Absence of galectin-3 does not affect the development of experimental tongue carcinomas in mice

Background: Galectin-3 is a lectin that presents pivotal roles in tumor biology and there are no studies evaluating their expression in dysplasias and carcinomas developed from tongue carcinogenesis models. Aims: To investigate the role of galectin-3 in the development of tongue carcinomas using a mouse model of oral carcinogenesis. Methods: Galectin-3-deficient (gal3(-/-)) and wild-type (gal3(+/+)) mice were challenged with 4-nitroquinoline-1-oxide in drinking water for 16 weeks and killed at different times. Tongues were removed and the number of dysplasias and carcinomas was counted. An immunohistochemical study for galectin-3 was performed only in the tongue from gal3(+/+) mice. Results: In both groups, a reduction of dysplasias and an increase of carcinomas from week 16 to week 32 (p > 0.05) were observed. A predominance of high cytoplasmic and nuclear galectin-3 expression was observed in carcinomas (64.7%) and dysplasias (55.5%), respectively (p > 0.05). The perilesional areas always presented a statistical cytoplasmic and nuclear galectin-3 overexpression. Conclusions: Absence of galectin-3 did not directly affect the process of carcinogenesis and a cytoplasm shift of galectin-3 seems to be associated with development of tongue carcinomas. (C) 2010 Elsevier Inc. All rights reserved.

Reciprocal changes in gene expression profiles of cocultured breast epithelial cells and primary fibroblasts

The importance of epithelial-stroma interaction in normal breast development and tumor progression has been recognized. To identify genes that were regulated by these reciprocal interactions, we cocultured a nonmalignant (MCF10A) and a breast cancer derived (MDA-MB231) basal cell lines, with fibroblasts isolated from breast benign-disease adjacent tissues (NAF) or with carcinoma-associated fibroblasts (CAF), in a transwell system. Gene expression profiles of each coculture pair were compared with the correspondent monocultures, using a customized microarray. Contrariwise to large alterations in epithelial cells genomic profiles, fibroblasts were less affected. In MDA-MB231 highly represented genes downregulated by CAF derived factors coded for proteins important for the specificity of vectorial transport between ER and golgi, possibly affecting cell polarity whereas the response of MCF10A comprised an induction of genes coding for stress responsive proteins, representing a prosurvival effect. While NAF downregulated genes encoding proteins associated to glycolipid and fatty acid biosynthesis in MDA-MB231, potentially affecting membrane biogenesis, in MCF10A, genes critical for growth control and adhesion were altered. NAFs responded to coculture with MDA-MB231 by a decrease in the expression of genes induced by TGF beta 1 and associated to motility. However, there was little change in NAFs gene expression profile influenced by MCF10A. CAFs responded to the presence of both epithelial cells inducing genes implicated in cell proliferation. Our data indicate that interactions between breast fibroblasts and basal epithelial cells resulted in alterations in the genomic profiles of both cell types which may help to clarify some aspects of this heterotypic signaling. (C) 2009 UICC

ADAM23 Negatively Modulates alpha(v)beta(3) Integrin Activation during Metastasis

