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Paxillin promotes colorectal tumor invasion and poor patient outcomes via ERK-mediated stabilization of Bcl-2 protein by phosphorylation at Serine 87.

Huang CC, Wu DW, Lin PL, Lee H - Oncotarget (2015)

Bottom Line: Stabilization of Bcl-2 protein by paxillin (PXN)-mediated ERK activation was recently reported to cause an unfavorable response to 5-Fluorouracil-based chemotherapy.Here, we present evidence from cell and animal models to demonstrate that stabilization of Bcl-2 protein by phosphorylation at Serine 87 (pBcl-2-S87) via PXN-mediated ERK activation is responsible for cancer cell invasiveness and occurs via upregulation of MMP2 expression.In conclusion, PXN promotes Bcl-2 phosphorylation at Serine 87 via PXN-mediated ERK activation, and its stabilization associated with increased tumor formation efficacy in mice and poor patient outcome in colorectal cancer patients.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Chung Shan Medical University, Taichung, Taiwan.

ABSTRACT
Stabilization of Bcl-2 protein by paxillin (PXN)-mediated ERK activation was recently reported to cause an unfavorable response to 5-Fluorouracil-based chemotherapy. Here, we present evidence from cell and animal models to demonstrate that stabilization of Bcl-2 protein by phosphorylation at Serine 87 (pBcl-2-S87) via PXN-mediated ERK activation is responsible for cancer cell invasiveness and occurs via upregulation of MMP2 expression. Immunostainings of 190 tumors resected from colorectal cancer patients indicated that PXN expression was positively correlated with Bcl-2, pBcl-2-S87, and MMP2 expression. A positive correlation of pBcl-2-S87 with Bcl-2 and MMP2 was also observed in this study population. Patients with high PXN, Bcl-2, pBcl-2-S87, and MMP2 had poor overall survival (OS) and shorter relapse free survival (RFS). In conclusion, PXN promotes Bcl-2 phosphorylation at Serine 87 via PXN-mediated ERK activation, and its stabilization associated with increased tumor formation efficacy in mice and poor patient outcome in colorectal cancer patients.

No MeSH data available.


Related in: MedlinePlus

Increased stabilization of Bcl-2 protein by PXN overexpression promotes cell invasion in colorectal cancer cells(A) HCT116 cells were transfected with two different PXN shRNAs. HT29 cells were transfected with two doses of WT PXN and cell lysates were separated by SDS-PAGE to evaluate Bcl-2 and PXN expression by western blotting using specific antibodies. (B) HCT116 cells were transfected with two different PXN shRNAs and then treated with 0 or 5 μmol/L MG132 for an additional 5 hours. Cell lysates were separated by SDS-PAGE to evaluate Bcl-2, pBcl-2-S87, and PXN expression by western blotting using specific antibodies. VC: vector control; NC: non-specific shRNA control. (C) HT29 cells were transfected with WT PXN or mutant PXN-Y31/118F for 36 hours and then treated with 0 or 0.2 μmol/L Src inhibitor (Dasatinib) for an additional 5 hours. The cells lysates were separated by SDS-PAGE to evaluate PXN, pPXN-Y31, pPXN-Y118, ERK, p-ERK, and Bcl-2 expression by western blotting using specific antibodies. (D) HCT116 and PXN-overexpressing HT29 cells were treated with two ERK inhibitors (10 μmol/L U0126 or 5 μmol/L AZD6244, AZD) for 5 hours. These cells were treated with 0 or 5 μmol/L MG132, and then cells were lysed. The cell lysates were separated by SDS-PAGE to evaluate Bcl-2 and pBcl-2-S87 expression by western blotting using specific antibodies. (E) HCT116 cells were transfected with PXN shRNA and then treated with 0 or 5 μmol/L MG132 for an additional 5 hours. HT29 cells were co-transfected with WT PXN and shBcl-2. Cell lysates were separated by SDS-PAGE to evaluate Bcl-2 and PXN expression by western blotting using specific antibodies. (F) Representative numbers of invading HCT116 and HT29 cells transfected with the indicated combinations of WT PXN, shPXN, or shBcl-2. The invasion capability is summarized for the indicated cells.
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Figure 1: Increased stabilization of Bcl-2 protein by PXN overexpression promotes cell invasion in colorectal cancer cells(A) HCT116 cells were transfected with two different PXN shRNAs. HT29 cells were transfected with two doses of WT PXN and cell lysates were separated by SDS-PAGE to evaluate Bcl-2 and PXN expression by western blotting using specific antibodies. (B) HCT116 cells were transfected with two different PXN shRNAs and then treated with 0 or 5 μmol/L MG132 for an additional 5 hours. Cell lysates were separated by SDS-PAGE to evaluate Bcl-2, pBcl-2-S87, and PXN expression by western blotting using specific antibodies. VC: vector control; NC: non-specific shRNA control. (C) HT29 cells were transfected with WT PXN or mutant PXN-Y31/118F for 36 hours and then treated with 0 or 0.2 μmol/L Src inhibitor (Dasatinib) for an additional 5 hours. The cells lysates were separated by SDS-PAGE to evaluate PXN, pPXN-Y31, pPXN-Y118, ERK, p-ERK, and Bcl-2 expression by western blotting using specific antibodies. (D) HCT116 and PXN-overexpressing HT29 cells were treated with two ERK inhibitors (10 μmol/L U0126 or 5 μmol/L AZD6244, AZD) for 5 hours. These cells were treated with 0 or 5 μmol/L MG132, and then cells were lysed. The cell lysates were separated by SDS-PAGE to evaluate Bcl-2 and pBcl-2-S87 expression by western blotting using specific antibodies. (E) HCT116 cells were transfected with PXN shRNA and then treated with 0 or 5 μmol/L MG132 for an additional 5 hours. HT29 cells were co-transfected with WT PXN and shBcl-2. Cell lysates were separated by SDS-PAGE to evaluate Bcl-2 and PXN expression by western blotting using specific antibodies. (F) Representative numbers of invading HCT116 and HT29 cells transfected with the indicated combinations of WT PXN, shPXN, or shBcl-2. The invasion capability is summarized for the indicated cells.

