Dual PI3K/mTOR Inhibitor NVP-BEZ235 Leads to a Synergistic Enhancement of Cisplatin and Radiation in Both HPV-Negative and -Positive HNSCC Cell Lines.

The standard of care for advanced head and neck cancers (HNSCCs) is radiochemotherapy, including cisplatin. This treatment results in a cure rate of approximately 85% for oropharyngeal HPV-positive HNSCCs, in contrast to only 50% for HPV-negative HNSCCs, and is accompanied by severe side effects for both entities. Therefore, innovative treatment modalities are required, resulting in a better outcome for HPV-negative HNSCCs, and lowering the adverse effects for both entities. The effect of the dual PI3K/mTOR inhibitor NVP-BEZ235 on a combined treatment with cisplatin and radiation was studied in six HPV-negative and six HPV-positive HNSCC cell lines. Cisplatin alone was slightly more effective in HPV-positive cells. This could be attributed to a defect in homologous recombination, as demonstrated by depleting RAD51. Solely for HPV-positive cells, pretreatment with BEZ235 resulted in enhanced cisplatin sensitivity. For the combination of cisplatin and radiation, additive effects were observed. However, when pretreated with BEZ235, this combination changed into a synergistic interaction, with a slightly stronger enhancement for HPV-positive cells. This increase could be attributed to a diminished degree of DSB repair in G1, as visualized via the detection of γH2AX/53BP1 foci. BEZ235 can be used to enhance the effect of combined treatment with cisplatin and radiation in both HPV-negative and -positive HNSCCs.

Dual PI3K/mTOR Inhibitor NVP-BEZ235 Enhances Radiosensitivity of Head and Neck Squamous Cell Carcinoma (HNSCC) Cell Lines Due to Suppressed Double-Strand Break (DSB) Repair by Non-Homologous End Joining.

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.

HPV-negative and HPV-positive HNSCC cell lines show similar numerical but different structural chromosomal aberrations.

BACKGROUND: It was tested whether the difference in carcinogenesis between noxa and human papillomavirus (HPV)-driven head and neck squamous cell carcinoma (HNSCC) is associated with a variation in genomic instability. METHODS: Conventional and molecular cytogenetics in HPV-positive and HPV-negative HNSCC cell lines. RESULTS: Numerical aneuploidy determined by multicolor fluorescence in situ hybridization and DNA ploidy was very similar for both entities with most chromosomes being present either in quadruplicate or triplicate, and only few are still diploid with, however, a striking similarity in the overall pattern. A clear difference was seen concerning the translocations formed, with no difference in the total amount but with a significantly higher genomic instability of HPV-positive cell lines at chromosome 3 as compared to HPV-negative cells. CONCLUSION: The different processes of carcinogenesis of HPV-positive and HPV-negative HNSCC appear to result in a similar pattern of numerical but a clear difference in structural chromosomal aberrations.

In HPV-Positive HNSCC Cells, Functional Restoration of the p53/p21 Pathway by Proteasome Inhibitor Bortezomib Does Not Affect Radio- or Chemosensitivity.

Human papillomavirus (HPV) associated squamous cell carcinomas of the head and neck region (HPV+ HNSCCs) harbor diverging biological features as compared to classical noxa-induced (HPV-) HNSCC. One striking difference between subtypes is that the tumor suppressor gene TP53 is usually not mutated in HPV+ HNSCCs. However, p53 is inhibited by viral oncoprotein E6, leading to premature proteasomal degradation. We asked whether bortezomib (BZM), a clinically approved inhibitor of the proteasome, can functionally restore p53 and investigated in how far this will result in an enhanced radio- or chemosensitivity of HPV+ HNSCC cell lines. For all four HPV+ cell lines tested, BZM led to functional restoration of p53 and transactivation of downstream protein p21. In HPV+ cells, BZM also restored the radiation-induced p53/p21 transactivation. Consistently, in HPV+ cells, a restored G1 arrest as well as enhanced apoptosis were seen when BZM was given prior to irradiation (IR) or cisplatin (CDDP). BZM alone reduced the clonogenic survival of both HPV- and HPV+ cells. However, if BZM was combined with IR or CDDP, BZM did not significantly enhance radio- or chemosensitivity of HPV+ or HPV- HNSCC cell lines.

Roscovitine strongly enhances the effect of olaparib on radiosensitivity for HPV neg. but not for HPV pos. HNSCC cell lines.

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.

Increased sensitivity of HPV-positive head and neck cancer cell lines to x-irradiation ± Cisplatin due to decreased expression of E6 and E7 oncoproteins and enhanced apoptosis.

