Inhibition of TGFß1 activation prevents radiation-induced lung fibrosis.

BACKGROUND: Radiotherapy is the main treatment modality for thoracic tumours, but it may induce pulmonary fibrosis. Currently, the pathogenesis of radiation-induced pulmonary fibrosis (RIPF) is unclear, and effective treatments are lacking. Transforming growth factor beta 1 (TGFß1) plays a central role in RIPF. We found that activated TGFß1 had better performance for radiation pneumonitis (RP) risk prediction by detecting activated and total TGFß1 levels in patient serum. αv integrin plays key roles in TGFß1 activation, but the role of αv integrin-mediated TGFß1 activation in RIPF is unclear. Here, we investigated the role of αv integrin-mediated TGFß1 activation in RIPF and the application of the integrin antagonist cilengitide to prevent RIPF. METHODS: ItgavloxP/loxP ;Pdgfrb-Cre mice were generated by conditionally knocking out Itgav in myofibroblasts, and wild-type mice were treated with cilengitide or placebo. All mice received 16 Gy of radiation or underwent a sham radiation procedure. Lung fibrosis was measured by a modified Ashcroft score and microcomputed tomography (CT). An enzyme-linked immunosorbent assay (ELISA) was used to measure the serum TGFß1 concentration, and total Smad2/3 and p-Smad2/3 levels were determined via Western blotting. RESULTS: Conditional Itgav knockout significantly attenuated RIPF (p < .01). Hounsfield units (HUs) in the lungs were reduced in the knockout mice compared with the control mice (p < .001). Conditional Itgav knockout decreased active TGFß1 secretion and inhibited fibroblast p-Smad2/3 expression. Exogenous active TGFß1, but not latent TGFß1, reversed these reductions. Furthermore, cilengitide treatment elicited similar results and prevented RIPF. CONCLUSIONS: The present study revealed that conditional Itgav knockout and cilengitide treatment both significantly attenuated RIPF in mice by inhibiting αv integrin-mediated TGFß1 activation. HIGHLIGHTS: Activated TGFß1 has a superior capacity in predicting radiation pneumonitis (RP) risk and plays a vital role in the development of radiation-induced pulmonary fibrosis (RIPF). Conditional knock out Itgav in myofibroblasts prevented mice from developing RIPF. Cilengitide alleviated the development of RIPF by inhibiting αv integrin-mediated TGFß1 activation and may be used in targeted approaches for preventing RIPF.

Kinsenoside Protects Against Radiation-Induced Liver Fibrosis via Downregulating Connective Tissue Growth Factor Through TGF-ß1 Signaling.

Radiation-induced liver fibrosis (RILF) is a serious complication of the radiotherapy of liver cancer, which lacks effective prevention and treatment measures. Kinsenoside (KD) is a monomeric glycoside isolated from Anoectochilus roxburghii, which has been reported to show protective effect on the early progression of liver fibrosis. However, the role of KD in affecting RILF remains unknown. Here, we found that KD alleviated RILF via downregulating connective tissue growth factor (CTGF) through TGF-ß1 signaling. Sprague-Dawley rats were administered with 20 mg/kg KD per day for 8 weeks after a single 30Gy irradiation on the right part of liver, and tumor-bearing nude mice were administered with 30 mg/kg KD per day after a single fraction of 10Gy on the tumor inoculation site. Twenty-four weeks postirradiation, we found that the administration of KD after irradiation resulted in decreased expression of α-SMA and fibronectin in the liver tissue while had no adverse effect on the tumor radiotherapy. Besides, KD inhibited the activation of hepatic stellate cells (HSCs) postirradiation via targeting CTGF as indicated by the transcriptome sequencing. Results of the pathway enrichment and immunohistochemistry suggested that KD reduced the expression of TGF-ß1 protein after radiotherapy, and exogenous TGF-ß1 induced HSCs to produce α-SMA and other fibrosis-related proteins. The content of activated TGF-ß1 in the supernatant decreased after treatment with KD. In addition, KD inhibited the expression of the fibrosis-related proteins by regulating the TGF-ß1/Smad/CTGF pathway, resulting in the intervention of liver fibrosis. In conclusion, this study revealed that KD alleviated RILF through the regulation of TGFß1/Smad/CTGF pathway with no side effects on the tumor therapy. KD, in combination with blocking the TGF-ß1 pathway and CTGF molecule or not, may become the innovative and effective treatment for RILF.

