A role for ceramide glycosylation in resistance to oxaliplatin in colorectal cancer

Abstract

There is growing evidence to support a role for the ceramide-metabolizing enzyme, glucosylceramide synthase (GCS), in resistance to a variety of chemotherapeutic agents. Whether GCS contributes to oxaliplatin resistance in colorectal cancer (CRC) has not yet been determined. We have addressed this potentially important clinical issue by examining GCS function in two panels of oxaliplatin-resistant, isogenic CRC cell lines. Compared to parental cell lines, oxaliplatin-resistant cells have increased expression of GCS protein associated with increased levels of the pro-survival ceramide metabolite, glucosylceramide (GlcCer). Inhibition of GCS expression by RNAi-mediated gene knockdown resulted in a reduction in cellular GlcCer levels, with restored sensitivity to oxaliplatin. Furthermore, oxaliplatin-resistant CRC cells displayed lower ceramide levels both basally and after treatment with oxaliplatin, compared to parental cells. GlcCer, formed by GCS-mediated ceramide glycosylation, is the precursor to a complex array of glycosphingolipids. Differences in cellular levels and species of gangliosides, a family of glycosphingolipids, were also seen between parental and oxaliplatin-resistant CRC cells. Increased Akt activation was also observed in oxaliplatin-resistant CRC cell lines, together with increased expression of the anti-apoptotic protein survivin. Finally, this study shows that GCS protein levels are greatly increased in human CRC specimens, compared to matched, normal colonic mucosa, and that high levels of UGCG gene expression are significantly associated with decreased disease-free survival in colorectal cancer patients. These findings uncover an important cellular role for GCS in oxaliplatin chemosensitivity and may provide a novel cellular target for augmenting chemotherapeutic drug effectiveness in CRC.

Keywords: Chemoresistance; Colorectal cancer; Glucosylceramide; Glucosylceramide synthase; Oxaliplatin.

Figure 1:. Increased expression of GCS protein and GlcCer levels in oxaliplatin-resistant CRC cells.

A. Ceramide levels are decreased by its conversion into glucosylceramide (GlcCer) by the enzyme, glucosylceramide synthase (GCS). GlcCer formation serves as the rate-limiting step in the synthesis of the glycosphingolipid family of sphingolipids. B. Immunoblot analysis demonstrates that oxaliplatin-resistant HT-29 and RKO cell lines have increased expression of GCS protein, compared to parental cell lines. C. Thin layer chromatography of cellular glycosphingolipids reveals that oxaliplatin-resistant HT-29 and RKO cell lines have increased GlcCer levels, compared to parental cells. D. Thin layer chromatography of oxaliplatin-resistant HT-29 cellular glycosphingolipids demonstrates that stable knockdown of GCS reduces levels of GlcCer. E. Thin layer chromatography of HT-29 cellular glycosphingolipids reveals that exogenous over-expression of GCS results in increased levels of GlcCer. Arrows denotes GlcCer.

Figure 2:. Expression levels of GCS correlate with cellular sensitivity towards oxaliplatin, through either oxaliplatin-dependent cell cycle arrest or apoptosis.

Cell viability and oxaliplatin IC₅₀ analysis of A. HT-29 cells and B. RKO cells (parental, oxaliplatin-resistant + NT-shRNA and oxaliplatin-resistant + GCS-shRNA). C. Cell viability and oxaliplatin IC₅₀ analysis of parental HT-29 cells stably expressing either vector-only or FLAG-tagged GCS. Data points (in A., B., and C.) are the mean ± SE of triplicate wells of a representative experiment (n = 3). D. Cell cycle analysis shows that increased concentrations of oxaliplatin are needed in oxaliplatin-resistant HT-29 cells, compared to parental oxaliplatin-sensitive cells, to cause an S phase arrest. Data is the mean ± SE of three separate experiments. E. Immunoblot analysis demonstrates that oxaliplatin-resistant RKO cells have reduced levels of apoptosis, compared to oxaliplatin-sensitive parental cells. Additionally, stable knockdown of GCS in oxaliplatin-resistant cells partially rescues oxaliplatin-dependent apoptosis

Figure 3:. Increased survivin expression in oxaliplatin-resistant CRC cells.

