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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

Metastatic settlement of HT29 cells in dependence on cld7 expressionSCID mice (5/group) were treated with anti-asialoGM1, 24h in advance of receiving 1×106 HT29 cells s.c. (A,B) or i.v. (C,D,E); (A) mean tumor diameter after s.c. injection of HT29wt, -cld7kd or holoclone-derived cells, survival time and rate and mean survival time; significant differences in the tumor growth rate and the mean survival time depending on cld7 expression: * and p-values. (B) mice were sacrificed, when the subcutaneous tumor reached a mean diameter of 1.5cm, but latest after 210d. All mice were bled, and single cell suspensions were prepared from bone marrow, spleen, lymph nodes, lung and liver; the presence of tumor cells was evaluated by flow cytometry after staining with anti-EpC and anti-Tspan8; dispersed organs also were cultured for up to 4wk to observe tumor cell outgrowth. The mean percent of tumor cells in dispersed organs and the number of mice with tumor cell outgrowth in ex vivo organ cultures is shown; significant differences between mice bearing HT29wt, -cld7kd or holoclone-derived cells: *. (C) Survival time, survival rate and mean survival time after i.v. tumor cell application; significant differences in the mean survival time depending on cld7 expression are indicated; (D) number of visible lung metastases (mean±SD) of mice that became sick after i.v. HT29 cell application; significant differences in the number of visible lung metastases: *; (E) All mice were bled, and single cell suspensions were prepared for flow cytometry and ex vivo cultures; the percent of tumor cells / organ and the number of mice with tumor cell outgrowth in ex vivo cultured organ suspension; significant differences between HT29wt, -cld7kd and holoclone-derived tumor bearing mice: *. HT29-cld7kd cell growth in vivo starts with delay and metastatic spread is impaired after s.c. and, less pronounced, i.v. application. HT29 holoclone-derived cells show a significantly accelerated growth rate and most efficiently settle and grow in draining lymph nodes after s.c. application.
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Figure 5: Metastatic settlement of HT29 cells in dependence on cld7 expressionSCID mice (5/group) were treated with anti-asialoGM1, 24h in advance of receiving 1×106 HT29 cells s.c. (A,B) or i.v. (C,D,E); (A) mean tumor diameter after s.c. injection of HT29wt, -cld7kd or holoclone-derived cells, survival time and rate and mean survival time; significant differences in the tumor growth rate and the mean survival time depending on cld7 expression: * and p-values. (B) mice were sacrificed, when the subcutaneous tumor reached a mean diameter of 1.5cm, but latest after 210d. All mice were bled, and single cell suspensions were prepared from bone marrow, spleen, lymph nodes, lung and liver; the presence of tumor cells was evaluated by flow cytometry after staining with anti-EpC and anti-Tspan8; dispersed organs also were cultured for up to 4wk to observe tumor cell outgrowth. The mean percent of tumor cells in dispersed organs and the number of mice with tumor cell outgrowth in ex vivo organ cultures is shown; significant differences between mice bearing HT29wt, -cld7kd or holoclone-derived cells: *. (C) Survival time, survival rate and mean survival time after i.v. tumor cell application; significant differences in the mean survival time depending on cld7 expression are indicated; (D) number of visible lung metastases (mean±SD) of mice that became sick after i.v. HT29 cell application; significant differences in the number of visible lung metastases: *; (E) All mice were bled, and single cell suspensions were prepared for flow cytometry and ex vivo cultures; the percent of tumor cells / organ and the number of mice with tumor cell outgrowth in ex vivo cultured organ suspension; significant differences between HT29wt, -cld7kd and holoclone-derived tumor bearing mice: *. HT29-cld7kd cell growth in vivo starts with delay and metastatic spread is impaired after s.c. and, less pronounced, i.v. application. HT29 holoclone-derived cells show a significantly accelerated growth rate and most efficiently settle and grow in draining lymph nodes after s.c. application.

