RESUMEN
Low 5-year survival rate in laryngeal squamous cell carcinoma (LSCC) is to large extent attributable to high rate of recurrences and metastases. Despite the importance of the latter process, its complex genetic background remains not fully understood. Recently, we identified two metastasis-related candidate genes, DIAPH2 and DIAPH3 to be frequently targeted by hemizygous/homozygous deletions, respectively, in LSCC cell lines. They physiologically regulate such processes as cell movement and adhesion, hence we found it as a rationale, to study if tumor LSCC specimens harbor mutations of these genes and whether the mutations are associated with metastasizing tumors. As a proof of concept, we sequenced both genes in five LSCC cell lines derived from lymph node metastases assuming there the highest probability of finding alterations. Indeed, we identified one hemizygous deletion (c.3116_3240del125) in DIAPH2 targeting the FH2 domain. Moreover, we analyzed 95 LSCC tumors (53 N0 and 42 N+) using the Illumina platform and identified three heterozygous single nucleotide variants in DIAPH2 targeting conserved domains exclusively in N+ tumors. By combining these results with cBioPortal data we showed significant enrichment of DIAPH2 mutations (P = 0.036) in N+ tumors. To demonstrate the consequences of DIAPH2 inactivation, CRISPR/Cas9 editing was used to obtain a heterozygous DIAPH2+/- mutant HEK-293T cell line. Importantly, the edited line shows a shift from 'proliferation' to 'migration' phenotype typically observed in metastasizing cells. In conclusion, we report that DIAPH2 alterations are present primarily in metastasizing specimens of LSCC and suggest that they may contribute to the metastatic potential of the tumor.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/secundario , Movimiento Celular , Forminas/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Laríngeas/patología , Apoptosis , Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Estudios de Casos y Controles , Proliferación Celular , Estudios de Seguimiento , Forminas/genética , Humanos , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/metabolismo , Metástasis Linfática , Pronóstico , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
PURPOSE: Late-stage OTSCC is associated with poor overall survival (OS). Non-curative treatment approach aims to improve quality of life and prolong survival of patients deemed incurable. The purpose of this study was to investigate the used non-curative treatment modalities for OTSSC and patient survival. METHODS: All patients diagnosed with OTSCC and treated with non-curative intent at the HUS Helsinki University Hospital (Helsinki, Finland) during the 12-year period of 2005-2016 were included. Survival analysis after the non-curative treatment decision was conducted using the Kaplan-Meier method in this population-based study. RESULTS: Eighty-two patients were identified. A non-curative treatment decision was made at presentation without any previous treatment in 26 patients (7% of all patients diagnosed with OTSCC during the study period). Palliative radiotherapy was administered to 24% of all patients. The average survival time after the non-curative treatment decision was 3.7 months (median 2 and range 0-26). CONCLUSIONS: Due to the short mean survival time after decision for treatment with non-curative intent, and the notable symptom burden in this patient population, a prompt initiation of all non-curative measures is warranted.
Asunto(s)
Cuidados Paliativos , Calidad de Vida , Carcinoma de Células Escamosas de Cabeza y Cuello , Neoplasias de la Lengua , Adulto , Anciano , Toma de Decisiones Clínicas , Femenino , Finlandia/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Cuidados Paliativos/métodos , Cuidados Paliativos/psicología , Estudios Retrospectivos , Carcinoma de Células Escamosas de Cabeza y Cuello/mortalidad , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/psicología , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Análisis de Supervivencia , Tasa de Supervivencia , Neoplasias de la Lengua/mortalidad , Neoplasias de la Lengua/patología , Neoplasias de la Lengua/psicología , Neoplasias de la Lengua/terapiaRESUMEN
In this study, we analyzed the expression profile of four genes (CCNA2, CCNB1, CCNB2, and CDK1) in laryngeal squamous cell carcinoma (LSCC) cell lines and tumor samples. With the application of microarray platform, we have shown the overexpression of these genes in all analyzed LSCC samples in comparison to non-cancer controls from head and neck region. We have selected CDK1 for further analysis, due to its leading role in cell cycle regulation. It is a member of the Ser/Thr protein kinase family of proven oncogenic properties. The results obtained for CDK1 were further confirmed with the application of reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique, Western blot, and immunohistochemistry (IHC). The observed upregulation of CDK1 in laryngeal squamous cell carcinoma has encouraged us to analyze for genetic mechanisms that can be responsible this phenomenon. Therefore, with the application of array-CGH, sequencing analysis and two methods for epigenetic regulation analysis (DNA methylation and miRNA expression), we tried to identify such potential mechanisms. Our attempts to identify the molecular mechanisms responsible for observed changes failed as we did not observe significant alterations neither in the DNA sequence nor in the gene copy number that could underline CDK1 upregulation. Similarly, the pyrosequencing and miRNA expression analyses did not reveal any differences in methylation level and miRNA expression, respectively; thus, these mechanisms probably do not contribute to elevation of CDK1 expression in LSCC. However, our results suggest that alteration of CDK1 expression on both mRNA and protein level probably appears on the very early step of carcinogenesis.
