Apoptotic signaling in salivary mucoepidermoid carcinoma.

BACKGROUND: Mucoepidermoid carcinoma is the most common malignant tumor of salivary glands. Apoptosis plays an important role in organogenesis of glandular structures, and aberrations of apoptotic mechanisms is associated with a wide array of pathologic conditions. METHODS: The immunoexpression of proteins associated with apoptosis and proliferation was evaluated in 40 mucoepidermoid carcinoma cases. RESULTS: Par-4, Survivin, MUC1, PHLDA1, Fas, and Ki-67 were predominantly expressed in mucoepidermoid carcinoma. FasL was rarely expressed, and Caspase-3 expression was observed in almost 50% of the cases. SPARC expression was associated with low-grade tumors, and Ki-67 expression was associated with lymph node metastasis. Expression of Fas and decreased expression of Ki-67 and Caspase-3 were associated with better overall cancer-specific survival rates. CONCLUSIONS: The association of SPARC and Ki-67 expression with pathological features and the association of Fas, Caspase-3, and Ki-67 with survival probabilities suggest that these proteins may be useful prognostic markers for mucoepidermoid carcinoma.

Apoptosis-associated protein expression in human salivary gland morphogenesis.

OBJECTIVE: Salivary gland (SG) development is based on branching morphogenesis, in which programmed cell death has been proposed to play a role in cell signalling and organ shaping. In the mouse salivary gland apoptosis has been suggested to play a key role in lumen formation, removing the central cells of the epithelial stalks. Here we analyse the expression of several anti- and pro-regulators of apoptosis during human SG development in a range of developmental stages. DESIGN: Foetal SGs obtained from the University of São Paulo were analysed by immunohistochemistry to assess the expression of apoptosis-associated proteins: caspases (caspase-6, -7, -9 and cleaved caspase-3), Bcl-2 family members (Bax, Bak, Bad, Bid, Bcl-2, Bcl-x and Bcl-xL), Survivin (BIRC5), Cytochrome C and Apaf-1. RESULTS: Nuclear expression of Bax and Bak was identified in presumptive luminal areas at initial stages, while Bcl-xL showed the most relevant anti-apoptotic activity. Caspase-6, -7 and -9 were expressed during all stages, while interestingly cleaved caspase-3 showed no prominent expression, indicating that caspase-7 is the main effector. Apoptosome complex components Apaf-1 and Cytochrome C, as well as survivin were all positive in developing glands. CONCLUSIONS: The particular expression pattern of several apoptotic regulators in human SG development suggests the existence of a fundamental role for apoptosis during duct formation. The absence of Bad and Bid expressions indicates that the instrinsic pathway is more active then the extrinsic during human gland formation. The subcellular localisation of intrinsic-apoptosis proteins correlated with apoptotic activity, but also suggested additional non-apoptotic functions.

Insulin-like Growth Factor II Messenger RNA-binding Protein 3 in Salivary Gland Tumors.

Insulin-like growth factor II mRNA-binding protein 3 (IMP3) is strongly expressed in malignant tumors and has been associated with their aggressive behavior. The aim of this study was to evaluate the presence of IMP3 in a series of salivary gland tumors. The sample consisted of 9 pleomorphic adenomas (PA), 14 adenoid cystic carcinomas (ACC), and 13 mucoepidermoid carcinomas (MEC) that were investigated by immunohistochemical technique. All cases of PA and MEC were positive for IMP3 particularly in the cytoplasm. PA showed 4 cases as high expression and 6 as low expression. MEC showed 10 cases as low expression and 3 as high expression. For ACC, 4 cases were high expression, whereas 6 cases were low expression. No significant difference was observed between tumors (P>0.05, Fisher's test) when both scores of IMP3 were compared. This study showed that MEC seems to be more sensitive to IMP3 than ACC and provided an insight into this protein in salivary gland tumors. Furthermore, although IMP3 is not a specific diagnostic marker to distinguish the tumors studied, it seems to mediate cell adhesion and migration in these tumors. Further studies should be performed to better understand the IMP3 biology in salivary gland tumors.

Head and neck mucosal melanoma: a review.

Head and neck mucosal melanoma (MM) is an aggressive and rare neoplasm of melanocytic origin. To date, few retrospective series and case reports have been reported on MM. This article reviews the current evidence on head and neck MM and the molecular pathways that mediate the pathogenesis of this disease. Head and neck MM accounts for 0.7%-3.8% of all melanomas and involve (in decreasing order of frequency) the sinonasal cavity, oral cavity, pharynx, larynx, and upper esophagus. Although many studies have examined MM of the head and neck and the underlying molecular pathways, individual genetic and molecular alterations were less investigated. Further studies are needed to complement existing data and to increase our understanding of melanocytes tumorigenesis.

