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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

Mutant MTSS1 inhibits PC3 and MDA-MB-231 cancer cell proliferation(A-D) PC3 (A, B) or MDA-MB-231 (C, D) cells were infected with pBabe-EV, pBabe-HA-wild-type-MTSS1 or pBabe-HA-S322A-MTSS1 retroviral vectors and photographs were taken after growing the generated stable cell lines for 3 days in puromycin (1 μg/ml) selection medium to eliminate the non-infected cells. The numbers of PC3 (B) or MDA-MB-231 (D) cells were quantified at the indicated time points. The number of cells was normalized against the number of cells in the corresponding pBabe-EV cells. The error bars represent mean ± SD (n= 3). (E-H) PC3 (E, F) or MDA-MB-231 (G, H) cells were infected with pBabe-EV, pBabe-HA-wild-type-MTSS1 or pBabe-HA-S322A-MTSS1 retroviral vectors and photographs were taken after growing the cells for 3 days in puromycin (1 μg/ml) selection medium to eliminate the non-infected cells. Furthermore, the generated cell lines were subjected to pulse of BrdU and then immunostained using anti-BrdU antibody as described in the methods section. Quantitative measurements of PC3 (F) or MDA-MB-231 (H) cells stained for BrdU were presented. The number of cells was normalized against the number of cells in the corresponding pBabe-EV cells. The error bars represent mean ± SD (n= 3) * p< 0.05.
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Figure 6: Mutant MTSS1 inhibits PC3 and MDA-MB-231 cancer cell proliferation(A-D) PC3 (A, B) or MDA-MB-231 (C, D) cells were infected with pBabe-EV, pBabe-HA-wild-type-MTSS1 or pBabe-HA-S322A-MTSS1 retroviral vectors and photographs were taken after growing the generated stable cell lines for 3 days in puromycin (1 μg/ml) selection medium to eliminate the non-infected cells. The numbers of PC3 (B) or MDA-MB-231 (D) cells were quantified at the indicated time points. The number of cells was normalized against the number of cells in the corresponding pBabe-EV cells. The error bars represent mean ± SD (n= 3). (E-H) PC3 (E, F) or MDA-MB-231 (G, H) cells were infected with pBabe-EV, pBabe-HA-wild-type-MTSS1 or pBabe-HA-S322A-MTSS1 retroviral vectors and photographs were taken after growing the cells for 3 days in puromycin (1 μg/ml) selection medium to eliminate the non-infected cells. Furthermore, the generated cell lines were subjected to pulse of BrdU and then immunostained using anti-BrdU antibody as described in the methods section. Quantitative measurements of PC3 (F) or MDA-MB-231 (H) cells stained for BrdU were presented. The number of cells was normalized against the number of cells in the corresponding pBabe-EV cells. The error bars represent mean ± SD (n= 3) * p< 0.05.

Mentions: These results indicated that the SCF complex consisting of Cullin 1 and β-TRCP might play a key role in the regulation of MTSS1 in both breast and prostate cancer cells. As β-TRCP is the first identified E3 ligase for MTSS1, to explore the biological significance for SCFβ-TRCP-mediated destruction of MTSS1, next we intended to examine how ectopic expression of a non-degradable mutant form of MTSS1 (S322A-MTSS1) or wild-type MTSS1 (as a control) in both PC3 and MDA-MB-231 cancer cells could affect cellular migration or proliferation (Supplementary Figure S4A-B). Empty vector (EV) expressing cells were also used as a negative control for this experimental system. Importantly, S322A-MTSS1 expressing PC3 and MDA-MB-231 cells exhibited significantly reduced growth potential compared to wild-type MTSS1 or EV infected cells (Figure 6A-6D). Consistent with this finding, ectopoic expression of S322A-MTSS1 exerted stronger ability than WT-MTSS1 or EV controls in decreasing cell entry into the S phase, as illustrated by reduced BrdU staining in both PC3 and MDA-MB-231 cells (Figure 6E-6H). This suggests that elevated MTSS1 expression, in part due to deficient destruction by the SCFβ-TRCP E3 ligase, might suppress tumorigenesis by reducing S phase entry and cellular proliferation.