Mentions: We explored the possibility that PXN promoted cell invasiveness via increased Bcl-2 protein stability via ERK-mediated Bcl-2 phosphorylation at Serine 87. High and low PXN-expressing HCT116 and HT29 cells were enrolled for knockdown and overexpression of PXN using its small hairpin (sh)RNAs and its expression vector. Bcl-2 expression was markedly decreased by PXN-knockdown in HCT116 cells when compared with HCT116 cells with non-specific shRNA transfection (NC) (Figure 1A left panel). Conversely, Bcl-2 expression was increased in a dose-dependent manner by PXN-overexpression in HT29 cells (Figure 1A right panel). Interestingly, Bcl-2 and pBcl-2-S87 expression were diminished by PXN-knockdown in HCT116 cells (Figure 1B left panel), but the decrease in Bcl-2 expression by PXN-knockdown was rescued by MG132 treatment (Figure 1B right panel). However, no restoration of pBcl-2-S87 was seen in PXN-knockdown HCT116 cells treated with MG132 (Figure 1B right panel).


Paxillin promotes colorectal tumor invasion and poor patient outcomes via ERK-mediated stabilization of Bcl-2 protein by phosphorylation at Serine 87.

Huang CC, Wu DW, Lin PL, Lee H - Oncotarget (2015)

Increased stabilization of Bcl-2 protein by PXN overexpression promotes cell invasion in colorectal cancer cells(A) HCT116 cells were transfected with two different PXN shRNAs. HT29 cells were transfected with two doses of WT PXN and cell lysates were separated by SDS-PAGE to evaluate Bcl-2 and PXN expression by western blotting using specific antibodies. (B) HCT116 cells were transfected with two different PXN shRNAs and then treated with 0 or 5 μmol/L MG132 for an additional 5 hours. Cell lysates were separated by SDS-PAGE to evaluate Bcl-2, pBcl-2-S87, and PXN expression by western blotting using specific antibodies. VC: vector control; NC: non-specific shRNA control. (C) HT29 cells were transfected with WT PXN or mutant PXN-Y31/118F for 36 hours and then treated with 0 or 0.2 μmol/L Src inhibitor (Dasatinib) for an additional 5 hours. The cells lysates were separated by SDS-PAGE to evaluate PXN, pPXN-Y31, pPXN-Y118, ERK, p-ERK, and Bcl-2 expression by western blotting using specific antibodies. (D) HCT116 and PXN-overexpressing HT29 cells were treated with two ERK inhibitors (10 μmol/L U0126 or 5 μmol/L AZD6244, AZD) for 5 hours. These cells were treated with 0 or 5 μmol/L MG132, and then cells were lysed. The cell lysates were separated by SDS-PAGE to evaluate Bcl-2 and pBcl-2-S87 expression by western blotting using specific antibodies. (E) HCT116 cells were transfected with PXN shRNA and then treated with 0 or 5 μmol/L MG132 for an additional 5 hours. HT29 cells were co-transfected with WT PXN and shBcl-2. Cell lysates were separated by SDS-PAGE to evaluate Bcl-2 and PXN expression by western blotting using specific antibodies. (F) Representative numbers of invading HCT116 and HT29 cells transfected with the indicated combinations of WT PXN, shPXN, or shBcl-2. The invasion capability is summarized for the indicated cells.
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Related In: Results  -  Collection

