Mean global DNA methylation serves as independent prognostic marker in IDH-wildtype glioblastoma.

BACKGROUND: The IDH-wildtype glioblastoma (GBM) patients have a devastating prognosis. Here, we analyzed the potential prognostic value of global DNA methylation of the tumors. METHODS: DNA methylation of 492 primary samples and 31 relapsed samples, each treated with combination therapy, and of 148 primary samples treated with radiation alone were compared with patient survival. We determined the mean methylation values and estimated the immune cell infiltration from the methylation data. Moreover, the mean global DNA methylation of 23 GBM cell lines was profiled and correlated to their cellular radiosensitivity as measured by colony formation assay. RESULTS: High mean DNA methylation levels correlated with improved survival, which was independent from known risk factors (MGMT promoter methylation, age, extent of resection; P = 0.009) and methylation subgroups. Notably, this correlation was also independent of immune cell infiltration, as higher number of immune cells indeed was associated with significantly better OS but lower mean methylation. Radiosensitive GBM cell lines had a significantly higher mean methylation than resistant lines (P = 0.007), and improved OS of patients treated with radiotherapy alone was also associated with higher DNA methylation (P = 0.002). Furthermore, specimens of relapsed GBM revealed a significantly lower mean DNA methylation compared to the matching primary tumor samples (P = 0.041). CONCLUSIONS: Our results indicate that mean global DNA methylation is independently associated with outcome in glioblastoma. The data also suggest that a higher DNA methylation is associated with better radiotherapy response and less aggressive phenotype, both of which presumably contribute to the observed correlation with OS.

G2 checkpoint targeting via Wee1 inhibition radiosensitizes EGFRvIII-positive glioblastoma cells.

BACKGROUND: The gene of the Epidermal growth factor receptor (EGFR) is one of the most frequently altered genes in glioblastoma (GBM), with deletions of exons 2-7 (EGFRvIII) being amongst the most common genomic mutations. EGFRvIII is heterogeneously expressed in GBM. We already showed that EGFRvIII expression has an impact on chemosensitivity, replication stress, and the DNA damage response. Wee1 kinase is a major regulator of the DNA damage induced G2 checkpoint. It is highly expressed in GBM and its overexpression is associated with poor prognosis. Since Wee1 inhibition can lead to radiosensitization of EGFRvIII-negative (EGFRvIII-) GBM cells, we asked, if Wee1 inhibition is sufficient to radiosensitize also EGFRvIII-positive (EGFRvIII+) GBM cells. METHODS: We used the clinically relevant Wee1 inhibitor adavosertib and two pairs of isogenetic GBM cell lines with and without endogenous EGFRvIII expression exhibiting different TP53 status. Moreover, human GBM samples displaying heterogenous EGFRvIII expression were analyzed. Expression of Wee1 was assessed by Western blot and respectively immunohistochemistry. The impact of Wee1 inhibition in combination with irradiation on cell cycle and cell survival was analyzed by flow cytometry and colony formation assay. RESULTS: Analysis of GBM cells and patient samples revealed a higher expression of Wee1 in EGFRvIII+ cells compared to their EGFRvIII- counterparts. Downregulation of EGFRvIII expression by siRNA resulted in a strong decrease in Wee1 expression. Wee1 inhibition efficiently abrogated radiation-induced G2-arrest and caused radiosensitization, without obvious differences between EGFRvIII- and EGFRvIII+ GBM cells. CONCLUSION: We conclude that the inhibition of Wee1 is an effective targeting approach for the radiosensitization of both EGFRvIII- and EGFRvIII+ GBM cells and may therefore represent a promising new therapeutic option to increase response to radiotherapy.

Src family kinase targeting in head and neck tumor cells using SU6656, PP2 and dasatinib.