The ADAM23 gene is frequently silenced in different types of tumors, and, in breast tumors, silencing is correlated with tumor progression, suggesting that it might be associated with the acquisition of a metastatic phenotype. ADAM23 exerts its function mainly through the disintegrin domain, because its metalloprotease domain is inactive. Analysis of ADAM23 binding to integrins has revealed a specific interaction with alpha(v)beta(3) integrin mediated by the disintegrin domain. Altered expression of alpha(v)beta(3) integrin has been observed in different types of tumors, and expression of this integrin in the activated form has been shown to promote metastasis formation. Here, we investigated the possibility that interaction between ADAM23 and alpha(v)beta(3) integrin might negatively modulate alpha(v)beta(3) activation during metastatic progression. ADAM23 expression was knocked down using short hairpin RNA in the MDA-MB-435 cell line, which has been extensively used as a model for alpha(v)beta(3) integrin activation. Ablation of ADAM23 enhanced alpha(v)beta(3) integrin activation by at least 2- to 4-fold and ADAM23 knockdown cells showed enhanced migration and adhesion to classic alpha(v)beta(3) integrin ligands. Ablation of ADAM23 expression also enhanced pulmonary tumor cell arrest in immunodeficient mice. To complement our findings with clinical evidence, we showed that silencing of ADAM23 gene by DNA promoter hypermethylation in a collection of 94 primary breast tumors was significantly associated with lower distant metastases-free and disease-specific survivals and was an independent prognostic factor for poor disease outcome. Our results strongly support a functional role of ADAM23 during metastatic progression by negatively modulating alpha(v)beta(3) integrin activation. [Cancer Res 2009;69(13):5546-52]

CYP1A2*1C, CYP2E1*5B, and GSTM1 polymorphisms are predictors of risk and poor outcome in head and neck squamous cell carcinoma patients

Head and neck squamous cell carcinoma (HNSCC) is associated with environmental factors, especially tobacco and alcohol consumption. Most of the carcinogens present in tobacco smoke are converted into DNA-reactive metabolites by cytochrome P450 (CYPs) enzymes and detoxification of these substances is performed by glutathione S-transferases (GSTs). It has been suggested that genetic alterations, such as polymorphisms, play an important role in tumorigenesis and HNSCC progression. The aim of this study was to investigate CYP1A1, CYP1A2, CYP2E1, GSTM1, and GSTT1 polymorphisms as risk factors in HNSCC and their association with clinicopathologic data. The patients comprised 153 individuals with HNSCC (cases) and 145 with no current or previous diagnosis of cancer (controls). Genotyping of the single nucleotide polymorphisms (SNPs) of the CYP1A1, CYP1A2, and CYP2E1 genes was performed by PCR-RFLP and the GSTM1 and GSTT1 copy number polymorphisms (CNPs) were analyzed by PCR-multiplex. As expected, a significant difference was detected for tobacco and alcohol consumption between cases and controls (P < 0.001). It was observed that the CYP1A2*1D (OR = 16.24) variant and GSTM1 null alleles (OR = 0.02) confer increased risk of HNSCC development (P < 0.001). In addition, head and neck cancer alcohol consumers were more frequently associated with the CYP2E1*5B variant allele than control alcohol users (P < 0.0001, OR = 190.6). The CYP1A2*1C polymorphism was associated with tumor recurrence (log-rank test, P = 0.0161). The CYP2E1*5B and GSTM1 null alleles were significantly associated with advanced clinical stages (T3 + T4; P = 0.022 and P = 0.028, respectively). Overall, the findings suggested that the genetic polymorphisms studied are predictors of risk and are also associated with tumor recurrence, since they are important for determining the parameters associated with tumor progression and poor outcomes in HNSCC. (C) 2009 Elsevier Ltd. All rights reserved.

Mitochondrial DNA depletion and its correlation with TFAM, TFB1M, TFB2M and POLG in human diffusely infiltrating astrocytomas

Mitochondrial DNA (mtDNA) alterations and their clinical and pathological implications have been analyzed in several human malignancies. A marked decrease in mtDNA copy number along with the increase in malignancy was observed in diffusely infiltrating astrocytomas (24 WHO grade II, 18 grade III, and 78 grade IV or GBM) compared to non-neoplastic brain tissues, being mostly depleted in GBM. Although high relative gene expression levels of mtDNA replication regulators (mitochondrial polymerase catalytic subunit (POLG), transcription factors A (TFAM), B1 (TFB1M) and B2 (TFB2M)) were detected, it cannot successfully revert the mtDNA depletion observed in our samples. On the other hand, a strong correlation among the expression levels of mitochondrial transcription factors corroborates with the TFAM role in the direct control of TFB1M and TFB2M during initiation of mtDNA replication. POLG expression was related to decreased mtDNA copy number, and its overexpression associated with TFAM expression levels also have an impact on long-term survival among GBM patients, interpreted as a potential predictive factor for better prognosis. (C) 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Collagen Type I may Influence the Expression of E-Cadherin and Beta-catenin in Carcinoma Ex-pleomorphic Adenoma