Mentions: Cld7-promoted motility and invasiveness supports metastasis formation. SCID mice received an s.c. or i.v. application of HT29 cells. Subcutaneous growth of HT29-cld7kd cells started with delay and the survival time was significantly prolonged. Instead, the survival time of mice receiving holoclones was significantly shortened as compared to that of mice receiving wt cells (Fig.5A). Macroscopic metastases were seen in the draining LN of all 5 mice receiving holoclones and in 3 of 5 mice receiving HT29wt, but not in the draining LN of mice receiving cld7kd cells. Nonetheless, as revealed by flow cytometry of dispersed organs after double staining with anti-EpC and anti-Tspan8, draining LN of HT29-cld7kd-bearing mice contained few tumor cells. Few HT29-cld7kd cells were also recovered in the peripheral blood and very few in lung, liver, BM and spleen. All these organs contained a significantly higher number of HT29wt cells. With exception of the lung, recovery was further increased in mice receiving HT29 holoclone-derived cells. In ex vivo cultures HT29wt cells grew in draining LN, the peripheral blood and the lung of all 5 mice; HT29-cld7kd cells grew only in draining LN, spleen, BM and peripheral blood of 1 or 2 mice. In lung cultures, HT29-cld7kd cells were recovered in 3 of 5 mice. With exception of the liver (3 of 5 mice), HT29 holoclone-derived cells were recovered in the organs of all 5 mice (Fig.5B). After i.v. tumor cell application, the survival time of HT29-cld7kd bearing mice was significantly prolonged and 5 of the 10 mice receiving HT29-cld7kd cells were still healthy 210d after tumor cell application. Mice that were sacrificed as they started to loose weight, showed lung metastases with the exception of one HT29-cld7kd bearing mouse. HT29wt bearing mice showed around 80 metastatic nodules, the 5 HT29-cld7kd bearing mice that became sick showed 0-25 metastatic nodules (Fig.5C,5D). The tumor-load in the BM did not significantly differ between mice bearing wt or cld7kd tumors, but was increased in mice receiving HT29 holoclone-derived cells. The tumor load in the peripheral blood, the spleen and the lung was reduced in cld7kd bearing animals. Ex vivo outgrowth of tumor cells from dispersed organs confirmed that HT29-cld7kd cells hardly settled and/or survived in liver and lung (Fig.5E).


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)

Metastatic settlement of HT29 cells in dependence on cld7 expressionSCID mice (5/group) were treated with anti-asialoGM1, 24h in advance of receiving 1×106 HT29 cells s.c. (A,B) or i.v. (C,D,E); (A) mean tumor diameter after s.c. injection of HT29wt, -cld7kd or holoclone-derived cells, survival time and rate and mean survival time; significant differences in the tumor growth rate and the mean survival time depending on cld7 expression: * and p-values. (B) mice were sacrificed, when the subcutaneous tumor reached a mean diameter of 1.5cm, but latest after 210d. All mice were bled, and single cell suspensions were prepared from bone marrow, spleen, lymph nodes, lung and liver; the presence of tumor cells was evaluated by flow cytometry after staining with anti-EpC and anti-Tspan8; dispersed organs also were cultured for up to 4wk to observe tumor cell outgrowth. The mean percent of tumor cells in dispersed organs and the number of mice with tumor cell outgrowth in ex vivo organ cultures is shown; significant differences between mice bearing HT29wt, -cld7kd or holoclone-derived cells: *. (C) Survival time, survival rate and mean survival time after i.v. tumor cell application; significant differences in the mean survival time depending on cld7 expression are indicated; (D) number of visible lung metastases (mean±SD) of mice that became sick after i.v. HT29 cell application; significant differences in the number of visible lung metastases: *; (E) All mice were bled, and single cell suspensions were prepared for flow cytometry and ex vivo cultures; the percent of tumor cells / organ and the number of mice with tumor cell outgrowth in ex vivo cultured organ suspension; significant differences between HT29wt, -cld7kd and holoclone-derived tumor bearing mice: *. HT29-cld7kd cell growth in vivo starts with delay and metastatic spread is impaired after s.c. and, less pronounced, i.v. application. HT29 holoclone-derived cells show a significantly accelerated growth rate and most efficiently settle and grow in draining lymph nodes after s.c. application.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4385835&req=5