Asunto(s)
Carcinoma de Células Escamosas/genética , Quinasas Ciclina-Dependientes/biosíntesis , Neoplasias de Cabeza y Cuello/genética , Neoplasias Laríngeas/genética , Recurrencia Local de Neoplasia/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Western Blotting , Proteína Quinasa CDC2 , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/análisis , Femenino , Neoplasias de Cabeza y Cuello/patología , Humanos , Inmunohistoquímica , Neoplasias Laríngeas/patología , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Carcinoma de Células Escamosas de Cabeza y Cuello , Transcriptoma , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Endotelio Linfático/inmunología , Selectina L/inmunología , Lectinas Tipo C , Linfocitos/inmunología , Lectinas de Unión a Manosa , Receptores de Superficie Celular/inmunología , Animales , Anticuerpos/inmunología , Glicosilación , Humanos , Ligandos , Macrófagos/inmunología , Receptor de Manosa , Ratones , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Tumour-specific expression of matrix metalloproteinase (MMP)-7 has been noted in cutaneous squamous cell carcinomas (SCCs) in patients with recessive dystrophic epidermolysis bullosa (RDEB). OBJECTIVES: To examine the potential role of MMP-7 in shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in RDEB-associated and sporadic SCCs. METHODS: Tissue microarrays of RDEB-associated SCC (n = 20), non-EB SCC (n = 60) and Bowen disease (n = 28) were immunostained for MMP-7, CD44 variant 3 (CD44v3) and HB-EGF. Shedding of HB-EGF was studied in vitro using two cutaneous SCC cell lines. RESULTS: Immunohistochemical analysis showed that HB-EGF was absent in tumour cells when MMP-7 and CD44v3 colocalized, and that the absence of HB-EGF was more pronounced in RDEB-associated SCCs than in non-EB SCCs. The loss of HB-EGF in MMP-7-CD44v3 double-positive areas was interpreted to indicate shedding and activation of HB-EGF; this was also detected in Bowen disease indicating its importance in the early phase of SCC development. Specific knockdown of MMP-7 expression in human cutaneous SCC cells by small interfering RNA inhibited shedding of HB-EGF and resulted in diminished activation of the EGF receptor (EGFR) and ERK1/2, and in reduced proliferation of SCC cells. CONCLUSIONS: These findings provide evidence for the role of MMP-7 in promoting the growth of cutaneous SCCs by shedding HB-EGF, and identify EGFR signalling as a potential therapeutic target in RDEB-associated SCC and unresectable sporadic cutaneous SCC.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Metaloproteinasa 7 de la Matriz/fisiología , Neoplasias Cutáneas/metabolismo , Adulto , Carcinoma de Células Escamosas/patología , Proliferación Celular/efectos de los fármacos , Dipéptidos/farmacología , Activación Enzimática , Receptores ErbB/fisiología , Femenino , Técnicas de Silenciamiento del Gen , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Receptores de Hialuranos/metabolismo , Masculino , Inhibidores de la Metaloproteinasa de la Matriz , Persona de Mediana Edad , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas de Neoplasias/metabolismo , Inhibidores de Proteasas/farmacología , ARN Interferente Pequeño/genética , Transducción de Señal/fisiología , Neoplasias Cutáneas/patología , Células Tumorales Cultivadas , Adulto JovenRESUMEN
Wood is a natural fiber reinforced composite. It structurally resembles bone tissue to some extent. Specially heat-treated birch wood has been used as a model material for further development of synthetic fiber reinforced composites (FRC) for medical and dental use. In previous studies it has been shown, that heat treatment has a positive effect on the osteoconductivity of an implanted wood. In this study the effects of two different heat treatment temperatures (140 and 200 degrees C) on wood were studied in vitro. Untreated wood was used as a control material. Heat treatment induced biomechanical changes were studied with flexural and compressive tests on dry birch wood as well as on wood after 63 days of simulated body fluid (SBF) immersion. Dimensional changes, SBF sorption and hydroxylapatite type mineral formation were also assessed. The results showed that SBF immersion decreases the biomechanical performance of wood and that the heat treatment diminishes the effect of SBF immersion on biomechanical properties. With scanning electron microscopy and energy dispersive X-ray analysis it was shown that hydroxylapatite type mineral precipitation formed on the 200 degrees C heat-treated wood. An increased weight gain of the same material during SBF immersion supported this finding. The results of this study give more detailed insight of the biologically relevant changes that heat treatment induces in wood material. Furthermore the findings in this study are in line with previous in vivo studies.
