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Expression and function of tight junction associated molecules in human breast tumor cells is not affected by the
R Macek1, K Swisshelm, M Kubbies
1Roche Diagnostics GmbH, Pharma Research Oncology, Dept. Cell Biology, Nonnenwald 2,82377 Penzberg, Germany. robert.macek@roche.com
Abstract:
Constitutive activation of Ras or Ras-mediated signaling pathways is one of the initial steps during tumorigenesis that promotes neoplastic transformation. Recently it was reported that in Ha-Ras overexpressing MDCK cells the tight junction proteins claudin-1, occludin and ZO-1 were absent at cell-cell contact sites but present in the cytoplasm. Inhibition of MEK1 activity recruited all three proteins to the cell membrane leading to a restoration of the tight junction barrier function in MDCK cells. In order to evaluate the relevance of the MEK1 pathway in tight junction regulation in breast cancer cells, we investigated the effect ofMEK1 inhibition on expression of claudin-1, occludin and ZO-1 in natively claudin-1 expressing T47-D cells (low Ras activity), claudin-1 negative MCF-7 cells (elevated Ras activity) as well as two retroviral claudin-1 transduced MCF-7 daughter cell lines with prominent membrane and cytoplasmic claudin-1 dominant homing, respectively. Although we effectively blocked phosphorylation of MAPKs ERK-1 and ERK-2 using the selective MEK1 inhibitor PD98059, no quantitative changes of mRNA or protein levels of claudin-1, occludin and ZO-1 could be detected in all cell lines investigated. Furthermore, immnfluorescence analysis of claudin-1 revealed that inhibition of the MAPK pathway did not alter th e subcellular cytoplasmic distribution of claudin-1 to be more membrane specific. Finally, the diffusion barrier properties of tight junctions as analyzed by transepithelial resistance (TER) or paracellular flux analysis of 3 and 40 kDa dextran of tight junctions were not altered in the claudin-1 positive T47-D and the MCF-7 cell lines. Our findings indicate that the proposed involvement of the Ras-MEK-ERK pathway is likely not involved in the dysregulated tight junction formation in breast tumor cells and indicates that elevated activity of Ras might not be of general importance for the disruption of tight junction structures in breast tumors.
Insights
Ras-MEK-ERK pathway inhibition did not affect tight junction proteins claudin-1, occludin, and ZO-1 in breast cancer cells. This suggests the Ras-MEK-ERK pathway is not crucial for tight junction disruption in breast tumors.
Area of Science:
- Cell Biology
- Cancer Research
- Molecular Biology
Background:
- Constitutive activation of Ras signaling pathways is an early event in tumorigenesis.
- Ras-mediated pathways influence neoplastic transformation and cell-cell adhesion.
- Tight junction proteins (claudin-1, occludin, ZO-1) are crucial for epithelial barrier function.
Purpose of the Study:
- To investigate the role of the MEK1 pathway in regulating tight junction proteins in breast cancer cells.
- To determine if MEK1 inhibition impacts claudin-1, occludin, and ZO-1 expression and localization.
- To assess the effect of MEK1 inhibition on tight junction barrier function in breast cancer models.
Main Methods:
- Utilized breast cancer cell lines (T47-D, MCF-7) with varying Ras activity and claudin-1 expression.
- Administered the selective MEK1 inhibitor PD98059 to block MAPK/ERK phosphorylation.
- Quantified mRNA and protein levels of tight junction proteins via RT-PCR and Western blotting.
- Analyzed protein localization using immunofluorescence microscopy.
- Assessed tight junction barrier function using transepithelial electrical resistance (TER) and dextran flux assays.
Main Results:
- MEK1 inhibition effectively blocked ERK-1 and ERK-2 phosphorylation in all tested cell lines.
- No significant changes in mRNA or protein levels of claudin-1, occludin, or ZO-1 were observed.
- Immunofluorescence showed that MEK1 inhibition did not alter the subcellular distribution of claudin-1.
- Transepithelial resistance and dextran flux assays indicated no alteration in tight junction barrier properties.
Conclusions:
- The Ras-MEK-ERK pathway does not appear to be involved in the dysregulation of tight junction formation in the studied breast cancer cells.
- Elevated Ras activity may not be a general factor in the disruption of tight junction structures in breast tumors.
- Targeting the MEK1 pathway is unlikely to restore tight junction integrity in these breast cancer models.