MUC1-Mediated Metabolic Alterations Regulate Response to Radiotherapy in Pancreatic Cancer

Abstract

Purpose:MUC1, an oncogene overexpressed in multiple solid tumors, including pancreatic cancer, reduces overall survival and imparts resistance to radiation and chemotherapies. We previously identified that MUC1 facilitates growth-promoting metabolic alterations in pancreatic cancer cells. The present study investigates the role of MUC1-mediated metabolism in radiation resistance of pancreatic cancer by utilizing cell lines and in vivo models.Experimental Design: We used MUC1-knockdown and -overexpressed cell line models for evaluating the role of MUC1-mediated metabolism in radiation resistance through in vitro cytotoxicity, clonogenicity, DNA damage response, and metabolomic evaluations. We also investigated whether inhibition of glycolysis could revert MUC1-mediated metabolic alterations and radiation resistance by using in vitro and in vivo models.Results: MUC1 expression diminished radiation-induced cytotoxicity and DNA damage in pancreatic cancer cells by enhancing glycolysis, pentose phosphate pathway, and nucleotide biosynthesis. Such metabolic reprogramming resulted in high nucleotide pools and radiation resistance in in vitro models. Pretreatment with the glycolysis inhibitor 3-bromopyruvate abrogated MUC1-mediated radiation resistance both in vitro and in vivo, by reducing glucose flux into nucleotide biosynthetic pathways and enhancing DNA damage, which could again be reversed by pretreatment with nucleoside pools.Conclusions: MUC1-mediated nucleotide metabolism plays a key role in facilitating radiation resistance in pancreatic cancer and targeted effectively through glycolytic inhibition. Clin Cancer Res; 23(19); 5881-91. ©2017 AACR.

Figure 1. MUC1-expression decreases cancer cell sensitivity to radiation

(a) Survival of control and experimental (MUC1-knockdown/MUC1-overexpression) pancreatic cancer cells at 72 h post-exposure to indicated radiation doses, relative to untreated cells. (b) Western blot analysis of MUC1 expression levels in pancreatic cancer cell lines used as MUC1-knock down and overexpression models. β-tubulin or actin were utilized as loading controls. (c) Representative images and relative quantification of clonogenicity, 21 days post-exposure to the indicated radiation doses. (d) Effect of MUC1 overexpression in U2OS osteosarcoma-cell survival upon treatment with the indicated doses of radiation and western blot analysis of MUC1 expression in U2OS cells. Values in bar graphs and line plots indicate mean ± SEM of three replicates. ‘*’ p<0.05, ‘#’ p<0.01, and ‘$’ p<0.001 obtained through Two-way ANOVA and Tukey’s multiple comparison tests.

Figure 2. MUC1 expression reduces radiation-induced DNA damage

(a) Representative immunofluorescence images indicating γH2AX staining (red) as a measure of DNA damage upon irradiation and treatment with NHEJ or RI inhibitors for 12h; DAPI staining (blue) indicates nuclei. (b) Bar charts representing relative cell survival upon irradiation in the presence of solvent control, either the HR or NHEJ repair inhibitors, as measured by MTT assay. Fluorescence images represent representative fields of respective treatments captured at 20X magnification. All the bars represent mean ± SEM of 3 replicates. ‘*’ p<0.05, ‘#’ p<0.01, and ‘$’ p<0.001 obtained through two-way ANOVA and Bonferroni method for multiple comparisons.

Figure 3. MUC1 facilitates radiation-induced carbon flux into glycolysis and de novo nucleotide biosynthesis pathways

Partial least squares discriminant analysis (PLS-DA) plots in panels a and b represent differential clustering of control and irradiated cells based on their total polar metabolite content. Each filled circle/square indicates an individual sample. The three closely clustered circles or squares represent three biological replicates of an individual group of samples extracted from one cell line. The axes of the PLS-DA plots represent principal component-1 (PC-1) and -2 (PC-2), which indicate the variance among the groups. PLS-DA analyses were performed utilizing Metaboanalyst algorithm with mean intensities and pareto scaling distribution. Bar charts in a and b show fold change in metabolite levels of glycolysis, pentose phosphate pathway (PPP), and purine and pyrimidine metabolic pathways upon irradiation in experimental cells compared to their respective wild-type cells. Bars represent mean ± SEM of three biological replicate values; *, # and $-indicate p<0.05, p<0.01, and p<0.001, respectively, obtained through one-way ANOVA and Benjamini-Hochberg’s procedure to control the false discovery rate.

Figure 4. MUC1 facilitates radiation resistance through nucleoside-mediated DNA damage repair

(a) Representative immunofluorescence images indicating γH2AX staining (red) as a measure of DNA damage upon irradiation and treatment with nucleosides for 12 h. DAPI staining (blue) indicates nuclei. Fluorescence images represent representative fields of respective treatments captured at 20X magnification. Bar charts indicate quantification of the number of nuclei with >10 foci from a total of 50 nuclei considered for each condition. (b) Bar charts show relative cell survival under radiation and/or nucleoside supplementation. All bars represent mean ± SEM of three biological replicates. */#, **/##, and ***/### indicate p<0.05, p<0.01, and p<0.001, respectively, obtained through one-way ANOVA with Tukey’s multiple comparison test. *, **, and *** indicate statistical significance in comparison to wild type or MUC1-kd cells, whereas, #, ##, and ### indicate statistical significance with respect to the MUC1-expressing cells.

Figure 5. BrPA abrogates MUC1-mediated radiation-resistance

(a) Bar charts represent relative cell survival in wild-type and experimental cells upon irradiation and treatments with either BrPA or 6-AN. (b) Representative immunofluorescence images indicating γH2AX staining (red) as a measure of DNA damage upon irradiation and treatment with solvent control or BrPA treatments of cells for 12 h. DAPI staining (blue) indicates nuclei. Bars represent mean±SEM of three biological replicate values. # and $-indicate p<0.01 and p<0.001, respectively, obtained through two-way ANOVA with Bonferroni posttests for cell survival analysis. Fluorescence images represent representative fields of respective treatments captured at 20X magnification.

Figure 6. BrPA inhibits MUC1-mediated radiation resistance

(a) Representative images (top panels) of crystal violet stained colonies at 21 days post treatment with radiation and/or BrPA treatment. Bar charts in bottom panels indicate the normalized relative clonogenicity levels in the HPAF2 and S2-013 cell line models. (b) Tumor growth rate plots indicate the percent change in S2-013.Neo and S2-013.MUC1 tumor growth in comparison to the day 0 of the treatment (BrPA treatment initiation was set as the day 0). Each value indicates mean±SEM of either eight or nine biological replicates; *, # and $ -indicate p<0.05, <0.01 and <0.001, respectively, obtained through t-test analysis.