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

Abstract

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 E2 (PGE2) on colorectal CSC development and metastasis in mice and the correlation between levels of PGE2 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-2Rγ(-/-) (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), PGE2, celecoxib, and/or Ono-AE3-208. PGE2 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 PGE2 correlated with colonic CSC markers (CD133, CD44, LRG5, and SOX2 messenger RNAs) in human colorectal carcinoma samples. Administration of PGE2 to Apc(Min/+) mice increased tumor stem cells and tumor burden, compared with controls. NSG mice given PGE2 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 PGE2 receptor 4 (EP4), phosphoinositide 3-kinase (PI3K) p85α, extracellular signal-regulated kinase 1 (ERK1), or nuclear factor (NF)-κB reduced PGE2-induced sphere formation and expansion of LS-174T and/or human primary CRC cells. Knockdown of ERK1 or PI3K p85α also attenuated PGE2-induced activation of NF-κB in LS-174T cells. An EP4 antagonist reduced the ability of PGE2 to induce CSC expansion in orthotopic tumors and to accelerate the formation of liver metastases. Knockdown experiments showed that NF-κB was required for PGE2 induction of CSCs and metastasis in mice.

Conclusions: PGE2 induces CSC expansion by activating NF-κB, via EP4-PI3K and EP4-mitogen-activated protein kinase signaling, and promotes the formation of liver metastases in mice. The PGE2 signaling pathway therefore might be targeted therapeutically to slow CSC expansion and colorectal cancer progression.

Keywords: Colon Cancer; Cyclooxygenase; NSAIDs; Tumorigenesis.

Figure 1. PGE₂ levels correlate with the expression of CSC markers in human CRC

The levels of PGE₂ are correlated with the expression of CD133 (panel A), CD44 (panel B) LRG5 (panel C), and SOX2 (panel D) at mRNA levels in 25 pairs of human colorectal carcinomas (T) with matched normal tissues (N). Data were presented as fold changes in cancer specimens as compared to matched normal tissues. Nonparametric Spearman correlation analysis (R value) was performed.

Figure 2. PGE₂ increases CSCs and promotes liver metastasis

(A) Left panel, the representative immunostaining of CD133 and CD44 (brown) in colonic adenomas taken from Apc^Min/+ mice treated with PGE₂ or vehicle (scale bar, 50 μm). Right panel, relative expression of CD133 and CD44 at mRNA levels in polyps. (B) Formation of spheres from colonic epithelial adenoma cells isolated from Apc^Min/+ mice treated with PGE₂ or vehicle. (C) The representative immunostaining of CD44v6 and CD133 (brown) in cecal tumors taken from indicated group of mice treated with PGE₂ or vehicle (scale bar, 50 μm). (D) Cells isolated from indicated cecal tumors were subjected into flow cytometry analysis. Data represents the percentage of EpCAM⁺CD44v6⁺ (top panel) and EpCAM⁺CD133⁺ (bottom panel) cells in total cells of cecal tumor taken from above groups. (E) Formation of spheres from cecal tumor cells isolated from mice treated with PGE₂ or vehicle in above groups. (F) Incidence (left panel) of liver metastatic tumors and average numbers of liver metastatic tumors (middle and right panels) in mice treated with PGE₂ or vehicle in above groups. The error bar indicates ± SEM. *p<0.05.

Figure 3. Reduction of PGE₂ suppresses CSCs and inhibits liver metastasis

(A) Formation of spheres from intestinal adenoma cells isolated from Apc^Min/+ mice treated with vehicle, celecoxib, or Ono-AE3-208. (B) Average numbers of polyps at different size and total that includes all sizes in above treatment groups. (C) Intestinal PGE₂ levels in above treatment groups. (D) Formation of spheres from cecal tumor cells isolated from NSG mice treated with vehicle, celecoxib, PGE₂, or celecoxib plus PGE₂ after primary carcinoma cells from human CRC specimen were injected into cecum. (E) Average numbers of liver metastatic tumors at different size and total that includes all sizes in mice as described in panel D. (F) Cecal PGE₂ levels in mice as described in panel D. The error bar indicates ± SEM. *p<0.05.

Figure 4. PGE₂ directly induces CSCs in tumor epithelial cells

(A–D) LS-174T cells (A), mouse colonic epithelial tumor cells (B), primary carcinoma cells isolated from three CRC patients (C), and both CD133^−/−CD44^−/− and CD133^+/+CD44^+/+ LS-174T cells (D) were treated with vehicle or PGE₂ during in vitro sphere-forming assays. Left panels are representative phase contrast photos in panels A–C (scale bar, 50μm). Data in right panels represent the average of sphere numbers from three independent experiments. (E) The LS-174T cells and primary carcinoma cells from human CRC specimens were pretreated with vehicle or PGE₂ in regular tissue culture plates for 48 h. After treatment, the cells were subject to in vitro sphere-forming assays without PGE₂ treatment. The error bar for panels C indicates ± SD. The error bar for other panels indicates ± SEM. * p<0.05.

Figure 5. PGE₂ induces CSCs by activating NF-κB via EP4-PI3K and EP4-MAPK-pathways

(A) Formation of spheres in LS-174T cells (left panel) and human primary colon carcinoma cells (right panel) treated with vehicle, PGE₂, ONO-AE-208 (Ono), PD98059 (PD), and/or LY294002 (Ly). (B) Formation of spheres in LS-174T/con-sh (non-silencing control shRNA), LS-174T/shERK1, and LS-174T/shp85α cells treated with PGE₂ or vehicle (left panel). The protein levels of ERK1 and p85α in indicated cells were measured by Western blotting (right panels). (C) The nuclear NF-κB p65 levels in LS-174T/con-sh, LS-174T/shp85α, and LS-174T/shERK1 cells treated with PGE₂ or vehicle for 24 hr. The band density was quantitated by densitometry scanning. The band density of p65 was normalized by the band density of Lamin A. The results were reported as a mean of fold-induction from three independent experiments (low panel). (D) Formation of spheres in LS-174T/con-sh and LS-174T/shp65 cells treated with PGE₂ or vehicle (left panel). The protein levels of p65 in these cells were measured as described above (right panel).

Fig. 6. EP4 and NF-κB are required for PGE₂ induction of CSCs and liver metastasis

(A–B) LS-174T/con-sh or LS-174T/shp65 cells were injected into the cecal walls of NSG mice. After injection, these mice were treated with vehicle or PGE₂. (A) The representative immunostaining of CD44v6 and CD133 (brown) in cecal tumors taken from mice treated with PGE₂ or vehicle in vector group (left panel) and shp65 group (right panel), scale bar, 50 μm. (B) Data represents the percentage of EpCAM⁺CD44v6⁺ (top panel) and EpCAM⁺CD133⁺ (bottom panel) cells in total cells of cecal tumor in indicated group of mice treated with PGE₂ or vehicle. (C) Formation of spheres from cecal tumor cells isolated from mice treated with PGE₂ or vehicle in above groups. (D) Average numbers of liver metastatic tumors in above groups. (E) Formation of spheres from cecal tumor cells isolated from mice treated with vehicle, Ono-AE3-208, PGE₂, or Ono-AE3-208 plus PGE₂ after LS-174T cells were injected into mouse cecum. (F) Average numbers of liver metastatic tumors at different size and total that includes all sizes in groups as described in panel E. The error bar indicates ± SEM. *p<0.05.