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SCF β-TRCP targets MTSS1 for ubiquitination-mediated destruction to regulate cancer cell proliferation and migration.

Zhong J, Shaik S, Wan L, Tron AE, Wang Z, Sun L, Inuzuka H, Wei W - Oncotarget (2013)

Bottom Line: Importantly, decreased MTSS1 expression is associated with more aggressive forms of breast and prostate cancers, and with poor survival rate.Importantly, introducing wild-type MTSS1 or a non-degradable MTSS1 (S322A) into breast or prostate cancer cells with low MTSS1 expression significantly inhibited cellular proliferation and migration.Moreover, S322A-MTSS1 exhibited stronger effects in inhibiting cell proliferation and migration when compared to ectopic expression of wild-type MTSS1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA.

ABSTRACT
Metastasis suppressor 1 (MTSS1) is an important tumor suppressor protein, and loss of MTSS1 expression has been observed in several types of human cancers. Importantly, decreased MTSS1 expression is associated with more aggressive forms of breast and prostate cancers, and with poor survival rate. Currently, it remains unclear how MTSS1 is regulated in cancer cells, and whether reduced MTSS1 expression contributes to elevated cancer cell proliferation and migration. Here we report that the SCFβ-TRCP regulates MTSS1 protein stability by targeting it for ubiquitination and subsequent destruction via the 26S proteasome. Notably, depletion of either Cullin 1 or β-TRCP1 led to increased levels of MTSS1. We further demonstrated a crucial role for Ser322 in the DSGXXS degron of MTSS1 in governing SCFβ-TRCP-mediated MTSS1 degradation. Mechanistically, we defined that Casein Kinase Iδ (CKIδ) phosphorylates Ser322 to trigger MTSS1's interaction with β-TRCP for subsequent ubiquitination and degradation. Importantly, introducing wild-type MTSS1 or a non-degradable MTSS1 (S322A) into breast or prostate cancer cells with low MTSS1 expression significantly inhibited cellular proliferation and migration. Moreover, S322A-MTSS1 exhibited stronger effects in inhibiting cell proliferation and migration when compared to ectopic expression of wild-type MTSS1. Therefore, our study provides a novel molecular mechanism for the negative regulation of MTSS1 by β-TRCP in cancer cells. It further suggests that preventing MTSS1 degradation could be a possible novel strategy for clinical treatment of more aggressive breast and prostate cancers.

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Related in: MedlinePlus

MTSS1 protein stability is controlled by the SCFβ-TRCPE3 ubiquitin ligase(A) Immunoblot (IB) analysis of whole cell lysates (WCL) derived from 293T cells infected with shRNA constructs specific for GFP, β-TRCP1 (four independent lentiviral β-TRCP1-targeting shRNA constructs namely, -A, -B, -C, -D), or β-TRCP1+2, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. B) IB analysis of WCL from 293T cells transfected with shRNA specific for GFP, or several shRNA constructs against Cullin 1 (five independent lentiviral Cullin 1-targeting shRNA constructs namely, -A, -B, -C, -D, -E) followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (C) 293T cells were infected with the indicated shRNA constructs followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. The generated stable cell lines were then split into 60-mm dishes. 20 hours later, cells were treated with 20 μg/ml CHX. At the indicated time points, WCL were prepared, and immunoblots were probed with the indicated antibodies. (D) Quantification of the band intensities in C. MTSS1 band intensity was normalized to tubulin, and then normalized to the t = 0 controls. The error bars represent mean ± SD (n= 3). (E-F) Relative mRNA levels of MTSS1 (E) or β-TRCP1 (F) in 293T cells infected with shRNA constructs specific for GFP, -TRCP1 (-A and -B) or β-TRCP1+2 followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. MTSS1 and β-TRCP1 mRNA levels were normalized to GAPDH, and then normalized to the control cells (shGFP). (G) IB analysis of WCL derived from 293T cells treated with vehicle or MG132 as indicated.
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Figure 2: MTSS1 protein stability is controlled by the SCFβ-TRCPE3 ubiquitin ligase(A) Immunoblot (IB) analysis of whole cell lysates (WCL) derived from 293T cells infected with shRNA constructs specific for GFP, β-TRCP1 (four independent lentiviral β-TRCP1-targeting shRNA constructs namely, -A, -B, -C, -D), or β-TRCP1+2, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. B) IB analysis of WCL from 293T cells transfected with shRNA specific for GFP, or several shRNA constructs against Cullin 1 (five independent lentiviral Cullin 1-targeting shRNA constructs namely, -A, -B, -C, -D, -E) followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (C) 293T cells were infected with the indicated shRNA constructs followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. The generated stable cell lines were then split into 60-mm dishes. 20 hours later, cells were treated with 20 μg/ml CHX. At the indicated time points, WCL were prepared, and immunoblots were probed with the indicated antibodies. (D) Quantification of the band intensities in C. MTSS1 band intensity was normalized to tubulin, and then normalized to the t = 0 controls. The error bars represent mean ± SD (n= 3). (E-F) Relative mRNA levels of MTSS1 (E) or β-TRCP1 (F) in 293T cells infected with shRNA constructs specific for GFP, -TRCP1 (-A and -B) or β-TRCP1+2 followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. MTSS1 and β-TRCP1 mRNA levels were normalized to GAPDH, and then normalized to the control cells (shGFP). (G) IB analysis of WCL derived from 293T cells treated with vehicle or MG132 as indicated.

