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Claudin-7 promotes the epithelial-mesenchymal transition in human colorectal cancer.

Philip R, Heiler S, Mu W, Büchler MW, Zöller M, Thuma F - Oncotarget (2015)

Bottom Line: In line with this, migratory and invasive potential of cld7kd clones is strongly impaired, migration being inhibited by anti-CD49c, but not anti-EpCAM, although motility is reduced in EpCAM siRNA-treated cells.This is due to claudin-7 recruiting EpCAM in glycolipid-enriched membrane fractions towards claudin-7-associated TACE and presenilin2, which cleave EpCAM.The cleaved intracellular domain, EpIC, promotes epithelial-mesenchymal transition (EMT)-associated transcription factor expression, which together with fibronectin and vimentin are reduced in claudin-7kd cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Cell Biology, University Hospital of Surgery, Heidelberg.

ABSTRACT
In colorectal cancer (CoCa) EpCAM is frequently associated with claudin-7. There is evidence that tumor-promoting EpCAM activities are modulated by the association with claudin-7. To support this hypothesis, claudin-7 was knocked-down (kd) in HT29 and SW948 cells. HT29-cld7kd and SW948-cld7kd cells display decreased anchorage-independent growth and the capacity for holoclone-, respectively, sphere-formation is reduced. Tumor growth is delayed and cld7kd cells poorly metastasize. In line with this, migratory and invasive potential of cld7kd clones is strongly impaired, migration being inhibited by anti-CD49c, but not anti-EpCAM, although motility is reduced in EpCAM siRNA-treated cells. This is due to claudin-7 recruiting EpCAM in glycolipid-enriched membrane fractions towards claudin-7-associated TACE and presenilin2, which cleave EpCAM. The cleaved intracellular domain, EpIC, promotes epithelial-mesenchymal transition (EMT)-associated transcription factor expression, which together with fibronectin and vimentin are reduced in claudin-7kd cells. But, uptake of HT29wt and SW948wt exosomes by the claudin-7kd lines sufficed for transcription factor upregulation and for restoring motility. Thus, claudin-7 contributes to motility and invasion and is required for recruiting EpCAM towards TACE/presenilin2. EpIC generation further supports motility by promoting a shift towards EMT. Notably, EMT features of cld7-competent metastatic CoCa cells can be transferred via exosomes to poorly metastatic cells.

