The interplay between autophagy and cGAS-STING signaling and its implications for cancer.

Autophagy is an intracellular process that targets various cargos for degradation, including members of the cGAS-STING signaling cascade. cGAS-STING senses cytosolic double-stranded DNA and triggers an innate immune response through type I interferons. Emerging evidence suggests that autophagy plays a crucial role in regulating and fine-tuning cGAS-STING signaling. Reciprocally, cGAS-STING pathway members can actively induce canonical as well as various non-canonical forms of autophagy, establishing a regulatory network of feedback mechanisms that alter both the cGAS-STING and the autophagic pathway. The crosstalk between autophagy and the cGAS-STING pathway impacts a wide variety of cellular processes such as protection against pathogenic infections as well as signaling in neurodegenerative disease, autoinflammatory disease and cancer. Here we provide a comprehensive overview of the mechanisms involved in autophagy and cGAS-STING signaling, with a specific focus on the interactions between the two pathways and their importance for cancer.

Cellular and Molecular Biological Alterations after Photon, Proton, and Carbon Ions Irradiation in Human Chondrosarcoma Cells Linked with High-Quality Physics Data.

Chondrosarcomas are particularly difficult to treat due to their resistance to chemotherapy and radiotherapy. However, particle therapy can enhance local control and patient survival rates. To improve our understanding of the basic cellular radiation response, as a function of dose and linear energy transfer (LET), we developed a novel water phantom-based setup for cell culture experiments and characterized it dosimetrically. In a direct comparison, human chondrosarcoma cell lines were analyzed with regard to their viability, cell proliferation, cell cycle, and DNA repair behavior after irradiation with X-ray, proton, and carbon ions. Our results clearly showed that cell viability and proliferation were inhibited according to the increasing ionization density, i.e., LET, of the irradiation modes. Furthermore, a prominent G2/M arrest was shown. Gene expression profiling proved the upregulation of the senescence genes CDKN1A (p21), CDKN2A (p16NK4a), BMI1, and FOXO4 after particle irradiation. Both proton or C-ion irradiation caused a positive regulation of the repair genes ATM, NBN, ATXR, and XPC, and a highly significant increase in XRCC1/2/3, ERCC1, XPC, and PCNA expression, with C-ions appearing to activate DNA repair mechanisms more effectively. The link between the physical data and the cellular responses is an important contribution to the improvement of the treatment system.

Ganetespib selectively sensitizes cancer cells for proximal and distal spread-out Bragg peak proton irradiation.

OBJECTIVE: Hypersensitivity towards proton versus photon irradiation was demonstrated in homologous recombination repair (HRR)-deficient cell lines. Hence, combined treatment concepts targeting HRR provide a rational for potential pharmaceutical exploitation. The HSP90 inhibitor ganetespib (STA-9090) downregulates a multitude of HRR-associated proteins and sensitizes for certain chemotherapeutics. Thus, the radiosensitizing effect of HSP90-inhibiting ganetespib was investigated for reference photon irradiation and proton irradiation at a proximal and distal position in a spread-out Bragg peak (SOBP). METHODS: A549 and FaDu cells were treated with low-dose (2 nM resp. 1 nM) ganetespib and irradiated with 200 kV photons. Proton irradiation was performed at a proximal and a distal position within a SOBP, with corresponding dose-averaged linear-energy transfer (LETD) values of 2.1 and 4.5 keV/µm, respectively. Cellular survival data was fitted to the linear-quadratic model to calculate relative biological effectiveness (RBE) and the dose-modifying factor (DMF). Additionally, A549 cells were treated with increasing doses of ganetespib and investigated by flow cytometry, immunoblotting, and immunofluorescence microscopy to investigate cell cycle distribution, Rad51 protein levels, and γH2AX foci, respectively. RESULTS: Low-dosed ganetespib significantly sensitized both cancer cell lines exclusively for proton irradiation at both investigated LETD, resulting in increased RBE values of 10-40%. In comparison to photon irradiation, the fraction of cells in S/G2/M phase was elevated in response to proton irradiation with 10 nM ganetespib consistently reducing this population. No changes in cell cycle distribution were detected in unirradiated cells by ganetespib alone. Protein levels of Rad51 are downregulated in irradiated A549 cells by 10 nM and also 2 nM ganetespib within 24 h. Immunofluorescence staining demonstrated similar induction and removal of γH2AX foci, irrespective of irradiation type or ganetespib administration. CONCLUSION: Our findings illustrate a proton-specific sensitizing effect of low-dosed ganetespib in both employed cell lines and at both investigated SOBP positions. We provide additional experimental data on cellular response and a rational for future combinatorial approaches with proton radiotherapy.

An external perpendicular magnetic field does not influence survival and DNA damage after proton and carbon ion irradiation in human cancer cells.

