Lupenone regulates LOXL2-mediated PANoptosis signaling through E3 ubiquitin ligases RNF168 to improve radiation-induced lung injury.

Insulin-Like Growth Factor-II Is Increased in Systemic Sclerosis-Associated Pulmonary Fibrosis and Contributes to the Fibrotic Process via Jun N-Terminal Kinase- and Phosphatidylinositol-3 Kinase-Dependent Pathways

Raspberry ketone alleviates radiation-induced lung injury through the STAT2-P2X7r/NLRP3 signaling pathway.

Objective To investigate the risk factors for acute radiation esophagitis andpneumonitis after radiation therapy in esophageal cancer (EC) patients with diabetes or hypertension. Methods A total of 373 EC patients receiving three dimensional conformal radiation therapy (3D-CRT) or intensity modulated radiation therapy (IMRT) were included in this study. Among these patients, 42 showed concurrent with diabetes and 99 with hypertension. Radiation esophagitis or pneumonitis in patients with or without diabetes, and with or without hypertension were monitored in the 1-year follow up, respectively. Results The prevalence of grade 1, 2, 3 and 4 radiation esophagitis in diabetes and non-diabetes patients was 40.5%, 38.1%, 14.3%, 4.8% and 66.2%, 27.8%, 2.7%, 1.8%, respectively, while that of the grade 1, 2 and 3 radiation pneumonitis in diabetes and non-diabetes patients was 31.0%, 16.7%, 9.5% and 30.8%, 15.7%, 1.2%, respectively. The prevalence of grade 3 or above radiation esophagitis and pneumonitis in patients with diabetes and was significantly higher than those with non-diabetes (χ2=13.573, 12.279, P<0.05). The prevalence of grade 1, 2, 3 and 4 radiation esophagitis in hypertension and non-hypertension patients was 49.5%, 38.4%, 8.1%, 3.0% and 68.2%, 25.5%, 2.6%, 1.8%, respectively, while that of the grade 1, 2 and 3 radiation pneumonitis in hypertension and non-hypertension patients were 30.3%, 18.2%, 5.1% and 31.0%, 15.0%, 1.1%, respectively. The prevalence of grade 3 or above radiation esophagitis and pneumonitis in patients with hypertension was significantly higher than those with non-hypertension (χ2=5.695、5.422, P<0.05). Diabetes is an independent risk factor for grade 3 or above acute radiation esophagitis and pneumonitis. Conclusions Diabetes or hypertension might be risk factors for severe radiation esophagitis and pneumonitis in EC patients receiving radiation therapy. Key words: Diabetes; Hypertension; Esophageal cancer; Radiation esophagitis; Radiation pneumonitis

BackgroundLung fibroblasts are involved in extracellular matrix homeostasis, which is mainly regulated by transforming growth factor-beta (TGF-β), and are therefore crucial in lung tissue repair and remodeling. Abnormal repair and remodeling has been observed in lung diseases like COPD. As miRNA levels can be influenced by TGF-β, we hypothesized that TGF-β influences miRNA expression in lung fibroblasts, thereby affecting their function.Materials and methodsWe investigated TGF-β1-induced miRNA expression changes in 9 control primary parenchymal lung fibroblasts using miRNA arrays. TGF-β1-induced miRNA expression changes were validated and replicated in an independent set of lung fibroblasts composted of 10 controls and 15 COPD patients using qRT-PCR. Ago2-immunoprecipitation followed by mRNA expression profiling was used to identify the miRNA-targetomes of unstimulated and TGF-β1-stimulated primary lung fibroblasts (n = 2). The genes affected by TGF-β1-modulated miRNAs were identified by comparing the miRNA targetomes of unstimulated and TGF-β1-stimulated fibroblasts.ResultsTwenty-nine miRNAs were significantly differentially expressed after TGF-β1 stimulation (FDR<0.05). The TGF-β1-induced miR-455-3p and miR-21-3p expression changes were validated and replicated, with in addition, lower miR-455-3p levels in COPD (p<0.05). We identified 964 and 945 genes in the miRNA-targetomes of unstimulated and TGF-β1-stimulated lung fibroblasts, respectively. The TGF-β and Wnt pathways were significantly enriched among the Ago2-IP enriched and predicted targets of miR-455-3p and miR-21-3p. The miR-455-3p target genes HN1, NGF, STRADB, DLD and ANO3 and the miR-21-3p target genes HHEX, CHORDC1 and ZBTB49 were consistently more enriched after TGF-β1 stimulation.ConclusionTwo miRNAs, miR-455-3p and miR-21-3p, were induced by TGF-β1 in lung fibroblasts. The significant Ago2-IP enrichment of targets of these miRNAs related to the TGF-β and/or Wnt pathways (NGF, DLD, HHEX) in TGF-β1-stimulated fibroblasts suggest a role for these miRNAs in lung diseases by affecting lung fibroblast function.

