Search

Simple Summary: In this study, a new subgroup of UCHL1-related cancers was identified across multiple solid tumor entities and was characterized by comprehensive multi-omics analysis. Common features of UCHL1-related cancers are an increased genomic instability and a vulnerability to DNA-damaging treatment, which is accompanied by a favorable prognosis after radiotherapy. A novel classification model has been established for accurate identification of UCHL1-related cancers, leveraging further basic and translational research on this novel cancer subgroup. Purpose: Identification of molecularly-defined cancer subgroups and targeting tumor-specific vulnerabilities have a strong potential to improve treatment response and patient outcomes but remain an unmet challenge of high clinical relevance, especially in head and neck squamous cell carcinoma (HNSC). Experimental design: We established a UCHL1-related gene set to identify and molecularly characterize a UCHL1-related subgroup within TCGA-HNSC by integrative analysis of multi-omics data. An extreme gradient boosting model was trained on TCGA-HNSC based on GSVA scores for gene sets of the MSigDB to robustly predict UCHL1-related cancers in other solid tumors and cancer cell lines derived thereof. Potential vulnerabilities of UCHL1-related cancer cells were elucidated by an in-silico drug screening approach. Results: We established a 497-gene set, which stratified the TCGA-HNSC cohort into distinct subgroups with a UCHL1-related or other phenotype. UCHL1-related HNSC were characterized by higher frequencies of genomic alterations, which was also evident for UCHL1-related cancers of other solid tumors predicted by the classification model. These data indicated an impaired maintenance of genomic integrity and vulnerability for DNA-damaging treatment, which was supported by a favorable prognosis of UCHL1-related tumors after radiotherapy, and a higher sensitivity of UCHL1-related cancer cells to irradiation or DNA-damaging compounds (e.g., Oxaliplatin). Conclusion: Our study established UCHL1-related cancers as a novel subgroup across most solid tumor entities with a unique molecular phenotype and DNA-damaging treatment as a specific vulnerability, which requires further proof-of-concept in pre-clinical models and future clinical trials. [ABSTRACT FROM AUTHOR]

Background: Treatment of locally advanced HPV-negative head and neck squamous cell carcinoma (HNSCC) with photon radiation is the standard of care but shows only moderate success. Alterations in response toward DNA DSB repair, apoptosis, and senescence are underlying determinants of radioresistance in the tumor cells. Recently, senescence and the associated secretory phenotype (SASP) came into the focus of research and raised the need to identify the tumor-promoting molecular mechanisms of the SASP. The aim of this project was to unravel more of this process and to understand the impact of the IL1 pathway, which plays a major role in SASP. The studies were performed for photon and 12C-ion irradiation, which strongly vary in their effect on radioresistance. Materials and Methods: A panel of five HPV-negative HNSCC cell lines was treated with photon and 12C-ion irradiation and examined for clonogenic survival, DNA DSB repair, and senescence. SASP and IL1 gene expressions were determined by RNA sequencing and activation of the IL1 pathway by ELISA. A functional impact of IL1A and IL1B was examined by specific siRNA knockdown. Results: Cell killing and residual DSBs were higher after 12C-ion than after photon irradiation. 12C-ion induced more senescence with a significant correlation with cell survival. The impact on radioresistance appears to be less than after photon irradiation. The expression of SASP-related genes and the IL1 pathway are strongly induced by both types of irradiation and correlate with radioresistance and senescence, especially IL1A and IL1B which exhibit excellent associations. Surprisingly, knockdown of IL1A and IL1B revealed that the IL1 pathway is functionally not involved in radioresistance, DSB repair, or induction of senescence. Conclusions: IL1A and IL1B are excellent indicators of cellular radioresistance and senescence in HNSCC cells without functional involvement in these processes. Clearly more research is needed to understand the molecular mechanisms of senescence and SASP and its impact on radioresistance. [ABSTRACT FROM AUTHOR]

