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2.  Prolyl Hydroxylase PHD3 Enhances the Hypoxic Survival and G1 to S Transition of Carcinoma Cells 
PLoS ONE  2011;6(11):e27112.
Hypoxia restricts cell proliferation and cell cycle progression at the G1/S interface but at least a subpopulation of carcinoma cells can escape the restriction. In carcinoma hypoxia may in fact select for cells with enhanced hypoxic survival and increased aggressiveness. The cellular oxygen sensors HIF proline hydroxylases (PHDs) adapt the cellular functions to lowered environmental oxygen tension. PHD3 isoform has shown the strongest hypoxic upregulation among the family members. We detected a strong PHD3 mRNA expression in tumors of head and neck squamous cell carcinoma (HNSCC). The PHD3 expression associated with expression of hypoxic marker gene. Using siRNA in cell lines derived from HNSCC we show that specific inhibition of PHD3 expression in carcinoma cells caused reduced cell survival in hypoxia. The loss of PHD3, but not that of PHD2, led to marked cell number reduction. Although caspase-3 was activated at early hypoxia no induction of apoptosis was detected. However, hypoxic PHD3 inhibition caused a block in cell cycle progression. Cell population in G1 phase was increased and the population in S phase reduced demonstrating a block in G1 to S transition under PHD3 inhibition. In line with this, the level of hyperphosphorylated retinoblastoma protein Rb was reduced by PHD3 knock-down in hypoxia. PHD3 loss led to increase in cyclin-dependent kinase inhibitor p27 expression but not that of p21 or p16. The data demonstrated that increased PHD3 expression under hypoxia enhances cell cycle progression and survival of carcinoma cells.
doi:10.1371/journal.pone.0027112
PMCID: PMC3210766  PMID: 22087251
3.  Absence of Dickkopf (Dkk)-3 protein expression is correlated with longer disease-free survival and lower incidence of metastasis in head and neck squamous cell carcinoma 
Oncology Letters  2011;3(2):273-280.
Head and neck squamous cell carcinoma (HNSCC) is one of the most frequently occurring types of cancer worldwide. We focused on the fact that the aberrant function of Wnt/β-catenin signaling is a frequent event in malignancies. Dickkopf (Dkk)-3 is a major negative regulator of Wnt/β-catenin signaling, which is a known tumor suppressor and is down-regulated in various types of cancer. However, the expression profile of the Dkk-3 protein in HNSCC has not yet been reported. The present study was conducted to investigate Dkk-3 protein expression in 90 cases of HNSCC tissue samples and HNSCC-derived cell lines. In contrast to findings available on other types of cancer, the Western blot analysis revealed that HNSCC cell lines expressed the Dkk-3 protein. In immunohistochemistry, 76 cases (84.4%) out of 90 tissue samples were Dkk-3-positive, whereas only 14 cases (15.6%) were negative. Notably, survival analysis showed that the Dkk-3 (−) group exhibited significantly longer disease-free survival (p=0.038), metastasis-free survival (p=0.013) and longer overall survival (p=0.155). The results showed that the Dkk-3 protein was dominantly expressed and may be involved in carcinogenesis and metastasis in HNSCC. Moreover, the findings suggest that the function of Dkk-3 differs depending on the tissue of origin, and that it may exert an oncogenic function in HNSCC.
doi:10.3892/ol.2011.473
PMCID: PMC3362421  PMID: 22740894
head and neck squamous cell carcinoma; Dkk-3; immunohistochemistry
4.  Epigenetic Inactivation of GALR1 in Head and Neck Cancer 
Purpose
One copy of the GALR1 locus on 18q is often deleted and expression is absent in some head and neck squamous cell carcinoma (HNSCC) cell lines. To determine if LOH and hypermethylation might silence the GALR1 gene, promoter methylation status and gene expression were assessed in a large panel of HNSCC cell lines and tumors.
Experimental Design
Promoter methylation of GALR1 in 72 cell lines and 100 primary tumor samples was analyzed using methylation-specific PCR (MSP). GALR1 expression and methylation status were analyzed further by real-time PCR and bisulfite sequencing analysis.
Results
The GALR1 promoter was fully or partially methylated in 38 of 72 HNSCC cell lines (52.7%) but not in the majority 18/20 (90.0%) of non-malignant lines. GALR1 methylation was also found in 38/100 (38%) primary tumor specimens. Methylation correlated with decreased GALR1 expression. In tumors methylation was significantly correlated with increased tumor size (P=0.0036), lymph-node status (P=0.0414), tumor stage (P=0.0037), cyclin D1 expression (P=0.0420), and p16 methylation (P=0.0494) and survival (P=0.045). Bisulfite sequencing of 36 CpG sites upstream of the transcription start site revealed that CpG methylation within transcription factor binding sites correlated with complete suppression of GALR1 mRNA. Treatment with TSA and 5-azacytidine restored GALR1 expression. In UM-SCC-23 cells that have total silencing of GALR1, exogenous GALR1 expression and stimulation with galanin suppressed cell proliferation.
