Saúl Álvarez-Teijeiro

Frequent aberrant expression of the human ether à go-go (hEAG1) potassium channel in head and neck cancer: pathobiological mechanisms and clinical implications.

Compelling evidence indicates that the human ether-à-go-go voltage-gated potassium channels (hEAG1) may represent new valuable membrane therapeutic targets and diagnostic/prognostic biomarkers in various cancers. This study is the first to investigate the expression of hEAG1 potassium channel subunit in both primary tumors and HNSCC-derived cell lines to ascertain its clinical and biological role in tumor progression. Our findings demonstrate that hEAG1 is frequently aberrantly expressed in a high percentage of primary tumors (83xa0%, 45/54 cases) and HNSCC-derived cell lines (83xa0%, 10/12 cell lines). hEAG1 expression increased during HNSCC progression and was more frequent in advanced tumors. Strikingly, hEAG1 expression was also detected in a notable proportion (39xa0%, 17/44 cases) of patient-matched normal adjacent mucosa, whereas no expression was detected in normal epithelia from non-oncologic patients without exposure to tobacco carcinogens. In an attempt to identify the underlying mechanisms of aberrant hEAG1 expression in HNSCC, we found that hEAG1 gene copy gain occurred at a low frequency (15xa0%, 13/88 cases) in primary tumors but was not observed in early stages of HNSCC tumorigenesis. Furthermore, this study provides original evidence supporting the involvement of histone acetylation (i.e., H3Ac and H4K16Ac activating marks) in the regulation of hEAG1 expression in HNSCC. In addition, functional studies in HNSCC cells further revealed that hEAG1 expression is a biologically relevant feature that promotes cell proliferation and invasion, although independently of its ion-conducting function. Our findings strongly support the notion that hEAG1 may represent a promising candidate as tumor marker and membrane therapeutic target for HNSCC treatment.

Impact of PI3K/AKT/mTOR pathway activation on the prognosis of patients with head and neck squamous cell carcinomas

The PI3K/AKT/mTOR signaling pathway has emerged as one of the most frequently deregulated in head and neck squamous cell carcinomas (HNSCC). Numerous alterations of various upstream and downstream components have been described; however, their prognostic significance and impact on HNSCC patient survival remains to be established. This was addressed using an unbiased cohort of 93 consecutive and homogeneous surgically treated HNSCC patients and results confirmed in 432 HNSCC patients. Our findings reveal the high prevalence of S6 phosphorylation, a surrogate marker of mTORC1 activation, in HNSCC specimens (>70%) and, more importantly, demonstrate its relevance on clinical outcome. Phosphorylation of ribosomal protein S6 on either Ser235/236 or Ser240/244 was consistently and significantly correlated with favorable prognosis, although with differences depending on the tumor site. Thus, p-S6 expression was significantly correlated with better disease-specific survival specifically in the subgroup of laryngeal carcinoma patients (P< 0.001). In addition, multivariate regression models revealed p-S6 to be an inverse and independent predictor of lymph-node metastasis (P= 0.004) and distant metastasis (P= 0.006). Taken together, this study unveils an unprecedented correlation of mTOR activation with improved clinical outcome in patients with laryngeal carcinomas and uncovers the potential of p-S6 expression as a good prognostic biomarker and an inverse predictor of lymph node and distant metastases. These results should be of broad interest as immunohistochemical detection of p-S6 may help to stratify patients and guide treatment decisions.

Role of HERG1 potassium channel in both malignant transformation and disease progression in head and neck carcinomas.