Biomarkers with relevance for loco‐regional therapy are needed in human papillomavirus negative aka HPV(−) head and neck squamous cell carcinoma (HNSCC). Based on the premise that DNA methylation pattern is highly conserved, we sought to develop a reliable and robust methylome‐based classifier identifying HPV(−) HNSCC patients at risk for loco‐regional recurrence (LR) and all‐event progression after postoperative radiochemotherapy (PORT‐C). The training cohort consisted of HPV‐DNA negative HNSCC patients (n = 128) homogeneously treated with PORT‐C in frame of the German Cancer Consortium—Radiation Oncology Group (DKTK‐ROG) multicenter biomarker trial. DNA Methylation analysis was performed using Illumina 450 K and 850 K‐EPIC microarray technology. The performance of the classifier was integrated with a series of biomarkers studied in the training set namely hypoxia‐, 5‐microRNA (5‐miR), stem‐cell gene‐expression signatures and immunohistochemistry (IHC)‐based immunological characterization of tumors (CD3/CD8/PD‐L1/PD1). Validation occurred in an independent cohort of HPV(−) HNSCC patients, pooled from two German centers (n = 125). We identified a 38‐methylation probe‐based HPV(−) Independent Classifier of disease Recurrence (HICR) with high prognostic value for LR, distant metastasis and overall survival (P < 10−9). HICR remained significant after multivariate analysis adjusting for anatomical site, lymph node extracapsular extension (ECE) and size (T‐stage). HICR high‐risk tumors were enriched for younger patients with hypoxic tumors (15‐gene signature) and elevated 5‐miR score. After adjustment for hypoxia and 5‐miR covariates, HICR maintained predicting all endpoints. HICR provides a novel mean for assessing the risk of LR in HPV(−) HNSCC patients treated with PORT‐C and opens a new opportunity for biomarker‐assisted stratification and therapy adaptation in these patients. [ABSTRACT FROM AUTHOR]

BACKGROUND: MicroRNAs constitute promising biomarkers. OBJECTIVE: The aim was to investigate diagnostic and prognostic implications of miR-182-5p and miR-205-5p in p16-positive and p16-negative oropharyngeal squamous cell carcinomas (OPSCCs). METHODS: Expression of miR-182-5p, miR-205-5p were determined via quantitative real-time-PCR in fresh frozen tissues of 26 p16-positive, 19 p16-negative OPSCCs and 18 HPV-negative oropharyngeal controls. Associations between miRNA-expression, clinicopathological characteristics and prognosis were analyzed. RESULTS: Higher miR-182-5p expression was associated with significant inferior disease-specific survival for p16-positive OPSCCs (HR = 1.98E + 09, 95% CI 0–Inf; P = 0.028) and a similar trend was observed for p16-negative OPSCCs (HR = 1.56E + 09, 95% CI 0–Inf; P = 0.051). Higher miR-205-5p expression was associated with an inferior progression-free survival (HR = 4.62, 95% CI 0.98–21.83; P = 0.034) and local control rate (HR = 2.18E + 09, 95% CI 0–Inf; P = 0.048) for p16-positive OPSCCs. CONCLUSIONS: Results indicate that miR-182-5p and miR-205-5p can further stratify patients with p16-positive OPSCC into prognostic groups. [ABSTRACT FROM AUTHOR]

Background: The prognostic impact of hsa‐miRNA‐182‐5p in oral cancer remains unexplored. Therefore, the aim of this study was to investigate the prognostic value of hsa‐miRNA‐182‐5p and its predicted target kinectin 1 (KTN1) in oral squamous cell carcinoma (OSCC). Method: Expression level of hsa‐miRNA‐182‐5p was analyzed in tumor tissue (n = 36) and healthy oral mucosal tissue (n = 17) using quantitative real‐time polymerase chain reaction. Protein level of the predicted target KTN1 was detected via immunohistochemistry. Results were validated in a cohort of The Cancer Genome Atlas (TCGA). Results: After dividing the data into a subgroup with high and low hsa‐miRNA‐182‐5p expression level, a significant better overall (p = 0.016), recurrence‐free (p = 0.009), and progression‐free survival (p = 0.004) was observed in an upregulation of hsa‐miRNA‐182‐5p. Staining intensity of KTN1 showed a reciprocal impact on the prognosis. Validation in a TCGA cohort confirmed these results. Conclusion: Our results indicate hsa‐miRNA‐182‐5p and KTN1 as potential biomarkers for OSCC. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNA) recently evolved as potential cancer biomarkers. Therefore, the aim of the present study was to evaluate the prognostic impact of eight miRNAs connected to oral squamous cell carcinoma (OSCC). Method: Expression levels of hsa‐mir‐21‐5p, hsa‐mir‐29b‐3p, hsa‐mir‐31‐5p, hsa‐mir‐99a‐5p, hsa‐mir‐99b‐3p, hsa‐mir‐100‐5p, hsa‐mir‐143‐3p and hsa‐mir‐155‐5p were analyzed in tumor tissue (n = 36) and healthy oral mucosal tissue (n = 17) and correlated with clinical variables. Results of the study cohort were validated in an OSCC cohort of The Cancer Genome Atlas. Results: Increased hsa‐mir‐99b‐3p expression level showed a tendency toward advanced tumor stages, and high levels of hsa‐mir‐100‐5p expression were associated with extracapsular extension. While a high expression level of hsa‐mir‐99b‐3p was associated with better survival, a high expression level of hsa‐mir‐100‐5p was correlated with a poorer survival in the study cohort. Conclusion: Our results indicate that hsa‐mir‐99b‐3p and hsa‐mir‐100‐5p may serve as novel prognostic biomarkers in OSCC. [ABSTRACT FROM AUTHOR]