Mentions: Consistent with the key role of the SCF complex in the regulation of MTSS1 stability, we also found interactions between MTSS1 and Rbx1 (Figure 1E and Supplementary Figure S1D) as well as between MTSS1 and Skp1 (Figure 1F). These findings together suggest that the SCF complex comprising of Cullin 1, Rbx1, Skp1, and β-TRCP is involved in the regulation of MTSS1 stability. In further support of the physiological roles of β-TRCP and Cullin 1 in the regulation of MTSS1, we found that depletion of endogenous β-TRCP or Cullin 1 significantly upregulated MTSS1 (Figure 2A, 2B and Supplementary Figure S2A, S2B). Importantly, depletion of β-TRCP caused a marked increase in MTSS1 half-life (Figure 2C and 2D), but not in MTSS1 mRNA levels (Figure 2E and 2F). Moreover, in support of the notion that SCFβ-TRCP might regulate MTSS1 abundance in a post-translational mechanism, treatment with the proteasome inhibitor, MG132, significantly upregulated MTSS1 protein levels, indicating the potential involvement of 26S proteasome in MTSS1 degradation (Figure 2G). These findings together suggest that a post-transcriptional regulatory mechanism such as the ubiquitin proteasome system may be involved in the regulation of MTSS1 stability.


SCF β-TRCP targets MTSS1 for ubiquitination-mediated destruction to regulate cancer cell proliferation and migration.

Zhong J, Shaik S, Wan L, Tron AE, Wang Z, Sun L, Inuzuka H, Wei W - Oncotarget (2013)