Cigarette smoke alters cell cycle and induces inflammation in lung fibroblasts.

Novel therapies are desperately needed for radiation-induced lung injury (RILI), which, despite aggressive corticosteroid therapy, remains a potentially fatal and dose-limiting complication of thoracic radiotherapy. We assessed the utility of simvastatin, an anti-inflammatory and lung barrier-protective agent, in a dose- and time-dependent murine model of RILI (18-(25 Gy). Simvastatin reduced multiple RILI indices, including vascular leak, leukocyte infiltration, and histological evidence of oxidative stress, while reversing RILI-associated dysregulated gene expression, including p53, nuclear factor-erythroid-2-related factor, and sphingolipid metabolic pathway genes. To identify key regulators of simvastatin-mediated RILI protection, we integrated whole-lung gene expression data obtained from radiated and simvastatin-treated mice with protein-protein interaction network analysis (single-network analysis of proteins). Topological analysis of the gene product interaction network identified eight top-prioritized genes (Ccna2a, Cdc2, fcer1 g, Syk, Vav3, Mmp9, Itgam, Cd44) as regulatory nodes within an activated RILI network. These studies identify the involvement of specific genes and gene networks in RILI pathobiology, and confirm that statins represent a novel strategy to limit RILI.

Subclinical interstitial lung disease: Is it a risk factor for fatal radiation pneumonitis following stereotactic body radiotherapy?

Background: Fibroblasts play a crucial role in extracellular matrix formation in the lung and are regulated by TGF-β. We showed that TGF-β induces miR-455-3p and miR-21-3p in primary lung fibroblasts with lower miR-455-3p levels in COPD. Aim: To identify miR-455-3p and miR-21-3p target genes and their potential function in lung fibroblasts. Methods: The miRNA-targetomes of unstimulated and TGF-β-stimulated primary parenchymal lung fibroblasts (n=2) were identified using Ago2-IP followed by mRNA profiling. We tested for significant enrichment of the miR-455-3p and miR-21-3p predicted targets within the 1500 most enriched transcripts compared to all expressed genes in all four IP experiments. To define the TGF-β-dependent miRNA-target gene interactions, we compared the miRNA target gene lists of the unstimulated with the stimulated fibroblasts. Results: The predicted miR-455-3p and miR-21-3p targets were significantly enriched in the targetomes of unstimulated and stimulated lung fibroblasts (p HN1 , NGF , STRADB , DLD and ANO3 and the miR-21-3p target genes HHEX , CHORDC1 and ZBTB49 were more enriched after TGF-β stimulation. Conclusion: We defined the miRNA-targetomes of primary lung fibroblasts and identified miR-455-3p and miR-21-3p specific targets. Several targets related to the TGF-β and/or Wnt pathways ( NGF , DLD and HHEX ) had a more pronounced enrichment in the TGF-β-stimulated fibroblasts. Thus miR-455-3p and miR-21-3p may influence lung fibroblast function by affecting the TGF-β pathway. This implies a possible role of these miRNAs in lung diseases like COPD.