Head and neck squamous cell carcinomas (HNSCCs) are characterized by outstanding molecular heterogeneity that results in severe therapy resistance and poor clinical outcome. Inter- and intratumoral heterogeneity in epithelial-mesenchymal transition (EMT) was recently revealed as a major parameter of poor clinical outcome. Here, we addressed the expression and function of the therapeutic target epidermal growth factor receptor (EGFR) and of the major determinant of epithelial differentiation epithelial cell adhesion molecule (EpCAM) in clinical samples and in vitro models of HNSCCs. We describe improved survival of EGFRlow/EpCAMhigh HNSCC patients (n = 180) and provide a molecular basis for the observed disparities in clinical outcome. EGF/EGFR have concentration-dependent dual capacities as inducers of proliferation and EMT through differential activation of the central molecular switch phosphorylated extracellular signal–regulated kinase 1/2 (pERK1/2) and EMT transcription factors (EMT-TFs) Snail, zinc finger E-box-binding homeobox 1 (Zeb1), and Slug. Furthermore, soluble ectodomain extracellular domain of EpCAM (EpEX) was identified as a ligand of EGFR that activates pERK1/2 and phosphorylated AKT (pAKT) and induces EGFR-dependent proliferation but represses EGF-mediated EMT, Snail, Zeb1, and Slug activation and cell migration. EMT repression by EpEX is realized through competitive modulation of pERK1/2 activation strength and inhibition of EMT-TFs, which is reflected in levels of pERK1/2 and its target Slug in clinical samples. Accordingly, high expression of pERK1/2 and/or Slug predicted poor outcome of HNSCCs. Hence, EpEX is a ligand of EGFR that induces proliferation but counteracts EMT mediated by the EGF/EGFR/pERK1/2 axis. Therefore, the emerging EGFR/EpCAM molecular cross talk represents a promising target to improve patient-tailored adjuvant treatment of HNSCCs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Acquired and inherent radioresistance of tumor cells is related to tumor relapse and poor prognosis - not only in head and neck squamous cell carcinoma (HNSCC). The underlying molecular mechanisms are largely unknown. Therefore, systemic in-depth analyses are needed to identify key regulators of radioresistance. In the present study, subclones of the CAL-33 HNSCC cell line with different radiosensitivity were analyzed to identify signaling pathways related to the different phenotypes.Methods: Subclones with altered radiosensitivity were generated by fractionated irradiation of the parental CAL-33 cells. Differences in radiosensitivity were confirmed in colony formation assays. Selected subclones were characterized at the genomic and transcriptomic level by SKY, array CGH, and mRNA-microarray analyses. Time-course gene expression analyses upon irradiation using a natural cubic spline regression model identified temporally differentially expressed genes. Moreover, early and late responding genes were identified. Gene association networks were reconstructed using partial correlation. The Reactome pathway database was employed to conduct pathway enrichment analyses.Results: The characterization of two subclones with enhanced radiation resistance (RP) and enhanced radiosensitivity (SP) revealed distinct genomic and transcriptomic changes compared to the parental cells. Differentially expressed genes after irradiation shared by both subclones pointed to important pathways of the early and late radiation response, including senescence, apoptosis, DNA repair, Wnt, PI3K/AKT, and Rho GTPase signaling. The analysis of the most important nodes of the gene association networks revealed pathways specific to the radiation response in different phenotypes of radiosensitivity. Exemplarily, for the RP subclone the senescence-associated secretory phenotype (SASP) together with GPCR ligand binding were considered as crucial. Also, the expression of endogenous retrovirus ERV3-1in response to irradiation has been observed, and the related gene association networks have been identified.Conclusions: Our study presents comprehensive gene expression data of CAL-33 subclones with different radiation sensitivity. The resulting networks and pathways associated with the resistant phenotype are of special interest and include the SASP. The radiation-associated expression of ERV3-1 also appears highly attractive for further studies of the molecular mechanisms underlying acquired radioresistance. The identified pathways may represent key players of radioresistance, which could serve as potential targets for molecularly designed, therapeutical intervention. [ABSTRACT FROM AUTHOR]

