A Five-MicroRNA Signature Predicts Survival and Disease Control of Patients with Head and Neck Cancer Negative for HPV Infection.

PURPOSE: Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is associated with unfavorable prognosis, while independent prognostic markers remain to be defined. EXPERIMENTAL DESIGN: We retrospectively performed miRNA expression profiling. Patients were operated for locally advanced HPV-negative HNSCC and had received radiochemotherapy in eight different hospitals (DKTK-ROG; n = 85). Selection fulfilled comparable demographic, treatment, and follow-up characteristics. Findings were validated in an independent single-center patient sample (LMU-KKG; n = 77). A prognostic miRNA signature was developed for freedom from recurrence and tested for other endpoints. Recursive-partitioning analysis was performed on the miRNA signature, tumor and nodal stage, and extracapsular nodal spread. Technical validation used qRT-PCR. An miRNA-mRNA target network was generated and analyzed. RESULTS: For DKTK-ROG and LMU-KKG patients, the median follow-up was 5.1 and 5.3 years, and the 5-year freedom from recurrence rate was 63.5% and 75.3%, respectively. A five-miRNA signature (hsa-let-7g-3p, hsa-miR-6508-5p, hsa-miR-210-5p, hsa-miR-4306, and hsa-miR-7161-3p) predicted freedom from recurrence in DKTK-ROG [hazard ratio (HR) 4.42; 95% confidence interval (CI), 1.98-9.88, P < 0.001], which was confirmed in LMU-KKG (HR 4.24; 95% CI, 1.40-12.81, P = 0.005). The signature also predicted overall survival (HR 3.03; 95% CI, 1.50-6.12, P = 0.001), recurrence-free survival (HR 3.16; 95% CI, 1.65-6.04, P < 0.001), and disease-specific survival (HR 5.12; 95% CI, 1.88-13.92, P < 0.001), all confirmed in LMU-KKG data. Adjustment for relevant covariates maintained the miRNA signature predicting all endpoints. Recursive-partitioning analysis of both samples combined classified patients into low (n = 17), low-intermediate (n = 80), high-intermediate (n = 48), or high risk (n = 17) for recurrence (P < 0.001). CONCLUSIONS: The five-miRNA signature is a strong and independent prognostic factor for disease recurrence and survival of patients with HPV-negative HNSCC.See related commentary by Clump et al., p. 1441.

Genomic amplification of Fanconi anemia complementation group A (FancA) in head and neck squamous cell carcinoma (HNSCC): Cellular mechanisms of radioresistance and clinical relevance.

Radio (chemo) therapy is a crucial treatment modality for head and neck squamous cell carcinoma (HNSCC), but relapse is frequent, and the underlying mechanisms remain largely elusive. Therefore, novel biomarkers are urgently needed. Previously, we identified gains on 16q23-24 to be associated with amplification of the Fanconi anemia A (FancA) gene and to correlate with reduced progression-free survival after radiotherapy. Here, we analyzed the effects of FancA on radiation sensitivity in vitro, characterized the underlying mechanisms, and evaluated their clinical relevance. Silencing of FancA expression in HNSCC cell lines with genomic gains on 16q23-24 resulted in significantly impaired clonogenic survival upon irradiation. Conversely, overexpression of FancA in immortalized keratinocytes conferred increased survival accompanied by improved DNA repair, reduced accumulation of chromosomal translocations, but no hyperactivation of the FA/BRCA-pathway. Downregulation of interferon signaling as identified by microarray analyses, enforced irradiation-induced senescence, and elevated production of the senescence-associated secretory phenotype (SASP) appeared to be candidate mechanisms contributing to FancA-mediated radioresistance. Data of the TCGA HNSCC cohort confirmed the association of gains on 16q24.3 with FancA overexpression and impaired overall survival. Importantly, transcriptomic alterations similar to those observed upon FancA overexpression in vitro strengthened the clinical relevance. Overall, FancA amplification and overexpression appear to be crucial for radiotherapeutic failure in HNSCC.

Simultaneous perturbation of the MAPK and the PI3K/mTOR pathways does not lead to increased radiosensitization.

