Snail heterogeneity in clear cell renal cell carcinoma

Background: Intratumor heterogeneity may be responsible of the unpredictable aggressive clinical behavior that some clear cell renal cell carcinomas display. This clinical uncertainty may be caused by insufficient sampling, leaving out of histological analysis foci of high grade tumor areas. Although molecular approaches are providing important information on renal intratumor heterogeneity, a focus on this topic from the practicing pathologist' perspective is still pending. Methods: Four distant tumor areas of 40 organ-confined clear cell renal cell carcinomas were selected for histopathological and immunohistochemical evaluation. Tumor size, cell type (clear/granular), Fuhrman's grade, Staging, as well as immunostaining with Snail, ZEB1, Twist, Vimentin, E-cadherin, beta-catenin, PTEN, p-Akt, p110 alpha, and SETD2, were analyzed for intratumor heterogeneity using a classification and regression tree algorithm. Results: Cell type and Fuhrman's grade were heterogeneous in 12.5 and 60 % of the tumors, respectively. If cell type was homogeneous (clear cell) then the tumors were low-grade in 88.57 % of cases. Immunostaining heterogeneity was significant in the series and oscillated between 15 % for p110a and 80 % for Snail. When Snail immunostaining was homogeneous the tumor was histologically homogeneous in 100 % of cases. If Snail was heterogeneous, the tumor was heterogeneous in 75 % of the cases. Average tumor diameter was 4.3 cm. Tumors larger than 3.7 cm were heterogeneous for Vimentin immunostaining in 72.5 % of cases. Tumors displaying negative immunostaining for both ZEB1 and Twist were low grade in 100 % of the cases. Conclusions: Intratumor heterogeneity is a common event in clear cell renal cell carcinoma, which can be monitored by immunohistochemistry in routine practice. Snail seems to be particularly useful in the identification of intratumor heterogeneity. The suitability of current sampling protocols in renal cancer is discussed.

RAN GTPase is an effector of the invasive/metastatic phenotype induced by osteopontin.

Osteopontin (OPN) is a phosphorylated glycoprotein that binds to alpha v-containing integrins and is important in malignant transformation and cancer. Previously, we have utilized suppressive subtractive hybridization between mRNAs isolated from the Rama 37 (R37) rat mammary cell line and a subclone rendered invasive and metastatic by stable transfection with an expression vector for OPN to identify RAN GTPase (RAN) as the most overexpressed gene, in addition to that of OPN. Here we show that transfection of noninvasive R37 cells with an expression vector for RAN resulted in increased anchorage-independent growth, cell attachment and invasion through Matrigel in vitro, and metastasis in syngeneic rats. This induction of a malignant phenotype was induced independently of the expression of OPN, and was reversed by specifically reducing the expression of RAN using small-interfering RNAs. By using a combination of mutant protein and inhibitors, it was found that RAN signal transduction occurred through the c-Met receptor and PI3 kinase. This study therefore identifies RAN as a novel effector of OPN-mediated malignant transformation and some of its downstream signaling events in a mammary epithelial model of cancer invasion/metastasis.

In vivo selection for metastasis promoting genes in the mouse

Here, we report the identification of a metastasis promoting factor by a forward genetic screen in mice. A retroviral cDNA library was introduced into the nonmetastatic cancer cell line 168FARN, which was then orthotopically transplanted into mouse mammary fat pads, followed by selection for cells that metastasize to the lung. The genes encoding the disulfide isomerase ERp5 and beta-catenin were found to promote breast cancer invasion and metastasis. Disulfide isomerases (thiol isomerases), which catalyze disulfide bond formation, reduction, and isomerization, have not previously been implicated in cancer cell signaling and tumor metastasis. Overexpression of ERp5 promotes both in vitro migration and invasion and in vivo metastasis of breast cancer cells. These effects were shown to involve activation of ErbB2 and phosphoinositicle 3-kinase (PI3K) pathways through dimerization of ErbB2. Activation of ErbB2 and PI3K subsequently stimulates RhoA and beta-catenin, which mediate the migration and invasion of tumor cells. Inhibition of ErbB2 and PI3K reverses the phenotypes induced by ERp5. Finally, ERp5 was shown to be up-regulated in human surgical samples of invasive breast cancers. These data identify a link between disulfide isomerases and tumor development, and provide a mechanism that modulates ErbB2 and PI3K signaling in the promotion of cancer progression.

Expression of beta-human chorionic gonadotropin (beta-hCG) in non-trophoblastic elements of transitional cell carcinoma of the bladder: possible relationship with the prognosis.

