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Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model
Published on: March 20, 2020
SUMOylation modulates FOXK2-mediated paclitaxel sensitivity in breast cancer cells
Gabriela Nestal de Moraes1,2, Zongling Ji3, Lavender Y-N Fan1
1Department of Surgery and Cancer, Imperial College London, Imperial Centre for Translational and Experimental Medicine (ICTEM), Du Cane Road, London, W12 0NN, UK.
Abstract:
The forkhead transcription factor FOXK2 plays a critical role in suppressing tumorigenesis and mediating cytotoxic drug action in breast cancer. However, the mechanism by which the biological function of FOXK2 is regulated remains poorly understood. Here, we investigated the role of SUMOylation in modulating FOXK2-mediated drug sensitivity. We identified SUMOylation consensus motifs within the FOXK2 sequence and constructed two SUMOylation-defective double mutants by converting lysine 527 and 633 to arginines and glutamic acid 529 and 635 to alanines, respectively. We found that both the FOXK2 SUMOylation-deficient (K527/633 R) and (E529/635 A) mutants were ineffective in mediating the cytotoxic function of paclitaxel when compared to the wild-type (WT) FOXK2. When overexpressed, unlike the wild-type (WT) FOXK2, the K527/633 R mutant had little effect on the sensitivity of MCF-7 and MDA-MB-231 cells to paclitaxel, as examined by cell viability and clonogenic assays. Our results also showed that MCF-7 cells overexpressing the K527/633 R mutant form of FOXK2 or the empty expression vector have lower protein and mRNA levels of its tumour suppressive transcriptional target FOXO3 compared to the wild-type FOXK2. Consistently, ChIP assays revealed that unlike wild-type FOXK2, the SUMOylation-defective (K527/633 R) mutant is unable to bind to the FOXO3 promoter, despite expressing comparable levels of protein and having the same subcellular localization as the wild-type FOXK2 in MCF-7 cells. Interestingly, expression of neither the wild-type nor the K527/633 R mutant FOXK2 had any effect on the proliferation and paclitaxel sensitivity of the MCF-7 TaxR paclitaxel-resistant cells. In agreement, both the wild-type and the (K527/633 R) mutant FOXK2 failed to bind to the endogenous FOXO3 promoter in these cells. Collectively, our results suggest that SUMOylation positively regulates FOXK2 transcriptional activity and has a role in mediating the cytotoxic response to paclitaxel through the tumour suppressor FOXO3.
Insights
SUMOylation is crucial for FOXK2
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- Forkhead transcription factor FOXK2 is vital in breast cancer, impacting tumorigenesis and drug response.
- Mechanisms regulating FOXK2's biological functions, particularly its role in drug sensitivity, are not fully understood.
- Post-translational modifications like SUMOylation are key regulators of protein function.
Purpose of the Study:
- To investigate the role of SUMOylation in regulating FOXK2 activity and its impact on drug sensitivity in breast cancer.
- To elucidate how SUMOylation affects FOXK2's interaction with its target genes, specifically FOXO3.
- To determine the therapeutic implications of FOXK2 SUMOylation in paclitaxel-treated breast cancer cells.
Main Methods:
- Identification of SUMOylation consensus motifs in FOXK2.
- Construction and characterization of SUMOylation-defective FOXK2 mutants (K527/633R and E529/635A).
- Assessment of mutant FOXK2's effect on paclitaxel sensitivity using cell viability and clonogenic assays.
- Analysis of FOXO3 expression and FOXK2 binding to the FOXO3 promoter via ChIP assays.
- Evaluation of FOXK2 function in paclitaxel-resistant cell lines.
Main Results:
- SUMOylation-deficient FOXK2 mutants (K527/633R and E529/635A) failed to mediate paclitaxel's cytotoxic effects.
- Overexpression of SUMOylation-defective FOXK2 (K527/633R) reduced paclitaxel sensitivity in MCF-7 and MDA-MB-231 cells.
- SUMOylation-defective FOXK2 (K527/633R) impaired binding to the FOXO3 promoter, leading to decreased FOXO3 expression.
- FOXK2's SUMOylation status did not affect paclitaxel sensitivity or FOXO3 promoter binding in paclitaxel-resistant cells.
Conclusions:
- SUMOylation positively regulates FOXK2's transcriptional activity.
- SUMOylation-mediated FOXK2 function is essential for mediating the cytotoxic response to paclitaxel via the tumor suppressor FOXO3.
- Targeting FOXK2 SUMOylation may offer a therapeutic strategy to enhance paclitaxel efficacy in breast cancer.
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