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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

Development and characterization of highly migratory variants of PCa cellsA. Schematic diagram of isolation of migratory variants using a modified Boyden chamber assay. B.In vitro migration and C. invasion after each selection was determined using the modified Boyden chamber (migration) or matrigel-coated modified Boyden chamber (invasion) for 24 hours. Migrated (or invaded) cells were counted microscopically in 5 optical fields per filter. Bars represent mean ± SD from triplicate assays. *p < 0.05, **p < 0.001, ***p < 0.0001 by Student's t-test. D. Attachment assay was performed for the indicated cells for 30 minutes. Bar graph represents mean ± SD from two independent assays performed in triplicate. *p < 0.05 by Student's t-test.
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Figure 1: Development and characterization of highly migratory variants of PCa cellsA. Schematic diagram of isolation of migratory variants using a modified Boyden chamber assay. B.In vitro migration and C. invasion after each selection was determined using the modified Boyden chamber (migration) or matrigel-coated modified Boyden chamber (invasion) for 24 hours. Migrated (or invaded) cells were counted microscopically in 5 optical fields per filter. Bars represent mean ± SD from triplicate assays. *p < 0.05, **p < 0.001, ***p < 0.0001 by Student's t-test. D. Attachment assay was performed for the indicated cells for 30 minutes. Bar graph represents mean ± SD from two independent assays performed in triplicate. *p < 0.05 by Student's t-test.

Mentions: One of the principal roles of FAK-SFK complexes is regulating migration [4, 46, 47]. As numerous FAK activators and downstream signaling molecules are involved in this process, we developed highly migratory sublines of the prostate cancer cell lines PC3 and DU145 by multiple cycles of in vitro selection for cells that had migrated in a modified Boyden chamber (see schema, Fig. 1A). As described in Materials and Methods, cells that had migrated through the Boyden Chamber were grown to confluency and re-migrated. This process was repeated three times. Migratory-selected cells were termed PC3 Mig-1, PC3 Mig-2, PC3 Mig-3, DU145 Mig-1, DU145 Mig-2, and DU145 Mig-3, reflecting each cycle of selection (Fig. 1A). In vitro migration of these subclones was increased at each of the first three cycles of selection (Fig. 1B), with no further increases observed following subsequent selections (data not shown). The phenotype of the migratory variants has remained stable for more than 30 passages, the longest time examined. PC3 Mig-3 was increased in migration by 20 fold relative to PC3-P (PC3 parental) cells (Fig. 1B, p < 0.0001); DU145 Mig-3 cells were increased in migration by 6 fold (Fig. 1B) relative to DU145-P (DU145 parental) cells (p < 0.0001). As an independent measure of migration, time-lapse microscopy was performed for PC3-P and PC3 Mig-3 isogenic cell lines, and the average speed of the populations is plotted (Fig. S1, upper panel) along with representative images indicating the distance traveled by the cell populations in 24 hours (lower panel). Time-lapse movies of migration are shown in Videos S1 and S2. The speed of migration of PC3 Mig-3 was 0.08 ± 0.01 μm/min, compared to 0.04 ± 0.006 μm/min in PC3-P cells (p < 0.001). These data confirm that PC3 Mig-3 cells are more migratory than PC3-P 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)

Development and characterization of highly migratory variants of PCa cellsA. Schematic diagram of isolation of migratory variants using a modified Boyden chamber assay. B.In vitro migration and C. invasion after each selection was determined using the modified Boyden chamber (migration) or matrigel-coated modified Boyden chamber (invasion) for 24 hours. Migrated (or invaded) cells were counted microscopically in 5 optical fields per filter. Bars represent mean ± SD from triplicate assays. *p < 0.05, **p < 0.001, ***p < 0.0001 by Student's t-test. D. Attachment assay was performed for the indicated cells for 30 minutes. Bar graph represents mean ± SD from two independent assays performed in triplicate. *p < 0.05 by Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Development and characterization of highly migratory variants of PCa cellsA. Schematic diagram of isolation of migratory variants using a modified Boyden chamber assay. B.In vitro migration and C. invasion after each selection was determined using the modified Boyden chamber (migration) or matrigel-coated modified Boyden chamber (invasion) for 24 hours. Migrated (or invaded) cells were counted microscopically in 5 optical fields per filter. Bars represent mean ± SD from triplicate assays. *p < 0.05, **p < 0.001, ***p < 0.0001 by Student's t-test. D. Attachment assay was performed for the indicated cells for 30 minutes. Bar graph represents mean ± SD from two independent assays performed in triplicate. *p < 0.05 by Student's t-test.
Mentions: One of the principal roles of FAK-SFK complexes is regulating migration [4, 46, 47]. As numerous FAK activators and downstream signaling molecules are involved in this process, we developed highly migratory sublines of the prostate cancer cell lines PC3 and DU145 by multiple cycles of in vitro selection for cells that had migrated in a modified Boyden chamber (see schema, Fig. 1A). As described in Materials and Methods, cells that had migrated through the Boyden Chamber were grown to confluency and re-migrated. This process was repeated three times. Migratory-selected cells were termed PC3 Mig-1, PC3 Mig-2, PC3 Mig-3, DU145 Mig-1, DU145 Mig-2, and DU145 Mig-3, reflecting each cycle of selection (Fig. 1A). In vitro migration of these subclones was increased at each of the first three cycles of selection (Fig. 1B), with no further increases observed following subsequent selections (data not shown). The phenotype of the migratory variants has remained stable for more than 30 passages, the longest time examined. PC3 Mig-3 was increased in migration by 20 fold relative to PC3-P (PC3 parental) cells (Fig. 1B, p < 0.0001); DU145 Mig-3 cells were increased in migration by 6 fold (Fig. 1B) relative to DU145-P (DU145 parental) cells (p < 0.0001). As an independent measure of migration, time-lapse microscopy was performed for PC3-P and PC3 Mig-3 isogenic cell lines, and the average speed of the populations is plotted (Fig. S1, upper panel) along with representative images indicating the distance traveled by the cell populations in 24 hours (lower panel). Time-lapse movies of migration are shown in Videos S1 and S2. The speed of migration of PC3 Mig-3 was 0.08 ± 0.01 μm/min, compared to 0.04 ± 0.006 μm/min in PC3-P cells (p < 0.001). These data confirm that PC3 Mig-3 cells are more migratory than PC3-P 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