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)

Mutant MTSS1 inhibits PC3 and MDA-MB-231 cancer cell proliferation(A-D) PC3 (A, B) or MDA-MB-231 (C, D) cells were infected with pBabe-EV, pBabe-HA-wild-type-MTSS1 or pBabe-HA-S322A-MTSS1 retroviral vectors and photographs were taken after growing the generated stable cell lines for 3 days in puromycin (1 μg/ml) selection medium to eliminate the non-infected cells. The numbers of PC3 (B) or MDA-MB-231 (D) cells were quantified at the indicated time points. The number of cells was normalized against the number of cells in the corresponding pBabe-EV cells. The error bars represent mean ± SD (n= 3). (E-H) PC3 (E, F) or MDA-MB-231 (G, H) cells were infected with pBabe-EV, pBabe-HA-wild-type-MTSS1 or pBabe-HA-S322A-MTSS1 retroviral vectors and photographs were taken after growing the cells for 3 days in puromycin (1 μg/ml) selection medium to eliminate the non-infected cells. Furthermore, the generated cell lines were subjected to pulse of BrdU and then immunostained using anti-BrdU antibody as described in the methods section. Quantitative measurements of PC3 (F) or MDA-MB-231 (H) cells stained for BrdU were presented. The number of cells was normalized against the number of cells in the corresponding pBabe-EV cells. The error bars represent mean ± SD (n= 3) * p< 0.05.
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Related In: Results  -  Collection

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Figure 6: Mutant MTSS1 inhibits PC3 and MDA-MB-231 cancer cell proliferation(A-D) PC3 (A, B) or MDA-MB-231 (C, D) cells were infected with pBabe-EV, pBabe-HA-wild-type-MTSS1 or pBabe-HA-S322A-MTSS1 retroviral vectors and photographs were taken after growing the generated stable cell lines for 3 days in puromycin (1 μg/ml) selection medium to eliminate the non-infected cells. The numbers of PC3 (B) or MDA-MB-231 (D) cells were quantified at the indicated time points. The number of cells was normalized against the number of cells in the corresponding pBabe-EV cells. The error bars represent mean ± SD (n= 3). (E-H) PC3 (E, F) or MDA-MB-231 (G, H) cells were infected with pBabe-EV, pBabe-HA-wild-type-MTSS1 or pBabe-HA-S322A-MTSS1 retroviral vectors and photographs were taken after growing the cells for 3 days in puromycin (1 μg/ml) selection medium to eliminate the non-infected cells. Furthermore, the generated cell lines were subjected to pulse of BrdU and then immunostained using anti-BrdU antibody as described in the methods section. Quantitative measurements of PC3 (F) or MDA-MB-231 (H) cells stained for BrdU were presented. The number of cells was normalized against the number of cells in the corresponding pBabe-EV cells. The error bars represent mean ± SD (n= 3) * p< 0.05.
Mentions: These results indicated that the SCF complex consisting of Cullin 1 and β-TRCP might play a key role in the regulation of MTSS1 in both breast and prostate cancer cells. As β-TRCP is the first identified E3 ligase for MTSS1, to explore the biological significance for SCFβ-TRCP-mediated destruction of MTSS1, next we intended to examine how ectopic expression of a non-degradable mutant form of MTSS1 (S322A-MTSS1) or wild-type MTSS1 (as a control) in both PC3 and MDA-MB-231 cancer cells could affect cellular migration or proliferation (Supplementary Figure S4A-B). Empty vector (EV) expressing cells were also used as a negative control for this experimental system. Importantly, S322A-MTSS1 expressing PC3 and MDA-MB-231 cells exhibited significantly reduced growth potential compared to wild-type MTSS1 or EV infected cells (Figure 6A-6D). Consistent with this finding, ectopoic expression of S322A-MTSS1 exerted stronger ability than WT-MTSS1 or EV controls in decreasing cell entry into the S phase, as illustrated by reduced BrdU staining in both PC3 and MDA-MB-231 cells (Figure 6E-6H). This suggests that elevated MTSS1 expression, in part due to deficient destruction by the SCFβ-TRCP E3 ligase, might suppress tumorigenesis by reducing S phase entry and cellular proliferation.

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