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Numerous natural substances have anti-cancer properties. Especially indigenous people use aqueous plant extracts for tea or ointments including Dioscorea sansibarensis Pax to treat various diseases. The aim of this study was to evaluate the cytotoxic and radiosensitizing potential of aqueous extracts from Dioscorea sansibarensis Pax collected from Kenya in a panel of HPV-negative and -positive head and neck squamous cell carcinoma (HNSCC) cells grown in three-dimensional laminin-rich extracellular matrix (3D lrECM). The results show cytotoxicity, radiosensitization and increased levels of residual double strand breaks (DBS) by Dioscorea sansibarensis Pax extracts in HPV-negative and -positive HNSCC models in a concentration- and cell model-dependent manner. Application of ROS scavengers indicated an association between ROS-induced DSB and radiosensitization through Dioscorea sansibarensis Pax pretreatment. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based characterization of Dioscorea sansibarensis Pax identified the main components of the extract including camptothecin. Overall, Dioscorea sansibarensis Pax aqueous extracts alone and in combination with X-ray irradiation showed effective anticancer properties, which are worthy of further mechanistic investigation.
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The incidence rate of human papillomavirus-driven oropharyngeal cancer (HPV-OPC) is increasing in countries with high human development index. HPV cell-free DNA (cfDNA) isolated from 3 to 4 mL blood plasma has been successfully used for therapy surveillance. A highly discussed application of HPV-cfDNA is early detection of HPV-OPC. This requires sensitive and specific cfDNA detection as cfDNA levels can be very low. To study the predictive power of pre-diagnostic HPV-cfDNA, archived samples from epidemiological cohorts with limited plasma volume are an important source. To establish a cfDNA detection workflow for low plasma volumes, we compared cfDNA purification methods [MagNA Pure 96 (MP96) and QIAamp ccfDNA/RNA] and digital PCR systems (Biorad QX200 and QIAGEN QIAcuity One). Final assay validation included 65 low-volume plasma samples from oropharyngeal cancer (OPC) patients with defined HPV status stored for 2-9 years. MP96 yielded a 28% higher cfDNA isolation efficiency in comparison to QIAamp. Both digital PCR systems showed comparable analytical sensitivity (6-17 copies for HPV16 and HPV33), but QIAcuity detected both types in the same assay. In the validation set, the assay had 80% sensitivity (n = 28/35) for HPV16 and HPV33 and a specificity of 97% (n = 29/30). In samples with ≥750 µL plasma, the sensitivity was 85% (n = 17/20), while in samples with <750 µL plasma, it was 73% (n = 11/15). Despite the expected drop in sensitivity with decreased plasma volume, the assay is sensitive and highly specific even in low-volume samples and thus suited for studies exploring HPV-cfDNA as an early HPV-OPC detection marker in low-volume archival material.IMPORTANCEHPV-OPC has a favorable prognosis compared to HPV-negative OPC. However, the majority of tumors is diagnosed after regional spread, thus making intensive treatment necessary. This can cause lasting morbidity with a large impact on quality of life. One potential method to decrease treatment-related morbidity is early detection of the cancer. HPV cfDNA has been successfully used for therapy surveillance and has also been detected in pre-diagnostic samples of HPV-OPC patients. These pre-diagnostic samples are only commonly available from biobanks, which usually only have small volumes of blood plasma available. Hence, we have developed a workflow optimized for small-volume archival samples. With this method, a high sensitivity can be achieved despite sample limitations, making it suitable to conduct further large-scale biobank studies of HPV-cfDNA as a prognostic biomarker for HPV-OPC.
