Skp2 is oncogenic and overexpressed in human cancers

Skp2 is a member of the F-box family of substrate-recognition subunits of SCF ubiquitin-protein ligase complexes that has been implicated in the ubiquitin-mediated degradation of several key regulators of mammalian G(1) progression, including the cyclin-dependent kinase inhibitor p27, a dosage-dependent tumor suppressor protein. In this study, we examined Skp2 and p27 protein expression by immunohistochemistry in normal oral epithelium and in different stages of malignant oral cancer progression, including dysplasia and oral squamous cell carcinoma. We found that increased levels of Skp2 protein are associated with reduced p27 in a subset of oral epithelial dysplasias and carcinomas compared with normal epithelial controls. Tumors with high Skp2 (>20% positive cells) expression invariably showed reduced or absent p27 and tumors with high p27 (>20% positive cells) expression rarely showed Skp2 positivity. Increased Skp2 protein levels were not always correlated with increased cell proliferation (assayed by Ki-67 staining), suggesting that alterations of Skp2 may contribute to the malignant phenotype without affecting proliferation. Skp2 protein overexpression may lead to accelerated p27 proteolysis and contribute to malignant progression from dysplasia to oral epithelial carcinoma. Moreover, we also demonstrate that Skp2 has oncogenic potential by showing that Skp2 cooperates with H-Ras(G12V) to malignantly transform primary rodent fibroblasts as scored by colony formation in soft agar and tumor formation in nude mice. The observations that Skp2 can mediate transformation and is up-regulated during oral epithelial carcinogenesis support a role for Skp2 as a protooncogene in human tumors.     

Ineffective oesophageal motility： Manometric subsets exhibit different symptom profiles

AIM： To compare the demographic and clinical features of different manometric subsets of ineffective oesophageal motility （IOM; defined as ≥ 30% wet swallows with distal contractile amplitude 〈 30 mmHg）, and to determine whether the prevalence of gastro-oesophageal reflux differs between IOM subsets.
METHODS： Clinical characteristics of manometric subsets were determined in 100 IOM patients （73 female, median age 58 years） and compared to those of 100 age-and gender-matched patient controls with oesophageal symptoms, but normal manometry. Supine oesophageal manometry was performed with an eight-channel DentSleeve water-perfused catheter, and an ambulatory pH study assessed gastrooesophageal reflux.
RESULTS： Patients in the IOM subset featuring a majority of low-amplitude simultaneous contractions （LASC） experienced less heartburn （prevalence 26%）, but more dysphagia （57%） than those in the IOM subset featuring low-amplitude propagated contractions （LAP; heartburn 70%, dysphagia 24%; both P ≤ 0.01）. LASC patients also experienced less heartburn and more dysphagia than patient controls （heartburn 68%, dysphagia 11%; both P 〈 0.001）. The prevalence of heartburn and dysphagia in IOM patients featuring a majority of non-transmitted sequences （NT） was 54% （P = 0.04 vs LASC） and 36% （P 〈 0.01 vs controls）, respectively. No differences in age and gender distribution, chest pain prevalence, acid exposure time （AET） and symptom/reflux association existed between IOM subsets, or between subsets and controls.
CONCLUSION： IOM patients with LASC exhibit a different symptom profile to those with LAP, but do not differ in gastro-oesophageal reflux prevalence. These findings raise the possibility of different pathophysiological mechanisms in IOM subsets, which warrants further investigation.     

Gene expression profiles at different stages of human esophageal squamous cell carcinoma

AIM: To characterize the gene expression profiles in different stages of carcinogenesis of esophageal epithelium. METHODS: A microarray containing 588 cancer related genes was employed to study the gene expression profile at different stages of esophageal squamous cell carcinoma including basal cell hyperplasia, high-grade dysplasia, carcinoma in situ, early and late cancer. Principle component analysis was performed to search the genes which were important in carcinogenesis. RESULTS: More than 100 genes were up or down regulated in esophageal epithelial cells during the stages of basal cell hyperplasia, high-grade dysplasia, carcinoma in situ, early and late cancer. Principle component analysis identified a set of genes which may play important roles in the tumor development. Comparison of expression profiles between these stages showed that some genes, such as P160ROCK, JNK2, were activated and may play an important role in early stages of carcinogenesis. CONCLUSION: These findings provided an esophageal cancer-specific and stage-specific expression profiles, showing that complex alterations of gene expression underlie the development of malignant phenotype of esophageal cancer cells.     

WAF1 expression andp53 mutations in human colorectal cancers

The expression of theWAF1 gene was examined in the tissues of primary colorectal cancers and of adjacent non-neoplastic mucosas by Western blot analysis.p53 mutations of these cancer tissues were also analyzed by the polymerase chain reaction/single-strand conformation polymorphism followed by direct sequencing. Missense mutations ofp53 were recognized in 19 out of 40 cases. Five cancers (12.5%) displayed much lower expression of WAF1 than did their corresponding mucosas, and all of them contained mutantp53. Fourteen cancers (35%) expressed the same level of WAF1 as their mucosas, and 5 of them had mutantp53. Twenty-one cancers (52.5%) had much higher WAF1 expression than their mucosas, and 9 of them had mutantp53. When Duke's classification was applied to these colorectal cancers, it was found that the cancers with reduced expression of WAF1 basically coincided with late (C or D) stages (4 out of 5 cases). while the cancers with higher WAF1 expression were consistent with early (A or B) stages (17 out of 21 cases). Down-regulation of WAF1 in colorectal cancer tissues may be implicated in tumor progression.Abbreviations PCR polymerase chain reaction - SSCP single-strand conformation polymorphism     

