Mechanisms of Normal Tissue Toxicity From Irradiation

Division Of Basic Sciences - Nci

Investigators

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Abstract

In 2013, my laboratory identified that premature senescence in lung contributes to late radiation injury. Since that time, the laboratory has explored this phenomenon in cultured cells, animal models, and human tissue samples to develop a deeper understanding of the mechanisms of radiation injury and to identify potential therapeutics. We have identified that IR-induced senescence in normal tissue stem cells is a key component of radiation injury that results in parenchymal depletion via loss of replicative potential and also caused secondary senescence via elaboration of the senescence associated secretory phenotype (SASP). The SASP is a mixture of cytokines and mitogenic molecules secreted by senescent cells. A number of SASP molecules, such as TGF-beta, VEGF, EGF, IL-6 and IL-1alpha, have been implicated in fibrosis. My laboratory's findings suggested that senescence in irradiated tissue contributes to fibrosis through loss of replicative reserve, enhanced production of mitogenic and pro-inflammatory cytokines, and induction of secondary senescence. The laboratory's efforts have focused on further exploring senescence in radiation injury with the aim of understanding the underlying mechanisms of injury and eventual clinical translation of effective therapies. The general approach has been to target senescence through three mechanisms: 1) targeting SASP family members, 2) preventing senescence after irradiation, and 3) clearing senescent cells. The rationale for this approach is to determine if senescence and members of the SASP are markers or targets (correlated versus causally related), and to clarify further how senescence perpetuates chronic injury. We began exploring candidate genes from the senescence signature used in our initial microarray data set (45 genes), which included genes encoding members of the SASP and genes identified in the AGEMAP database. Using this gene set, we identified candidates for further evaluation based on novelty, evidence of inhibition of senescence in vitro, and supporting scientific rationale. Additional candidates have been identified through analysis of gene expression in mouse and human tissues after radiation exposure. The aging/senescence signature we identified in irradiated fibrotic lung included several SASP molecules not previously implicated in radiation lung injury. We have had promising success with the several targets we have tested, such as PAI-1 and 12-HETE. Our strategy is centered around the careful study of the most activated SASP pathways in our models which may provide important new targets, biomarkers of toxicity, and/or deeper mechanistic understanding. PAI-1. We used a recombinant truncated PAI-1 protein (rPAI-123) a that competitively inhibits endogenous PAI-1 binding on tPA and reduces PAI-1 activity to probe the importance of PAI-1 in radiation fibrosis and senescence. Treatment of mice with rPAI-123 was highly effective in preventing AECII senescence, reducing macrophage accumulation, and preventing lung fibrosis. We have since confirmed increased PAI-1 expression in a cutaneous fibrosis model and in irradiated human lung. 12-HETE (via 12-LOX). We hypothesized that 12-LOX activity after IR enhances the fibrotic response in lung via 12-Hydroxyeicosatetraenoic acid (12-HETE), the product of 12-LOX. Using primary pneumocyte cultures, we described that 12-HETE is capable of directly inducing senescence in AECII in a NOX4 dependent manner. Further, 12-HETE treated pneumocytes secreted increased levels of IL-13 and induced M2 polarization in co-cultured macrophages. ALOX12 deficient mice treated with thoracic IR were resistant to AECII senescence, fibrosis, and M2 macrophage accumulation, emphasizing the importance of 12-LOX in radiation fibrosis and inflammation. The laboratory subsequently demonstrated that 12-LOX is a critical component of accelerated tumor growth in senescent, irradiated tissue. NOX4. Studies in irradiated primary AECII cultures demonstrated that inhibition of NOX4 with diphenylene iodonium (DPI) inhibited superoxide production and prevented IR-induced AECII senescence. Treatment of mice exposed to 5x6 Gy thoracic IR with DPI inhibited senescence of AECII, reduced AECII depletion, and mitigated radiation-induced lung fibrosis. These data support the hypothesis that oxidative stress plays a critical role in radiation injury and AECII senescence in vivo. These data support the concept that AECII senescence and chronic oxidative stress mediated by NOX4 plays an important role in radiation lung injury. mTOR. In collaboration with Jim Mitchell (RBB), we explored mTOR inhibition with rapamycin as a method to prevent AECII senescence and lung injury. The mTOR signaling pathway plays an essential role in aging and senescence. Rapamycin prevents cellular senescence induced by cellular stress as well as oncogene-induced and replicative senescence. Delivery of rapamycin (low dose: 14 mg/kg/day) to C57BL/6NCr mice reduced lung fibrosis and significantly prolonged survival after thoracic IR (6Gy x 5). Treatment with rapamycin inhibited IR-induced signaling downstream of mTOR and reduced expression of pro-fibrotic, immunomodulatory, and senescence associated cytokines in irradiated lungs, including TGF-beta and IL-1beta. Lung tissue of rapamycin treated irradiated mice exhibited a marked reduction in macrophage accumulation, collagen content, and AECII senescence (linked publication). The laboratory later demonstrated that mTOR inhibition (rapamycin, INK-128) was capable of preventing accelerated tumor growth in senescent, irradiated lung. IGF1. Treatment of primary pneumocyte cultures with an IGF-1 neutralizing antibody prevented IR induced senescence. To restrict IGF-1R signaling inhibition to AECII, we crossed C57BL6 mice with AECII restricted CreER expression (Sftpc-CreERT2) to mice with loxP sites flanking exon 3 of the igf1r gene (Igf1rF/F). Treatment of the progeny (Sftpc-CreERT2;Igf1rF/F) with Tamoxifen two weeks prior to IR resulted in no detectable pIGF-1R in AECII in irradiated mouse lung tissue with retained IGF-1R phosphorylation in macrophages. Tamoxifen treated Sftpc-CreERT2;Igf1rF/F mice had significantly reduced collagen accumulation, histologic evidence of fibrosis, AECII senescence, and accumulation of alternatively activated macrophages (both infiltrating: Cd11b+, F4/80+, Cd206+ and residential: Cd11b+, F4/80+, Cd206+) after thoracic IR compared to corn oil treated controls. Conditioned media from irradiated primary pneumocyte cultures derived from Sftpc-CreERT2;Igf1r(F/F) mice treated with Tamoxifen or corn oil demonstrated that irradiated, senescent AECII with intact IGF-1R were capable of inducing M2 polarization in an IL-13 dependent manner, whereas IGF-1R deficient AECII cultures were resistant to radiation-induced senescence and IL-13 production, and incapable of inducing M2 polarization in macrophages. Importantly, the protein expression of p21, IGF-1, IL-13, CD-68, and CD-163 were found to be increased in surgically resected irradiated human lung compared to unirradiated human lung, highlighting the potential relevance of these targets in radiation induced fibrosis in humans. In connected studies, we identified that IL-13Ralpha2 is predominantly expressed in macrophages within irradiated lung and plays a crucial role in CCL2 expression, macrophage polarization, and transforming growth factor-beta expression in response to IL-13. These studies demonstrate an unexpected profibrotic role of IL-13Ralpha2 in RILI and suggest that strategies targeting IL-13Ralpha2 may ameliorate chronic inflammation and fibrosis.