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Introduction and Objectives: Dysfunction of the E-cadherin/catenin complex is directly related to carcinogenesis and metastases development. The aim of this paper is to investigate the prognostic significance of E-cadherin and β-catenin expression in surgically treated laryngeal and hypopharyngeal squamous cell carcinomas., Material and Methods: Tumour tissue samples were obtained from 133 consecutive patients with squamous cell carcinomas of the head and neck: 68 of the larynx and 65 hypopharyngeal carcinomas, who underwent surgical treatment in our hospital between 2000 and 2005. E-cadherin and β-catenin expression was analysed by immunohistochemistry, quantifying the percentage of stained cells and the intensity of staining., Results: E-cadherin and β-catenin expression was evaluable in 59 laryngeal carcinomas and in 58 cases of hypopharyngeal carcinomas. In the laryngeal tumours, a significant association was found between the low expression of membrane β-catenin with T4 tumours and tumour recurrence. In the hypopharynx there was a significant association between positive expression of nuclear β-catenin and poor histological differentiation (P=.02). In the multivariate analysis, only the presence of lymph node metastases was an independent predictive factor of decreased disease-specific survival in laryngeal squamous cell carcinomas., Conclusions: The expression of E-cadherin and β-catenin does not show prognostic significance in laryngeal and hypopharyngeal squamous cell carcinomas over the TNM classification., (Copyright © 2020 Sociedad Española de Otorrinolaringología y Cirugía de Cabeza y Cuello. Publicado por Elsevier España, S.L.U. All rights reserved.)

Aberrant SRC expression and activation is frequently detected in multiple cancers, and hence, targeting SRC has emerged as a promising therapeutic strategy. Different SRC inhibitors have demonstrated potent anti-tumor activity in preclinical models, although they largely lack clinical efficacy as monotherapy in late-stage solid tumors, including head and neck squamous cell carcinomas (HNSCC). Adequate selection and stratification of patients who may respond to and benefit from anti-SRC therapies is therefore needed to guide clinical trials and treatment efficacy. This study investigates the prognostic significance of active SRC expression in a homogeneous cohort of 122 human papillomavirus (HPV)-negative, surgically treated HNSCC patients. Immunohistochemical evaluation of the active form of SRC by means of anti-SRC Clone 28 monoclonal antibody was specifically performed and subsequently correlated with clinical data. The expression of p-SRC (Tyr419), total SRC, and downstream SRC effectors was also analyzed. Our results uncovered striking differences in the prognostic relevance of SRC expression in HNSCC patients depending on the tumor site. Active SRC expression was found to significantly associate with advanced disease stages, presence of lymph node metastasis, and tumor recurrences in patients with laryngeal tumors, but not in the pharyngeal subgroup. Multivariate Cox analysis further revealed active SRC expression as an independent predictor of cancer-specific mortality in patients with laryngeal carcinomas. Concordantly, expression of p-SRC (Tyr419) and the SRC substrates focal adhesion kinase (FAK) and the Arf GTPase-activating protein ASAP1 also showed specific associations with poor prognosis in the larynx. These findings could have important implications in ongoing Src family kinase (SFK)-based clinical trials, as these new criteria could help to improve patient selection and develop biomarker-stratified trials.

The frequent dysregulation of SRC family kinases (SFK) in multiple cancers prompted various inhibitors to be actively tested in preclinical and clinical trials. Disappointingly, dasatinib and saracatinib failed to demonstrate monotherapeutic efficacy in patients with head and neck squamous cell carcinomas (HNSCC). Deeper functional and mechanistic knowledge of the actions of these drugs is therefore needed to improve clinical outcome and to develop more efficient combinational strategies. Even though the SFK inhibitors dasatinib and saracatinib robustly blocked cell migration and invasion in HNSCC cell lines, this study unveils undesirable stem cell-promoting functions that could explain the lack of clinical efficacy in HNSCC patients. These deleterious effects were targeted by the mithramycin analog EC-8042 that efficiently eliminated cancer stem cells (CSC)-enriched tumorsphere cultures as well as tumor bulk cells and demonstrated potent antitumor activity in vivo. Furthermore, combination treatment of dasatinib with EC-8042 provided favorable complementary anti-proliferative, anti-invasive, and anti-CSC functions without any noticeable adverse interactions of both agents. These findings strongly support combinational strategies with EC-8042 for clinical testing in HNSCC patients. These data may have implications on ongoing dasatinib-based trials.