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Ácidos Nucleicos Livres , DNA Viral , Neoplasias Orofaríngeas , Infecções por Papillomavirus , Reação em Cadeia da Polimerase , Humanos , DNA Viral/sangue , DNA Viral/genética , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/sangue , Infecções por Papillomavirus/virologia , Ácidos Nucleicos Livres/sangue , Reação em Cadeia da Polimerase/métodos , Neoplasias Orofaríngeas/virologia , Neoplasias Orofaríngeas/sangue , Neoplasias Orofaríngeas/diagnóstico , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Feminino , Sensibilidade e Especificidade , Masculino , Pessoa de Meia-Idade , Idoso , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus HumanoRESUMO
PURPOSE: Human papilloma virus (HPV) positive head and neck squamous cell carcinoma (HNSCC) tumors respond significantly better to anticancer treatments. It is assumed to be due to a better response to radiotherapy (RT), and presumably to an increased immunogenicity. However, little is known how the immune phenotype of HNSCC tumor cells is modulated by standard treatment, namely by radiochemotherapy (RCT). METHODS: Therefore, we aimed to examine the impact of the HPV status on the immune phenotype of HNSCC cell lines following RCT with 5 × 3Gy or 1 × 19.3Gy and/or docetaxel, by analyzing cell death, release of damage-associated molecular patterns (DAMPs), surface expression of immune checkpoint molecules (ICMs) and the impact on activation of human monocyte-derived dendritic cells (hmDCs). RESULTS: Cell death induction and Hsp70 release following RCT was independent of the HPV status, and RCT significantly increased the expression of the immune suppressive ICMs PD-L1, PD-L2 and HVEM. An immune stimulatory ICM, CD137, was significantly increased following RCT only on HPV-positive cell lines, as well as the release of HMGB1. Although the treatment increased cell death and modulated ICM expression in HNSCC, the hmDCs were not activated after co-incubation with treated tumor cells. CONCLUSION: Our data with the HPV-dependent release of HMGB1 and increased expression of CD137 following RCT provide a hint for increased immunogenicity underlining the better prognosis for HPV positive tumors following RCT.
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Carcinoma de Células Escamosas , Proteína HMGB1 , Neoplasias de Cabeça e Pescoço , Infecções por Papillomavirus , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Docetaxel/farmacologia , Docetaxel/uso terapêutico , Proteína HMGB1/genética , Proteína HMGB1/uso terapêutico , Infecções por Papillomavirus/complicações , Carcinoma de Células Escamosas/metabolismo , FenótipoRESUMO
Objectives: In head and neck squamous cell carcinoma (HNSCC), tumors negative for Human Papillomavirus (HPV) remain a difficult to treat entity and the morbidity of current multimodal treatment is high. Radiotherapy in combination with molecular targeting could represent suitable, less toxic treatment options especially for cisplatin ineligible patients. Therefore, we tested dual targeting of PARP and the intra-S/G2 checkpoint through Wee1 inhibition for its radiosensitizing capacity in radioresistant HPV-negative HNSCC cells. Materials and methods: Three radioresistant HPV-negative cell lines (HSC4, SAS, UT-SCC-60a) were treated with olaparib, adavosertib and ionizing irradiation. The impact on cell cycle, G2 arrest and replication stress was assessed through flow cytometry after DAPI, phospho-histone H3 and γH2AX staining. Long term cell survival after treatment was determined through colony formation assay and DNA double-strand break (DSB) levels were assessed through quantification of nuclear 53BP1 foci in cell lines and patient-derived HPV± tumor slice cultures. Results: Wee1 and dual targeting induced replication stress but failed to effectively inhibit radiation-induced G2 cell cycle arrest. Single as well as combined inhibition increased radiation sensitivity and residual DSB levels, with the largest effects induced through dual targeting. Dual targeting also enhanced residual DSB levels in patient-derived slice cultures from HPV-negative but not HPV+ HNSCC (5/7 vs. 1/6). Conclusion: We conclude that the combined inhibition of PARP and Wee1 results in enhanced residual DNA damage levels after irradiation and effectively sensitizes radioresistant HPV-negative HNSCC cells. Ex vivo tumor slice cultures may predict the response of individual patients with HPV-negative HNSCC to this dual targeting approach.