Evidence indicates that human ether à-go-go-related gene 1 (HERG1) voltage-gated potassium channels could represent new valuable membrane therapeutic targets and diagnostic/prognostic biomarkers in various cancers. This study is the first to investigate the expression pattern of HERG1 potassium channel subunit in both primary tumors and precancerous lesions to establish its clinical and biological role during the development and progression of head and neck squamous cell carcinomas. HERG1 protein expression was evaluated by immunohistochemistry in paraffin-embedded tissue specimens from 133 patients with laryngeal/hypopharyngeal squamous cell carcinomas and 75 patients with laryngeal dysplasia, and correlated with clinical data. Our findings demonstrate that HERG1 is frequently aberrantly expressed in a high percentage of primary tumors (87%), whereas expression was negligible in both stromal cells and normal-adjacent epithelia. HERG1 expression increased during head and neck squamous cell carcinoma progression and was significantly associated with lymph node metastasis (P=0.04), advanced disease stages (P<0.001), regional tumor recurrence (P=0.004), distant metastasis (P=0.03) and reduced disease-specific survival (P=0.012, log-rank test). HERG1-positive expression was also detected in 31 (41%) of 75 laryngeal dysplasias. Interestingly, HERG1 expression increased with the grade of dysplasia; however, HERG1 expression but not histology correlated significantly with increased laryngeal cancer risk (P=0.007). In addition, functional studies in head and neck squamous cell carcinoma-derived cell lines further revealed that HERG1 expression promotes anchorage-dependent and -independent cell growth and invasive capability, although independently of its ion-conducting function. Our data demonstrate that HERG1 expression is a biologically and clinically relevant feature in head and neck squamous cell carcinoma progression and also during malignant transformation, and a promising candidate as cancer risk marker and therapeutic target for head and neck squamous cell carcinoma prevention and treatment.

Clinical significance of Anoctamin-1 gene at 11q13 in the development and progression of head and neck squamous cell carcinomas.

This study investigates the clinical significance of Anoctamin-1 gene mapping at 11q13 amplicon in both the development and progression of head and neck squamous cell carcinomas (HNSCC). ANO1 protein expression was evaluated by immunohistochemistry in a cohort of 372 surgically treated HNSCC patients and also in 35 laryngeal precancerous lesions. ANO1 gene amplification was determined by real-time PCR in all the laryngeal premalignancies and 60 of the HNSCCs, and molecular data correlated with clinical outcome. ANO1 gene amplification was frequently detected in both premalignant lesions (63%) and HNSCC tumours (58%), whereas concomitant ANO1 expression occurred at a much lower frequency (20 and 22%). Interestingly, laryngeal dysplasias harbouring ANO1 gene amplification showed a higher risk of malignant transformation (HRu2009=u20093.62; 95% CI 0.79–16.57; Pu2009=u20090.097; Cox regression). ANO1 expression and gene amplification showed no significant associations with clinicopathological parameters in HNSCC. However, remarkably ANO1 expression differentially influenced patient survival depending on the tumour site. Collectively, this study provides original evidence demonstrating the distinctive impact of ANO1 expression on HNSCC prognosis depending on the tumour site.

Dysregulation of Mir-196b in Head and Neck Cancers Leads to Pleiotropic Effects in the Tumor Cells and Surrounding Stromal Fibroblasts

The miR-196 family members have been found dysregulated in different cancers. Therefore, they have been proposed as promising biomarkers and therapeutic targets. This study is the first to investigate the role of miR-196b in the development and progression of head and neck squamous cell carcinomas (HNSCC), and also the impact on the surrounding tumor microenvironment. Increased miR-196b levels were detected in 95% of primary tumors and precancerous lesions, although no significant differences were observed between non-progressing versus progressing dysplasias. Furthermore, increased levels of both miR-196a and miR-196b were successfully detected in saliva samples from HNSCC patients. The functional consequences of altered miR-196 expression were investigated in both HNSCC cell lines and cancer-associated fibroblasts (CAFs) by transfection with specific pre-miR precursors. Results showed that both miR-196a and miR-196b elicit cell-specific responses in target genes and downstream regulatory pathways, and have a distinctive impact on cell proliferation, migration and invasion. These data reveal the early occurrence and prevalence of miR-196b dysregulation in HNSCC tumorigenesis, suggesting its utility for early diagnosis and/or disease surveillance and also as a non-invasive biomarker in saliva. The pleiotropic effects of miR-196a/b in HNSCC cell subpopulations and surrounding CAFs may complicate a possible therapeutic application.