BACKGROUND: The mitogen-activated protein kinases (MAPK) and the phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways are intertwined on various levels and simultaneous inhibition reduces tumorsize and prolonges survival synergistically. Furthermore, inhibiting these pathways radiosensitized cancer cells in various studies. To assess, if phenotypic changes after perturbations of this signaling network depend on the genetic background, we integrated a time series of the signaling data with phenotypic data after simultaneous MAPK/ERK kinase (MEK) and PI3K/mTOR inhibition and ionizing radiation (IR). METHODS: The MEK inhibitor AZD6244 and the dual PI3K/mTOR inhibitor NVP-BEZ235 were tested in glioblastoma and lung carcinoma cells, which differ in their mutational status in the MAPK and the PI3K/mTOR pathways. Effects of AZD6244 and NVP-BEZ235 on the proliferation were assessed using an ATP assay. Drug treatment and IR effects on the signaling network were analyzed in a time-dependent manner along with measurements of phenotypic changes in the colony forming ability, apoptosis, autophagy or cell cycle. RESULTS: Both inhibitors reduced the tumor cell proliferation in a dose-dependent manner, with NVP-BEZ235 revealing the higher anti-proliferative potential. Our Western blot data indicated that AZD6244 and NVP-BEZ235 perturbed the MAPK and PI3K/mTOR signaling cascades, respectively. Additionally, we confirmed crosstalks and feedback loops in the pathways. As shown by colony forming assay, the AZD6244 moderately radiosensitized cancer cells, whereas NVP-BEZ235 caused a stronger radiosensitization. Combining both drugs did not enhance the NVP-BEZ235-mediated radiosensitization. Both inhibitors caused a cell cycle arrest in the G1-phase, whereas concomitant IR and treatment with the inhibitors resulted in cell line- and drug-specific cell cycle alterations. Furthermore, combining both inhibitors synergistically enhanced a G1-phase arrest in sham-irradiated glioblastoma cells and induced apoptosis and autophagy in both cell lines. CONCLUSION: Perturbations of the MEK and the PI3K pathway radiosensitized tumor cells of different origins and the combination of AZD6244 and NVP-BEZ235 yielded cytostatic effects in several tumor entities. However, this is the first study assessing, if the combination of both drugs also results in synergistic effects in terms of radiosensitivity. Our study demonstrates that simultaneous treatment with both pathway inhibitors does not lead to synergistic radiosensitization but causes cell line-specific effects.

Influence of hypoxia and irradiation on osteopontin expression in head and neck cancer and glioblastoma cell lines.

BACKGROUND: Tumor hypoxia is a known risk factor for reduced response to radiotherapy. The evaluation of noninvasive methods for the detection of hypoxia is therefore of interest. Osteopontin (OPN) has been discussed as an endogenous hypoxia biomarker. It is overexpressed in many cancers and is involved in tumor progression and metastasis. METHODS: To examine the influence of hypoxia and irradiation on osteopontin expression we used different cell lines (head and neck cancer (Cal27 and FaDu) and glioblastoma multiforme (U251 and U87)). Cells were treated with hypoxia for 24 h and were then irradiated with doses of 2 and 8 Gy. Osteopontin expression was analyzed on mRNA level by quantitative real-time RT-PCR (qPCR) and on protein level by western blot. Cell culture supernatants were evaluated for secreted OPN by ELISA. RESULTS: Hypoxia caused an increase in osteopontin protein expression in all cell lines. In Cal27 a corresponding increase in OPN mRNA expression was observed. In contrast the other cell lines showed a reduced mRNA expression under hypoxic conditions. After irradiation OPN mRNA expression raised slightly in FaDu and U87 cells while it was reduced in U251 and stable in Cal27 cells under normoxia. The combined treatment (hypoxia and irradiation) led to a slight increase of OPN mRNA after 2 Gy in U251 (24 h) and in U87 (24 and 48 h) cell lines falling back to base line after 8 Gy. This effect was not seen in Cal27 or in FaDu cells. Secreted OPN was detected only in the two glioblastoma cell lines with reduced protein levels under hypoxic conditions. Again the combined treatment resulted in a minor increase in OPN secretion 48 hours after irradiation with 8 Gy. CONCLUSION: Osteopontin expression is strongly modulated by hypoxia and only to a minor extent by irradiation. Intracellular OPN homeostasis seems to vary considerably between cell lines. This may explain the partly conflicting results concerning response prediction and prognosis in the clinical setting.

Novel PI3K and mTOR Inhibitor NVP-BEZ235 Radiosensitizes Breast Cancer Cell Lines under Normoxic and Hypoxic Conditions.

In the present study, we assessed, if the novel dual phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor NVP-BEZ235 radiosensitizes triple negative (TN) MDA-MB-231 and estrogen receptor (ER) positive MCF-7 cells to ionizing radiation under various oxygen conditions, simulating different microenvironments as occurring in the majority of breast cancers (BCs). Irradiation (IR) of BC cells cultivated in hypoxic conditions revealed increased radioresistance compared to normoxic controls. Treatment with NVP-BEZ235 completely circumvented this hypoxia-induced effects and radiosensitized normoxic, reoxygenated, and hypoxic cells to similar extents. Furthermore, NVP-BEZ235 treatment suppressed HIF-1α expression and PI3K/mTOR signaling, induced autophagy, and caused protracted DNA damage repair in both cell lines in all tested oxygen conditions. Moreover, after incubation with NVP-BEZ235, MCF-7 cells revealed depletion of phospho-AKT and considerable signs of apoptosis, which were significantly enhanced by radiation. Our findings clearly demonstrate that NVP-BEZ235 has a clinical relevant potential as a radiosensitizer in BC treatment.

