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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

CD44ICD interacts with the stemness factors, Nanog, Sox2, and Oct4 and regulates the nuclear-localization(A) MDA-MB-231 cells were transfected with CD44ICD. The interaction between CD44 and Nanog, Sox2, and Oct4 were detected with an immunoprecipitation assay described in “materials and methods”. (B) HEK293 cells were transfected with CD44ICD or the truncated mutant constructs, ICD_ΔN17, ICD_ΔN35, ICD_ΔC19, and co-transfected with Sox2 and Oct4 expression vectors for 36 hr as described in “materials methods”. Their interaction was detected with an immunoprecipitation assay. (C) Endogenous CD44 stable knockdown MDA-MB-231 and MCF-7 cells were transfected with the control and CD44ICD and C-terminal region truncated CD44 vectors. The changes in the localization of CD44ICD and stemness factors were detected by western blot. GAPDH and Lamin A/C were used as loading controls.
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Figure 5: CD44ICD interacts with the stemness factors, Nanog, Sox2, and Oct4 and regulates the nuclear-localization(A) MDA-MB-231 cells were transfected with CD44ICD. The interaction between CD44 and Nanog, Sox2, and Oct4 were detected with an immunoprecipitation assay described in “materials and methods”. (B) HEK293 cells were transfected with CD44ICD or the truncated mutant constructs, ICD_ΔN17, ICD_ΔN35, ICD_ΔC19, and co-transfected with Sox2 and Oct4 expression vectors for 36 hr as described in “materials methods”. Their interaction was detected with an immunoprecipitation assay. (C) Endogenous CD44 stable knockdown MDA-MB-231 and MCF-7 cells were transfected with the control and CD44ICD and C-terminal region truncated CD44 vectors. The changes in the localization of CD44ICD and stemness factors were detected by western blot. GAPDH and Lamin A/C were used as loading controls.

Mentions: Next, we performed an immunoprecipitation assay and investigated whether CD44ICD interacted directly with Nanog, Sox2, and Oct4 using MDA-MB-231 cell lysates (Figure 5A). We generated an N-terminal truncated mutant CD44-ICD expressing vector (ICD_ΔN35), and a NLS truncated mutant construct, CD44-ICD_ΔN17, because the NLS of CD44ICD is located in the N-terminal region [20] and the C-terminal truncated mutant CD44ICD_ΔC19, as indicated in Figure 5B. We then, co-transfected these mutant vectors and Sox2- or Oct4-expressing vectors to detect their interaction site. The C-terminal truncated mutant CD44ICD_ΔC19 did not interact with Sox2 and Oct4 (Figure 5B). This suggests that the essential region for the interaction with Sox2 and Oct4 is located in the C-terminal region of CD44ICD, including the PDZ domain [5]. We overexpressed the C-terminal truncated mutant CD44IC_ΔC19 and fractionated the nucleus and cytosol (Figure 5C). As expected, the reduced nuclear localization of both CD44ICD and the stemness factors following the overexpression of the C-terminal truncated mutant CD44IC_ΔC19 was detected in MDA-MB-231 and MCF-7 cells (Figure 5C). Taken together, our results suggest that CD44ICD regulates the nuclear localization and transcriptional activation of stemness factors through an interaction with its C-terminal domain.


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)

CD44ICD interacts with the stemness factors, Nanog, Sox2, and Oct4 and regulates the nuclear-localization(A) MDA-MB-231 cells were transfected with CD44ICD. The interaction between CD44 and Nanog, Sox2, and Oct4 were detected with an immunoprecipitation assay described in “materials and methods”. (B) HEK293 cells were transfected with CD44ICD or the truncated mutant constructs, ICD_ΔN17, ICD_ΔN35, ICD_ΔC19, and co-transfected with Sox2 and Oct4 expression vectors for 36 hr as described in “materials methods”. Their interaction was detected with an immunoprecipitation assay. (C) Endogenous CD44 stable knockdown MDA-MB-231 and MCF-7 cells were transfected with the control and CD44ICD and C-terminal region truncated CD44 vectors. The changes in the localization of CD44ICD and stemness factors were detected by western blot. GAPDH and Lamin A/C were used as loading controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: CD44ICD interacts with the stemness factors, Nanog, Sox2, and Oct4 and regulates the nuclear-localization(A) MDA-MB-231 cells were transfected with CD44ICD. The interaction between CD44 and Nanog, Sox2, and Oct4 were detected with an immunoprecipitation assay described in “materials and methods”. (B) HEK293 cells were transfected with CD44ICD or the truncated mutant constructs, ICD_ΔN17, ICD_ΔN35, ICD_ΔC19, and co-transfected with Sox2 and Oct4 expression vectors for 36 hr as described in “materials methods”. Their interaction was detected with an immunoprecipitation assay. (C) Endogenous CD44 stable knockdown MDA-MB-231 and MCF-7 cells were transfected with the control and CD44ICD and C-terminal region truncated CD44 vectors. The changes in the localization of CD44ICD and stemness factors were detected by western blot. GAPDH and Lamin A/C were used as loading controls.
Mentions: Next, we performed an immunoprecipitation assay and investigated whether CD44ICD interacted directly with Nanog, Sox2, and Oct4 using MDA-MB-231 cell lysates (Figure 5A). We generated an N-terminal truncated mutant CD44-ICD expressing vector (ICD_ΔN35), and a NLS truncated mutant construct, CD44-ICD_ΔN17, because the NLS of CD44ICD is located in the N-terminal region [20] and the C-terminal truncated mutant CD44ICD_ΔC19, as indicated in Figure 5B. We then, co-transfected these mutant vectors and Sox2- or Oct4-expressing vectors to detect their interaction site. The C-terminal truncated mutant CD44ICD_ΔC19 did not interact with Sox2 and Oct4 (Figure 5B). This suggests that the essential region for the interaction with Sox2 and Oct4 is located in the C-terminal region of CD44ICD, including the PDZ domain [5]. We overexpressed the C-terminal truncated mutant CD44IC_ΔC19 and fractionated the nucleus and cytosol (Figure 5C). As expected, the reduced nuclear localization of both CD44ICD and the stemness factors following the overexpression of the C-terminal truncated mutant CD44IC_ΔC19 was detected in MDA-MB-231 and MCF-7 cells (Figure 5C). Taken together, our results suggest that CD44ICD regulates the nuclear localization and transcriptional activation of stemness factors through an interaction with its C-terminal domain.

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