Figure 5: Metastatic settlement of HT29 cells in dependence on cld7 expressionSCID mice (5/group) were treated with anti-asialoGM1, 24h in advance of receiving 1×106 HT29 cells s.c. (A,B) or i.v. (C,D,E); (A) mean tumor diameter after s.c. injection of HT29wt, -cld7kd or holoclone-derived cells, survival time and rate and mean survival time; significant differences in the tumor growth rate and the mean survival time depending on cld7 expression: * and p-values. (B) mice were sacrificed, when the subcutaneous tumor reached a mean diameter of 1.5cm, but latest after 210d. All mice were bled, and single cell suspensions were prepared from bone marrow, spleen, lymph nodes, lung and liver; the presence of tumor cells was evaluated by flow cytometry after staining with anti-EpC and anti-Tspan8; dispersed organs also were cultured for up to 4wk to observe tumor cell outgrowth. The mean percent of tumor cells in dispersed organs and the number of mice with tumor cell outgrowth in ex vivo organ cultures is shown; significant differences between mice bearing HT29wt, -cld7kd or holoclone-derived cells: *. (C) Survival time, survival rate and mean survival time after i.v. tumor cell application; significant differences in the mean survival time depending on cld7 expression are indicated; (D) number of visible lung metastases (mean±SD) of mice that became sick after i.v. HT29 cell application; significant differences in the number of visible lung metastases: *; (E) All mice were bled, and single cell suspensions were prepared for flow cytometry and ex vivo cultures; the percent of tumor cells / organ and the number of mice with tumor cell outgrowth in ex vivo cultured organ suspension; significant differences between HT29wt, -cld7kd and holoclone-derived tumor bearing mice: *. HT29-cld7kd cell growth in vivo starts with delay and metastatic spread is impaired after s.c. and, less pronounced, i.v. application. HT29 holoclone-derived cells show a significantly accelerated growth rate and most efficiently settle and grow in draining lymph nodes after s.c. application.
Mentions: Cld7-promoted motility and invasiveness supports metastasis formation. SCID mice received an s.c. or i.v. application of HT29 cells. Subcutaneous growth of HT29-cld7kd cells started with delay and the survival time was significantly prolonged. Instead, the survival time of mice receiving holoclones was significantly shortened as compared to that of mice receiving wt cells (Fig.5A). Macroscopic metastases were seen in the draining LN of all 5 mice receiving holoclones and in 3 of 5 mice receiving HT29wt, but not in the draining LN of mice receiving cld7kd cells. Nonetheless, as revealed by flow cytometry of dispersed organs after double staining with anti-EpC and anti-Tspan8, draining LN of HT29-cld7kd-bearing mice contained few tumor cells. Few HT29-cld7kd cells were also recovered in the peripheral blood and very few in lung, liver, BM and spleen. All these organs contained a significantly higher number of HT29wt cells. With exception of the lung, recovery was further increased in mice receiving HT29 holoclone-derived cells. In ex vivo cultures HT29wt cells grew in draining LN, the peripheral blood and the lung of all 5 mice; HT29-cld7kd cells grew only in draining LN, spleen, BM and peripheral blood of 1 or 2 mice. In lung cultures, HT29-cld7kd cells were recovered in 3 of 5 mice. With exception of the liver (3 of 5 mice), HT29 holoclone-derived cells were recovered in the organs of all 5 mice (Fig.5B). After i.v. tumor cell application, the survival time of HT29-cld7kd bearing mice was significantly prolonged and 5 of the 10 mice receiving HT29-cld7kd cells were still healthy 210d after tumor cell application. Mice that were sacrificed as they started to loose weight, showed lung metastases with the exception of one HT29-cld7kd bearing mouse. HT29wt bearing mice showed around 80 metastatic nodules, the 5 HT29-cld7kd bearing mice that became sick showed 0-25 metastatic nodules (Fig.5C,5D). The tumor-load in the BM did not significantly differ between mice bearing wt or cld7kd tumors, but was increased in mice receiving HT29 holoclone-derived cells. The tumor load in the peripheral blood, the spleen and the lung was reduced in cld7kd bearing animals. Ex vivo outgrowth of tumor cells from dispersed organs confirmed that HT29-cld7kd cells hardly settled and/or survived in liver and lung (Fig.5E).

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