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mPGES-1 in prostate cancer controls stemness and amplifies epidermal growth factor receptor-driven oncogenicity.

Finetti F, Terzuoli E, Giachetti A, Santi R, Villari D, Hanaka H, Radmark O, Ziche M, Donnini S - Endocr. Relat. Cancer (2015)

Bottom Line: There is evidence that an inflammatory microenvironment is associated with the development and progression of prostate cancer (PCa), although the determinants of intrinsic inflammation in PCa cells are not completely understood.They also show increased capacity to survive irrespective of anchorage condition, and overexpress EGFR compared to mPGES-1(KD) cells. mPGES-1 expression correlates with increased in vivo tumour growth and metastasis.Although EGFR inhibition reduces mPGES-1(SC) and mPGES-1(KD) cell xenograft tumour growth, we show that mPGES-1/PGE2 signalling sensitizes tumour cells to EGFR inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Life SciencesUniversity of Siena, Via Aldo Moro 2, 53100 Siena, ItalyDepartment of Surgery and Translational MedicineUniversity of Florence, Largo Brambilla 3, 50134 Firenze, ItalyDepartment of Clinical and Experimental MedicineUniversity of Florence, Viale Pieraccini 18, 50139 Firenze, ItalyDepartment of Medical Biochemistry and BiophysicsKarolinska Institutet, SE-171 77 Stockholm, SwedenIstituto Toscano Tumori (ITT)Firenze, Italy.

No MeSH data available.


Related in: MedlinePlus

EGFR activation mediates mPGES-1/PGE2-dependent EMT of DU145 cells. (A) FACS analysis for CD24 and CD44 expression in DU145 mPGES-1SC-and mPGES-1KD cells, and (B) western blot analysis and quantification of α6 and β1-integrin expression in mPGES-1SC and mPGES-1KD cells. Data are representative of three different experiments and quantification was performed by Image J. *P<0.01 vs DU145 mPGES-1SC (C) RT-PCR analysis of Nanog and Oct4 expression in mPGES-1SC and mPGES-1KD DU145 cells. Data are reported as fold increase vs mPGES-1SC cells. **P<0.01 vs DU145 mPGES-1SC. (D) Cell viability of DU145 mPGES-1SC and mPGES-1KD in suspension in 0.1% of serum. Results are expressed as % of dead cells. **P<0.01 and ***P<0.001 vs DU145 mPGES-1SC. (E) Western blot analysis of caspase 3 activation in DU145 cells grown in suspension for the indicated time. (F) Adhesion of DU145 mPGES-1SC and mPGES-1KD untreated or pretreated with PGE2 (1 μmol/l, 96 h) on fibronectin coated-96 well plate. Cell adhesion was evaluated after 2 h of incubation in 1% serum. Results (three experiments in triplicate) are expressed as % of adherent cells relative to untreated mPGES-1SC. **P<0.01 vs mPGES-1SC; ###P<0.01 vs mPGES-1KD. (G) CytoTracker labeled DU145 mPGES-1SCor mPGES-1KD cells were allowed to attach to untreated or TNFα-pre-treated HUVEC monolayers in 48 well plates for 1 h. Adherent cells were lysed and quantified. Results (three experiments in duplicate) are expressed as fold increase to the adhesion of mPGES-1SC on untreated HUVEC. **P<0.01 vs mPGES-1SC. (H) Migration of DU145 mPGES-1SC and mPGES-1KD toward HUVEC monolayer. CytoTracker labeled tumor cells were seeded in the upper side of 48 well-transwell and migration was evaluated after 2 h by measuring the fluorescence in the lower side of the well. Data are reported as relative fluorescence unit of three experiments run in duplicate. **P<0.01 vs DU145 mPGES-1SC.
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fig3: EGFR activation mediates mPGES-1/PGE2-dependent EMT of DU145 cells. (A) FACS analysis for CD24 and CD44 expression in DU145 mPGES-1SC-and mPGES-1KD cells, and (B) western blot analysis and quantification of α6 and β1-integrin expression in mPGES-1SC and mPGES-1KD cells. Data are representative of three different experiments and quantification was performed by Image J. *P<0.01 vs DU145 mPGES-1SC (C) RT-PCR analysis of Nanog and Oct4 expression in mPGES-1SC and mPGES-1KD DU145 cells. Data are reported as fold increase vs mPGES-1SC cells. **P<0.01 vs DU145 mPGES-1SC. (D) Cell viability of DU145 mPGES-1SC and mPGES-1KD in suspension in 0.1% of serum. Results are expressed as % of dead cells. **P<0.01 and ***P<0.001 vs DU145 mPGES-1SC. (E) Western blot analysis of caspase 3 activation in DU145 cells grown in suspension for the indicated time. (F) Adhesion of DU145 mPGES-1SC and mPGES-1KD untreated or pretreated with PGE2 (1 μmol/l, 96 h) on fibronectin coated-96 well plate. Cell adhesion was evaluated after 2 h of incubation in 1% serum. Results (three experiments in triplicate) are expressed as % of adherent cells relative to untreated mPGES-1SC. **P<0.01 vs mPGES-1SC; ###P<0.01 vs mPGES-1KD. (G) CytoTracker labeled DU145 mPGES-1SCor mPGES-1KD cells were allowed to attach to untreated or TNFα-pre-treated HUVEC monolayers in 48 well plates for 1 h. Adherent cells were lysed and quantified. Results (three experiments in duplicate) are expressed as fold increase to the adhesion of mPGES-1SC on untreated HUVEC. **P<0.01 vs mPGES-1SC. (H) Migration of DU145 mPGES-1SC and mPGES-1KD toward HUVEC monolayer. CytoTracker labeled tumor cells were seeded in the upper side of 48 well-transwell and migration was evaluated after 2 h by measuring the fluorescence in the lower side of the well. Data are reported as relative fluorescence unit of three experiments run in duplicate. **P<0.01 vs DU145 mPGES-1SC.

Mentions: We also observed a prevalence of stem-cell-like markers, regarded as indicators of tumour invasiveness, in DU145 mPGES-1SC compared to mPGES-1KD cells. This was indicated by the large increase in the CD44+/CD24− ratio, the decrease in α6-integrin and the increase in β1 integrin, as well as transcription factors Nanog and Oct4 in mPGES-1SC (Fig. 3A, B and C), all pluripotency maintaining factors overexpressed in PCa stem cells (Klarmann et al. 2009, Rentala et al. 2010, Marthick & Dickinson 2012, Nanta et al. 2013, Hoogland et al. 2014).Similar results were obtained for the PC-3 cell line. PC-3 cells expressed constitutive mPGES-1, and knocking down the enzyme significantlyreduced PGE2 output (>60%, P<0.01 vs mPGES-1SC), promoted epithelial phenotype, decreased cell clonogenicity and reduced the expression of stem cell markers (Supplementary Figure 3A, B, C, D, E and F, see section on supplementary data given at the end of this article). We also recorded anchorage-independent cell viability in mPGES-1SC cells, in contrast to the drastic decline seen in mPGES-1KD cells, associated with caspase-3 activation (Fig. 3D and E, and Supplementary Figure 3G and H). Adhesion studies with fibronectin coated-wells or endothelial cells revealed that mPGES-1SC cells rapidly adhered (2 h) to the matrix or endothelium (whether or not it had been activated by TNFα to favour cell–cell interaction and transmigration), whereas mPGES-1KD cells displayed a significantly delayed adhesion (F and G). Since knock down of mPGES-1 in cells does not affect cell survival in vitro, as measured by MTT assay (Abs 0.95±0.08 and 0.88±0.11 for DU145 mPGES-1SC and mPGES-1KD cells; Abs 0.74±0.06 and 0.79±0.09 for PC-3 mPGES-1SC and mPGES-1KD cells respectively), or cell apoptosis in suspension up to 24–48 h, we conclude that the inhibition of adhesion in mPGES-1KD cells depends on the modification of cytoskeletal organization, for example of vimentin and fibronectin. Finally, by measuring trans-endothelial migration, we showed a far greater ability (nearly threefold) of mPGES-1SC than mPGES-1KD cells to cross cell layers (Fig. 3H), indicating the involvement of mPGES-1/PGE2 signalling in prostate tumour invasiveness.


mPGES-1 in prostate cancer controls stemness and amplifies epidermal growth factor receptor-driven oncogenicity.

Finetti F, Terzuoli E, Giachetti A, Santi R, Villari D, Hanaka H, Radmark O, Ziche M, Donnini S - Endocr. Relat. Cancer (2015)

EGFR activation mediates mPGES-1/PGE2-dependent EMT of DU145 cells. (A) FACS analysis for CD24 and CD44 expression in DU145 mPGES-1SC-and mPGES-1KD cells, and (B) western blot analysis and quantification of α6 and β1-integrin expression in mPGES-1SC and mPGES-1KD cells. Data are representative of three different experiments and quantification was performed by Image J. *P<0.01 vs DU145 mPGES-1SC (C) RT-PCR analysis of Nanog and Oct4 expression in mPGES-1SC and mPGES-1KD DU145 cells. Data are reported as fold increase vs mPGES-1SC cells. **P<0.01 vs DU145 mPGES-1SC. (D) Cell viability of DU145 mPGES-1SC and mPGES-1KD in suspension in 0.1% of serum. Results are expressed as % of dead cells. **P<0.01 and ***P<0.001 vs DU145 mPGES-1SC. (E) Western blot analysis of caspase 3 activation in DU145 cells grown in suspension for the indicated time. (F) Adhesion of DU145 mPGES-1SC and mPGES-1KD untreated or pretreated with PGE2 (1 μmol/l, 96 h) on fibronectin coated-96 well plate. Cell adhesion was evaluated after 2 h of incubation in 1% serum. Results (three experiments in triplicate) are expressed as % of adherent cells relative to untreated mPGES-1SC. **P<0.01 vs mPGES-1SC; ###P<0.01 vs mPGES-1KD. (G) CytoTracker labeled DU145 mPGES-1SCor mPGES-1KD cells were allowed to attach to untreated or TNFα-pre-treated HUVEC monolayers in 48 well plates for 1 h. Adherent cells were lysed and quantified. Results (three experiments in duplicate) are expressed as fold increase to the adhesion of mPGES-1SC on untreated HUVEC. **P<0.01 vs mPGES-1SC. (H) Migration of DU145 mPGES-1SC and mPGES-1KD toward HUVEC monolayer. CytoTracker labeled tumor cells were seeded in the upper side of 48 well-transwell and migration was evaluated after 2 h by measuring the fluorescence in the lower side of the well. Data are reported as relative fluorescence unit of three experiments run in duplicate. **P<0.01 vs DU145 mPGES-1SC.
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fig3: EGFR activation mediates mPGES-1/PGE2-dependent EMT of DU145 cells. (A) FACS analysis for CD24 and CD44 expression in DU145 mPGES-1SC-and mPGES-1KD cells, and (B) western blot analysis and quantification of α6 and β1-integrin expression in mPGES-1SC and mPGES-1KD cells. Data are representative of three different experiments and quantification was performed by Image J. *P<0.01 vs DU145 mPGES-1SC (C) RT-PCR analysis of Nanog and Oct4 expression in mPGES-1SC and mPGES-1KD DU145 cells. Data are reported as fold increase vs mPGES-1SC cells. **P<0.01 vs DU145 mPGES-1SC. (D) Cell viability of DU145 mPGES-1SC and mPGES-1KD in suspension in 0.1% of serum. Results are expressed as % of dead cells. **P<0.01 and ***P<0.001 vs DU145 mPGES-1SC. (E) Western blot analysis of caspase 3 activation in DU145 cells grown in suspension for the indicated time. (F) Adhesion of DU145 mPGES-1SC and mPGES-1KD untreated or pretreated with PGE2 (1 μmol/l, 96 h) on fibronectin coated-96 well plate. Cell adhesion was evaluated after 2 h of incubation in 1% serum. Results (three experiments in triplicate) are expressed as % of adherent cells relative to untreated mPGES-1SC. **P<0.01 vs mPGES-1SC; ###P<0.01 vs mPGES-1KD. (G) CytoTracker labeled DU145 mPGES-1SCor mPGES-1KD cells were allowed to attach to untreated or TNFα-pre-treated HUVEC monolayers in 48 well plates for 1 h. Adherent cells were lysed and quantified. Results (three experiments in duplicate) are expressed as fold increase to the adhesion of mPGES-1SC on untreated HUVEC. **P<0.01 vs mPGES-1SC. (H) Migration of DU145 mPGES-1SC and mPGES-1KD toward HUVEC monolayer. CytoTracker labeled tumor cells were seeded in the upper side of 48 well-transwell and migration was evaluated after 2 h by measuring the fluorescence in the lower side of the well. Data are reported as relative fluorescence unit of three experiments run in duplicate. **P<0.01 vs DU145 mPGES-1SC.
Mentions: We also observed a prevalence of stem-cell-like markers, regarded as indicators of tumour invasiveness, in DU145 mPGES-1SC compared to mPGES-1KD cells. This was indicated by the large increase in the CD44+/CD24− ratio, the decrease in α6-integrin and the increase in β1 integrin, as well as transcription factors Nanog and Oct4 in mPGES-1SC (Fig. 3A, B and C), all pluripotency maintaining factors overexpressed in PCa stem cells (Klarmann et al. 2009, Rentala et al. 2010, Marthick & Dickinson 2012, Nanta et al. 2013, Hoogland et al. 2014).Similar results were obtained for the PC-3 cell line. PC-3 cells expressed constitutive mPGES-1, and knocking down the enzyme significantlyreduced PGE2 output (>60%, P<0.01 vs mPGES-1SC), promoted epithelial phenotype, decreased cell clonogenicity and reduced the expression of stem cell markers (Supplementary Figure 3A, B, C, D, E and F, see section on supplementary data given at the end of this article). We also recorded anchorage-independent cell viability in mPGES-1SC cells, in contrast to the drastic decline seen in mPGES-1KD cells, associated with caspase-3 activation (Fig. 3D and E, and Supplementary Figure 3G and H). Adhesion studies with fibronectin coated-wells or endothelial cells revealed that mPGES-1SC cells rapidly adhered (2 h) to the matrix or endothelium (whether or not it had been activated by TNFα to favour cell–cell interaction and transmigration), whereas mPGES-1KD cells displayed a significantly delayed adhesion (F and G). Since knock down of mPGES-1 in cells does not affect cell survival in vitro, as measured by MTT assay (Abs 0.95±0.08 and 0.88±0.11 for DU145 mPGES-1SC and mPGES-1KD cells; Abs 0.74±0.06 and 0.79±0.09 for PC-3 mPGES-1SC and mPGES-1KD cells respectively), or cell apoptosis in suspension up to 24–48 h, we conclude that the inhibition of adhesion in mPGES-1KD cells depends on the modification of cytoskeletal organization, for example of vimentin and fibronectin. Finally, by measuring trans-endothelial migration, we showed a far greater ability (nearly threefold) of mPGES-1SC than mPGES-1KD cells to cross cell layers (Fig. 3H), indicating the involvement of mPGES-1/PGE2 signalling in prostate tumour invasiveness.

Bottom Line: There is evidence that an inflammatory microenvironment is associated with the development and progression of prostate cancer (PCa), although the determinants of intrinsic inflammation in PCa cells are not completely understood.They also show increased capacity to survive irrespective of anchorage condition, and overexpress EGFR compared to mPGES-1(KD) cells. mPGES-1 expression correlates with increased in vivo tumour growth and metastasis.Although EGFR inhibition reduces mPGES-1(SC) and mPGES-1(KD) cell xenograft tumour growth, we show that mPGES-1/PGE2 signalling sensitizes tumour cells to EGFR inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Life SciencesUniversity of Siena, Via Aldo Moro 2, 53100 Siena, ItalyDepartment of Surgery and Translational MedicineUniversity of Florence, Largo Brambilla 3, 50134 Firenze, ItalyDepartment of Clinical and Experimental MedicineUniversity of Florence, Viale Pieraccini 18, 50139 Firenze, ItalyDepartment of Medical Biochemistry and BiophysicsKarolinska Institutet, SE-171 77 Stockholm, SwedenIstituto Toscano Tumori (ITT)Firenze, Italy.

No MeSH data available.


Related in: MedlinePlus