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Recent advances in understanding the tumor's biology in line with a constantly growing number of innovative technologies have prompted characterization of patients' individual malignancies and may display a prerequisite to treat cancer at its patient individual tumor vulnerability. In recent decades, radiation- induced signaling and tumor promoting local events for radiation sensitization were explored in detail, resulting the development of novel molecular targets. A multitude of pharmacological, genetic, and immunological principles, including small molecule- and antibody-based targeted strategies, have been developed that are suitable for combined concepts with radiation (RT) or chemoradiation therapy (CRT). Despite a plethora of promising experimental and preclinical findings, however, so far, only a very limited number of clinical trials have demonstrated a better outcome and/or patient benefit when RT or CRT are combined with targeted agents. The current review aims to summarize recent progress in molecular therapies targeting oncogenic drivers, DNA damage and cell cycle response, apoptosis signaling pathways, cell adhesion molecules, hypoxia, and the tumor microenvironment to impact therapy refractoriness and to boost radiation response. In addition, we will discuss recent advances in nanotechnology, e.g., RNA technologies and protein-degrading proteolysis-targeting chimeras (PROTACs) that may open new and innovative ways to benefit from molecular-targeted therapy approaches with improved efficacy.
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Antineoplásicos , Neoplasias , Humanos , Terapia de Alvo Molecular , Neoplasias/radioterapia , Neoplasias/tratamento farmacológico , Antineoplásicos/uso terapêutico , Transdução de Sinais , Microambiente TumoralRESUMO
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.
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Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/radioterapia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Neoplasias Encefálicas/radioterapia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/uso terapêuticoRESUMO
PURPOSE: Immune checkpoint inhibition is a therapeutic option in many cancer entities. In head and neck squamous cell carcinoma (HNSCC) targeting of the PD-1/PD-L1 (B7-H1) axis is approved in recurrent/metastatic disease and is being explored in the curative setting. Here, we evaluated two related members of the B7 family, B7-H3 & B7-H4, for their prognostic impact under standard treatment. METHODS: A tissue microarray (TMA) of a single center HNSCC cohort was stained for B7-H3 and B7-H4. Staining intensity and the number of tumor cells stained were assessed, and the expression was scored according to an established algorithm. Staining scores were correlated with clinicopathological parameters and associated with patient survival. mRNA levels of both proteins were associated with patient outcome using the TCGA dataset. RESULTS: mRNA levels of B7-H3 and B7-H4 were not significantly associated with patient survival. TMA analysis revealed interpretable protein staining in 408 samples. Strong staining was the most frequent category for B7-H3 and no staining for B7-H4. In patients with p16-negative oropharyngeal SCC (OPSCC) and in a pooled cohort consisting of p16-negative OPSCC, laryngeal, hypopharyngeal and oral cavity SCC, strong B7-H3 expression was associated with better overall survival. For the latter cohort, this was in part due to reduced lymph node involvement. B7-H3 expression in p16-positive OPSCC and B7-H4 expression were not associated with outcome. CONCLUSION: Despite a possible role in tumor immune escape, B7-H3 was associated with favorable prognosis in HPV-negative HNSCC in our cohort. The underlying mechanisms and a potential impact for B7-H3 targeting remain to be elucidated.
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Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Prognóstico , Antígeno B7-H1/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/genética , RNA Mensageiro , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/análiseRESUMO
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.