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Related in: MedlinePlus

EMT induction by exosomes(A) WB of EpC and cld7 expression in HT29wt and SW948wt and -cld7kd exosomes. (B-F) HT29-cld7kd and SW948-cld7kd cells were cultured for 48h with 10μg/ml exosomes derived from HT29wt and SW948wt, -cld7kd or sphere- / holoclone-derived cells. (B) Flow cytometry analysis of cld7, EpC and EMT gene expression; the % stained cells (mean±SD, triplicates) and representative examples are shown; significant differences between wt, cld7kd and holoclone- / sphere-derived cells: *; (C) confocal microscopy of EMT gene expression in HT29-cld7kd cells cocultured with cld7kd or holoclone-derived exosomes; single fluorescence and overlays with anti-EpC are shown (scale bar: 10μm); (D) light microscopy of HT29-cld7kd and SW948-cld7kd cells after coculture with cld7kd or holoclone- / sphere-derived exosomes (scale bar: 25μm); (E) confocal microscopy of HT29-cld7kd cells cultured with/without cld7kd or holoclone-derived exosomes; EpC (red), phalloidin-FITC and overlays are shown (scale bar: 10μm); (F) HT29-cld7kd and SW948-cld7kd cells were seeded in 24w plates; subconfluent cultures were scratched with a pipette tip. Wound healing was observed for 40h or 72h in the presence of medium with exosome-depleted FCS, or with exosomes from wt, cld7kd or holoclone / sphere-derived cells; representative examples (scale bar: 250μm) and the wound area (mean±SD, triplicates) are shown; significant differences between wt versus cld7kd cells: *, significant differences by coculture with wt, cld7kd and holoclone- / sphere-derived exosomes: s. (G) HT29-cld7kd and SW948-cld7kd cells were seeded in the upper part of a Boyden chamber. The lower chamber contained 20% exosome-depleted FCS with/without 10μg/ml cld7kd, wt or holoclone- / sphere-derived exosomes; the percent migrating cells is shown; significant differences in the presence of exosomes: *; (H) SW948-cld7kd and HT29-cld7kd cells were seeded on matrigel-coated inserts of a Boyden chamber; the lower chamber contained RPMI supplemented with 20% exosome-depleted FCS with/without 10μg/ml cld7kd, wt or holoclone- / sphere-derived exosomes. After overnight incubation, the matrigel block was stained with crystal violet and the cells that had invaded or penetrated the matrigel were counted; representative examples (scale bar: 250μm) and the number of invading and penetrating cells (mean±SD, triplicates) are shown; significant differences in the presence of cld7kd, wt, or sphere- / holoclone-derived exosomes: *. Exosomes from cld7 competent cells suffice for upregulated expression of mesenchymal markers and genes in cld7kd cells, which is accompanied by changes in cell shape as well as increased motility and invasiveness.
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Figure 8: EMT induction by exosomes(A) WB of EpC and cld7 expression in HT29wt and SW948wt and -cld7kd exosomes. (B-F) HT29-cld7kd and SW948-cld7kd cells were cultured for 48h with 10μg/ml exosomes derived from HT29wt and SW948wt, -cld7kd or sphere- / holoclone-derived cells. (B) Flow cytometry analysis of cld7, EpC and EMT gene expression; the % stained cells (mean±SD, triplicates) and representative examples are shown; significant differences between wt, cld7kd and holoclone- / sphere-derived cells: *; (C) confocal microscopy of EMT gene expression in HT29-cld7kd cells cocultured with cld7kd or holoclone-derived exosomes; single fluorescence and overlays with anti-EpC are shown (scale bar: 10μm); (D) light microscopy of HT29-cld7kd and SW948-cld7kd cells after coculture with cld7kd or holoclone- / sphere-derived exosomes (scale bar: 25μm); (E) confocal microscopy of HT29-cld7kd cells cultured with/without cld7kd or holoclone-derived exosomes; EpC (red), phalloidin-FITC and overlays are shown (scale bar: 10μm); (F) HT29-cld7kd and SW948-cld7kd cells were seeded in 24w plates; subconfluent cultures were scratched with a pipette tip. Wound healing was observed for 40h or 72h in the presence of medium with exosome-depleted FCS, or with exosomes from wt, cld7kd or holoclone / sphere-derived cells; representative examples (scale bar: 250μm) and the wound area (mean±SD, triplicates) are shown; significant differences between wt versus cld7kd cells: *, significant differences by coculture with wt, cld7kd and holoclone- / sphere-derived exosomes: s. (G) HT29-cld7kd and SW948-cld7kd cells were seeded in the upper part of a Boyden chamber. The lower chamber contained 20% exosome-depleted FCS with/without 10μg/ml cld7kd, wt or holoclone- / sphere-derived exosomes; the percent migrating cells is shown; significant differences in the presence of exosomes: *; (H) SW948-cld7kd and HT29-cld7kd cells were seeded on matrigel-coated inserts of a Boyden chamber; the lower chamber contained RPMI supplemented with 20% exosome-depleted FCS with/without 10μg/ml cld7kd, wt or holoclone- / sphere-derived exosomes. After overnight incubation, the matrigel block was stained with crystal violet and the cells that had invaded or penetrated the matrigel were counted; representative examples (scale bar: 250μm) and the number of invading and penetrating cells (mean±SD, triplicates) are shown; significant differences in the presence of cld7kd, wt, or sphere- / holoclone-derived exosomes: *. Exosomes from cld7 competent cells suffice for upregulated expression of mesenchymal markers and genes in cld7kd cells, which is accompanied by changes in cell shape as well as increased motility and invasiveness.