Asunto(s)
Fenómenos Biomecánicos/fisiología , Precipitación Química , Calor , Minerales/química , Madera/química , Adsorción/fisiología , Líquidos Corporales/metabolismo , Líquidos Corporales/fisiología , Fuerza Compresiva , Durapatita , Inmersión , Técnicas In Vitro , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Minerales/metabolismo , Propiedades de Superficie , Resistencia a la Tracción , Madera/metabolismoRESUMEN
CD44 is a family of glycoproteins involved in cell-cell and cell-matrix interactions. In addition to the major 90-kD form present on most hematopoietic cells, larger 140-230 kD forms are found on keratinocytes and carcinoma cell lines. These bigger isoforms of CD44 arise by alternative splicing that results in insertion of one or more of the "variant" exons into the extracellular part of the 90-kD constant form of the molecule. In rat, v6 (variant exon v6) containing form of CD44 confers metastatic potential to carcinoma cells, and therefore, it is of interest to study the distribution of this isoform in humans. We raised antibodies against a synthetic peptide containing a sequence encoded by the exon v6. A mAb thus obtained (designated Var3.1) strongly reacted with the plasma membranes of squamous cells in upper layers of skin and tonsil surface epithelia. Weaker staining was seen in germinal centers, vascular endothelia and enterocytes. Exon v6 containing forms of CD44 (CD44v6) were absent from tissue leukocytes and connective tissue components. In comparison, Hermes-3 epitope (on the constant part) containing forms of CD44 were preferentially localized in basal layers of epithelia, present on the surface on most leukocytes and connective tissue cells, and undetectable on the luminal surface of high endothelial venules. In benign neoplasms, epithelial cells stained with mAb Var3.1 like in normal tissues. In contrast, immunostaining of 30 squamous carcinoma specimens (both primary and metastatic lesions) revealed that malignant transformation resulted in downregulation or disappearance of Var3.1 epitope, but in majority of cases, not in diminished synthesis of the Hermes-3 epitope. Biochemical analyses showed that mAb Var3.1 recognized two major forms of CD44 (220 and 300 kD). In conclusion, epitopes on exon v6 and constant part of CD44 are differentially synthesized and regulated during normal and malignant growth of cells in man.
Asunto(s)
Carcinoma de Células Escamosas/inmunología , Transformación Celular Neoplásica , Exones , Papiloma/inmunología , Receptores Mensajeros de Linfocitos/genética , Adulto , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Secuencia de Bases , Carcinoma de Células Escamosas/genética , Línea Celular Transformada , Tejido Conectivo/inmunología , Células del Tejido Conectivo , Regulación hacia Abajo , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/inmunología , Humanos , Leucocitos/inmunología , Linfocitos/inmunología , Datos de Secuencia Molecular , Papiloma/genética , Receptores Mensajeros de Linfocitos/análisis , Receptores Mensajeros de Linfocitos/química , Receptores Mensajeros de Linfocitos/inmunología , Solubilidad , Células Tumorales CultivadasRESUMEN
BACKGROUND: Carcinomas of the salivary glands are uncommon and morphologically a diverse group of malignancies. To evaluate the prognostic value of CD34 immunostaining of the vessels in adenoid cystic carcinoma (AdCC) and mucoepidermoid carcinoma (MEC), an automated image analysis method was used. METHOD: In a nationwide study, covering salivary gland cancer (SGC) patients in Finland 1991-1996, 37 AdCC and 18 MEC patients (M 25, F 30, age 25-90, mean 63) were included. In addition to clinical characteristics the size, shape, staining intensity and vessel density in CD34 immunostained histologic samples were measured. RESULTS: Altogether 4433 vessels were measured from AdCC and 2615 from MEC tumor. Of the total tumor vessels measured, 2651 were from patients who deceased with disease (Group I) and 4397 were from specimens derived from those who did not die of disease (Group II) during the 10-year follow-up. The staining intensity was significantly higher in MEC than in AdCC tumor (P = 0.0005). In MEC, the Group I patients had a higher staining intensity among high-grade patients compared with patients with low grade disease, whereas the tumors in Group II had a lower staining intensity among the high-grade compared with the low grade tumors (P = 0.018). A higher vessel density was found in patients with MEC in group II compared with group I (P = 0.017). CONCLUSIONS: The staining intensity of CD34 positive vessels in MEC was higher than in AdCC. In MEC, higher staining intensity of vessels in high-grade tumors and lower vessel density in all MEC patients, predicted poor survival.