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Neoplasias de Cabeça e Pescoço , Quinases da Família src , Humanos , Dasatinibe/farmacologia , Quinases da Família src/metabolismo , Pirimidinas/farmacologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular TumoralRESUMO
Background: The lack of detectable precancerous lesions poses challenges to the early detection of human papillomavirus-driven oropharyngeal cancer (HPV-OPC). Antibodies against HPV16 early proteins, especially E6, are uniquely sensitive and specific biomarkers detectable years prior to HPV-OPC diagnosis. Thus, HPV16 early protein serology warrants clinical investigation for HPV-OPC screening. Methods: Using multiplex serology, we analyzed HPV16 serum antibodies of the first 5000 participants (n=4,424 sera, recruited 2016-2017) of the Hamburg City Health Study, a population-based prospective cohort (45-74 years). Participants seropositive for HPV16 E6 and at least one additional early protein (E1, E2, E7) were considered at high risk for HPV-OPC development and invited to six-monthly non-invasive head and neck follow-up (FU) examinations (visual inspection, endoscopy, ultrasonography, performed 2019-2020). Participants with suspicious lesions were examined by magnetic resonance imaging and panendoscopy with biopsy. Histologically confirmed OPC cases were treated according to standard of care. Findings: In total, 35 out of 4,424 study participants (0·8%, 95% confidence interval (CI) 0·6-1·1%) were HPV16 E6 seropositive. Among these, eleven (0·3%, 95%CI 0·1-0·5%) were considered at high risk for HPV-OPC of which nine were successfully re-contacted and invited to regular clinical FU examinations. Two males and one female were diagnosed with stage I HPV-OPC within 1·3 years of clinical FU (3-4 years after initial blood draw), representing one diagnosis of prevalent advanced disease, one incident diagnosis of advanced disease, and one incident diagnosis of early disease. The remaining participants showed no detectable signs of cancer, and undergo regular examinations (median clinical FU: 1·0 years, median total FU from blood draw to last clinical FU visit: 4·7 years). Interpretation: HPV16 early antibodies allowed identifying three asymptomatic stage I HPV-OPC patients, out of eleven participants considered at high risk. However, two of the three cases already showed signs of advanced disease at diagnosis. Targeting multiple early proteins may considerably improve the positive predictive value of HPV16 serology and may have clinical utility for HPV-OPC screening. Funding: This work was funded by DKFZ and UKE intramural funding.
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Patients with human papillomavirus-positive squamous cell carcinoma of the head and neck (HPV+ HNSCC) have a favorable prognosis compared to those with HPV-negative (HPV-) ones. We have shown previously that HPV+ HNSCC cell lines are characterized by enhanced radiation sensitivity and impaired DNA double-strand break (DSB) repair. Since then, various publications have suggested a defect in homologous recombination (HR) and dysregulated expression of DSB repair proteins as underlying mechanisms, but conclusions were often based on very few cell lines. When comparing the expression levels of suggested proteins and other key repair factors in 6 HPV+ vs. 5 HPV- HNSCC strains, we could not confirm most of the published differences. Furthermore, HPV+ HNSCC strains did not demonstrate enhanced sensitivity towards PARP inhibition, questioning a general HR defect. Interestingly, our expression screen revealed minimal levels of the central DNA damage response kinase ATM in the two most radiosensitive HPV+ strains. We therefore tested whether insufficient ATM activity may contribute to the enhanced cellular radiosensitivity. Irrespective of their ATM expression level, radiosensitive HPV+ HNSCC cells displayed DSB repair kinetics similar to ATM-deficient cells. Upon ATM inhibition, HPV+ cell lines showed only a marginal increase in residual radiation-induced γH2AX foci and induction of G2 cell cycle arrest as compared to HPV- ones. In line with these observations, ATM inhibition sensitized HPV+ HNSCC strains less towards radiation than HPV- strains, resulting in similar levels of sensitivity. Unexpectedly, assessment of the phosphorylation kinetics of the ATM targets KAP-1 and Chk2 as well as ATM autophosphorylation after radiation did not indicate directly compromised ATM activity in HPV-positive cells. Furthermore, ATM inhibition delayed radiation induced DNA end resection in both HPV+ and HPV- cells to a similar extent, further suggesting comparable functionality. In conclusion, DNA repair kinetics and a reduced effectiveness of ATM inhibition clearly point to an impaired ATM-orchestrated DNA damage response in HPV+ HNSCC cells, but since ATM itself is apparently functional, the molecular mechanisms need to be further explored.