Triple negative breast cancer (TNBC) carries a poorer prognosis than other breast cancer subtypes due to high metastatic incidence, lack of molecular drug targets, and rapid development of chemoresistance. Cancer-associated fibroblasts (CAFs) are key extrinsic drivers of chemoresistance in primary TNBC tumors, but it is unknown whether resident fibroblasts in distant metastatic tissues also influence TNBC drug sensitivity. Since the lung is a top metastatic site in TNBC, we developed a breast cancer cell (BCC) - primary human donor-derived lung fibroblast (LF) co-culture model to identify therapeutic vulnerabilities and mechanisms of fibroblast-mediated extrinsic chemoresistance in lung-metastatic TNBC. We characterized the growth and chemoresistance phenotypes of BCC co-cultured with primary LFs via bioluminescence and fluorescence imaging and evaluated their transcriptional and secretory profiles using bulk and scRNAseq and multiplex cytokine analysis. Co-culture of human BCC and primary LFs resulted in BCC molecular subtype-dependent changes in cell growth and drug resistance compared with conventional monoculture. Moreover, we also observed increased LF secretion of the immunomodulatory cytokines IL-6 and CCL2 in response to both BCC co-culture and BCC-conditioned media. Transcriptomic analysis of LFs sorted from co-culture identified upregulation of pro-inflammatory and pro-fibrotic iCAF and myCAF-like gene signatures, while co-cultured TNBC cells upregulated interferon response-mediated multi-therapy resistance genes and cellular dormancy signatures. We applied this model in a high throughput screen of 846 kinase inhibitor compounds, which identified reproducible differential BCC responses to kinase inhibitors between monoculture and co-culture conditions. When co-cultured with LFs, BCC were broadly more resistant to kinase inhibitor compounds but showed increased sensitivity to inhibitors of the lipid kinase VPS34, an important initiator of autophagy. We subsequently established that autophagy is increased in co-cultured TNBC cells and contributes to LF-mediated resistance to kinase inhibitors. Moreover, we determined that while paracrine signals from BCC can promote LF activation indicated by enhanced cytokine secretion, cell-cell contact is required for LF modulation of TNBC therapy responses in co-culture. Thus, we conclude that breast cancer cell co-culture with primary lung fibroblasts is a scalable in vitro model that can facilitate therapeutic discovery and mechanistic evaluation of extrinsic modulation of therapy responses in metastatic breast cancer, and further demonstrate that BCC can promote paracrine activation of co-cultured LFs in association with juxtacrine induction of BCC autophagy and extrinsic therapy resistance. Citation Format: Marika L. Klosowski, Kathryn Cronise, Claire Stratton, Qiong Zhou, Hector Esquer, Daniel LaBarbera, Daniel Regan. TNBC cell co-culture with primary human lung fibroblasts identifies a paracrine-juxtacrine signaling loop promoting fibroblast activation and autophagy-dependent breast cancer cell therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB317.

