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MUC4 stabilizes HER2 expression and maintains the cancer stem cell population in ovarian cancer cells.

Ponnusamy MP, Seshacharyulu P, Vaz A, Dey P, Batra SK - J Ovarian Res (2011)

Bottom Line: MUC4 overexpressed SKOV3 cells showed an increased expression of HER2 compared to control cells.These studies demonstrate that MUC4 overexpression leads to an enriched ovarian cancer stem cell population either directly or indirectly through HER2.In future, this study would be helpful for MUC4-directed therapy for the ovarian cancer stem cell population.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA. sbatra@unmc.edu.

ABSTRACT

Background: Recent evidence has suggested that the capability of cancer to grow, propagate and relapse after therapy is dependent on a small subset of the cell population within the tumor, called cancer stem cells. Therefore, this subpopulation of cells needs to be targeted with different approaches by identification of unique stem-cell specific target antigens. One of the well known tumor antigens is the epithelial cell mucin MUC4, which is aberrantly expressed in ovarian cancer as compared to the normal ovary and plays a pivotal role in the aggressiveness and metastasis of ovarian cancer cells. In the present study, we aimed to analyze the cancer stem cell population in MUC4 overexpressed ovarian cancer cells.

Methods: MUC4 was ectopically overexpressed in SKOV3 ovarian cancer cells. Western blot analysis was performed for MUC4, HER2, CD133, ALDH1 and Shh expression in MUC4 overexpressed cells. Confocal analysis of MUC4, HER2 and CD133 was also done in the MUC4 overexpressed cells. CD133 and Hoechst33342 dye staining was used to analyze the cancer stem cell population via FACS method in SKOV3-MUC4 cells.

Results: MUC4 overexpressed SKOV3 cells showed an increased expression of HER2 compared to control cells. MUC4 overexpression leads to increased (0.1%) side population (SP) and CD133-positive cancer stem cells compared to the control cells. Interestingly, the tumor sphere type circular colony formation was observed only in the MUC4 overexpressed ovarian cancer cells. Furthermore, the cancer stem cell marker CD133 was expressed along with MUC4 in the isolated circular colonies as analyzed by both confocal and western blot analysis. HER2 and cancer stem cell specific marker ALDH1 along with Shh, a self-renewal marker, showed increased expression in the isolated circular colonies compared to MUC4-transfected cells.

Conclusion: These studies demonstrate that MUC4 overexpression leads to an enriched ovarian cancer stem cell population either directly or indirectly through HER2. In future, this study would be helpful for MUC4-directed therapy for the ovarian cancer stem cell population.

No MeSH data available.


Related in: MedlinePlus

Expression of cancer stem cell markers in circular colonies. (A) Confocal analysis showed significant expression of CD133 (Red) in isolated colonies compared to SKOV3-MUC4 cells. MUC4 (Green) expression was seen in both isolated colonies and SKOV3-MUC4 cells. DAPI (Blue) was used as nuclear counter staining. (B) Western blot analysis showed MUC4, HER2, ALDH1, CD133 and Shh expression in SKOV3-MUC4 and isolated colonies from MUC4 overexpressed SKOV3 cells. β-actin served as a loading control. DIC - differential interference contrast and staining (Scale bar-20 μm).
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Figure 4: Expression of cancer stem cell markers in circular colonies. (A) Confocal analysis showed significant expression of CD133 (Red) in isolated colonies compared to SKOV3-MUC4 cells. MUC4 (Green) expression was seen in both isolated colonies and SKOV3-MUC4 cells. DAPI (Blue) was used as nuclear counter staining. (B) Western blot analysis showed MUC4, HER2, ALDH1, CD133 and Shh expression in SKOV3-MUC4 and isolated colonies from MUC4 overexpressed SKOV3 cells. β-actin served as a loading control. DIC - differential interference contrast and staining (Scale bar-20 μm).

Mentions: Cancer stem cells express numerous universal markers such as CD133, CD44, CD24, ESA and ALDH1 in different cancers [1]. Few of these markers were used for the confirmation of an MUC4 enriched cancer stem cell population. Stem cells and cancer stem cells are known to possess the phenomenal property of self-renewal which is maintained by few specific pathways such as Shh, Wnt and Notch [1]. The circular colonies or tumor spheres from MUC4-transfected SKOV3 cells were isolated and grown in a separate glass cover slip for the cancer stem cell marker analysis by confocal microscopy. The confocal results showed immunofluorescence staining of CD133 marker expression (Red) in the isolated colonies and SKOV3-MUC4 cells (Figure 4A). On the other hand, MUC4 (green) immunofluorescence staining is almost equal in both isolated colonies and SKOV3-MUC4 cells (Figure 4A). In our study we have also analyzed MUC4, HER2, ALDH1 and CD133 for the cancer stem cells and Shh for the self-renewal pathway in the isolated colonies from MUC4 overexpressed SKOV3 cells and SKOV3-MUC4. MUC4 expression was observed at an almost equal level in both SKOV3-MUC4 and isolated colonies although there was a minor molecular weight change in isolated colonies (Figure 4B). Interestingly, increased expression of HER2 was seen in isolated colonies compared to SKOV3-MUC4 cells. Expression of CD133 was also shown in both SKOV3-MUC4 and isolated colonies, whereas ALDH1 showed an increased expression in isolated colonies compared to MUC4 overexpressed SKOV3 cells (Figure 4B). In addition, the Shh self-renewal protein expression was observed only in isolated colonies, while there was no expression in SKOV3-MUC4 (Figure 4B). This suggests that the isolated colonies from MUC4 overexpressed cells behave like cancer stem cells which are capable of maintaining the self-renewal property (Figure 5).