Cilengitide, an αvß3-integrin inhibitor, enhances the efficacy of anti-programmed cell death-1 therapy in a murine melanoma model.

Integrins play an important role in multiple stages of tumor progression and metastasis. Previous studies have shown synergistic effects of combined αvß6-integrin and αvß8-integrin inhibitors with immunotherapy. However, the role of αvß3-integrin inhibitor in tumor immunity is still unclear. In this study, we aimed to elucidate the impact of the αvß3-integrin inhibitor on PD-L1 expression and sensitivity to immune checkpoint blockade in melanoma. We investigated the effects of cilengitide, an αvß3-integrin inhibitor, on cell viability and apoptosis of melanoma cell lines. And we explored how cilengitide regulated the expression of PD-L1 in melanoma cells in vitro and in vivo, using immunofluorescence, flow cytometry, Western blotting, and immunohistochemistry. A subcutaneous B16 murine melanoma model was utilized to determine whether combining cilengitide with anti-PD1 therapy inhibited tumor growth and positively regulated tumor microenvironment (TME). Our results showed that cilengitide inhibited cell viability and induced apoptosis in B16 and A375 cell lines. Furthermore, cilengitide decreased PD-L1 expression by reducing STAT3 phosphorylation in two melanoma cell lines. Cilengitide also reduced subcutaneous tumor PD-L1 expression in the B16 murine melanoma model. Accordingly, cilengitide positively regulated antitumor immune responses and provided durable therapy when combined with anti-PD1 monoclonal antibody in the murine melanoma model. This combination therapy reduced tumor growth and extended survival. Our study highlights that cilengitide enhances the efficacy of anti-PD1 therapy and produces a stronger antitumor immune response. This combination therefore represents a novel therapeutic regimen that may improve immunotherapy treratment.

Dexrazoxane ameliorates radiation-induced heart disease in a rat model.

Treatment of thoracic tumors with radiotherapy can lead to severe cardiac injury. We investigated the effects of dexrazoxane, a USFDA-approved cardioprotective drug administered with chemotherapy, on radiation-induced heart disease (RIHD) in a rat model. Male Sprague-Dawley rats were irradiated with a single dose of 20 Gy to the heart and treated with dexrazoxane at the time of irradiation and for 12 subsequent weeks. Dexrazoxane suppressed radiation-induced myocardial apoptosis and significantly reversed changes in serum cardiac troponin I levels and histopathological characteristics six months post-radiation. Treatment with dexrazoxane did not alter the radiosensitivity of thoracic tumors in a tumor formation experiment using male nude Balb/C mice with tumors generated by H292 cells. Dexrazoxane reduced the accumulation of reactive oxygen species in rat cardiac tissues, but not in tumors in nude mice. Transcriptome sequencing showed that IKBKE, MAP3K8, NFKBIA, and TLR5, which are involved in Toll-like receptor signaling, may be associated with the anti-RIHD effects of dexrazoxane. Immunohistochemistry revealed that dexrazoxane significantly decreased NF-κB p65 expression in cardiomyocytes. These findings suggest dexrazoxane may protect against RIHD by suppressing apoptosis and oxidative stress in cardiomyocytes.

A Prognostic Nomogram Model Based on mRNA Expression of DNA Methylation-Driven Genes for Gastric Cancer.

PURPOSE: The exploration and interpretation of DNA methylation-driven genes might contribute to molecular classification, prognostic prediction and therapeutic choice. In this study, we built a prognostic risk model via integrating analysis of the transcriptome and methylation profile for patients with gastric cancer (GC). METHODS: The mRNA expression profiles, DNA methylation profiles and corresponding clinicopathological information of 415 GC patients were downloaded from The Cancer Genome Atlas (TCGA). Differential expression and correlation analysis were performed to identify DNA methylation-driven genes. The candidate genes were selected by univariate Cox regression analyses followed by the least absolute shrinkage and selection operator (LASSO) regression. A prognostic risk nomogram model was then built together with clinicopathological parameters. RESULTS: 5 DNA methylation-driven genes (CXCL3, F5, GNAI1, GAMT and GHR) were identified by integrated analyses and selected to construct the prognostic risk model with clinicopathological parameters. High expression and low DNA hypermethylation of F5, GNAI1, GAMT and GHR, as well as low expression and high DNA hypomethylation of CXCL3 were significantly associated with poor prognosis rates, respectively. The high-risk group showed a significantly shorter prognosis than the low-risk group in the TCGA dataset (HR = 0.212, 95% CI = 0.139-0.322, P = 2e-15). The final nomogram model showed high predictive efficiency and consistency in the training and validation group. CONCLUSION: We construct and validate a prognostic nomogram model for GC based on five DNA methylation-driven genes with high performance and stability. This nomogram model might be a powerful tool for prognosis evaluation in the clinic and also provided novel insights into the epigenetics in GC.

