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Identification of beta-2 as a key cell adhesion molecule in PCa cell neurotropic behavior: a novel ex vivo and biophysical approach.

Jansson KH, Castillo DG, Morris JW, Boggs ME, Czymmek KJ, Adams EL, Schramm LP, Sikes RA - PLoS ONE (2014)

Bottom Line: We overexpressed beta-2 as a fusion protein with enhanced cyan fluorescence protein (ECFP) in weakly aggressive LNCaP cells and observed neurotropic effects utilizing our novel ex vivo organotypic spinal cord co-culture model, and performed functional assays with neural matrices and atomic force microscopy.On laminin, a neural CAM, overexpression of beta-2 enhances PCa cell migration, invasion, and growth. 2BECFP cells exhibit marked binding affinity to laminin relative to LNECFP controls, and recombinant beta-2 ectodomain elicits more binding events to laminin than BSA control.Functional overexpression of VSSC beta subunits in PCa may mediate PCa metastatic behavior through association with neural matrices.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Cancer Ontogeny and Therapeutics, Department of Biological Sciences, University of Delaware, Newark, Delaware, United States of America; The Center for Translational Cancer Research, University of Delaware, Newark, Delaware, United States of America.

ABSTRACT
Prostate cancer (PCa) is believed to metastasize through the blood/lymphatics systems; however, PCa may utilize the extensive innervation of the prostate for glandular egress. The interaction of PCa and its nerve fibers is observed in 80% of PCa and is termed perineural invasion (PNI). PCa cells have been observed traveling through the endoneurium of nerves, although the underlying mechanisms have not been elucidated. Voltage sensitive sodium channels (VSSC) are multimeric transmembrane protein complexes comprised of a pore-forming α subunit and one or two auxiliary beta (β) subunits with inherent cell adhesion molecule (CAM) functions. The beta-2 isoform (gene SCN2B) interacts with several neural CAMs, while interacting putatively with other prominent neural CAMs. Furthermore, beta-2 exhibits elevated mRNA and protein levels in highly metastatic and castrate-resistant PCa. When overexpressed in weakly aggressive LNCaP cells (2BECFP), beta-2 alters LNCaP cell morphology and enhances LNCaP cell metastasis associated behavior in vitro. We hypothesize that PCa cells use beta-2 as a CAM during PNI and subsequent PCa metastasis. The objective of this study was to determine the effect of beta-2 expression on PCa cell neurotropic metastasis associated behavior. We overexpressed beta-2 as a fusion protein with enhanced cyan fluorescence protein (ECFP) in weakly aggressive LNCaP cells and observed neurotropic effects utilizing our novel ex vivo organotypic spinal cord co-culture model, and performed functional assays with neural matrices and atomic force microscopy. With increased beta-2 expression, PCa cells display a trend of enhanced association with nerve axons. On laminin, a neural CAM, overexpression of beta-2 enhances PCa cell migration, invasion, and growth. 2BECFP cells exhibit marked binding affinity to laminin relative to LNECFP controls, and recombinant beta-2 ectodomain elicits more binding events to laminin than BSA control. Functional overexpression of VSSC beta subunits in PCa may mediate PCa metastatic behavior through association with neural matrices.

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Representative confocal images from novel ex vivo organotypic spinal cord slice and co-culture model with C4-2B4 cells.A. Tile scan where YFP spinal cord is clearly positive as well as outgrowth of neurons with multiple nodal appearance (scale bar  =  500 µm) B. Higher magnification image of neuronal outgrowths from A. In this image C4-2B cells appear to associate with YFP+ nerve axons (red arrows, scale bar  =  100 µm).
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pone-0098408-g002: Representative confocal images from novel ex vivo organotypic spinal cord slice and co-culture model with C4-2B4 cells.A. Tile scan where YFP spinal cord is clearly positive as well as outgrowth of neurons with multiple nodal appearance (scale bar  =  500 µm) B. Higher magnification image of neuronal outgrowths from A. In this image C4-2B cells appear to associate with YFP+ nerve axons (red arrows, scale bar  =  100 µm).

Mentions: To examine whether PCa cells have an affinity for spinal cord nerve axons, a novel ex vivo organotypic spinal cord co-culture model was developed. Within this model, PCa cells of varying aggressiveness and levels of beta-2 expression were examined for their association with nerve axons. A representative tile scan displays 2BECFP cells in association with YFP+ nerve axons (Figure 1A). At a higher magnification LNECFP cells exhibit close proximity with a YFP+ nerve axon (Figure 1B). 2BECFP cells (Figure 1C) appear to have similar proximity to the YFP+ nerve axon as LNECFP cells (Figure 1B); however, they generate beta-2 specific microvilli that are unique to the 2BECFP cell type (Figure 1C, red arrows). Indeed, LNECFP cells do not produce visible microvilli. C4-2B4 cells transfected with the ECFP vector were observed in propinquity with nerve axons, as exhibited by tile scan (Figure 2A) and high magnification representative images (Figure 2B, red arrows).


