Prostaglandin E2 Promotes Colorectal Cancer Stem Cell Expansion and Metastasis in Mice

BACKGROUND & AIMS: Inflammation may contribute to the formation, maintenance, and expansion of cancer stem cells (CSCs), which have the capacity for self-renewal, differentiation, and resistance to cytotoxic agents. We investigated the effects of the inflammatory mediator prostaglandin E-2 (PGE(2)) on colorectal CSC development and metastasis in mice and the correlation between levels of PGE(2) and CSC markers in human colorectal cancer (CRC) specimens. METHODS: Colorectal carcinoma specimens and matched normal tissues were collected from patients at the Mayo Clinic (Scottsdale, AZ) and analyzed by mass spectrometry and quantitative polymerase chain reaction. Human primary CRC cells and mouse tumor cells were isolated using microbeads or flow cytometry and analyzed for sphere-formation and by flow cytometry assays. LS-174T cells were sorted by flow cytometry (for CD133(+)CD44(+) and CD133(-)CD44(-) cells) and also used in these assays. NOD-scidIL-2R gamma(-/-) (NSG) mice were given cecal or subcutaneous injections of LS-174T or human primary CRC cells. Apc(Min/+) mice and NSG mice with orthotopic cecal tumors were given vehicle (controls), PGE(2), celecoxib, and/or Ono-AE3-208. PGE(2) downstream signaling pathways were knocked down with small hairpin RNAs, expressed from lentiviral vectors in LS-174T cells, or blocked with inhibitors in human primary CRC cells. RESULTS: Levels of PGE(2) correlated with colonic CSC markers (CD133, CD44, LRG5, and SOX2 messenger RNAs) in human colorectal carcinoma samples. Administration of PGE(2) to Apc(Min/+) mice increased tumor stem cells and tumor burden, compared with controls. NSG mice given PGE(2) had increased numbers of cecal CSCs and liver metastases compared with controls after intracecal injection of LS-174T or human primary CRC cells. Alternatively, celecoxib, an inhibitor of prostaglandin-endoperoxide synthase 2, reduced polyp numbers in Apc(Min/+) mice, liver metastasis in NSG mice with orthotopic tumors, and numbers of CSCs in Apc(Min/+) and NSG mice. Inhibitors or knockdown of PGE(2) receptor 4 (EP4), phosphoinositide 3-kinase (PI3K) p85 alpha, extracellular signal-regulated kinase 1 (ERK1), or nuclear factor (NF)-kappa B reduced PGE(2)-induced sphere formation and expansion of LS-174T and/or human primary CRC cells. Knockdown of ERK1 or PI3K p85 alpha also attenuated PGE(2)-induced activation of NF-kappa B in LS-174T cells. An EP4 antagonist reduced the ability of PGE(2) to induce CSC expansion in orthotopic tumors and to accelerate the formation of liver metastases. Knockdown experiments showed that NF-kappa B was required for PGE(2) induction of CSCs and metastasis in mice. CONCLUSIONS: PGE(2) induces CSC expansion by activating NF-kappa B, via EP4-PI3K and EP4-mitogen-activated protein kinase signaling, and promotes the formation of liver metastases in mice. The PGE(2) signaling pathway therefore might be targeted therapeutically to slow CSC expansion and colorectal cancer progression.