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Cleaved CD44 intracellular domain supports activation of stemness factors and promotes tumorigenesis of breast cancer.

Cho Y, Lee HW, Kang HG, Kim HY, Kim SJ, Chun KH - Oncotarget (2015)

Bottom Line: We have found that the overexpression of CD44ICD increased mammosphere formation in breast cancer cells.Interestingly, CD44ICD decreased the expression levels and nuclear localization of stemness factors, but overexpression of CD44ICD reversed these effects.We suggest that the prevention of cleavage and nuclear-translocation of CD44ICD is a potential target in treating breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, Yonsei University College of Medicine, Seodaemun-gu, Seoul 120-752, Korea.

ABSTRACT
CD44 plays a role in the progression of tumors and is expressed in cancer stem cells (CSCs). However, the mechanisms underlying the crosstalk of CD44 with stemness genes in CSC maintenance remains unclear. In this study, we demonstrated how the cleaved intracellular domain of CD44 (CD44ICD) activates stemness factors such as Nanog, Sox2 and Oct4, and contributes to the tumorigenesis of breast cancer. We have found that the overexpression of CD44ICD increased mammosphere formation in breast cancer cells. Treatment with a γ-secretase inhibitor (GSI), which blocks the cleavage of CD44ICD, interfered with mammosphere formation. Interestingly, CD44ICD decreased the expression levels and nuclear localization of stemness factors, but overexpression of CD44ICD reversed these effects. In addition, we showed that nuclear localization of CD44ICD is important for transcriptional activation of the stemness factors. Furthermore, CD44ICD-overexpressed cells exhibited strong tumorigenecity and greater metastatic potential than did the control cells or CD44-depleted cells in vivo in mice models. Taken together, it was supposed that CD44 promotes tumorigenesis through the interaction and nuclear-translocation of its intracellular domain and stemness factors. We suggest that the prevention of cleavage and nuclear-translocation of CD44ICD is a potential target in treating breast cancer.

No MeSH data available.


Related in: MedlinePlus

Overexpression of CD44ICD increases the transcriptional activation of the stemness factors, Sox2, and Oct4(A) The transcriptional activation was measured with a reporter assay in wild-type (diagonal bars) and CD44KD MCF-7 (solid bars) cells. Cells were transfected with a Sox2 and Oct4 reporter vector alone or co-transfected with CD44 siRNA, CD44 or CD44ICD expression vectors. Following transfection, the cells were incubated for 12 hr and a vehicle (−) or 5 μM of GSI (+) were added. The cells were incubated for an additional 24 hr. The transcriptional activity was measured by luciferase activity described in “materials and methods”. (B) The expression of CD44ICD or of the truncated mutant constructs, ICD_ΔN17 and ICD_ΔC19 were detected with a western blot. (C) MCF-7 cells were transfected with the reporter vector alone or co-transfected with CD44-ICD or CD44-ICD_ΔN17 expression vectors for 36 hr. The luciferase activity was measured as described in “materials methods”. (D) MCF-7 cells were transfected with the reporter vector alone or co-transfected with CD44ICD or CD44ICD_ΔC19 expression vectors for 36 hr. The luciferase activity was measured. The data are presented as the mean ± SD (n = 3). Significant differences are indicated by an asterisk (*p < 0.05), and the p values were calculated using the Student's t test.
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Figure 6: Overexpression of CD44ICD increases the transcriptional activation of the stemness factors, Sox2, and Oct4(A) The transcriptional activation was measured with a reporter assay in wild-type (diagonal bars) and CD44KD MCF-7 (solid bars) cells. Cells were transfected with a Sox2 and Oct4 reporter vector alone or co-transfected with CD44 siRNA, CD44 or CD44ICD expression vectors. Following transfection, the cells were incubated for 12 hr and a vehicle (−) or 5 μM of GSI (+) were added. The cells were incubated for an additional 24 hr. The transcriptional activity was measured by luciferase activity described in “materials and methods”. (B) The expression of CD44ICD or of the truncated mutant constructs, ICD_ΔN17 and ICD_ΔC19 were detected with a western blot. (C) MCF-7 cells were transfected with the reporter vector alone or co-transfected with CD44-ICD or CD44-ICD_ΔN17 expression vectors for 36 hr. The luciferase activity was measured as described in “materials methods”. (D) MCF-7 cells were transfected with the reporter vector alone or co-transfected with CD44ICD or CD44ICD_ΔC19 expression vectors for 36 hr. The luciferase activity was measured. The data are presented as the mean ± SD (n = 3). Significant differences are indicated by an asterisk (*p < 0.05), and the p values were calculated using the Student's t test.

Mentions: We measured the luciferase activity of the Sox2 and Oct4 promoter reporters to check whether CD44ICD also regulates the transcriptional activation of Sox2 and Oct4 (Figure 6A). In control cells, transcriptional activation of the Sox2 promoter (left graph) and the Oct4 promoter (right graph) was repressed by both GSI and CD44 siRNA treatment (diagonal bars in Figure 5A). The overexpression of CD44ICD enhanced the transcriptional activation of the Sox2 and Oct4 promoters more than in control cells. In CD44-depleted stable cells, the overexpression of CD44ICD alone induced the transcriptional activation of Sox2 and Oct4 more than in cells that overexpressed the full-length form of CD44 (solid bars in Figure 5A).


