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Androgen receptor non-nuclear regulation of prostate cancer cell invasion mediated by Src and matriptase.

Zarif JC, Lamb LE, Schulz VV, Nollet EA, Miranti CK - Oncotarget (2015)

Bottom Line: Matriptase activation and shedding induced by cytoplasmic AR is dependent on Src.However, only inhibition of Matriptase, but not CDCP1, suppresses the AR/Src-dependent increase in invasion.Thus, invasion is stimulated by a rapid but sustained increase in Src activity, mediated non-genomically by cytoplasmic AR, leading to rapid activation and shedding of the laminin protease Matriptase.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Integrin Signaling and Tumorigenesis, Van Andel Research Institute, Grand Rapids, MI 49503, USA.

ABSTRACT
Castration-resistant prostate cancers still depend on nuclear androgen receptor (AR) function despite their lack of dependence on exogenous androgen. Second generation anti-androgen therapies are more efficient at blocking nuclear AR; however resistant tumors still develop. Recent studies indicate Src is highly active in these resistant tumors. By manipulating AR activity in several different prostate cancer cell lines through RNAi, drug treatment, and the use of a nuclear-deficient AR mutant, we demonstrate that androgen acting on cytoplasmic AR rapidly stimulates Src tyrosine kinase via a non-genomic mechanism. Cytoplasmic AR, acting through Src enhances laminin integrin-dependent invasion. Active Matriptase, which cleaves laminin, is elevated within minutes after androgen stimulation, and is subsequently shed into the medium. Matriptase activation and shedding induced by cytoplasmic AR is dependent on Src. Concomitantly, CDCP1/gp140, a Matriptase and Src substrate that controls integrin-based migration, is activated. However, only inhibition of Matriptase, but not CDCP1, suppresses the AR/Src-dependent increase in invasion. Matriptase, present in conditioned medium from AR-stimulated cells, is sufficient to enhance invasion in the absence of androgen. Thus, invasion is stimulated by a rapid but sustained increase in Src activity, mediated non-genomically by cytoplasmic AR, leading to rapid activation and shedding of the laminin protease Matriptase.

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CDCP1 activity is regulated by AR and Src(A) PC3-Puro (PP) or PC3-AR cells were treated with scrambled siRNA (Sc), AR- or Src-specific siRNA (sAR, sSr), or 10 nM dasatinib for 24 hours. Tyrosine phosphorylation and expression of full length (140kDa) and cleaved (70kDa) CDCP1 from immunoprecipitates were measured by immunoblotting with anti-phosphotyrosine antibody or CDCP1 antibody respectively. (B, C) PC3-AR cells were treated with scrambled siRNA (Scr) or CDCP1-specific siRNA (siCP1). (B) CDCP1 expression was measured by immunoblotting and (C) Matrigel invasion was quantified.
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Figure 5: CDCP1 activity is regulated by AR and Src(A) PC3-Puro (PP) or PC3-AR cells were treated with scrambled siRNA (Sc), AR- or Src-specific siRNA (sAR, sSr), or 10 nM dasatinib for 24 hours. Tyrosine phosphorylation and expression of full length (140kDa) and cleaved (70kDa) CDCP1 from immunoprecipitates were measured by immunoblotting with anti-phosphotyrosine antibody or CDCP1 antibody respectively. (B, C) PC3-AR cells were treated with scrambled siRNA (Scr) or CDCP1-specific siRNA (siCP1). (B) CDCP1 expression was measured by immunoblotting and (C) Matrigel invasion was quantified.

Mentions: Cub Domain Containing Protein 1 (CDCP1/gp140/Trask) is a transmembrane glycoprotein that facilitates integrin-dependent migration and invasion and whose elevated expression in primary tumors correlates with metastasis in several cancers [38]. CDCP1 is cleaved by several serine proteases, including Matriptase [39]. Full-length CDCP1 (140 kDa) and its cleaved form (72 kDa) are direct Src substrates and phosphorylation by Src is required for promoting cellular de-adhesion from matrix and invasion [40–42]. AR expression in PC3 cells increased the expression, cleavage, and tyrosine phosphorylation of cleaved CDCP1 (Figure 5A). Inhibition of AR or Src expression with siRNA, or treatment with the Src inhibitor dasatinib prevented cleavage and tyrosine phosphorylation of the cleaved fragment (Figure 5A). While dasatinib can target other Src-related tyrosine kinases as well as some receptor kinases, it abrogated generation of the CDCP1 cleavage product (72kDa) and its phosphorylation to a similar extent as the Src siRNA (Figure 5A). Conversely, treatment of C4–2 cells with R1881 rapidly increased CDCP1 cleavage and tyrosine phosphorylation (not shown). However, inhibition of CDCP1 expression (Figure 5B) had no effect on invasion (Figure 5C). Although Src activation by AR is able to induce cleavage and phosphorylation of CDCP1, this target is not critical for AR-dependent invasion of Matrigel.


