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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

Syntenin loss-of-function leads to reduced proliferation in different cancer cell models. (A) Western blots illustrating syntenin expression levels, at different time point, in B16F10, HT29, and MCF7 cells, after transfection with non-targeting (Si Ctrl) or syntenin (Si Syntenin) siRNAs. Tubulin was used as a loading control. (B) Bar graphs indicate the absolute number of living cells measured after different days of culture, as indicated. Note that significant differences were already observed at day 2. n = 3, bars represent mean value ± SD, n.s., non-significant, ∗P < 0.05 (Student’s t-test). (C) Western blots illustrating Syntenin expression levels in MCF7 cells 48 h after transfection with different constructs, as indicated. SiRNA Syntenin (Si Syntenin); non-targeting siRNA (Si Ctrl); expression vector for human Syntenin non-tagged and mutated for the siRNA targeting sequence (Syntenin OE); empty expression vector (Empty vector). Tubulin was used as loading control. (D) Bar graph indicating the absolute number of living MCF7 cells in Syntenin rescue experiments after different days in culture. n = 3, bars represent mean value ± SD, n.s., non-significant, ∗P < 0.05, ∗∗P < 0.01 (Student’s t-test).
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Figure 4: Syntenin loss-of-function leads to reduced proliferation in different cancer cell models. (A) Western blots illustrating syntenin expression levels, at different time point, in B16F10, HT29, and MCF7 cells, after transfection with non-targeting (Si Ctrl) or syntenin (Si Syntenin) siRNAs. Tubulin was used as a loading control. (B) Bar graphs indicate the absolute number of living cells measured after different days of culture, as indicated. Note that significant differences were already observed at day 2. n = 3, bars represent mean value ± SD, n.s., non-significant, ∗P < 0.05 (Student’s t-test). (C) Western blots illustrating Syntenin expression levels in MCF7 cells 48 h after transfection with different constructs, as indicated. SiRNA Syntenin (Si Syntenin); non-targeting siRNA (Si Ctrl); expression vector for human Syntenin non-tagged and mutated for the siRNA targeting sequence (Syntenin OE); empty expression vector (Empty vector). Tubulin was used as loading control. (D) Bar graph indicating the absolute number of living MCF7 cells in Syntenin rescue experiments after different days in culture. n = 3, bars represent mean value ± SD, n.s., non-significant, ∗P < 0.05, ∗∗P < 0.01 (Student’s t-test).

Mentions: We also tested the effect of syntenin depletion by transiently downregulating syntenin expression, using small interfering RNA (siRNA). Effective syntenin knockdown by mouse and human syntenin siRNAs over a period of 4 days was validated by Western blot analysis (Figure 4A). In these experiments, syntenin expression was downregulated by 90–95% compared to controls. We tested the proliferation of B16F10, HT29, and MCF7 cells transiently transfected with non-targeting control siRNA (si Ctrl) and syntenin siRNA (si Syntenin) by counting the number of viable cells every day over a period of 5 days (Figure 4B). Control B16F10 and HT29 cells (si Ctrl) showed a 30-fold increase in cell number after 5 days, while control MCF7 cells showed a 20-fold increase. Syntenin-depleted B16F10 and HT29 cells (siSyntenin) showed a 15-fold increase in cell number after 5 days, while syntenin-depleted MCF7 cells showed a 10-fold increase. A significant difference between controls and syntenin-depleted cells was observed in all models at day 2 and later times (Figure 4B). An effect of syntenin depletion on cell death could be ruled out because we observed in controls and syntenin depleted cells a similar extremely low number of Trypan blue (Supplementary Figure 2) and annexin-V positive cells (Supplementary Figure 3). To further validate syntenin effects on cellular proliferation, we also performed rescue experiments with MCF7 cells. Cells were treated with control (si Ctrl) and syntenin siRNAs (si Syntenin) for 24 h and then transiently transfected with an expression vector for wild-type syntenin mutated for the siRNA targeting sequence (syntenin OE) or the empty vector as a control. Total cell lysates analyzed by Western blot indicated rescue of syntenin expression above the control levels in MCF7 cells at 48h (Figure 4C), but these levels were still in the physiological range commonly observed in cell cultures. Rescued MCF7 cells showed a significant improvement of proliferation at day 2 and later, and 30-fold increase in cell number after 5 days of culture (Figure 4D). We assume that this increase in proliferation (by a factor 1.5 at day 5 when compared to control cells-Si Ctrl in Figure 4B), might result from the slight gain of syntenin expression in rescued cells. Altogether, the above data indicate that syntenin supports the capacity of single cells to form colonies, anchorage-independent cell growth, and proliferation in B16F10, HT29, and MCF7 cells and that cellular proliferation might be directly correlated to syntenin expression levels.