CD24 and CD44 in salivary gland pleomorphic adenoma and in human salivary gland morphogenesis: differential markers of glandular structure or stem cell indicators?

AIMS: Salivary gland neoplasms originate from salivary gland compartments, to which they are histologically related. Pleomorphic adenoma (PA) is a benign salivary gland neoplasm that comprises epithelial and myoepithelial cells and a complex stroma, whose structure, architecture and origin (from intercalated ducts) suggest stem cell participation. We compared the expression of CD24 and CD44 in PA and in developing human salivary glands to investigate whether these markers can be considered as cancer stem cell markers. METHODS AND RESULTS: One hundred and one cases of PA and salivary gland specimens from 20 human fetuses were examined by immunohistochemistry and real-time reverse transcription polymerase chain reaction (RT-PCR). All PAs were positive for CD24 and CD44 by immunohistochemistry: neoplastic luminal structures were positive for CD24; modified myoepithelial cells were positive for CD44. In fetal salivary glands, these markers were restricted to the intercalated duct region. Real-time RT-PCR assays detected increased expression of CD44, but not CD24, in PA specimens in comparison with normal salivary gland controls. CONCLUSIONS: PA and stem cells share the expression of CD24 and CD44; their value as markers of neoplastic cell multipotency and the implications of their expression for tumour behaviour are yet to be determined.

A comparison between manual and automated evaluations of tissue microarray patterns of protein expression.

Tissue microarray technology enables us to evaluate the pattern of protein expression in large numbers of samples. However, manual data acquisition and analysis still represent a challenge because they are subjective and time-consuming. Automated analysis may thus increase the speed and reproducibility of evaluation. However, the reliability of automated analysis systems should be independently evaluated. Herein, the expression of phosphorylated AKT and mTOR was determined by ScanScope XT (Aperio; Vista, CA) and ACIS III (Dako; Glostrup, Denmark) and compared with the manual analysis by two observers. The percentage of labeled pixels or nuclei analysis had a good correlation between human observers and automated systems (κ = 0.855 and 0.879 for ScanScope vs. observers and κ = 0.765 and 0.793 for ACIS III vs. observers). The intensity of labeling determined by ScanScope was also correlated with that found by the human observers (correlation index of 0.946 and 0.851 for pAKT and 0.851 and 0.875 for pmTOR). However, the correlation between ACIS III and human observation varied for labeling intensity and was considered poor in some cases (correlation index of 0.718 and 0.680 for pAKT and 0.223 and 0.225 for pmTOR). Thus, the percentage of positive pixels or nuclei determination was satisfactorily performed by both systems; however, labeling intensity was better identified by ScanScope XT.

The CDKN2A and MAP kinase pathways: molecular roads to primary oral mucosal melanoma.

The etiology and pathogenesis of oral mucosal melanomas are poorly understood, and no intraoral risk factors have been identified. Recent studies have postulated that DNA repair mechanisms and cell growth pathways are involved in the development of melanoma-particularly changes in the CDKN2A (p16-cyclinD-Cdk-pRb) and MAPK pathways (RAS, BRAF, MEK 1/2, and ERK 1/2 proteins). We examined the central components of the CDKN2A and RAS-RAF-MEK-ERK cascades by immunohistochemistry in a series of 35 primary oral melanomas by tissue microarray (TMA). We noted altered expression of the CDKN2A cascade proteins, although these modulations did not correlate significantly with clinical and pathological parameters. The expression of MAP kinase cascade proteins changed in most cases. We observed that 28.57% of cases were RAS-positive and that 82.85% and 74.28% of cases were positive for BRAF and ERK2, respectively; MEK2 and ERK1 were not expressed in 48.57% and 80% of cases, and all cases were negative for MEK1. The absence of RAS and ERK1 and positivity for BRAF and ERK2 were associated with higher histological grade, vascular invasion, and metastasis. Expression of MEK2 was significantly linked to vascular invasion (P = 0.043). The CDKN2A and MAPK pathways require further study in mucosal melanomas, but our results highlight the significance of important alterations, particularly with regard to histological indicators of poor prognosis in primary oral mucosal melanomas, independent of UV exposure.

