Due to the large number of putative microRNA gene targets predicted by sequence-alignment databases and the relative low accuracy of such predictions which are conducted independently of biological context by design, systematic experimental identification and validation of every functional microRNA target is currently challenging. Consequently, biological studies have yet to identify, on a genome scale, key regulatory networks perturbed by altered microRNA functions in the context of cancer. In this report, we demonstrate for the first time how phenotypic knowledge of inheritable cancer traits and of risk factor loci can be utilized jointly with gene expression analysis to efficiently prioritize deregulated microRNAs for biological characterization. Using this approach we characterize miR-204 as a tumor suppressor microRNA and uncover previously unknown connections between microRNA regulation, network topology, and expression dynamics. Specifically, we validate 18 gene targets of miR-204 that show elevated mRNA expression and are enriched in biological processes associated with tumor progression in squamous cell carcinoma of the head and neck (HNSCC). We further demonstrate the enrichment of bottleneckness, a key molecular network topology, among miR-204 gene targets. Restoration of miR-204 function in HNSCC cell lines inhibits the expression of its functionally related gene targets, leads to the reduced adhesion, migration and invasion in vitro and attenuates experimental lung metastasis in vivo. As importantly, our investigation also provides experimental evidence linking the function of microRNAs that are located in the cancer-associated genomic regions (CAGRs) to the observed predisposition to human cancers. Specifically, we show miR-204 may serve as a tumor suppressor gene at the 9q21.1–22.3 CAGR locus, a well established risk factor locus in head and neck cancers for which tumor suppressor genes have not been identified. This new strategy that integrates expression profiling, genetics and novel computational biology approaches provides for improved efficiency in characterization and modeling of microRNA functions in cancer as compared to the state of art and is applicable to the investigation of microRNA functions in other biological processes and diseases.

No abstract

Hepatocyte growth factor receptor (HGFR), the product of the MET gene, plays an important role in normal cellular function and oncogenesis. In cancer, HGFR has been implicated in cellular proliferation, cell survival, invasion, cell motility, metastasis and angiogenesis. Activation of HGFR can occur through binding to its ligand, hepatocyte growth factor (HGF), overexpression/amplification, mutation, and/or decreased degradation. Amplification of HGFR can occur de novo or in resistance to therapy. Mutations of HGFR have been described in the tyrosine kinase domain, juxtamembrane domain, or semaphorin domain in a number of tumors. These mutations appear to have gain of function, and also reflect differential sensitivity to therapeutic inhibition. There have been various drugs developed to target HGFR, including antibodies to HGFR/HGF, small-molecule inhibitors against the tyrosine kinase domain of HGFR and downstream targets. Different HGFR inhibitors are currently in clinical trials in lung cancer and a number of solid tumors. Several phase I trials have already been completed, and two specific trials have been reported combining HGFR with epidermal growth factor receptor (EGFR) inhibition in non-small cell lung cancer. In particular, trials involving MetMAb and ARQ197 (tivantinib) have gained interest. Ultimately, as individualized therapies become a reality for cancers, HGFR will be an important molecular target.

BackgroundNon-small cell lung cancer (NSCLC) is a heterogeneous group of disorders with a number of genetic and proteomic alterations. c-CBL is an E3 ubiquitin ligase and adaptor molecule important in normal homeostasis and cancer. We determined the genetic variations of c-CBL, relationship to receptor tyrosine kinases (EGFR and MET), and functionality in NSCLC.Methods and FindingsUsing archival formalin-fixed paraffin embedded (FFPE) extracted genomic DNA, we show that c-CBL mutations occur in somatic fashion for lung cancers. c-CBL mutations were not mutually exclusive of MET or EGFR mutations; however they were independent of p53 and KRAS mutations. In normal/tumor pairwise analysis, there was significant loss of heterozygosity (LOH) for the c-CBL locus (22%, n = 8/37) and none of these samples revealed any mutation in the remaining copy of c-CBL. The c-CBL LOH also positively correlated with EGFR and MET mutations observed in the same samples. Using select c-CBL somatic mutations such as S80N/H94Y, Q249E and W802* (obtained from Caucasian, Taiwanese and African-American samples, respectively) transfected in NSCLC cell lines, there was increased cell viability and cell motility.ConclusionsTaking the overall mutation rate of c-CBL to be a combination as somatic missense mutation and LOH, it is clear that c-CBL is highly mutated in lung cancers and may play an essential role in lung tumorigenesis and metastasis.

The association between the expression of MOR and the progression of the tumour is consistent with the hypothesis of a direct effect of MOR on cancer progression.

