of stem cells (iPSCs), lending to tumor growth. Inhibition of glucosylceramide synthase (GCS)-catalyzed ceramide glycosylation with PDMP increases cellular ceramide levels, bring about restoration of wt p53 protein expression, in turn increasing p53-responsive proteins, including p21 and Puma, thereby suppressing tumor progression.