Aerosolized Thyroid Hormone Prevents Radiation Induced Lung Fibrosis.

Radiation induced lung fibrosis (RILF) is a common late complication after radiotherapy without effective treatment. Thyroid hormone (TH) is known to reverse bleomycin-induced pulmonary fibrosis in recent study. We therefore sought to examine TH effect in RILF. Aerosolized TH delivery prevented pulmonary fibrosis according to either micro-computed tomography scans or histological evaluations, without significant changes in serum THs in a murine model of RILF by attenuating TGF-ß1 and phosphorylated Smad2/3 expressions and reducing the accumulation of M2-like macrophages. Furthermore, hypothyroidism was significantly correlated with RILF in a retrospectively analyzed data from nasopharyngeal carcinoma patients treated by intensity-modulated radiation therapy with a median follow-up time of 25.5 months. Together, aerosolized TH may prevent RILF by inhibiting the TGF-ß1/SMADs signaling pathway.

IEO model: A novel concept describing the complete metastatic process in the liver microenvironment.

Metastasis is a characteristic behavior of malignant tumor cells. It is determined by the mutual interaction between primary tumor cells and the state of the microenvironment at sites of metastasis, particularly the liver, bone, lungs and brain. In the present review, a novel pattern is defined and termed the IEO model (prI-, prE- and pOst-metastatic niche), for the hepatic metastatic microenvironment which characterizes the complete metastatic process. In the IEO model, the components of the hepatic metastatic niche, including the extracellular matrix, hepatocytes, mesenchymal cells, Kupffer cells, hepatic sinusoidal endothelial cells, hepatic stellate cells and immunocytes are continually remodelled by tumor cells to form various microenvironments during different stages of hepatic metastasis. The IEO model explains the plasticity of the hepatic microenvironment and provides novel insights into the role of different stages of the metastatic niche. This novel concept may provide a basis for advances in theoretical cancer research and for improvements in the complete course management of malignant tumors.

The Intestinal Microbiota Plays as a Protective Regulator Against Radiation Pneumonitis.

Radiation pneumonitis is a common complication of thoracic irradiation for lung cancer patients. The healthy gut microbiota plays an important role in the local mucosal defense process as well as pulmonary immunomodulation of the host. However, the effect of the intestinal microbiota on radiation pneumonitis is not well understood. Here we studied how the intestinal microbiota affected the host response to radiation pneumonitis. C57BL/6 mice were administered antibiotics to induce disequilibrium in the gut microbiota, and subsequently irradiated. We found that the intestinal microbiota served as a protective mediator against radiation pneumonitis, as indicated by decreased body weight and increased mortality in antibiotic-treated mice. In mice with gut microbiota disequilibrium, more serious pathological lung damage was observed at two and four weeks postirradiation. Fecal microbiota transplantation into irradiated mice led to improvement from radiation-induced inflammation two weeks postirradiation. High-throughput sequencing of murine feces displayed conversion of flora diversity, bacterial composition and community structure in the absence of normal intestinal flora. We filtered the potentially important species among the gut microbiota and considered that the tissue-type plasminogen activator might be involved in the inflammatory process. This study reveals that the gut microbiota functions as a protective regulator against radiation pneumonitis. Additionally, fecal microbiota transplantation was shown to alleviate lung injury in the irradiated model. The protective role of the healthy gut microbiota and the utilization of the gut-lung axis show potential for innovative therapeutic strategies in radiation-induced lung injury.

Validation study of the association between genetic variant of IL4 and severe radiation pneumonitis in lung cancer patients treated with radiation therapy.