Annexin A1 down-regulation in head and neck squamous cell carcinoma is mediated via transcriptional control with direct involvement of miR-196a/b

Annexin A1 (ANXA1) down-regulation is an early and frequent event in the development of head and neck squamous cell carcinomas (HNSCC). In an attempt to identify the underlying mechanisms of reduced ANXA1 protein expression, this study investigated ANXA1 mRNA expression in HNSCC specimens by both in situ hybridization and RT-qPCR. Results showed a perfect concordance between the pattern of ANXA1 mRNA and protein detected by immunofluorescence in tumors, precancerous lesions and normal epithelia, reflecting that ANXA1 down-regulation occurs at transcriptional level. We also found that both miR-196a and miR-196b levels inversely correlated with ANXA1 mRNA levels in paired HNSCC tissue samples and patient-matched normal mucosa. In addition, endogenous levels of ANXA1 mRNA and protein were consistently and significantly down-regulated upon miR-196a and miR-196b over-expression in various HNSCC-derived cell lines. The direct interaction of both mature miR-196a and miR-196b was further confirmed by transfection with Anxa1 3′UTR constructs. Combined bioinformatics and functional analysis of ANXA1 promoter activity contributed to identify key regions and potential mediators of ANXA1 transcriptional control. This study unveils that, in addition to miR-196a, miR-196b also directly targets ANXA1 in HNSCC.

HERG1A potassium channel is the predominant isoform in head and neck squamous cell carcinomas: evidence for regulation by epigenetic mechanisms

Evidences indicate that HERG1 voltage-gated potassium channel is frequently aberrantly expressed in various cancers including head and neck squamous cell carcinomas (HNSCC), representing a clinically and biologically relevant feature during disease progression and a potential therapeutic target. The present study further and significantly extends these data investigating for the first time the expression and individual contribution of HERG1 isoforms, their clinical significance during disease progression and also the underlying regulatory mechanisms. Analysis of HERG1A and HERG1B expression using real-time RT-PCR consistently showed that HERG1A is the predominant isoform in ten HNSCC-derived cell lines tested. HERG2 and HERG3 were also detected. Immunohistochemical analysis of HERG1A expression on 133 HNSCC specimens demonstrated that HERG1A expression increased during tumour progression and correlated significantly with reduced disease-specific survival. Furthermore, our study provides original evidence supporting the involvement of histone acetylation (i.e. H3Ac and H4K16Ac activating marks) in the regulation of HERG1 expression in HNSCC. Interestingly, this mechanism was also found to regulate the expression of another oncogenic channel (Kv3.4) as well as HERG2 and HERG3. These data demonstrate that HERG1A is the predominant and disease-relevant isoform in HNSCC progression, while histone acetylation emerges as an important regulatory mechanism underlying Kv gene expression.

Cleavage of Fibulin-2 by the aggrecanases ADAMTS-4 and ADAMTS-5 contributes to the tumorigenic potential of breast cancer cells

Fibulin-2 participates in the assembly of extracellular matrix components through interactions with multiple ligands and promotes contacts between cells and their surrounding environment. Consequently, identification of processes that could lead to an altered Fibulin-2 could have a major impact not only in the maintenance of tissue architecture and morphogenesis but also in pathological situations including cancer. Herein, we have investigated the ability of the secreted metalloproteases ADAMTS-4 and ADAMTS-5 to digest Fibulin-2. Using in vitro approaches and cultured breast cancer cell lines we demonstrate that Fibulin-2 is a better substrate for ADAMTS-5 than it is for ADAMTS-4. Moreover, Fibulin-2 degradation is associated to an enhancement of the invasive potential of T47D, MCF-7 and SK-BR-3 cells. We have also found that conditioned medium from MCF-7 cells that simultaneously overexpress Fibulin-2 and ADAMTS-5 significantly induced the migratory and invasive ability of normal breast fibroblasts using 3D collagen matrices. Immunohistochemical analysis highlights the close proximity or partial overlap of both Fibulin-2 and ADAMTS-5 in breast tumor samples. Additionally, proteolytic products derived from a potential degradation of Fibulin-2 by ADAMTS-5 were also identified in these samples. Finally, we also show that the cleavage of Fibulin-2 by ADAMTS-5 is counteracted by ADAMTS-12, a metalloprotease that interacts with Fibulin-2. Overall, our results provide direct evidence indicating that Fibulin-2 is a novel substrate of ADAMTS-5 and that this proteolysis could alter the cellular microenvironment affecting the balance between protumor and antitumor effects associated to both Fibulin-2 and the ADAMTSs metalloproteases.