Mentions: EpC and cld7 are recovered at a high level in exosomes. EpC expression was slightly reduced in exosomes from cld7kd cells (Fig.8A), which fits the requirement of cld7 for the recruitment of EpC towards internalization prone GEM [10]. When HT29-cld7kd and SW948-cld7kd cells were cultured with exosomes derived from HT29wt and SW948wt cells, expression of N-cadherin, vimentin and FN (only SW948-cld7kd cells) as well as of Snail, Twist, ZEB1 and TCF4 became upregulated, though more pronounced in HT29-cld7kd than SW948-cld7kd cells. Upregulated expression became more pronounced after coculture of cld7kd cells with sphere- or holoclone-derived exosomes. This accounted particularly for vimentin and Notch. Instead, none of these effects where seen, when cld7kd cells were cocultured with cld7kd exosomes (Fig.8B), which was confirmed by confocal microscopy (Fig.8C). Furthermore, upon coculture with exosomes derived from holoclones or spheres the round / epitheloid shape of HT29-cld7kd cells changed towards fibroid and SW948-cld7kd cells again formed most tightly packed clusters (Fig.8D). Changes in cell shape were accompanied by reorganization of the actin cytoskeleton with actin bundle formation (Fig8E). Finally, HT29- and SW498-cld7kd cells treated with wt exosomes regained full motility. Wound closure was further accelerated by coculture with exosomes from holoclones or spheres. Coculture with exosomes from cld7kd cells exerted no effect (Fig.8F,Suppl.Fig.4). Treatment with wt or holoclone- / sphere-derived exosome, but not with cld7kd-derived exosomes also restored transwell migration (Fig.8G) and matrigel invasion, the efficacy of holoclone- / sphere-derived exosomes exceeding that of wt exosomes (Fig.8H). Thus, cld7-competent exosomes from metastasizing tumor cells suffice to initiate EMT in poorly metastasizing cld7kd cells.


Claudin-7 promotes the epithelial-mesenchymal transition in human colorectal cancer.

Philip R, Heiler S, Mu W, Büchler MW, Zöller M, Thuma F - Oncotarget (2015)