Carcinoma ex-pleomorphic adenoma (CXPA) is an aggressive salivary gland malignancy, usually derived from a long-standing or a recurrent benign tumor, the pleomorphic adenoma (PA). In the context of dynamic reciprocity, changes in the composition and structure of extracellular matrix proteins and cell surface receptors have been frequently associated with dysfunctional adhesion and invasive behavior of tumor cells. It is not fully understood if these changes are involved in the conversion of PA to CXPA. In this study, different progression stages of CXPA were investigated regarding the expression of the major extracellular matrix proteins, collagen type I, and of E-cadherin and beta-catenin, the components of adherens junctions. By immunohistochemical analysis, we have demonstrated that direct contact of tumor cells with fibrillar type I collagen, particularly near the invasive front and in invasive areas prevailing small nests of CXPA cells, could be associated with reduced expression of the E-cadherin and beta-catenin adhesion molecules and with invasive behavior of epithelial; but not of CXPA with myoepithelial component. Our results also suggested that this association could depend on the organization of collagen molecules, being prevented by high-order polymeric structures. These findings could implicate the local microenvironment in the transition from the premalignant PA to invasive CXPA.

Tenascin and fibronectin expression in carcinoma ex pleomorphic adenoma

This study was conducted to analyze the participation of tenascin and fibronectin, components of the extracellular matrix. in different types of carcinoma ex pleomorphic adenoma (CXPA). Seventeen cases of CXPA, classified according to the presence of epithelial and myoepithelial cells and the degree of invasion-intracapsular, minimally, and frankly invasive carcinoma-were immunohistochemically labeled for tenascin and fibronectin. Normal salivary gland included in the specimens showed tenascin only around the excretory duct, and fibronectin slightly expressed all over the stroma of the gland. In reminiscent pleomorphic adenoma, tenascin and fibronectin were observed around tubular structures and in the stroma. Both tenascin and fibronectin were expressed in all the CXPA studied. In areas of in situ carcinoma of the intracapsular type, the expression of these extracellular matrix proteins was enhanced compared with areas of residual pleomorphic adenoma. In intracapsular and minimally invasive types of CXPA, some areas of the tumor border presented tenascin and no fibronectin, pattern that may represent the real invasive front. In frankly invasive CXPA type with only epithelial component, fibronectin was strongly observed in a fibrillar network pattern, and tenascin was only focal. In frankly invasive type with myoepithelial component, tenascin staining was very strong and diffuse. This study showed different patterns of expression of tenascin and fibronectin along the process of tumorigenesis and tumor progression in CXPA, a fact that might play a role in invasion properties of these tumors.

Association Between Integrin Expression and Prognosis in Localized Prostate Cancer

BACKGROUND. Integrins and other adhesion molecules are essential for maintaining the epithelial phenotype. Some studies have reported correlations between abnormalities in their expression and carcinogenesis, but their role in prostate cancer is unclear. Our aim was to study the expression profile of integrins in surgical specimens of prostate cancer and associate their expression patterns with patient outcomes. METHODS. We selected 111 patients with localized prostate cancer who had undergone radical prostatectomy. Of these patients, 60 had no tumor recurrence after a median follow-up of 123 months. Integrin expression was evaluated by immunohistochemistry in a tissue microarray containing two tumor samples per patient. A semiquantitative analysis was employed. We measured the association between the expression of eight integrins and tumor recurrence. RESULTS. Multivariate analysis showed that expression of alpha 3 and alpha 3 beta 1 was related to worse outcome. When alpha 3 expression was strong and alpha 3 beta 1 expression was positive, the odds of recurrence were 3.0- and 2.5-fold higher, respectively. Only 19% and 28% of patients were recurrence-free in a mean period of 123 months of follow up when their tumors showed strong alpha 3 or positive alpha 3 beta 1 immuno-expression, respectively. CONCLUSIONS. We have shown that the expression of integrin alpha 3 beta 1 was independently associated with tumor recurrence after radical prostatectomy, suggesting that this integrin is a potential prognostic marker. Prostate 70: 1189-1195, 2010. (C) 2010 Wiley-Liss, Inc.

E-cadherin and beta-catenin Loss of Expression Related to Bone Metastasis in Prostate Cancer

Objectives: E-cadherin and beta-catenin are adhesion molecules responsible for the maintenance of normal epithelial cell phenotype. A disturbance in epithelial cell adhesion, which leads to a more invasive and metastatic phenotype, is a hallmark of tumor progression. Several immunohistochemical studies have reported a strong correlation between loss of their expression to higher stage and grade in prostate carcinoma, but their influence in metastatic process is not yet known. The aim of this study is to verify the role of adhesion molecules in the progression of prostate cancer (PC), assessing the expression of E-cadherin and beta-catenin in bone metastasis. Materials and Methods: Twenty-eight bone metastases of prostate carcinoma were submitted to immunohistochemistry analysis for E-cadherin and beta-catenin expression. In 6 patients, we were able to assess the expression of the adhesion molecules in the primary tumors and their respective metastases. The definition of normal expression for both antibodies was strong and diffuse expression in more than 70% of tumor cells. Results: In bone metastases, there was loss of expression of E-cadherin and beta-catenin in 86% and 82%, respectively. Among the primary tumors, E-cadherin and beta-catenin expression was normal in 83% and 50% cases, respectively. Considering the 6 patients with paired primary and bone metastasis, we found loss of expression for both E-cadherin and beta-catenin in most of the cases. Conclusions: Comparing primary PC and its metastasis, we showed persistent loss of E-cadherin and beta-catenin expression. This phenomenon may be related to metastatic potential in PC, because we have shown underexpression for E-cadherin and beta-catenin in 86% and 82% of bone metastases.

Combination of Multiple Molecular Markers Can Improve Prognostication in Patients With Locally Advanced and Lymph Node Positive Bladder Cancer

Purpose: We tested whether the combination of 4 established cell cycle regulators (p53, pRB, p21 and p27) could improve the ability to predict clinical outcomes in a large multi-institutional collaboration of patients with pT3-4N0 or pTany Npositive urothelial carcinoma of the bladder. We also assessed whether the combination of molecular markers is superior to any individual biomarker. Materials and Methods: The study comprised 692 patients with pT3-4N0 or pTany Npositive urothelial carcinoma of the bladder treated with radical cystectomy and bilateral lymphadenectomy (median followup 5.3 years). Scoring was performed using advanced cell imaging and color detection software. The base model incorporated patient age, gender, stage, grade, lymphovascular invasion, number of lymph nodes removed, number of positive lymph nodes, concomitant carcinoma in situ and adjuvant chemotherapy. Results: Individual molecular markers did not improve the predictive accuracy for disease recurrence and cancer specific mortality. Combination of all 4 molecular markers into number of altered molecular markers resulted in significantly 1 higher predictive accuracy than any single biomarker (p < 0.001.). Moreover addition of number of altered molecular markers to the base model significantly improved the predictive accuracy for disease recurrence (3.9%, p < 0.001) and cancer specific mortality (4.3%, p < 0.001). Addition of number of altered molecular markers retained statistical significance for improving the prediction of clinical outcomes in the subgroup of patients with pT3N0 (280), pT4N0 (83) and pTany Npositive (329) disease (p < 0.001). Conclusions: While the status of individual molecular markers does not add sufficient value to outcome prediction in patients with advanced urothelial carcinoma of the bladder, combinations of molecular markers may improve molecular staging, prognostication and possibly prediction of response to therapy.