Radiosensitization of Glioblastoma Cell Lines by the Dual PI3K and mTOR Inhibitor NVP-BEZ235 Depends on Drug-Irradiation Schedule.

Previous studies have shown that the dual phosphatidylinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) inhibitor NVP-BEZ235 radiosensitizes tumor cells if added shortly before ionizing radiation (IR) and kept in culture medium thereafter. The present study explores the impact of inhibitor and IR schedule on the radiosensitizing ability of NVP-BEZ235 in four human glioblastoma cell lines. Two different drug-IR treatment schedules were compared. In schedule I, cells were treated with NVP-BEZ235 for 24 hours before IR and the drug was removed before IR. In schedule II, the cells were exposed to NVP-BEZ235 1 hour before, during, and up to 48 hours after IR. The cellular response was analyzed by colony counts, expression of marker proteins of the PI3K/AKT/mTOR pathway, cell cycle, and DNA damage. We found that under schedule I, NVP-BEZ235 did not radiosensitize cells, which were mostly arrested in G1 phase during IR exposure. In addition, the drug-pretreated and irradiated cells exhibited less DNA damage but increased expressions of phospho-AKT and phospho-mTOR, compared to controls. In contrast, NVP-BEZ235 strongly enhanced the radiosensitivity of cells treated according to schedule II. Possible reasons of radiosensitization by NVP-BEZ235 under schedule II might be the protracted DNA repair, prolonged G2/M arrest, and, to some extent, apoptosis. In addition, the PI3K pathway was downregulated by the NVP-BEZ235 at the time of irradiation under schedule II, as contrasted with its activation in schedule I. We found that, depending on the drug-IR schedule, the NVP-BEZ235 can act either as a strong radiosensitizer or as a cytostatic agent in glioblastoma cells.

Hsp90 inhibition by NVP-AUY922 and NVP-BEP800 decreases migration and invasion of irradiated normoxic and hypoxic tumor cell lines.

This study explores the impact of Hsp90 inhibitors NVP-AUY922 and NVP-BEP800 in combination with ionizing radiation (IR) on the migration and invasion of lung carcinoma A549 and glioblastoma SNB19 cells, under normoxia or hypoxia. Independent of oxygen concentration, both drugs decreased the migration and invasion rates of non-irradiated tumor cells. Combined drug-IR treatment under hypoxia inhibited cell invasion to a greater extent than did each treatment alone. Decreased migration of cells correlated with altered expression of several matrix-associated proteins (FAK/p-FAK, Erk2, RhoA) and impaired F-actin modulation. The anti-metastatic efficacy of the Hsp90 inhibitors could be useful in combinational therapies of cancer.

Hsp90 inhibitor NVP-AUY922 enhances radiation sensitivity of tumor cell lines under hypoxia.

NVP-AUY922, a novel inhibitor of Hsp90, was shown to enhance the effect of ionizing radiation (IR) on tumor cells under normoxic conditions. Since low oxygen tension is a common feature of solid tumors, we explore in the present study the impact of hypoxia on the combined treatment of lung carcinoma A549 and glioblastoma SNB19 cell lines with NVP-AUY922 and IR. Cellular analysis included the colony-forming ability, expression of CAIX, Hsp90, Hsp70, Raf-1, Akt, cell cycle progression and associated proteins, as well as DNA damage measured by histone γH2AX.   The clonogenic assay revealed that in both cell lines NVP-AUY922 enhanced the radiotoxicity under hypoxic exposure to a level similar to that observed under oxic conditions. Irrespective of oxygen supply during drug treatment, NVP-AUY922 also reduced the expression of anti-apoptotic proteins Raf-1 and Akt. As judged by the levels of histone γH2AX, drug-treated hypoxic cells exhibited a lower repair rate of DNA double-strand breaks than normoxic cells. The drug-IR mediated changes in the cell cycle, i.e., S-phase depletion and G 2/M arrest, developed not directly during hypoxic exposure but first upon 24 h reoxygenation. Under both oxygen tensions, Hsp90 inhibition downregulated the cell cycle-associated proteins, Cdk1, Cdk4 and pRb. The finding that NVP-AUY922 can enhance the in vitro radiosensitivity of hypoxic tumor cells may have implications for the combined modality treatment of solid tumors.