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)

Syntenin loss-of-function leads to reduced proliferation in different cancer cell models. (A) Western blots illustrating syntenin expression levels, at different time point, in B16F10, HT29, and MCF7 cells, after transfection with non-targeting (Si Ctrl) or syntenin (Si Syntenin) siRNAs. Tubulin was used as a loading control. (B) Bar graphs indicate the absolute number of living cells measured after different days of culture, as indicated. Note that significant differences were already observed at day 2. n = 3, bars represent mean value ± SD, n.s., non-significant, ∗P < 0.05 (Student’s t-test). (C) Western blots illustrating Syntenin expression levels in MCF7 cells 48 h after transfection with different constructs, as indicated. SiRNA Syntenin (Si Syntenin); non-targeting siRNA (Si Ctrl); expression vector for human Syntenin non-tagged and mutated for the siRNA targeting sequence (Syntenin OE); empty expression vector (Empty vector). Tubulin was used as loading control. (D) Bar graph indicating the absolute number of living MCF7 cells in Syntenin rescue experiments after different days in culture. n = 3, bars represent mean value ± SD, n.s., non-significant, ∗P < 0.05, ∗∗P < 0.01 (Student’s t-test).
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Figure 4: Syntenin loss-of-function leads to reduced proliferation in different cancer cell models. (A) Western blots illustrating syntenin expression levels, at different time point, in B16F10, HT29, and MCF7 cells, after transfection with non-targeting (Si Ctrl) or syntenin (Si Syntenin) siRNAs. Tubulin was used as a loading control. (B) Bar graphs indicate the absolute number of living cells measured after different days of culture, as indicated. Note that significant differences were already observed at day 2. n = 3, bars represent mean value ± SD, n.s., non-significant, ∗P < 0.05 (Student’s t-test). (C) Western blots illustrating Syntenin expression levels in MCF7 cells 48 h after transfection with different constructs, as indicated. SiRNA Syntenin (Si Syntenin); non-targeting siRNA (Si Ctrl); expression vector for human Syntenin non-tagged and mutated for the siRNA targeting sequence (Syntenin OE); empty expression vector (Empty vector). Tubulin was used as loading control. (D) Bar graph indicating the absolute number of living MCF7 cells in Syntenin rescue experiments after different days in culture. n = 3, bars represent mean value ± SD, n.s., non-significant, ∗P < 0.05, ∗∗P < 0.01 (Student’s t-test).
Mentions: We also tested the effect of syntenin depletion by transiently downregulating syntenin expression, using small interfering RNA (siRNA). Effective syntenin knockdown by mouse and human syntenin siRNAs over a period of 4 days was validated by Western blot analysis (Figure 4A). In these experiments, syntenin expression was downregulated by 90–95% compared to controls. We tested the proliferation of B16F10, HT29, and MCF7 cells transiently transfected with non-targeting control siRNA (si Ctrl) and syntenin siRNA (si Syntenin) by counting the number of viable cells every day over a period of 5 days (Figure 4B). Control B16F10 and HT29 cells (si Ctrl) showed a 30-fold increase in cell number after 5 days, while control MCF7 cells showed a 20-fold increase. Syntenin-depleted B16F10 and HT29 cells (siSyntenin) showed a 15-fold increase in cell number after 5 days, while syntenin-depleted MCF7 cells showed a 10-fold increase. A significant difference between controls and syntenin-depleted cells was observed in all models at day 2 and later times (Figure 4B). An effect of syntenin depletion on cell death could be ruled out because we observed in controls and syntenin depleted cells a similar extremely low number of Trypan blue (Supplementary Figure 2) and annexin-V positive cells (Supplementary Figure 3). To further validate syntenin effects on cellular proliferation, we also performed rescue experiments with MCF7 cells. Cells were treated with control (si Ctrl) and syntenin siRNAs (si Syntenin) for 24 h and then transiently transfected with an expression vector for wild-type syntenin mutated for the siRNA targeting sequence (syntenin OE) or the empty vector as a control. Total cell lysates analyzed by Western blot indicated rescue of syntenin expression above the control levels in MCF7 cells at 48h (Figure 4C), but these levels were still in the physiological range commonly observed in cell cultures. Rescued MCF7 cells showed a significant improvement of proliferation at day 2 and later, and 30-fold increase in cell number after 5 days of culture (Figure 4D). We assume that this increase in proliferation (by a factor 1.5 at day 5 when compared to control cells-Si Ctrl in Figure 4B), might result from the slight gain of syntenin expression in rescued cells. Altogether, the above data indicate that syntenin supports the capacity of single cells to form colonies, anchorage-independent cell growth, and proliferation in B16F10, HT29, and MCF7 cells and that cellular proliferation might be directly correlated to syntenin expression levels.

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