Limits...
Syntenin controls migration, growth, proliferation, and cell cycle progression in cancer cells.

Kashyap R, Roucourt B, Lembo F, Fares J, Carcavilla AM, Restouin A, Zimmermann P, Ghossoub R - Front Pharmacol (2015)

Bottom Line: In human adulthood, syntenin gain-of-function is increasingly associated with various cancers and poor prognosis.We observed decreased migration, growth, and proliferation of the mouse melanoma cell line B16F10, the human colon cancer cell line HT29 and the human breast cancer cell line MCF7.We further documented that syntenin controls the presence of active β1 integrin at the cell membrane and G1/S cell cycle transition as well as the expression levels of CDK4, Cyclin D2, and Retinoblastoma proteins.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Signal Integration in Cell Fate Decision, Department of Human Genetics, KU Leuven Leuven, Belgium ; Centre de Recherche en Cancérologie de Marseille, Aix-Marseille Université Marseille, France ; Inserm U1068, Institut Paoli-Calmettes Marseille, France ; Centre National de la Recherche Scientifique, UMR7258 Marseille, France.

ABSTRACT
The scaffold protein syntenin abounds during fetal life where it is important for developmental movements. In human adulthood, syntenin gain-of-function is increasingly associated with various cancers and poor prognosis. Depending on the cancer model analyzed, syntenin affects various signaling pathways. We previously have shown that syntenin allows syndecan heparan sulfate proteoglycans to escape degradation. This indicates that syntenin has the potential to support sustained signaling of a plethora of growth factors and adhesion molecules. Here, we aim to clarify the impact of syntenin loss-of-function on cancer cell migration, growth, and proliferation, using cells from various cancer types and syntenin shRNA and siRNA silencing approaches. We observed decreased migration, growth, and proliferation of the mouse melanoma cell line B16F10, the human colon cancer cell line HT29 and the human breast cancer cell line MCF7. We further documented that syntenin controls the presence of active β1 integrin at the cell membrane and G1/S cell cycle transition as well as the expression levels of CDK4, Cyclin D2, and Retinoblastoma proteins. These data confirm that syntenin supports the migration and growth of tumor cells, independently of their origin, and further highlight the attractiveness of syntenin as potential therapeutic target.

No MeSH data available.


Related in: MedlinePlus

Downregulation of syntenin impairs cell cycle G1/S transition in MCF7 cells. (A) Bar graph representing the percentage of cells in G1, S, and G2/M cell cycle phases after synchronization. Cells were transfected with non-targeting siRNA (Si Ctrl) or Syntenin siRNA (Si Syntenin). n = 5, bars represent mean value ± SD, ∗P < 0.05 (Student’s t-test). (B) Bar graphs illustrate one kinetic experiment indicating the percentage of cells in G1, S, and G2/M cell cycle phases after serum stimulation, at different time points, as indicated, in cells transfected with non-targeting siRNA (top) or Syntenin si RNA (bottom). (C) Western blot comparing the expression levels of different cell cycle markers, as indicated, in control cells (Si Ctrl) and Syntenin-depleted (si Syntenin) cells. Tubulin was used as loading control. Note that the expression of Cyclin D2, CDK4, and Rb is downregulated in syntenin-depleted cells. (D) Bar graph indicating the expression level of cell cycle markers in Syntenin-depleted cells relative to controls (taken as 100%). n = 3, 4, and 5 for Cyclin D2, Rb, and CDK4, respectively, bars represent mean value ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (Student’s t-test).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4612656&req=5

Figure 5: Downregulation of syntenin impairs cell cycle G1/S transition in MCF7 cells. (A) Bar graph representing the percentage of cells in G1, S, and G2/M cell cycle phases after synchronization. Cells were transfected with non-targeting siRNA (Si Ctrl) or Syntenin siRNA (Si Syntenin). n = 5, bars represent mean value ± SD, ∗P < 0.05 (Student’s t-test). (B) Bar graphs illustrate one kinetic experiment indicating the percentage of cells in G1, S, and G2/M cell cycle phases after serum stimulation, at different time points, as indicated, in cells transfected with non-targeting siRNA (top) or Syntenin si RNA (bottom). (C) Western blot comparing the expression levels of different cell cycle markers, as indicated, in control cells (Si Ctrl) and Syntenin-depleted (si Syntenin) cells. Tubulin was used as loading control. Note that the expression of Cyclin D2, CDK4, and Rb is downregulated in syntenin-depleted cells. (D) Bar graph indicating the expression level of cell cycle markers in Syntenin-depleted cells relative to controls (taken as 100%). n = 3, 4, and 5 for Cyclin D2, Rb, and CDK4, respectively, bars represent mean value ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (Student’s t-test).