Development of human minor salivary glands: expression of mucins according to stage of morphogenesis.

The formation of salivary glands entails the proliferation of epithelial cells from the stomatodeum into the underlying ectomesenchyme, culminating in a complex network of ducts and acinar bulbs. The extent to which mucins regulate this process is unknown, but they appear to mediate luminal space formation and maturation. Our aim was to examine mucin expression patterns during the morphogenesis of human salivary glands. Mucin expression - MUC1, 2, 3, 4, 5AC, 5B, 6, and 16 - was analyzed in specimens of developing human salivary glands, obtained from fetuses at 4-24 weeks' gestation, and fully developed salivary glands by immunohistochemistry. Expression patterns were analyzed qualitatively according to the development stage of the salivary glands. Mucins 1, 3, 4, 5B, and 16 were expressed during salivary gland development - being stronger in all ductal segments by the final phases of branching morphogenesis and in mature glands. Acinar cells were negative for most mucins, including MUC1 in mature salivary glands. Mucins 2, 5AC, and 6 were not expressed. Mucins MUC1, 3, 4, 5B, and 16 are expressed in developing human salivary glands and in mature glands, suggesting important roles in the maturation and maintenance of the ductal network.

Human salivary gland morphogenesis: myoepithelial cell maturation assessed by immunohistochemical markers.

AIMS: Myoepithelial cells are important components of salivary gland structure, aiding the expulsion of saliva from acinar lobules. The aim was to evaluate the expression of smooth muscle actin (SMA), calponin, caldesmon, CD10, CD29, S100 protein, glial fibrillary acidic protein (GFAP) and p63 in myoepithelial cells during salivary gland morphogenesis to understand the maturation process of these cells and their possible use in the diagnosis of salivary gland lesions. METHODS AND RESULTS: Major and minor human salivary glands at various stages of development, derived from fetuses at 8-26 weeks of gestation, were studied immunohistochemically. Fully developed salivary glands were used as controls. The protein p63 was present in all stages of salivary gland morphogenesis from initial bud to terminal bud stage. CD29, S100 and calponin were detected increasingly as salivary gland structure matured and in fully developed salivary gland. Proteins GFAP, CD10 and caldesmon were not observed in myoepithelial cells of salivary glands. CONCLUSIONS: The proteins SMA, calponin, CD29, S100 and p63, which are present from the earliest stages of salivary gland maturation, are valuable myoepithelial markers but, although very specific, are not exclusive markers for this cell type.

Oral squamous cell carcinoma: status of tight junction claudins in the different histopathological patterns and relationship with clinical parameters. A tissue-microarray-based study of 136 cases.

AIMS: Claudins are integral transmembrane proteins of the tight junctions, critical for maintaining cell adhesion and polarity. Alterations in the expression of individual claudins have been detected in carcinomas and appear to correlate with tumour progression. METHODS: In this study, a panel of anti-claudin antibodies (anti-claudins 1, 2, 3, 4, 5 and 7) was employed to map claudin expression in 136 cases of oral squamous cell carcinoma (OSCC) organised in a tissue microarray. RESULTS: Claudins were expressed in a reticular pattern up to the prickle layer in normal mucosal epithelium. In OSCC, claudins were strongly present in well-differentiated tumours, they presented mild and low expression in moderately differentiated OSCC, and were negative in poorly differentiated OSCC; the absences of claudin 1 (p=0.002) and claudin 4 (p<0.001) were associated with moderately/poorly differentiated tumours. Strong expression of claudin 4 was associated with decreased perineural infiltration (p=0.024). Claudins 5 and 7 were mostly negative or weakly expressed in all cases studied. Expression of claudin 7 was associated with the early clinical stages of the disease, whereas loss of claudin 7 tended to be more frequent in advanced stages of OSCC (p=0.054). Absence of claudin 7 was also associated with absent vascular infiltration (p=0.045) and with presence of recurrence (p=0.052). CONCLUSIONS: Claudin expression patterns showed a strong correlation with histological type of OSCC; claudin expression was decreased in areas of invasion, and negative in poorly differentiated tumours. This pattern may be related to evolution and prognosis of these tumours, especially in the case of claudin 7, which seems to be associated with a poor prognosis in OSCC.

Genetic alterations in juvenile nasopharyngeal angiofibromas.

