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Published on: April 25, 2017
Loss of MCL1 function sensitizes the MDA-MB-231 breast cancer cells to rh-TRAIL by increasing DR4 levels
Anna De Blasio1,2, Giovanni Pratelli1,2, Rosa Drago-Ferrante1,2
1Laboratory of Biochemistry, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Polyclinic, Palermo, Italy.
Abstract:
Triple-negative breast cancer (TNBC) is a form of BC characterized by high aggressiveness and therapy resistance probably determined by cancer stem cells. MCL1 is an antiapoptotic Bcl-2 family member that could limit the efficacy of anticancer agents as recombinant human tumor necrosis factor related apoptosis-inducing ligand (rh-TRAIL). Here, we investigated MCL1 expression in TNBC tissues and cells. We found MCL1 differentially expressed (upregulated or downregulated) in TNBC tissues. Furthermore, in comparison to the human mammary epithelial cells, we found that MDA-MB-231 cells show similar messenger RNA levels but higher MCL1 protein levels, whereas it resulted downregulated in MDA-MB-436 and BT-20 cells. We evaluated the effects of rh-TRAIL and A-1210477, a selective MCL1 inhibitor, on cell viability and growth of MDA-MB-231 cells. We demonstrated that the drug combination reduced the cell growth and activated the apoptotic pathway. Similar effects were observed on three-dimensional cultures and tertiary mammospheres of MDA-MB-231 cells. In MDA-MB-231 cells, after MCL1 silencing, rh-TRAIL confined the cell population in the sub-G0/G1 phase and induced a drop in the mitochondrial transmembrane potential. To understand the molecular mechanism by which the loss of MCL1 function sensitizes the MDA-MB-231 cells to rh-TRAIL, we analyzed by real-time reverse transcription polymerase chain reaction, the expression of genes related to apoptosis, stemness, cell cycle, and those involved in epigenetic regulation. Interestingly, among the upregulated genes through MCL1 silencing or inhibition, there was TNFRSF10A (DR4). Moreover, MCL1 inhibition increased DR4 protein levels and its cell surface expression. Finally, we demonstrated MCL1-DR4 interaction and dissociation of this complex after A-1210477 treatment. Overall, our findings highlight the potential MCL1-roles in MDA-MB-231 cells and suggest that MCL1 targeting could be an effective strategy to overcome TNBC's rh-TRAIL resistance.
Insights
Targeting MCL1, an anti-apoptotic protein, can overcome resistance to recombinant human tumor necrosis factor related apoptosis-inducing ligand (rh-TRAIL) therapy in triple-negative breast cancer (TNBC). Inhibiting MCL1 increases DR4 expression, sensitizing cells to rh-TRAIL and promoting apoptosis.
Area of Science:
- Molecular Biology
- Cancer Research
- Cellular Biology
Background:
- Triple-negative breast cancer (TNBC) exhibits high aggressiveness and therapy resistance, potentially linked to cancer stem cells.
- MCL1, an anti-apoptotic protein, can limit the efficacy of anticancer agents like recombinant human tumor necrosis factor related apoptosis-inducing ligand (rh-TRAIL).
- MCL1 expression is variable in TNBC tissues and cell lines, with higher protein levels observed in MDA-MB-231 cells compared to normal mammary epithelial cells.
Purpose of the Study:
- To investigate MCL1 expression in TNBC.
- To evaluate the effects of rh-TRAIL combined with a selective MCL1 inhibitor (A-1210477) on TNBC cell viability and growth.
- To elucidate the molecular mechanisms underlying MCL1's role in rh-TRAIL resistance.
Main Methods:
- Analysis of MCL1 expression in TNBC tissues and cell lines (MDA-MB-231, MDA-MB-436, BT-20).
- Treatment of MDA-MB-231 cells with rh-TRAIL and A-1210477, assessing cell viability, apoptosis, and cell cycle.
- Gene expression analysis (RT-PCR) and protein level assessment of apoptosis-related genes (e.g., DR4) and MCL1-DR4 interaction studies.
Main Results:
- The combination of rh-TRAIL and A-1210477 significantly reduced MDA-MB-231 cell growth and induced apoptosis in both 2D and 3D cultures.
- MCL1 inhibition or silencing sensitized cells to rh-TRAIL, leading to cell cycle arrest and decreased mitochondrial potential.
- MCL1 inhibition upregulated TNFRSF10A (DR4) expression, increased DR4 protein levels and cell surface expression, and disrupted MCL1-DR4 complex formation.
Conclusions:
- MCL1 plays a significant role in mediating resistance to rh-TRAIL in MDA-MB-231 cells.
- Targeting MCL1, in combination with rh-TRAIL, represents a promising therapeutic strategy to overcome resistance in triple-negative breast cancer.
- The observed increase in DR4 expression and cell surface localization upon MCL1 inhibition is a key mechanism sensitizing TNBC cells to rh-TRAIL.
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