Combined PIK3CA and SOX2 Gene Amplification Predicts Laryngeal Cancer Risk beyond Histopathological Grading.

The PIK3CA and SOX2 genes map at 3q26, a chromosomal region frequently amplified in head and neck cancers, which is associated with poor prognosis. This study explores the clinical significance of PIK3CA and SOX2 gene amplification in early tumorigenesis. Gene copy number was analyzed by real-time PCR in 62 laryngeal precancerous lesions and correlated with histopathological grading and laryngeal cancer risk. Amplification of the SOX2 and PIK3CA genes was frequently detected in 19 (31%) and 32 (52%) laryngeal dysplasias, respectively, and co-amplification in 18 (29%) cases. The PIK3CA and SOX2 amplifications were predominant in high-grade dysplasias and significantly associated with laryngeal cancer risk beyond histological criteria. Multivariable Cox analysis further revealed PIK3CA gene amplification as an independent predictor of laryngeal cancer development. Interestingly, combined PIK3CA and SOX2 amplification allowed us to distinguish three cancer risk subgroups, and PIK3CA and SOX2 co-amplification was found the strongest predictor by ROC analysis. Our data demonstrate the clinical relevance of PIK3CA and SOX2 amplification in early laryngeal tumorigenesis. Remarkably, PIK3CA amplification was found to be an independent cancer predictor. Furthermore, combined PIK3CA and SOX2 amplification is emerging as a valuable and easy-to-implement tool for cancer risk assessment in patients with laryngeal precancerous lesions beyond current WHO histological grading.

Dissecting the functions of cancer-associated fibroblasts to therapeutically target head and neck cancer microenvironment.

Head and neck cancers (HNC) are a diverse group of aggressive malignancies with high morbidity and mortality, leading to almost half-million deaths annually worldwide. A better understanding of the molecular processes governing tumor formation and progression is crucial to improve current diagnostic and prognostic tools as well as to develop more personalized treatment strategies. Tumors are highly complex and heterogeneous structures in which growth and dissemination is not only governed by the cancer cells intrinsic mechanisms, but also by the surrounding tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) emerge as predominant TME components and key players in the generation of permissive conditions that ultimately impact in tumor progression and metastatic dissemination. Although CAFs were initially considered a consequence of tumor development, it is now well established that they actively contribute to numerous cancer hallmarks i.e., tumor cell growth, migration and invasion, cancer cell stemness, angiogenesis, metabolic reprograming, inflammation, and immune system modulation. In this scenario, therapeutic strategies targeting CAF functions could potentially have a major impact in cancer therapeutics, providing avenues for new treatment options or for improving efficacy in established approaches. This review is focused on thoroughly dissecting existing evidences supporting the contribution of CAFs in HNC biology with an emphasis on current knowledge of the key molecules and pathways involved in CAF-tumor crosstalk, and their potential as novel biomarkers and/or therapeutic targets to effectively interfere the tumor-stroma crosstalk for HNC patients benefit. involved in CAF-tumor crosstalk, and their potential as novel biomarkers and/or therapeutic targets to effec- tively interfere the tumor-stroma crosstalk for HNC patients benefit.

Driving role of head and neck cancer cell secretome on the invasion of stromal fibroblasts: Mechanistic insights by phosphoproteomics.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are major players in tumor-stroma communication, and participate in several cancer hallmarks to drive tumor progression and metastatic dissemination. This study investigates the driving effects of tumor-secreted factors on CAF biology, with the ultimate goal of identifying effective therapeutic targets/strategies for head and neck squamous cell carcinomas (HNSCC). METHODS: Functionally, conditioned media (CM) from different HNSCC-derived cell lines and normal keratinocytes (Kc) were tested on the growth and invasion of populations of primary CAFs and normal fibroblasts (NFs) using 3D invasion assays in collagen matrices. The changes in MMPs expression were evaluated by RT-qPCR and kinase enrichment was analyzed using mass spectrometry phosphoproteomics. RESULTS: Our results consistently demonstrate that HNSCC-secreted factors (but not Kc CM) specifically and robustly promoted pro-invasive properties in both CAFs and NFs, thereby reflecting the plasticity of fibroblast subtypes. Concomitantly, HNSCC-secreted factors massively increased metalloproteinases levels in CAFs and NFs. By contrast, HNSCC CM and Kc CM exhibited comparable growth-promoting effects on stromal fibroblasts. Mechanistically, phosphoproteomic analysis predominantly revealed phosphorylation changes in fibroblasts upon treatment with HNSCC CM, and various promising kinases were identified: MKK7, MKK4, ASK1, RAF1, BRAF, ARAF, COT, PDK1, RSK2 and AKT1. Interestingly, pharmacologic inhibition of RAF1/BRAF using sorafenib emerged as the most effective drug to block tumor-promoted fibroblast invasion without affecting fibroblast viability CONCLUSIONS: Our findings demonstrate that HNSCC-secreted factors specifically fine tune the invasive potential of stromal fibroblasts, thereby generating tumor-driven pro-invasive niches, which in turn to ultimately facilitate cancer cell dissemination. Furthermore, the RAF/BRAF inhibitor sorafenib was identified as a promising candidate to effectively target the onset of pro-invasive clusters of stromal fibroblasts in the HNSCC microenvironment.