Conclusions
Frequent promoter hypermethylation, gene silencing, association with prognosis, and growth suppression after re-expression support the hypothesis that GALR1 is a tumor suppressor gene in HNSCC.
doi:10.1158/1078-0432.CCR-07-4673
PMCID: PMC3189853  PMID: 19047085
Gene silencing; tumor suppressor gene; human chromosome 18; G-protein coupled receptor; promoter hypermethylation; transcription factor binding sites; methyltransferase inhibitor; histone deacetylase inhibitor
5.  Differences in the Nemosis Response of Normal and Cancer-Associated Fibroblasts from Patients with Oral Squamous Cell Carcinoma 
PLoS ONE  2009;4(9):e6879.
Background
Tumor-stroma reaction is associated with activation of fibroblasts. Nemosis is a novel type of fibroblast activation. It leads to an increased production of growth factors and proinflammatory and proteolytic proteins, while at the same time cytoskeletal proteins are degraded. Here we used paired normal skin fibroblasts and cancer-associated fibroblasts (CAF) and primary and recurrent oral squamous cell carcinoma (SCC) cells to study the nemosis response.
Principal Findings
Fibroblast nemosis was analyzed by protein and gene expression and the paracrine regulation with colony formation assay. One of the normal fibroblast strains, FB-43, upregulated COX-2 in nemosis, but FB-74 cells did not. In contrast, CAF-74 spheroids expressed COX-2 but CAF-43 cells did not. Alpha-SMA protein was expressed in both CAF strains and in FB-74 cells, but not in FB-43 fibroblasts. Its mRNA levels were downregulated in nemosis, but the CAFs started to regain the expression. FSP1 mRNA was downregulated in normal fibroblasts and CAF-74 cells, but not in CAF-43 fibroblasts. Serine protease FAP was upregulated in all fibroblasts, more so in nemotic CAFs. VEGF, HGF/SF and FGF7 mRNA levels were upregulated to variable degree in nemosis. CAFs increased the colony formation of primary tumor cell lines UT-SCC-43A and UT-SCC-74A, but normal fibroblasts inhibited the anchorage-independent growth of recurrent UT-SCC-43B and UT-SCC-74B cells.
Conclusions
Nemosis response, as observed by COX-2 and growth factor induction, and expression of CAF markers α-SMA, FSP1 and FAP, varies between fibroblast populations. The expression of CAF markers differs between normal fibroblasts and CAFs in nemosis. These results emphasize the heterogeneity of fibroblasts and the evolving tumor-promoting properties of CAFs.
doi:10.1371/journal.pone.0006879
PMCID: PMC2730537  PMID: 19721715
6.  Pim-1 Kinase Expression Predicts Radiation Response in Squamocellular Carcinoma of Head and Neck and Is under the Control of Epidermal Growth Factor Receptor12 
Neoplasia (New York, N.Y.)  2009;11(7):629-636.
Pim-1 is an oncogenic serine/threonine kinase with poorly defined function in epithelial cancers. In this study, we determined 1) associations of Pim-1 expression with clinicopathological parameters including responsiveness to irradiation in squamocellular cancers of head and neck and 2) how Pim-1 expression is controlled subsequent to irradiation. Moderate to high expression of Pim-1 correlated to poor response to radiation therapy (P = .003). It is also associated to the expression of epidermal growth factor receptor (EGFR, P < .0001), which has been shown to be activated by irradiation. In radioresistant tumors, irradiation promoted nuclear translocation of Pim-1 (P < .005). When directly testing EGFR dependence of Pim-1 expression, up-regulation and nuclear translocation of Pim-1 could be induced through stimulation of EGFR with its ligands EGF or transforming growth factor α. Both ligand- and irradiation-induced changes in Pim-1 expression and localization could be inhibited by the monoclonal anti-EGFR antibody cetuximab and by the tyrosine kinase inhibitor gefitinib also targeting EGFR. These results suggest that irradiation-induced activation of EGFR upregulates Pim-1, and Pim-1 may be used as a novel predictive marker of radiation response in patients with squamocellular cancers of head and neck.
PMCID: PMC2697349  PMID: 19568408
7.  Mannose Receptor Is a Novel Ligand for L-Selectin and Mediates Lymphocyte Binding to Lymphatic Endothelium 
The Journal of Experimental Medicine  2001;194(8):1033-1042.
Continuous lymphocyte recirculation between blood and lymphoid tissues forms a basis for the function of the immune system. Lymphocyte entrance from the blood into the tissues has been thoroughly characterized, but mechanisms controlling lymphocyte exit from the lymphoid tissues via efferent lymphatics have remained virtually unknown. In this work we have identified mannose receptor (MR) on human lymphatic endothelium and demonstrate its involvement in binding of lymphocytes to lymphatic vessels. We also show that the binding requires L-selectin, and L-selectin and MR form a receptor–ligand pair. On the other hand, L-selectin binds to peripheral lymph node addressins (PNAds) on high endothelial venules (HEVs) that are sites where lymphocytes enter the lymphatic organs. Interestingly, MR is absent from HEVs and PNAds from lymphatic endothelium. Thus, lymphocyte L-selectin uses distinct ligand molecules to mediate binding at sites of lymphocyte entrance and exit within lymph nodes. Taken together, interaction between L-selectin and MR is the first molecularly defined mechanism mediating lymphocyte binding to lymphatic endothelium.
PMCID: PMC2193520  PMID: 11602634
lymphatics; lymphocyte recirculation; cancer metastasis; endothelium; lymph node

Results 1-7 (7)