GDF15 Contributes to Radioresistance by Mediating the EMT and Stemness of Breast Cancer Cells.

Radiotherapy is one of the conventional methods for the clinical treatment of breast cancer. However, radioresistance has an adverse effect on the prognosis of breast cancer patients after radiotherapy. In this study, using bioinformatic analysis of GSE59732 and GSE59733 datasets in the Gene Expression Omnibus (GEO) database together with the prognosis database of breast cancer patients after radiotherapy, the GDF15 gene was screened out to be related to the poor prognosis of breast cancer after radiotherapy. Compared with radiosensitive parental breast cancer cells, breast cancer cells with acquired radioresistance exhibited a high level of GDF15 expression and enhanced epithelial-to-mesenchymal transition (EMT) properties of migration and invasion, as well as obvious stem-like traits, including the increases of mammosphere formation ability, the proportion of stem cells (CD44+ CD24- cells), and the expressions of stem cell-related markers (SOX2, NANOG). Moreover, knockdown of GDF15 sensitized the radioresistance cells to irradiation and significantly inhibited their EMT and stem-like traits, indicating that GDF15 promoted the radioresistance of breast cancer by enhancing the properties of EMT and stemness. Conclusively, GDF15 may be applicable as a novel prognosis-related biomarker and a potential therapeutic target for breast cancer radiotherapy.

Irradiation combined with PD-L1-/- and autophagy inhibition enhances the antitumor effect of lung cancer via cGAS-STING-mediated T cell activation.

Radiotherapy combined with immune checkpoint blockade has gradually revealed the superiority in the antitumor therapy; however, the contribution of host PD-L1 remains elusive. In this study, we found that the activation of CD8+ T cells was strikingly increased in both irradiated PD-L1-expressing primary tumor and distant non-irradiated syngeneic tumor in PD-L1-deficient mouse host, and thus enhanced radiation-induced antitumor abscopal effect (ATAE) by activating cGAS-STING pathway. Notably, the autophagy inhibitors distinctively promoted dsDNA aggregation in the cytoplasm and increased the release of cGAS-STING-regulated IFN-ß from irradiated cells, which further activated bystander CD8+ T cells to release IFN-γ and contributed to ATAE. These findings revealed a signaling cascade loop that the cytokines released from irradiated tumor recruit CD8+ T cells that in turn act on the tumor cells with amplified immune responses in PD-L1-deficient host, indicating a potential sandwich therapy strategy of RT combined with PD-L1 blockage and autophagy inhibition.

MMP14 Contributes to HDAC Inhibition-Induced Radiosensitization of Glioblastoma.

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Radiotherapy has long been an important treatment method of GBM. However, the intrinsic radioresistance of GBM cells is a key reason of poor therapeutic efficiency. Recently, many studies have shown that using the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) in radiotherapy may improve the prognosis of GBM patients, but the underlying molecular mechanisms remain unclear. In this study, Gene Expression Omnibus (GEO) datasets GSE153982 and GSE131956 were analyzed to evaluate radiation-induced changes of gene expression in GBM without or with SAHA treatment, respectively. Additionally, the survival-associated genes of GBM patients were screened using the Chinese Glioma Genome Atlas (CGGA) database. Taking the intersection of these three datasets, 11 survival-associated genes were discovered to be activated by irradiation and regulated by SAHA. The expressions of these genes were further verified in human GBM cell lines U251, T98G, and U251 homologous radioresistant cells (U251R) by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). It was found that MMP14 mRNA was considerably highly expressed in the radioresistant cell lines and was reduced by SAHA treatment. Transfection of MMP14 siRNA (siMMP14) suppressed cell survivals of these GBM cells after irradiation. Taken together, our results reveal for the first time that the MMP14 gene contributed to SAHA-induced radiosensitization of GBM.

Fractionated Irradiation of Right Thorax Induces Abscopal Damage on Bone Marrow Cells via TNF-α and SAA.