MTSS1 protein stability is controlled by the SCFβ-TRCPE3 ubiquitin ligase(A) Immunoblot (IB) analysis of whole cell lysates (WCL) derived from 293T cells infected with shRNA constructs specific for GFP, β-TRCP1 (four independent lentiviral β-TRCP1-targeting shRNA constructs namely, -A, -B, -C, -D), or β-TRCP1+2, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. B) IB analysis of WCL from 293T cells transfected with shRNA specific for GFP, or several shRNA constructs against Cullin 1 (five independent lentiviral Cullin 1-targeting shRNA constructs namely, -A, -B, -C, -D, -E) followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (C) 293T cells were infected with the indicated shRNA constructs followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. The generated stable cell lines were then split into 60-mm dishes. 20 hours later, cells were treated with 20 μg/ml CHX. At the indicated time points, WCL were prepared, and immunoblots were probed with the indicated antibodies. (D) Quantification of the band intensities in C. MTSS1 band intensity was normalized to tubulin, and then normalized to the t = 0 controls. The error bars represent mean ± SD (n= 3). (E-F) Relative mRNA levels of MTSS1 (E) or β-TRCP1 (F) in 293T cells infected with shRNA constructs specific for GFP, -TRCP1 (-A and -B) or β-TRCP1+2 followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. MTSS1 and β-TRCP1 mRNA levels were normalized to GAPDH, and then normalized to the control cells (shGFP). (G) IB analysis of WCL derived from 293T cells treated with vehicle or MG132 as indicated.
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Figure 2: MTSS1 protein stability is controlled by the SCFβ-TRCPE3 ubiquitin ligase(A) Immunoblot (IB) analysis of whole cell lysates (WCL) derived from 293T cells infected with shRNA constructs specific for GFP, β-TRCP1 (four independent lentiviral β-TRCP1-targeting shRNA constructs namely, -A, -B, -C, -D), or β-TRCP1+2, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. B) IB analysis of WCL from 293T cells transfected with shRNA specific for GFP, or several shRNA constructs against Cullin 1 (five independent lentiviral Cullin 1-targeting shRNA constructs namely, -A, -B, -C, -D, -E) followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (C) 293T cells were infected with the indicated shRNA constructs followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. The generated stable cell lines were then split into 60-mm dishes. 20 hours later, cells were treated with 20 μg/ml CHX. At the indicated time points, WCL were prepared, and immunoblots were probed with the indicated antibodies. (D) Quantification of the band intensities in C. MTSS1 band intensity was normalized to tubulin, and then normalized to the t = 0 controls. The error bars represent mean ± SD (n= 3). (E-F) Relative mRNA levels of MTSS1 (E) or β-TRCP1 (F) in 293T cells infected with shRNA constructs specific for GFP, -TRCP1 (-A and -B) or β-TRCP1+2 followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. MTSS1 and β-TRCP1 mRNA levels were normalized to GAPDH, and then normalized to the control cells (shGFP). (G) IB analysis of WCL derived from 293T cells treated with vehicle or MG132 as indicated.
Mentions: Consistent with the key role of the SCF complex in the regulation of MTSS1 stability, we also found interactions between MTSS1 and Rbx1 (Figure 1E and Supplementary Figure S1D) as well as between MTSS1 and Skp1 (Figure 1F). These findings together suggest that the SCF complex comprising of Cullin 1, Rbx1, Skp1, and β-TRCP is involved in the regulation of MTSS1 stability. In further support of the physiological roles of β-TRCP and Cullin 1 in the regulation of MTSS1, we found that depletion of endogenous β-TRCP or Cullin 1 significantly upregulated MTSS1 (Figure 2A, 2B and Supplementary Figure S2A, S2B). Importantly, depletion of β-TRCP caused a marked increase in MTSS1 half-life (Figure 2C and 2D), but not in MTSS1 mRNA levels (Figure 2E and 2F). Moreover, in support of the notion that SCFβ-TRCP might regulate MTSS1 abundance in a post-translational mechanism, treatment with the proteasome inhibitor, MG132, significantly upregulated MTSS1 protein levels, indicating the potential involvement of 26S proteasome in MTSS1 degradation (Figure 2G). These findings together suggest that a post-transcriptional regulatory mechanism such as the ubiquitin proteasome system may be involved in the regulation of MTSS1 stability.

Bottom Line: Importantly, decreased MTSS1 expression is associated with more aggressive forms of breast and prostate cancers, and with poor survival rate.Importantly, introducing wild-type MTSS1 or a non-degradable MTSS1 (S322A) into breast or prostate cancer cells with low MTSS1 expression significantly inhibited cellular proliferation and migration.Moreover, S322A-MTSS1 exhibited stronger effects in inhibiting cell proliferation and migration when compared to ectopic expression of wild-type MTSS1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA.

ABSTRACT
Metastasis suppressor 1 (MTSS1) is an important tumor suppressor protein, and loss of MTSS1 expression has been observed in several types of human cancers. Importantly, decreased MTSS1 expression is associated with more aggressive forms of breast and prostate cancers, and with poor survival rate. Currently, it remains unclear how MTSS1 is regulated in cancer cells, and whether reduced MTSS1 expression contributes to elevated cancer cell proliferation and migration. Here we report that the SCFβ-TRCP regulates MTSS1 protein stability by targeting it for ubiquitination and subsequent destruction via the 26S proteasome. Notably, depletion of either Cullin 1 or β-TRCP1 led to increased levels of MTSS1. We further demonstrated a crucial role for Ser322 in the DSGXXS degron of MTSS1 in governing SCFβ-TRCP-mediated MTSS1 degradation. Mechanistically, we defined that Casein Kinase Iδ (CKIδ) phosphorylates Ser322 to trigger MTSS1's interaction with β-TRCP for subsequent ubiquitination and degradation. Importantly, introducing wild-type MTSS1 or a non-degradable MTSS1 (S322A) into breast or prostate cancer cells with low MTSS1 expression significantly inhibited cellular proliferation and migration. Moreover, S322A-MTSS1 exhibited stronger effects in inhibiting cell proliferation and migration when compared to ectopic expression of wild-type MTSS1. Therefore, our study provides a novel molecular mechanism for the negative regulation of MTSS1 by β-TRCP in cancer cells. It further suggests that preventing MTSS1 degradation could be a possible novel strategy for clinical treatment of more aggressive breast and prostate cancers.

Show MeSH
Related in: MedlinePlus