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The receptor tyrosine kinase Axl is described to promote migration, metastasis and resistance against molecular targeting, radiotherapy, and chemotherapy in various tumor entities, including head and neck squamous cell carcinoma (HNSCC). Since clinical data on Axl and its ligand Gas6 in HNSCC are sparse, we assessed the association of Axl and Gas6 expression with patient survival in a single center retrospective cohort in a tissue microarray format. Expression was evaluated manually using an established algorithm and correlated with clinicopathological parameters and patient survival. A number of 362 samples yielded interpretable staining, which did not correlate with T- and N-stage. Protein expression levels were not associated with the survival of patients with p16-positive oropharyngeal SCC. In HPV-negative tumors, Axl expression did not impact patients treated with primary or adjuvant radio(chemo)therapy, but was significantly associated with inferior overall and recurrence-free survival in patients treated with surgery alone. Gas6 was a positive predictor of survival in patients whose treatment included radiotherapy. Associations remained significant in multivariable analysis. Our data question a meaningful contribution of the Axl/Gas6 pathway to radio-resistance in HNSCC and instead suggest that strong Axl expression identifies tumors requiring adjuvant radio(chemo)therapy after surgery.
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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.
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Since the introduction of Cetuximab as a biological molecule against Epidermal Growth Factor Receptor (EGFR), its use in the cancers of head and neck region is widely explored. With the recognition that EGFR expression is associated with radioresistance and poor prognosis, incorporation of an anti-EGFR agent along with Radiotherapy (RT) is a logical and attractive option. Cetuximab in combination with RT as Bio-Radiotherapy (BRT) is considered one of the standard treatment modalities in Locally Advanced Head and Neck Squamous Cell Cancers (LA-HNSCC). Many important phase-III clinical trials were undertaken simultaneously, where the use of Cetuximab BRT was tested in various clinical scenarios with different hypothesis. With the studies still ongoing and the results awaited, its use was continued in clinical practice. Today the results are out and definitely not encouraging. After the initial success, Cetuximab has miserably failed to win over cisplatin based chemoradiation which is the current standard of care in LA-HNSCC. Hence, it is the need of the hour to re-evaluate and define the present role of Cetuximab in the definitive management of LA-HNSCC in the light of the latest clinical evidence..
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BACKGROUND: HPV-positive head and neck squamous cell carcinoma of the oropharynx (OPSCC) are more sensitive towards radiation than HPV-negative OPSCC. Two main theories exist regarding the underlying mechanism. Stronger lymphocyte infiltration points to an enhanced immunogenicity, whereas data from HPV-positive HNSCC cell lines suggest an enhanced cellular radiosensitivity based on a defect in DNA double-strand break (DSB) repair. The critical limitation of the latter theory is that the evidence was largely derived from a small number of established HPV-positive HNSCC cell lines. METHODS AND MATERIALS: Fresh patient-derived OPSCC samples were cut in 400 µm sections and cultured on cell culture inserts. Slice cultures were irradiated, in part combined with ATM inhibition, and fixed and frozen after 2 and 24 h. DSBs were analyzed by quantification of 53BP1 foci in nuclei co-stained with the SCC marker p63 via immunofluorescence microscopy. RESULTS: Ex vivo OPSCC tumor slice cultures maintained stable oxygenation and proliferation characteristics for at least 3 days. Areas of p63-positivity in immunofluorescence microscopy matched histologically confirmed tumor cell areas in serial sections, indicating the suitability of p63 as a tumor cell marker. p63-positive nuclei in HPV-positive OPSCC tissues (n = 14) showed profoundly elevated numbers of residual radiation-induced DSBs as compared to those from HPV-negative OPSCC (n = 12) (3 Gy: on average 4.9 vs. 1.2 foci per nucleus; p < 0.0001). Within the HPV-positive subgroup, samples derived from patients with a smoking history of less than 10 pack years demonstrated higher residual DSBs as compared to those derived from patients with 10 or more pack years (3 Gy: on average 6.5 vs. 3.2 foci per nucleus; p = 0.0105). Additional ATM inhibition resulted in a substantial increase in residual foci in all 4 HPV-negative samples tested but strikingly only in 2 out of 11 HPV-positive samples. CONCLUSIONS: In summary, our data provide robust, cell line-independent experimental evidence for an intrinsic DSB repair deficiency in HPV-positive OPSCC, strongly suggesting a meaningful contribution to the enhanced clinical radiosensitivity. The reduced effectiveness of ATM inhibition indicates a defect in the ATM-orchestrated DNA damage response. Lower numbers of residual 53BP1 nuclear foci in the ex vivo assay may identify HPV-positive patients with effective DSB repair who should potentially be excluded from de-intensification approaches.
