Modeling bystander effects that cause growth delay of breast cancer xenografts in bone marrow of mice treated with radium-223.

RATIONALE: The role of radiation-induced bystander effects in cancer therapy with alpha-particle emitting radiopharmaceuticals remains unclear. With renewed interest in using alpha-particle emitters to sterilize disseminated tumor cells, micrometastases, and tumors, a better understanding of the direct effects of alpha particles and the contribution of the bystander responses they induce is needed to refine dosimetric models that help predict clinical benefit. Accordingly, this work models and quantifies the relative importance of direct effects (DE) and bystander effects (BE) in the growth delay of human breast cancer xenografts observed previously in the tibiae of mice treated with 223RaCl2. METHODS: A computational model of MDA-MB-231 and MCF-7 human breast cancer xenografts in the tibial bone marrow of mice administered 223RaCl2 was created. A Monte Carlo radiation transport simulation was performed to assess individual cell absorbed doses. The responses of the breast cancer cells to direct alpha particle irradiation and gamma irradiation were needed as input data for the model and were determined experimentally using a colony-forming assay and compared to the responses of preosteoblast MC3T3-E1 and osteocyte-like MLO-Y4 bone cells. Using these data, a scheme was devised to simulate the dynamic proliferation of the tumors in vivo, including DE and BE propagated from the irradiated cells. The parameters of the scheme were estimated semi-empirically to fit experimental tumor growth. RESULTS: A robust BE component, in addition to a much smaller DE component, was required to simulate the in vivo tumor proliferation. We also found that the relative biological effectiveness (RBE) for cell killing by alpha particle radiation was greater for the bone cells than the tumor cells. CONCLUSION: This modeling study demonstrates that DE of radiation alone cannot explain experimental observations of 223RaCl2-induced growth delay of human breast cancer xenografts. Furthermore, while the mechanisms underlying BE remain unclear, the addition of a BE component to the model is necessary to provide an accurate prediction of the growth delay. More complex models are needed to further comprehend the extent and complexity of 223RaCl2-induced BE.

Radium-223-Induced Bystander Effects Cause DNA Damage and Apoptosis in Disseminated Tumor Cells in Bone Marrow.

Radiation-induced bystander effects have been implicated in contributing to the growth delay of disseminated tumor cells (DTC) caused by 223RaCl2, an alpha particle-emitting radiopharmaceutical. To understand how 223RaCl2 affects the growth, we have quantified biological changes caused by direct effects of radiation and bystander effects caused by the emitted radiations on DTC and osteocytes. Characterizing these effects contribute to understanding the efficacy of alpha particle-emitting radiopharmaceuticals and guide expansion of their use clinically. MDA-MB-231 or MCF-7 human breast cancer cells were inoculated intratibially into nude mice that were previously injected intravenously with 50 or 600 kBq/kg 223RaCl2. At 1-day and 3-days postinoculation, tibiae were harvested and examined for DNA damage (γ-H2AX foci) and apoptosis in osteocytes and cancer cells located within and beyond the range (70 µm) of alpha particles emitted from the bone surface. Irradiated and bystander MDA-MB-231 and MCF-7 cells harbored DNA damage. Bystander MDA-MB-231 cells expressed DNA damage at both treatment levels while bystander MCF-7 cells required the higher administered activity. Osteocytes also had DNA damage regardless of inoculated cancer cell line. The extent of DNA damage was quantified by increases in low (1-2 foci), medium (3-5 foci), and high (5+ foci) damage. MDA-MB-231 but not MCF-7 bystander cells showed increases in apoptosis in 223RaCl2-treated animals, as did irradiated osteocytes. In summary, radiation-induced bystander effects contribute to DTC cytotoxicity caused by 223RaCl2. IMPLICATIONS: This observation supports clinical investigation of the efficacy of 223RaCl2 to prevent breast cancer DTC from progressing to oligometastases.

Dose-Dependent Growth Delay of Breast Cancer Xenografts in the Bone Marrow of Mice Treated with 223Ra: The Role of Bystander Effects and Their Potential for Therapy.

The role of radiation-induced bystander effects in radiation therapy remains unclear. With renewed interest in therapy with α-particle emitters, and their potential for sterilizing disseminated tumor cells (DTCs), it is critical to determine the contribution of bystander effects to the overall response so they can be leveraged for maximum clinical benefit. Methods: Female Foxn1nu athymic nude mice were administered 0, 50, or 600 kBq/kg 223RaCl2 to create bystander conditions. At 24 hours after administration, MDA-MB-231 or MCF-7 human breast cancer cells expressing luciferase were injected into the tibial marrow compartment. Tumor burden was tracked weekly via bioluminescence. Results: The MDA-MB-231 xenografts were observed to have a 10-day growth delay in the 600 kBq/kg treatment group only. In contrast, MCF-7 cells had 7- and 65-day growth delays in the 50 and 600 kBq/kg groups, respectively. Histologic imaging of the tibial marrow compartment, α-camera imaging, and Monte Carlo dosimetry modeling revealed DTCs both within and beyond the range of the α-particles emitted from 223Ra in bone for both MCF-7 and MDA-MB-231 cells. Conclusion: Taken together, these results support the participation of 223Ra-induced antiproliferative/cytotoxic bystander effects in delayed growth of DTC xenografts. They indicate that the delay depends on the injected activity and therefore is dose-dependent. They suggest using 223RaCl2 as an adjuvant treatment for select patients at early stages of breast cancer.

RNF11 sequestration of the E3 ligase SMURF2 on membranes antagonizes SMAD7 down-regulation of transforming growth factor ß signaling.

The activity of the E3 ligase, SMURF2, is antagonized by an intramolecular, autoinhibitory interaction between its C2 and Hect domains. Relief of SMURF2 autoinhibition is induced by TGFß and is mediated by the inhibitory SMAD, SMAD7. In a proteomic screen for endomembrane interactants of the RING-domain E3 ligase, RNF11, we identified SMURF2, among a cohort of Hect E3 ligases previously implicated in TGFß signaling. Reconstitution of the SMURF2·RNF11 complex in vitro unexpectedly revealed robust SMURF2 E3 ligase activity, with biochemical properties previously restricted to the SMURF2·SMAD7 complex. Using in vitro binding assays, we find that RNF11 can directly compete with SMAD7 for SMURF2 and that binding is mutually exclusive and dependent on a proline-rich domain. Moreover, we found that co-expression of RNF11 and SMURF2 dramatically reduced SMURF2 ubiquitylation in the cell. This effect is strictly dependent on complex formation and sorting determinants that regulate the association of RNF11 with membranes. RNF11 is overexpressed in certain tumors, and, importantly, we found that depletion of this protein down-regulated gene expression of several TGFß-responsive genes, dampened cell proliferation, and dramatically reduced cell migration in response to TGFß. Our data suggest for the first time that the choice of binding partners for SMURF2 can sustain or repress TGFß signaling, and RNF11 may promote TGFß-induced cell migration.