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Carbon ion irradiation is an emerging therapeutic option for various tumor entities. Radiation resistance of solid tumors toward photon irradiation is caused by attenuation of DNA damage in less oxygenated tumor areas and by increased hypoxia-inducible factor (HIF)-1 signaling. Carbon ion irradiation acts independently of oxygen; however, the role of HIF-1 is unclear. We analyzed the effect of HIF-1 signaling after carbon ions in comparison to photons by using biological equivalent radiation doses in a human non-small-cell cancer model. The studies were performed in cultured A549 and H1299 cell lines and in A549 xenografts. Knockdown of HIF-1α in vivo combined with photon irradiation delayed tumor growth (23 vs. 13 d; P<0.05). Photon irradiation induced HIF-1α and target genes, predominantly in oxygenated cells (1.6-fold; P<0.05), with subsequent enhanced tumor angiogenesis (1.7-fold; P<0.05). These effects were not observed after carbon ion irradiation. Micro-DNA array analysis indicated that photons, but not carbon ions, significantly induced components of the mTOR (mammalian target of rapamycin) pathway (gene set enrichment analysis; P<0.01) as relevant for HIF-1α induction. After carbon ion irradiation in vivo, we observed substantially decreased HIF-1α levels (8.9-fold; P<0.01) and drastically delayed tumor growth (P<0.01), an important finding that indicates a higher relative biological effectiveness (RBE) than anticipated from the cell survival data. Taken together, the evidence showed that carbon ions mediate an improved therapeutic effectiveness without tumor-promoting HIF-1 signaling. [ABSTRACT FROM AUTHOR]

Background: Surgery and radiotherapy are current therapeutic options for malignant tumors involving the nasal vestibule. Depending on the location, organ-preserving resection is not always possible, even for small tumors. Definitive radiotherapy is an alternative as an organ-preserving procedure. Carbon ion beam radiotherapy offers highly conformal dose distributions and more complex biological radiation effects eventually resulting in optimized normal tissue sparing and improved outcome. The aim of the current study was to analyze toxicity, local control (LC), and organ preserving survival (OPS) after irradiation of carcinoma of the nasal vestibule with raster-scanned carbon ion radiotherapy boost (CIRT-B) combined with volumetric intensity modulated arc therapy (VMAT) with photons., Methods: Between 12/2015 and 05/2021, 21 patients with malignant tumors involving the nasal vestibule were irradiated with CIRT-B combined with VMAT and retrospectively analyzed. Diagnosis was based on histologic findings. A total of 17 patients had squamous cell carcinoma (SCC) and 4 had other histologies. In this series, 10%, 67%, and 24% of patients had Wang stages 1, 2, and 3 tumors, respectively. Three patients had pathologic cervical nodes on MRI. The median CIRT-B dose was 24 Gy(RBE), while the median VMAT dose was 50 Gy. All patients with pathologic cervical nodes received simultaneously integrated boost with photons (SIB) up to a median dose of 62.5 Gy to the pathological lymph nodes. Eight patients received cisplatin chemotherapy. All patients received regular follow-up imaging after irradiation. Kaplan-Meier estimation was used for statistical assessment., Results: The median follow-up after irradiation was 18.9 months. There were no common toxicity criteria grade 5 or 4 adverse events. A total of 20 patients showed grade 3 adverse events mainly on skin and mucosa. All patients were alive at the end of follow-up. The median OPS after treatment was 56.5 months. The 6- and 24-month OPS were 100% and 83.3%, respectively. All local recurrences occurred within 12 months after radiotherapy. The median progression free survival (PFS) after treatment was 52.4 months. The 6-, 12-, and 24-month PFS rates were 95%, 83.6%, and 74.3%, respectively., Conclusion: CIRT-B combined with VMAT in malignant tumors of the nasal vestibule is safe and feasible, results in high local control rates, and thus is a good option as organ-preserving therapy. No radiation-associated grade 4 or 5 acute or late AE was documented., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Eberle, Engenhart-Cabillic, Schymalla, Dumke, Schötz, Subtil, Baumann, Stuck, Langer, Jensen, Hauswald and Lautenschläger.)

