BackgroundPrevious studies have demonstrated that a number of HOX genes, a family of transcription factors with key roles in early development, are up-regulated in head and neck squamous cell carcinoma (HNSCC) and other cancers. The loci of several Homeobox (HOX) genes also contain microRNAs (miRs), including miR-196a.MethodsGlobal miR expression and expression of all 39 HOX genes in normal oral keratinocytes (NOKs), oral pre-malignant (OPM) and HNSCC cells was assessed by expression microarray and qPCR and in tissues by immunohistochemistry (IHC) and qPCR of laser microdissected (LCM) tissues. Expression of miR196a and HOXB9 was reduced using anti-miR-196a and siRNA, respectively. Expression microarray profiles of anti-miR196a and pre-miR196a transfected cells were compared to parental cells in order to identify novel targets of miR-196a. Putative miR196a targets were validated by qPCR and were confirmed as binding to the 3’UTR of miR196a by a dual luciferase reporter assay combined with mutational analysis of the miR-196a binding site.ResultsmiR-196a and HOXB9 are highly expressed in HNSCC compared to NOKs, a pattern also seen in HNSCC tissues by HOXB9 IHC and qPCR of miR-196a in LCM tissue. Knock-down of miR-196a expression decreased HNSCC cell migration, invasion and adhesion to fibronectin, but had no effect on proliferation. Furthermore, knock-down of HOXB9 expression decreased migration, invasion and proliferation but did not alter adhesion. We identified a novel primary mRNA transcript containing HOXB9 and miR196a-1 as predicted from in-silico analysis. Expression array analysis identified a number of miR196a targets, including MAMDC2 and HOXC8. We confirmed that MAMDC2 is a novel miR-196a target using a dual luciferase reporter assay with the effect abolished on mutation of the binding site.ConclusionsThese results show that miR-196a and HOXB9 are overexpressed, perhaps co-ordinately, as HNSCC develops and exert a pro-tumourigenic phenotype in HNSCC and OPM cells.

Background:HOX gene expression is altered in many cancers; previous microarray revealed changes in HOX gene expression in head and neck squamous cell carcinoma (HNSCC), particularly HOXD10.Methods:HOXD10 expression was assessed by qPCR and immunoblotting in vitro and by immunohistochemistry (IHC) in tissues. Low-expressing cells were stably transfected with HOXD10 and the phenotype assessed with MTS, migration and adhesion assays and compared with the effects of siRNA knockdown in high-HOXD10-expressing cells. Novel HOXD10 targets were identified using expression microarrays, confirmed by reporter assay, and validated in tissues using IHC.Results:HOXD10 expression was low in NOKs, high in most primary tumour cells, and low in lymph node metastasis cells, a pattern confirmed using IHC in tissues. Overexpression of HOXD10 decreased cell invasion but increased proliferation, adhesion and migration, with knockdown causing reciprocal effects. There was no consistent effect on apoptosis. Microarray analysis identified several putative HOXD10-responsive genes, including angiomotin (AMOT-p80) and miR-146a. These were confirmed as HOXD10 targets by reporter assay. Manipulation of AMOT-p80 expression resulted in phenotypic changes similar to those on manipulation of HOXD10 expression.Conclusions:HOXD10 expression varies by stage of disease and produces differential effects: high expression giving cancer cells a proliferative and migratory advantage, and low expression may support invasion/metastasis, in part, by modulating AMOT-p80 levels.

Recent decades have witnessed the publication of numerous studies reporting alterations in the genome and transcriptome of head and neck squamous cell carcinoma (HNSCC). Currently, the utilisation of these alterations as biomarkers and targets for therapy is limited and new, useful molecular characteristics are being sought. Many of the published HNSCC gene expression profiles demonstrate alterations in the expression of HOX genes. These are a family of Homeobox-containing genes which are involved in developmental patterning and morphogenesis in the embryo, and which are often aberrantly expressed in cancer. The 39 HOX genes found in the human genome are arranged in four paralogous groups at different chromosomal loci. These control a wide range of cellular processes, including proliferation and migration, which are relevant in the context of cancer development. In this review article, we will outline the biology of HOX genes in relation to cancer and summarise the accumulating evidence for their role in the development of HNSCC and the possibility that they could be a therapeutic target in this malignancy. We will also identify areas where our current understanding is weak to focus future work and appraise the ongoing strategies for pharmacological intervention.

There is compelling evidence that senescent cells, through the senescence-associated secretory phenotype (SASP), can promote malignant transformation and invasion. IL-1 is a key mediator of this cytokine network, but the control of its activity in the senescence program has not been elucidated. IL-1 signalling is regulated by IL-1RA, which has four variants. Here, we show that expression of intracellular IL-1RA type 1 (icIL-1RA1), which competitively inhibits binding of IL-1 to its receptor, is progressively lost during oral carcinogenesis ex vivo and that the pattern of expression is associated with keratinocyte replicative fate in vitro. We demonstrate icIL-1RA1 is an important regulator of the SASP in mortal cells, as CRISPR-CAS9 mediated icIL-1RA1 knockdown in normal and mortal dysplastic oral keratinocytes is followed by increased IL-6 and IL-8 secretion, and rapid senescence following release from ROCK inhibition. Thus, we suggest that downregulation of icIL-1RA1 in early stages of the carcinogenesis process can enable the development of a premature and de-regulated SASP, creating a pro-inflammatory state in which cancer is more likely to arise.

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