Squamous cell carcinoma of the head and neck region (HNSCC), which is related to an infection with human papilloma virus (HPV), responds better to simultaneous radio-chemotherapy with Cisplatin based regimens than HPV-negative tumors. The underlying molecular mechanisms for this clinical observation are not fully understood. Therefore, the response of four HPV-positive (HPV+) (UM-SCC-47, UM-SCC-104, 93-VU-147T, UPCI:SCC152) and four HPV-negative (HPV-) (UD-SCC-1, UM-SCC-6, UM-SCC-11b, UT-SCC-33) HNSCC cell lines to x-irradiation ± Cisplatin incubation in terms of clonogenic survival, cell cycle progression, protein expression (cyclin A2, cyclin E2, E6, E7, p53) and induction of apoptosis, was investigated. HPV+ cells were more radio- and chemosensitive and were more effectively sensitized to x-irradiation by simultaneous Cisplatin incubation than HPV- cell lines. HPV+ cell lines revealed an increased and prolonged G2/M arrest after irradiation, whereas Cisplatin induced a blockage of cells in S phase. In comparison to irradiation only, addition of Cisplatin significantly enhanced apoptosis especially in HPV+ cell lines. While irradiation alone increased the amount of HPV E6 and E7 proteins, both were down-regulated by Cisplatin incubation either alone or in combination with x-rays, which however did not increase the expression of endogenous p53. Our results demonstrate that cell cycle deregulation together with downregulation of HPV E6 and E7 proteins facilitating apoptosis after Cisplatin incubation promote the enhanced sensitivity of HPV+ HNSCC cells to simultaneous radio-chemotherapy. Combined effects of irradiation and Cisplatin appear to be relevant in mediating the enhanced therapeutic response of HPV-related HNSCC and are indicative of the benefit of combined modality approaches in future treatment optimization strategies.

Increased radiosensitivity of HPV-positive head and neck cancer cell lines due to cell cycle dysregulation and induction of apoptosis.

BACKGROUND AND PURPOSE: Human Papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) respond favourably to radiotherapy as compared to HPV-unrelated HNSCC. We investigated DNA damage response in HPV-positive and HPV-negative HNSCC cell lines aiming to identify mechanisms, which illustrate reasons for the increased sensitivity of HPV-positive cancers of the oropharynx. METHODS: Radiation response including clonogenic survival, apoptosis, DNA double-strand break (DSB) repair, and cell cycle redistribution in four HPV-positive (UM-SCC-47, UM-SCC-104, 93-VU-147T, UPCI:SCC152) and four HPV-negative (UD-SCC-1, UM-SCC-6, UM-SCC-11b, UT-SCC-33) cell lines was evaluated. RESULTS: HPV-positive cells were more radiosensitive (mean SF2: 0.198 range: 0.22-0.18) than HPV-negative cells (mean SF2: 0.34, range: 0.45-0.27; p = 0.010). Irradiated HPV-positive cell lines progressed faster through S-phase showing a more distinct accumulation in G2/M. The abnormal cell cycle checkpoint activation was accompanied by a more pronounced increase of cell death after x-irradiation and a higher number of residual and unreleased DSBs. CONCLUSIONS: The enhanced responsiveness of HPV-related HNSCC to radiotherapy might be caused by a higher cellular radiosensitivity due to cell cycle dysregulation and impaired DNA DSB repair.

Comparison of the effects of carbon ion and photon irradiation on the angiogenic response in human lung adenocarcinoma cells.

PURPOSE: Radiotherapy resistance is a commonly encountered problem in cancer treatment. In this regard, stabilization of endothelial cells and release of angiogenic factors by cancer cells contribute to this problem. In this study, we used human lung adenocarcinoma (A549) cells to compare the effects of carbon ion and X-ray irradiation on the cells' angiogenic response. METHODS AND MATERIALS: A549 cells were irradiated with biologically equivalent doses for cell survival of either carbon ions (linear energy transfer, 170 keV/µm; energy of 9.8 MeV/u on target) or X-rays and injected with basement membrane matrix into BALB/c nu/nu mice to generate a plug, allowing quantification of angiogenesis by blood vessel enumeration. The expression of angiogenic factors (VEGF, PlGF, SDF-1, and SCF) was assessed at the mRNA and secreted protein levels by using real-time reverse transcription-PCR and enzyme-linked immunosorbent assay. Signal transduction mediated by stem cell factor (SCF) was assessed by phosphorylation of its receptor c-Kit. For inhibition of SCF/c-Kit signaling, a specific SCF/c-Kit inhibitor (ISCK03) was used. RESULTS: Irradiation of A549 cells with X-rays (6 Gy) but not carbon ions (2 Gy) resulted in a significant increase in blood vessel density (control, 20.71 ± 1.55; X-ray, 36.44 ± 3.44; carbon ion, 16.33 ± 1.03; number per microscopic field). Concordantly, irradiation with X-rays but not with carbon ions increased the expression of SCF and subsequently caused phosphorylation of c-Kit in endothelial cells. ISCK03 treatment of A549 cells irradiated with X-rays (6 Gy) resulted in a significant decrease in blood vessel density (X-ray, 36.44 ± 3.44; X-ray and ISCK03, 4.33 ± 0.71; number of microscopic field). These data indicate that irradiation of A549 cells with X-rays but not with carbon ions promotes angiogenesis. CONCLUSIONS: The present study provides evidence that SCF is an X-ray-induced mediator of angiogenesis in A549 cells, a phenomenon that could not be observed with carbon ion irradiation. Thus, in this model system evaluating angiogenesis, carbon ion irradiation may have a therapeutic advantage. This observation should be confirmed in orthotopic lung tumor models.

