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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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Beta-2 expression increases PCa cell association with nerve.A. The association of PCa cells with unmyelinated nerve axons is associated directly with metastatic potential of the PCa cells and increased beta-2 expression. B. PCa cell association with nerve displays a similar trend when axons are myelinated. C. Following 2 hours in co-culture with neuronally differentiated F11 cells, 2BECFP cells have established more stable associations as compared to LNECFP cells (Per 10x field, 6,156.27 µm2). Graph represents the mean ± SEM and the asterisks denote statistical significance (P< 0.05, n = 3).
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pone-0098408-g003: Beta-2 expression increases PCa cell association with nerve.A. The association of PCa cells with unmyelinated nerve axons is associated directly with metastatic potential of the PCa cells and increased beta-2 expression. B. PCa cell association with nerve displays a similar trend when axons are myelinated. C. Following 2 hours in co-culture with neuronally differentiated F11 cells, 2BECFP cells have established more stable associations as compared to LNECFP cells (Per 10x field, 6,156.27 µm2). Graph represents the mean ± SEM and the asterisks denote statistical significance (P< 0.05, n = 3).

Mentions: Regardless of their aggressiveness and/or beta-2 expression, all PCa cells tested in this system associated and/or localized with YFP+ nerve axons (Figure 1 and 2). However, quantitative analysis of tile scans reveals beta-2 levels may affect the ability of PCa cells to associate with YFP+ nerve axons after 2 hours in co-culture. A relative increase in association with YFP+ nerve axons was observed as cells became more aggressive and metastatic and/or expressed more beta-2 (Figure 3A, black bars). To amplify the presence of myelin protein on YFP+ nerve axons, a putative binding partner of beta-2, the cultures were placed under myelinating conditions. Under these conditions and following 6 hours in co-culture, a trend of increased association with enhanced metastatic potential and expression levels of beta-2 was observed (Figure 3B). Utilizing a reductionist approach towards PCa cell association with nerve cells, we examined LNECFP and 2BECFP association with differentiated F11 cells after 2 hours of co-culture. Beta-2 overexpressing 2BECFP cells have a significantly enhanced ability (129.3 cells per 10x field) to remain in co-culture with F11 cells following 2 hours co-culture and subsequent media exchange relative to LNECFP cells (43.3 cells per 10x field) (Figure 3C, P<0.05). As PCa cells become more aggressive and metastatic and express more beta-2, they associate more frequently with both unmyelinated and myelinated spinal cord YFP+ nerve axons and have a significantly enhanced ability to remain in co-culture with differentiated F11 cells.


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)

Beta-2 expression increases PCa cell association with nerve.A. The association of PCa cells with unmyelinated nerve axons is associated directly with metastatic potential of the PCa cells and increased beta-2 expression. B. PCa cell association with nerve displays a similar trend when axons are myelinated. C. Following 2 hours in co-culture with neuronally differentiated F11 cells, 2BECFP cells have established more stable associations as compared to LNECFP cells (Per 10x field, 6,156.27 µm2). Graph represents the mean ± SEM and the asterisks denote statistical significance (P< 0.05, n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098408-g003: Beta-2 expression increases PCa cell association with nerve.A. The association of PCa cells with unmyelinated nerve axons is associated directly with metastatic potential of the PCa cells and increased beta-2 expression. B. PCa cell association with nerve displays a similar trend when axons are myelinated. C. Following 2 hours in co-culture with neuronally differentiated F11 cells, 2BECFP cells have established more stable associations as compared to LNECFP cells (Per 10x field, 6,156.27 µm2). Graph represents the mean ± SEM and the asterisks denote statistical significance (P< 0.05, n = 3).
Mentions: Regardless of their aggressiveness and/or beta-2 expression, all PCa cells tested in this system associated and/or localized with YFP+ nerve axons (Figure 1 and 2). However, quantitative analysis of tile scans reveals beta-2 levels may affect the ability of PCa cells to associate with YFP+ nerve axons after 2 hours in co-culture. A relative increase in association with YFP+ nerve axons was observed as cells became more aggressive and metastatic and/or expressed more beta-2 (Figure 3A, black bars). To amplify the presence of myelin protein on YFP+ nerve axons, a putative binding partner of beta-2, the cultures were placed under myelinating conditions. Under these conditions and following 6 hours in co-culture, a trend of increased association with enhanced metastatic potential and expression levels of beta-2 was observed (Figure 3B). Utilizing a reductionist approach towards PCa cell association with nerve cells, we examined LNECFP and 2BECFP association with differentiated F11 cells after 2 hours of co-culture. Beta-2 overexpressing 2BECFP cells have a significantly enhanced ability (129.3 cells per 10x field) to remain in co-culture with F11 cells following 2 hours co-culture and subsequent media exchange relative to LNECFP cells (43.3 cells per 10x field) (Figure 3C, P<0.05). As PCa cells become more aggressive and metastatic and express more beta-2, they associate more frequently with both unmyelinated and myelinated spinal cord YFP+ nerve axons and have a significantly enhanced ability to remain in co-culture with differentiated F11 cells.

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