15-Hydroxyprostaglandin dehydrogenase is down-regulated in colorectal cancer

Abstract

Prostaglandin E2 (PGE2) can stimulate tumor progression by modulating several proneoplastic pathways, including proliferation, angiogenesis, cell migration, invasion, and apoptosis. Although steady-state tissue levels of PGE2 stem from relative rates of biosynthesis and breakdown, most reports examining PGE2 have focused solely on the cyclooxygenase-dependent formation of this bioactive lipid. Enzymatic degradation of PGE2 involves the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The present study examined a range of normal tissues in the human and mouse and found high levels of 15-PGDH in the large intestine. By contrast, the expression of 15-PGDH is decreased in several colorectal carcinoma cell lines and in other human malignancies such as breast and lung carcinomas. Consistent with these findings, we observe diminished 15-Pgdh expression in ApcMin+/- mouse adenomas. Enzymatic activity of 15-PGDH correlates with expression levels and the genetic disruption of 15-Pgdh completely blocks production of the urinary PGE2 metabolite. Finally, 15-PGDH expression and activity are significantly down-regulated in human colorectal carcinomas relative to matched normal tissue. In summary, these results suggest a novel tumor suppressive role for 15-PGDH due to loss of expression during colorectal tumor progression.

Fig. 1. 15-PGDH expression in normal and pathologic tissues

FirstBlot membranes blotted with RNA from normal human (A) and mouse (B) tissues were probed for expression of 15-PGDH. Each blot contains 2 μg of poly(A) RNA/lane. C, a Cancer Profiling Array II was hybridized with a 15-PGDH cDNA probe and examined for differential expression in paired normal (N) and tumor (T) samples from the following tissues: breast (n = 10), colon (n = 10), stomach (n = 10), lung (n = 10), skin (n = 10), kidney (n = 9), small intestine (n = 7), pancreas (n = 7), and liver (n = 3). Nine cancer cell lines were included as follows (from top to bottom): HeLa (cervical carcinoma), Daudi (Burkitt’s lymphoma), K562 (CML), HL60 (promyelocytic leukemia), G361 (melanoma), A549 (lung carcinoma), MOLT4 (ALL), SW480 (colorectal carcinoma), and Raji (Burkitt’s lymphoma).

Fig. 2. Expression and modulation of 15-PGDH in multiple colorectal cell lines

A, total RNA was isolated following harvest of several colorectal carcinoma cell lines. 5 μg of RNA were loaded, and the levels of 15-PGDH mRNA were determined by Northern blot analysis. B, colorectal carcinoma cells were grown for 24 h (HCT-15, HCT-116, and HCA-7) and 48 h (LS-174T) in serum-free media prior to the addition of indomethacin (10 μM) for 72 h. Following the isolation of total cellular protein, 30 μg of protein were separated by SDS-PAGE and visualized with 15-PGDH-specific antibody and β-actin. Data from three independent experiments were quantified by densitometry for the LS-174T, HCT-15, and HCA-7 cell lines and are shown as the means ± S.E. Indomethacin-treated samples were normalized to untreated controls. C, colorectal carcinoma cells were grown for 24 h (HCT-15, HCT-116, and HCA-7) and 48 h (LS-174T) in serum-free media prior to the addition of EGF (100 ng/ml) for 24 h with or without 1 h of pretreatment with 5 μM erlotinib. Following the isolation of total cellular protein, 30 μg of protein were separated by SDS-PAGE and visualized with 15-PGDH-specific antibody and β-actin. Data from three independent experiments were quantified by densitometry for both the HCT-15 and HCA-7 cell lines and are shown as the means ± S.E. EGF- and erlotinib-treated samples were normalized to untreated control samples.

Fig. 3. Loss of 15-PGDH decreases PGE₂ catabolism and contributes to carcinogenesis in vivo

A, Northern analysis of 15-Pgdh and Cox-2 expression in the small intestine of control C57BL/6 and Apc^Min+/− mice at 15 weeks of age. N, normal mucosa; P, polyp tissue. B, Western analysis of Cox-2 expression in the small intestine of Apc^Min+/− mice at 15 weeks of age. C, determination of 15-PGDH activity in HCT-15 cells treated with and without EGF (100 ng/ml) for 24 h (n = 6). ***, p < 0.001. D, comparative urinalysis of pooled Pgdh^+/+ and Pgdh^−/− mice for the PGE₂ metabolite PGE-M (n = 3). ***, p < 0.001.

Fig. 4. Loss of 15-PGDH expression and activity in human colorectal cancer tissues

A, total RNA and protein were isolated from 23 individual human colorectal cancer tissues and matched normal mucosa. Equal amounts of RNA and protein were analyzed for 15-PGDH expression. N, normal mucosa; T, tumor tissue. B, determination of 15-PGDH activity is shown in eight human colorectal cancer tissues and matched normal mucosa.