Presenter(s)
M. S. Francis, Z. Wang, and E. J. Moding; Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA
Purpose/Objective(s): Radiation therapy (RT) plays a key role in achieving local control of soft tissue sarcomas (STS), and new approaches to widen the therapeutic index will be crucial to minimize side effects and improve responses to RT. Calcium (Ca) is a second messenger implicated in cell survival, proliferation, and stress responses, and dysregulation of Ca signaling has been linked to cancer progression and metastasis. Mutations affecting Ca signaling were previously shown to be enriched in STS subclones that contract during RT, but the underlying molecular mechanisms remain to be further explored. We hypothesized that RT alters intracellular Ca levels by modulating the activity of Ca transporters and that targeting Ca signaling can improve the therapeutic index of RT.
Materials/Methods: The effect of RT on intracellular Ca levels and the impact of blocking Ca signaling on radiosensitivity were investigated in human STS cell lines (HT1080-fibrosarcoma, SW872-liposarcoma, RD-rhabdomyosarcoma, SW982-synovial sarcoma, and GCT-undifferentiated pleomorphic sarcoma) and normal cell lines (skin fibroblasts, TIME-microvascular endothelial cells, and HUVEC-macrovascular endothelial cells). Intracellular Ca levels were measured using FLUO-4-AM. RT-induced changes in protein expression of plasma membrane (PMCA2) and endoplasmic reticulum (SERCA2) Ca ATPase pumps and the Ca-binding proteins calnexin and annexin VI (ANXA6) were assessed by Western blot. RT response was assessed by MTT and clonogenic assays with and without thapsigargin (TG), a SERCA2 inhibitor, or verapamil (VP), an L-type Ca channel blocker, administered 6h before RT.
Results: RT universally increased intracellular Ca levels, but the time course varied across cell lines. SW872, GCT, and HUVEC cells exhibited a transient elevation in Ca levels 24h to 48h post-RT, followed by a return to baseline. In contrast, intracellular Ca levels continued to increase up to 72h post-RT in HT1080, RD, SW982, skin fibroblast, and TIME cells. These changes aligned with the timeline of RT-induced changes in PMCA2, SERCA2, calnexin, and ANXA6 protein levels across these cell lines, leading to adaptation and clearance or accumulation of intracellular Ca. VP (50 to 100 µM) or low doses of TG (2.5 to 5 nM) in combination with RT significantly increased cytotoxicity in STS but not in normal cells.
Conclusion: Radiation-induced increases in intracellular Ca levels varied across tumor and normal tissue cell lines. Blocking Ca transport across the endoplasmic reticulum or plasma membranes selectively radiosensitized STS cells, suggesting that targeting Ca signaling may improve the therapeutic index of RT.