Immune response to RB1-regulated senescence limits radiation-induced osteosarcoma formation
Maya Kansara, Huei San Leong, Dan Mei Lin, Sophie Popkiss, Puiyi Pang, Dale W. Garsed, Carl R. Walkley, Carleen Cullinane, Jason Ellul, Nicole M. Haynes, Rod Hicks, Marieke L. Kuijjer, Anne-Marie Cleton-Jansen, Philip W. Hinds, Mark J. Smyth, David M. Thomas
Maya Kansara, Huei San Leong, Dan Mei Lin, Sophie Popkiss, Puiyi Pang, Dale W. Garsed, Carl R. Walkley, Carleen Cullinane, Jason Ellul, Nicole M. Haynes, Rod Hicks, Marieke L. Kuijjer, Anne-Marie Cleton-Jansen, Philip W. Hinds, Mark J. Smyth, David M. Thomas
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Research Article Oncology

Immune response to RB1-regulated senescence limits radiation-induced osteosarcoma formation

Abstract

Ionizing radiation (IR) and germline mutations in the retinoblastoma tumor suppressor gene (RB1) are the strongest risk factors for developing osteosarcoma. Recapitulating the human predisposition, we found that Rb1+/– mice exhibited accelerated development of IR-induced osteosarcoma, with a latency of 39 weeks. Initial exposure of osteoblasts to carcinogenic doses of IR in vitro and in vivo induced RB1-dependent senescence and the expression of a panel of proteins known as senescence-associated secretory phenotype (SASP), dominated by IL-6. RB1 expression closely correlated with that of the SASP cassette in human osteosarcomas, and low expression of both RB1 and the SASP genes was associated with poor prognosis. In vivo, IL-6 was required for IR-induced senescence, which elicited NKT cell infiltration and a host inflammatory response. Mice lacking IL-6 or NKT cells had accelerated development of IR-induced osteosarcomas. These data elucidate an important link between senescence, which is a cell-autonomous tumor suppressor response, and the activation of host-dependent cancer immunosurveillance. Our findings indicate that overcoming the immune response to senescence is a rate-limiting step in the formation of IR-induced osteosarcoma.

Authors

Maya Kansara, Huei San Leong, Dan Mei Lin, Sophie Popkiss, Puiyi Pang, Dale W. Garsed, Carl R. Walkley, Carleen Cullinane, Jason Ellul, Nicole M. Haynes, Rod Hicks, Marieke L. Kuijjer, Anne-Marie Cleton-Jansen, Philip W. Hinds, Mark J. Smyth, David M. Thomas

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Figure 2

RNAi-mediated knockdown of RB1 attenuates senescence response following IR and reveals an immune/inflammatory signature.

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RNAi-mediated knockdown of RB1 attenuates senescence response following ...
(A) Verification of RB1 knockdown. Western blotting was carried out using hOBs stably transfected with shRB1 or shEV at 24 hours after exposure to 0, 1, and 4 Gy IR. (B) Clonogenic assay for cell fitness. shEV and shRB1 knockdown cells exposed to 0, 1, 2, 4, and 8 Gy IR were seeded at 1,000 cells per 6-well plate, and colonies consisting of >10 cells were counted at days 9–11. Values represent the mean and SD of at least 3 separate experiments. (C) Quantification of SA-β-Gal staining of hOB shEV cells and hOB shRB1 cells 1, 5, and 8 days after exposure to 0, 1, 4 Gy IR. Values represent the mean and SEM of 3 independent experiments. *P-value < 0.05, 2-tailed Student’s t test. (D) Global expression profiling of shEV and shRB1 hOBs 0, 8, 16, and 24 hours after 4 Gy IR shows expression of differentially regulated SASP genes analyzed by GSEA. (E) IL-6 and IL-8 protein levels measured using CB bead arrays 10 days following IR. Data are expressed as fold difference between hOB shEV and hOB shRB1 in at least 3 independent experiments. (F) Effect of expression of RB1 and SASP on metastasis-free survival in primary osteosarcoma. High expression of RB1 and SASP profile was associated with better metastasis-free survival than low expression (P = 0.03, Mantel-Cox).