Transitional cell carcinoma (TCC) of the bladder is a neoplasm with variability in its clinical behavior. Although there are several studies correlating stage and ABO isoantigen expression with invasiveness, there is no single predictor factor to assess the potential invasiveness, especially in the low grade, non-invasive TCC. In the present study we evaluated the correlation of histological grade plus stage and the expression of beta human chorionic gonadotropin (beta-hCG), in 100 cases of TCC, with the clinical behavior. These features were correlated with tumor progression in patients with at least two years of follow up. We observed more aggressiveness in G4 group (high grade and invasive) (93% had tumor progression) when compared to G1 group (low grade and superficial) (11% had tumor progression). However in 25.5% of the TCC cases (groups G2: low grade and invasive and G3: high grade and superficial) the clinical behavior was intermediate, showing some limitation in using grading and staging only, as a predictive factor. There was an expression of beta-hCG in 21.4% of the cases in up to 25% of the tumor cells without any trophoblastic morphology. These beta-hCG producing TCC had a strong correlation with aggressiveness: 39.1% and 12.8% of the TCC expressed beta-hCG with and without tumor progression, respectively.

Frequent p53 mutations and occasional loss of chromosome 4 in invasive bladder carcinoma induced by N-butyl-N-(4-hydroxybutil)nitrosamine in B6D2F1 mice

B6D2F1 mice (45/group) were treated with N-butyl-N-(4- hydroxybutyl)nitrosamine (BBN) or uracil as follows: Group 1 received 0.05% BBN in drinking water for the entire experiment, Group 2 received 5 mg of BBN by gastric gavage in 0.1 mL of 20% ethanol twice per week for 10 wk, Group 3 received a 2.5% uracil-containing diet for the entire experiment, and Group 4 was controls (received 0.1 mL of 20% ethanol by gavage twice per week for 10 wk). The surviving mice in Group 1 were killed after week 26 and those in the other groups after week 30. By week 15, three of 11 Group 1 and one of 15 Group 2 mice had bladder carcinoma. By 26 and 30 wk, respectively, invasive carcinomas were observed in 33 of 34 and six of 21 mice in Groups 1 and 2 and renal pelvic carcinomas in 11 of 34 and three of 21 mice in Groups 1 and 2. Four of 19 uracil-treated mice had bladder nodular hyperplasia. By polymerase chain reaction-single-strand conformation polymorphism and sequence analyses, 16 of 20 and two of five bladder carcinomas from Groups 1 and 2, respectively, showed mutations in the p53 gene. Ha-ras mutation was present in one case. Loss of heterozygosity analysis with simple-sequence length polymorphism markers for chromosome 4 showed that 10 of 21, two of 15, and nine of 13 mice in Groups 1-3, respectively, had heterozygous or homozygous deletions. B6D2F1 mice are therefore susceptible to the urothelial carcinogenic effects of BBN and develop frequent p53 mutations and chromosome 4 deletions. Chromosome 4 deletions were also seen with uracil.

Estudo comparativo dos métodos de ultra-sonografia, tomografia computadorizada e ressonância magnética no estadiamento e invasão das estruturas adjacentes por tumores renais.

BACKGROUND: Ultrasonography (US), Computed Tomography (CT), and Magnetic Resonance imaging (MR) were compared for the staging of renal tumors. The differences between these imaging techniques were also studied for their ability to detect adenopathies, vascular invasion, distant intra-abdominal metastases, and particularly adjacent organ invasion. METHODS: Thirty-one patients with solid or complex renal masses were prospectively studied using US, CT, and MR. Differences between the results obtained were studied using the COCHRAN G test and the McNEMAR test. The sensitivity and specificity of each diagnostic technique were compared against a gold standard of the surgical and histopathological findings. RESULTS: The following sensitivities were obtained: For the detection of adenopathy, US 63.6%, CT and MR 90.9%. For vascular invasion, US 42.8%, CT and MR 85.7%. For the adjacent organ invasion, US 28.5%, CT 85.7%, and MR 71.4%. Some of the criteria that suggest invasion of adjacent structures include: the envelopment of the adjacent structures by the tumor, tumor extension into the adjacent structures with an irregular appearance, and alterations in shape, size, and density of adjacent structures. Loss of fat planes between the tumor and adjacent structures is not a sign of tumor invasion. CONCLUSIONS: Significant differences were found in the detection capacity of US in relation to CT and MR, which were similar. All three techniques were highly sensitive and specific only in the detection of distant abdominal metastases. In addition to the accuracy of these diagnostic modalities for the detection and staging of tumors, invasiveness, risks and cost should be considered in relation to relative costs and benefits.

Papilomavírus humano (HPV) em lesões benignas e malignas de mucosa oral pelos métodos de imuno-histoquímica e hibridização in situ

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Expression of the nonclassical HLA-G and HLA-E molecules in laryngeal lesions as biomarkers of tumor invasiveness