Mentions: To better understand the effect of syntenin loss-of-function on cell growth and proliferation, we tested whether cells were arrested in a particular phase of the cell cycle. Syntenin-depleted (si Syntenin) and control (si Ctrl) MCF7 cells were synchronized by serum starvation for 24 h. Cells were then serum-stimulated for 24 h and analyzed for different cell cycle phases by flow cytometry. Syntenin-depleted cells showed a significant increase in G1 phase and a significant decrease in S and G2/M phases when compared to control cells (Figure 5A). The effects of serum-stimulation on the different phases of the cell cycle were also analyzed at different time points over a period of 2 days in two independent experiments; see Figure 5B for one illustration. At early time points (from 0 to 12 h), control and syntenin-depleted cells did not show drastic differences in G1 phase (on average 73% in controls and 71% in depleted cells) but the percentage of S phase in control cells was twice more important compared to syntenin-depleted cells (on average 5.5% in controls and 2.5% in depleted cells). Starting from 18 h, S phase was increased to reach 19% at 48 h in control cells, while in syntenin-depleted cells, S phase poorly increased over time to reach at maximum 6% after 48h of serum stimulation. Moreover, the ratio of cells in G1, S, and G2/M stayed quite constant all along the experiment in syntenin depletion conditions on the contrary to what we observed in controls (Figure 5B). Taken together, these data suggest that syntenin depletion induces a defect in G1/S cell cycle transition. Of prime importance in this process is cyclin D2, which binds and activates cyclin-dependent kinases 4 (CDK4), thereby phosphorylating Retinoblastoma protein (Rb) and promoting progression from mid to late G1 (Sherr, 1994). We therefore tested for the expression levels of these three cell cycle regulators of G1/S transition by Western blot and observed that they are all significantly downregulated in syntenin-depleted cells compared to control cells (Figures 5C,D). Altogether, our data indicate that syntenin might control cell growth by acting on G1/S cell cycle transition.


Syntenin controls migration, growth, proliferation, and cell cycle progression in cancer cells.

Kashyap R, Roucourt B, Lembo F, Fares J, Carcavilla AM, Restouin A, Zimmermann P, Ghossoub R - Front Pharmacol (2015)

Downregulation of syntenin impairs cell cycle G1/S transition in MCF7 cells. (A) Bar graph representing the percentage of cells in G1, S, and G2/M cell cycle phases after synchronization. Cells were transfected with non-targeting siRNA (Si Ctrl) or Syntenin siRNA (Si Syntenin). n = 5, bars represent mean value ± SD, ∗P < 0.05 (Student’s t-test). (B) Bar graphs illustrate one kinetic experiment indicating the percentage of cells in G1, S, and G2/M cell cycle phases after serum stimulation, at different time points, as indicated, in cells transfected with non-targeting siRNA (top) or Syntenin si RNA (bottom). (C) Western blot comparing the expression levels of different cell cycle markers, as indicated, in control cells (Si Ctrl) and Syntenin-depleted (si Syntenin) cells. Tubulin was used as loading control. Note that the expression of Cyclin D2, CDK4, and Rb is downregulated in syntenin-depleted cells. (D) Bar graph indicating the expression level of cell cycle markers in Syntenin-depleted cells relative to controls (taken as 100%). n = 3, 4, and 5 for Cyclin D2, Rb, and CDK4, respectively, bars represent mean value ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (Student’s t-test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4612656&req=5