MUC4 stabilizes HER2 expression and maintains the cancer stem cell population in ovarian cancer cells.

Ponnusamy MP, Seshacharyulu P, Vaz A, Dey P, Batra SK - J Ovarian Res (2011)

Expression of cancer stem cell markers in circular colonies. (A) Confocal analysis showed significant expression of CD133 (Red) in isolated colonies compared to SKOV3-MUC4 cells. MUC4 (Green) expression was seen in both isolated colonies and SKOV3-MUC4 cells. DAPI (Blue) was used as nuclear counter staining. (B) Western blot analysis showed MUC4, HER2, ALDH1, CD133 and Shh expression in SKOV3-MUC4 and isolated colonies from MUC4 overexpressed SKOV3 cells. β-actin served as a loading control. DIC - differential interference contrast and staining (Scale bar-20 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3111401&req=5

Figure 4: Expression of cancer stem cell markers in circular colonies. (A) Confocal analysis showed significant expression of CD133 (Red) in isolated colonies compared to SKOV3-MUC4 cells. MUC4 (Green) expression was seen in both isolated colonies and SKOV3-MUC4 cells. DAPI (Blue) was used as nuclear counter staining. (B) Western blot analysis showed MUC4, HER2, ALDH1, CD133 and Shh expression in SKOV3-MUC4 and isolated colonies from MUC4 overexpressed SKOV3 cells. β-actin served as a loading control. DIC - differential interference contrast and staining (Scale bar-20 μm).
Mentions: Cancer stem cells express numerous universal markers such as CD133, CD44, CD24, ESA and ALDH1 in different cancers [1]. Few of these markers were used for the confirmation of an MUC4 enriched cancer stem cell population. Stem cells and cancer stem cells are known to possess the phenomenal property of self-renewal which is maintained by few specific pathways such as Shh, Wnt and Notch [1]. The circular colonies or tumor spheres from MUC4-transfected SKOV3 cells were isolated and grown in a separate glass cover slip for the cancer stem cell marker analysis by confocal microscopy. The confocal results showed immunofluorescence staining of CD133 marker expression (Red) in the isolated colonies and SKOV3-MUC4 cells (Figure 4A). On the other hand, MUC4 (green) immunofluorescence staining is almost equal in both isolated colonies and SKOV3-MUC4 cells (Figure 4A). In our study we have also analyzed MUC4, HER2, ALDH1 and CD133 for the cancer stem cells and Shh for the self-renewal pathway in the isolated colonies from MUC4 overexpressed SKOV3 cells and SKOV3-MUC4. MUC4 expression was observed at an almost equal level in both SKOV3-MUC4 and isolated colonies although there was a minor molecular weight change in isolated colonies (Figure 4B). Interestingly, increased expression of HER2 was seen in isolated colonies compared to SKOV3-MUC4 cells. Expression of CD133 was also shown in both SKOV3-MUC4 and isolated colonies, whereas ALDH1 showed an increased expression in isolated colonies compared to MUC4 overexpressed SKOV3 cells (Figure 4B). In addition, the Shh self-renewal protein expression was observed only in isolated colonies, while there was no expression in SKOV3-MUC4 (Figure 4B). This suggests that the isolated colonies from MUC4 overexpressed cells behave like cancer stem cells which are capable of maintaining the self-renewal property (Figure 5).

Bottom Line: MUC4 overexpressed SKOV3 cells showed an increased expression of HER2 compared to control cells.These studies demonstrate that MUC4 overexpression leads to an enriched ovarian cancer stem cell population either directly or indirectly through HER2.In future, this study would be helpful for MUC4-directed therapy for the ovarian cancer stem cell population.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA. sbatra@unmc.edu.

ABSTRACT

Background: Recent evidence has suggested that the capability of cancer to grow, propagate and relapse after therapy is dependent on a small subset of the cell population within the tumor, called cancer stem cells. Therefore, this subpopulation of cells needs to be targeted with different approaches by identification of unique stem-cell specific target antigens. One of the well known tumor antigens is the epithelial cell mucin MUC4, which is aberrantly expressed in ovarian cancer as compared to the normal ovary and plays a pivotal role in the aggressiveness and metastasis of ovarian cancer cells. In the present study, we aimed to analyze the cancer stem cell population in MUC4 overexpressed ovarian cancer cells.

Methods: MUC4 was ectopically overexpressed in SKOV3 ovarian cancer cells. Western blot analysis was performed for MUC4, HER2, CD133, ALDH1 and Shh expression in MUC4 overexpressed cells. Confocal analysis of MUC4, HER2 and CD133 was also done in the MUC4 overexpressed cells. CD133 and Hoechst33342 dye staining was used to analyze the cancer stem cell population via FACS method in SKOV3-MUC4 cells.

Results: MUC4 overexpressed SKOV3 cells showed an increased expression of HER2 compared to control cells. MUC4 overexpression leads to increased (0.1%) side population (SP) and CD133-positive cancer stem cells compared to the control cells. Interestingly, the tumor sphere type circular colony formation was observed only in the MUC4 overexpressed ovarian cancer cells. Furthermore, the cancer stem cell marker CD133 was expressed along with MUC4 in the isolated circular colonies as analyzed by both confocal and western blot analysis. HER2 and cancer stem cell specific marker ALDH1 along with Shh, a self-renewal marker, showed increased expression in the isolated circular colonies compared to MUC4-transfected cells.

Conclusion: These studies demonstrate that MUC4 overexpression leads to an enriched ovarian cancer stem cell population either directly or indirectly through HER2. In future, this study would be helpful for MUC4-directed therapy for the ovarian cancer stem cell population.

No MeSH data available.


Related in: MedlinePlus