The role of DPYD and the effects of DPYD suppressor luteolin combined with 5-FU in pancreatic cancer

Abstract

Background: Despite advances in the treatment of cancer, pancreatic ductal adenocarcinoma (PDAC) remains highly lethal due to the lack of effective therapies. Our previous study showed that Luteolin (Lut), a flavonoid, suppressed pancreatocarcinogenesis and reduced the expression of dihydropyrimidine dehydrogenase (DPYD), an enzyme that degrades pyrimidines such as 5-fluorouracil (5-FU), in PDACs. In this study, we investigated the role of DPYD and evaluated the therapeutic potential of combining 5-FU with Lut in PDACs.

Methods and results: PDAC cells overexpressing DPYD showed increased proliferation, and invasiveness, adding to the resistance to 5-FU. The xenograft tumors of DPYD-overexpressing PDAC cells also exhibit enhanced growth and invasion compared to the control xenograft tumors. RNA-seq analysis of the DPYD-overexpressing PDAC xenograft tumors revealed an upregulation of genes associated with metallopeptidase activity-MMP9 and MEP1A. Furthermore, the overexpression of MEP1A in PDAC was associated with invasion. Next, we investigated the combined effects of Lut, a DPYD suppressor, and 5-FU on DPYD-overexpressing xenograft tumors and PDAC of Pdx1-Cre; LSL-KrasG12D/+; Trp53flox/flox(KPPC) mice. Neither single administration of 5-FU nor Lut showed significant inhibitory effects; however, the combined administration of 5-FU and Lut exhibited a significant tumor-suppressive effect in both the xenograft tumors and KPPC models.

Conclusion: We have elucidated that DPYD expression contributes to proliferation, invasiveness, and 5-FU resistance, in PDACs. The combination therapy of Lut and 5-FU holds the potential for enhanced efficacy against PDACs.

Keywords: 5‐fluorouracil; combination drug therapy; dihydropyrimidine dehydrogenase; luteolin; pancreatic cancer.

FIGURE 1

DPYD‐overexpressing PDAC cells show increased proliferation, invasiveness, angiogenesis, and 5‐FU resistance. AsPC1‐ and PK1‐DPYD cells overexpress protein (A) of DPYD. AsPC1‐ and PK1‐DPYD cells show increased proliferation (n = 4) (B), resistance to low doses of 5‐FU (n = 4) (C), and invasiveness by invasion assay (n = 3–4) (D) compared to AsPC1‐ and PK1‐LacZ cells in vitro. In the xenograft model, AsPC1‐ and PK1‐DPYD tumors show increased volume (E), weight (F), Ki67 labeling index (G), muscle invasion (H), compared to AsPC1‐ and PK1‐LacZ tumors (n = 8, 6). Data are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 statistically significant compared with AsPC1‐ or PK1‐LacZ.

FIGURE 2

DPYD‐overexpressing PDAC cells show enhanced metallopeptidase expression. Heat map of RNA‐seq on AsPC1‐DPYD (n = 3) and AsPC1‐LacZ xenograft tumors (n = 3) (A), and its Gene Ontology analysis with respect to molecular function (B). AsPC1‐DPYD xenograft tumors (n = 7) show upregulation of genes associated with metallopeptidase activity, including MEP1A and MMP9 (C). Immunohistochemical analysis of MEP1A (D) and MMP9 (E) indicate that these proteins are secreted into intraductal lesions (yellow arrowheads) and are upregulated in AsPC1‐DPYD compared to AsPC1‐LacZ xenograft tumors (n = 8). Data are represented as mean ± SD. *p < 0.05, **p < 0.01, statistically significant compared with AsPC1‐LacZ.

FIGURE 3

MEP1A overexpression in PDAC cells does not affect proliferation but promotes invasiveness. MEP1A‐overexpressing AsPC1 cells secrete MEP1A into the medium (A). MEP1A expression does not contribute to proliferation (n = 4) (B) but promotes invasiveness (n = 4) (C). Data are represented as mean ± SD. *p < 0.05 statistically significant compared with AsPC1‐LacZ.

FIGURE 4

Effect of combination therapy of 5‐FU and Luteolin on AsPC1‐DPYD xenograft tumors. Combination index of 5‐FU and Lut in KP4 cells (n = 3) (A). DPYD inhibitory effects of Lut and other flavonoids in KP4 cells (B). The schematic of treatment for AsPC1‐DPYD xenograft tumors (C), and combination treatment of 5‐FU and Lut significantly decreased tumor volume (D) and weights (E) compared to untreated control (n = 6–7). Viable cell areas of tumors on HE staining (F) and DPYD relative intensity to control (G) are significantly decreased in 5‐FU and Lut groups compared to the 5‐FU group and the control group (n = 6–7). Data are represented as mean ± SD. *p < 0.05, **p < 0.01, as compared to the control group. ^# p < 0.05, ^## p < 0.01, as compared to the 5‐FU group.

FIGURE 5

Effect of combination therapy of 5‐FU and Luteolin effect on KPPC mice. The schematic of treatment for PDACs of KPPC mice (A). Combination therapy of 5‐FU and Lut significantly decreased pancreas weights (B, C) and the proportion of PDAC in the pancreatic sections (D) compared to untreated control and Lut single‐treatment (yellow circle: PDAC area, blue circle: non‐cancerous lesion) (n = 13–15). DPYD mRNA levels are decreased in 5‐FU, Lut, and 5‐FU + Lut groups compared to the control group (n = 13–15) (E). Data are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 as compared to the control group. ^† p < 0.05, ^††† p < 0.001, as compared to the Lut group.

FIGURE 6

DPYD expression in human PDAC tissue microarray is associated with poor prognosis. Five‐year overall survival (OS) (A) and 3‐year recurrence‐free survival (RFS) (B) of PDAC patients with low‐DPYD (n = 91) and high‐DPYD expression (n = 41). Five‐year overall OS (C) and three‐year RFS (D) on PDAC patients with 5‐FU chemo adjuvant treatment, classified into low‐DPYD (n = 45), and high‐DPYD (n = 21) PDAC groups. The graph of recurrence‐free duration versus DPYD expression score in PDAC patients who received adjuvant 5‐FU chemotherapy but experienced PDAC recurrence (n = 49) (Spearman p < 0.01, r = −0.4365) (E). Five years (OS) in PDAC patients classified into MEP1A low (n = 97) and MEP1A high (n = 35) groups (F). *p < 0.05 statistically significant difference compared by using the Grehan–Breslow–Wilcoxon test.