Patients with human papillomavirus (HPV)-related oropharyngeal tumors display improved prognosis. The biological basis of this tumor phenotype is poorly understood. We investigated whether increased lymphocyte infiltrate in HPV-positive oropharyngeal squamous cell carcinomas could account for better prognosis. We previously identified, in an Affymetrix GeneChip analysis of 83 HPV-unrelated and 11 HPV-related squamous cell carcinoma of the oropharynx, several candidate genes, including CD8a and CD3f. Their expression was validated in this study by qRT-PCR on an independent clinical series of 144 oropharyngeal tumors. Immunohistochemical staining of tumor specimens was performed to evaluate infiltration of tumor stroma by CD81 and CD41 lymphocytes. The prognostic value of CD8a and CD3f expression levels was measured by Kaplan-Meier and Cox regression model analyses. Immune response-related signaling pathways were found to be deregulated in HPV-positive oropharyngeal tumors. Expression of CD8a, CD3f, granzyme K, CD28 and integrin aL RNAs was upregulated in HPV-positive lesions when compared with HPV-unrelated tumors (p < 0.05). Stroma of HPV-positive tumors was frequently and strongly infiltrated by CD8a-and CD3f-positive T cells. CD8a RNA expression correlated with both improved global (Kaplan-Meier; p 5 0.005; Cox regression: p 5 0.003) and disease-free (Cox regression: p 5 0.04) survival. CD3f RNA expression correlated with improved overall survival (Cox regression: p 5 0.024). These results suggest that an increased cytotoxic T-cell-based antitumor immune response is involved in improved prognosis of patients with HPV-positive tumors.The presence of human papillomaviruses (HPVs; mainly HPV type 16) correlates with the onset and development of about 25% of head and neck squamous cell carcinomas (HNSCC). 1 HPV-positive cancers of the head and neck arise mainly in the oropharynx and define a subgroup of radioand chemosensitive tumors with distinct clinical, pathological and molecular features and improved prognosis with respect to their HPV-negative counterparts. [2][3][4][5] The majority of HPVrelated oropharyngeal squamous cell carcinomas (OSCC) express wild-type TP53, 6,7 which might be involved in the improved response of tumors to treatment. Despite thorough analysis of gene expression profiles of HPV-positive OSCC by several groups, [8][9][10][11] no novel prognostic or predictive biomarker has been identified, and the molecular mechanisms that underlie improved prognosis of HPV-related OSCC are poorly understood.The immune response has an important impact in many HPV-associated tumors and can have either favorable or unfavorable consequences. 12 The importance of the immune response in HPV-related OSCC is less well established. An increased T-cell response against HPV E7 epitopes has been detected in the blood of patients with HPV-positive HNSCC. 13-15 A more recent study has shown that T cells that proliferate and synthesize inflammatory cytokines upon HPV16 E6 and E7 oncoprotein recognition can be isolate...

The DNA repair protein damaged DNA-binding 2 (DDB2) has been implicated in promoting cell-cycle progression by regulating gene expression. DDB2 is selectively overexpressed in breast tumor cells that are noninvasive, but not in those that are invasive. We found that its overexpression in invasive human breast tumor cells limited their motility and invasiveness in vitro and blocked their ability to colonize lungs in vivo, defining a new function for DDB2 in malignant progression. DDB2 overexpression attenuated the activity of NF-kB and the expression of its target matrix metalloprotease 9 (MMP9). Mechanistic investigations indicated that DDB2 decreased NF-kB activity by upregulating expression of IkBa by binding the proximal promoter of this gene. This effect was causally linked to invasive capacity. Indeed, knockdown of DDB2-induced IkBa gene expression restored NF-kB activity and MMP9 expression, along with the invasive properties of breast tumor cells overexpressing DDB2. Taken together, our findings enlighten understanding of how breast cancer cells progress to an invasive phenotype and underscore potential clinical interest in DDB2 as a prognostic marker or therapeutic target in this setting. Cancer Res; 73(16); 5040-52. Ó2013 AACR.

Purpose: Distant metastasis after treatment is observed in about 20% of squamous cell carcinoma of the head and neck (HNSCC). In the absence of any validated robust biomarker, patients at higher risk for metastasis cannot be provided with tailored therapy. To identify prognostic HNSCC molecular subgroups and potential biomarkers, we have conducted genome-wide integrated analysis of four omic sets of data.Experimental Design: Using state-of-the-art technologies, a core set of 45 metastasizing and 55 nonmetastasizing human papillomavirus (HPV)-unrelated HNSCC patient samples were analyzed at four different levels: gene expression (transcriptome), DNA methylation (methylome), DNA copy number (genome), and microRNA (miRNA) expression (miRNome). Molecular subgroups were identified by a model-based clustering analysis. Their clinical relevance was evaluated by survival analysis, and functional significance by pathway enrichment analysis.Results: Patient subgroups selected by transcriptome, methylome, or miRNome integrated analysis are associated with shorter metastasis-free survival (MFS). A common subgroup, R1, selected by all three omic approaches, is statistically more significantly associated with MFS than any of the single omic-selected subgroups. R1 and non-R1 samples display similar DNA copy number landscapes, but more frequent chromosomal aberrations are observed in the R1 cluster (especially loss at 13q14.2-3). R1 tumors are characterized by alterations of pathways involved in cell-cell adhesion, extracellular matrix (ECM), epithelial-to-mesenchymal transition (EMT), immune response, and apoptosis.Conclusions: Integration of data across several omic profiles leads to better selection of patients at higher risk, identification of relevant molecular pathways of metastasis, and potential to discover biomarkers and drug targets. Clin Cancer Res; 19(15); 4174-84. Ó2013 AACR.

