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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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β-TRCP levels inversely correlate with MTSS1 abundance in several cancer cell lines(A) Whole cell lysates (WCL) prepared from the indicated cancer cell lines were analyzed by immunoblot (IB) analysis. (B) IB analysis of WCL prepared from PC3 cells that were infected with shRNA constructs specific for GFP or Cullin 1, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (C) IB analysis of WCL prepared from PC3 cells that were infected with shRNA constructs specific for GFP, β-TRCP1 (A, B), or β-TRCP1+2, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (D) IB analysis of WCL prepared from MDA-MB-231 cells that were infected with shRNA constructs specific for GFP or Cullin 1, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (E) IB analysis of WCL prepared from MDA-MB-231 cells that were infected with shRNA constructs specific for GFP, β-TRCP1 (A, B), or β-TRCP1+2, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells.
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Figure 5: β-TRCP levels inversely correlate with MTSS1 abundance in several cancer cell lines(A) Whole cell lysates (WCL) prepared from the indicated cancer cell lines were analyzed by immunoblot (IB) analysis. (B) IB analysis of WCL prepared from PC3 cells that were infected with shRNA constructs specific for GFP or Cullin 1, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (C) IB analysis of WCL prepared from PC3 cells that were infected with shRNA constructs specific for GFP, β-TRCP1 (A, B), or β-TRCP1+2, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (D) IB analysis of WCL prepared from MDA-MB-231 cells that were infected with shRNA constructs specific for GFP or Cullin 1, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (E) IB analysis of WCL prepared from MDA-MB-231 cells that were infected with shRNA constructs specific for GFP, β-TRCP1 (A, B), or β-TRCP1+2, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells.

Mentions: Given that a significant decrease in MTSS1 abundance is frequently observed in both prostate and breast cancers [7, 10], we sought to investigate whether MTSS1 expression in these cancer cells inversely correlates with cellular proliferation and migration. To begin this investigation, we first analyzed MTSS1 protein levels in various prostate and breast cancer cell lines. Notably, we found that the PC3 prostate cancer cells and the MDA-MB-231 breast cancer cells displayed a significantly reduced expression of MTSS1, whereas DU145 and MCF-7 cells expressed relatively high MTSS1 levels (Figure 5A). Furthermore, we noticed that the MTSS1 levels inversely correlate with the endogenous β-TRCP1 levels, arguing that β-TRCP1 expression levels might dictate the abundance of MTSS1 in this experimental setting. To further examine this hypothesis, we depleted endogenous Cullin 1 or β-TRCP via lentiviral shRNA infection to examine its effects on MTSS1 abundance. In keeping with a critical role for SCFβ-TRCP in governing MTSS1 stability, we found that depletion of either Cullin 1 or both β-TRCP isoforms led to a signficant upregulation of MTSS1 in both PC3 and MDA-MB-231 cells (Figure 5 B-E).


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)

β-TRCP levels inversely correlate with MTSS1 abundance in several cancer cell lines(A) Whole cell lysates (WCL) prepared from the indicated cancer cell lines were analyzed by immunoblot (IB) analysis. (B) IB analysis of WCL prepared from PC3 cells that were infected with shRNA constructs specific for GFP or Cullin 1, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (C) IB analysis of WCL prepared from PC3 cells that were infected with shRNA constructs specific for GFP, β-TRCP1 (A, B), or β-TRCP1+2, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (D) IB analysis of WCL prepared from MDA-MB-231 cells that were infected with shRNA constructs specific for GFP or Cullin 1, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (E) IB analysis of WCL prepared from MDA-MB-231 cells that were infected with shRNA constructs specific for GFP, β-TRCP1 (A, B), or β-TRCP1+2, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells.
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Related In: Results  -  Collection

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Figure 5: β-TRCP levels inversely correlate with MTSS1 abundance in several cancer cell lines(A) Whole cell lysates (WCL) prepared from the indicated cancer cell lines were analyzed by immunoblot (IB) analysis. (B) IB analysis of WCL prepared from PC3 cells that were infected with shRNA constructs specific for GFP or Cullin 1, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (C) IB analysis of WCL prepared from PC3 cells that were infected with shRNA constructs specific for GFP, β-TRCP1 (A, B), or β-TRCP1+2, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (D) IB analysis of WCL prepared from MDA-MB-231 cells that were infected with shRNA constructs specific for GFP or Cullin 1, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (E) IB analysis of WCL prepared from MDA-MB-231 cells that were infected with shRNA constructs specific for GFP, β-TRCP1 (A, B), or β-TRCP1+2, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells.
Mentions: Given that a significant decrease in MTSS1 abundance is frequently observed in both prostate and breast cancers [7, 10], we sought to investigate whether MTSS1 expression in these cancer cells inversely correlates with cellular proliferation and migration. To begin this investigation, we first analyzed MTSS1 protein levels in various prostate and breast cancer cell lines. Notably, we found that the PC3 prostate cancer cells and the MDA-MB-231 breast cancer cells displayed a significantly reduced expression of MTSS1, whereas DU145 and MCF-7 cells expressed relatively high MTSS1 levels (Figure 5A). Furthermore, we noticed that the MTSS1 levels inversely correlate with the endogenous β-TRCP1 levels, arguing that β-TRCP1 expression levels might dictate the abundance of MTSS1 in this experimental setting. To further examine this hypothesis, we depleted endogenous Cullin 1 or β-TRCP via lentiviral shRNA infection to examine its effects on MTSS1 abundance. In keeping with a critical role for SCFβ-TRCP in governing MTSS1 stability, we found that depletion of either Cullin 1 or both β-TRCP isoforms led to a signficant upregulation of MTSS1 in both PC3 and MDA-MB-231 cells (Figure 5 B-E).

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