EMT induction by exosomes(A) WB of EpC and cld7 expression in HT29wt and SW948wt and -cld7kd exosomes. (B-F) HT29-cld7kd and SW948-cld7kd cells were cultured for 48h with 10μg/ml exosomes derived from HT29wt and SW948wt, -cld7kd or sphere- / holoclone-derived cells. (B) Flow cytometry analysis of cld7, EpC and EMT gene expression; the % stained cells (mean±SD, triplicates) and representative examples are shown; significant differences between wt, cld7kd and holoclone- / sphere-derived cells: *; (C) confocal microscopy of EMT gene expression in HT29-cld7kd cells cocultured with cld7kd or holoclone-derived exosomes; single fluorescence and overlays with anti-EpC are shown (scale bar: 10μm); (D) light microscopy of HT29-cld7kd and SW948-cld7kd cells after coculture with cld7kd or holoclone- / sphere-derived exosomes (scale bar: 25μm); (E) confocal microscopy of HT29-cld7kd cells cultured with/without cld7kd or holoclone-derived exosomes; EpC (red), phalloidin-FITC and overlays are shown (scale bar: 10μm); (F) HT29-cld7kd and SW948-cld7kd cells were seeded in 24w plates; subconfluent cultures were scratched with a pipette tip. Wound healing was observed for 40h or 72h in the presence of medium with exosome-depleted FCS, or with exosomes from wt, cld7kd or holoclone / sphere-derived cells; representative examples (scale bar: 250μm) and the wound area (mean±SD, triplicates) are shown; significant differences between wt versus cld7kd cells: *, significant differences by coculture with wt, cld7kd and holoclone- / sphere-derived exosomes: s. (G) HT29-cld7kd and SW948-cld7kd cells were seeded in the upper part of a Boyden chamber. The lower chamber contained 20% exosome-depleted FCS with/without 10μg/ml cld7kd, wt or holoclone- / sphere-derived exosomes; the percent migrating cells is shown; significant differences in the presence of exosomes: *; (H) SW948-cld7kd and HT29-cld7kd cells were seeded on matrigel-coated inserts of a Boyden chamber; the lower chamber contained RPMI supplemented with 20% exosome-depleted FCS with/without 10μg/ml cld7kd, wt or holoclone- / sphere-derived exosomes. After overnight incubation, the matrigel block was stained with crystal violet and the cells that had invaded or penetrated the matrigel were counted; representative examples (scale bar: 250μm) and the number of invading and penetrating cells (mean±SD, triplicates) are shown; significant differences in the presence of cld7kd, wt, or sphere- / holoclone-derived exosomes: *. Exosomes from cld7 competent cells suffice for upregulated expression of mesenchymal markers and genes in cld7kd cells, which is accompanied by changes in cell shape as well as increased motility and invasiveness.
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Figure 8: EMT induction by exosomes(A) WB of EpC and cld7 expression in HT29wt and SW948wt and -cld7kd exosomes. (B-F) HT29-cld7kd and SW948-cld7kd cells were cultured for 48h with 10μg/ml exosomes derived from HT29wt and SW948wt, -cld7kd or sphere- / holoclone-derived cells. (B) Flow cytometry analysis of cld7, EpC and EMT gene expression; the % stained cells (mean±SD, triplicates) and representative examples are shown; significant differences between wt, cld7kd and holoclone- / sphere-derived cells: *; (C) confocal microscopy of EMT gene expression in HT29-cld7kd cells cocultured with cld7kd or holoclone-derived exosomes; single fluorescence and overlays with anti-EpC are shown (scale bar: 10μm); (D) light microscopy of HT29-cld7kd and SW948-cld7kd cells after coculture with cld7kd or holoclone- / sphere-derived exosomes (scale bar: 25μm); (E) confocal microscopy of HT29-cld7kd cells cultured with/without cld7kd or holoclone-derived exosomes; EpC (red), phalloidin-FITC and overlays are shown (scale bar: 10μm); (F) HT29-cld7kd and SW948-cld7kd cells were seeded in 24w plates; subconfluent cultures were scratched with a pipette tip. Wound healing was observed for 40h or 72h in the presence of medium with exosome-depleted FCS, or with exosomes from wt, cld7kd or holoclone / sphere-derived cells; representative examples (scale bar: 250μm) and the wound area (mean±SD, triplicates) are shown; significant differences between wt versus cld7kd cells: *, significant differences by coculture with wt, cld7kd and holoclone- / sphere-derived exosomes: s. (G) HT29-cld7kd and SW948-cld7kd cells were seeded in the upper part of a Boyden chamber. The lower chamber contained 20% exosome-depleted FCS with/without 10μg/ml cld7kd, wt or holoclone- / sphere-derived exosomes; the percent migrating cells is shown; significant differences in the presence of exosomes: *; (H) SW948-cld7kd and HT29-cld7kd cells were seeded on matrigel-coated inserts of a Boyden chamber; the lower chamber contained RPMI supplemented with 20% exosome-depleted FCS with/without 10μg/ml cld7kd, wt or holoclone- / sphere-derived exosomes. After overnight incubation, the matrigel block was stained with crystal violet and the cells that had invaded or penetrated the matrigel were counted; representative examples (scale bar: 250μm) and the number of invading and penetrating cells (mean±SD, triplicates) are shown; significant differences in the presence of cld7kd, wt, or sphere- / holoclone-derived exosomes: *. Exosomes from cld7 competent cells suffice for upregulated expression of mesenchymal markers and genes in cld7kd cells, which is accompanied by changes in cell shape as well as increased motility and invasiveness.
Mentions: EpC and cld7 are recovered at a high level in exosomes. EpC expression was slightly reduced in exosomes from cld7kd cells (Fig.8A), which fits the requirement of cld7 for the recruitment of EpC towards internalization prone GEM [10]. When HT29-cld7kd and SW948-cld7kd cells were cultured with exosomes derived from HT29wt and SW948wt cells, expression of N-cadherin, vimentin and FN (only SW948-cld7kd cells) as well as of Snail, Twist, ZEB1 and TCF4 became upregulated, though more pronounced in HT29-cld7kd than SW948-cld7kd cells. Upregulated expression became more pronounced after coculture of cld7kd cells with sphere- or holoclone-derived exosomes. This accounted particularly for vimentin and Notch. Instead, none of these effects where seen, when cld7kd cells were cocultured with cld7kd exosomes (Fig.8B), which was confirmed by confocal microscopy (Fig.8C). Furthermore, upon coculture with exosomes derived from holoclones or spheres the round / epitheloid shape of HT29-cld7kd cells changed towards fibroid and SW948-cld7kd cells again formed most tightly packed clusters (Fig.8D). Changes in cell shape were accompanied by reorganization of the actin cytoskeleton with actin bundle formation (Fig8E). Finally, HT29- and SW498-cld7kd cells treated with wt exosomes regained full motility. Wound closure was further accelerated by coculture with exosomes from holoclones or spheres. Coculture with exosomes from cld7kd cells exerted no effect (Fig.8F,Suppl.Fig.4). Treatment with wt or holoclone- / sphere-derived exosome, but not with cld7kd-derived exosomes also restored transwell migration (Fig.8G) and matrigel invasion, the efficacy of holoclone- / sphere-derived exosomes exceeding that of wt exosomes (Fig.8H). Thus, cld7-competent exosomes from metastasizing tumor cells suffice to initiate EMT in poorly metastasizing cld7kd cells.