Clear differences have been established between head and neck squamous cell carcinomas (HNSCC) depending on human papillomavirus (HPV) infection status. This study specifically investigated the status of the CTTN , CCND1 and ANO1 genes mapping at the 11q13 amplicon in relation to the HPV status in HNSCC patients. CTTN, CCND1 and ANO1 protein expression and gene amplification were respectively analyzed by immunohistochemistry and real-time PCR in a homogeneous cohort of 392 surgically treated HNSCC patients. The results were further confirmed using an independent cohort of 279 HNSCC patients from The Cancer Genome Atlas (TCGA). The impact on patient survival was also evaluated. CTTN , CCND1 and ANO1 gene amplification and protein expression were frequent in HPV-negative tumors, while absent or rare in HPV-positive tumors. Using an independent validation cohort of 279 HNSCC patients, we consistently found that these three genes were frequently co-amplified (28%) and overexpressed (39⁻46%) in HPV-negative tumors, whereas almost absent in HPV-positive tumors. Remarkably, these alterations (in particular CTTN and ANO1 overexpression) were associated with poor prognosis. Taken together, the distinctive expression and amplification of these genes could cooperatively contribute to the differences in prognosis and clinical outcome between HPV-positive and HPV-negative tumors. These findings could serve as the basis to design more personalized therapeutic strategies for HNSCC patients.

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.

Background: Cortactin ( CTTN ) and the focal adhesion kinase ( FAK ) are two major candidate genes to, respectively, drive 11q13- and 8q24-associated aggressive behavior in various cancers. Recent evidence uncovered their clinical relevance in early stages of tumorigenesis as promising biomarkers for cancer risk assessment. Methods: Using a multicenter validation study, CTTN and FAK expression was evaluated by immunohistochemistry (IHC) in a cohort of 109 patients with laryngeal precancerous lesions, and correlated with clinicopathologic parameters and laryngeal cancer risk. The pathophysiologic role of CTTN and FAK was further investigated using functional studies in cellular models. Results: Positive CTTN and FAK expression (scores 2 and 3) was detected in 49 (41%) and 35 (32%) laryngeal dysplasias, respectively. Univariate Cox analysis showed that CTTN and FAK expression but not histologic grading was significantly associated with both recurrence risk and laryngeal cancer risk. Patients carrying strong CTTN- or FAK-expressing lesions (score 3) experienced the highest laryngeal cancer incidence (log-rank P < 0.001). In multivariate stepwise analysis, FAK expression [HR = 13.91; 95% CI, 4.82-40.15; P < 0.001] and alcohol consumption (HR = 2.22; 95% confidence interval, 1.17-4.20; P = 0.014) were significant independent predictors of laryngeal cancer development. Targeting FAK by either RNAi or pharmacologic inhibitors effectively blocked cell growth, colony formation, and invasion into 3D collagen matrices. Conclusions: CTTN and FAK emerge as powerful predictors of laryngeal cancer risk and recurrence risk beyond histologic grading. Impact: Our work supports the applicability of IHC CTTN and FAK as complementary markers for risk stratification in patients with laryngeal precancerous lesions. Cancer Epidemiol Biomarkers Prev; 27(7); 805-13. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Tumor growth and progression is the result of a complex process controlled not only by malignant cancer cells but also by the surrounding tumor microenvironment (TME). Cancer associated fibroblasts (CAFs), the most abundant cellular component of TME, play an active role in tumor invasion and metastasis by promoting cancer cell invasion through cell-cell interactions and secretion of pro-invasive factors such as extracellular matrix (ECM)-degrading proteases. Due to their tumor-promoting activities, there is an emerging interest in investigating CAFs biology and its potential as drug targets for cancer therapies. Here we describe an easy and highly reproducible quantitative method to analyze CAF invasive activity by forming multicellular spheroids embedded into a three-dimensional (3D) matrix that mimics in vivo ECM. Subsequently, invasion is monitored over time using a time-lapse microscope. We also provide an automated image analysis system that enables the rapid quantification of the spheroid area increase (invasive area) over time. The use of a 96-well plate format with one CAF spheroid per well and the automated analysis provides a method suitable for drug screening test, such as protease inhibitors.

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.