Radiation-induced abscopal effect (RIAE) outside of radiation field is becoming more attractive. However, the underlying mechanisms are still obscure. This work investigated the deleterious effect of thoracic irradiation (Th-IR) on distant bone marrow and associated signaling factors by irradiating the right thorax of mice with fractionated doses (8 Gy × 3). It was found that this localized Th-IR increased apoptosis of bone marrow cells and micronucleus formation of bone marrow polychromatic erythrocytes after irradiation. Tandem mass tagging (TMT) analysis and ELISA assay showed that the concentrations of TNF-α and serum amyloid A (SAA) in the mice were significantly increased after Th-IR. An immunohistochemistry assay revealed a robust increase in SAA expression in the liver rather than in the lungs after Th-IR. In vitro experiments demonstrated that TNF-α induced SAA expression in mouse hepatoma Hepa1-6 cells, and these two signaling factors induced DNA damage in bone marrow mesenchymal stem cells (BMSCs) by increasing reactive oxygen species (ROS). On the other hand, injection with TNF-α inhibitor before Th-IR reduced the secretion of SAA and attenuated the abscopal damage in bone marrow. ROS scavenger NAC could also mitigated Th-IR/SAA-induced bone marrow damage in mice. Our findings indicated that Th-IR triggered TNF-α release from lung, which further promoted SAA secretion from liver in a manner of cascade reaction. Consequently, these signaling factors resulted in induction of abscopal damage on bone marrow of mice.

LACTB2 renders radioresistance by activating PINK1/Parkin-dependent mitophagy in nasopharyngeal carcinoma.

Radiotherapy is a standard and conventional treatment strategy for nasopharyngeal carcinoma (NPC); however, radioresistance remains refractory to clinical outcomes. Understanding the molecular mechanism of radioresistance is crucial for advancing the efficacy of radiotherapy and improving the prognosis of NPC. In this study, ß-lactamase-like-protein 2 (LACTB2) was identified as a potential biomarker for radioresistance using tandem mass tag proteomic analysis of NPC cells, gene chip analysis of NPC tissues, and differential gene analysis between NPC and normal nasopharyngeal tissues from the Gene Expression Omnibus database GSE68799. Meanwhile, LACTB2 levels were elevated in the serum of patients with NPC after radiotherapy. Inhibiting LACTB2 levels and mitophagy can sensitize NPC cells to ionizing radiation. In NPC cells, LACTB2 was augmented at the transcription and protein levels after radiation rather than nucleus-cytoplasm-mitochondria transposition to activate PTEN-induced kinase 1 (PINK1) and mitophagy. In addition, LACTB2 was first authenticated to co-locate with PINK1 by interacting with its N-terminal domain. Together, our findings indicate that overexpressed LACTB2 provoked PINK1-dependent mitophagy to promote radioresistance and thus might serve as a prognostic biomarker for NPC radiotherapy.

Macrophage contributes to radiation-induced anti-tumor abscopal effect on transplanted breast cancer by HMGB1/TNF-α signaling factors.

Objectives: The roles of innate immunity including macrophages in radiation-induced abscopal effect (RIAE) are ambiguous. In this study, we evaluated the role of macrophage in RIAE and the interaction of cytokines in tumor microenvironment after irradiation. Materials and Methods: Transplanted tumor of breast cancer cells in BalB/C mice, severe combined immunodeficiency (SCID) mice and non-obese diabetic (NOD)-SCID mice were irradiated with fractionation doses to observe anti-tumor abscopal effect. The underlying mechanism of RIAE was investigated by treating the mice with TNF-α inhibitor or macrophage depletion drug and analyzing the alteration of macrophage distribution in tumors. A co-culture system of breast cancer cells and macrophages was applied to disclose the signaling factors and related pathways involved in the RIAE. Results: The growth of nonirradiated tumor was effectively suppressed in mice with normal or infused macrophages but not in mice with insufficiency/depletion of macrophage or TNF-α inhibition, where M1-macrophage was mainly involved. Investigation of the bystander signaling factors in vitro demonstrated that HMGB1 released from irradiated breast cancer cells promoted bystander macrophages to secret TNF-α through TLR-4 pathway and further inhibited the proliferation and migration of non-irradiated cancer cells by PI3K-p110γ suppression. Conclusions: HMGB1 and TNF-α contributes to M1-macrophages facilitated systemic anti-tumor abscopal response triggered by radiotherapy in breast cancer, indicating that the combination of immunotherapy and radiotherapy may has important implication in enhancing the efficiency of tumor treatment.

