Strahlensensitivierung von Kopf-Hals-Tumorzellen durch den dualen PI3K/mTOR-Inhibitor NVP-BEZ235

SUMMARY For years, squamous cell carcinoma of the head and neck region (HNSCC) have been among the six most frequent malignant diseases worldwide. Besides alcohol and nicotine consumption, local infection with human papilloma viruses (mainly HPV-16 and HPV-18) contributes to oncogenesis. They establish their own molecular tumor entity with rising incidence. Radiation therapy is, along with surgical resection, one of the most important therapeutic options in the multimodal treatment of these tumors and represents an effective way to treat the tumor in a way that preserves the integrity of the organs. As these tumors are often detected very late and treated at an advanced stage, and have a relatively high rate of recurrences and secondary tumors, the disease is usually associated with a poor prognosis. Only for HPV-associated carcinomas a significantly better survival is observed, which is mainly due to an enhanced radiation sensitivity. However, despite much progress, intensive therapy is associated with high normal tissue toxicity. For this reason, efforts have been underway for years to improve the therapy in order to achieve better cure rates while reducing normal tissue toxicity. The scientific focus is primarily on molecular approaches that specifically intervene in specific tumor processes. One of the most promising target structures in HNSCC tumors is the PI3K/mTOR/Akt signaling pathway, as it is misregulated in a large number of these tumors mostly resulting in an over-activation. This pathway influences proliferation, metabolism and DNA repair via a large number of effectors. Therefore, it was expected, that the inhibition of this signaling pathway may lead to a targeted radiation sensitization. Within the scope of this work, HPV-positive as well as HPV-negative HNSCC cell lines were examined for the first time to determine whether treatment with BEZ235 effectively suppresses the repair of radiation-induced DNA double-strand breaks (DSB) and thereby achieves a significant radiation sensitivity. Four HPV-negative HNSCC cell lines (UM-SCC-3, UM-SCC-6, UM-SCC-11b, UM-SCC-33) and four HPV-positive lines (UM-SCC-104, UD-SCC-2, UM-SCC-47, 93VU-147T) were used. The effect on cell survival was determined using the colony formation assay. The detection of DSB was performed with the very sensitive gH2AX foci technique. The proportion of cells with ≥5 gH2AX-Foci was determined. To distinguish between G1- and G2-phase cells, the cell cycle-dependent expression of the centromere specific protein F (CenpF) was used. Overall, the following observations were made: 1. Treatment with 50 nM BEZ235 alone has little or no effect on cell survival. 2. In combination with irradiation, BEZ235 causes a significant radiation sensitization, whereby this effect is independent of the HPV-status of the HNSCC cells. 3. In untreated HPV-negative as well as -positive HNSCC cell lines, the proportion of cells with ≥5 gH2AX foci is very low, averaging only 1-2%. 4. When irradiated with 2 Gy, the proportion of cells with ≥5 gH2AX foci is significantly higher in the HPV-positive cells when compared to HPV-negative cells, which - in accordance with previous data - indicates a defective DSB repair in HPV-positive cells. 5. In both HPV-negative and HPV-positive HNSCC cells, BEZ235 inhibits DSB repair, resulting in a significant increase in the proportion of cells with ≥5 gH2AX foci. 6. This increase is only seen in G1-phase cells and not in G2-phase cells This indicates that BEZ235 primarily suppresses non-homologous end joining (NHEJ), which is the most important repair process in G1-phase. 7. When BEZ235 is combined with irradiation, the proportion of cells with ≥5 gH2AX foci as measured in G1-phase cells correlates very well with the corresponding decrease in survival rate. This finding indicates that the radiation sensitization by BEZ235 primarily results from an inhibition of DSB repair. Overall, this work demonstrates for the first time that a strong radiation sensitization can be achieved in all HPV-negative and HPV-positive HNSCC cell lines by effective suppression of the PI3k/mTOR/Akt signaling pathway using the dual inhibitor BEZ235. In the long term, this result offers the possibility to increase the overall survival of HNSCC by a combined treatment with a targeted radiation sensitization while reducing normal tissue toxicity. The data of this work were published together with other results in the journal Cancers: Schötz U, Balzer V, Brandt FW, Ziemann F, Subtil FSB, Rieckmann T, Köcher S, Engenhart-Cabillic R, Dikomey E, Wittig A, Arenz A. 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. Cancers (Basel). 2020 Feb 18;12(2).