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Figure 1: Increased stabilization of Bcl-2 protein by PXN overexpression promotes cell invasion in colorectal cancer cells(A) HCT116 cells were transfected with two different PXN shRNAs. HT29 cells were transfected with two doses of WT PXN and cell lysates were separated by SDS-PAGE to evaluate Bcl-2 and PXN expression by western blotting using specific antibodies. (B) HCT116 cells were transfected with two different PXN shRNAs and then treated with 0 or 5 μmol/L MG132 for an additional 5 hours. Cell lysates were separated by SDS-PAGE to evaluate Bcl-2, pBcl-2-S87, and PXN expression by western blotting using specific antibodies. VC: vector control; NC: non-specific shRNA control. (C) HT29 cells were transfected with WT PXN or mutant PXN-Y31/118F for 36 hours and then treated with 0 or 0.2 μmol/L Src inhibitor (Dasatinib) for an additional 5 hours. The cells lysates were separated by SDS-PAGE to evaluate PXN, pPXN-Y31, pPXN-Y118, ERK, p-ERK, and Bcl-2 expression by western blotting using specific antibodies. (D) HCT116 and PXN-overexpressing HT29 cells were treated with two ERK inhibitors (10 μmol/L U0126 or 5 μmol/L AZD6244, AZD) for 5 hours. These cells were treated with 0 or 5 μmol/L MG132, and then cells were lysed. The cell lysates were separated by SDS-PAGE to evaluate Bcl-2 and pBcl-2-S87 expression by western blotting using specific antibodies. (E) HCT116 cells were transfected with PXN shRNA and then treated with 0 or 5 μmol/L MG132 for an additional 5 hours. HT29 cells were co-transfected with WT PXN and shBcl-2. Cell lysates were separated by SDS-PAGE to evaluate Bcl-2 and PXN expression by western blotting using specific antibodies. (F) Representative numbers of invading HCT116 and HT29 cells transfected with the indicated combinations of WT PXN, shPXN, or shBcl-2. The invasion capability is summarized for the indicated cells.
Mentions: We explored the possibility that PXN promoted cell invasiveness via increased Bcl-2 protein stability via ERK-mediated Bcl-2 phosphorylation at Serine 87. High and low PXN-expressing HCT116 and HT29 cells were enrolled for knockdown and overexpression of PXN using its small hairpin (sh)RNAs and its expression vector. Bcl-2 expression was markedly decreased by PXN-knockdown in HCT116 cells when compared with HCT116 cells with non-specific shRNA transfection (NC) (Figure 1A left panel). Conversely, Bcl-2 expression was increased in a dose-dependent manner by PXN-overexpression in HT29 cells (Figure 1A right panel). Interestingly, Bcl-2 and pBcl-2-S87 expression were diminished by PXN-knockdown in HCT116 cells (Figure 1B left panel), but the decrease in Bcl-2 expression by PXN-knockdown was rescued by MG132 treatment (Figure 1B right panel). However, no restoration of pBcl-2-S87 was seen in PXN-knockdown HCT116 cells treated with MG132 (Figure 1B right panel).

Bottom Line: Stabilization of Bcl-2 protein by paxillin (PXN)-mediated ERK activation was recently reported to cause an unfavorable response to 5-Fluorouracil-based chemotherapy.Here, we present evidence from cell and animal models to demonstrate that stabilization of Bcl-2 protein by phosphorylation at Serine 87 (pBcl-2-S87) via PXN-mediated ERK activation is responsible for cancer cell invasiveness and occurs via upregulation of MMP2 expression.In conclusion, PXN promotes Bcl-2 phosphorylation at Serine 87 via PXN-mediated ERK activation, and its stabilization associated with increased tumor formation efficacy in mice and poor patient outcome in colorectal cancer patients.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Chung Shan Medical University, Taichung, Taiwan.

ABSTRACT
Stabilization of Bcl-2 protein by paxillin (PXN)-mediated ERK activation was recently reported to cause an unfavorable response to 5-Fluorouracil-based chemotherapy. Here, we present evidence from cell and animal models to demonstrate that stabilization of Bcl-2 protein by phosphorylation at Serine 87 (pBcl-2-S87) via PXN-mediated ERK activation is responsible for cancer cell invasiveness and occurs via upregulation of MMP2 expression. Immunostainings of 190 tumors resected from colorectal cancer patients indicated that PXN expression was positively correlated with Bcl-2, pBcl-2-S87, and MMP2 expression. A positive correlation of pBcl-2-S87 with Bcl-2 and MMP2 was also observed in this study population. Patients with high PXN, Bcl-2, pBcl-2-S87, and MMP2 had poor overall survival (OS) and shorter relapse free survival (RFS). In conclusion, PXN promotes Bcl-2 phosphorylation at Serine 87 via PXN-mediated ERK activation, and its stabilization associated with increased tumor formation efficacy in mice and poor patient outcome in colorectal cancer patients.

No MeSH data available.


Related in: MedlinePlus