Background: 100% of cancer survivors who undergo thoracic radiation (TR) treatment are at risk of developing radiation-induced lung injury (RILI). Progressive RILI can lead to radiation-induced lung fibrosis (scar) a permanent diagnosis with no FDA-approved curative strategy. 90% of thoracic cancer survivors have CT-detectable RILI every year. Current techniques to limit RILI may make radiotherapy less effective at treating cancer. Our lab was the first to publish metabolomic findings from the exhaled breath condensate (EBC) of patients with diagnosed RILI. EBC is a natural matrix of the respiratory tract that represents the composition of the lung lining fluid. We reported that RILI is associated with global biosynthetic and bioenergetic metabolic defects including increased glycolysis and excess lactate production. Here, we hypothesized that targeting a key glycolytic enzyme, pyruvate kinase M2, will protect against radiation-induced myofibroblast differentiation. Radiation can transform lung fibroblasts into myofibroblasts. Myofibroblasts are the major scar-producing cells in fibrosis and thus the main effectors in the disease. Methods: In this study, primary human lung fibroblasts (pHLFs) were irradiated to induce myofibroblast differentiation and harvested after 5 days. We measured protein expression by western blot, and we performed targeted metabolomics analysis on cell lysates by liquid chromatography/tandem mass spectrometry. Glycolytic rate was measured by Seahorse assay. Results: Radiation markedly increases extracellular lactate (Fig1A). Radiation also induces alpha-smooth muscle actin, (α-SMA – myofibroblast marker), and other key proteins that make up scar (Fig1B). Radiation-induced myofibroblasts accelerate both basal and compensatory glycolysis shown (p = &amp;lt;0.0001) (Fig1C). Targeted metabolomics showed that major glycolytic intermediates accumulate significantly in radiation-induced myofibroblasts compared to non-irradiated controls namely fructose-1,6-bisphosphate (p = &amp;lt;0.0001), phosphoglycerate (p = &amp;lt;0.0001), and phosphoenolpyruvate (p = &amp;lt;0.0001) (Fig1D). Using both a genetic and pharmacologic inhibitor of pyruvate kinase M2, we blunted the final glycolysis reaction before irradiating the pHLFs. This inhibition significantly decreased radiation-induced myofibroblast differentiation (α-SMA, p = &amp;lt;0.0001) (Fig1E). Conclusions: We observed metabolic reprogramming associated with radiation-induced myofibroblast differentiation. Increased glycolysis and diversion to lactate were consistent with the metabolomic signature observed in patients who develop RILI and a mouse model of RILI (not shown here). Importantly, these results are comparable to metabolic reprogramming in cancer (Warburg). Targeting glycolysis disrupted a radiation-induced profibrotic phenotype.

Radiation-induced lung injury (RILI) is one of the most common and fatal complications of thoracic radiotherapy. Inflammatory cell infiltration, imbalance of inflammatory cytokines, and oxidative damage were reported to be involved during RILI pathogenesis, especially in the early phase of RILI. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidative cascades, and regulates life span of mice after administration of thoracic irradiation. We investigated the effects of Nrf2 on RILI and inflammation using Nrf2-knockout, Nrf2-overexpression and wild-type mice with or without 15 Gy ionizing radiation to thorax. Our results showed that Nrf2 deficiency aggravated radiation-induced histopathological changes, macrophage and neutrophil infiltration, serum levels of pro-inflammatory cytokines (IL-6, MCP-1, IFN-γ, TNF, and IL-12p70), and the levels of peroxidation products in the mouse lung. Moreover, loss of Nrf2 reduced radiation-induced serum levels of anti-inflammatory cytokine, IL-10, and antioxidative proteins. Nrf2 overexpression significantly alleviated radiation-induced histopathological changes, macrophages and neutrophils infiltration, serum levels of pro-inflammatory cytokines, and the levels of peroxidation products in lung tissues. Nrf2 overexpression also increased the serum levels of IL-10 and antioxidative proteins. These results indicated that Nrf2 had a protective role against radiation-induced acute lung injury and inflammation, and that antioxidative therapy might be a promising treatment for RILI.

Postradiation White Blood Cell Count Is a Novel Marker of Radiation Pneumonitis in Non-Small Cell Lung Cancer

Chronic obstructive pulmonary disease (COPD) is characterized by the abnormal and chronic lung inflammation. We hypothesized that lung fibroblasts could contribute to the local inflammation and investigated whether low dose theophylline had a beneficial effect on fibroblasts inflammation. Subjects undergoing lobectomy for bronchial carcinoma were enrolled and divided into COPD and control groups according to spirometry. Primary human lung fibroblasts were cultured from peripheral lung tissue distant to tumor tissue. There was a significant increase in both the mRNA expressions and protein levels for IL-6 and IL-8 in fibroblasts in COPD group, and the values were negatively correlated with lung function (P < 0.05). For COPD fibroblasts, the protein levels of IL-6 and IL-8 decreased from 993.0 ± 738.9 pg/mL to 650.1 ± 421.9 pg/mL (P = 0.014) and from 703.1 ± 278.0 pg/mL to 492.0 ± 214.9 pg/mL (P = 0.001), respectively, with 5 μg/mL theophylline treatment. In addition, theophylline at the dose of 5 μg/mL reduced the increased production of IL-6 and IL-8 induced by 1 μg/mL LPS in primary human lung fibroblasts. Our data suggest that lung fibroblasts participate in the chronic inflammation in COPD by releasing IL-6 and IL-8, and low dose theophylline can alleviate the proinflammatory mediators' production by fibroblasts.