Cleaved CD44 intracellular domain supports activation of stemness factors and promotes tumorigenesis of breast cancer.

Cho Y, Lee HW, Kang HG, Kim HY, Kim SJ, Chun KH - Oncotarget (2015)

Overexpression of CD44ICD increases the transcriptional activation of the stemness factors, Sox2, and Oct4(A) The transcriptional activation was measured with a reporter assay in wild-type (diagonal bars) and CD44KD MCF-7 (solid bars) cells. Cells were transfected with a Sox2 and Oct4 reporter vector alone or co-transfected with CD44 siRNA, CD44 or CD44ICD expression vectors. Following transfection, the cells were incubated for 12 hr and a vehicle (−) or 5 μM of GSI (+) were added. The cells were incubated for an additional 24 hr. The transcriptional activity was measured by luciferase activity described in “materials and methods”. (B) The expression of CD44ICD or of the truncated mutant constructs, ICD_ΔN17 and ICD_ΔC19 were detected with a western blot. (C) MCF-7 cells were transfected with the reporter vector alone or co-transfected with CD44-ICD or CD44-ICD_ΔN17 expression vectors for 36 hr. The luciferase activity was measured as described in “materials methods”. (D) MCF-7 cells were transfected with the reporter vector alone or co-transfected with CD44ICD or CD44ICD_ΔC19 expression vectors for 36 hr. The luciferase activity was measured. The data are presented as the mean ± SD (n = 3). Significant differences are indicated by an asterisk (*p < 0.05), and the p values were calculated using the Student's t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496178&req=5

Figure 6: Overexpression of CD44ICD increases the transcriptional activation of the stemness factors, Sox2, and Oct4(A) The transcriptional activation was measured with a reporter assay in wild-type (diagonal bars) and CD44KD MCF-7 (solid bars) cells. Cells were transfected with a Sox2 and Oct4 reporter vector alone or co-transfected with CD44 siRNA, CD44 or CD44ICD expression vectors. Following transfection, the cells were incubated for 12 hr and a vehicle (−) or 5 μM of GSI (+) were added. The cells were incubated for an additional 24 hr. The transcriptional activity was measured by luciferase activity described in “materials and methods”. (B) The expression of CD44ICD or of the truncated mutant constructs, ICD_ΔN17 and ICD_ΔC19 were detected with a western blot. (C) MCF-7 cells were transfected with the reporter vector alone or co-transfected with CD44-ICD or CD44-ICD_ΔN17 expression vectors for 36 hr. The luciferase activity was measured as described in “materials methods”. (D) MCF-7 cells were transfected with the reporter vector alone or co-transfected with CD44ICD or CD44ICD_ΔC19 expression vectors for 36 hr. The luciferase activity was measured. The data are presented as the mean ± SD (n = 3). Significant differences are indicated by an asterisk (*p < 0.05), and the p values were calculated using the Student's t test.
Mentions: We measured the luciferase activity of the Sox2 and Oct4 promoter reporters to check whether CD44ICD also regulates the transcriptional activation of Sox2 and Oct4 (Figure 6A). In control cells, transcriptional activation of the Sox2 promoter (left graph) and the Oct4 promoter (right graph) was repressed by both GSI and CD44 siRNA treatment (diagonal bars in Figure 5A). The overexpression of CD44ICD enhanced the transcriptional activation of the Sox2 and Oct4 promoters more than in control cells. In CD44-depleted stable cells, the overexpression of CD44ICD alone induced the transcriptional activation of Sox2 and Oct4 more than in cells that overexpressed the full-length form of CD44 (solid bars in Figure 5A).

Bottom Line: We have found that the overexpression of CD44ICD increased mammosphere formation in breast cancer cells.Interestingly, CD44ICD decreased the expression levels and nuclear localization of stemness factors, but overexpression of CD44ICD reversed these effects.We suggest that the prevention of cleavage and nuclear-translocation of CD44ICD is a potential target in treating breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, Yonsei University College of Medicine, Seodaemun-gu, Seoul 120-752, Korea.

ABSTRACT
CD44 plays a role in the progression of tumors and is expressed in cancer stem cells (CSCs). However, the mechanisms underlying the crosstalk of CD44 with stemness genes in CSC maintenance remains unclear. In this study, we demonstrated how the cleaved intracellular domain of CD44 (CD44ICD) activates stemness factors such as Nanog, Sox2 and Oct4, and contributes to the tumorigenesis of breast cancer. We have found that the overexpression of CD44ICD increased mammosphere formation in breast cancer cells. Treatment with a γ-secretase inhibitor (GSI), which blocks the cleavage of CD44ICD, interfered with mammosphere formation. Interestingly, CD44ICD decreased the expression levels and nuclear localization of stemness factors, but overexpression of CD44ICD reversed these effects. In addition, we showed that nuclear localization of CD44ICD is important for transcriptional activation of the stemness factors. Furthermore, CD44ICD-overexpressed cells exhibited strong tumorigenecity and greater metastatic potential than did the control cells or CD44-depleted cells in vivo in mice models. Taken together, it was supposed that CD44 promotes tumorigenesis through the interaction and nuclear-translocation of its intracellular domain and stemness factors. We suggest that the prevention of cleavage and nuclear-translocation of CD44ICD is a potential target in treating breast cancer.

No MeSH data available.


Related in: MedlinePlus