The oncogene epidermal growth factor receptor variant III (EGFRvIII) is frequently expressed in glioblastomas (GBM) but its impact on therapy response is still under controversial debate. Here we wanted to test if EGFRvIII influences the sensitivity towards the alkylating agent temozolomide (TMZ). Therefore, we retrospectively analyzed the survival of 336 GBM patients, demonstrating that under standard treatment, which includes TMZ, EGFRvIII expression is associated with prolonged survival, but only in patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated tumors. Using isogenic GBM cell lines with endogenous EGFRvIII expression we could demonstrate that EGFRvIII increases TMZ sensitivity and results in enhanced numbers of DNA double-strand breaks and a pronounced S/G2-phase arrest after TMZ treatment. We observed a higher expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cells and patient tumor samples, which was most pronounced for MSH2 and MSH6. EGFRvIII-specific knockdown reduced MMR protein expression thereby increasing TMZ resistance. Subsequent functional kinome profiling revealed an increased activation of p38-and ERK1/2-dependent signaling in EGFRvIII expressing cells, which regulates MMR protein expression downstream of EGFRvIII. In summary, our results demonstrate that the oncoprotein EGFRvIII sensitizes a fraction of GBM to current standard of care treatment through the upregulation of DNA MMR.

Overexpression of the epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas (HNSCC) is considered to cause increased EGFR activity, which adds to tumorigenicity and therapy resistance. Since it is still unclear, whether EGFR expression is indeed associated with increased activity in HNSCC, we analyzed the relationship between EGFR expression and auto-phosphorylation as a surrogate marker for activity. We used a tissue micro array, fresh frozen HNSCC tumor and corresponding normal tissue samples and a large panel of HNSCC cell lines. While we observed substantial overexpression only in approximately 20% of HNSCC, we also observed strong discrepancies between EGFR protein expression and auto-phosphorylation in HNSCC cell lines as well as in tumor specimens using Western blot and SH2-profiling; for the majority of HNSCC EGFR expression therefore seems not to be correlated with EGFR auto-phosphorylation. Blocking of EGFR activity by cetuximab and erlotinib points to increased EGFR activity in samples with increased basal auto-phosphorylation. However, we could also identify cells with low basal phosphorylation but relevant EGFR activity. In summary, our data demonstrate that EGFR expression and activity are not well correlated. Therefore EGFR positivity is no reliable surrogate marker for EGFR activity, arguing the need for alternative biomarkers or functional predictive tests.

Inhibition of homologous recombination (HR) is believed to be a transactivation-independent function of p53 that protects from genetic instability. Misrepair by HR can lead to genetic alterations such as translocations, duplications, insertions and loss of heterozygosity, which all bear the risk of driving oncogenic transformation. Regulation of HR by wild-type p53 (wtp53) should prevent these genomic rearrangements. Mutation of p53 is a frequent event during carcinogenesis. In particular, dominantnegative mutants inhibiting wtp53 expressed from the unperturbed allel can drive oncogenic transformation by disrupting the p53-dependent anticancer barrier. Here, we asked whether the hot spot mutants R175H and R273H relax HR control in p53proficient cells. Utilizing an I-SceI-based reporter assay, we observed a moderate (1.5 Â) stimulation of HR upon expression of the mutant proteins in p53-proficient CV-1, but not in p53-deficient H1299 cells. Importantly, the stimulatory effect was exactly paralleled by an increase in the number of HR competent Sand G2-phase cells, which can well explain the enhanced recombination frequencies. Furthermore, the impact on HR exerted by the transactivation domain double-mutant L22Q/W23S and mutant R273P, both of which were reported to regulate HR independently of G1-arrest execution, is also exactly mirrored by cell-cycle behavior. These results are in contrast to previous concepts stating that the transactivation-independent impact of p53 on HR is a general phenomenon valid for replication-associated and also for directly induced double-strand break. Our data strongly suggest that the latter is largely mediated by cell-cycle regulation, a classical transactivation-dependent function of p53.