Asunto(s)
Antígenos CD34/inmunología , Carcinoma Adenoide Quístico/irrigación sanguínea , Carcinoma Mucoepidermoide/irrigación sanguínea , Procesamiento de Imagen Asistido por Computador , Microvasos/inmunología , Neovascularización Patológica/inmunología , Neoplasias de las Glándulas Salivales/irrigación sanguínea , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD34/análisis , Carcinoma Adenoide Quístico/inmunología , Carcinoma Adenoide Quístico/patología , Carcinoma Mucoepidermoide/inmunología , Carcinoma Mucoepidermoide/patología , Femenino , Humanos , Técnicas para Inmunoenzimas , Masculino , Microvasos/patología , Persona de Mediana Edad , Pronóstico , Neoplasias de las Glándulas Salivales/inmunología , Neoplasias de las Glándulas Salivales/patologíaRESUMEN
Recent studies indicate that the specificity of p38 mitogen-activated protein kinase (MAPK)-mediated cellular stress responses is determined by the expression pattern of the distinct p38 isoforms. Here, we have analysed the function of distinct p38 isoforms in the growth and invasion of head and neck squamous cell carcinomas (HNSCCs). Activation of p38 MAPK by arsenite resulted in inactivation of the ERK1,2 signaling pathway by dephosphorylation of MEK1,2 in primary human epidermal keratinocytes (HEKs), whereas in HNSCC cells this p38-mediated inhibition of the ERK1,2 pathway was absent. Quantitation of p38 pathway component mRNA expression in HNSCC cell lines (n=42) compared to HEKs (n=8) revealed that p38alpha and p38delta isoforms are predominantly expressed in both cell types and that MKK3 is the primary upstream activator expressed. Inhibition of endogenous p38alpha or p38delta activity by adenoviral delivery of corresponding dominant-negative p38 isoforms potently reduced MMP-13 and MMP-1 expressions, and suppressed the invasion of HNSCC cells through collagen. Dominant-negative p38alpha and p38delta inhibited squamous cell carcinoma (SCC) cell proliferation and inhibition of p38alpha activity also compromised survival of SCC cells. p38alpha and p38delta were predominantly expressed in HNSCCs (n=24) and nonneoplastic epithelium in vivo (n=6), with MKK3 being the primary upstream activator. Activation and expression of p38alpha and p38delta by tumor cells was detected in HNSCCs in vivo (n=16). Adenoviral expression of dominant-negative p38alpha or p38delta in cutaneous SCC cells potently inhibited their implantation in skin of severe combined immunodeficiency mice and growth of xenografts in vivo. Our results indicate that p38alpha and p38delta specifically promote the malignant phenotype of SCC cells by regulating cell survival, proliferation and invasion, suggesting these p38 MAPK isoforms as potential therapeutic targets in HNSCCs.
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Carcinoma de Células Escamosas/patología , División Celular/fisiología , Neoplasias de Cabeza y Cuello/patología , Isoenzimas/fisiología , Invasividad Neoplásica , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Secuencia de Bases , Western Blotting , Carcinoma de Células Escamosas/enzimología , Línea Celular Tumoral , Cartilla de ADN , Activación Enzimática , Citometría de Flujo , Neoplasias de Cabeza y Cuello/enzimología , Humanos , Inmunohistoquímica , Isoenzimas/metabolismo , Queratinocitos/enzimología , Metaloproteinasas de la Matriz/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
B-RAF is one of the most commonly mutated oncogenes in human cancer. However, the mutation status of B-RAF has not been established completely in HNSCC. We have analysed the mutation status of the kinase domain of the B-RAF gene (exons 11 and 15) in 91 Japanese HNSCC patients as well as 12 HNSCC cell lines. DNA was extracted and amplified by PCR. Mutations were then analysed by SSCP mutation detection method. Since V600EB-RAF constitutes 90 % of the mutations identified in B-RAF in human cancers, we also used MASA analysis to specifically detect this mutation in exon 15 of B-RAF. Using both methods, no mutation was found in both exon 11 and 15 in all patients and cell lines. Mu tations are absent or rare in the kinase domain of B-RAF in Japanese HNSCC. However, more studies are still needed to determine its usefulness as a target for molecular therapy in these patients.