NANOG is a master regulator of embryonic stem cell pluripotency, found to be frequently aberrantly expressed in a variety of cancers, including laryngeal carcinomas. This study investigates for the first time the role of NANOG expression in early stages of laryngeal tumourigenesis and its potential utility as cancer risk marker. NANOG protein expression was evaluated by immunohistochemistry using two large independent cohorts of patients with laryngeal precancerous lesions, and correlated with clinicopathological parameters and laryngeal cancer risk. NANOG expression was detected by immunohistochemistry in 49 (60%) of 82 laryngeal dysplasias, whereas expression was negligible in patient-matched normal epithelia. Strong NANOG expression was found in 22 (27%) lesions and was established as cut-off point, showing the most robust association with laryngeal cancer risk (P = 0.003) superior to the histological classification (P = 0.320) the current gold standard in the clinical practice. Similar trends were obtained using a multicenter validation cohort of 86 patients with laryngeal dysplasia. Our findings uncover a novel role for NANOG expression in laryngeal tumourigenesis, and its unprecedented application as biomarker for cancer risk assessment.

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.

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.

Background: HERG1 potassium channel plays a critical role in the cell proliferation., Methods: HERG1 protein expression was analyzed by immunohistochemistry (IHC) in 62 patients with oral leukoplakias and 100 patients with oral squamous cell carcinomas (OSCC). HERG1 mRNA levels were assessed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in 22 patients with primary head and neck squamous cell carcinoma (HNSCC)., Results: Statistically significant associations were found between HERG1 expression and tobacco consumption, disease stage, tumor differentiation, tumor recurrence, and reduced survival. There was no association between HERG1 expression and the risk of progression from oral leukoplakia to OSCC. In addition, a high proportion of tumors (80%) showed increased HERG1 mRNA levels compared to normal mucosa from nononcologic patients., Conclusion: Aberrant HERG1 expression increases as oral tumorigenesis progresses from oral hyperplasia to OSCC. Increased HERG1 mRNA levels were also frequently detected in OSCC and other HNSCC subsites. HERG1 expression emerges as a clinically relevant feature during tumor progression and a potential poor prognostic biomarker for OSCC. © 2016 Wiley Periodicals, Inc. Head Neck 38: 1708-1716, 2016., (© 2016 Wiley Periodicals, Inc.)

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., Competing Interests: The authors declare no conflicts of interest.

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.

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 (HR = 3.62; 95% CI 0.79-16.57; P = 0.097; 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.

β-Lapachone (β-lap) is a novel anticancer agent that selectively induces cell death in human cancer cells, by activation of the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation. We characterized the gene expression profile of budding yeast cells treated with β-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were differentially expressed in β-lap treated cells. β-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, an inhibitor of NADH dehydrogenases. A yeast mutant in the mitochondrial NADH dehydrogenase Nde2p was found to be resistant to β-lap treatment, despite inducing ROS production in a WT manner. Most interestingly, DNA damage responses triggered by β-lap were abolished in the nde2Δ mutant. Amino acid biosynthesis genes were also induced in β-lap treated cells, suggesting that β-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Accordingly, β-lap treatment increased phosphorylation of eIF2α subunit in a manner dependent on the Gcn2p kinase. eIF2α phosphorylation required Gcn1p, Gcn20p and Nde2p. Gcn2p was also required for cell survival upon exposure to β-lap and to elicit checkpoint responses. Remarkably, β-lap treatment increased phosphorylation of eIF2α in breast tumor cells, in a manner dependent on the Nde2p ortholog AIF, and the eIF2 kinase PERK. These findings uncover a new target pathway of β-lap in yeast and human cells and highlight a previously unknown functional connection between Nde2p, Gcn2p and DNA damage responses.

CREBZF is a member of the mammalian ATF/CREB family of transcription factors. Here, we describe a novel functional interaction between CREBZF and the tumor suppressor p53. CREBZF was identified in a yeast two-hybrid screen using HEY1, recently characterized as an indirect p53 activator, as bait. CREBZF interacts in vitro with both HEY1 and p53, and CREBZF expression stabilizes and activates p53. Moreover, CREBZF cooperates synergistically with HEY1 to enhance p53 transcriptional activity. On the other hand, partial depletion of endogenous CREBZF diminishes p53 protein levels and inhibits HEY1-mediated activation of p53. CREBZF-positive effects on p53 signaling may reflect, at least in part, an observed induction of posttranslational modifications in p53 known to prevent its degradation. CREBZF expression protects HCT116 cells from UV radiation-induced cell death. In addition, CREBZF expression confers sensitivity to 5-fluorouracil, a p53-activating chemotherapeutic drug. Our study suggests that CREBZF may participate in the modulation of p53 tumor suppressor function.

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.