u-0126 and Head-and-Neck-Neoplasms

Galanin and its receptors, GALR1 and GALR2, are tumor suppressors and represent therapeutic targets in head and neck squamous cell carcinoma (HNSCC). In the present study, it was demonstrated that the re‑expression of GALR1 in GALR1 and GALR2‑negative HNSCC cells suppresses tumor cell proliferation. This is mediated via extracellular‑regulated protein kinase‑1/2 (ERK1/2)‑dependent effects on the cyclin‑dependent kinase inhibitors (CKI) and cyclin D1. In combination with galanin, GALR2 also suppressed proliferation by increasing CKI and decreasing cyclin D1 levels. In contrast to GALR1, overexpression of GALR2 also induced caspase‑3‑dependent apoptosis. It was identified that in GALR2‑transfected cells, galanin induced activation of ERK1/2 and suppressed cell proliferation. Galanin stimulation also decreased the expression of cyclin D1 and induced apoptotic DNA ladder formation in GALR2‑transfected cells. Pretreatment with the ERK1/2‑specific inhibitor U0126 and pertussis toxin prevented the suppression of cyclin D1 expression, however did not affect DNA ladder formation. In conclusion, GALR2 expression in the presence of galanin exerts antitumor effects via cell cycle arrest and apoptotic pathways, and reactivation of these pathways may have therapeutic benefits in HNSCC.

Topics: Antineoplastic Agents; Apoptosis; Butadienes; Carcinoma, Squamous Cell; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Galanin; Gene Expression Regulation; Head and Neck Neoplasms; Humans; MAP Kinase Signaling System; Nitriles; Receptor, Galanin, Type 2; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

Kinases downstream of growth factor receptors have been implicated in radioresistance and are, therefore, attractive targets to improve radiotherapy outcome in head and neck squamous cell carcinoma (HNSCC) patients.. An antibody-based array was used to quantify the expression levels of multiple phospho-kinases involved in growth factor signaling in nine untreated or irradiated HNSCC lines. Radiosensitivity was assessed with clonogenic cell survival assays and correlated with the expression levels of the phospho-kinases. Inhibitors of the kinases that were associated with radiosensitivity were tested for their ability to increase radiosensitivity in the 3 most radioresistant HNSCC lines.. The basal expression of phosphorylated Yes, Src and STAT5A, and the expression after radiotherapy of phosphorylated AKT, MSK1/2, Src, Lyn, Fyn, Hck, and STAT6, were correlated with radiosensitivity in the panel of HNSCC lines. In combination with radiotherapy, inhibitors of AKT, p38 and Src Family Kinases (SFK) were variably able to reduce survival, whereas MEK1/2, STAT5 and STAT6 inhibition reduced survival in all cell lines. The combined effect of radiotherapy and the kinase inhibitors on cell survival was mostly additive, although also supra-additive effects were observed for AKT, MEK1/2, p38 and STAT5 inhibition.. Kinases of the AKT, MAPK, STAT and SFK pathways correlated with radiosensitivity in a panel of HNSCC lines. Particularly inhibitors against MEK1/2, STAT5 and STAT6 were able to decrease survival in combination with radiotherapy. Hence, inhibitors against these kinases have the potential to improve radiotherapy outcome in HNSCC patients and further research is warranted to confirm this in vivo.

Topics: Antineoplastic Agents; Butadienes; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Chemoradiotherapy; Dasatinib; Drug Screening Assays, Antitumor; Head and Neck Neoplasms; Humans; Imidazoles; Isoxazoles; Leflunomide; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Nitriles; Protein Kinase Inhibitors; Pyridines; Pyrimidines; Radiation Tolerance; STAT5 Transcription Factor; STAT6 Transcription Factor; Thiazoles; Tumor Suppressor Proteins

