sphingosine-kinase and Head-and-Neck-Neoplasms

Sphingosine-1-phosphate (S1P) signaling has been widely explored as a therapeutic target in cancer. Sphingosine kinase 2 (SK2), one of the kinases that phosphorylate sphingosine, has a cell type and cell location-dependent mechanism of action, so the ability of SK2 to induce cell cycle arrest, apoptosis, proliferation, and survival is strongly influenced by the cell-context. In contrast to SK1, which is widely studied in different types of cancer, including head and neck cancer, the role of SK2 in the development and progression of oral cancer is still poorly understood. In order to elucidate SK2 role in oral cancer, we performed the overexpression of SK2 in non-tumor oral keratinocyte cell (NOK SK2) and in oral squamous cell carcinoma (HN12 SK2), and RNA interference for SK2 in another oral squamous cell carcinoma (HN13 shSK2). In our study we demonstrate for the first time that accumulation of SK2 can be a starting point for oncogenesis and transforms a non-tumor oral keratinocyte (NOK-SI) into highly aggressive tumor cells, even acting on cell plasticity. Furthermore, in oral metastatic cell line (HN12), SK2 contributed even more to the tumorigenesis, inducing proliferation and tumor growth. Our work reveals the intriguing role of SK2 as an oral tumor promoter and regulator of different pathways and cellular processes.

Topics: Autophagy; Carcinogenesis; Carcinoma, Squamous Cell; Cell Proliferation; Cell Transformation, Neoplastic; Head and Neck Neoplasms; Humans; Keratinocytes; Mouth Neoplasms; Squamous Cell Carcinoma of Head and Neck

By analyzing the expression profile of microRNAs in head and neck squamous cell carcinomas (HNSCC), we found that the expression level of miR-124 was 4.59-fold lower in tumors than in normal tissues. To understand its functions, we generated a miR-124-expressing subline (JHU-22miR124) and a mock vector-transfected subline (JHU-22vec) by transfecting the mimic of miR-124 into JHU-22 cancer cells. Restored expression of miR-124 in JHU-22miR124 cells led to reduced cell proliferation, delayed colony formation, and decreased tumor growth, indicating a tumor-suppressive effect of miR-124. Subsequent target search revealed that the 3'-UTR of SphK1 mRNA carries a complementary site for the seed region of miR-124. SphK1 was also detected to be overexpressed in HNSCC cell lines, but down-expressed in JHU-22miR124 cells and tumor xenografts. These results suggest that SphK1 is a target of miR-124. To confirm this finding, we constructed a 3'-UTR-Luc-SphK1 vector and a binding site-mutated luciferase reporter vector. Co-transfection of 3'-UTR-Luc-SphK1 with miR-124 expression vector exhibited a 9-fold decrease in luciferase activity compared with mutated vector, suggesting that miR-124 inhibits SphK1 activity directly. Further studies on downstream signaling demonstrated accumulation of ceramide, increased expression of the pro-apoptotic Bax, BAD and PARP, decreased expression of the anti-apoptotic Bcl-2 and Bcl-xL, and enhanced expression of cytochrome c and caspase proteins in JHU-22miR124 compared with JHU-22vec cells and tumor xenografts. We conclude that miR-124 acts as a tumor suppressor in HNSCC by directly inhibiting SphK1 activity and its downstream signals.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Genes, Tumor Suppressor; Head and Neck Neoplasms; Heterografts; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Transfection

SphK1 is known to play a role in tumor progression, resistance to radiochemotherapy, and migration patterns. As the overall survival rates of squamous cell carcinoma of the head and neck (HNSCC) remain poor due to limitations in surgery and irradiation and chemotherapy resistance, SphK1 is an important enzyme to investigate. The purpose of this study was to elucidate the impact of SphK1 on irradiation efficacy of HNSCC in-vitro with emphasis on EGFR signaling. By immunhistochemical staining we found a positive correlation between EGFR and SphK1 expression in patient specimens. In colony formation assays irradiation sensitive cell lines showed a poor response to cetuximab, an EGFR inhibitor, and SKI-II, a SphK1 inhibitor, and vice versa. In irradiation sensitive cells an enhanced reduction of cell migration and survival was found upon simultaneous targeting of EGFR and SphK1. In the present study, we elucidated a linkage between the two signaling pathways with regard to the efficacy of cetuximab treatment and the impact on the migration behavior of tumor cells. We investigated the biological impact of inhibiting these pathways and examined the biochemical implications after different treatments. An understanding of the processes involved could help to improve the treatment of patients with HNSCC.