MicroRNAs represent ~22 nt long endogenous small RNA molecules that have been experimentally shown to regulate gene expression post-transcriptionally. One main interest in miRNA research is the investigation of their functional roles, which can typically be accomplished by identification of mi-/mRNA interactions and functional annotation of target gene sets. We here present a novel method "miRlastic", which infers miRNA-target interactions using transcriptomic data as well as prior knowledge and performs functional annotation of target genes by exploiting the local structure of the inferred network. For the network inference, we applied linear regression modeling with elastic net regularization on matched microRNA and messenger RNA expression profiling data to perform feature selection on prior knowledge from sequence-based target prediction resources. The novelty of miRlastic inference originates in predicting data-driven intra-transcriptome regulatory relationships through feature selection. With synthetic data, we showed that miRlastic outperformed commonly used methods and was suitable even for low sample sizes. To gain insight into the functional role of miRNAs and to determine joint functional properties of miRNA clusters, we introduced a local enrichment analysis procedure. The principle of this procedure lies in identifying regions of high functional similarity by evaluating the shortest paths between genes in the network. We can finally assign functional roles to the miRNAs by taking their regulatory relationships into account. We thoroughly evaluated miRlastic on a cohort of head and neck cancer (HNSCC) patients provided by The Cancer Genome Atlas. We inferred an mi-/mRNA regulatory network for human papilloma virus (HPV)-associated miRNAs in HNSCC. The resulting network best enriched for experimentally validated miRNA-target interaction, when compared to common methods. Finally, the local enrichment step identified two functional clusters of miRNAs that were predicted to mediate HPV-associated dysregulation in HNSCC. Our novel approach was able to characterize distinct pathway regulations from matched miRNA and mRNA data. An R package of miRlastic was made available through: http://icb.helmholtz-muenchen.de/mirlastic. [ABSTRACT FROM AUTHOR]

Background: Head and neck squamous cell carcinoma (HNSCC) is a very heterogeneous disease resulting in huge differences in the treatment response. New individualized therapy strategies including molecular targeting might help to improve treatment success. In order to identify potential targets, we developed a HNSCC radiochemotherapy cell culture model of primary HNSCC cells derived from two different patients (HN1957 and HN2092) and applied an integrative microRNA (miRNA) and mRNA analysis in order to gain information on the biological networks and processes of the cellular therapy response. We further identified potential target genes of four therapy-responsive miRNAs detected previously in the circulation of HNSCC patients by pathway enrichment analysis. Results: The two primary cell cultures differ in global copy number alterations and P53 mutational status, thus reflecting heterogeneity of HNSCC. However, they also share many copy number alterations and chromosomal rearrangements as well as deregulated therapy-responsive miRNAs and mRNAs. Accordingly, six common therapy-responsive pathways (direct P53 effectors, apoptotic execution phase, DNA damage/telomere stress induced senescence, cholesterol biosynthesis, unfolded protein response, dissolution of fibrin clot) were identified in both cell cultures based on deregulated mRNAs. However, inflammatory pathways represented an important part of the treatment response only in HN1957, pointing to differences in the treatment responses of the two primary cultures. Focused analysis of target genes of four therapy-responsive circulating miRNAs, identified in a previous study on HNSCC patients, revealed a major impact on the pathways direct P53 effectors, the E2F transcription factor network and pathways in cancer (mainly represented by the PTEN/AKT signaling pathway). Conclusions: The integrative analysis combining miRNA expression, mRNA expression and the related cellular pathways revealed that the majority of radiochemotherapy-responsive pathways in primary HNSCC cells are related to cell cycle, proliferation, cell death and stress response (including inflammation). Despite the heterogeneity of HNSCC, the two primary cell cultures exhibited strong similarities in the treatment response. The findings of our study suggest potential therapeutic targets in the E2F transcription factor network and the PTEN/AKT signaling pathway. [ABSTRACT FROM AUTHOR]

