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Collagen XV inhibits epithelial to mesenchymal transition in pancreatic adenocarcinoma cells.

Clementz AG, Mutolo MJ, Leir SH, Morris KJ, Kucybala K, Harris H, Harris A - PLoS ONE (2013)

Bottom Line: Collagen XV (COLXV) is a secreted non-fibrillar collagen found within basement membrane (BM) zones of the extracellular matrix (ECM).Here we show that COLXV significantly reduces invasion of pancreatic adenocarcinoma cells through a collagen I (COLI) matrix.In the presence of COLXV, the intracellular redistribution of E-Cad from the cell periphery, which is associated with COLI-activated EMT, is inhibited and concurrently, DDR1 signaling is suppressed.

View Article: PubMed Central - PubMed

Affiliation: Human Molecular Genetics Program, Lurie Children's Research Center, Chicago, Illinois, USA.

ABSTRACT
Collagen XV (COLXV) is a secreted non-fibrillar collagen found within basement membrane (BM) zones of the extracellular matrix (ECM). Its ability to alter cellular growth in vitro and to reduce tumor burden and increase survival in vivo support a role as a tumor suppressor. Loss of COLXV during the progression of several aggressive cancers precedes basement membrane invasion and metastasis. The resultant lack of COLXV subjacent to the basement membrane and subsequent loss of its interactions with other proteins in this zone may directly impact tumor progression. Here we show that COLXV significantly reduces invasion of pancreatic adenocarcinoma cells through a collagen I (COLI) matrix. Moreover, we demonstrate that epithelial to mesenchymal transition (EMT) in these cells, which is recapitulated in vitro by cell scattering on a COLI substrate, is inhibited by over-expression of COLXV. We identify critical collagen-binding surface receptors on the tumor cells, including the discoidin domain receptor 1 (DDR1) and E-Cadherin (E-Cad), which interact with COLXV and appear to mediate its function. In the presence of COLXV, the intracellular redistribution of E-Cad from the cell periphery, which is associated with COLI-activated EMT, is inhibited and concurrently, DDR1 signaling is suppressed. Furthermore, continuous exposure of the pancreatic adenocarcinoma cells to high levels of COLXV suppresses endogenous levels of N-Cadherin (N-Cad). These data reveal a novel mechanism whereby COLXV can function as a tumor suppressor in the basement membrane zone.

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Collagen XV inhibits invasion of BxPC cells though a collagen I-coated membrane.A) Stable expression of collagen XV in BxPC-3 and S2-013 pancreatic adenocarcinoma cells. Western blot probed with anti-COLXV antibody or anti-ß-tubulin loading control. Clones Bx15.1-15.40 express variable levels of COLXV. Also shown is one vector control clone (BxVC1). Note: the anti-COLXV antibody cross-reacts with an irrelevant higher MW protein that is seen in many cell types, irrespective of COLXV expression. B) The COLXV is secreted from BxPC-3 cells into the cell culture media. C) COLXV expression in a representative stable clone of S2-013 cells (S2.15.10), also shown is a vector control (S2.VC3). D) Growth rates of 4 BxPC-3 vector control clones and 4 COLXV-expressing clones are independent of COLXV expression. E, F) Invasion assay: E) vector (BxVC1, BxVC3) or COLXV (Bx15.14 and Bx15.23) cells plated on a membrane coated with COLI. After 72 hr cells invading through the membrane were stained, visualized and counted at 40X magnification. Random fields were selected from three independent experiments representing n = 16 vector plates (BxVC1, BxVC3) and n = 15 COLXV plates (Bx15.14 and Bx15.23). F) The average of each independent experiment is shown #1, #2, #3, (Left, V = vector control; C = COLXV) and the cumulative average of all triplicates (right).
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pone-0072250-g001: Collagen XV inhibits invasion of BxPC cells though a collagen I-coated membrane.A) Stable expression of collagen XV in BxPC-3 and S2-013 pancreatic adenocarcinoma cells. Western blot probed with anti-COLXV antibody or anti-ß-tubulin loading control. Clones Bx15.1-15.40 express variable levels of COLXV. Also shown is one vector control clone (BxVC1). Note: the anti-COLXV antibody cross-reacts with an irrelevant higher MW protein that is seen in many cell types, irrespective of COLXV expression. B) The COLXV is secreted from BxPC-3 cells into the cell culture media. C) COLXV expression in a representative stable clone of S2-013 cells (S2.15.10), also shown is a vector control (S2.VC3). D) Growth rates of 4 BxPC-3 vector control clones and 4 COLXV-expressing clones are independent of COLXV expression. E, F) Invasion assay: E) vector (BxVC1, BxVC3) or COLXV (Bx15.14 and Bx15.23) cells plated on a membrane coated with COLI. After 72 hr cells invading through the membrane were stained, visualized and counted at 40X magnification. Random fields were selected from three independent experiments representing n = 16 vector plates (BxVC1, BxVC3) and n = 15 COLXV plates (Bx15.14 and Bx15.23). F) The average of each independent experiment is shown #1, #2, #3, (Left, V = vector control; C = COLXV) and the cumulative average of all triplicates (right).