Bottom Line: In line with this, migratory and invasive potential of cld7kd clones is strongly impaired, migration being inhibited by anti-CD49c, but not anti-EpCAM, although motility is reduced in EpCAM siRNA-treated cells.This is due to claudin-7 recruiting EpCAM in glycolipid-enriched membrane fractions towards claudin-7-associated TACE and presenilin2, which cleave EpCAM.The cleaved intracellular domain, EpIC, promotes epithelial-mesenchymal transition (EMT)-associated transcription factor expression, which together with fibronectin and vimentin are reduced in claudin-7kd cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Cell Biology, University Hospital of Surgery, Heidelberg.

ABSTRACT
In colorectal cancer (CoCa) EpCAM is frequently associated with claudin-7. There is evidence that tumor-promoting EpCAM activities are modulated by the association with claudin-7. To support this hypothesis, claudin-7 was knocked-down (kd) in HT29 and SW948 cells. HT29-cld7kd and SW948-cld7kd cells display decreased anchorage-independent growth and the capacity for holoclone-, respectively, sphere-formation is reduced. Tumor growth is delayed and cld7kd cells poorly metastasize. In line with this, migratory and invasive potential of cld7kd clones is strongly impaired, migration being inhibited by anti-CD49c, but not anti-EpCAM, although motility is reduced in EpCAM siRNA-treated cells. This is due to claudin-7 recruiting EpCAM in glycolipid-enriched membrane fractions towards claudin-7-associated TACE and presenilin2, which cleave EpCAM. The cleaved intracellular domain, EpIC, promotes epithelial-mesenchymal transition (EMT)-associated transcription factor expression, which together with fibronectin and vimentin are reduced in claudin-7kd cells. But, uptake of HT29wt and SW948wt exosomes by the claudin-7kd lines sufficed for transcription factor upregulation and for restoring motility. Thus, claudin-7 contributes to motility and invasion and is required for recruiting EpCAM towards TACE/presenilin2. EpIC generation further supports motility by promoting a shift towards EMT. Notably, EMT features of cld7-competent metastatic CoCa cells can be transferred via exosomes to poorly metastatic cells.

Show MeSH
Related in: MedlinePlus