Limits...
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.

Show MeSH

Related in: MedlinePlus

Fold binding force (A, B, C) and total binding events (D, E, F) of perlecan domain IV (A, D), laminin (B, E), and fibronectin (C, F) attached to an AFM tip engaged to either LNECFP or 2BECFP cells plated on a 60 mm tissue culture dish.A. When the AFM tip is coated with perlecan domain IV, vector control 2BECFP cells exhibit a 40% decrease in binding force relative to LNECFP cells (top panel, P<0.05), and sustain nearly 700 less binding events than their LNECFP counterparts (bottom panel). B. LNCaP cells that overexpress beta-2 have a nearly 26 fold increase in average binding force to laminin over LNECFP cells (top panel, P<0.05) and incur a massive amount of binding events compared to LNECFP cells (bottom panel). C. LNECFP cells have an increased average binding force relative to 2BECFP cells (top panel, P<0.05) but exhibit a diminutive number of total events relative to 2BECFP cells when the AFM tip is coated with fibronectin (bottom panel). Fold binding force data presented as fold relative to LNECFP ± normalized SEM (n = 3).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4043823&req=5

pone-0098408-g006: Fold binding force (A, B, C) and total binding events (D, E, F) of perlecan domain IV (A, D), laminin (B, E), and fibronectin (C, F) attached to an AFM tip engaged to either LNECFP or 2BECFP cells plated on a 60 mm tissue culture dish.A. When the AFM tip is coated with perlecan domain IV, vector control 2BECFP cells exhibit a 40% decrease in binding force relative to LNECFP cells (top panel, P<0.05), and sustain nearly 700 less binding events than their LNECFP counterparts (bottom panel). B. LNCaP cells that overexpress beta-2 have a nearly 26 fold increase in average binding force to laminin over LNECFP cells (top panel, P<0.05) and incur a massive amount of binding events compared to LNECFP cells (bottom panel). C. LNECFP cells have an increased average binding force relative to 2BECFP cells (top panel, P<0.05) but exhibit a diminutive number of total events relative to 2BECFP cells when the AFM tip is coated with fibronectin (bottom panel). Fold binding force data presented as fold relative to LNECFP ± normalized SEM (n = 3).

Mentions: As demonstrated previously, 2BECFP cells have altered adhesion profiles to vitronectin, fibronectin, Matrigel and Matrigel growth factor reduced relative to LNECFP cells [62]. With this in mind, we were curious as to whether 2BECFP cells would also have distinctive binding profiles to various extracellular matrix molecules via atomic force microscopy (AFM) binding assays. LNECFP control cells exhibited a 40% increase in binding force (Figure 6A top panel, P<0.05) and a major uptick in binding events (852 total, 0.47 average) (Figure 6A bottom panel) to perlecan domain IV relative to 2BECFP cells (145 total, 0.08 average). 2BECFP, beta-2 overexpressing LNCaP cells had dramatically increased binding force to laminin (Figure 6B top panel, P<0.05) and a similarly dramatic boost in total binding events (3973 total, 1.47 average) over LNECFP control cells (34 total, 0.037 average) (Figure 6B bottom panel). Fibronectin-coated AFM tips engaged to LNECFP control cells elicited a nearly 70% enhanced binding force (Figure 6C top panel, P<0.05) but exhibited a substantial reduction in binding events (7 total, 0.004 average) (Figure 6C bottom panel) relative to 2BECFP cells (2354 total, 1.30 average). By overexpressing beta-2 in LNCaP cells, binding affinity for different extracellular matrix molecules is remarkably altered.


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)

Fold binding force (A, B, C) and total binding events (D, E, F) of perlecan domain IV (A, D), laminin (B, E), and fibronectin (C, F) attached to an AFM tip engaged to either LNECFP or 2BECFP cells plated on a 60 mm tissue culture dish.A. When the AFM tip is coated with perlecan domain IV, vector control 2BECFP cells exhibit a 40% decrease in binding force relative to LNECFP cells (top panel, P<0.05), and sustain nearly 700 less binding events than their LNECFP counterparts (bottom panel). B. LNCaP cells that overexpress beta-2 have a nearly 26 fold increase in average binding force to laminin over LNECFP cells (top panel, P<0.05) and incur a massive amount of binding events compared to LNECFP cells (bottom panel). C. LNECFP cells have an increased average binding force relative to 2BECFP cells (top panel, P<0.05) but exhibit a diminutive number of total events relative to 2BECFP cells when the AFM tip is coated with fibronectin (bottom panel). Fold binding force data presented as fold relative to LNECFP ± normalized SEM (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098408-g006: Fold binding force (A, B, C) and total binding events (D, E, F) of perlecan domain IV (A, D), laminin (B, E), and fibronectin (C, F) attached to an AFM tip engaged to either LNECFP or 2BECFP cells plated on a 60 mm tissue culture dish.A. When the AFM tip is coated with perlecan domain IV, vector control 2BECFP cells exhibit a 40% decrease in binding force relative to LNECFP cells (top panel, P<0.05), and sustain nearly 700 less binding events than their LNECFP counterparts (bottom panel). B. LNCaP cells that overexpress beta-2 have a nearly 26 fold increase in average binding force to laminin over LNECFP cells (top panel, P<0.05) and incur a massive amount of binding events compared to LNECFP cells (bottom panel). C. LNECFP cells have an increased average binding force relative to 2BECFP cells (top panel, P<0.05) but exhibit a diminutive number of total events relative to 2BECFP cells when the AFM tip is coated with fibronectin (bottom panel). Fold binding force data presented as fold relative to LNECFP ± normalized SEM (n = 3).
Mentions: As demonstrated previously, 2BECFP cells have altered adhesion profiles to vitronectin, fibronectin, Matrigel and Matrigel growth factor reduced relative to LNECFP cells [62]. With this in mind, we were curious as to whether 2BECFP cells would also have distinctive binding profiles to various extracellular matrix molecules via atomic force microscopy (AFM) binding assays. LNECFP control cells exhibited a 40% increase in binding force (Figure 6A top panel, P<0.05) and a major uptick in binding events (852 total, 0.47 average) (Figure 6A bottom panel) to perlecan domain IV relative to 2BECFP cells (145 total, 0.08 average). 2BECFP, beta-2 overexpressing LNCaP cells had dramatically increased binding force to laminin (Figure 6B top panel, P<0.05) and a similarly dramatic boost in total binding events (3973 total, 1.47 average) over LNECFP control cells (34 total, 0.037 average) (Figure 6B bottom panel). Fibronectin-coated AFM tips engaged to LNECFP control cells elicited a nearly 70% enhanced binding force (Figure 6C top panel, P<0.05) but exhibited a substantial reduction in binding events (7 total, 0.004 average) (Figure 6C bottom panel) relative to 2BECFP cells (2354 total, 1.30 average). By overexpressing beta-2 in LNCaP cells, binding affinity for different extracellular matrix molecules is remarkably altered.

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