Asunto(s)
Carcinoma de Células Escamosas/genética , Neoplasias de Cabeza y Cuello/genética , Mutación/genética , Proteínas Proto-Oncogénicas B-raf/genética , Alelos , Línea Celular Tumoral , Análisis Mutacional de ADN , Exones/genética , Humanos , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
Squamous cell carcinoma (SCC) cells of the head and neck specifically express collagenase-3 (matrix metalloproteinase-13 (MMP-13)), the expression of which correlates with their invasion capacity. Transforming growth factor-beta (TGF-beta) enhances MMP-13 and collagenase-1 (MMP-1) expression and invasion of SCC cells via p38 mitogen-activated protein kinase. Here, we have examined the role of Smad signaling in regulating MMP-13 expression and in invasion of head and neck SCC cells. Treatment with TGF-beta resulted in activation of Smad2 and Smad3 in SCC cells, but had no effect on their proliferation or viability. Basal activation of Smad3 and p38 was noted in SCC cells without exogenous TGF-beta stimulation, and adenoviral delivery of Smad7 and dominant-negative Smad3 inhibited p38 activation in these cells. Adenoviral overexpression of Smad3 augmented the upregulatory effect of TGF-beta on MMP-13 expression by SCC cells. Disruption of Smad signaling by adenoviral expression of kinase-defective TGF-beta type I receptor (activin-receptor-like kinase-5), Smad7, and dominant-negative Smad3 potently suppressed the basal and TGF-beta-induced expression of MMP-13 and MMP-1 in SCC cells, and inhibited their basal and TGF-beta-induced invasion through Matrigel and type I collagen. Adenoviral overexpression of Smad7 in cutaneous and oral SCC cells significantly inhibited their implantation in skin of SCID mice and growth of xenografts in vivo, as compared to LacZ adenovirus-transduced control cells. Together, these results show that Smad signaling plays an important role in promoting the invasive phenotype of human head and neck SCC cells by upregulating their collagenase expression.
Asunto(s)
Carcinoma de Células Escamosas/enzimología , Colagenasas/metabolismo , Neoplasias de Cabeza y Cuello/enzimología , Transducción de Señal/fisiología , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Proteína smad7/metabolismo , Adenoviridae/genética , Animales , Northern Blotting , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/prevención & control , Proliferación Celular , Colágeno Tipo I/metabolismo , Colagenasas/genética , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/prevención & control , Humanos , Metaloproteinasa 13 de la Matriz , Ratones , Ratones SCID , Invasividad Neoplásica , Factor de Crecimiento Transformador beta/farmacología , Trasplante Heterólogo , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Molecular mechanisms contributing to initiation and progression of head and neck squamous cell carcinoma are still poorly known. Numerous genetic alterations have been described, but molecular consequences of such alterations in most cases remain unclear. Here, we performed an integrated high-resolution microarray analysis of gene copy number and expression in 20 laryngeal cancer cell lines and primary tumors. Our aim was to identify genetic alterations that play a key role in disease pathogenesis and pinpoint genes whose expression is directly impacted by these events. Integration of DNA level data from array-based comparative genomic hybridization with RNA level information from oligonucleotide microarrays was achieved with custom-developed bioinformatic methods. High-level amplifications had a clear impact on gene expression. Across the genome, overexpression of 739 genes could be attributed to gene amplification events in cell lines, with 325 genes showing the same phenomenon in primary tumors including FADD and PPFIA1 at 11q13. The analysis of gene ontology and pathway distributions further pinpointed genes that may identify potential targets of therapeutic intervention. Our data highlight genes that may be critically important to laryngeal cancer progression and offer potential therapeutic targets.
Asunto(s)
Carcinoma de Células Escamosas/genética , Dosificación de Gen , Perfilación de la Expresión Génica/métodos , Neoplasias Laríngeas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Línea Celular Tumoral , Expresión Génica , Humanos , InmunohistoquímicaRESUMEN
PURPOSE: Syndecan-1 is a multifunctional transmembrane heparan sulfate proteoglycan present on a variety of cell types that mediates basic fibroblast growth factor (bFGF) and other growth factor binding. High serum syndecan-1 (S-syndecan-1) ectodomain levels have been found to be associated with poor outcome in lung cancer and myeloma, but little is known about the effect of cancer treatment on S-syndecan-1 levels. We studied S-syndecan-1 levels longitudinally in a series of patients diagnosed with locoregional squamous cell larynx or hypopharynx carcinoma (n=44) and who we treated with surgery and/or radiation therapy. METHODS: S-syndecan-1 and S-bFGF levels were measured with ELISA prior to, during, and following primary treatment of patients. Syndecan-1 expression was assessed from formalin-fixed and paraffin-embedded tumour samples using immunohistochemistry. RESULTS: S-syndecan-1 levels tended to correlate positively with S-bFGF levels, and the pretreatment levels decreased from a median value of 75 to 58 ng/ml 3 months following treatment (P<0.0001). Patients treated with radiation therapy had a transient increase in S-syndecan-1 during the course of radiation therapy. Patients whose S-syndecan-1 decreased >or=10% from the pretreatment level had more favourable survival than those whose levels remained stable or increased (P=0.0069). Recurred cancer was associated with elevated S-syndecan-1 as compared to the levels measured 3 months following completion of primary therapy. CONCLUSIONS: These findings suggest that a part of S-syndecan-1 originates from the cancerous tissue, and that S-syndecan-1 levels generally decrease following successful cancer treatment.
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Biomarcadores de Tumor/sangre , Carcinoma de Células Escamosas/terapia , Neoplasias Hipofaríngeas/terapia , Neoplasias Laríngeas/terapia , Glicoproteínas de Membrana/sangre , Proteoglicanos/sangre , Anciano , Carcinoma de Células Escamosas/sangre , Carcinoma de Células Escamosas/secundario , Ensayo de Inmunoadsorción Enzimática , Femenino , Factor 2 de Crecimiento de Fibroblastos/sangre , Humanos , Neoplasias Hipofaríngeas/sangre , Neoplasias Hipofaríngeas/patología , Inmunohistoquímica , Neoplasias Laríngeas/sangre , Neoplasias Laríngeas/patología , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Sindecano-1 , SindecanosRESUMEN
Standard therapy for squamous cell carcinoma (SCC) of the vulva consists of radical surgery and inguinal node dissection. Radiation therapy has been used for preoperative treatment in advanced cases to reduce the size of the tumor, and also as the only treatment in inoperable or recurrent disease. To study the inherent radiation sensitivity of vulvar carcinoma, we tested three new vulvar carcinoma cell lines and the long-established cell line A-431 by using a 96-well plate clonogenic assay, earlier shown by us to be suitable for survival studies of SCC. SCC and adenocarcinoma cell lines derived from other sites were used as a reference. Cells were irradiated with a 4-MeV linear accelerator at a dose rate of 2.0 Gy/min. The vulvar cell lines were found to be highly resistant to radiation with the average mean inactivation dose of 3.44 +/- 0.34 Gy as calculated from the area under the curve. The results were consistent in repeated experiments and for all cell lines. The average value for area under the curve was 1.79 +/- 0.30 for the other SCC lines tested. The values for area under the curve differed significantly (P less than 0.0001) between the vulvar lines and reference SCC lines. These results indicate that vulvar SCC cells in vitro express exceptional inherent radioresistance, and thus development of other forms of additional treatment would be more advantageous in advanced cases.
Asunto(s)
Carcinoma de Células Escamosas/radioterapia , Neoplasias de la Vulva/radioterapia , Supervivencia Celular/efectos de la radiación , Femenino , Humanos , Dosis de Radiación , Tolerancia a Radiación , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
Collagenase-1 [matrix metalloproteinase (MMP)-1] is expressed by stromal fibroblasts of various invasive malignant tumors. Here, we have examined the molecular mechanisms of tumor-induced expression of MMP-1 by stromal fibroblasts. Treatment of fibroblasts with conditioned media of tumor cells derived from squamous cell carcinomas (SCCs) of the oral cavity and larynx resulted in activation of fibroblast MMP-1 expression at the transcriptional level. The induction of MMP-1 expression correlates with activation of c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase and phosphorylation of c-Jun and activating transcription factor-2 (ATF-2) and is dependent on the activity of p38 mitogen-activated protein kinase. Furthermore, using fibroblasts derived from JNK2-/- mice, we show that JNK2 is required for induction of fibroblast collagenase-3 expression in response to conditioned SCC tumor cell medium. Together, these results provide evidence that stress-activated p38 and JNK pathways play a crucial role in paracrine regulation of collagenolytic capacity of stromal fibroblasts in SCCs and suggest JNK2 as a novel target for inhibition of MMP-1 expression and tumor invasion.