At the time of diagnosis, 60% of patients with head and neck squamous cell carcinoma (HNSCC) present tumors in an advanced stage (III-IV) of disease and 80% will relapse within the first two years post-treatment, due to their frequent radio(chemo)resistance. To identify new molecular targets and companion biomarkers, we have investigated the miRNome of 75 stage III-IV oropharynx tumors without relapse (R) or with loco-regional relapse (non-responder, NR) within two years post-treatment. Interestingly, miR-422a was significantly downregulated in NR tumors, in agreement with the increase in cell proliferation and adhesion induced by miR-422a inhibition in vitro. Furthermore, we identified CD73/NT5E oncogene as target of miR-422a. Indeed, modulation of the endogenous level of miR-422a inversely influences the expression and the enzymatic activity of CD73. Moreover, knocking down CD73 mimics the effects of miR-422a upregulation. Importantly, in tumors, miR-422a and CD73 expression levels are inversely correlated, and both are predictive of relapse free survival - especially considering loco(regional) recurrence - in vitro two independent cohorts of advanced oropharynx or HNSCC (N=255) tumors. In all, we reported, for the first time, that MiR-422a and its target CD73 are involved in early loco(regional) recurrence of HNSCC tumors and are new targets for personalized medicine.

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BackgroundEstrogen receptor alpha36 (ERalpha36), a variant of estrogen receptor alpha (ER) is expressed in about half of breast tumors, independently of the [ER+]/[ER-] status. In vitro, ERalpha36 triggers mitogenic non-genomic signaling and migration ability in response to 17beta-estradiol and tamoxifen. In vivo, highly ERalpha36 expressing tumors are of poor outcome especially as [ER+] tumors are submitted to tamoxifen treatment which, in turn, enhances ERalpha36 expression.ResultsOur study aimed to validate ERalpha36 expression as a reliable prognostic factor for cancer progression from an estrogen dependent proliferative tumor toward an estrogen dispensable metastatic disease. In a retrospective study, we tried to decipher underlying mechanisms of cancer progression by using an original modeling of the relationships between ERalpha36, other estrogen and growth factor receptors and metastatic marker expression. Nonlinear correlation analyses and mutual information computations led to characterize a complex network connecting ERalpha36 to either non-genomic estrogen signaling or to metastatic process.ConclusionsThis study identifies ERalpha36 expression level as a relevant classifier which should be taken into account for breast tumors clinical characterization and [ER+] tumor treatment orientation, using a generic approach for the rapid, cheap and relevant evaluation of any candidate gene expression as a predictor of a complex biological process.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-015-0178-7) contains supplementary material, which is available to authorized users.

The management of locally advanced head and neck squamous cell carcinoma (HNSCC) with Cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), achieves only moderate response rates, and clinical trials that evaluated EGFR-blockade with tyrosine kinase inhibitors (TKI) yielded disappointing results. Inter-tumor heterogeneity may hinder the therapeutic efficiency of anti-EGFR treatments. HNSCC heterogeneity was addressed in several studies, which all converged towards the definition of molecular subgroups. They include the basal subgroup, defined by the deregulated expression of factors involved in the EGFR signaling pathway, including the epiregulin EGFR ligand encoded by the EREG gene. These observations indicate that basal tumors could be more sensitive to anti-EGFR treatments. To test this hypothesis, we performed a screen of a representative collection of basal versus non-basal HNSCC cell lines for their sensitivity to several anti-EGFR drugs (Cetuximab, Afatinib, and Gefitinib), tested as monotherapy or in combination with drugs that target closely-linked pathways [Mitogen-activated protein kinase kinase/extracellular signal–regulated kinases (MEK), mammalian Target of Rapamycine (mTOR) or Human Epidermal growth factor Receptor 2 (HER2)]. Basal-like cell lines were found to be more sensitive to EGFR blockade alone or in combination with treatments that target MEK, mTOR, or HER2. Strikingly, the basal-like status was found to be a better predictor of cell response to EGFR blockade than clinically relevant mutations [e.g., cyclin-dependent kinase Inhibitor 2A (CDKN2A)]. Interestingly, we show that EGFR blockade inhibits EREG expression, and that EREG knock-down decreases basal cell clonogenic survival, suggesting that EREG expression could be a predictive functional marker of sensitivity to EGFR blockade in basal-like HNSCC.

The extracellular matrix (ECM) molecule Tenascin-C (TNC) is well-known to promote tumor progression by multiple mechanisms. However, reliable TNC detection in tissues of tumor banks remains limited. Therefore, we generated dromedary single-domain nanobodies Nb3 and Nb4 highly specific for human TNC (hTNC) and characterized the interaction with TNC by several approaches including ELISA, western blot, isothermal fluorescence titration and negative electron microscopic imaging. Our results revealed binding of both nanobodies to distinct sequences within fibronectin type III repeats of hTNC. By immunofluroescence and immunohistochemical imaging we observed that both nanobodies detected TNC expression in PFA and paraffin embedded human tissue from ulcerative colitis, solid tumors and liver metastasis. As TNC impairs cell adhesion to fibronectin we determined whether the nanobodies abolished this TNC function. Indeed, Nb3 and Nb4 restored adhesion of tumor and mesangial cells on a fibronectin/TNC substratum. We recently showed that TNC orchestrates the immune-suppressive tumor microenvironment involving chemoretention, causing tethering of CD11c+ myeloid/dendritic cells in the stroma. Here, we document that immobilization of DC2.4 dendritic cells by a CCL21 adsorbed TNC substratum was blocked by both nanobodies. Altogether, our novel TNC specific nanobodies could offer valuable tools for detection of TNC in the clinical practice and may be useful to inhibit the immune-suppressive and other functions of TNC in cancer and other diseases.