Focal adhesion kinase (FAK), a main regulator of integrin signaling and cell migration, is frequently overexpressed and hyperphosphorylated in human head-and-neck squamous cell carcinoma (HNSCC). We have previously shown that pharmacologic FAK inhibition leads to radiosensitization of 3-dimensionally grown HNSCC cell lines. To further evaluate the role of FAK in radioresistance and as a potential cancer target, we examined FAK and FAK downstream signaling in HNSCC cell lines grown in more physiologic extracellular matrix-based 3-dimensional cell cultures.. Seven HNSCC cell lines were grown in 3-dimensional extracellular matrix and the clonogenic radiation survival, expression, and phosphorylation of FAK, paxillin, Akt1, extracellular signal-regulated kinase (ERK)1/2, and MEK1/2 were analyzed after siRNA-mediated knockdown of FAK, Akt1, MEK1, FAK+Akt1, or FAK+MEK1 compared with controls or stable overexpression of FAK. The role of MEK1/2 for clonogenic survival and signaling was investigated using the MEK inhibitor U0126 with or without irradiation.. FAK knockdown moderately or significantly enhanced the cellular radiosensitivity of 3-dimensionally grown HNSCC cells. The FAK downstream targets paxillin, Akt1, and ERK1/2 were substantially dephosphorylated under FAK depletion. FAK overexpression, in contrast, increased radiation survival and paxillin, Akt1, and ERK1/2 phosphorylation. The degree of radiosensitization upon Akt1, ERK1/2, or MEK1 depletion or U0126 was superimposable to FAK knockdown. Combination knockdown conditions (ie, Akt1/FAK, MEK1/FAK, or U0126/FAK) failed to provide additional radiosensitization.. Our data provide further evidence for FAK as important determinant of radiation survival, which acts in the same signaling axis as Akt1 and ERK1/2. These data strongly support our hypothesis that FAK is a relevant molecular target for HNSCC radiotherapy.

Topics: Butadienes; Carcinoma, Squamous Cell; Cell Culture Techniques; Cell Line, Tumor; Cell Survival; Focal Adhesion Kinase 1; Head and Neck Neoplasms; Humans; MAP Kinase Kinase Kinase 1; MAP Kinase Kinase Kinase 2; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Molecular Targeted Therapy; Neoplasm Proteins; Nitriles; Paxillin; Phosphorylation; Proto-Oncogene Proteins c-akt; Radiation Tolerance; Tumor Stem Cell Assay

Irradiation plays a pivotal role in head and neck squamous cell carcinoma (HNSCC) treatment. However, especially recurrent tumors frequently show increased radioresistance. We analyzed irradiation-stimulated mitogen-activated protein kinase (MAPK) signaling pathways to define cellular rescue mechanisms.. Irradiated HNSCC cells were screened for MAPK activation and results were confirmed and refined by functional analyses. Extracellular signal-regulated kinase (ERK) inhibitor U0126 application enabled us to specify postradiogenic cellular responses. Vascular endothelial growth factor (VEGF) levels were analyzed additionally.. We observed a pronounced and time-dependent ERK stimulation. Pathway inhibition resulted in decreased radioresistance. Likewise, we found a decrease of VEGF release after inhibitor treatment. ERK activation was confirmed in xenotransplants showing elevated postradiogenic phospho-ERK (pERK) and VEGF levels.. Our data give evidence for induction of ERK and successive VEGF release in HNSCC during radiotherapy, which might be partially explained by autoregulated cytoprotection maintained by pERK and potentially VEGF. In conclusion, targeting the ERK-VEGF axis might enhance the efficiency of radiotherapy.

Topics: Animals; Butadienes; Carcinoma, Squamous Cell; Cell Line, Tumor; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Head and Neck Neoplasms; Humans; Immunohistochemistry; MAP Kinase Signaling System; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Neoplasm Transplantation; Nitriles; Radiotherapy Dosage; Signal Transduction; Up-Regulation; Vascular Endothelial Growth Factor A