BACKGROUND: We have recently shown a frequent upregulation of Src-family kinases (SFK) in head and neck squamous cell carcinoma (HNSCC). Here we tested, if SFK targeting is effective especially in HNSCC cells with upregulated SFK signaling. METHODS: The impact of SFK inhibitors SU6656, PP2 and dasatinib on three HNSCC cell lines with different SFK activity levels was analyzed using proliferation and colony formation assays, Western blot and functional kinomics. RESULTS: Proliferation was blocked by all inhibitors in a micro-molar range. With respect to cell kill, dasatinib was most effective, while SU6656 showed moderate and PP2 minor effects. Cellular signaling was affected differently, with PP2 having no effect on SFK signaling while dasatinib probably has non-SFK specific effects. Only SU6656 showed clear SFK specific effects on signaling. CONCLUSION: The results demonstrate potential benefit of SFK inhibition in HNSCC but they also highlight challenges due to non-specificities of the different drugs.

Cytotoxic N-Methylpretrichodermamide B Reveals Anticancer Activity and Inhibits P-Glycoprotein in Drug-Resistant Prostate Cancer Cells.

N-methylpretrichodermamide B (NB) is a biologically active epidithiodiketopiperazine isolated from several strains of the algae-derived fungus Penicillium sp. Recently, we reported the first data on its activity in human cancer cells lines in vitro. Here, we investigated the activity, selectivity, and mechanism of action of NB in human prostate cancer cell lines, including drug-resistant subtypes. NB did not reveal cross-resistance to docetaxel in the PC3-DR cell line model and was highly active in hormone-independent 22Rv1 cells. NB-induced cell death was stipulated by externalization of phosphatidylserine and activation of caspase-3. Moreover, inhibition of caspase activity by z-VAD(OMe)-fmk did not affect NB cytotoxicity, suggesting a caspase-independent cell death induced by NB. The compound has a moderate p-glycoprotein (p-gp) substrate-like affinity and can simultaneously inhibit p-gp at nanomolar concentrations. Therefore, NB resensitized p-gp-overexpressing PC3-DR cells to docetaxel. A kinome profiling of the NB-treated cells revealed, among other things, an induction of mitogen-activated protein kinases JNK1/2 and p38. Further functional analysis confirmed an activation of both kinases and indicated a prosurvival role of this biological event in the cellular response to the treatment. Overall, NB holds promising anticancer potential and further structure-activity relationship studies and structural optimization are needed in order to improve its biological properties.

Increased replication stress and R-loop accumulation in EGFRvIII-expressing glioblastoma present new therapeutic opportunities.

Background: The oncogene epidermal growth factor receptor variant III (EGFRvIII) is expressed in approximately one-third of all glioblastomas (GBMs). So far it is not clear if EGFRvIII expression induces replication stress in GBM cells, which might serve as a therapeutical target. Methods: Isogenetic EGFRvIII- and EGFRvIII+ cell lines with endogenous EGFRvIII expression were used. Markers of oncogenic and replication stress such as γH2AX, RPA, 53BP1, ATR, and CHK1 were analyzed using western blot, immunofluorescence, and flow cytometry. The DNA fiber assay was performed to analyze replication, transcription was measured by incorporation of EU, and genomic instability was investigated by micronuclei and CGH-Array analysis. Immunohistochemistry staining was used to detect replication stress markers and R-loops in human GBM samples. Results: EGFRvIII+ cells exhibit an activated replication stress response, increased spontaneous DNA damage, elevated levels of single-stranded DNA, and reduced DNA replication velocity, which are all indicative characteristics of replication stress. Furthermore, we show here that EGFRvIII expression is linked to increased genomic instability. EGFRvIII-expressing cells display elevated RNA synthesis and R-loop formation, which could also be confirmed in EGFRvIII-positive GBM patient samples. Targeting replication stress by irinotecan resulted in increased sensitivity of EGFRvIII+ cells. Conclusion: This study demonstrates that EGFRvIII expression is associated with increased replication stress, R-loop accumulation, and genomic instability. This might contribute to intratumoral heterogeneity but may also be exploited for individualized therapy approaches.

Kinomic comparison of snap frozen and ex vivo-cultured head and neck tumors.