Radiation-induced abscopal reproductive effect is driven by TNF-α/p38 MAPK/Rac1 axis in Sertoli cells.

Rationale: Radiotherapy has become a mainstay for tumor management, and more than 50% of patients with thoracic tumor need to be treated with radiotherapy. However, the potential adverse effects of thoracic radiotherapy on the reproductive system remain elusive. Methods: Western blot analysis, immunofluorescence assay and transmission electron microscopy (TEM) analysis were performed to investigate the integrity of blood-testis barrier (BTB) in male mice after hypofractionated irradiation (IR) on the right thorax. RNA sequencing, co-immunoprecipitation (IP), Duolink PLA and inhibitor experiments were carried out to demonstrate the molecular mechanisms of the BTB dynamics changes and the subsequent reproductive effect. Results: It was found that the hypofractionated IR on right thorax evoked ultrastructural destruction in distant testes, and thus caused radiation-induced abscopal reproductive effect (RIARE) in male mice. Mechanistically, thoracic IR induced significant nuclear translocation of Rac Family Small GTPase 1 (Rac1) in abscopal Sertoli cells, which closely correlated with the activation of TNF-α/p38 mitogen activated protein kinase (MAPK) pathway. Of note, YWHAZ, a critical polarity protein, was found to be co-localized with Rac1 in Sertoli cells, and this interaction was indispensable for thoracic IR-induced Rac1 nuclear translocation and subsequent degradation of BTB-associated proteins. Conclusions: Our findings imply for the first time that YWHAZ-mediated Rac1 nuclear translocation plays central roles in RIARE, and TNF-α/p38 MAPK/Rac1 axis can be employed as a therapeutic target against RIARE for young male patients receiving hypofractionated radiotherapy.

CD81 Enhances Radioresistance of Glioblastoma by Promoting Nuclear Translocation of Rad51.

Glioblastoma (GBM) is the most common type of primary tumor in central nervous system in adult with a 5-year survival rate of ≤5%. Despite of recent advances in tumor radiotherapy, the prognosis of GBM remains to be dismal due to radioresistance. In this study, we identified CD81 as a potential biomarker of GBM radioresistance with the analysis of upregulated genes in human glioma radioresistant cell lines U251R and T98G in comparison with U251 cells. In vitro and in vivo experiments demonstrated that suppressing CD81 by siRNA/shRNA enhanced radiation-induced cell killing and DNA damage of γ-H2AX formation, and delayed tumor xenograft growth of GBM. Mechanistically, we found that knockdown of CD81 significantly decreased radiation-induced expression of nuclear Rad51, a key protein involved in homologous recombination repair (HRR) of DNA, suggesting that CD81 is essential for DNA damage response. Meanwhile, when the cells were treated with B02, a Rad51 inhibitor, silencing CD81 would not sensitize GBM cells to radiation, which further illustrates that Rad51 acts as an effector protein of CD81 in tumor radioresistance. Dual immunofluorescence staining of CD81 and Rad51 illustrated that nuclear membrane CD81 contributed to the nuclear transport of Rad51 after irradiation. In conclusion, we demonstrated for the first time that CD81 not only played a vital role in DNA repair through regulating Rad51 nuclear transport, but also might serve as a potential target of GBM radiotherapy.

TIGAR/AP-1 axis accelerates the division of Lgr5- reserve intestinal stem cells to reestablish intestinal architecture after lethal radiation.

During radiologic or nuclear accidents, high-dose ionizing radiation (IR) can cause gastrointestinal syndrome (GIS), a deadly disorder that urgently needs effective therapy. Unfortunately, current treatments based on natural products and antioxidants have shown very limited effects in alleviating deadly GIS. Reserve intestinal stem cells (ISCs) and secretory progenitor cells are both reported to replenish damaged cells and contribute to crypt regeneration. However, the suppressed ß-catenin/c-MYC axis within these slow-cycling cells leads to limited regenerative response to restore intestinal integrity during fatal accidental injury. Current study demonstrates that post-IR overexpression of TIGAR, a critical downstream target of c-MYC in mouse intestine, mounts a hyperplastic response in Bmi1-creERT+ reserve ISCs, and thus rescues mice from lethal IR exposure. Critically, by eliminating damaging reactive oxygen species (ROS) yet retaining the proliferative ROS signals, TIGAR-overexpression enhances the activity of activator protein 1, which is indispensable for initiating reserve-ISC division after lethal radiation. In addition, it is identified that TIGAR-induction exclusively gears the Lgr5- subpopulation of reserve ISCs to regenerate crypts, and intestinal TIGAR-overexpression displays equivalent intestinal reconstruction to reserve-ISC-restricted TIGAR-induction. Our findings imply that precise administrations toward Lgr5- reserve ISCs are promising strategies for unpredictable lethal injury, and TIGAR can be employed as a therapeutic target for unexpected radiation-induced GIS.