A. Oxaliplatin-resistant HT-29 with stable GCS knockdown undergoing mitotic catastrophe. Arrows denote cells actively undergoing mitotic catastrophe; large, single cells with multiple nuclei. B. Immunoblot analysis of HT-29 cell lysate reveals that both survivin and GCS protein expression increase as the relative cellular oxaliplatin resistance levels increase. C. Immunoblot analysis of cellular lysates of RKO cells with various GCS protein expression levels. Survivin expression levels are increased in oxaliplatin-resistant cells, compared to parental cells. Stable knockdown of GCS results in decreased expression of survivin protein. D. Immunoblot analysis of HT-29 cell lysates demonstrates that exogenous over-expression of GCS increases survivin protein levels. Immunoblot analysis of oxaliplatin-resistant HT-29 (E.) or RKO (F.) cells treated with or without oxaliplatin for 48-hours in the presence or absence of the GCS inhibitor, PPMP. For both cell lines, oxaliplatin treatment of oxaliplatin-resistant cells results in increased expression of survivin protein and chemical inhibition of GCS causes a decrease in survivin protein expression.

Figure 4:. Oxaliplatin-resistant CRC cells have increased levels of activated Akt.

A. Immunoblot analysis of cellular lysates of HT-29 and RKO cells demonstrates that increased levels of activated Akt (P-S473) are seen in oxaliplatin-resistant CRC cells and that stable knockdown of GCS results in a decrease in Akt phosphorylation. Immunoblot analysis of oxaliplatin-resistant HT-29 (B.) or RKO (C.) cells treated with the GCS inhibitor, PPMP, shows that inhibition of GCS results in decreased levels of activated Akt (P-S473). D. Immunoblot analysis of oxaliplatin-resistant HT-29 and RKO cells treated with or without the PI3K inhibitor, LY294002, for 16-hours demonstrates that survivin protein levels are regulated in a PI3K-Akt-dependent manner. As a control, levels of activated Akt (P-S473) are decreased after treatment with LY294002.

Figure 5:. Determination of ceramide levels after oxaliplatin-treatment of CRC cells.

A. Total ceramide levels of various RKO cells, treated with or without 2.5 μM oxaliplatin were determined quantitatively by lipid mass spectrometry analysis and normalized to cellular phosphate. Total ceramide data points are the mean ± SE of 3 experimental replicates. (* = P<0.01, ** = P<0.0001) B. C16-ceramide levels of various HT-29 cells, treated with or without 10 μM oxaliplatin, were determined quantitatively by lipid mass spectrometry analysis and normalized to cellular phosphate. C16-ceramide data points are the mean ± SE of 3 experimental replicates. (* = P<0.05, ** = P<0.01, *** = P<0.0001) For both A. and B., statistical analysis was performed using linear modeling and P values < 0.05 were considered statistically significant.

Figure 6:. Oxaliplatin-resistant CRC cells have increased levels of cellular gangliosides.

Thin layer chromatography of cellular gangliosides from parental, OxR + NT-shRNA and OxR + GCS-shRNA HT-29 (A.) and RKO (B.) cells. Levels of select gangliosides are increased in oxaliplatin-resistant CRC cell lines and stable knockdown of GCS ablates this effect. C. Thin layer chromatography of cellular gangliosides from HT-29 cells demonstrates that exogenous over-expression of GCS results in increased levels of select cellular gangliosides.

Figure 7:. Human CRC tumor specimens have increased expression of GCS protein and increased UGCG message correlates with decreased CRC patient survival.

A. Immunoblot analysis of 14 matched pairs of CRC adenocarcinoma and normal adjacent colon tissue demonstrates that increased expression of GCS protein is seen in tumor, compared to normal tissue. N = normal colon tissue, T = colon tumor tissue. B. Kaplan-Meier survival analysis, of gene expression data matrixes deposited to the GEO database, demonstrates that patients with high UGCG gene expression have poor disease-free survival, compared to patients with low UGCG gene expression. P value < 0.01 was considered statistically significant.