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. 2012 Sep;2(9):840-55.
doi: 10.1158/2159-8290.CD-12-0101. Epub 2012 Jul 3.

Cancer-stimulated mesenchymal stem cells create a carcinoma stem cell niche via prostaglandin E2 signaling

Hua-Jung Li  1 , Ferenc Reinhardt, Harvey R Herschman, Robert A Weinberg
Affiliations

Cancer-stimulated mesenchymal stem cells create a carcinoma stem cell niche via prostaglandin E2 signaling

Hua-Jung Li et al. Cancer Discov. 2012 Sep.

Abstract

Mesenchymal cells of the tumor-associated stroma are critical determinants of carcinoma cell behavior. We focus here on interactions of carcinoma cells with mesenchymal stem cells (MSC), which are recruited to the tumor stroma and, once present, are able to influence the phenotype of the carcinoma cells. We find that carcinoma cell-derived interleukin-1 (IL-1) induces prostaglandin E(2) (PGE(2)) secretion by MSCs. The resulting PGE(2) operates in an autocrine manner, cooperating with ongoing paracrine IL-1 signaling, to induce expression of a group of cytokines by the MSCs. The PGE(2) and cytokines then proceed to act in a paracrine fashion on the carcinoma cells to induce activation of β-catenin signaling and formation of cancer stem cells. These observations indicate that MSCs and derived cell types create a cancer stem cell niche to enable tumor progression via release of PGE(2) and cytokines.

Significance: Although PGE2 has been implicated time and again in fostering tumorigenesis, its effects on carcinoma cells that contribute specifically to tumor formation are poorly understood. Here we show that tumor cells are able to elicit a strong induction of the COX-2/microsomal prostaglandin-E synthase-1 (mPGES-1)/PGE(2) axis in MSCs recruited to the tumor-associated stroma by releasing IL-1, which in turn elicits a mesenchymal/stem cell–like phenotype in the carcinoma cells.

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Conflict of interest statement

Conflict of interest statement: No potential conflicts of interest were disclosed.

Figures

Fig. 1
Fig. 1. Carcinoma cell secreted IL-1 induces PGE2 production in MSCs
(A) PGE2 (Panel a) and COX-2 (Panel b) were measured in the indicated CM or cultures. PGE2 data are means ± SE, n = 3. ***p < 0.005 (vs that in LoVo medium). LoVo+MSC; LoVo lysate and MSC lysate mixed in equal amounts; LoVoMSC, lysate of LoVoMSC coculture. The same nomenclature applies to the SW1116 cells. (B) Panel (a) MSCs were treated with LoVoCM, LoVoCM with neutralizing antibodies (500 ng/ml), or IL-1 only. Panel (b) MSCs were cocultured with LoVo cells expressing shRNAs against IL1α+IL1β (LoVoshIL1αβ-1, LoVoshIL1αβ-2), IL1β (LoVoshIL1β-1, LoVoshIL1β-2), or a scrambled sequence (LoVoshsc). Panel (c) MSCs were treated with DME, LoVoCM, or LoVoCM+IL-1ra. After incubation, media were collected and assayed for PGE2. Data are means ± SE, n = 3. (C) Panel (a) IL-1α, IL-1β, and IL-1ra protein levels in CM of the carcinoma cells. Data are means ± SE, n = 3. Panel (b) PGE2 levels in carcinoma cells, MSCs (M), and co-cultures of the carcinoma cells with MSCs. Data are means ± SE, n = 3. ***p < 0.005 (vs that in MSC culture).
Fig. 2
Fig. 2. IL-1 and PGE2 mediate GRO-α, IL-6 and IL-8, but not RANTES induction in carcinoma cell-MSC coculture
(A) Levels of PGE2 and the cytokines were measured in CM from the cultures. Data are means ± SE, n = 3. **p < 0.01, ***p < 0.005 (vs that in MSC culture). (B) mRNA levels of the enzymes governing PGE2 production and the cytokines (upper panel) and COX2 protein levels (lower panel) in MSCs treated with IL-1 as indicated. Data are means ± SE, n = 3. *p < 0.05, **p < 0.01, ***p < 0.005 (vs control). (C) Cytokine levels in CM from MSCs, LoVo cells and LoVoMSC cocultures, in the presence of indomethacin (indo; 100 µM), PGE2 (100 nM), or Indo + PGE2. Data are means ± SE, n = 3. (D) IL-6, IL-8 and Gro-α mRNA induction in MSCs either by LoVoCM or by IL-1 in the presence of EP2 and EP4 antagonists (AH6809 15 µM + GW627368X 20 µM). mRNA levels are set as 0% for vehicle-treated MSCs and 100% for LoVoCM-treated or IL-1-treated MSCs. Data are means ± SE, n = 3. *p < 0.05, **p < 0.01 (vs MSC treated with LoVoCM or IL-1 without inhibitors). (E) The proposed interactions for PGE2 and cytokine induction from MSCs. AA: arachidonic acid.
Fig. 3
Fig. 3. MSCs elict EMT, invasion of carcinoma cells, and increased tumor initiation of xenografts
(A) E-cadherin (E-cad), vimentin (VIM), fibronectin, Snail, and β-actin protein expression in carcinoma cells cultured either alone or with MSCs. The numbers indicate relative protein levels. (B) EMT markers and EMT-TFs were measured in LoVo cells treated with vehicle, PGE2 (100 nM) or the cytokines (100 ng/ml IL-6, 100 ng/ml IL-8, 100 ng/ml GRO-α, and/or 10 ng/ml RANTES) as indicated for 6 days. (C) (Left panel) LoVo-tdTomato cells were cultured either alone or with MSCs in the upper wells of Boyden chambers. The presence or absence of NS-398 (50 µM) or NS-398 + PGE2 (100 nM) is indicated for each panel. The images show the LoVo-tdTomato cells that migrated through the Matrigel-coated membranes in 72 h. Scale bar = 100 µm. (Right panel), Data are means ± SE, n = 3. (D) LoVo cell migration in LoVoMSC coculture treated with cytokine neutralizing antibodies as indicated. Data are means ± SE, n = 5. (E) MSCs increase invasion of LoVo tumors. LoVo (5 × 105 cells/injection) were injected subcutanously into SCID mice either alone (a, b, c) or with MSCs (5 × 105 cell/injection, d, e, f). After 8 weeks, tumors of comparable size were isolated. H&E staining was performed on the tumor sections. Scale bar = 100 µm. (F and G) Weights of tumors derived from (F) carcinoma cells (5 × 104 cells/injection) and (G) LoVo cells injected into SCID mice, either alone or with MSCs. Filled circles indicate individual tumor weights; Open circles indicate no tumor grew at the site of injection. Bars are means ± SE. (H) The ranges of the estimated tumor-initiating frequencies in panel G evaluated by ELDA (with 95% confidence).
Fig. 4
Fig. 4. MSC-induced increase in tumor initiation is reflected in an increase in ALDHhigh CSC-enriched populations
(A) ALDH1 protein expression in LoVo cells cultured either alone or with MSCs. The numbers indicate relative protein levels. (B) Unsorted (whole), ALDHhigh and ALDHlow LoVo cells were cultured either alone or with dTtomato-MSCs. After 5 days, ALDH activity of LoVo cells was analyzed by flow cytometry (panel a) and dTtomato-MSCs were removed from the cultures by flow sorting. After removing MSCs, the LoVo cells were cultured alone for another five days. The ALDH activities were again analyzed by flow cytometry (panel b). The percentages indicate the percentage of ALDHhigh LoVo cells; i.e., percent of LoVo cells with ALDH activity beyond the indicated thresholds. (C) a: Weights of tumors derived from ALDHhigh or ALDHlow LoVo cells injected into SCID mice, either alone or with MSCs. Solid-filled and hash-filled circles indicate individual tumor weights; open circles indicate no tumor grew at the site of injection. Red circles: injection of ALDHhigh LoVo cells. Blue circles: injection of ALDHlow LoVo cells. Hash-filled circles: injection of LoVo cells, Solid-filled circles: injection of LoVo cells and MSCs. Bars are means ± SE, b: The ranges of the estimated tumor-initiating frequencies evaluated by ELDA. (D) LoVo cells cultured with tdTomato-MSCs have increased numbers of TICs. Cells were cultured as in panel B. After isolating LoVo cells by sorting, cells (5 × 104 cells/injection) were injected into mice. After 6 weeks, the tumors were isolated and weighted. Filled circles indicate individual tumor weights; Open circles indicate no tumor grew at the site of injection. Bars are means ± SE.
Fig. 5
Fig. 5. COX2-PGE2 signaling is required for MSC-induced increase in ALDHhigh CSC-enriched population and tumor initiation
(A) ALDH1 protein expression in LoVo cells treated as indicated for 5 days. (B) ALDH activities of LoVo cells treated with vehicle, PGE2 (100 nM) or GW627368X (20 µM) was analyzed by flow cytometry. The percentages indicate the percentage of ALDHhigh LoVo cells; i.e., LoVo cells with ALDH activity beyond the indicated thresholds. The gray line at the right side of the plot indicates the threshhold of the high ALDH activitiy. (C) ALDH1 protein levels in various carcinoma cells treated with vehicle or PGE2. The numbers indicate relative protein levels. (D) PGE2 increases LoVo TICs. LoVo cells pre-treated with vehicle or PGE2 (100 nM) were injected into SCID mice (5 × 104 cells/injection). After 6 weeks, tumors were isolated and weighted. Bars are means ± SE. (E) LoVo cells were cultured with tdTomato-MSCs, PGE2, NS398 or GW627368X, as indicated. After 5 days, the LoVo cells were isolated by cell sorting and injected into SCID mice (5 × 104 cells/injection). After 7 weeks, the tumors were isolated and weighted. Filled circles indicate individual tumor weights; Open circles indicate no tumor grew at the site of injection. Bars are means ± SE. (F) Panel (a), levels of PGE2 secreted by LoVoCM-treated MSCshsc and MSCshcox2. Data are means ± SE, n = 3. Panel (b), Weights of tumors derived from LoVo cells (5 × 104 cells/injection) injected into SCID mice either alone, with MSCshsc or with MSCshcox-2 (2 × 105 cells/injection). Filled circles indicate individual tumor weights; Open circles indicate no tumor grew at the site of injection. Bars are means ± SE.
Fig. 6
Fig. 6. PGE2 induces ALDHhigh cancer cells through the Akt/GSK-3/β-catenin signaling axis
(A) Activation of Akt/GSK3/β-catenin signaling in LoVo cells treated as indicated for one hour was analyzed by Western blots for phosphorylated Akt, total Akt, phosphorylated GSK-3α, phosphorylated GSK-3β, total GSK-3β, phosphorylated β-catenin, total β-catenin and GAPDH. The numbers indicate relative protein levels. (B) The distribution of E-cadherin (red), ZO-1 (green) and β-catenin (red) in LoVo cells treated with vehicle or PGE2 (100 nM) for 48 h was analyzed by immunofluorescence. Cell nuclei were stained with DAPI (in blue). The graphs show the fluorescence intensities along the dashed lines in the images of β-catenin staining. β-catenin intensities are in red lines and DAPI intensities are in blue lines. (C) Quantification of the levels of E-cadherin, ZO-1 and β-catenin associated with membrane and of nuclear β-catenin. The fluorescence intensities for the staining of these proteins in randomly selected cells in images (e.g. the cells crossed by dashed lines in panel B) were quantified. Bars are means ± SE, n > 50 for each bar. M β-catenin: membrane-bound β-catenin. N β-catenin; nuclear β-catenin. (D) Nuclear/cytosolic distribution of β-catenin in LoVo cells treated as indicated was analyzed by Western blot of β-catenin, GAPDH, and lamin B1, a nuclear envelope marker, in nuclear and cytosolic fractions. (E) mRNA expression of selected β-catenin/TCF dependent genes in LoVo cells treated with vehicle or PGE2 for 7 h. The mRNA levels of these genes were normalized to GAPDH mRNA. Data are fold induction by PGE2 (vs that of vehicle-treated LoVo cells). Data are means ± SE, n = 3. (F) ALDH activities of ALDHhigh LoVo cells treated with vehicle, PGE2, PGE2 + GW627368X (GW; 20µM), PGE2 + LY294002 (0.5 µM) or PGE2 + FH535 (6 µM) for 5 days (a–c). Percentages of ALDHhigh LoVo cells are presented in the table (d). The gray lines at the right side of the plots indicate the thersholds of the high ALDH activitiy.
Fig. 7
Fig. 7. MSCs in tumor stroma serve as an ALDHhigh cancer stem cell niche
(A) Immunofluorescence analyses were performed on tumors derived from LoVo cells (5 × 104 cells/injection) injected along with tdTomato-MSCs (5 × 105 cells/injection), using antibodies against tdTomato-RFP (in green), ALDH1 (in red, panel a), Fibroblast Surface Protein FSP (in red, panel b), and COX2 (cyan). Panels a and b are serial sections from one tumor. (B) IL-1 and COX2 mRNA expression in human colon and breast carcinoma. The “Fold Change” indicates the average mRNA levels of the three genes in colon mucinous carcinoma samples, compared to that of normal colon samples (panel a) and in TNBC samples, compared to that of non-TNBC samples (panel b). (C) The proposed interactions for induction and maintenance of EMT, cancer cell stemness and invasiveness by MSCs. AA: arachidonic acid
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