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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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Binding force distribution of perlecan domain IV (A), laminin (B), and fibronectin (C) attached to an AFM tip engaged to either LNECFP or 2BECFP cells plated on a 60 mm tissue culture dish.A. Binding between a perlecan domain IV-coated AFM tip and an LNECFP cell displays a more broad binding force distribution than their 2BECFP counterparts. B. 2BECFP LNCaP cells display a very wide force distribution relative to LNECFP cells with a laminin-treated AFM tip. C. When the tip is coated with fibronectin, LNECFP cells display a narrow force distribution compared to 2BECFP cells (n = 3).
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pone-0098408-g007: Binding force distribution of perlecan domain IV (A), laminin (B), and fibronectin (C) attached to an AFM tip engaged to either LNECFP or 2BECFP cells plated on a 60 mm tissue culture dish.A. Binding between a perlecan domain IV-coated AFM tip and an LNECFP cell displays a more broad binding force distribution than their 2BECFP counterparts. B. 2BECFP LNCaP cells display a very wide force distribution relative to LNECFP cells with a laminin-treated AFM tip. C. When the tip is coated with fibronectin, LNECFP cells display a narrow force distribution compared to 2BECFP cells (n = 3).

Mentions: Analysis of binding force distribution allows examination of the frequency of binding events at specific binding forces. We examined whether the binding force distribution of LNECFP and 2BECFP cells was different and whether these differences were matrix-dependent. LNECFP cells displayed a dynamic binding force distribution relative to 2BECFP cells when interacting with perlecan domain IV (Figure 7A), laminin (Figure 7B), and fibronectin (Figure 7C) coated AFM cantilevers. Perlecan domain IV induced a myriad range of binding events with LNECFP cells but a very narrow and shallow binding force distribution for 2BECFP cells (Figure 7A). When coated with laminin, AFM tips educed a large number of binding events through a wide range of binding forces with 2BECFP cells while LNECFP cells incur merely 34 binding events within the 0.1nN range (Figure 7B). Despite the increased average binding force relative to 2BECFP cells measured when LNECFP cells bind to a fibronectin-coated tip, LNECFP binding force distribution was miniscule relative to 2BECFP cells (Figure 7C). Overexpression of beta-2 dramatically alters binding force distribution in LNCaP cells, signifying beta-2 as an influential cog in transient cell adhesion.


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)

Binding force distribution of perlecan domain IV (A), laminin (B), and fibronectin (C) attached to an AFM tip engaged to either LNECFP or 2BECFP cells plated on a 60 mm tissue culture dish.A. Binding between a perlecan domain IV-coated AFM tip and an LNECFP cell displays a more broad binding force distribution than their 2BECFP counterparts. B. 2BECFP LNCaP cells display a very wide force distribution relative to LNECFP cells with a laminin-treated AFM tip. C. When the tip is coated with fibronectin, LNECFP cells display a narrow force distribution compared to 2BECFP cells (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098408-g007: Binding force distribution of perlecan domain IV (A), laminin (B), and fibronectin (C) attached to an AFM tip engaged to either LNECFP or 2BECFP cells plated on a 60 mm tissue culture dish.A. Binding between a perlecan domain IV-coated AFM tip and an LNECFP cell displays a more broad binding force distribution than their 2BECFP counterparts. B. 2BECFP LNCaP cells display a very wide force distribution relative to LNECFP cells with a laminin-treated AFM tip. C. When the tip is coated with fibronectin, LNECFP cells display a narrow force distribution compared to 2BECFP cells (n = 3).
Mentions: Analysis of binding force distribution allows examination of the frequency of binding events at specific binding forces. We examined whether the binding force distribution of LNECFP and 2BECFP cells was different and whether these differences were matrix-dependent. LNECFP cells displayed a dynamic binding force distribution relative to 2BECFP cells when interacting with perlecan domain IV (Figure 7A), laminin (Figure 7B), and fibronectin (Figure 7C) coated AFM cantilevers. Perlecan domain IV induced a myriad range of binding events with LNECFP cells but a very narrow and shallow binding force distribution for 2BECFP cells (Figure 7A). When coated with laminin, AFM tips educed a large number of binding events through a wide range of binding forces with 2BECFP cells while LNECFP cells incur merely 34 binding events within the 0.1nN range (Figure 7B). Despite the increased average binding force relative to 2BECFP cells measured when LNECFP cells bind to a fibronectin-coated tip, LNECFP binding force distribution was miniscule relative to 2BECFP cells (Figure 7C). Overexpression of beta-2 dramatically alters binding force distribution in LNCaP cells, signifying beta-2 as an influential cog in transient cell adhesion.

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