Androgen receptor non-nuclear regulation of prostate cancer cell invasion mediated by Src and matriptase.

Zarif JC, Lamb LE, Schulz VV, Nollet EA, Miranti CK - Oncotarget (2015)

CDCP1 activity is regulated by AR and Src(A) PC3-Puro (PP) or PC3-AR cells were treated with scrambled siRNA (Sc), AR- or Src-specific siRNA (sAR, sSr), or 10 nM dasatinib for 24 hours. Tyrosine phosphorylation and expression of full length (140kDa) and cleaved (70kDa) CDCP1 from immunoprecipitates were measured by immunoblotting with anti-phosphotyrosine antibody or CDCP1 antibody respectively. (B, C) PC3-AR cells were treated with scrambled siRNA (Scr) or CDCP1-specific siRNA (siCP1). (B) CDCP1 expression was measured by immunoblotting and (C) Matrigel invasion was quantified.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4466655&req=5

Figure 5: CDCP1 activity is regulated by AR and Src(A) PC3-Puro (PP) or PC3-AR cells were treated with scrambled siRNA (Sc), AR- or Src-specific siRNA (sAR, sSr), or 10 nM dasatinib for 24 hours. Tyrosine phosphorylation and expression of full length (140kDa) and cleaved (70kDa) CDCP1 from immunoprecipitates were measured by immunoblotting with anti-phosphotyrosine antibody or CDCP1 antibody respectively. (B, C) PC3-AR cells were treated with scrambled siRNA (Scr) or CDCP1-specific siRNA (siCP1). (B) CDCP1 expression was measured by immunoblotting and (C) Matrigel invasion was quantified.
Mentions: Cub Domain Containing Protein 1 (CDCP1/gp140/Trask) is a transmembrane glycoprotein that facilitates integrin-dependent migration and invasion and whose elevated expression in primary tumors correlates with metastasis in several cancers [38]. CDCP1 is cleaved by several serine proteases, including Matriptase [39]. Full-length CDCP1 (140 kDa) and its cleaved form (72 kDa) are direct Src substrates and phosphorylation by Src is required for promoting cellular de-adhesion from matrix and invasion [40–42]. AR expression in PC3 cells increased the expression, cleavage, and tyrosine phosphorylation of cleaved CDCP1 (Figure 5A). Inhibition of AR or Src expression with siRNA, or treatment with the Src inhibitor dasatinib prevented cleavage and tyrosine phosphorylation of the cleaved fragment (Figure 5A). While dasatinib can target other Src-related tyrosine kinases as well as some receptor kinases, it abrogated generation of the CDCP1 cleavage product (72kDa) and its phosphorylation to a similar extent as the Src siRNA (Figure 5A). Conversely, treatment of C4–2 cells with R1881 rapidly increased CDCP1 cleavage and tyrosine phosphorylation (not shown). However, inhibition of CDCP1 expression (Figure 5B) had no effect on invasion (Figure 5C). Although Src activation by AR is able to induce cleavage and phosphorylation of CDCP1, this target is not critical for AR-dependent invasion of Matrigel.

Bottom Line: Matriptase activation and shedding induced by cytoplasmic AR is dependent on Src.However, only inhibition of Matriptase, but not CDCP1, suppresses the AR/Src-dependent increase in invasion.Thus, invasion is stimulated by a rapid but sustained increase in Src activity, mediated non-genomically by cytoplasmic AR, leading to rapid activation and shedding of the laminin protease Matriptase.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Integrin Signaling and Tumorigenesis, Van Andel Research Institute, Grand Rapids, MI 49503, USA.

ABSTRACT
Castration-resistant prostate cancers still depend on nuclear androgen receptor (AR) function despite their lack of dependence on exogenous androgen. Second generation anti-androgen therapies are more efficient at blocking nuclear AR; however resistant tumors still develop. Recent studies indicate Src is highly active in these resistant tumors. By manipulating AR activity in several different prostate cancer cell lines through RNAi, drug treatment, and the use of a nuclear-deficient AR mutant, we demonstrate that androgen acting on cytoplasmic AR rapidly stimulates Src tyrosine kinase via a non-genomic mechanism. Cytoplasmic AR, acting through Src enhances laminin integrin-dependent invasion. Active Matriptase, which cleaves laminin, is elevated within minutes after androgen stimulation, and is subsequently shed into the medium. Matriptase activation and shedding induced by cytoplasmic AR is dependent on Src. Concomitantly, CDCP1/gp140, a Matriptase and Src substrate that controls integrin-based migration, is activated. However, only inhibition of Matriptase, but not CDCP1, suppresses the AR/Src-dependent increase in invasion. Matriptase, present in conditioned medium from AR-stimulated cells, is sufficient to enhance invasion in the absence of androgen. Thus, invasion is stimulated by a rapid but sustained increase in Src activity, mediated non-genomically by cytoplasmic AR, leading to rapid activation and shedding of the laminin protease Matriptase.

Show MeSH
Related in: MedlinePlus