Background and Purpose: HPV positive (pos.) HNSCC cells are significantly more radiosensitive to photon irradiation as compared to HPV negative (neg.) cells. Functionally, this is considered to result from a reduced DSB repair capacity. It was now tested, whether such a difference is also observed when using carbon ion ( 12 C) irradiation., Material and Methods: Five HPV pos. and five HPV neg. HNSCC cell lines were irradiated with photons or 12 C-ions using 2D or 3D cell culture conditions. Clonogenic survival was determined by colony formation assay and DSB repair by immunofluorescence using co-staining of γH2AX and 53BP1 foci., Results: The pronounced difference in radiosensitivity known for these two entities when exposed to photons in 2D cell culture, was reduced when treated under 3D conditions. Irradiation with 12 C-ions strongly enhanced cell killing, whereby increase was more pronounced for the HPV neg. when compared to the HPV pos. cell line (RBE = 2.81 vs. 2.14). As a consequence, after 12 C-irradiation clonogenic survival was almost identical for the two entities as was demonstrated for all cell lines at a dose of 3 Gy. In line with this, the significant difference in DSB repair capacity between HPV pos. and neg. HNSCC cells, as seen after photon irradiation, was abrogated after 12 C-irradiation., Conclusion: While HPV pos. cells are significantly more radiosensitive to photons than HPV neg. cells, no significant difference was seen after 12 C-irradiation. This needs to be considered when planning new clinical protocols for the treatment of HPV neg. and pos. tumors with 12 C-ions., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The PI3K/Akt/mTOR pathway is frequently altered in human papillomavirus (HPV)-positive and negative squamous cell carcinoma of the head and neck (HNSCC) and overstimulation is associated with poor prognosis. PI3K drives Akt activation and constitutive signaling acts pro-proliferative, supports cell survival, DNA repair, and contributes to radioresistance. Since the small molecule NVP-BEZ235 (BEZ235) is a potent dual inhibitor of this pathway, we were interested whether BEZ235 could be an efficient radiosensitizer. The 50 nM BEZ235 was found to abrogate endogenous and irradiation-induced phosphorylation of Akt (Ser473). The anti-proliferative capacity of the drug resulted in an increase in G1-phase cells. Repair of radiation-induced DNA double-strand breaks (DSBs) was strongly suppressed. Reduction in DSB repair was only apparent in G1- but not in G2-phase cells, suggesting that BEZ235 primarily affects non-homologous end joining. This finding was confirmed using a DSB repair reporter gene assay and could be attributed to an impaired phosphorylation of DNA-PKcs (S2056). Cellular radiosensitivity increased strongly after BEZ235 addition in all HNSCC cell lines used, especially when irradiated in the G0 or G1 phase. Our data indicate that targeting the PI3K/Akt/mTOR pathway by BEZ235 with concurrent radiotherapy may be considered an effective strategy for the treatment of HNSCC, regardless of the HPV and Akt status., Competing Interests: The authors declare no conflict of interest.

Non-small cell lung cancer (NSCLC) has a very poor prognosis even when treated with the best therapies available today often including radiation. NSCLC is frequently complicated by pulmonary infections which appear to impair prognosis as well as therapy, whereby the underlying mechanisms are still not known. It was investigated here, whether the bacterial lipopolysaccharides (LPS) might alter the tumor cell radiosensitivity. LPS were found to induce a radioresistance but solely in cells with an active TLR-4 pathway. Proteome profiling array revealed that LPS combined with irradiation resulted in a strong phosphorylation of cAMP response element-binding protein (CREB). Inhibition of CREB binding protein (CBP) by the specific inhibitor ICG-001 not only abrogated the LPS-induced radioresistance but even led to an increase in radiosensitivity. The sensitization caused by ICG-001 could be attributed to a reduction of DNA double-strand break (DSB) repair. It is shown that in NSCLC cells LPS leads to a CREB dependent radioresistance which is, however, reversible through CBP inhibition by the specific inhibitor ICG-001. These findings indicate that the combined treatment with radiation and CBP inhibition may improve survival of NSCLC patients suffering from pulmonary infections., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