BACKGROUND AND PURPOSE: Magnetic field effects on the radiobiological effectiveness during treatment of magnetic resonance (MRI) guided particle therapy are being debated. This study aims at assessing the influence of a perpendicular magnetic field on the biological effects in two human cancer cell lines irradiated with proton or carbon ions. METHODS AND MATERIALS: In vitro cell irradiations were performed in water inside a perpendicular magnetic field of 0 and 1T for both protons and carbon ions. Samples were located in the center of a spread-out Bragg peak at 8cm water equivalent depth with a dose averaged linear energy transfer (LETd) of 4.2 or 83.4keV/µm for protons and carbon ions, respectively. Physical dose levels of 0, 0.5, 1, 2, 4 and 6Gy were employed. The irradiation field was shifted and laterally enlarged, to compensate for the beam deflection due to the magnetic field and ensure consistent and homogenous irradiations of the flasks. The human cancer cell lines SKMel (Melanoma) and SW1353 (chondrosarcoma) were selected which represent a high and a low (α/ß)x ratio cell type. Cell survival curves were generated applying a linear-quadratic curve fit. DNA damage and DNA damage clearance were assessed via γH2AX foci quantification at 1 and 24h post radiation treatment. RESULTS: Without a magnetic field, RBE10 values of 1.04±0.03 (SW1353) and 1.51±0.06 (SKMel) as well as RBE80 values of 0.93±0.15 (SW1353) and 2.28±0.40 (SKMel) were calculated for protons. Carbon treatments yielded RBE10 values of 1.68±0.04 (SW1353) and 2.30±0.07 (SKMel) and RBE80 values of 2.19±0.24 (SW1353) and 4.06±0.33 (SKMel). For a field strength of B=1T, RBE10 values of 1.06±0.03 (SW1353) and 1.47±0.06 (SKMel) resulted from protons, while RBE10 values of 1.70±0.05 (SW1353) and 2.37±0.08 (SKMel) were obtained for carbon ions. RBE80 values were calculated to be 1.06±0.12 (SW1353) and 2.33±0.40 (SKMel) following protons and 2.13±0.25 (SW1353) and 4.29±0.35 (SKMel) following carbon treatments. Substantially increased γH2AX foci per nucleus were found in both cell lines 1h after radiation with both ion species. At the 24h time point only carbon treated samples of both cell lines showed increased γH2AX levels. The presence of the magnetic field did neither influence the survival parameters of either cell line, nor initial DNA damage and DNA damage clearance. CONCLUSIONS: Applying a perpendicular magnetic field did not influence the cell survival, DNA repair, nor the biological effectiveness of protons or carbon ions in two human cancer cell lines.

Activation of efficient DNA repair mechanisms after photon and proton irradiation of human chondrosarcoma cells.

Although particle therapy with protons has proven to be beneficial in the treatment of chondrosarcoma compared to photon-based (X-ray) radiation therapy, the cellular and molecular mechanisms have not yet been sufficiently investigated. Cell viability and colony forming ability were analyzed after X-ray and proton irradiation (IR). Cell cycle was analyzed using flow cytometry and corresponding regulator genes and key players of the DNA repair mechanisms were measured using next generation sequencing, protein expression and immunofluorescence staining. Changes in metabolic phenotypes were determined with nuclear magnetic resonance spectroscopy. Both X-ray and proton IR resulted in reduced cell survival and a G2/M phase arrest of the cell cycle. Especially 1 h after IR, a significant dose-dependent increase of phosphorylated γH2AX foci was observed. This was accompanied with a reprogramming in cellular metabolism. Interestingly, within 24 h the majority of clearly visible DNA damages were repaired and the metabolic phenotype restored. Involved DNA repair mechanisms are, besides the homology directed repair (HDR) and the non-homologous end-joining (NHEJ), especially the mismatch mediated repair (MMR) pathway with the key players EXO1, MSH3, and PCNA. Chondrosarcoma cells regenerates the majority of DNA damages within 24 h. These molecular mechanisms represent an important basis for an improved therapy.

Effects of a combined therapy of bortezomib and ionizing radiation on chondrosarcoma three-dimensional spheroid cultures.

Chondrosarcomas represent a heterogeneous group of primary bone cancers that are characterized by hyaline cartilaginous neoplastic tissue and are predominantly resistant to radiation and chemotherapy. However, adjuvant radiotherapy is often recommended in inoperable cases or after incomplete resections. To improve the efficiency of treatment, the present study tested a combination therapy with ionizing radiation (IR) and the proteasome inhibitor bortezomib. Using a three-dimensional (3D) spheroid model, 0-20 Gy of IR was applied to chondrosarcoma cells and healthy human chondrocytes. Following combined treatment with IR and bortezomib, the cell cycle distribution, apoptotic induction, the survivin pathway, autophagy and DNA damage were evaluated. Both cell types exhibited a slight decrease in viability following increasing doses of IR; the chondrosarcoma cells demonstrated a significant dose-dependent increase in the expression levels of the DNA damage marker histone H2AX phosphorylation at serine 139 (γH2AX). The combination treatment with bortezomib significantly decreased the cell viability after 48 h compared with that in irradiated cells. High-dose IR induced a G2/M phase arrest, which was accompanied by a decrease in the number of cells at the G1 and S phase. Co-treatment with bortezomib changed the distribution of the cell cycle phases. The mRNA expression levels of the proapoptotic genes Bcl-2-associated X protein (Bax) and Bak were significantly increased by bortezomib treatment and combination therapy with IR. In addition, the combination therapy resulted in a synergistic decrease of the expression levels of survivin and its corresponding downstream pathway molecules, including heat shock protein 90, X-linked inhibitor of apoptosis protein, smad 2 and smad 3. Comparative analyses of γH2AX at 1 and 24 h post-IR revealed efficient DNA repair in human chondrosarcoma cells. Therefore, additional bortezomib treatment may only temporarily improve the radiation sensitivity of chondrosarcoma cells. However, the inhibition of the survivin pathway by the combined treatment with IR and bortezomib, observed in the present study, revealed a novel aspect in the tumor biology of chondrosarcoma 3D spheroid cultures and may represent a potential target for therapy.