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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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E-Cadherin is stabilized at the cell periphery in collagen XV expressing cells.Confocal microscopy with an antibody specific for the extracellular domain of E-Cad (green) and nuclei stained with DAPI (blue). BxVC1 vector clone and Bx15.23 COLXV clone grown on plastic A) or COLI B). E-Cad is most abundant at the cell surface in both clones on plastic. On COLI, E-Cad moves from the cell periphery into the cytoplasm in BxVC1, but this redistribution is inhibited in the presence of COLXV (BX15.23). C) EEA1 (red) is found in the endoplasmic reticulum (ER)/Golgi zone of the cells grown on plastic, while E-Cad is at the cell periphery. D) After relocation of E-Cad on COLI, EEA1 colocalizes with E-Cad (white arrowheads) in BxVC1 cells but not Bx15.23 cells. Images are representative of several clones. E) Flow cytometry after staining cells with an E-Cad antibody shows increased cell-surface expression of E-Cad in cells with COLXV (Bx15.5 and 15.24) in comparison to vector controls (BxVC4 and BxVC1). All experiments performed a minimum of 3 times with consistent results.
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pone-0072250-g003: E-Cadherin is stabilized at the cell periphery in collagen XV expressing cells.Confocal microscopy with an antibody specific for the extracellular domain of E-Cad (green) and nuclei stained with DAPI (blue). BxVC1 vector clone and Bx15.23 COLXV clone grown on plastic A) or COLI B). E-Cad is most abundant at the cell surface in both clones on plastic. On COLI, E-Cad moves from the cell periphery into the cytoplasm in BxVC1, but this redistribution is inhibited in the presence of COLXV (BX15.23). C) EEA1 (red) is found in the endoplasmic reticulum (ER)/Golgi zone of the cells grown on plastic, while E-Cad is at the cell periphery. D) After relocation of E-Cad on COLI, EEA1 colocalizes with E-Cad (white arrowheads) in BxVC1 cells but not Bx15.23 cells. Images are representative of several clones. E) Flow cytometry after staining cells with an E-Cad antibody shows increased cell-surface expression of E-Cad in cells with COLXV (Bx15.5 and 15.24) in comparison to vector controls (BxVC4 and BxVC1). All experiments performed a minimum of 3 times with consistent results.

Mentions: Since DDR1 and E-Cad were shown previously to be in complex [24] and we demonstrate here that COLXV is in complex with both receptors, we next asked whether COLXV stabilized these receptors at the cell surface. When BxPC-3 cells are grown on plastic substrate E-Cad is largely cell surface-associated but becomes cytosolic when the cells scatter on COLI. We demonstrated above (Fig. 2A) that COLXV inhibits this scatter on COLI. Using confocal microscopy, we next observed that COLXV expressing BxPC-3 cells grown on a COLI substrate showed marked stabilization of E-Cad at the cell surface in comparison to the punctate, cytosolic distribution seen in the vector only cells (Fig. 3A, B). Moreover, colocalization of Early Endosome Antigen I (EEAI) with E-Cad when vector- control cells were grown on COLI demonstrated endocytosis and potential recycling of E-Cad to early endosomes (Fig. 3C, D). In contrast, COLXV expressing BxPC-3 cells showed no distinct colocalization of E-Cad and EEAI on COLI and the distribution of these two markers, appeared similar to cells grown on plastic.


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)

E-Cadherin is stabilized at the cell periphery in collagen XV expressing cells.Confocal microscopy with an antibody specific for the extracellular domain of E-Cad (green) and nuclei stained with DAPI (blue). BxVC1 vector clone and Bx15.23 COLXV clone grown on plastic A) or COLI B). E-Cad is most abundant at the cell surface in both clones on plastic. On COLI, E-Cad moves from the cell periphery into the cytoplasm in BxVC1, but this redistribution is inhibited in the presence of COLXV (BX15.23). C) EEA1 (red) is found in the endoplasmic reticulum (ER)/Golgi zone of the cells grown on plastic, while E-Cad is at the cell periphery. D) After relocation of E-Cad on COLI, EEA1 colocalizes with E-Cad (white arrowheads) in BxVC1 cells but not Bx15.23 cells. Images are representative of several clones. E) Flow cytometry after staining cells with an E-Cad antibody shows increased cell-surface expression of E-Cad in cells with COLXV (Bx15.5 and 15.24) in comparison to vector controls (BxVC4 and BxVC1). All experiments performed a minimum of 3 times with consistent results.
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Related In: Results  -  Collection

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pone-0072250-g003: E-Cadherin is stabilized at the cell periphery in collagen XV expressing cells.Confocal microscopy with an antibody specific for the extracellular domain of E-Cad (green) and nuclei stained with DAPI (blue). BxVC1 vector clone and Bx15.23 COLXV clone grown on plastic A) or COLI B). E-Cad is most abundant at the cell surface in both clones on plastic. On COLI, E-Cad moves from the cell periphery into the cytoplasm in BxVC1, but this redistribution is inhibited in the presence of COLXV (BX15.23). C) EEA1 (red) is found in the endoplasmic reticulum (ER)/Golgi zone of the cells grown on plastic, while E-Cad is at the cell periphery. D) After relocation of E-Cad on COLI, EEA1 colocalizes with E-Cad (white arrowheads) in BxVC1 cells but not Bx15.23 cells. Images are representative of several clones. E) Flow cytometry after staining cells with an E-Cad antibody shows increased cell-surface expression of E-Cad in cells with COLXV (Bx15.5 and 15.24) in comparison to vector controls (BxVC4 and BxVC1). All experiments performed a minimum of 3 times with consistent results.
Mentions: Since DDR1 and E-Cad were shown previously to be in complex [24] and we demonstrate here that COLXV is in complex with both receptors, we next asked whether COLXV stabilized these receptors at the cell surface. When BxPC-3 cells are grown on plastic substrate E-Cad is largely cell surface-associated but becomes cytosolic when the cells scatter on COLI. We demonstrated above (Fig. 2A) that COLXV inhibits this scatter on COLI. Using confocal microscopy, we next observed that COLXV expressing BxPC-3 cells grown on a COLI substrate showed marked stabilization of E-Cad at the cell surface in comparison to the punctate, cytosolic distribution seen in the vector only cells (Fig. 3A, B). Moreover, colocalization of Early Endosome Antigen I (EEAI) with E-Cad when vector- control cells were grown on COLI demonstrated endocytosis and potential recycling of E-Cad to early endosomes (Fig. 3C, D). In contrast, COLXV expressing BxPC-3 cells showed no distinct colocalization of E-Cad and EEAI on COLI and the distribution of these two markers, appeared similar to cells grown on plastic.

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