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Neoplasias de Cabeça e Pescoço , Neoplasias Orofaríngeas , Infecções por Papillomavirus , Linhagem Celular Tumoral , DNA , Reparo do DNA , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Neoplasias Orofaríngeas/radioterapia , Orofaringe/metabolismo , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/metabolismoRESUMO
PURPOSE: The incidence of head and neck squamous cell carcinomas (HNSCC) is increasing worldwide, especially when triggered by the human papilloma virus (HPV). Radiotherapy has immune-modulatory properties, but the role of macrophages present in HNSCC and having contact with irradiated tumor cells remains unclear. The influence of irradiated (2â¯× 5Gy) HNSCC cells on the (re-)polarization and phagocytosis of human macrophages, either non-polarized or with a more M1 or M2 phenotype, was therefore investigated. METHODS: Human monocytes were differentiated with the hematopoietic growth factors MCSF (m) or GM-CSF (g) and additionally pre-polarized with either interleukin (IL)-4 and IL-10 or interferon (IFN)-γ and lipopolysaccharides (LPS), respectively. Subsequently, they were added to previously irradiated (2â¯× 5Gy) and mock-treated HPV-positive (UD-SCC-2) and HPV-negative (Cal33) HNSCC cells including their supernatants. RESULTS: The HNSCC cells treated with hypofractionated irradiation died via apoptosis and were strongly phagocytosed by M0m and M2 macrophages. M0g and M1 macrophages phagocytosed the tumor cells to a lesser extent. Irradiated HNSCC cells were better phagocytosed by M1 macrophages compared to mock-treated controls. The polarization status of the macrophages was not significantly changed, except for the expression of CD206 on M2 macrophages, which was reduced after phagocytosis of irradiated HPV-negative cells. Further, a significant increase in the uptake of irradiated HPV-positive cells by M0g macrophages when compared to HPV-negative cells was observed. CONCLUSION: HNSCC cells treated with hypofractionated irradiation foster phagocytosis by anti-tumorigenic M1 macrophages. The data provide the first evidence on the impact of the HPV status of HNSCC cells on the modulation of the macrophage response to irradiated tumor cells.
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Neoplasias de Cabeça e Pescoço , Macrófagos , Apoptose , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Macrófagos/metabolismo , Papillomaviridae , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapiaRESUMO
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.