Topics: Aged; Aged, 80 and over; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cetuximab; Disease Progression; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Male; Middle Aged; Paraffin Embedding; Phosphotransferases (Alcohol Group Acceptor); Receptor Cross-Talk; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Thiazoles

Radiation therapy (RT) is an important treatment modality used against a number of human cancers, including head and neck squamous cell carcinoma (HNSCC). However, most of these cancers have an inherent anti-apoptotic mechanism that makes them resistant to radiation therapy. This radioresistance of cancer cells necessitates the irradiation of tumor areas with extremely high doses of radiation to achieve effective therapy, resulting in damage to normal tissues and leading to several treatment related side effects. These side effects significantly impair the quality of life of treated patients, and preclude the possibility of repeat radiation treatment in patients with tumor recurrence. Our previous research has correlated the upregulation of the anti-apoptotic sphingosine kinase (SphK1) gene in HNSCC cells with their radioresistance properties. In the current study, we hypothesized that by downregulating the SphK1 gene using nanotechnology mediated gene silencing, we can render these cells more vulnerable to radiation therapy by enabling apoptosis at lower radiation doses. We have employed biocompatible gold nanorods (GNRs) as carriers of short interfering RNA (siRNA) targeting the SphK1 gene. GNRs play a critical role in protecting the siRNA molecules against physiological degradation, as well as delivering them inside target cells. Following their synthesis and characterization, these nanoplexes were applied to HNSCC cells in culture, resulting in the radiosensitization of the treated cells. Furthermore, the GNR-siRNA nanoplexes were injected intratumorally into subcutaneous HNSCC tumors grown in mice, prior to the initiation of radiation therapy in vivo. Subsequent exposure of GNR-SphK1siRNA nanoplex-treated tumors to radiation (GNR-SphK1siRNA + IRRA) resulted in over 50% tumor regression compared to control GNR-GFPsiRNA nanoplex and radiation treated tumors (GNR-GFPsiRNA + IRRA). In addition, we were able to induce this tumor regression in nanoplex treated tumors with radiation doses much lower than those commonly required in clinical RT. These experiments lay the foundation for the development of a nanotechnology-mediated gene silencing tool for more potent radiation therapy of a number of human cancers, with minimal, if any, toxic side effects.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cell Survival; Gold; Head and Neck Neoplasms; Immunohistochemistry; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Confocal; Microscopy, Electron, Transmission; Nanotubes; Phosphotransferases (Alcohol Group Acceptor); Radiation-Sensitizing Agents; RNA, Small Interfering; Squamous Cell Carcinoma of Head and Neck

Sphingosine kinase 1 (SphK1) is an important regulator of apoptosis, survival, and proliferation in cancer cells. SphK1 expression in head and neck squamous cell cancer (HNSCC) cell lines and tumor tissue was assessed, and the efficacy of SphK1 knockdown in increasing tumor radiosensitivity was evaluated in vitro and in vivo.. Expression of SphK1 was determined by immunohistochemistry, Western blot, and real-time polymerase chain reaction (RT-PCR) in 34 prospectively collected HNSCC tumor samples. HNSCC cell lines squamous cell carcinoma (SCC)-15 and SCC-25 were treated with SphK1 inhibitor SKI-II and siRNA targeting SphK1 with and without radiation, and the cell viability was assessed. SCC-15 cells with and without transfection of SphK1 siRNA were then injected into athymic nude mice to develop tumor xenografts, and these 2 groups were further divided into 1 group that received radiation and 1 group that did not. Tumor size was measured over 18 days, when the animals were killed and the tumors were evaluated by immunohistochemistry.. SphK1 is found in both HNSCC cell lines and human tumor samples, with higher expression correlated with advanced tumor stage, nodal involvement, and recurrence. In vitro, both SCC-15 and SCC-25 were found to be radioresistant; however, they were sensitized by administration of SKI-II and transfection with siRNA targeting SphK1. In vivo, SphK1-siRNA transfected xenografts were decreased in size compared with both nonradiated control and radiated control mice, whereas mice with both SphK1-siRNA and radiation treatment showed a synergistic reduction in tumor volume. Histopathologic analysis demonstrated a decreased proliferative state in SphK1-siRNA transfected tumors.. SphK1 is upregulated in HNSCC, and inhibition of SphK1 sensitizes HNSCC to radiation-induced cytotoxicity.