Mentions: To investigate the mechanism of action of COLXV in modulating pancreatic tumor cell growth in vitro, we stably expressed a human COLXV cDNA construct in the BxPC-3 and S2-013 pancreatic adenocarcinoma cell lines. Vector (pcDNA3.1) control clones of each cell line were also generated. Unlike most pancreatic adenocarcinoma cell lines, BxPC-3 has wild-type KRas, while S2-013 carries the common KRasG12D mutation. However, unlike S2-013, BxPC-3 demonstrates a marked scatter/EMT phenotype when grown on COLI substrates [19] and is thus an excellent model to study EMT in pancreatic cancer. Multiple clones were produced with a range of COLXV expression and 4 clones with high levels of COLXV were analyzed further (Bx15.5, Bx15.14, Bx15.23 and Bx15.24, Fig. 1A). Moreover, we showed that a substantial amount of COLXV is secreted from these clones into the cell culture medium (Fig. 1B). An equivalent series of cell clones expressing the COLXV transgene was generated from the S2-013 pancreatic adenocarcinoma cell line (Fig. 1C) and high-expressing clones S2.15.5 and S2.15.10 were taken forward for further analysis together with vector control clones.


Collagen XV inhibits epithelial to mesenchymal transition in pancreatic adenocarcinoma cells.

Clementz AG, Mutolo MJ, Leir SH, Morris KJ, Kucybala K, Harris H, Harris A - PLoS ONE (2013)

Collagen XV inhibits invasion of BxPC cells though a collagen I-coated membrane.A) Stable expression of collagen XV in BxPC-3 and S2-013 pancreatic adenocarcinoma cells. Western blot probed with anti-COLXV antibody or anti-ß-tubulin loading control. Clones Bx15.1-15.40 express variable levels of COLXV. Also shown is one vector control clone (BxVC1). Note: the anti-COLXV antibody cross-reacts with an irrelevant higher MW protein that is seen in many cell types, irrespective of COLXV expression. B) The COLXV is secreted from BxPC-3 cells into the cell culture media. C) COLXV expression in a representative stable clone of S2-013 cells (S2.15.10), also shown is a vector control (S2.VC3). D) Growth rates of 4 BxPC-3 vector control clones and 4 COLXV-expressing clones are independent of COLXV expression. E, F) Invasion assay: E) vector (BxVC1, BxVC3) or COLXV (Bx15.14 and Bx15.23) cells plated on a membrane coated with COLI. After 72 hr cells invading through the membrane were stained, visualized and counted at 40X magnification. Random fields were selected from three independent experiments representing n = 16 vector plates (BxVC1, BxVC3) and n = 15 COLXV plates (Bx15.14 and Bx15.23). F) The average of each independent experiment is shown #1, #2, #3, (Left, V = vector control; C = COLXV) and the cumulative average of all triplicates (right).
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getmorefigures.php?uid=PMC3750028&req=5

