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, 71 (11), 3980-90

Phosphorylation of Serine 68 of Twist1 by MAPKs Stabilizes Twist1 Protein and Promotes Breast Cancer Cell Invasiveness

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Phosphorylation of Serine 68 of Twist1 by MAPKs Stabilizes Twist1 Protein and Promotes Breast Cancer Cell Invasiveness

Jun Hong et al. Cancer Res.

Abstract

Twist1, a basic helix-loop-helix transcription factor, promotes breast tumor cell epithelial-mesenchymal transition (EMT), invasiveness, and metastasis. However, the mechanisms responsible for regulating Twist1 stability are unknown in these cells. We identified the serine 68 (Ser 68) as a major phosphorylation site of Twist1 by mass spectrometry and with specific antibodies. This Ser 68 is phosphorylated by p38, c-Jun N-terminal kinases (JNK), and extracellular signal-regulated kinases1/2 in vitro, and its phosphorylation levels positively correlate with Twist1 protein levels in human embryonic kidney 293 and breast cancer cells. Prevention of Ser 68 phosphorylation by an alanine (A) mutation (Ser 68A) dramatically accelerates Twist1 ubiquitination and degradation. Furthermore, activation of mitogen-activated protein kinases (MAPK) by an active Ras protein or TGF-β treatment significantly increases Ser 68 phosphorylation and Twist1 protein levels without altering Twist1 mRNA expression, whereas blocking of MAPK activities by either specific inhibitors or dominant negative inhibitory mutants effectively reduces the levels of both induced and uninduced Ser 68 phosphorylation and Twist protein. Accordingly, the mammary epithelial cells expressing Twist1 exhibit much higher degrees of EMT and invasiveness on stimulation with TGF-β or the active Ras and paclitaxel resistance compared with the same cells expressing the Ser 68A-Twist1 mutant. Importantly, the levels of Ser 68 phosphorylation in the invasive human breast ductal carcinomas positively correlate with the levels of Twist1 protein and JNK activity and are significantly higher in progesterone receptor-negative and HER2-positive breast cancers. These findings suggest that activation of MAPKs by tyrosine kinase receptors and Ras signaling pathways may substantially promote breast tumor cell EMT and metastasis via phoshorylation and stabilization of Twist1.