Topics: Animals; Apoptosis; Blotting, Western; Carcinoma; Carcinoma, Squamous Cell; Disease Models, Animal; Enzyme Inhibitors; Head and Neck Neoplasms; Humans; Immunohistochemistry; In Vitro Techniques; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms, Squamous Cell; Phosphotransferases (Alcohol Group Acceptor); Polymerase Chain Reaction; Radiation Tolerance; Squamous Cell Carcinoma of Head and Neck; Thiazoles; Treatment Outcome

It is important to identify novel and effective targets for cancer prevention and therapy against head and neck squamous cell carcinoma (HNSCC), one of the most lethal cancers. Accumulating evidence suggests that the bioactive sphingolipids, such as sphingosine-1-phosphate (S1P) and its generating enzyme, sphingosine kinase 1 (SphK1) play pivotal roles in several important biological functions including promoting tumor growth and carcinogenesis. However, roles of SphK1/S1P in HNSCC development and/or progression have not been defined previously. Therefore, in this study, we first analyzed the expression of SphK1 in human HNSCC tumor samples and normal head & neck tissues (n = 78 and 17, respectively) using immunohistochemistry. The data showed that SphK1 is overexpressed in all of the HNSCC tumors tested (stages I-IV). We next investigated whether SphK1 is necessary for HNSCC development. To define the role of SphK1/S1P in HNSCC development, we utilized 4-nitroquinoline-1-oxide (4-NQO)-induced HNSCC model in wild-type mice compared with SphK1(-/-) knockout (KO) mice. Remarkably, we found that the genetic loss of SphK1, which reduced S1P generation, significantly prevented 4-NQO-induced HNSCC carcinogenesis, with decreased tumor incidence, multiplicity, and volume when compared with controls. Moreover, our data indicated that prevention of 4-NQO-induced HNSCC development in SphK1(-/-) KO mice might be associated with decreased cell proliferation, increased levels of cleaved (active) caspase 3, and downregulation of phospho (active) AKT expression. Thus, these novel data suggest that SphK1/S1P signaling may play important roles in HNSCC carcinogenesis, and that targeting SphK1/S1P might provide a novel strategy for chemoprevention and treatment against HNSCC.

Topics: Animals; Cell Proliferation; Disease Progression; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Genetic; Oligonucleotide Array Sequence Analysis; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt

Sphingosine kinase-1 (SPHK1) modulates the proliferation, apoptosis and differentiation of keratinocytes through the regulation of ceramide and sphingosine-1-phosphate levels. However, studies on the expression of SPHK1 in human head and neck squamous cell carcinoma (HNSCC) specimens are lacking. Therefore, the aim of the present work was to evaluate SPHK1 expression in human primary HNSCCs and to correlate the results with clinical and anatomopathological parameters. We investigated the expression of this protein by immunohistochemistry performed in tissue microarrays of HNSCC and in an independent cohort of 37 paraffin-embedded specimens. SPHK1 expression was further validated by real-time PCR performed on laser capture-microdissected tissue samples. The positive rate of SPHK1 protein in the cancerous tissues was significantly higher (74%) than that in the nontumor oral tissues (23%), and malignant tissues showed stronger immunoreactivity for SPHK1 than normal matching samples. These results were confirmed by real-time PCR quantification of SPHK1 mRNA. Interestingly, the positive expression of SPHK1 was associated with shorter patient survival time (Kaplan-Meier survival curves) and with the loss of p21 expression. Taken together, these results demonstrate that SPHK1 is upregulated in HNSCC and provide clues of the role SPHK1 might play in tumor progression.

Topics: Biomarkers, Tumor; Blotting, Western; Carcinoma, Squamous Cell; Disease Progression; Gene Expression; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Microarray Analysis; Phosphotransferases (Alcohol Group Acceptor); Polymerase Chain Reaction; Prognosis; RNA, Messenger; Sphingolipids; Up-Regulation