Introduction: HLA-G and HLA-E are two nonclassical class I molecules, which have been well recognized as modulators of innate and adaptive immune responses, and the expression of these molecules in virus infected cells has been associated with subversion of the immune response. Objective: In this study we performed a cross-sectional study, systematically comparing the expression of HLA-G and HLA-E in benign, premalignant and malignant laryngeal lesions, correlating with demographic and clinical variables and with the presence of high-risk and low-risk HPV types. Materials and methods: Laryngeal lesions were collected from 109 patients and stratified into 27 laryngeal papillomas, 17 dysplasias, 10 in situ laryngeal carcinomas, 27 laryngeal carcinomas without metastases, 28 laryngeal carcinomas with metastasis along with their respective draining cervical lymph nodes, and 10 normal larynx specimens. The expression of HLA-G and HLA-E molecules was determined by immunohistochemistry. HPV DNA detection and typing was performed using generic and specific primers. Results: HLA nonclassical molecules showed a distinct distribution pattern, according to the larynx lesion grade. HLA-G expression increased in benign and premalignant lesions, and gradually decreased in invasive carcinomas and in respective draining cervical lymph nodes. Conversely, HLA-E expression increased as far as lesion grade increased, including increased molecule expression in the draining lymph nodes of malignant lesions. Only 17 (15.6%) patients were HPV DNA positive. Conclusions: Overexpression of HLA-E and underexpression of HLAG appear to be good markers for malignant larynx lesion.

Tumor-associated macrophages and the profile of inflammatory cytokines in oral squamous cell carcinoma

Objective: To evaluate and characterize macrophage populations (M1/M2) in the tumor microenvironment of oral cavity squamous cell carcinoma (OCSCC). The relationship between macrophages and clinicopathological factors, such as survival data, lymph node metastasis, tumoral proliferation, and WHO histological grading are also analyzed. Materials and methods: The samples consisted of surgically excised specimens from patients with non-metastatic and metastatic OCSCC and normal oral mucosa (control). Immunohistochemistry, flow cytometry, and qRT-PCR were used to evaluate macrophage populations and the expression of pro- (IL-12, IL-23, and INF-γ) and anti-inflammatory (IL-10 and TGF-β) cytokines. The level required for statistical significance was defined as p < 0.05. Results: The data showed a predominance of M2 phenotype (high percentage of IL-10+TGF-β+) macrophages in the tumor microenvironment of OCSCC. A higher percentage of macrophages expressing TGF-β was seen in the OCSCC group when compared with healthy individuals. The assessment of mRNA expression also presented a greater expression of anti-inflammatory cytokines TGFβ and IL10 in OCSCC when compared with the control group. The percentage of macrophages, demonstrated by immunohistochemistry, was significantly higher in the metastatic OCSCC group than in the non-metastatic and control groups. The log-rank test also showed that the mean survival time for patients with high levels of macrophages was less (44 months) when compared with patients with a low percentage of such cells (93 months). Conclusion: A predominance of the M2 phenotype in the tumor microenvironment of OCSCC could contribute to local immunosuppression, via TGF-β production, and consequently greater lymph node involvement and reduced patient survival time. © 2012 Elsevier Ltd. All rights reserved.

Avaliação da associação entre a presença de polimorfismo no gene da metaloproeinase-9 e o desenvolvimento do carcinoma espinocelular quimicamente induzido pelo DMBA na pele de camundongos hairless

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

PURINERGIC SIGNALING REGULATES FILOPODIA-INDUCED ZIPPERING

The molecular mechanisms that mediate endometrial cancer invasion and metastasis remain poorly understood. This is a significant clinical problem, as there is no definitive cure for metastatic disease. The purinergic pathway’s generation of adenosine and its activation of the adenosine receptor A2B (A2BR) induces cell-cell adhesion to promote barrier function. This barrier function is known to be important in maintaining homeostasis during hypoxia, trauma, and sepsis. Loss of this epithelial barrier function provides a considerable advantage for carcinoma progression, as loss of cell-cell adhesions supports proliferation, aberrant signaling, epithelial-to-mesenchymal transition, invasion, and metastasis. The present work provides strong evidence that CD73-generated adenosine actively promotes cell-cell adhesion in carcinoma cells by filopodia-induced zippering. Adenosine-generating ecto-enzyme, CD73, was down-regulated in moderately- and poorly-differentiated, invasive, and metastatic endometrial carcinomas. CD73 expression and enzyme activity in normal endometrium and endometrial carcinomas was significantly correlated to the epithelial phenotype. Barrier function in normal epithelial cells of the endometrium was dependent on stress-induced generation of adenosine by CD73 and adenosine’s activation of A2BR. This same mechanism inhibited endometrial carcinoma cell migration and invasion. Finally, adenosine’s activation of A2BR induced the formation of filopodia that promoted the re-forming of cell-cell adhesions in carcinoma cells. Overall, these studies identified purinergic pathway-induced filopodia to be a novel mechanism of adenosine’s barrier function and a mechanism that has to be avoided/down-regulated by endometrial carcinoma cells attempting to lose attachment with their neighboring cells. These results provide insight into the molecular mechanisms of endometrial cancer invasion. In addition, because loss of cell-cell adhesions has been closely linked to therapy resistance in cancer, these results provide a rational clinical strategy for the re-establishment of cell-cell adhesions to potentially increase therapeutic sensitivity. In contrast to other molecular mechanisms regulating cell-cell adhesions, the purinergic pathway is clinically druggable, with agonists and antagonists currently being tested in clinical trials of various diseases.

Cysteine protease inhibitors as chemotherapy: Lessons from a parasite target

Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite’s lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.