BACKGROUND AND PURPOSE: Recent researches demonstrated that single nucleotide polymorphisms (SNPs) of genes involving inflammation, DNA repair, etc. were associated with risk of radiation pneumonitis (RP). However, these studies were single-centered, from single ethnic origin, without validation from independent cohort studies from other populations. In order to identify clinical valuable SNPs for RP, in this study we selected 19 RP-related SNPs candidates previously published before 2016 for validation in our cohort. MATERIAL AND METHODS: 359 lung cancer patients with radiotherapy were included in our prospective study (NCT02490319). Peripheral blood samples from these patients were genotyped by MassArray and Sanger Sequence method. Multivariate Cox hazard and other analyses were applied to estimate the hazard ratio (HR) and 95% confidence intervals (CIs) of all factors possibly related to the risk of RP. RESULTS: Patients with elder age, MLD ≥15 Gy, V20 ≥24% had higher risk of RP ≥grade 3 compared with their counterparts (HR = 2.020, 95% CI: 1.045-3.906, P = 0.037; HR = 2.502, 95% CI: 1.346-4.652, P = 0.004; HR = 2.256, 95% CI: 1.191-4.272, P = 0.013, respectively). Moreover, patients receiving IMRT were associated with decreased incidence of RP (HR = 0.520, 95% CI: 0.280-0.963, P = 0.037). Importantly, CT + TT genotype of IL4: rs2243250 was strongly related to decreased risk of RP ≥grade 3 (HR = 0.195, 95% CI: 0.090-0.424, P = 0.000037, Pc = 0.0006). CONCLUSION: IL4: rs2243250 was validated to be significantly related to RP of grade ≥3 in our cohort. Our results further emphasized the prevalence and clinical value of IL4: rs2243250 on RP, and may thus be one of the important predictors of severe RP before radiotherapy.

Phosphorylated-Akt overexpression is associated with a higher risk of brain metastasis in patients with non-small cell lung cancer.

Brain metastasis (BM) of non-small cell lung cancer (NSCLC) is relatively common and has a poor prognosis. Moreover, identifying which patients are more likely to develop BM is challenging. Akt, a serine/threonine-specific protein kinase, can be activated in various tumors, including lung cancer, and may be associated with poor prognosis. Here, we used immunohistochemistry to evaluate phosphorylated-Akt (p-Akt) expression in tumor tissues of 99 NSCLC patients. We also analyzed the genotype of the patients for two single nucleotide polymorphisms (SNPs) of the AKT1 gene, rs2498804 and rs2494732. We found that p-Akt expression differs between NSCLC patients and correlates with the risk of BM. Indeed, patients exhibiting medium to high p-Akt expression had a higher incidence of BM than those exhibiting low to no p-Akt expression (39% vs 16%). Our data also show that patients with the rs2498804 GT/GG and rs2494732 CT/TT variant genotypes were more likely to exhibit higher levels of p-Akt expression than those with the rs2498804 TT and rs2494732 CC variant genotypes (35% vs. 24% and 37% vs. 25%, respectively). Our results suggest that the level of expression of p-Akt, which may be affected by the AKT1 genotype, is correlated with the risk of BM. However, further studies are needed to establish p-Akt as a predictive marker for BM in NSCLC patients.

Antifibrotic Agent Pirfenidone Protects against Development of Radiation-Induced Pulmonary Fibrosis in a Murine Model.

Radiation-induced complications of the respiratory system are a common side effect of thoracic radiotherapy with no viable treatment option. Here, we investigated the potential therapeutic effect of the orphan drug pirfenidone for treating radiation-induced pulmonary fibrosis. C57BL/6 mice received a single fraction of 16 Gy to the thorax and were subsequently treated with 300 mg/kg/day pirfenidone for four weeks. Survival and body weight of the mice were quantified. Micro-CT in vivo lung imaging was performed to dynamically observe the developmental process of pulmonary fibrosis. The lungs were excised at the end of the experiment and evaluated for histological changes. Compared to the irradiated mice that received no pirfenidone, mice treated with pirfenidone after irradiation had an extended median survival time (>140 days vs. 73 days, P < 0.01). The accumulation of collagen and fibrosis in lung tissues after irradiation was decreased with pirfenidone treatment. Pirfenidone also reduced the expression of TGF-ß1 and phosphorylation of Smad3 in lung tissues. The dose level of Pirfenidone used in this study attenuated pulmonary fibrosis and prolonged the life span of irradiated mice. It may offer a promising approach to treat or minimize radiation-induced pulmonary fibrosis.

Irradiated Human Umbilical Vein Endothelial Cells Undergo Endothelial-Mesenchymal Transition via the Snail/miR-199a-5p Axis to Promote the Differentiation of Fibroblasts into Myofibroblasts.