PurposeThoracic radiotherapy is a major treatment modality of stage III non-small cell lung cancer. The normal lung tissue is sensitive to radiation and radiation pneumonitis is the most important dose-limiting complication of thoracic radiation therapy. This study was performed to identify the clinical and dosimetric parameters related to the risk of radiation pneumonitis after definitive radiotherapy in stage III non-small cell cancer patients.Materials and MethodsThe medical records were reviewed for 49 patients who completed definitive radiation therapy for locally advanced non-small cell lung cancer from August 2000 to February 2010. Radiation therapy was delivered with the daily dose of 1.8 Gy to 2.0 Gy and the total radiation dose ranged from 50.0 Gy to 70.2 Gy (median, 61.2 Gy). Elective nodal irradiation was delivered at a dose of 45.0 Gy to 50.0 Gy. Seven patients (14.3%) were treated with radiation therapy alone and forty two patients (85.7%) were treated with chemotherapy either sequentially or concurrently.ResultsTwenty-five cases (51.0%) out of 49 cases experienced radiation pneumonitis. According to the radiation pneumonitis grade, 10 (20.4%) were grade 1, 9 (18.4%) were grade 2, 4 (8.2%) were grade 3, and 2 (4.1%) were grade 4. In the univariate analyses, no clinical factors including age, sex, performance status, smoking history, underlying lung disease, tumor location, total radiation dose and chemotherapy were associated with grade ≥2 radiation pneumonitis. In the subgroup analysis of the chemotherapy group, concurrent rather than sequential chemotherapy was significantly related to grade ≥2 radiation pneumonitis comparing sequential chemotherapy. In the univariate analysis with dosimetric factors, mean lung dose (MLD), V20, V30, V40, MLDipsi, V20ipsi, V30ipsi, and V40ipsi were associated with grade ≥2 radiation pneumonitis. In addition, multivariate analysis showed that MLD and V30 were independent predicting factors for grade ≥2 radiation pneumonitis.ConclusionConcurrent chemotherapy, MLD and V30 were statistically significant predictors of grade ≥2 radiation pneumonitis in patients with stage III non-small cell lung cancer undergoing definitive radiotherapy. The cutoff values for MLD and V30 were 16 Gy and 18%, respectively.

Objective To study the related factors of radiation induced lung injury(RILI) after three-dimensional conformal radiotherapy(3D-CRT) in the elderly patients with advance esophageal cancer, thus to provide reference for radiotherapy planning. Methods The clinical data and physical parameters of 72 elderly patients with advance esophageal cancer were analyzed, including gender, age, performance status scoring, smoking history, tumor location, T staging, underlying disease of the lung, radiation dose, the whole lung accepted 5 Gy exposure volume(V5), V10, V15, V20, V25, V30, V40 and mean lung dose(MLD). Results Among 72 patients, 15 cases developed RILI.It was revealed by univariate analysis that there were statistically significant differences in advanced age, underlying disease of the lung, V5, V10, V15, V20, V25, MLD with RILI(χ2=5.098, P=0.026; χ2=3.598, P=0.030; t=3.854, P=0.034; t=4.901, P=0.022, t=4.638, P=0.029, t=5.122, P=0.015, t=3.652, P=0.041, t=5.760, P=0.010). Conclusion It should be payed more attention to factors such as advanced age, underlying disease of the lung, V5, V10, V15, V20, V25 and MLD when elderly patients with advance esophageal cancer patients were treated with 3D-CRT in order to prevent and decrease the risks of RILI. Key words: Esophageal tumor; Radiotherapy, conformal; Lung injury; Aged