Juvenile nasopharyngeal angiofibroma (JNA) is a rare benign neoplasm of the nasopharynx that accounts for 0.5% of all head and neck tumors. Although histologically benign in appearance, JNAs are locally aggressive and destructive, spreading from the nasal cavity to the nasopharynx, paranasal sinuses, and orbit skull base with intracranial extension. The gender selectivity of JNA and the relatively young age at diagnosis suggest hormone-dependent development. Hormonal disorders have been reported in patients with JNA, and androgen and estrogen receptors have been identified in tumor tissue; however, a hormonal influence on JNA is controversial. Recent studies have attempted to further delineate the pathogenesis of JNA through analysis of genetic and molecular changes. Understanding of the molecular mechanisms involved in JNA might improve prevention, prognosis, and treatment of this tumor. In this review, we discuss published studies addressing the possible molecular pathways that might be involved in the development of JNA.

Human salivary gland branching morphogenesis: morphological localization of claudins and its parallel relation with developmental stages revealed by expression of cytoskeleton and secretion markers.

Development of salivary glands is a highly complex and dynamic process termed branching morphogenesis, where branched structures differentiate into mature glands. Tight junctions (TJ) are thought to play critical roles in physiological functions of tubular organs, contributing to cell polarity and preventing lateral movement of membrane proteins. Evidence demonstrated that claudins are directly involved in TJ formation and function. Using immunohistochemistry and immunofluorescence we have mapped the distribution of claudins-1, 2, 3, 4, 5, 7 and 11 and compared it with the expression of differentiation markers in human salivary glands obtained from foetuses ranging from weeks 4 to 24 of gestation. Expression of all claudins, except claudin-2 was detected in the various phases of human salivary gland development, up to fully mature salivary gland. The expression of all claudins increased according to the progression of salivary gland maturation evidenced by the classical markers-cytokeratin 14, cytokeratin low molecular weight, smooth muscle actin and human secretory component. Tight junction proteins-claudins appear to be important in the final shape and physiological functions of human salivary glands and are parallel related with markers of salivary gland differentiation.

Differentially expressed genes in the prostate cancer cell line LNCaP after exposure to androgen and anti-androgen.

Androgens play an important role in growth and maintenance of prostate cells. The actions of androgens are mediated by the androgen receptor (AR), a transcription factor member of the super-family of nuclear hormone receptors. Androgen regulated genes (ARGs) are potential markers for early diagnosis and treatment of prostate cancer patients. In the present study, we used DDRT-PCR (differential display reverse transcriptase polymerase chain reaction) technique in order to investigate differentially expressed genes in the prostate cancer cell line LNCaP after treatment with dihydrotestosterone and bicalutamide for 6, 24, and 48 hours. Fifty-five differentially expressed fragments were isolated, cloned, and sequenced. Sequencing analysis of these fragments revealed 56 different transcripts that showed homology to transcription factors, cell cycle regulators, metabolic enzymes, and hypothetical proteins. Among the differentially expressed genes, SPA17 and DDEF2 were further validated using quantitative real time RT-PCR (qPCR) in a series of 25 prostate tumor samples. The DDEF2 gene is involved in adhesion and cell migration of monocytes, and the SPA17 gene might be involved in cellular signal transduction. The transcripts of both, SPA17 and DDEF2 genes, showed altered pattern of expression in the group of prostate tumors analyzed by qPCR. The differentially expressed genes identified in this study might provide new insights into the androgen signaling pathways in prostate cells.

Relaxation of imprinting of IGFII gene in juvenile nasopharyngeal angiofibromas.

IGFII and H19 genes are expressed only from one allele due to genomic imprinting, biallelic expression (loss of imprinting) being associated with the tumorigenic process of different types of tumors. The mechanism responsible for genomic imprinting is not yet determined, although DNA methylation has been considered the main genetic event for an imprinted mark. In the current study, the authors analyzed the imprinting status and expression levels of the IGFII and H19 genes in 27 cases of Juvenile Nasopharyngeal Angiofibroma (JNA) using RFLPs, RT-PCR, and Southern and Northern Blots. The authors found that four out of eight informative cases (50%) for ApaI/IFGII polymorphism showed biallelic expression of IFGII whereas none of the nine informative cases for the polymorphism showed biallelic expression of the H19 gene. Overexpression of IFGII was observed in 8 out of 22 cases (36.4%), and 7 out of 19 cases (36.8%) showed H19 overexpression. Hypomethylation was found only in the H19 gene in six out of eight cases analyzed. Therefore, our results demonstrate that alterations in the IFGII/H19 imprinted region occur in JNA.