Protein kinase C (PKC) zeta has been implicated as a mediator of epidermal growth factor (EGF) receptor (EGFR) signaling in certain cell types. Because EGFR is ubiquitously expressed in squamous cell carcinomas of the head and neck (SCCHN) and plays a key role in tumor progression, we determined whether PKCzeta is required for tumor cell proliferation and viability. Examination of total and phosphorylated PKCzeta expression in normal oral mucosa, dysplasia, and carcinoma as well as SCCHN tumor cell lines revealed a significant increase in activated PKCzeta expression from normal to malignant tissue. PKCzeta activity is required for EGF-induced extracellular signal-regulated kinase (ERK) activation in both normal human adult epidermal keratinocytes and five of seven SCCHN cell lines. SCCHN cells express constitutively activated EGFR family receptors, and inhibition of either EGFR or mitogen-activated protein kinase (MAPK) activity suppressed DNA synthesis. Consistent with this observation, inhibition of PKCzeta using either kinase-dead PKCzeta mutant or peptide inhibitor suppressed autocrine and EGF-induced DNA synthesis. Finally, PKCzeta inhibition enhanced the effects of both MAPK/ERK kinase (U0126) and broad spectrum PKC inhibitor (chelerythrine chloride) and decreased cell proliferation in SCCHN cell lines. The results indicate that (a) PKCzeta is associated with SCCHN progression, (b) PKCzeta mediates EGF-stimulated MAPK activation in keratinocytes and SCCHN cell lines, (c) PKCzeta mediates EGFR and MAPK-dependent proliferation in SCCHN cell lines; and (d) PKCzeta inhibitors function additively with other inhibitors that target similar or complementary signaling pathways.

Topics: Alkaloids; Amino Acid Sequence; Benzophenanthridines; Butadienes; Carcinoma, Squamous Cell; Cell Growth Processes; Cell Line, Tumor; Cell Transformation, Neoplastic; DNA, Neoplasm; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Keratinocytes; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Mouth Mucosa; Mouth Neoplasms; Nitriles; Phenanthridines; Protein Kinase C; Protein Kinase Inhibitors

Interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF) promote tumor angiogenesis, growth, and metastasis and are coexpressed by human head and neck squamous cell carcinomas (HNSCCs) and a variety of other cancers. The promoters of the IL-8 and VEGF genes contain different recognition sites for transcription factors nuclear factor (NF)-kappaB and activator protein-1 (AP-1), which we showed previously are coactivated in HNSCCs. NF-kappaB and AP-1 may be modulated by the inhibitor kappaB kinase (IKK) and mitogen-activated protein kinase (MAPK) signal pathways, but the contribution of these pathways to expression of IL-8 and VEGF and as potential targets for antiangiogenesis therapy in HNSCC is not known. In this study, we examined the effects of modulation of the MAPK and IKK pathways on expression of IL-8 and VEGF by UM-SCC-9 and UM-SCC-11B cell lines. Interruption of IKK-mediated activation of NF-kappaB by expression of an inhibitor kappaB alpha mutant (IkappaB alphaM) in UM-SCC-9 cells resulted in partial inhibition of expression of IL-8 but not VEGF. Analysis of possible alternative pathways for induction of these genes revealed activation of the MAPK extracellular signal-regulated kinase (ERK1/2) in cell lines UM-SCC-9 and UM-SCC-11B. Basal and tumor necrosis factor-alpha-inducible phosphorylation of ERK1/2 and secretion of IL-8 and VEGF could be specifically inhibited by a MEK inhibitor, U0126. Expression of IL-8 and VEGF in the cell lines was associated with coactivation of both NF-kappaB and AP-1, and U0126 inhibited both NF-kappaB and AP-1 reporter activity in UM-SCC-9 and UM-SCC-11B cells. The ERK pathway appears to contribute to expression of IL-8 and VEGF and transactivation of NF-kappaB as well as AP-1 in HNSCC. Combined inhibition of both MAPK and IKK pathways may be needed for suppression of the signal transduction mechanism(s) regulating VEGF and IL-8 secretion and angiogenesis by human HNSCC.

Topics: Butadienes; Carcinoma, Squamous Cell; Endothelial Growth Factors; Enzyme Inhibitors; Head and Neck Neoplasms; Humans; I-kappa B Kinase; Interleukin-8; Lymphokines; MAP Kinase Kinase Kinase 1; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; Protein Serine-Threonine Kinases; Signal Transduction; Transcription Factor AP-1; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors