Genetic deletion of microsomal prostaglandin E synthase-1 suppresses mouse mammary tumor growth and angiogenesis

Abstract

The cyclooxygenase/prostaglandin (COX/PG) signaling pathway is of central importance in inflammation and neoplasia. COX inhibitors are widely used for analgesia and also have demonstrated activity for cancer prophylaxis. However, cardiovascular toxicity associated with this drug class diminishes their clinical utility and motivates the development of safer approaches both for pain relief and cancer prevention. The terminal synthase microsomal PGE synthase-1 (mPGES-1) has attracted considerable attention as a potential target. Overexpression of mPGES-1 has been observed in both colorectal and breast cancers, and gene knockout and overexpression approaches have established a role for mPGES-1 in gastrointestinal carcinogenesis. Here we evaluate the contribution of mPGES-1 to mammary tumorigenesis using a gene knockout approach. Mice deficient in mPGES-1 were crossed with a strain in which breast cancer is driven by overexpression of human epidermal growth factor receptor 2 (HER2/neu). Loss of mPGES-1 was associated with a substantial reduction in intramammary PGE2 levels, aromatase activity, and angiogenesis in mammary glands from HER2/neu transgenic mice. Consistent with these findings, we observed a significant reduction in multiplicity of tumors ≥1mm in diameter, suggesting that mPGES-1 contributes to mammary tumor growth. Our data identify mPGES-1 as a potential anti-breast cancer target.

Keywords: Angiogenesis; Aromatase; Breast cancer; Mouse; PGE(2); mPGES-1.

Fig. 1

Mammary PGE₂ levels are markedly decreased in mPGES-1 knockout MMTV/NDL mice. MGs were harvested from 20 week old virgin female mice that were MMTV/NDL, mPGES-1 +/+ (WT) or MMTV/NDL, mPGES-1 −/− (KO), and PGE₂ levels were assayed by ELISA. Mammary PGE₂ levels were reduced from 18.0 [14.5, 23.0] ng/mg protein (median [range], n=6) in MMTV/NDL, mPGES-1 +/+ samples to 7.4 [4.5, 12.3] ng/mg protein in MMTV/NDL, mPGES-1 −/− samples (P=0.002; Wilcoxon rank-sum test).

Fig. 2

Mammary gland vascularization is reduced in mPGES-1-null MMTV/NDL mice. (A, B) Microvessel density is reduced in mPGES-1-null tissue. MG tissue sections from 20 week old virgin MMTV/NDL females that were mPGES-1 wildtype (WT) or mPGES-1 null (KO) were immunohistochemically stained with anti-CD31 antibody. Several microscopic fields were evaluated for each animal. The number of CD31-positive blood vessels associated with a ductal or lobular unit was scored in each microscopic field, and a mean value was calculated for each mouse. The mean CD31-positive blood vessel count in WT mice was significantly greater than that in KO mice: 9.45+/−2.05 (mean+/−SD, n=10) vs 6.26+/−1.07 (mean+/−SD, n=8); P<0.001 (Student t-test). Panel A shows representative images for mPGES-1 wildtype (WT) and mPGES-1 null (KO) mammary glands. Examples of CD31-positive blood vessels are indicated by arrows. Panel B shows the data obtained from numerical evaluation of anti-CD31-stained tissue sections. (C) VEGF levels are strikingly reduced in mPGES-1-null tissue. Transcript levels of VEGF-A were assayed in MGs harvested from 20 week old virgin female mice that were MMTV/NDL, mPGES-1 +/+ (WT) or MMTV/NDL, mPGES-1 −/− (KO). Relative VEGF-A transcript levels (normalized to GAPDH) were reduced from 1.33 [0.28, 1.96] (median [range], n=10) in MMTV/NDL, mPGES-1 +/+ samples to 0.42 [0.06, 1.15] in MMTV/NDL, mPGES-1 −/− samples (P=0.02; Wilcoxon rank-sum test).

Fig. 3

Aromatase activity is substantially reduced in MGs from mPGES-1-deficient MMTV/NDL mice. Aromatase activity was assayed in microsomes prepared from MGs harvested from 20 week old virgin female mice that were MMTV/NDL, mPGES-1 +/+ (WT) or MMTV/NDL, mPGES-1 −/− (KO). Aromatase activity was assayed by measuring tritiated water release from 1β-[³H]-androstenedione. Mammary aromatase activity was reduced from 328 [234, 534] fmoles/μg protein/hr (median [range], n=6) in MMTV/NDL, mPGES-1 +/+ samples to 182 [98, 234] fmoles/μg protein/hr in MMTV/NDL, mPGES-1 −/− samples (P=0.006; Wilcoxon rank-sum test).