Introduction: Circulating microRNAs (miRNAs) are easily accessible and have already proven to be useful as prognostic markers in cancer patients. However, their origin and function in the circulation is still under discussion. In the present study we analyzed changes in the miRNAs in blood plasma of head and neck squamous cell carcinoma (HNSCC) patients in response to radiochemotherapy and compared them to the changes in a cell culture model of primary HNSCC cells undergoing simulated anti-cancer therapy.Materials and Methods: MiRNA-profiles were analyzed by qRT-PCR arrays in paired blood plasma samples of HNSCC patients before therapy and after two days of treatment. Candidate miRNAs were validated by single qRT-PCR assays. An in vitro radiochemotherapy model using primary HNSCC cell cultures was established to test the possible tumor origin of the circulating miRNAs. Microarray analysis was performed on primary HNSCC cell cultures followed by validation of deregulated miRNAs via qRT-PCR.Results: Unsupervised clustering of the expression profiles using the six most regulated miRNAs (miR-425-5p, miR-21-5p, miR-106b-5p, miR-590-5p, miR-574-3p, miR-885-3p) significantly (p = 0.012) separated plasma samples collected prior to treatment from plasma samples collected after two days of radiochemotherapy. MiRNA profiling of primary HNSCC cell cultures treated in vitro with radiochemotherapy revealed differentially expressed miRNAs that were also observed to be therapy-responsive in blood plasma of the patients (miR-425-5p, miR-21-5p, miR-106b-5p, miR-93-5p) and are therefore likely to stem from the tumor. Of these candidate marker miRNAs we were able to validate by qRT-PCR a deregulation of eight plasma miRNAs as well as miR-425-5p and miR-93-5p in primary HNSCC cultures after radiochemotherapy.Conclusion: Changes in the abundance of circulating miRNAs during radiochemotherapy reflect the therapy response of primary HNSCC cells after an in vitro treatment. Therefore, the responsive miRNAs (miR-425-5p, miR-93-5p) may represent novel biomarkers for therapy monitoring. The prognostic value of this exciting observation requires confirmation using an independent patient cohort that includes clinical follow-up data. [ABSTRACT FROM AUTHOR]

Background: Signaling from integrins and receptor tyrosine kinases (RTKs) contributes substantially to therapy resistance of malignant tumors. We investigated simultaneous β1 integrin-epidermal growth factor receptor (EGFR) targeting plus radiotherapy in human head and neck squamous cell carcinomas (HNSCCs). Methods: Ten HNSCC cell lines were grown in three-dimensional laminin-rich extracellular matrix cell cultures and two of them as tumor xenografts in nude mice (n = 12–16 per group). Targeting of β1 integrin and EGFR with monoclonal inhibitory antibodies (AIIB2 and cetuximab, respectively) was combined with x-ray irradiation. Clonogenic survival, tumor growth, and tumor control (evaluated by Kaplan-Meier analysis), apoptosis, phosphoproteome (interactome, network betweeness centrality analysis), receptor expression (immunohistochemistry), and downstream signaling (western blotting) were assessed. Various mutants of the integrin signaling mediator focal adhesion kinase (FAK) were employed for mechanistic studies. All statistical tests were two-sided. Results: Compared with β1 integrin or EGFR single inhibition, combined β1 integrin-EGFR targeting resulted in enhanced cytotoxicity and radiosensitization in eight out of 10 tested HNSCC cell lines, which responded with an FAK dephosphorylation after β1 integrin inhibition. In vivo, simultaneous anti-β1 integrin/anti-EGFR treatment and radiotherapy of UTSCC15 responder xenografts enabled better tumor control compared with anti-EGFR monotherapy and irradiation (hazard ratio [HR] = 6.9, 95% confidence interval [CI] = 1.6 to 30.9, P = .01), in contrast to the SAS nonresponder tumor model (HR = 0.9, 95% CI = 0.4 to 2.3, P = .83). Mechanistically, a protein complex consisting of FAK- and Erk1-mediated prosurvival signals for radiation resistance, which was effectively compromised by β1 integrin and EGFR blocking. Conclusions: Concomitant targeting of β1 integrin and EGFR seems a powerful and promising approach to overcome radioresistance of HNSCCs. [ABSTRACT FROM PUBLISHER]