Radiation induced pulmonary fibrosis (RIPF) is one of the major side effects of radiotherapy for lung cancer. Previous studies have shown that endothelial cells and activated myofibroblasts play a key role in RIPF. However, the interaction between irradiated endothelial cells and activation of myofibroblasts has not been reported. The aim of the present study was to examine whether irradiated endothelial cells would affect the differentiation of fibroblasts into myofibroblasts in the process of RIPF. In the current study, we used a coculture system that allowed direct contact between human fetal lung fibroblasts (MRC-5) and irradiated human umbilical vein endothelial cells (HUVECs). After 24 or 48 h, cells were sorted by flow cytometry. Radiation induced endothelial-mesenchymal transition (EndMT) by significantly increasing the expression of Snail and vimentin and reducing the expression of CD31 in HUVECs. In addition, irradiation of HUVECs induced the expression of collagen type I and α-smooth muscle actin (α-SMA) in MRC-5 cells. Further investigation indicated that irradiation of HUVECs induced the differentiation of fibroblasts into myofibroblasts through the Snail/miR-199a-5p axis. We conclude that irradiated endothelial cells undergo EndMT to promote differentiation of fibroblasts into myofibroblasts via the Snail/miR-199a-5p axis.

Genetic variants in the plasminogen activator inhibitor-1 gene are associated with an increased risk of radiation pneumonitis in lung cancer patients.

Plasminogen activator inhibitor-1 (PAI-1) plays a crucial role in the process of lung injury, although its association with radiation pneumonitis (RP) is unclear. We hypothesized that genetic variants in PAI-1 may influence the risk of RP. In this study, 169 lung cancer patients were genotyped for six single-nucleotide polymorphisms in PAI-1 using the Sequenom MassARRAY system. The risk of RP was evaluated by Cox proportional hazards analyses. The cumulative RP probabilities by genotype were assessed using Kaplan-Meier analyses. Univariate and multivariate analyses revealed that PAI-1:rs7242 GT/GG was correlated with an increased occurrence of grade ≥3 RP (crude hazard ratio = 3.331; 95% confidence interval, 1.168-9.497; P = 0.024). Our results indicated that PAI-1:rs7242 in the 3'-untranslated region of PAI-1 can be a predictor of grade ≥3 RP before radiotherapy.

MMP-1 promoter polymorphism is associated with risk of radiation-induced lung injury in lung cancer patients treated with radiotherapy.

Matrix metalloproteinase-1 (MMP-1) has been implicated in several inflammatory and fibrotic diseases. We hypothesized that genetic variations in the MMP1 promoter region are associated with risk of radiation-induced lung injury (RILI). A cohort of 251 lung cancer patients was genotyped for five single nucleotide polymorphisms in the MMP1 promoter region. We found that rs1144393 AG/GG was strongly correlated with an increased occurrence of grade ≥ 2 RILI (p = 0.002). Additionally, patients with the rs1144393 AG/GG genotypes exhibited higher MMP-1 expression than patients with the AA genotype in lung tissues (n = 28, p = 0.022) and plasma samples (n = 40, p = 0.018). Our results indicated that rs1144393 in the MMP1 promoter region can be a predictor of grade ≥ 2 RILI in lung cancer patients treated with thoracic radiation.

Genetic variants in the ITGB6 gene is associated with the risk of radiation pneumonitis in lung cancer patients treated with thoracic radiation therapy.

The αvß6 integrin is a cell surface adhesion molecule that plays critical roles in the injury and inflammation processes. We hypothesized that polymorphisms in αvß6 integrin genes (ITGAV and ITGB6) might be associated with the risk of radiation pneumonitis (RP) in lung cancer patients treated with thoracic radiation. We genotyped three potentially functional αvß6 integrin single-nucleotide polymorphisms (rs1839123 and rs4129787 in ITGAV, and rs4665162 in ITGB6) and found that the genotypes of rs4665162 single-nucleotide polymorphisms were predictors of RP development (for grade ≥2 RP, HR = 1.505, 95 % CI 1.069-2.119, P = 0.019; for grade ≥3 RP, HR = 3.006, 95 % CI 1.431-6.313, P = 0.004). Our results showed that AG/GG genotypes of ITGB6 rs4665162 gene were associated with a higher risk of RP in patients with lung cancer receiving radiotherapy and thus may serve as a reliable predictor of RP. Further studies will be needed to confirm these findings.