pone-0072250-g001: Collagen XV inhibits invasion of BxPC cells though a collagen I-coated membrane.A) Stable expression of collagen XV in BxPC-3 and S2-013 pancreatic adenocarcinoma cells. Western blot probed with anti-COLXV antibody or anti-ß-tubulin loading control. Clones Bx15.1-15.40 express variable levels of COLXV. Also shown is one vector control clone (BxVC1). Note: the anti-COLXV antibody cross-reacts with an irrelevant higher MW protein that is seen in many cell types, irrespective of COLXV expression. B) The COLXV is secreted from BxPC-3 cells into the cell culture media. C) COLXV expression in a representative stable clone of S2-013 cells (S2.15.10), also shown is a vector control (S2.VC3). D) Growth rates of 4 BxPC-3 vector control clones and 4 COLXV-expressing clones are independent of COLXV expression. E, F) Invasion assay: E) vector (BxVC1, BxVC3) or COLXV (Bx15.14 and Bx15.23) cells plated on a membrane coated with COLI. After 72 hr cells invading through the membrane were stained, visualized and counted at 40X magnification. Random fields were selected from three independent experiments representing n = 16 vector plates (BxVC1, BxVC3) and n = 15 COLXV plates (Bx15.14 and Bx15.23). F) The average of each independent experiment is shown #1, #2, #3, (Left, V = vector control; C = COLXV) and the cumulative average of all triplicates (right).
Mentions: To investigate the mechanism of action of COLXV in modulating pancreatic tumor cell growth in vitro, we stably expressed a human COLXV cDNA construct in the BxPC-3 and S2-013 pancreatic adenocarcinoma cell lines. Vector (pcDNA3.1) control clones of each cell line were also generated. Unlike most pancreatic adenocarcinoma cell lines, BxPC-3 has wild-type KRas, while S2-013 carries the common KRasG12D mutation. However, unlike S2-013, BxPC-3 demonstrates a marked scatter/EMT phenotype when grown on COLI substrates [19] and is thus an excellent model to study EMT in pancreatic cancer. Multiple clones were produced with a range of COLXV expression and 4 clones with high levels of COLXV were analyzed further (Bx15.5, Bx15.14, Bx15.23 and Bx15.24, Fig. 1A). Moreover, we showed that a substantial amount of COLXV is secreted from these clones into the cell culture medium (Fig. 1B). An equivalent series of cell clones expressing the COLXV transgene was generated from the S2-013 pancreatic adenocarcinoma cell line (Fig. 1C) and high-expressing clones S2.15.5 and S2.15.10 were taken forward for further analysis together with vector control clones.

Bottom Line: Collagen XV (COLXV) is a secreted non-fibrillar collagen found within basement membrane (BM) zones of the extracellular matrix (ECM).Here we show that COLXV significantly reduces invasion of pancreatic adenocarcinoma cells through a collagen I (COLI) matrix.In the presence of COLXV, the intracellular redistribution of E-Cad from the cell periphery, which is associated with COLI-activated EMT, is inhibited and concurrently, DDR1 signaling is suppressed.

View Article: PubMed Central - PubMed

Affiliation: Human Molecular Genetics Program, Lurie Children's Research Center, Chicago, Illinois, USA.

ABSTRACT
Collagen XV (COLXV) is a secreted non-fibrillar collagen found within basement membrane (BM) zones of the extracellular matrix (ECM). Its ability to alter cellular growth in vitro and to reduce tumor burden and increase survival in vivo support a role as a tumor suppressor. Loss of COLXV during the progression of several aggressive cancers precedes basement membrane invasion and metastasis. The resultant lack of COLXV subjacent to the basement membrane and subsequent loss of its interactions with other proteins in this zone may directly impact tumor progression. Here we show that COLXV significantly reduces invasion of pancreatic adenocarcinoma cells through a collagen I (COLI) matrix. Moreover, we demonstrate that epithelial to mesenchymal transition (EMT) in these cells, which is recapitulated in vitro by cell scattering on a COLI substrate, is inhibited by over-expression of COLXV. We identify critical collagen-binding surface receptors on the tumor cells, including the discoidin domain receptor 1 (DDR1) and E-Cadherin (E-Cad), which interact with COLXV and appear to mediate its function. In the presence of COLXV, the intracellular redistribution of E-Cad from the cell periphery, which is associated with COLI-activated EMT, is inhibited and concurrently, DDR1 signaling is suppressed. Furthermore, continuous exposure of the pancreatic adenocarcinoma cells to high levels of COLXV suppresses endogenous levels of N-Cadherin (N-Cad). These data reveal a novel mechanism whereby COLXV can function as a tumor suppressor in the basement membrane zone.

Show MeSH
Related in: MedlinePlus