OBJECTIVES: The use of primary tumor tissue in experimental and pre-clinical cancer research is becoming increasingly important. Especially the use of tissue slice cultures of tumor specimen, so called ex vivo cultures or tumor explants, promises functional analysis under approximate physiological conditions. This includes screening and testing of targeted therapeutics directed against deregulated protein kinases. However, it is unclear if ex vivo cultures indeed represent the in situ situation especially with respect to very sensitive and transient molecular processes such as kinase dependent signaling. We now asked here, if and to what extent ex vivo culturing affects kinase activity. MATERIALS AND METHODS: We analyzed the activity of protein tyrosine kinases (PTK) using functional kinome profiling of either snap frozen or ex vivo-cultured tumor tissue samples of head and neck cancer patients. RESULTS: Although we observed a quantitative decline in overall kinase activity after 24 h or 48 h of ex vivo cultivation, we most importantly noticed that the signaling characteristics were conserved in most samples; approximately two thirds of all ex vivo-cultured samples displayed a signaling pattern which was qualitatively comparable to the parental tumor. We could also demonstrate kinase inhibition by treatment of ex vivo slice cultures with the multi-kinase inhibitor staurosporine, although higher concentrations were needed compared to cell cultures. CONCLUSION: We here demonstrate that the tyrosine kinase dependent signaling is conserved under exvivo culturing conditions in the majority of samples, which highlights the power of this method in experimental and pre-clinical cancer research.

Analyzing tyrosine kinase activity in head and neck cancer by functional kinomics: Identification of hyperactivated Src family kinases as prognostic markers and potential targets.

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 test-functional 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.

Efficacy and Mechanism of Action of Marine Alkaloid 3,10-Dibromofascaplysin in Drug-Resistant Prostate Cancer Cells.

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.

Marine alkaloid monanchoxymycalin C: a new specific activator of JNK1/2 kinase with anticancer properties.

Monanchoxymycalin C (MomC) is a new marine pentacyclic guanidine alkaloid, recently isolated from marine sponge Monanchora pulchra by us. Here, anticancer activity and mechanism of action was investigated for the first time using a human prostate cancer (PCa) model. MomC was active in all PCa cell lines at low micromolar concentrations and induced an unusual caspase-independent, non-apoptotic cell death. Kinase activity screening identified activation of mitogen-activated protein kinase (MAPK) c-Jun N-terminal protein kinase (JNK1/2) to be one of the primary molecular mechanism of MomC anticancer activity. Functional assays demonstrated a specific and selective JNK1/2 activation prior to the induction of other cell death related processes. Inhibition of JNK1/2 by pretreatment with the JNK-inhibitor SP600125 antagonized cytotoxic activity of the marine compound. MomC caused an upregulation of cytotoxic ROS. However, in contrast to other ROS-inducing agents, co-treatment with PARP-inhibitor olaparib revealed antagonistic effects indicating an active PARP to be necessary for MomC activity. Interestingly, although no direct regulation of p38 and ERK1/2 were detected, active p38 kinase was required for MomC efficacy, while the inhibition of ERK1/2 increased its cytotoxicity. In conclusion, MomC shows promising activity against PCa, which is exerted via JNK1/2 activation and non-apoptotic cell death.

Mass Spectrometric Comparison of HPV-Positive and HPV-Negative Oropharyngeal Cancer.