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Carcinoma de Células Escamosas/enzimología , Colagenasas/metabolismo , Fibroblastos/enzimología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Transcripción Activador 2 , Animales , Northern Blotting , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Activación Enzimática , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Recién Nacido , Neoplasias Laríngeas/enzimología , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 13 de la Matriz , Ratones , Proteína Quinasa 9 Activada por Mitógenos , Neoplasias de la Boca/enzimología , Fenotipo , Fosforilación , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN/metabolismo , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo , Transcripción Genética , Transfección , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Loss of heterozygosity (LOH) on 18q predicts poor survival in head and neck squamous cell carcinomas (HNSCCs). Several putative tumor suppressor genes, such as DCC, DPC4/Smad4, and MADR2/Smad2, are mapped to 18q, but thus far, the important gene locus in HNSCC is not known. To identify possible gene loci on 18q, we performed LOH studies using tumor DNA from 57 HNSCC primary tumor cell lines and DNA isolated from fibroblasts or lymphoblastoid cells from the same patients. Forty-two highly polymorphic microsatellite markers spaced not more than 5 cM apart (mean distance, 1.82 cM) spanning the region from D18S44 in 18q11.1 to D18S1141 in 18q23 were used. D18S71 in 18p11.21 on 18p was also used to determine whether the short arm was retained. Forty-three of 57 (75%) HNSCC lines showed LOH or isolated allelic imbalance (AI) for at least one locus on 18q. Although many of the cell lines had large distal 18q deletions with a breakpoint between 18q11.1 and 18q12.2 to qter, three loci were identified that were lost in 70% or more of the cases. The minimally lost regions (MLRs) range in size from 1.5-15.79 cM. The most proximal is centered on D18S39 (1.56 cM) in band 18q21.1, with LOH or isolated AI in 28 of 38 (74%) of informative cases. The largest (15.8 cM) begins at D18S61 (28 of 40; 70%) in band 18q22.2 and extends through D18S50 in 18q23. The third is centered on D18S70 (30 of 40; 75%) in band 18q23 (3.67 cM). Of these MLRs, only the one centered on D18S39 has been implicated previously in HNSCC. D18S70, the most frequently lost marker, was the only marker consistently lost in three tumor cell lines with very minimal losses, UM-SCC-19, UM-SCC-67, and UM-SCC-73A. In addition, UM-SCC-91 exhibited AI only at this locus, and UT-SCC-4 had AI at D18S70 and D18S39 only. Close physical mapping of these three regions may pinpoint one or more previously unidentified tumor suppressor genes.
Asunto(s)
Carcinoma de Células Escamosas/genética , Cromosomas Humanos Par 18 , Neoplasias de Cabeza y Cuello/genética , Pérdida de Heterocigocidad , Carcinoma de Células Escamosas/patología , Centrómero/genética , Mapeo Cromosómico , Fibroblastos/patología , Marcadores Genéticos , Neoplasias de Cabeza y Cuello/patología , Humanos , Repeticiones de Microsatélite , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
CD44 is a family of molecules involved in cell-cell and cell-matrix interactions. Various isoforms of CD44 arise by insertion of one or more of the variant exons into the common backbone shared by all forms of CD44. In this work, we studied the expression of CD44 and exon v6-containing CD44 isoforms (CD44v6) in several nonmalignant and malignant conditions and the possibilities for regulating the expression of CD44v6. In primary squamocellular carcinomas of the head and neck, CD44 and CD44v6 were down-regulated in poorly differentiated tumors, whereas these molecules were uniformly expressed in the normal squamocellular epithelium, in proliferating skin diseases, and in nonmalignant tumors. When CD44v6 expression of original tumors and that of squamocellular carcinoma cell lines derived from them were compared, no CD44v6 up-regulation could be observed on in vitro growing cells. Moreover, several regulators were unable to up-regulate CD44v6 expression on cultured cell lines in vitro. When the same cell lines formed tumors after s.c. injection into severe combined immunodeficient mice, some of them up-regulated their CD44v6 expression. These data suggest that cell lines at certain differentiation stages can be induced to express CD44v6. Our results further indicate that CD44v6 positivity cannot be used as a universal indicator of tumor metastasis. Instead, the down-regulation of CD44v6 in squamocellular tumors is a sign of malignant transformation of the epithelium.
