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Identification of mitogen-activated protein kinase kinase as a chemoresistant pathway in MCF-7 cells by using gene
Christopher B Weldon1, Ali B Scandurro, Kevin W Rolfe
1Department of Surgery, Tulane University School of Medicine and Tulane University School of Public Health & Tropical Medicine, New Orleans 70112, USA.
Background:
Components of the mitogen-activated protein kinase (MAPK) cascade have been implicated in apoptotic regulation. This study used gene expression profiling analysis to identify and implicate mitogen-activated protein kinase kinase (MEK5)-BMK1 (big mitogen-activated kinase-1)/extracellular signal related protein kinase (ERK5) pathway as a novel target involved in chemoresistance.
Methods:
Differential gene expression between apoptotically sensitive (APO+) and apoptotically resistant (APO-) MCF-7 cell variants was determined by using microarray and confirmed by reverse transcriptase- polymerase chain reaction (RT-PCR). An apoptotic/viability reporter gene assay was used to deter-mine the effects of the transfection of a dominant-negative mutant of BMK1 (BMK1/DN) in conjunction with apoptotic-inducing agents (etoposide, tumor necrosis factor-alpha [TNF], or TNF-related apoptosis-inducing ligand [TRAIL]), with or without phorbol ester (PMA).
Results:
Of the 1186 genes detected through microarray analysis, MEK5 was increased 22-fold in APO- cells. Overexpression of MEK5 was confirmed by using RT-PCR analysis. Expression of BMK1/DN alone resulted in a dose-dependent increase in cell death versus control (P <.05). In addition, BMK1/DN enhanced the sensitivity of MCF-7 cells to treatment-induced cell death (P <.05). The ability of PMA to partially suppress TRAIL- and TNF-induced cell death was inhibited by BMK1/DN. However, only TRAIL-induced activity suppression reached statistical significance (P <.05).
Conclusions:
The overexpression of MEK5 in APO- MCF-7 breast carcinoma cells shows that this MAPK signaling protein represents a potent survival molecule. Molecular inhibition of MEK5 signaling may represent a mechanism for sensitizing cancer cells to chemotherapeutic regimens.
Insights
The mitogen-activated protein kinase kinase (MEK5)-big mitogen-activated kinase-1 (BMK1) pathway is involved in chemoresistance. Inhibiting this pathway may sensitize cancer cells to chemotherapy.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Mitogen-activated protein kinase (MAPK) cascades are involved in apoptosis regulation.
- This study investigates the role of the MEK5-BMK1/ERK5 pathway in chemoresistance.
Purpose of the Study:
- To identify novel targets involved in chemoresistance using gene expression profiling.
- To elucidate the role of the MEK5-BMK1/ERK5 pathway in mediating chemoresistance in breast carcinoma cells.
Main Methods:
- Differential gene expression analysis using microarrays and reverse transcriptase-polymerase chain reaction (RT-PCR).
- Apoptotic/viability reporter gene assays to assess the effects of dominant-negative BMK1 (BMK1/DN) transfection on cell death.
- Treatment of cells with apoptotic-inducing agents (etoposide, TNF, TRAIL) with or without phorbol ester (PMA).
Main Results:
- MEK5 was significantly overexpressed (22-fold) in chemoresistant MCF-7 cells (APO-).
- BMK1/DN expression increased cell death and enhanced sensitivity to chemotherapy agents.
- BMK1/DN inhibited the suppressive effects of PMA on TNF- and TRAIL-induced cell death.
Conclusions:
- Overexpression of MEK5 in breast cancer cells acts as a survival molecule, contributing to chemoresistance.
- Molecular inhibition of MEK5 signaling presents a potential strategy to sensitize cancer cells to chemotherapeutic regimens.