Figure 5: Downregulation of syntenin impairs cell cycle G1/S transition in MCF7 cells. (A) Bar graph representing the percentage of cells in G1, S, and G2/M cell cycle phases after synchronization. Cells were transfected with non-targeting siRNA (Si Ctrl) or Syntenin siRNA (Si Syntenin). n = 5, bars represent mean value ± SD, ∗P < 0.05 (Student’s t-test). (B) Bar graphs illustrate one kinetic experiment indicating the percentage of cells in G1, S, and G2/M cell cycle phases after serum stimulation, at different time points, as indicated, in cells transfected with non-targeting siRNA (top) or Syntenin si RNA (bottom). (C) Western blot comparing the expression levels of different cell cycle markers, as indicated, in control cells (Si Ctrl) and Syntenin-depleted (si Syntenin) cells. Tubulin was used as loading control. Note that the expression of Cyclin D2, CDK4, and Rb is downregulated in syntenin-depleted cells. (D) Bar graph indicating the expression level of cell cycle markers in Syntenin-depleted cells relative to controls (taken as 100%). n = 3, 4, and 5 for Cyclin D2, Rb, and CDK4, respectively, bars represent mean value ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (Student’s t-test).
Mentions: To better understand the effect of syntenin loss-of-function on cell growth and proliferation, we tested whether cells were arrested in a particular phase of the cell cycle. Syntenin-depleted (si Syntenin) and control (si Ctrl) MCF7 cells were synchronized by serum starvation for 24 h. Cells were then serum-stimulated for 24 h and analyzed for different cell cycle phases by flow cytometry. Syntenin-depleted cells showed a significant increase in G1 phase and a significant decrease in S and G2/M phases when compared to control cells (Figure 5A). The effects of serum-stimulation on the different phases of the cell cycle were also analyzed at different time points over a period of 2 days in two independent experiments; see Figure 5B for one illustration. At early time points (from 0 to 12 h), control and syntenin-depleted cells did not show drastic differences in G1 phase (on average 73% in controls and 71% in depleted cells) but the percentage of S phase in control cells was twice more important compared to syntenin-depleted cells (on average 5.5% in controls and 2.5% in depleted cells). Starting from 18 h, S phase was increased to reach 19% at 48 h in control cells, while in syntenin-depleted cells, S phase poorly increased over time to reach at maximum 6% after 48h of serum stimulation. Moreover, the ratio of cells in G1, S, and G2/M stayed quite constant all along the experiment in syntenin depletion conditions on the contrary to what we observed in controls (Figure 5B). Taken together, these data suggest that syntenin depletion induces a defect in G1/S cell cycle transition. Of prime importance in this process is cyclin D2, which binds and activates cyclin-dependent kinases 4 (CDK4), thereby phosphorylating Retinoblastoma protein (Rb) and promoting progression from mid to late G1 (Sherr, 1994). We therefore tested for the expression levels of these three cell cycle regulators of G1/S transition by Western blot and observed that they are all significantly downregulated in syntenin-depleted cells compared to control cells (Figures 5C,D). Altogether, our data indicate that syntenin might control cell growth by acting on G1/S cell cycle transition.

Bottom Line: In human adulthood, syntenin gain-of-function is increasingly associated with various cancers and poor prognosis.We observed decreased migration, growth, and proliferation of the mouse melanoma cell line B16F10, the human colon cancer cell line HT29 and the human breast cancer cell line MCF7.We further documented that syntenin controls the presence of active β1 integrin at the cell membrane and G1/S cell cycle transition as well as the expression levels of CDK4, Cyclin D2, and Retinoblastoma proteins.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Signal Integration in Cell Fate Decision, Department of Human Genetics, KU Leuven Leuven, Belgium ; Centre de Recherche en Cancérologie de Marseille, Aix-Marseille Université Marseille, France ; Inserm U1068, Institut Paoli-Calmettes Marseille, France ; Centre National de la Recherche Scientifique, UMR7258 Marseille, France.

ABSTRACT
The scaffold protein syntenin abounds during fetal life where it is important for developmental movements. In human adulthood, syntenin gain-of-function is increasingly associated with various cancers and poor prognosis. Depending on the cancer model analyzed, syntenin affects various signaling pathways. We previously have shown that syntenin allows syndecan heparan sulfate proteoglycans to escape degradation. This indicates that syntenin has the potential to support sustained signaling of a plethora of growth factors and adhesion molecules. Here, we aim to clarify the impact of syntenin loss-of-function on cancer cell migration, growth, and proliferation, using cells from various cancer types and syntenin shRNA and siRNA silencing approaches. We observed decreased migration, growth, and proliferation of the mouse melanoma cell line B16F10, the human colon cancer cell line HT29 and the human breast cancer cell line MCF7. We further documented that syntenin controls the presence of active β1 integrin at the cell membrane and G1/S cell cycle transition as well as the expression levels of CDK4, Cyclin D2, and Retinoblastoma proteins. These data confirm that syntenin supports the migration and growth of tumor cells, independently of their origin, and further highlight the attractiveness of syntenin as potential therapeutic target.

No MeSH data available.


Related in: MedlinePlus