CLIC4 regulates radioresistance of nasopharyngeal carcinoma by iNOS after γ-rays but not carbon ions irradiation.

Nasopharyngeal carcinoma (NPC) is a major health problem in the East and Southeast Asia, and the intensity modulated radiotherapy (IMRT) is the current preferred treatment method of NPC, but radioresistance-induced residual and recurrent tumors are the main cause of treatment failure. Till now, the mechanism of radioresistance and prognostic biomarkers of NPC are still unrevealed. In this study, we collected clinical NPC samples and established radioresistant NPC-R cell lines by irradiating NPC cells with fractionation doses of γ-rays. Using genechip assay between radioresistance and radiosensitive clinical samples and TMT assay between NPC and NPC-R cells, differential expressed genes were examined and the potential biomarker of radioresistance was screened. Immunohistochemical assay of NPC clinical specimens showed that CLIC4 was significantly up-regulated in radioresistance tumor tissues. In vitro studies confirmed that up-regulation of CLIC4 gene enhanced radioresistance in comparison with the alterations of intracellular oxidative metabolism of reactive oxygen species (ROS) and nitric oxide (NO) in an opposite way. Correspondingly, inhibition of CLIC4 sensitized NPC cells to irradiation and decreased nuclear translocation of iNOS and intracellular level of NO in NPC cells. Interestingly, the capacity for DNA repair had no difference between NPC and NPC-R cells. Moreover, because of great interests in using carbon ion irradiation to treat NPC effectively, we demonstrated that, after carbon ion irradiation, NPC-R and NPC cells had similar survival even under the status of up- or down-regulation of CLIC4. Conclusively, CLIC4 contributes to radioresistance of NPC to γ-rays but not carbon ions by regulating intracellular oxidative metabolism of nuclear translocation of iNOS.

ANXA6 Contributes to Radioresistance by Promoting Autophagy via Inhibiting the PI3K/AKT/mTOR Signaling Pathway in Nasopharyngeal Carcinoma.

Radiotherapy is a conventional and effective treatment method for nasopharyngeal carcinoma (NPC), although it can fail, mainly because radioresistance results in residual or recurrent tumors. However, the mechanisms and predictive markers of NPC radioresistance are still obscure. In this study, we identified Annexin A6 (ANXA6) as a candidate radioresistance marker by using Tandem Mass Tag quantitative proteomic analysis of NPC cells and gene chip analysis of NPC clinical samples with different radiosensitivities. It was observed that a high expression level of ANXA6 was positively correlated with radioresistance of NPC and that inhibition of ANXA6 by siRNA increased the radiosensitivity. The incidence of autophagy was enhanced in the established radioresistant NPC cells in comparison with their parent cells, and silencing autophagy with LC3 siRNA (siLC3) sensitized NPC cells to irradiation. Furthermore, ANXA6 siRNA (siANXA6) suppressed cellular autophagy by activating the PI3K/AKT/mTOR pathway, ultimately leading to radiosensitization. The combination of siANXA6 and CAL101 (an inhibitor of PI3K, p-AKT, and mTOR, concurrently) significantly reversed the above siANAX6-reduced autophagy. Suppression of PI3K/AKT/mTOR by CAL101 also increased the expression of ANXA6 in a negative feedback process. In conclusion, this study revealed for the first time that ANXA6 could promote autophagy by inhibiting the PI3K/AKT/mTOR pathway and that it thus contributes to radioresistance of NPC. The significance of this is that ANXA6 could be applied as a new predictive biomarker of NPC prognosis after radiotherapy.