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Antineoplásicos , Neoplasias de Cabeça e Pescoço , Antineoplásicos/uso terapêutico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina QuinasesRESUMO
In head and neck squamous cell carcinoma (HNSCC), tumors positive for human papillomavirus (HPV) represent a distinct biological entity with favorable prognosis. An enhanced radiation sensitivity of these tumors is evident in the clinic and on the cellular level when comparing HPV-positive and HPV-negative HNSCC cell lines. We could show that the underlying mechanism is a defect in DNA double-strand break repair associated with a profound and sustained G2 arrest. This defect can be exploited by molecular targeting approaches additionally compromising the DNA damage response to further enhance their radiation sensitivity, which may offer new opportunities in the setting of future de-intensified regimes. Against this background, we tested combined targeting of PARP and the DNA damage-induced intra-S/G2 cell cycle checkpoints to achieve effective radiosensitization. Enhancing CDK1/2 activity through the Wee1 inhibitor adavosertib or a combination of Wee1 and Chk1 inhibition resulted in an abrogation of the radiation-induced G2 cell cycle arrest and induction of replication stress as assessed by γH2AX and chromatin-bound RPA levels in S phase cells. Addition of the PARP inhibitor olaparib had little influence on these endpoints, irrespective of checkpoint inhibition. Combined PARP/Wee1 targeting did not result in an enhancement in the absolute number of residual, radiation induced 53BP1 foci as markers of DNA double-strand breaks but it induced a shift in foci numbers from S/G2 to G1 phase cells. Most importantly, while sole checkpoint or PARP inhibition induced moderate radiosensitization, their combination was clearly more effective, while exerting little effect in p53/G1 arrest proficient normal human fibroblasts, thus indicating tumor specificity. We conclude that the combined inhibition of PARP and the intra-S/G2 checkpoint is a highly effective approach for the radiosensitization of HPV-positive HNSCC cells and may represent a viable alternative for the current standard of concomitant cisplatin-based chemotherapy. In vivo studies to further evaluate the translational potential are highly warranted.
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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.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Quinases da Família src/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Fosforilação , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Estudos Retrospectivos , Taxa de Sobrevida , Análise Serial de Tecidos , Células Tumorais Cultivadas , Quinases da Família src/antagonistas & inibidoresRESUMO
Chemoradiation, either alone or in combination with surgery or induction chemotherapy, is the current standard of care for most locally advanced solid tumors. Though chemoradiation is usually performed at the maximum tolerated doses of both chemotherapy and radiation, current cure rates are not satisfactory for many tumor entities, since tumor heterogeneity and plasticity result in chemo- and radioresistance. Advances in the understanding of tumor biology, a rapidly growing number of molecular targeting agents and novel technologies enabling the in-depth characterization of individual tumors, have fuelled the hope of entering an era of precision oncology, where each tumor will be treated according to its individual characteristics and weaknesses. At present though, molecular targeting approaches in combination with radiotherapy or chemoradiation have not yet proven to be beneficial over standard chemoradiation treatment in the clinical setting. A promising approach to improve efficacy is the combined usage of two targeting agents in order to inhibit backup pathways or achieve a more complete pathway inhibition. Here we review preclinical attempts to utilize such dual targeting strategies for future tumor radiosensitization.
RESUMO
Trifluridine/tipiracil (FTD/TPI; marketed as Lonsurf®) has shown clinically relevant activity after fluoropyrimidine failure in colorectal cancer and may thus be of increased efficacy compared with current standard capecitabine chemoradiation. Here we investigated the colorectal cancer cell lines HT29, HCT116, SW48 and Caco-2 to provide a preclinical rationale for FTD/TPI-based chemoradiation treatment. All lines incorporated similar amounts of FTD, irrespective of treatment concentration and duration, then arrested in S phase, showed persistent γH2AX induction and eventually underwent endoreplication, resulting in polyploidy. Clonogenic assays performed for four combined treatment schedules demonstrated additivity for treatments given within 6 h of each other. However, 24 h FTD/TPI treatment prior to irradiation caused 1.6-2.4 fold radiosensitisation. Combined in vivo treatment was well tolerated and caused a marked tumour growth delay, similar to capecitabine radiochemotherapy regimes. Prolonged S phase arrest, persistent γH2AX signalling, endoreplication and polyploidy may contribute to the cytotoxicity of FTD/TPI. The strong radiosensitising effect observed in vitro after prolonged treatment with FTD/TPI and equivalence with capecitabine-based chemoradiation in vivo support a daily fractionated combined regime of FTD/TPI and radiation in rectal cancer treatment. This is now being tested in a phase I/II clinical trial (NCT04177602).