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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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An AFM tip coated with either serum-free CHO cell conditioned media (CHO), serum-free CHO conditioned media containing beta-2 myc-His ectodomain (CHO Beta-2), 100 µg/mL BSA (BSA), or 100 µg/mL purified beta-2 myc-His ectodomain (Purified beta-2) was engaged to a 60 mm plate coated with 50 µg/mL laminin.CHO Beta-2 exhibits a statistically significant reduction in binding force (A, P<0.05) relative to control CHO but generates a slight increase average binding events (B) and force distribution (C,D) when engaged to a laminin-coated dish. Purified beta-2 displays a 40% decrease in binding force relative to BSA (A) but elicits a significant increase in average binding events per engagement (B) and a more extensive force distribution (C,D) than BSA. Specifically, purified beta-2 elicits a significant increase in binding events between 10–200 pN compared to BSA (D). Fold binding force data presented as fold relative to BSA ± normalized SEM (n = 3).
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pone-0098408-g008: An AFM tip coated with either serum-free CHO cell conditioned media (CHO), serum-free CHO conditioned media containing beta-2 myc-His ectodomain (CHO Beta-2), 100 µg/mL BSA (BSA), or 100 µg/mL purified beta-2 myc-His ectodomain (Purified beta-2) was engaged to a 60 mm plate coated with 50 µg/mL laminin.CHO Beta-2 exhibits a statistically significant reduction in binding force (A, P<0.05) relative to control CHO but generates a slight increase average binding events (B) and force distribution (C,D) when engaged to a laminin-coated dish. Purified beta-2 displays a 40% decrease in binding force relative to BSA (A) but elicits a significant increase in average binding events per engagement (B) and a more extensive force distribution (C,D) than BSA. Specifically, purified beta-2 elicits a significant increase in binding events between 10–200 pN compared to BSA (D). Fold binding force data presented as fold relative to BSA ± normalized SEM (n = 3).

Mentions: Based on the whole cell atomic force studies, it was apparent that beta-2 overexpression induced a strong affinity for laminin by LNCaP cells. To ascertain, at least partly, the cause of the increased binding force and binding events between laminin and 2BECFP cells, we generated a recombinant beta-2 ectodomain. The ectodomain of beta-2 contains the V-set Ig domain including the putative residues for binding to extracellular proteins (Table 1). Atomic force studies utilizing this domain allowed us to determine if beta-2 would bind to laminin directly, or if it was enhancing LNCaP binding to laminin indirectly. Due to the entirely nonspecific nature of BSA interaction with laminin: CHO, CHO Beta-2, and Purified Beta-2 all had a vastly lower fold binding force relative to the control BSA-coated tips (Figure 8A, P<0.05). However, purified beta-2 elicited considerably more binding events (30,856 total, 17.46 average) than CHO (5694 total, 2.95 average), CHO Beta-2 (6229 total, 3 average), or BSA (5804 total, 3.23 average) (Figure 8B). This data is consistent with the observed binding force distribution observed in figure 7, as purified beta-2 had a very large binding force distribution between 0.1nN and 1nN, while the treatments yielded very few interactions after 0.1nN (Figure 8C). This difference is more pronounced when examined between 10 and 200 pN, as purified beta-2 has a radically different binding profile than all other treatments (Figure 8D). Employing purified beta-2 ectodomain AFM binding assays to laminin engenders weak binding forces and a myriad of binding events that is unique from the CHO, CHO Beta-2, and BSA binding profiles. In part, these data may suggest a mechanism for the weak and transient binding force observed at lower amplitudes between 2BECFP cells and laminin.


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)

An AFM tip coated with either serum-free CHO cell conditioned media (CHO), serum-free CHO conditioned media containing beta-2 myc-His ectodomain (CHO Beta-2), 100 µg/mL BSA (BSA), or 100 µg/mL purified beta-2 myc-His ectodomain (Purified beta-2) was engaged to a 60 mm plate coated with 50 µg/mL laminin.CHO Beta-2 exhibits a statistically significant reduction in binding force (A, P<0.05) relative to control CHO but generates a slight increase average binding events (B) and force distribution (C,D) when engaged to a laminin-coated dish. Purified beta-2 displays a 40% decrease in binding force relative to BSA (A) but elicits a significant increase in average binding events per engagement (B) and a more extensive force distribution (C,D) than BSA. Specifically, purified beta-2 elicits a significant increase in binding events between 10–200 pN compared to BSA (D). Fold binding force data presented as fold relative to BSA ± normalized SEM (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098408-g008: An AFM tip coated with either serum-free CHO cell conditioned media (CHO), serum-free CHO conditioned media containing beta-2 myc-His ectodomain (CHO Beta-2), 100 µg/mL BSA (BSA), or 100 µg/mL purified beta-2 myc-His ectodomain (Purified beta-2) was engaged to a 60 mm plate coated with 50 µg/mL laminin.CHO Beta-2 exhibits a statistically significant reduction in binding force (A, P<0.05) relative to control CHO but generates a slight increase average binding events (B) and force distribution (C,D) when engaged to a laminin-coated dish. Purified beta-2 displays a 40% decrease in binding force relative to BSA (A) but elicits a significant increase in average binding events per engagement (B) and a more extensive force distribution (C,D) than BSA. Specifically, purified beta-2 elicits a significant increase in binding events between 10–200 pN compared to BSA (D). Fold binding force data presented as fold relative to BSA ± normalized SEM (n = 3).
Mentions: Based on the whole cell atomic force studies, it was apparent that beta-2 overexpression induced a strong affinity for laminin by LNCaP cells. To ascertain, at least partly, the cause of the increased binding force and binding events between laminin and 2BECFP cells, we generated a recombinant beta-2 ectodomain. The ectodomain of beta-2 contains the V-set Ig domain including the putative residues for binding to extracellular proteins (Table 1). Atomic force studies utilizing this domain allowed us to determine if beta-2 would bind to laminin directly, or if it was enhancing LNCaP binding to laminin indirectly. Due to the entirely nonspecific nature of BSA interaction with laminin: CHO, CHO Beta-2, and Purified Beta-2 all had a vastly lower fold binding force relative to the control BSA-coated tips (Figure 8A, P<0.05). However, purified beta-2 elicited considerably more binding events (30,856 total, 17.46 average) than CHO (5694 total, 2.95 average), CHO Beta-2 (6229 total, 3 average), or BSA (5804 total, 3.23 average) (Figure 8B). This data is consistent with the observed binding force distribution observed in figure 7, as purified beta-2 had a very large binding force distribution between 0.1nN and 1nN, while the treatments yielded very few interactions after 0.1nN (Figure 8C). This difference is more pronounced when examined between 10 and 200 pN, as purified beta-2 has a radically different binding profile than all other treatments (Figure 8D). Employing purified beta-2 ectodomain AFM binding assays to laminin engenders weak binding forces and a myriad of binding events that is unique from the CHO, CHO Beta-2, and BSA binding profiles. In part, these data may suggest a mechanism for the weak and transient binding force observed at lower amplitudes between 2BECFP cells and laminin.

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