Efficacy and mechanism of action of marine alkaloid 3,10-dibromofascaplysin (DBF) were investigated in human prostate cancer (PCa) cells harboring different levels of drug resistance. Anticancer activity was observed across all cell lines examined without signs of cross-resistance to androgen receptor targeting agents (ARTA) or taxane based chemotherapy. Kinome analysis followed by functional investigation identified JNK1/2 to be one of the molecular targets of DBF in 22Rv1 cells. In contrast, no activation of p38 and ERK1/2 MAPKs was observed. Inhibition of the drug-induced JNK1/2 activation or of the basal p38 activity resulted in increased cytotoxicity of DBF, whereas an active ERK1/2 was identified to be important for anticancer activity of the alkaloid. Synergistic effects of DBF were observed in combination with PARP-inhibitor olaparib most likely due to the induction of ROS production by the marine alkaloid. In addition, DBF intensified effects of platinum-based drugs cisplatin and carboplatin, and taxane derivatives docetaxel and cabazitaxel. Finally, DBF inhibited AR-signaling and resensitized AR-V7-positive 22Rv1 prostate cancer cells to enzalutamide, presumably due to AR-V7 down-regulation. These findings propose DBF to be a promising novel drug candidate for the treatment of human PCa regardless of resistance to standard therapy.

Despite aggressive chemoradiation (CRT) protocols in the treatment of patients with head and neck squamous cell carcinomas (HNSCC), the outcome is still unfavorable. To improve therapy efficacy we had already successfully tested the multikinase inhibitor sorafenib in combination with irradiation (IR) in previous studies on HNSCC cell lines. In this study we investigated its effect on combined CRT treatment using cisplatin.Radio- and chemosensitivity with and without sorafenib was measured in four HNSCC cell lines and normal fibroblasts (NF) by colony formation assay. Apoptosis and cell cycle analysis were performed by flow cytometry.In HNSCC cells, sorafenib enhanced the antiproliferative effect of cisplatin without affecting apoptosis induction and with only minor effects on cell inactivation. Sorafenib added prior to irradiation enhanced cellular radiosensitivity in three of the tested HNSCC cell lines and caused massive overall cell inactivation when combined with CRT. In contrast, sorafenib did not radiosensitize NF and reduced cisplatin-induced cell inactivation. Cell inactivation by IR and cisplatin is further increased by the addition of sorafenib in HNSCC, but not in NF cells. Therefore, sorafenib is a promising candidate to improve therapy efficacy for HNSCC.

Signal transduction via protein kinases is of central importance in cancer biology and treatment. However, the clinical success of kinase inhibitors is often hampered by a lack of robust predictive biomarkers, which is also caused by the discrepancy between kinase expression and activity. Therefore, there is a need for functional tests to identify aberrantly activated kinases in individual patients. Here we present a systematic analysis of the tyrosine kinases in head and neck cancer using such a testfunctional kinome profiling. We detected increased tyrosine kinase activity in tumors compared with their corresponding normal tissue. Moreover, we identified members of the family of Src kinases (Src family kinases [SFK]) to be aberrantly activated in the majority of the tumors, which was confirmed by additional methods. We could also show that SFK hyperphosphorylation is associated with poor prognosis, while inhibition of SFK impaired cell proliferation, especially in cells with hyperactive SFK. In summary, functional kinome profiling identified SFK to be frequently hyperactivated in head and neck squamous cell carcinoma. SFK may therefore be potential therapeutic targets. These results furthermore demonstrate how functional tests help to increase our understanding of cancer biology and support the expansion of precision oncology.

The increase in cellular radiosensitivity by EGF receptor (EGFR) inhibition has been shown to be attributable to the induction of a G1-arrest in p53-proficient cells. Because EGFR targeting in combination with radiotherapy is used to treat head and neck squamous cell carcinomas (HNSCC) which are predominantly p53 mutated, we tested the effects of EGFR targeting on cellular radiosensitivity, proliferation, apoptosis, DNA repair and cell cycle control using a large panel of HNSCC cell lines. In these experiments EGFR targeting inhibited signal transduction, blocked proliferation and induced radiosensitization but only in some cell lines and only under normal (pre-plating) conditions. This sensitization was not associated with impaired DNA repair (53BP1 foci) or induction of apoptosis. However, it was associated with the induction of a lasting G2-arrest. Both, the radiosensitization and the G2-arrest were abrogated if the cells were re-stimulated (delayed plating) with actually no radiosensitization being detectable in any of the 14 tested cell lines. Therefore we conclude that EGFR targeting can induce a reversible G2 arrest in p53 deficient HNSCC cells, which does not consequently result in a robust cellular radiosensitization. Together with recent animal and clinical studies our data indicate that EGFR inhibition is no effective strategy to increase the radiosensitivity of HNSCC cells.

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