Identification of beta-2 as a key cell adhesion molecule in PCa cell neurotropic behavior: a novel ex vivo and biophysical approach.

Jansson KH, Castillo DG, Morris JW, Boggs ME, Czymmek KJ, Adams EL, Schramm LP, Sikes RA - PLoS ONE (2014)

Representative confocal images from novel ex vivo organotypic spinal cord slice and co-culture model with C4-2B4 cells.A. Tile scan where YFP spinal cord is clearly positive as well as outgrowth of neurons with multiple nodal appearance (scale bar  =  500 µm) B. Higher magnification image of neuronal outgrowths from A. In this image C4-2B cells appear to associate with YFP+ nerve axons (red arrows, scale bar  =  100 µm).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043823&req=5

pone-0098408-g002: Representative confocal images from novel ex vivo organotypic spinal cord slice and co-culture model with C4-2B4 cells.A. Tile scan where YFP spinal cord is clearly positive as well as outgrowth of neurons with multiple nodal appearance (scale bar  =  500 µm) B. Higher magnification image of neuronal outgrowths from A. In this image C4-2B cells appear to associate with YFP+ nerve axons (red arrows, scale bar  =  100 µm).
Mentions: To examine whether PCa cells have an affinity for spinal cord nerve axons, a novel ex vivo organotypic spinal cord co-culture model was developed. Within this model, PCa cells of varying aggressiveness and levels of beta-2 expression were examined for their association with nerve axons. A representative tile scan displays 2BECFP cells in association with YFP+ nerve axons (Figure 1A). At a higher magnification LNECFP cells exhibit close proximity with a YFP+ nerve axon (Figure 1B). 2BECFP cells (Figure 1C) appear to have similar proximity to the YFP+ nerve axon as LNECFP cells (Figure 1B); however, they generate beta-2 specific microvilli that are unique to the 2BECFP cell type (Figure 1C, red arrows). Indeed, LNECFP cells do not produce visible microvilli. C4-2B4 cells transfected with the ECFP vector were observed in propinquity with nerve axons, as exhibited by tile scan (Figure 2A) and high magnification representative images (Figure 2B, red arrows).

Bottom Line: We overexpressed beta-2 as a fusion protein with enhanced cyan fluorescence protein (ECFP) in weakly aggressive LNCaP cells and observed neurotropic effects utilizing our novel ex vivo organotypic spinal cord co-culture model, and performed functional assays with neural matrices and atomic force microscopy.On laminin, a neural CAM, overexpression of beta-2 enhances PCa cell migration, invasion, and growth. 2BECFP cells exhibit marked binding affinity to laminin relative to LNECFP controls, and recombinant beta-2 ectodomain elicits more binding events to laminin than BSA control.Functional overexpression of VSSC beta subunits in PCa may mediate PCa metastatic behavior through association with neural matrices.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Cancer Ontogeny and Therapeutics, Department of Biological Sciences, University of Delaware, Newark, Delaware, United States of America; The Center for Translational Cancer Research, University of Delaware, Newark, Delaware, United States of America.

ABSTRACT
Prostate cancer (PCa) is believed to metastasize through the blood/lymphatics systems; however, PCa may utilize the extensive innervation of the prostate for glandular egress. The interaction of PCa and its nerve fibers is observed in 80% of PCa and is termed perineural invasion (PNI). PCa cells have been observed traveling through the endoneurium of nerves, although the underlying mechanisms have not been elucidated. Voltage sensitive sodium channels (VSSC) are multimeric transmembrane protein complexes comprised of a pore-forming α subunit and one or two auxiliary beta (β) subunits with inherent cell adhesion molecule (CAM) functions. The beta-2 isoform (gene SCN2B) interacts with several neural CAMs, while interacting putatively with other prominent neural CAMs. Furthermore, beta-2 exhibits elevated mRNA and protein levels in highly metastatic and castrate-resistant PCa. When overexpressed in weakly aggressive LNCaP cells (2BECFP), beta-2 alters LNCaP cell morphology and enhances LNCaP cell metastasis associated behavior in vitro. We hypothesize that PCa cells use beta-2 as a CAM during PNI and subsequent PCa metastasis. The objective of this study was to determine the effect of beta-2 expression on PCa cell neurotropic metastasis associated behavior. We overexpressed beta-2 as a fusion protein with enhanced cyan fluorescence protein (ECFP) in weakly aggressive LNCaP cells and observed neurotropic effects utilizing our novel ex vivo organotypic spinal cord co-culture model, and performed functional assays with neural matrices and atomic force microscopy. With increased beta-2 expression, PCa cells display a trend of enhanced association with nerve axons. On laminin, a neural CAM, overexpression of beta-2 enhances PCa cell migration, invasion, and growth. 2BECFP cells exhibit marked binding affinity to laminin relative to LNECFP controls, and recombinant beta-2 ectodomain elicits more binding events to laminin than BSA control. Functional overexpression of VSSC beta subunits in PCa may mediate PCa metastatic behavior through association with neural matrices.

Show MeSH
Related in: MedlinePlus