Limits...
Yes-mediated phosphorylation of focal adhesion kinase at tyrosine 861 increases metastatic potential of prostate cancer cells.

Chatterji T, Varkaris AS, Parikh NU, Song JH, Cheng CJ, Schweppe RE, Alexander S, Davis JW, Troncoso P, Friedl P, Kuang J, Lin SH, Gallick GE - Oncotarget (2015)

Bottom Line: In human specimens, Yes expression was increased in lymph node metastases relative to paired primary tumors from the same patient, and increased pFAK Y861 expression in lymph node metastases correlated with poor prognosis.These results demonstrate a unique role for Yes in phosphorylation of FAK and in promoting PCa metastasis.Therefore, phosphorylated FAK Y861 and increased Yes expression may be predictive markers for PCa metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Genitourinary Medical Oncology, The David Koch Center for Applied Research in Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
To study the role of FAK signaling complexes in promoting metastatic properties of prostate cancer (PCa) cells, we selected stable, highly migratory variants, termed PC3 Mig-3 and DU145 Mig-3, from two well-characterized PCa cell lines, PC3 and DU145. These variants were not only increased migration and invasion in vitro, but were also more metastatic to lymph nodes following intraprostatic injection into nude mice. Both PC3 Mig-3 and DU145 Mig-3 were specifically increased in phosphorylation of FAK Y861. We therefore examined potential alterations in Src family kinases responsible for FAK phosphorylation and determined only Yes expression was increased. Overexpression of Yes in PC3 parental cells and src-/-fyn-/-yes-/- fibroblasts selectively increased FAK Y861 phosphorylation, and increased migration. Knockdown of Yes in PC3 Mig-3 cells decreased migration and decreased lymph node metastasis following orthotopic implantation of into nude mice. In human specimens, Yes expression was increased in lymph node metastases relative to paired primary tumors from the same patient, and increased pFAK Y861 expression in lymph node metastases correlated with poor prognosis. These results demonstrate a unique role for Yes in phosphorylation of FAK and in promoting PCa metastasis. Therefore, phosphorylated FAK Y861 and increased Yes expression may be predictive markers for PCa metastasis.

No MeSH data available.


Related in: MedlinePlus

Expression and Activity of Yes in PCa cellsA. Expression of SFKs in the migratory variants of PC3 cells was determined by immunoblotting (left panel). Quantification of expression of Yes, Lyn, Src, Fyn normalized to vinculin (right panel). B. Identical analyses on DU145 cells; immunoblot (left panel); quantification (right panel). Representative data are shown from three independent assays. C. mRNA expression of c-yes in indicated cells normalized to actin as a control. The data are represented as mean ± SD. **p < 0.001, *p < 0.05 by Student's t-test of three independent assays performed in triplicate. D. Activity of the SFK's was estimated by immunoprecipitation with specific antibodies for individual SFKs as indicated, followed by immunoblotting for the Src autophosphorylation site (pSrc Y416). Representative data are shown from three independent assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4496348&req=5

Figure 4: Expression and Activity of Yes in PCa cellsA. Expression of SFKs in the migratory variants of PC3 cells was determined by immunoblotting (left panel). Quantification of expression of Yes, Lyn, Src, Fyn normalized to vinculin (right panel). B. Identical analyses on DU145 cells; immunoblot (left panel); quantification (right panel). Representative data are shown from three independent assays. C. mRNA expression of c-yes in indicated cells normalized to actin as a control. The data are represented as mean ± SD. **p < 0.001, *p < 0.05 by Student's t-test of three independent assays performed in triplicate. D. Activity of the SFK's was estimated by immunoprecipitation with specific antibodies for individual SFKs as indicated, followed by immunoblotting for the Src autophosphorylation site (pSrc Y416). Representative data are shown from three independent assays.

Mentions: Src family kinases (SFK's) catalyze phosphorylation of all of the FAK tyrosine phosphorylation sites, excluding the autophosphorylation site (FAK Y397) [29]. Hence, we investigated the expression and activity of SFK's in PC3 Mig-3 and DU145 Mig-3 cells relative to PC3-P cells. As shown in Fig. 4A, no increased expression of Src, Fyn and Lyn was observed (immunoblot, left panel; quantification, right panel). However, a 2.5 fold increase in Yes expression was observed in PC3 Mig-3 cells relative to PC3-P cells. Similarly, Yes expression was increased by 2 fold in DU145 Mig-3 relative to DU145-P cells (Fig. 4B, immunoblot, left panel; quantification, right panel), with no increase in other Src family kinase members. As determined by qRT-PCR, c-yes mRNA was increased 2.3 fold (p < 0.001) in the PC3 Mig-3 and 2 fold (p < 0.05) in DU145 Mig-3 cells relative to their respective parental cells (Fig. 4C). We next examined the kinase activity of the SFKs expressed in PC3 cells. For these studies, immunoprecipitation of individual SFKs was performed using specific antibodies to each protein followed by immunoblotting with an antibody to chicken Src pY416 that recognizes the autophosphorylation sites (indicative of the activated form of the kinases) of all the SFKs examined. As shown in Fig. 4D, no increase in expression or autophosphorylation was observed for Src, Lyn and Fyn in PC3 Mig-3 cells relative to the PC3-P cells. However, Yes activity was increased by 3 fold in the more migratory PC3 Mig-3 cells relative to the PC3-P cells (Fig. 4D). These results suggest that increased expression of Yes is accompanied with increased total kinase activity in PC3 Mig-3 cells.