Squamous cell carcinoma of the head and neck (HNSCC) consist of two distinct biological entities. While the numbers of classical, tobacco-induced HNSCC are declining, tumors caused by human papillomavirus (HPV) infection are increasing in many countries. HPV-positive HNSCC mostly arise in the oropharynx and are characterized by an enhanced sensitivity towards radiotherapy and a favorable prognosis. To identify molecular differences between both entities on the protein level, we conducted a mass spectrometric comparison of eight HPV-positive and nine HPV-negative oropharyngeal tumors (OPSCC). Overall, we identified 2051 proteins, of which 31 were found to be differentially expressed. Seventeen of these can be assorted to three functional groups, namely DNA replication, nuclear architecture and cytoskeleton regulation, with the differences in the last group potentially reflecting an enhanced migratory and invasive capacity. Furthermore, a number of identified proteins have been described to directly impact on DNA double-strand break repair or radiation sensitivity (e.g., SLC3A2, cortactin, RBBP4, Numa1), offering explanations for the differential prognosis. The unequal expression of three proteins (SLC3A2, MCM2 and lamin B1) was confirmed by immunohistochemical staining using a tissue microarray containing 205 OPSCC samples. The expression levels of SLC3A2 and lamin B1 were found be of prognostic relevance in patients with HPV-positive and HPV-negative OPSCC, respectively.

EGFRvIII upregulates DNA mismatch repair resulting in increased temozolomide sensitivity of MGMT promoter methylated glioblastoma.

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.

Analyzing expression and phosphorylation of the EGF receptor in HNSCC.

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.

Receptor tyrosine kinase MET as potential target of multi-kinase inhibitor and radiosensitizer sorafenib in HNSCC.

BACKGROUND: The multi-kinase inhibitor sorafenib displays antitumoral effects in head and neck squamous cell carcinoma (HNSCC); however, the targeted kinases are unknown. Here we aimed to identify those kinases to determine the mechanism of sorafenib-mediated effects and establish candidate biomarkers for patient stratification. METHODS: The effects of sorafenib and MET inhibitors crizotinib and SU11274 were analyzed using a slide-based antibody array, Western blotting, proliferation, and survival assays. X-rays were used for irradiations. RESULTS: Sorafenib inhibited auto-phosphorylation of epidermal growth factor receptor and MET, which has not been described previously. MET expression in HNSCC cells was not always associated with activity/phosphorylation. Furthermore, sorafenib-dependent cell kill and radiosensitization was not associated with MET level. Although MET inhibitors blocked proliferation, they caused only mild cytotoxicity and no radiosensitization. CONCLUSION: We identified MET as a new potential target of sorafenib. However, MET inhibition is not the cause for sorafenib-mediated cytotoxicity or radiosensitization.

Radiosensitization of HNSCC cells by EGFR inhibition depends on the induction of cell cycle arrests.

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.

Effect of sorafenib on cisplatin-based chemoradiation in head and neck cancer cells.

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.

Quantitative proteomics unveiled: Regulation of DNA double strand break repair by EGFR involves PARP1.

BACKGROUND: EGFR inhibition blocks DNA double strand break (DSB) repair but the detailed mechanisms are still unclear. We asked whether EGFR inhibition blocks DSB repair by reducing the X-ray-induced phosphorylation of repair proteins using a phosphoproteomic approach. MATERIALS AND METHODS: Using UT-SCC5 and SAS head and neck cancer cells we established a differential phosphoproteomic approach for quantitative analysis of DNA repair proteins by stable isotope labeling with amino acids. Nuclear phosphoproteins were isolated and analyzed by liquid chromatography/tandem mass spectrometry. Erlotinib, PD98059 and olaparib were used to inhibit EGFR, MEK1/2 and PARP1, respectively. PARP1 was knocked down by siRNA. DSB repair was measured by quantifying residual 53BP1 foci. RESULTS: Over 150 nuclear phosphoproteins were quantified after irradiation, including 24 DNA repair proteins. Two of these, including PARP1, were consistently reduced in both cell lines upon erlotinib treatment. PARP1 inhibition or knock-down and EGFR inhibition resulted in an analog number of residual foci which was not further increased by combination of both strategies. MEK1/2 inhibition with or without blockage of EGFR or PARP1 caused similar effects. CONCLUSION: We have established a powerful, quantitative phosphoproteomic approach to investigate regulatory mechanisms in DSB repair, dependent on protein phosphorylation after irradiation. Using this approach we have identified PARP1 as a mediator of EGFR/MEK-dependent regulation of DSB repair.