Figures

Fig. 1
Fig. 1. Twist1 expression, purification, phosphorylation and stability assays
A. Immunoprecipitated F-Twist1 (F-T) was analyzed by immunoblotting (IB) with antibodies against Flag, p-Serine (pSer) and p-Tyrosine (pTyr). Immunoprecipitation from F cells served as a negative control. IgG-HC and IgG-LC, IgG heavy and light chains. B1. 293 cells were transfected with the indicated plasmids. Cell lysates were assayed by IB with the indicated antibodies. HA-T, HA-tagged Twist1; p-S68, pS68-Twist1. B2. The cell lysates were prepared from the indicated cell lines and analyzed by IB with antibodies against Twist1, pS68-Twist and β-actin. C. 293 cells were transfected with HA-Twist1 or HA-S68A-Twist1 plasmids. After 12 hours, cells were treated with cycloheximide for time periods as indicated. IB was performed with HA and tubulin antibodies. Densitometric values were determined and presented. The half lives (50%) of HA-Twist1 and HA-S68A-Twist1 are indicated. D. F (-), F-Twist1 (W), F-S68A-Twist1 (A) and F-S68E-Twist1 (E) inducible 293 cells were transfected with mock plasmids or HA-ubiquitin expression plasmids as indicated. After 12 hours of transfection, cells were treated with Dox for 6 hours before cells were treated with a vehicle or MG132 for another 6 hours. Immunoprecipitation was performed with Flag antibody, followed by IB with HA and Flag antibodies as indicated. ns, non-specific band.
Fig. 2
Fig. 2. MAPKs phosphorylate Twist1 at Sre68 in vitro and Ras activation stabilizes Twist1 in HEK293 cells
A. GST control and GST-Twist1-N Proteins were incubated with MAPKs as indicated for in vitro phosphorylation assays. Immunoblotting (IB) was performed with pS68-Twist1 antibody. Coomassie blue staining of GST and GST-Twist1-N Proteins served as the loading control. SB203580, a p38 MAPK inhibitor; SP60125, a JNK inhibitor. B. 293 cells were transfected with HA-Twist1 plasmids in combination with H-RasV12 (+) plasmids or its mock vector (-). H-Ras, Twist1 and GAPDH mRNAs were measured by RT-PCR. H-Ras, pS68-Twist1 and tubulin proteins were measured by IB. C. 293 cells were transfected with H-RasV12 plasmids in combination with HA-Twist1 or HA-S68A-Twist1 plasmids. After 12 hours, cells were treated with cycloheximide as indicated. IB was performed with antibodies against HA (for Twist1), H-Ras and tubulin. Densitometric values were determined and plotted.
Fig. 3
Fig. 3. Inhibition of MAPKs reduces pS68-Twist1 and Twist1 proteins
A. 293 cells were transfected with Twist1 plasmids and H-RasV12 or mock plasmids as indicated. After 12 hours, cells were treated with a vehicle, SB203580, SP60125 or PD98059. Immunoblotting was performed with the indicated antibodies. B. 293 cells were transfected with Twist1, in combination with mock vectors (-), H-RasV12 expression plasmids (+) and dominant negative (DN) forms of Flag-DN-JNK (JN), Flag-DN-p38 MAPK (p) or DN-MEK (ME) as indicated. Immunoblotting was performed with the indicated antibodies. Note that the p38 antibody recognized both endogenous and DN-p38. The anti-Flag antibody recognized both Flag-DN-JNK and Flag-DN-p38. C. 4T1 cells were treated with a vehicle or different MAPK inhibitors for 8 hours. Immunoblotting was performed with the antibodies indicated to detect the indicated endogenous proteins.
Fig. 4
Fig. 4. Ser 68 phosphorylation is required for Ras-stimulated and Twist1-promoted breast cancer cell invasion
A. Stable MCF-10A cell lines with mock control, Twist1 expression or Twist1-S68A expression were assayed by immunoblotting (IB) with antibodies indicated. Twist1, S68A-Twist1 and GAPDH mRNAs were measured by RT-PCR. B. The above cells were immunostained by the E-cadherin antibody (green color in all panels). Cell nuclei were stained by DAPI (right panels). C. Morphologies of MCF-10A cell lines expressing H-RasV12 and Twist1, S68A-Twist1 or a mock control as indicated (upper panel). Real-time cell invasion assays (lower panel) were performed with the 3 cell lines in the upper panel and the 3 cell lines in panel A.
Fig. 5
Fig. 5. TGF-β induces pS68-Twist1 and Twist1 proteins and cell invasion
A. MDA-MB-231 cells were treated with TGF-β as indicated. Twist1 mRNA levels were measured by real time RT-PCR and normalized to 18S RNA levels (upper panel). Lysates were prepared from TGF-β-treated MDA-MB-231 cells and subjected to immunoblotting with antibodies against Twist1, pS68-Twist1 and β-actin (lower panel). B. MDA-MB-231 cells were treated with TGF-β and/or different MAPK inhibitors. Cell lysates were analyzed by immunoblotting with the indicated antibodies. C. MCF-10A cells with mock vector (-), Twist1 (WT) expression or S68A-Twist1 (S68A) expression were treated with a vehicle (-) or TGF-β as indicated. Immunoblotting was performed using antibodies against Twist1, pS68-Twist1 and β-actin (upper panel). Cell invasion assays were performed in the absence or presence of TGF-β as indicated. D. Summary diagram of working model. If S68-Twist is not phosphorylated by MAPKs, it will subject to ubiquitination and degradation. If phosphorylated, the pS68-Twist1 is resistant to ubiquitination and degradation and becomes stabilized, so its function for promotion of EMT, cell invasion and cell survival will be enhanced.
Fig. 6
Fig. 6. Analyses of pS68-Twist1, Twist1 and MAPK levels in human primary breast tumors
A. Immunoblotting analyses of breast tumors using antibodies against pS68-Twist1, Twist1, p-JNK, p-p44/42 ERKs, p-p38 MAPK and β-actin. The values of band intensity measured by densitometry are listed. The tumor types are indicated as invasive ductal carcinoma (idc), invasive lobular andenocarcinoma (ila) and lipid-rich epithelia carcinoma (lrc). Tumors were divided into 3 groups according to the immunostaining results of ERα, PR and HER2. The percentage of Ki67-positive cell numbers is listed. For ERα and PR, the staining intensities were graded as 0 (negative), 1 (weak staining), 2 (medium staining) and 3 (strong staining), and the ratios of positive cells were graded as 1 (0-25%), 2 (26-50%), 3 (51-75%) and 4 (76-100%). The final scores indicated here were determined according to the sum of staining intensity and positive cell ratio grades: 0, ≤ 2 sum points; 1, 3-4 sum points; 2, 5-6 sum points; and 3, 7 sum points. For HER2, score 0 was designated as negative staining or <10% of tumor cells with weak membrane staining; score 1 was designated as >10% of tumor cells with weak staining and partial membrane staining; score 2 was designated as >10% of tumor cells with weak to medium membrane staining; and score 3 was designated as >10% of tumor cells with strong membrane staining. B. The correlation relationship between Twist1 and pS68-Twist1 levels in breast tumors assayed in panel A. The correlation was statistically calculated by Spearman rank correlation analysis. C. The correlation relationships between p-JNK and pS68-Twist1 or Twist1 levels in breast tumors assayed in panel A. D. The relative levels of pS68-Twist1, Twist1 and p-JNK in ERα+;PR+;HER2+, ERα+;PR-;HER2+, and ERa-;PR-;HER2+ breast tumors assayed in panel A. The data are presented in a box plot, where the upper limit of the rectangle is the third quartile and the bottom limit is the first quartile. *, p < 0.01; **, p< 0.001 by t test.

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