Childhood radiation-associated atypical meningioma with novel complex rearrangements involving chromosomes 1 and 12

Meningiomas are recognized as the most common late complication following radiotherapy. However, cytogenetic studies in childhood atypical radiation-induced meningioma are sporadic, mainly because this condition generally occurs after a long latent period. In the present study we show the results of conventional and molecular cytogenetics in a 14-year-old boy with a secondary atypical meningioma. Apart from numerical changes, we found complex aberrations with the participation of chromosomes 1, 6 and 12. The invariable presence of loss of 1p was demonstrated by fluorescent in situ hybridization (FISH) analysis with probes directed to telomeric regions and by comparative genome hybridization (CGH). Previous cytogenetic studies on adult spontaneous and radiation-associated meningiomas showed loss of chromosome 22 as the most frequent change, followed by loss of the short arm of chromosome 1. To the best of our knowledge this is the first report of highly complex chromosome aberrations in the pediatric setting of meningioma.

Galectin-3 Overexpression in Invasive Laryngeal Carcinoma, Assessed by Computer-assisted Analysis

The larynx is the most common site of malignancy in the upper aerodigestive tract. In Brazil, malignant laryngeal lesions represent 2% of all cancers, with similar to 3000 annual deaths. The association between human papillomavirus (HPV) and laryngeal cancer is still controversial. The aim of the present retrospective study was to determine the expression of galectin-3 immunoperoxidase in laryngeal carcinoma by examining paraffin-em bedded larynx biopsies from 65 patients, 10 in situ laryngeal carcinomas, 27 laryngeal carcinomas without metastases, and 28 with metastases. Twenty-eight cervical lymph nodes from patients with metastatic lesions were also evaluated. Nested PCR was performed to detect and type HPV DNA. Galectin-3 expression was assessed by immunohistochemistry using a computer-assisted system. Among 65 patients, 55 (84.6%)were positive to beta-globin (internal control); 10 (15.4%) patients were beta-globin negative and were excluded from the HPV evaluation. Thus, 7 (12.7%) out of 55 patients were HPV positive and 48 (87.3%) out of 55 patients were HPV negative. High expression of galectin-3 was observed in invasive laryngeal tumors, suggesting that galectin-3 could be associated with the invasiveness and aggressiveness of laryngeal carcinomas. (J Histochem Cytochem 57:665-673, 2009)

Genomic deletions at 1p and 14q are associated with an abnormal cDNA microarray gene expression pattern in meningiomas but not in schwannomas

The molecular pathology of meningiomas and shwannomas involve the inactivation of the NF2 gene to generate grade I tumors. Genomic losses at 1p and 14q are observed in both neoplasms, although more frequently in meningiomas. The inactivation of unidentified genes located in these regions appears associated with tumor progression in meningiomas, but no clues to its molecular/clinical meaning are available in schwannomas. Recent microarray gene expression studies have demonstrated the existence of molecular subgroups in both entities. In the present study, we correlated the presence of genomic deletions at 1p, 14q, and 22q with the expression patterns of 96 tumor-related genes obtained by cDNA low-density microarrays in a series of 65 tumors including 42 meningiomas and 23 schwannomas. Two expression pattern groups were identified by cDNA mycroarray analysis when compared to the expression pattern in normal control RNA in both meningiomas and schwannomas, each one with patterns similar and different from the normal control. Meningioma and schwannoma subgroups differed in the expression of 38 and 16 genes, respectively. Using MLPA and microsatellites, we identified genomic losses at 1p, 14q, and 22q at nonrandom frequencies (12.5-69%) in meningiomas and schwannomas. Losses at 22q were almost equally frequent in both molecular expression subgroups in both neoplasms. However, deletions at 1p and 14q accumulated in meningiomas with a gene expression pattern different from the normal pattern, whereas the inverse situation occurred in schwannomas. Those anomalies characterized the schwannomas with expression pattern similar to the normal control. These findings suggest that deletions at 1p and 14q enhance the development of an abnormal tumor-related gene expression pattern in meningiomas, but this fact is not corroborated in schwannomas. (C) 2010 Elsevier Inc. All rights reserved.