At present, advanced stage human Papillomavirus (HPV) negative and positive head and neck squamous cell carcinoma (HNSCC) are treated by intense multimodal therapy that includes radiochemotherapy, which are associated with relevant side effects. Patients with HPV positive tumors possess a far better prognosis than those with HPV negative cancers. Therefore, new therapeutic strategies are needed to improve the outcome especially of the latter one as well as quality of life for all HNSCC patients. Here we tested whether roscovitine, an inhibitor of cyclin-dependent kinases (CDKs), which hereby also blocks homologous recombination (HR), can be used to enhance the radiation sensitivity of HNSCC cell lines. In all five HPV negative and HPV positive cell lines tested, roscovitine caused inhibition of CDK1 and 2. Surprisingly, all HPV positive cell lines were found to be defective in HR. In contrast, HPV negative strains demonstrated efficient HR, which was completely suppressed by roscovitine. In line with this, for HPV negative but not for HPV positive cell lines, treatment with roscovitine resulted in a pronounced enhancement of the radiation-induced G2 arrest as well as a significant increase in radiosensitivity. Due to a defect in HR, all HPV positive cell lines were efficiently radiosensitized by the PARP-1 inhibitor olaparib. In contrast, in HPV negative cell lines a significant radiosensitization by olaparib was only achieved when combined with roscovitine., Competing Interests: CONFLICTS OF INTEREST The authors have no conflicts of interest to declare.

Background and Purpose: The SCF/c-Kit pathway is often overexpressed in human tumors leading to an enhanced tumorigenesis, proliferation and migration. It was now tested for NSCLC and prostate cancer cells growing in 2D and 3D whether the inhibition of this pathway can be used to achieve a significant radiosensitization and whether a respective biomarker may be identified., Material and Methods: Experiments were performed with different cancer cell lines (NSCLC: H23, H520, H226, H1975 and PrCa: DU145) growing either under 2D or 3D conditions. Expression of SCF and c-Kit was determined by RT-PCR and Western blot, SCF was knocked down by siRNA, c-Kit was inhibited by ISCK03 inhibitor and cell survival was determined by colony formation assay., Results: There is a profound variation in the expression of both c-Kit and SCF with no association between each other. Neither levels did correlate with the respective cellular radiosensitivity determined for 2D or 3D with only a trend seen for SCF. Knock-down of SCF was generally found to result in no or only minor reduction of plating efficiency or cellular radioresistance. A significant reduction was only obtained for H520 cells characterized by an extreme over-expression of SCF. The inhibition of c-Kit by a specific inhibitor was also found to result only in minor radiosensitization., Conclusion: Generally, the SCF/c-Kit pathway does not have a dominant effect on both, cell survival and radioresponse and, as a consequence, knockdown of this pathway does not result in a strong effect on radioresistance, except when SCF is strongly over-expressed.

Background and Purpose: Success of radiotherapy is often limited by therapy resistance and metastasis resulting from cancer cell motility. It was tested in vitro whether this cancer cell motility is affected by growth condition, active SCF/c-Kit pathway or X-irradiation., Materials and Methods: Cell motility was measured with BioCoat™ Matrigel™ invasion chamber using four different cancer cell lines (NSCLC: H23, H520, H226 and PrCa: DU145). Cells were grown in 2D or 3D, SCF was knocked down by siRNA and cells were irradiated with 2 or 6Gy., Results: All cell lines except H520 showed a 2-3-fold increase in cell motility when grown in 3D. This effect was considered to result from the EMT-like change seen when cells were grown in 3D as indicated by the enhanced expression of vimentin and N-cadherin and reduction of E-cadherin. Just the opposite trends were found for H520 cells. Knockdown of SCF was found to result in reduced cell motility for both 2D and 3D. In contrast, X-irradiation did not modulate cell motility neither under 2D nor 3D. In line with this, X-irradiation did neither induce the expression of EMT-associated genes nor SCF., Conclusion: X-irradiation affects neither the expression of important EMT genes such as vimentin, E-cadherin and N-cadherin nor SCF/c-Kit signaling and, as a consequence, does not alter cell motility., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)