Gene expression modulation in A549 human lung cells in response to combustion-generated nano-sized particles.

High levels of ambient air pollution are associated in humans with aggravation of asthma and of respiratory and cardiopulmonary morbidity; long-term exposures to particulate matter (PM) have been linked to possible increases in lung cancer risk, chronic respiratory disease, and increased death rates. The Biodiagnostics Group of the DLR Institute of Aerospace Medicine develops cellular test systems capable of monitoring the biological consequences of environmental conditions on humans already on cellular and molecular level. Such bioassays rely on the receptor-reporter principle, where cell lines are transfected with plasmids carrying a reporter gene under control of environment-dependent promoters (receptor), which play a key role in regulating gene expressions in response to extracellular signals. We developed the recombinant human lung epithelial cell line A549-NF-kappaB-EGFP/Neo carrying a genetically encoded fluorescent indicator for monitoring activation of the NF-kappaB signaling pathway in living cells in response to genotoxic and cytotoxic environmental influences. With this cell line we screened several candidate human radiation-responsive genes (GADD45beta, CDKN1A) and NF-kappaB-dependent genes (IL-6, NFkappaBIA, and pNF-kappaB-EGFP) for gene expression changes by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay, using cDNA obtained from total RNA isolated at various time points after exposure to combustion generated nano-sized particle samples.

Activation of nuclear factor kappa B by different agents: influence of culture conditions in a cell-based assay.

The transcription factor nuclear factor kappaB (NF-kappaB) or other components of this pathway have been identified as possible therapeutic targets in inflammatory processes, cancer, and autoimmune diseases. In order to clarify the role of NF-kappaB in epithelial cells in response to different stresses, a cell-based screening assay for activation of NF-kappaB-dependent gene transcription in human embryonic kidney cells (HEK/293) was developed. This assay allows detection of NF-kappaB activation by measurement of the fluorescence of the reporter protein destabilized enhanced green fluorescent protein (d2EGFP). For characterization of the cell-based assay, activation of the pathway by several agents, for example, tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), lipopolysaccharide (LPS), camptothecin and phorbol ester (PMA), and the influence of the culture conditions on NF-kappaB activation by TNF-alpha were examined. NF-kappaB was activated by TNF-alpha, IL-1beta, PMA, and camptothecin in a dose-dependent manner, but not by LPS. TNF-alpha results in the strongest induction of NF-kappaB-dependent gene expression. However, this response fluctuated from 30 to 90% of the cell population showing d2EGFP expression. This variation can be explained by differences in growth duration and cell density at the time of treatment. With increasing confluence of the cells, the activation potential decreased. In a confluent cell layer, only 20-35% of the cell population showed d2EGFP expression. The underlying mechanism of this phenomenon can be the production of soluble factors by the cells inhibiting the NF-kappaB activation or direct communication via gap junctions in the cell layer diminishing the TNF-alpha response.

Cellular monitoring of the nuclear factor kappaB pathway for assessment of space environmental radiation.

A screening assay for the detection of NF-kappaB-dependent gene induction using the destabilized variant of the reporter protein enhanced green fluorescent protein (d2EGFP) is used for assessing the biological effects of accelerated heavy ions as a model of space environmental radiation conditions. The time course of d2EGFP expression and therefore of activation of NF-kappaB-dependent gene expression was measured after treatment with TNFA or after heavy-ion exposure using flow cytometry. The reported experiments clearly show that accelerated argon ions (95 MeV/nucleon, LET 230 keV/microm) induce the NF-kappaB pathway at low particle densities (1-2 particle hits per nucleus), which result in as few as 5-50 induced DSBs per cell.