Downregulation of CD9 protein expression is associated with aggressive behavior of oral squamous cell carcinoma

Squamous cell carcinoma of the oral cavity (OSCC) is a malignancy characterized by a high degree of local aggression and metastasis to cervical lymph nodes. Tetraspanins are proteins with functional roles in a wide array of cellular processes and are reported to be associated with tumor progression. The present study investigated the expression of the CD9, CD37, CD63, CD81 and CD82 tetraspanins in OSCC using immunohistochemistry (IHC) and quantitative Real Time-PCR (qRT-PCR). Tissue microarray (TMA) analysis of samples from 179 cases of OSCC and 10 normal samples oral mucosa were evaluated immunomorphologically. We analyzed CD9 and CD82 expression by qRT-PCR in 66 OSCC cases and 4 normal samples of oral mucosa. Expression of CD63, CD37 and CD81 was not detected in the samples studied. CD82 was downregulated or negative in 127 of 179 (80%) specimens; no correlation was observed between CD82 expression, clinicopathological parameters, disease-free survival and 5-year overall survival. CD9 expression was downregulated or negative in 75 of 129 (42%) OSCC samples. Loss of CD9 expression in OSCC samples correlated with the incidence of lymph node metastasis (p = 0.017). Disease-free survival and the 5-year overall survival of patients with downregulated or negative CD9 expression were significantly lower than in patients with positive CD9 expression (p = 0.010 and p = 0.071, respectively). No correlation was found between CD9 or CD82 expression and clinicopathological parameters by qRT-PCR. Our results suggest that the downregulation or lack of expression of the CD9 protein might indicate a more aggressive of OSCC. (C) 2009 Elsevier Ltd. All rights reserved.

Differential expression of galectin-3, beta-catenin, and cyclin D1 in adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma of salivary glands

Background: Galectin-3 has been implicated in tumor progression of some malignancies as thyroid, prostate, and salivary gland tumors. Recently, it has been suggested that this protein may be an important mediator of the beta-catenin/Wnt pathway. Moreover, nuclear galectin-3 expression has been implicated in cell proliferation, promoting cyclin D1 activation. Thus, the present study aimed to correlate galectin-3 expression with beta-catenin and cyclin D1 expressions in adenoid cystic carcinoma (ACC) and in polymorphous low-grade adenocarcinoma (PLGA). Methods: Fifteen formalin-fixed paraffin-embedded cases of each tumor were retrieved from the files of the Surgical Oral Pathology Service at the University of Sao Paulo and the proteins were analyzed by immunohistochemistry. Results: Adenoid cystic carcinoma showed galectin-3 immunostaining mainly in the nuclei, while PLGA revealed a positive mostly cytoplasmic reaction to galectin-3 in the largest part of tumor cells. Both tumors showed intense cytoplasmic/nuclear staining for beta-catenin in majority of cases. Cyclin D1 immunoreactivity was not detected in 14/15 PLGA and showed specific nuclear staining in 10/15 cases of ACC in more than 5% of the neoplastic cells. Cyclin D1 expression was correlated with cytoplasmic and nuclear galectin-3 expression in ACC (P < 0.05). Conclusions: These results suggest that in ACC galectin-3 may play a role in cellular proliferation through cyclin D1 activation. In addition, nuclear expression of galectin-3 in ACC may be related to a more aggressive behavior of this lesion. Although beta-catenin seems to play a role in carcinogenesis in both lesions, it seems that it does not bind to galectin-3 for cyclin D1 stimulation.