Asunto(s)
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Isomerismo , Adulto , Anciano , Animales , Regulación hacia Abajo , Epitelio/metabolismo , Exones , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Receptores de Hialuranos/inmunología , Inmunohistoquímica , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Enfermedades de la Piel/genética , Enfermedades de la Piel/metabolismo , Células Tumorales Cultivadas , Rayos Ultravioleta , Regulación hacia ArribaRESUMEN
Collagenase-3 (MMP-13) is characterized by an exceptionally wide substrate specificity and restricted expression. MMP-13 is specifically expressed by transformed human keratinocytes in squamous cell carcinomas in vivo and its expression correlates with their invasion capacity. Here, we show, that interferon-gamma (IFN-gamma) markedly inhibits expression of MMP-13 by human cutaneous SCC cells (UT-SCC-7) and by ras-transformed human epidermal keratinocytes (A-5 cells) at the transcriptional level. In addition, IFN-gamma inhibits collagenase-1 (MMP-1) expression in these cells. IFN-gamma abolished the enhancement of MMP-13 and MMP-1 expression by transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha), and inhibited invasion of A-5 cells through type I collagen. IFN-gamma also rapidly and transiently activates extracellular signal-regulated kinase 1,2 (ERK1,2) and blocking ERK1,2 pathway (Raf/MEK1,2/ERK1,2) by specific MEK1,2 inhibitor PD98059 partially (by 50%) prevents Ser-727 phosphorylation of STAT1 and suppression of MMP-13 expression by IFN-gamma. Furthermore, Ser-727 phosphorylation of STAT1 by ERK1,2, or independently of ERK1,2 activation is associated with marked reduction in MMP-13 expression. These observations identify a novel role for IFN-gamma as a potent inhibitor of collagenolytic activity and invasion of transformed squamous epithelial cells, and show that inhibition of MMP-13 expression by IFN-gamma involves activation of ERK1,2 and STAT1.
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Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Colagenasas/biosíntesis , Proteínas de Unión al ADN/metabolismo , Interferón gamma/farmacología , Queratinocitos/enzimología , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/biosíntesis , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transactivadores/metabolismo , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Colagenasas/genética , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Metaloproteinasa 13 de la Matriz , Metaloproteinasas de la Matriz/genética , Proteína Quinasa 3 Activada por Mitógenos , Especificidad de Órganos/genética , Proteínas Recombinantes , Factor de Transcripción STAT1 , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
The prognostication of patient outcome is one of the greatest challenges in the management of early stage oral tongue squamous cell carcinoma (OTSCC). This study introduces a simple histopathological model for the prognostication of survival in patients with early OTSCC. A total of 311 cases (from Finland and Brazil) with clinically evaluated early stage OTSCC (cT1-T2cN0cM0) were included in this multicentre retrospective study. Tumour budding (B) and depth of invasion (D) were scored on haematoxylin-eosin-stained cancer slides. The cut-off point for tumour budding was set at 5 buds (low <5; high ≥5) and for depth of invasion at 4mm (low <4mm; high ≥4mm). The scores of B and D were combined into one model: the BD predictive model. On multivariate analysis, a high risk score (BD score 2) correlated significantly with loco-regional recurrence (P=0.033) and death due to OTSCC (P<0.001) in early stage OTSCC. The new BD model is a promising prognostic tool to identify those patients with aggressive cases of early stage OTSCC who might benefit from multimodality treatment.
Asunto(s)
Carcinoma de Células Escamosas/patología , Neoplasias de la Lengua/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Brasil/epidemiología , Carcinoma de Células Escamosas/mortalidad , Niño , Femenino , Finlandia/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Pronóstico , Estudios Retrospectivos , Medición de Riesgo , Análisis de Supervivencia , Neoplasias de la Lengua/mortalidadRESUMEN
Syndecans comprise a family of integral membrane proteoglycans that presumably participate in cell-matrix interactions and the modulation of growth factor response. Expression of syndecan-1, a cell surface proteoglycan that binds basic fibroblast growth factor (bFGF) and extracellular matrix components, was studied in cultured human keratinocytes from the oral mucosa and in tissue sections derived from various epithelia and carcinomas of the head and neck. For the study, polyclonal antibodies were raised against the core protein of human syndecan-1. The affinity-purified antibody (designated anti-P117) was shown to react specifically with syndecan-1. Abundant expression of syndecan-1 was detected in frozen sections of various stratified epithelia as well as in cultured keratinocytes. Keratinocytes located in the spinous cell layers showed intense immunoreactivity for syndecan-1, while basal cells stained rather weakly, suggesting that the expression of syndecan-1 could be stimulated during the differentiation of keratinocytes. Cultured human keratinocytes were induced to terminally differentiate by increasing the extracellular calcium concentration of the medium. Parallel to the induction of involucrin expression, the mRNA levels of syndecan-1 were found to increase, suggesting that syndecan-1 is indeed induced during keratinocyte differentiation. The molecular mass and glycosaminoglycan composition of syndecan-1 did not change markedly during calcium-induced differentiation. Malignant transformation was associated with marked reduction of syndecan-1 expression, based on the immunoreactivity of anti-P117 in frozen sections from squamous cell carcinomas (SCCs) of the head and neck.(ABSTRACT TRUNCATED AT 250 WORDS)