Loss of MK2 Enhances Radiation-Mediated Apoptosis in Bladder Cancer
Deri Morgan1, Kiersten L Berggren1,2, Grace Millington1
1Department of Radiation Oncology, University of Kansas Medical Center, Kansas City, KS, USA.
Background:
Bladder cancer patients unable to receive cystectomy or who choose to pursue organ-sparing approach are managed with definitive (chemo)radiotherapy. However, this standard of care has not evolved in decades and disease recurrence and survival outcomes remain poor. Identifying novel therapies to combine with radiotherapy (RT) is therefore paramount to improve overall patient outcomes and survival. One approach is to find cellular mechanisms that can be targeted to increase the radiosensitivity of bladder cancer. The stress-activated kinase directly downstream from p38 mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase activated protein kinase 2 (MAPKAPK2 or MK2), has been shown to enhance cancer-mediated inflammation, mesenchymal gene expression, and in vivo tumor growth. Here we examined the impact that MK2 knockdown (KD) has on bladder cancer cell radiosensitivity.
Methods:
We utilized short hairpin RNA (shRNA) KD of MK2 using lentiviral transfection in the bladder cancer cell lines, T24 and HTB9. We compared the growth of KD cells to wild type using colony formation assays, proliferation assays and cell counts to determine differences in cell growth. Apoptosis was examined by annexin-based flow cytometry and western blots. Flow cytometry was also used for cell cycle analysis.
Results:
KD clones showed a greater than 90% inhibition of MK2 expression as determined by western blot. Clonogenic assays exhibited an increase in radiosensitivity among the MK2 KD bladder cancer cells. These data were supported with proliferation assays that displayed a greater reduction in cell number following RT in MK2 KD bladder cancer cells. Annexin V binding in bladder cancer cells suggested increased apoptosis in MK2 KD cells. This was confirmed by comparing the amount of cleaved caspase products for the caspases 3 and 8 to scrambled control (SCR), and the release of cytochrome C into the cytosol. Both cell types showed disruptions in the cell cycle but at different points in the cycle.
Conclusion:
These results show that MK2 controls irradiation-induced apoptosis in bladder cancer cells.
Insights
Targeting mitogen-activated protein kinase activated protein kinase 2 (MK2) enhances bladder cancer radiosensitivity. MK2 knockdown increases tumor cell apoptosis and improves outcomes when combined with radiotherapy (RT).
Area of Science:
- Oncology
- Molecular Biology
- Radiotherapy Research
Background:
- Definitive (chemo)radiotherapy is standard for bladder cancer patients ineligible for or avoiding cystectomy.
- Current bladder cancer radiotherapy approaches have stagnant outcomes, with high recurrence and poor survival rates.
- Novel therapeutic strategies are needed to enhance radiosensitivity and improve patient survival.
Purpose of the Study:
- To investigate the role of mitogen-activated protein kinase activated protein kinase 2 (MAPKAPK2 or MK2) in bladder cancer radiosensitivity.
- To determine if MK2 inhibition can sensitize bladder cancer cells to radiotherapy (RT).
- To explore MK2's impact on cellular mechanisms, including apoptosis and cell cycle, following RT.
Main Methods:
- Utilized short hairpin RNA (shRNA) for MK2 knockdown (KD) in T24 and HTB9 bladder cancer cell lines via lentiviral transfection.
- Assessed cell growth and proliferation using colony formation and proliferation assays.
- Analyzed apoptosis via annexin V flow cytometry and western blots for cleaved caspases and cytochrome C release; performed cell cycle analysis.
Main Results:
- MK2 knockdown achieved >90% inhibition of MK2 expression, confirmed by western blot.
- MK2 KD bladder cancer cells demonstrated increased radiosensitivity in clonogenic assays and reduced cell numbers post-RT.
- MK2 KD cells exhibited elevated apoptosis, evidenced by increased Annexin V binding, cleaved caspases 3/8, and cytosolic cytochrome C release.
Conclusions:
- Mitogen-activated protein kinase activated protein kinase 2 (MK2) plays a critical role in regulating irradiation-induced apoptosis in bladder cancer cells.
- MK2 inhibition represents a promising strategy to enhance bladder cancer cell radiosensitivity.
- Targeting MK2 may improve therapeutic efficacy when combining radiotherapy with novel agents for bladder cancer treatment.
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