Yes-mediated phosphorylation of focal adhesion kinase at tyrosine 861 increases metastatic potential of prostate cancer cells.

Chatterji T, Varkaris AS, Parikh NU, Song JH, Cheng CJ, Schweppe RE, Alexander S, Davis JW, Troncoso P, Friedl P, Kuang J, Lin SH, Gallick GE - Oncotarget (2015)

Expression and Activity of Yes in PCa cellsA. Expression of SFKs in the migratory variants of PC3 cells was determined by immunoblotting (left panel). Quantification of expression of Yes, Lyn, Src, Fyn normalized to vinculin (right panel). B. Identical analyses on DU145 cells; immunoblot (left panel); quantification (right panel). Representative data are shown from three independent assays. C. mRNA expression of c-yes in indicated cells normalized to actin as a control. The data are represented as mean ± SD. **p < 0.001, *p < 0.05 by Student's t-test of three independent assays performed in triplicate. D. Activity of the SFK's was estimated by immunoprecipitation with specific antibodies for individual SFKs as indicated, followed by immunoblotting for the Src autophosphorylation site (pSrc Y416). Representative data are shown from three independent assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496348&req=5

Figure 4: Expression and Activity of Yes in PCa cellsA. Expression of SFKs in the migratory variants of PC3 cells was determined by immunoblotting (left panel). Quantification of expression of Yes, Lyn, Src, Fyn normalized to vinculin (right panel). B. Identical analyses on DU145 cells; immunoblot (left panel); quantification (right panel). Representative data are shown from three independent assays. C. mRNA expression of c-yes in indicated cells normalized to actin as a control. The data are represented as mean ± SD. **p < 0.001, *p < 0.05 by Student's t-test of three independent assays performed in triplicate. D. Activity of the SFK's was estimated by immunoprecipitation with specific antibodies for individual SFKs as indicated, followed by immunoblotting for the Src autophosphorylation site (pSrc Y416). Representative data are shown from three independent assays.
Mentions: Src family kinases (SFK's) catalyze phosphorylation of all of the FAK tyrosine phosphorylation sites, excluding the autophosphorylation site (FAK Y397) [29]. Hence, we investigated the expression and activity of SFK's in PC3 Mig-3 and DU145 Mig-3 cells relative to PC3-P cells. As shown in Fig. 4A, no increased expression of Src, Fyn and Lyn was observed (immunoblot, left panel; quantification, right panel). However, a 2.5 fold increase in Yes expression was observed in PC3 Mig-3 cells relative to PC3-P cells. Similarly, Yes expression was increased by 2 fold in DU145 Mig-3 relative to DU145-P cells (Fig. 4B, immunoblot, left panel; quantification, right panel), with no increase in other Src family kinase members. As determined by qRT-PCR, c-yes mRNA was increased 2.3 fold (p < 0.001) in the PC3 Mig-3 and 2 fold (p < 0.05) in DU145 Mig-3 cells relative to their respective parental cells (Fig. 4C). We next examined the kinase activity of the SFKs expressed in PC3 cells. For these studies, immunoprecipitation of individual SFKs was performed using specific antibodies to each protein followed by immunoblotting with an antibody to chicken Src pY416 that recognizes the autophosphorylation sites (indicative of the activated form of the kinases) of all the SFKs examined. As shown in Fig. 4D, no increase in expression or autophosphorylation was observed for Src, Lyn and Fyn in PC3 Mig-3 cells relative to the PC3-P cells. However, Yes activity was increased by 3 fold in the more migratory PC3 Mig-3 cells relative to the PC3-P cells (Fig. 4D). These results suggest that increased expression of Yes is accompanied with increased total kinase activity in PC3 Mig-3 cells.

Bottom Line: In human specimens, Yes expression was increased in lymph node metastases relative to paired primary tumors from the same patient, and increased pFAK Y861 expression in lymph node metastases correlated with poor prognosis.These results demonstrate a unique role for Yes in phosphorylation of FAK and in promoting PCa metastasis.Therefore, phosphorylated FAK Y861 and increased Yes expression may be predictive markers for PCa metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Genitourinary Medical Oncology, The David Koch Center for Applied Research in Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
To study the role of FAK signaling complexes in promoting metastatic properties of prostate cancer (PCa) cells, we selected stable, highly migratory variants, termed PC3 Mig-3 and DU145 Mig-3, from two well-characterized PCa cell lines, PC3 and DU145. These variants were not only increased migration and invasion in vitro, but were also more metastatic to lymph nodes following intraprostatic injection into nude mice. Both PC3 Mig-3 and DU145 Mig-3 were specifically increased in phosphorylation of FAK Y861. We therefore examined potential alterations in Src family kinases responsible for FAK phosphorylation and determined only Yes expression was increased. Overexpression of Yes in PC3 parental cells and src-/-fyn-/-yes-/- fibroblasts selectively increased FAK Y861 phosphorylation, and increased migration. Knockdown of Yes in PC3 Mig-3 cells decreased migration and decreased lymph node metastasis following orthotopic implantation of into nude mice. In human specimens, Yes expression was increased in lymph node metastases relative to paired primary tumors from the same patient, and increased pFAK Y861 expression in lymph node metastases correlated with poor prognosis. These results demonstrate a unique role for Yes in phosphorylation of FAK and in promoting PCa metastasis. Therefore, phosphorylated FAK Y861 and increased Yes expression may be predictive markers for PCa metastasis.

No MeSH data available.


Related in: MedlinePlus