PO-143 Frequent loss of annexin a1 expression in head and neck squamous cell carcinomas by transcriptional regulatory mechanisms

Introduction Annexins are frequently deregulated in many cancers (‘annexinopathies’). Annexin A1 (ANXA1) overexpression has been reported in gastric, pancreatic and hepatocellular carcinoma; however, it is markedly down-regulated in breast, prostate, esophageal and head and neck cancers. The contrasting patterns of ANXA1 expression in different tumours types is just one of the enigmas in deciphering the underlying regulatory mechanisms and phenotypic specificity of ANXA1. We have previously demonstrated that ANXA1 down-regulation is an early and frequent event in the development of head and neck squamous cell carcinomas (HNSCC). Material and methods In an attempt to identify the underlying mechanisms of reduced ANXA1 protein expression, this study investigated ANXA1 protein expression and ANXA1 mRNA levels in HNSCC tissue specimens by immunofluoresce, in situ hybridization and RT-qPCR. Subsequently, the relative levels of miR-196a and miR-196b were quantified in paired HNSCC specimens using Taqman miRNA assays and compared to ANXA1 mRNA levels. Functional studies were performed in HNSCC cells by transfection with specific pre-miR precursors, and also ANXA1 promoter activity using deletion constructs from the 5’ ANXA1 promoter region. Results and discussion Results showed a perfect concordance between the pattern of ANXA1 mRNA and protein detected by immunofluorescence in tumours, precancerous lesions and normal epithelia, reflecting that ANXA1 down-regulation occurs at transcriptional level. We also found that both miR-196a and miR-196b levels inversely correlated with ANXA1 mRNA levels in paired HNSCC tissue samples and patient-matched normal mucosa. In addition, endogenous levels of ANXA1 mRNA and protein were consistently and significantly down-regulated upon miR-196a and miR-196b over-expression in various HNSCC-derived cell lines. The direct interaction of both mature miR-196a and miR-196b was further confirmed by transfection with Anxa1 3’UTR constructs. Combined bioinformatics and functional analysis of ANXA1 promoter activity contributed to identify key regions and potential mediators of ANXA1 transcriptional control. Conclusion These findings demonstrate that ANXA1 down-regulation in head and neck squamous cell carcinomas occurs via transcriptional control with direct involvement of miR-196a/b.

Factors Secreted by Cancer-Associated Fibroblasts that Sustain Cancer Stem Properties in Head and Neck Squamous Carcinoma Cells as Potential Therapeutic Targets

This study investigates for the first time the crosstalk between stromal fibroblasts and cancer stem cell (CSC) biology in head and neck squamous cell carcinomas (HNSCC), with the ultimate goal of identifying effective therapeutic targets. The effects of conditioned media from cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs) on the CSC phenotype were assessed by combining functional and expression analyses in HNSCC-derived cell lines. Further characterization of CAFs and NFs secretomes by mass spectrometry was followed by pharmacologic target inhibition. We demonstrate that factors secreted by CAFs but not NFs, in the absence of serum/supplements, robustly increased anchorage-independent growth, tumorsphere formation, and CSC-marker expression. Modulators of epidermal growth factor receptor (EGFR), insulin-like growth factor receptor (IGFR), and platelet-derived growth factor receptor (PDGFR) activity were identified as paracrine cytokines/factors differentially secreted between CAFs and NFs, in a mass spectrometry analysis. Furthermore, pharmacologic inhibition of EGFR, IGFR, and PDGFR significantly reduced CAF-induced tumorsphere formation and anchorage-independent growth suggesting a role of these receptor tyrosine kinases in sustaining the CSC phenotype. These findings provide novel insights into tumor stroma–CSC communication, and potential therapeutic targets to effectively block the CAF-enhanced CSC niche signaling circuit.