Pathobiological functions and clinical implications of annexin dysregulation in human cancers.

Annexins are an extensive superfamily of structurally related calcium- and phospholipid-binding proteins, largely conserved and widely distributed among species. Twelve human annexins have been identified, referred to as Annexin A1-13 (A12 remains as of yet unassigned), whose genes are spread throughout the genome on eight different chromosomes. According to their distinct tissue distribution and subcellular localization, annexins have been functionally implicated in a variety of biological processes relevant to both physiological and pathological conditions. Dysregulation of annexin expression patterns and functions has been revealed as a common feature in multiple cancers, thereby emerging as potential biomarkers and molecular targets for clinical application. Nevertheless, translation of this knowledge to the clinic requires in-depth functional and mechanistic characterization of dysregulated annexins for each individual cancer type, since each protein exhibits varying expression levels and phenotypic specificity depending on the tumor types. This review specifically and thoroughly examines the current knowledge on annexin dysfunctions in carcinogenesis. Hence, available data on expression levels, mechanism of action and pathophysiological effects of Annexin A1-13 among different cancers will be dissected, also further discussing future perspectives for potential applications as biomarkers for early diagnosis, prognosis and molecular-targeted therapies. Special attention is devoted to head and neck cancers (HNC), a complex and heterogeneous group of aggressive malignancies, often lately diagnosed, with high mortality, and scarce therapeutic options.

Lectin-Like Transcript 1 (LLT1) Checkpoint: A Novel Independent Prognostic Factor in HPV-Negative Oropharyngeal Squamous Cell Carcinoma.

Lectin-like transcript 1 (LLT1) expression by tumor cells contributes to immune evasion, thereby emerging as a natural killer (NK) cell-mediated immunotherapeutic target. This study is the first to investigate LLT1 expression (encoded by CLEC2D gene) in head and neck cancers to ascertain its impact on patient prognosis. LLT1 expression was analyzed by immunohistochemistry in a homogeneous cohort of human papillomavirus (HPV)-negative oropharyngeal squamous cell carcinomas (OPSCC), and correlated with clinical data. Results were further validated using transcriptomic data from the TCGA database. Tumoral LLT1 expression was detected in 190/221 (86%) OPSCC specimens, whereas normal pharyngeal epithelium was negative. Patients harboring LLT1-positive tumors showed significantly lower disease-specific (DSS) and overall survival (OS) (p = 0.049 and p = 0.036, respectively, log-rank test). High density of LLT1-positive tumor-infiltrating lymphocytes (TIL) was also frequently detected in 160 (73%) OPSCC samples, and significantly associated with better DSS and OS (p < 0.001 and p = 0.007, respectively). Multivariate Cox analysis further revealed that tumoral LLT1 expression and infiltration of LLT1-positive TIL were independent prognostic factors for DSS and OS. CLEC2D mRNA levels are also significantly increased in primary tumors compared to normal tissue. Strikingly, the prognostic impact of CLEC2D mRNA levels varied depending on HPV status in OPSCC, and among distinct cancer types. CLEC2D expression was significantly correlated with NK cell infiltration using the MCP-counter model. These findings uncover LLT1/CLEC2D as an independent prognostic factor in HPV-negative OPSCC, and a potential novel target for immunotherapy.

Prognostic Significance of the Pluripotency Factors NANOG, SOX2, and OCT4 in Head and Neck Squamous Cell Carcinomas.

Cancer stem cells (CSCs) play major roles in tumor initiation, progression, and resistance to cancer therapy. Several CSC markers have been studied in head and neck squamous cell carcinomas (HNSCC), including the pluripotency factors NANOG, SOX2, and OCT4; however, their clinical significance is still unclear. NANOG, SOX2, and OCT4 expression was evaluated by immunochemistry in 348 surgically-treated HNSCC, and correlated with clinicopathological parameters and patient outcomes. mRNA expression was further analyzed in 530 The Cancer Genome Atlas (TCGA) HNSCC. NANOG protein expression was detected in 250 (72%) cases, more frequently in patients with lymph node metastasis (p = 0.003), and was an independent predictor of better survival in multivariate analysis. While OCT4 expression was undetectable, SOX2 expression was observed in 105 (30%) cases, and strongly correlated with NANOG expression. Combined expression of both proteins showed the highest survival rates, and double-negative cases the worst survival. Strikingly, the impact of NANOG and SOX2 on outcome varied depending on tumor site and lymph node infiltration, specifically showing prognostic significance in pharyngeal tumors. Correlation between NANOG and SOX2 at mRNA and protein was specifically observed in node positive (N+) patients, and consistently correlated with better survival rates. According to our findings, NANOG protein expression is frequent in HNSCC, thereby emerging as an independent predictor of better prognosis in pharyngeal tumors. Moreover, this study uncovers a differential impact of NANOG and SOX2 expression on HNSCC prognosis, depending on tumor site and lymph node infiltration, which could facilitate high-risk patient stratification.