Angiogenesis, an essential step in the development of neoplasia, is a complex process that involves the interaction of tumor cells with stromal cells. Tumor-associated macrophages (TAMs) can participate in the induction of tumor angiogenesis and are thought to be of prognostic value in some neoplasms. We have investigated how macrophages contribute to angiogenesis in head-and-neck squamous-cell carcinoma (HNSCC) and have found that tumor cells attract monocytes and activate them to secrete angiogenic factors. The attraction of macrophages was due to the secretion of monocyte chemotactic protein-1 and TGF-␤1 by tumor cells, while tumor production of TGF-␤1 was responsible for activating macrophages. In addition, activated macrophages produced cytokines that acted in a paracrine fashion by secreting both TNF-␣ and IL-1, which in turn stimulated tumor cells to secrete increased levels of IL-8 and VEGF. These data demonstrate that TAMs play an important role in the in vivo induction of angiogenesis in HNSCC and suggest that antiangiogenic therapies for HNSCC and perhaps other neoplasms must include strategies that will block the ability of tumor cells to recruit macrophages into the tumor microenvironment.Key words : squamous-cell carcinoma; angiogenesis; macrophages; angiogenic loop; head-and-neck cancer Angiogenesis, the growth of new blood vessels from pre-existing ones, is an essential phenotype for tumor formation. 1 Expression of the angiogenic phenotype in the tumor micro-environment is a complex process involving the interaction of many different cell types. The interplay between tumor cells and the various constituents of the surrounding stroma is thought to be critically important in various aspects of tumor biology, including angiogenesis. 2 Macrophages, part of the tumor stroma, are members of the mononuclear phagocyte system of inflammatory cells. A very heterogeneous group, they perform a wide variety of functions depending on the physiologic or pathophysiologic condition to which they are recruited. Given the remarkable versatility of macrophages, it is not surprising that they participate in the induction of tumor angiogenesis. 3 There is a great deal of interest in developing chemotherapeutic clinical trials that are based on anti-angiogenic therapies. 4 However, one of the major challenges for designing such therapies is that there are numerous direct and indirect mechanisms by which tumors can induce new blood vessel growth, including eliciting the help of various stromal cells. Therefore, it is reasonable to assume that successful anti-angiogenic protocols must address each of the possible mechanisms by which tumors can induce new blood vessel growth. Our aim was to identify additional potential mechanisms by which human head-and-neck squamous-cell carcinoma (HNSCC) may induce neovascularization in vivo. Specifically, we investigated how macrophages contribute to angiogenesis in HNSCC and show that HNSCC tumor cells attract monocytes and activate them to secrete angiogenic factors. In additio...

Despite numerous advances, the 5-year survival rate for head and neck squamous cell cancer (HNSCC) has remained largely unchanged. This poor outcome is due to several variables, including the development of multiple primary tumors. Therefore, it is essential to supplement early detection with preventive strategies. Using the 4-nitroquinoline 1-oxide (4-NQO) mouse model, we sought to define an appropriate dose and duration of administration that would predict the histologic timeline of HNSCC progression. Additionally, we sought to determine the timing of the onset of the angiogenic phenotype. Finally, using ABT-510 as a proof-of-principle drug, we tested the hypothesis that inhibitors of angiogenesis can slow/delay the development of HNSCC. We determined that 8 weeks of 100 μg/mL 4-NQO in the drinking water was the optimal dosage and duration to cause a sufficient incidence of hyperkeratoses, dysplasias, and HNSCC over a period of 32 weeks with minimal morbidity and mortality. Increased microvessel density and vascular endothelial growth factor expression in hyperkeratotic lesions provided evidence that the initiation of the angiogenic phenotype occurred before the development of dysplasia. Importantly, ABT-510 significantly decreased the overall incidence of HNSCC from 37.3% to 20.3% (P = 0.021) as well as the combined incidence of dysplasia and HNSCC from 82.7% to 50.6% (P < 0.001). These findings suggest that our refinement of the 4-NQO model allows for the investigation of the histologic, molecular, and biological alterations that occur during the premalignant phase of HNSCC. In addition, these data support the hypothesis that inhibitors of angiogenesis may be promising chemopreventive agents.

Malignant pleural mesothelioma (MPM) is an aggressive disease with a poor prognosis. Studies have shown that both MET and its key downstream intracellular signaling partners, PI3K and mTOR, are overexpressed in MPM. Here we determined the combinatorial therapeutic efficacy of a new generation small molecule inhibitor of MET, ARQ 197, and dual PI3K/mTOR inhibitors NVP-BEZ235 and GDC-0980 in mesothelioma cell and mouse xenograft models. Cell viability results show that mesothelioma cell lines were sensitive to ARQ 197, NVP-BEZ235 and GDC-0980 inhibitors. The combined use of ARQ 197 with either NVP-BEZ235 or GDC-0980, was synergistic (CI<1). Significant delay in wound healing was observed with ARQ 197 (p<0.001) with no added advantage of combining it with either NVP-BEZ235 or GDC-0980. ARQ 197 alone mainly induced apoptosis (20±2.36%) that was preceded by suppression of MAPK activity, while all the three suppressed cell cycle progression. Both GDC-0980 and NVP-BEZ235 strongly inhibited activities of PI3K and mTOR as evidenced from the phosphorylation status of AKT and S6 kinase. The above observation was further substantiated by the finding that a majority of the MPM archival samples tested revealed highly active AKT. While the single use of ARQ 197 and GDC-0980 inhibited significantly the growth of MPM xenografts (p<0.05, p<0.001 respectively) in mice, the combination of the above two drugs was highly synergistic (p<0.001). Our results suggest that the combined use of ARQ 197/NVP-BEZ235 and ARQ 197/GDC-0980 is far more effective than the use of the drugs singly in suppressing MPM tumor growth and motility and therefore merit further translational studies.