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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 in the absence of CD44 increases the ability of mamosphere formation in breast cancer cells(A) The mammosphere-forming ability of the indicated stable lines was measured under sphere forming conditions for 15 days. (B) The number of spheres was quantified in the experiments (A) (C) Cells were transfected with scRNA and CD44 siRNA. Following transfection, the cells were incubated for 48 hr, and cell proliferation was detected with a WST assay. (D) and (E) Cells were transfected with control and Oct4 expression vectors (D), and with control and Oct4 expression vectors (E) Following transfection, the cells were incubated for 48 hr, and the cell proliferation detected with a WST assay. 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 2: Overexpression of CD44ICD in the absence of CD44 increases the ability of mamosphere formation in breast cancer cells(A) The mammosphere-forming ability of the indicated stable lines was measured under sphere forming conditions for 15 days. (B) The number of spheres was quantified in the experiments (A) (C) Cells were transfected with scRNA and CD44 siRNA. Following transfection, the cells were incubated for 48 hr, and cell proliferation was detected with a WST assay. (D) and (E) Cells were transfected with control and Oct4 expression vectors (D), and with control and Oct4 expression vectors (E) Following transfection, the cells were incubated for 48 hr, and the cell proliferation detected with a WST assay. 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: To test whether CD44ICD regulates the potential of breast cancer cells for mammosphere formation, we selected MDA-MB-231 and MCF-7 cells and generated stable cell lines, including CD44-depleted cells (CD44KD), CD44-depleted cells with overexpression of CD44ICD (CD44KD/ICDover), and wild-type cells with overexpression of CD44ICD (ICDover), using a lentiviral vector with a genotype that was confirmed by RT-PCR (upper panel) and western blotting (lower panel) (Supplementary Figure S1). These cells were mammosphere-cultured for 15 days (Figure 2A and 2B). The size and number of mammospheres were significantly reduced in CD44-depleted MDA-MB-231 and MCF-7 cells. However, CD44-depleted and CD44ICD-overexpressed cells showed an increased size and number of mammospheres. Additionally, the size and number of spheres were greater in cells with CD44ICD overexpression than in wild-type (WT) cells. Treatment with GSI also significantly inhibited the formation of mammospheres in MCF-7 cells (Supplementary Figure S2). We also determined that the cell proliferation of breast cancer MDA-MB231 and MCF-7 cells was significantly reduced by CD44 ablation (Figure 2C). Taken together, our results suggest that CD44ICD might regulate mammosphere formation in breast cancer cells.


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 in the absence of CD44 increases the ability of mamosphere formation in breast cancer cells(A) The mammosphere-forming ability of the indicated stable lines was measured under sphere forming conditions for 15 days. (B) The number of spheres was quantified in the experiments (A) (C) Cells were transfected with scRNA and CD44 siRNA. Following transfection, the cells were incubated for 48 hr, and cell proliferation was detected with a WST assay. (D) and (E) Cells were transfected with control and Oct4 expression vectors (D), and with control and Oct4 expression vectors (E) Following transfection, the cells were incubated for 48 hr, and the cell proliferation detected with a WST assay. 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 2: Overexpression of CD44ICD in the absence of CD44 increases the ability of mamosphere formation in breast cancer cells(A) The mammosphere-forming ability of the indicated stable lines was measured under sphere forming conditions for 15 days. (B) The number of spheres was quantified in the experiments (A) (C) Cells were transfected with scRNA and CD44 siRNA. Following transfection, the cells were incubated for 48 hr, and cell proliferation was detected with a WST assay. (D) and (E) Cells were transfected with control and Oct4 expression vectors (D), and with control and Oct4 expression vectors (E) Following transfection, the cells were incubated for 48 hr, and the cell proliferation detected with a WST assay. 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: To test whether CD44ICD regulates the potential of breast cancer cells for mammosphere formation, we selected MDA-MB-231 and MCF-7 cells and generated stable cell lines, including CD44-depleted cells (CD44KD), CD44-depleted cells with overexpression of CD44ICD (CD44KD/ICDover), and wild-type cells with overexpression of CD44ICD (ICDover), using a lentiviral vector with a genotype that was confirmed by RT-PCR (upper panel) and western blotting (lower panel) (Supplementary Figure S1). These cells were mammosphere-cultured for 15 days (Figure 2A and 2B). The size and number of mammospheres were significantly reduced in CD44-depleted MDA-MB-231 and MCF-7 cells. However, CD44-depleted and CD44ICD-overexpressed cells showed an increased size and number of mammospheres. Additionally, the size and number of spheres were greater in cells with CD44ICD overexpression than in wild-type (WT) cells. Treatment with GSI also significantly inhibited the formation of mammospheres in MCF-7 cells (Supplementary Figure S2). We also determined that the cell proliferation of breast cancer MDA-MB231 and MCF-7 cells was significantly reduced by CD44 ablation (Figure 2C). Taken together, our results suggest that CD44ICD might regulate mammosphere formation in breast cancer cells.

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