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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 scatter of BxPC-3 cells on a collagen I substrate and interacts directly with DDR1 and E-Cadherin.A) Vector control (BxVC3) and COLXV expressing (Bx15.1, Bx15.14) clones are shown grown on plastic substrate and on COLI coated substrate. Phase contrast microscopy, all panels 100X magnification. 3 vector clones and 5 COLXV expressing clones (2 with low expression and 3 with high expression) were analyzed at least 3 times and the results were consistent. B) Immunoprecipitation (IP) of DDR1 from vector clone (BxVC3) and COLXV clone (Bx15.14), followed by probing of western blots of the IP material with antibodies specific for COLXV, DDR1 and E-Cad. C) IP of FLAG-tagged COLXV (FCOLXV) from the BxF15.3 clone using the M2 antibody. D) IP of COLXV from clone Bx15.3 with M2 after depletion of DDR1 with a specific siRNA (DDR1i) or transfection of scrambled control siRNA (Scrbi). Depletion of DDR1 reduced the amount of DDR1, but not E-Cad, interacting with COLXV. E) IP of COLXV from clone BxF15.3 with M2 after depletion of E-Cad with a specific siRNA (E-Cadi) or transfection of scrambled control siRNA (Scrbi). Depletion of E-Cad reduced the amounts interacting with COLXV. All experiments were performed a minimum of 3 times with consistent results.
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pone-0072250-g002: Collagen XV inhibits scatter of BxPC-3 cells on a collagen I substrate and interacts directly with DDR1 and E-Cadherin.A) Vector control (BxVC3) and COLXV expressing (Bx15.1, Bx15.14) clones are shown grown on plastic substrate and on COLI coated substrate. Phase contrast microscopy, all panels 100X magnification. 3 vector clones and 5 COLXV expressing clones (2 with low expression and 3 with high expression) were analyzed at least 3 times and the results were consistent. B) Immunoprecipitation (IP) of DDR1 from vector clone (BxVC3) and COLXV clone (Bx15.14), followed by probing of western blots of the IP material with antibodies specific for COLXV, DDR1 and E-Cad. C) IP of FLAG-tagged COLXV (FCOLXV) from the BxF15.3 clone using the M2 antibody. D) IP of COLXV from clone Bx15.3 with M2 after depletion of DDR1 with a specific siRNA (DDR1i) or transfection of scrambled control siRNA (Scrbi). Depletion of DDR1 reduced the amount of DDR1, but not E-Cad, interacting with COLXV. E) IP of COLXV from clone BxF15.3 with M2 after depletion of E-Cad with a specific siRNA (E-Cadi) or transfection of scrambled control siRNA (Scrbi). Depletion of E-Cad reduced the amounts interacting with COLXV. All experiments were performed a minimum of 3 times with consistent results.

Mentions: BxPC-3 cells were previously shown to upregulate N-Cad and scatter, when plated on a COLI substrate. Scattering is an in vitro process that mimics epithelial to mesenchymal transition (EMT) during tumor progression [37], [38]. Since COLXV is lost from the basement membrane zone prior to metastasis of several tumor types, we asked whether it might interfere with COLI-mediated scatter and thus prevent EMT. BxPC-3 clones expressing COLXV and vector-only control clones were plated on untreated plastic or on COLI-coated plastic and grown for 48 to 72 hours. As observed previously [19], vector control clones scattered on the COLI substrate; however, in contrast, high levels of COLXV expression (Bx15.14, Bx15.24) inhibited the scatter response (Bx15.14 shown in Fig. 2A). Moreover, this effect appeared to correlate with COLXV expression levels since clones Bx15.1 (shown in Fig. 2A) and Bx15.40, which have very little COLXV (Fig. 1A), showed minimal inhibition of scatter.


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 scatter of BxPC-3 cells on a collagen I substrate and interacts directly with DDR1 and E-Cadherin.A) Vector control (BxVC3) and COLXV expressing (Bx15.1, Bx15.14) clones are shown grown on plastic substrate and on COLI coated substrate. Phase contrast microscopy, all panels 100X magnification. 3 vector clones and 5 COLXV expressing clones (2 with low expression and 3 with high expression) were analyzed at least 3 times and the results were consistent. B) Immunoprecipitation (IP) of DDR1 from vector clone (BxVC3) and COLXV clone (Bx15.14), followed by probing of western blots of the IP material with antibodies specific for COLXV, DDR1 and E-Cad. C) IP of FLAG-tagged COLXV (FCOLXV) from the BxF15.3 clone using the M2 antibody. D) IP of COLXV from clone Bx15.3 with M2 after depletion of DDR1 with a specific siRNA (DDR1i) or transfection of scrambled control siRNA (Scrbi). Depletion of DDR1 reduced the amount of DDR1, but not E-Cad, interacting with COLXV. E) IP of COLXV from clone BxF15.3 with M2 after depletion of E-Cad with a specific siRNA (E-Cadi) or transfection of scrambled control siRNA (Scrbi). Depletion of E-Cad reduced the amounts interacting with COLXV. All experiments were performed a minimum of 3 times with consistent results.
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Related In: Results  -  Collection

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pone-0072250-g002: Collagen XV inhibits scatter of BxPC-3 cells on a collagen I substrate and interacts directly with DDR1 and E-Cadherin.A) Vector control (BxVC3) and COLXV expressing (Bx15.1, Bx15.14) clones are shown grown on plastic substrate and on COLI coated substrate. Phase contrast microscopy, all panels 100X magnification. 3 vector clones and 5 COLXV expressing clones (2 with low expression and 3 with high expression) were analyzed at least 3 times and the results were consistent. B) Immunoprecipitation (IP) of DDR1 from vector clone (BxVC3) and COLXV clone (Bx15.14), followed by probing of western blots of the IP material with antibodies specific for COLXV, DDR1 and E-Cad. C) IP of FLAG-tagged COLXV (FCOLXV) from the BxF15.3 clone using the M2 antibody. D) IP of COLXV from clone Bx15.3 with M2 after depletion of DDR1 with a specific siRNA (DDR1i) or transfection of scrambled control siRNA (Scrbi). Depletion of DDR1 reduced the amount of DDR1, but not E-Cad, interacting with COLXV. E) IP of COLXV from clone BxF15.3 with M2 after depletion of E-Cad with a specific siRNA (E-Cadi) or transfection of scrambled control siRNA (Scrbi). Depletion of E-Cad reduced the amounts interacting with COLXV. All experiments were performed a minimum of 3 times with consistent results.
Mentions: BxPC-3 cells were previously shown to upregulate N-Cad and scatter, when plated on a COLI substrate. Scattering is an in vitro process that mimics epithelial to mesenchymal transition (EMT) during tumor progression [37], [38]. Since COLXV is lost from the basement membrane zone prior to metastasis of several tumor types, we asked whether it might interfere with COLI-mediated scatter and thus prevent EMT. BxPC-3 clones expressing COLXV and vector-only control clones were plated on untreated plastic or on COLI-coated plastic and grown for 48 to 72 hours. As observed previously [19], vector control clones scattered on the COLI substrate; however, in contrast, high levels of COLXV expression (Bx15.14, Bx15.24) inhibited the scatter response (Bx15.14 shown in Fig. 2A). Moreover, this effect appeared to correlate with COLXV expression levels since clones Bx15.1 (shown in Fig. 2A) and Bx15.40, which have very little COLXV (Fig. 1A), showed minimal inhibition of scatter.

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