MicroRNA expression analysis of mammospheres cultured from human breast cancers

Purpose

There is accumulating evidence suggests that tumors are initiated and maintained by a small fraction of tumor-initiating cells (TICs). TICs can be enriched by mammospheres culturing without surface markers. MicroRNAs participated in many important processes of life including regulating tumorigenicity of TICs. However, roles of miRNAs in TICs of breast cancer are still unknown.

Methods

We compared mammospheres formation of four breast cancer cell lines, cultured mammospheres from breast cancers specimens of three patients and compared microRNAs profiling of mammospheres cultured from breast cancer specimens with differentiated progenies.

Results

Three of the four breast cancer cell lines showed the ability of mammospheres formation. The proportions of CD24^?cells in mammospheres were increased significantly in the three cell lines. The expression of genes associated with stem cells and chemoresistance increased significantly after mammospheres formation. Breast cancer cells isolated from patients’ specimens survived in nonadherent culture conditions generated mammospheres with ability of self-renewal and bilineage potential. MicroRNA expression profiling of mammospheres compared with differentiated progenies isolated and propagated from the three patients identified 17 microRNAs. And the target genes of these miRNAs are involved in several key signaling pathways.

Conclusions

The results suggested that mammospheres were enriched in TICs and proved a valuable model for studies of breast cancer TICs in vitro, microRNAs played critical roles in maintenance of stemness properties of mammospheres and provided novel insights into breast cancer therapy.     

Valence-sensitive neurons exhibit divergent functional profiles in gregarious and asocial species

The medial bed nucleus of the stria terminalis (BSTm) influences both social approach and social aversion, suggesting that this structure may play an important role in generating motivational and behavioral differences between gregarious and asocial species. However, no specific neurons have been identified within the BSTm that influence species-typical levels of sociality or that mediate approach and avoidance. Using five songbird species that differ selectively in their species-typical group sizes, we now demonstrate that vasotocin-immunoreactive (VT-ir) neurons of the BSTm exhibit very different immediate early gene responses to same-sex stimuli in gregarious and asocial species. Exposure to a same-sex conspecific increases VT-Fos colocalization in gregarious species while decreasing colocalization in relatively asocial species. We additionally demonstrate that these neurons are selectively activated by social stimuli that normally elicit affiliation (positively valenced social stimuli) but not by stimuli that elicit aversion (negatively valenced social stimuli). Constitutive Fos activity of the VT-ir neurons is also significantly greater in the gregarious species, and the two most social species express significantly more VT-ir neurons. These findings demonstrate that the properties of valence-sensitive neurons evolve in relation to sociality and indicate that gregarious species accentuate positive stimulus properties during social interactions.     

Cyclooxygenase-2 expression in primary human colorectal cancers and bone marrow micrometastases

BACKGROUND: Both the expressions of the inducible form of cyclooxygenase-2 and the presence of bone marrow micrometastases are poor prognostic markers in patients with colorectal carcinoma. AIMS: As cyclooxygenase-2 expression in these tumours is associated with increased metastatic potential in vitro, our objectives were to determine the relationship between cyclooxygenase-2 and haematogenous spread to bone marrow. PATIENTS AND METHODS: Thirty-two patients with resection of colorectal carcinoma were evaluated (median age: 69.5 years). Bone marrow was obtained from all patients from both iliac crests before manipulation of the primary tumour. The tumours were of varying stages at diagnosis (5 Dukes' A, 14 Dukes' B, 11 Dukes' C and 2 Dukes' D). Tumour sections were stained for cyclooxygenase-2 using the avidin-biotin immunohistochemical technique. Extent of staining was graded depending on the percentage of epithelial cells staining positive for cyclooxygenase-2. Micrometastases were detected by staining contaminant cytokeratin-18 positive cells in the bone marrow aspirates by either immunohistochemical (ARAAP) or immunological (flow cytometry) methods. Fisher's exact probability test was used to calculate statistical significance. RESULTS: Cyclooxygenase-2 expression in the primary tumour was detected in 72% of the patients. Twelve (38%) patients had bone marrow micrometastases detected by either immunohistochemistry or flow cytometry. Of the 12 patients who had bone marrow micrometastases, 8 tumours demonstrated increased expression of cyclooxygenase-2 protein (66.6%). In contrast, 9 out of the 20 (45%) patients in whom micrometastases were not detected expressed increased levels of cyclooxygenase-2 (P = 0.29). When dividing the patients into subgroups of localised (Dukes' A and B) versus disseminated (Dukes' C and D) disease, there was no further association between cyclooxygenase-2 expression and bone marrow micrometastases (P = 0.179 and 1.0). CONCLUSION: In this pilot study, there was no association between cyclooxygenase-2 expression and bone marrow micrometastases in patients with otherwise localised or disseminated disease.     

Gene expression profiles in stages II and III colon cancers: application of a 128-gene signature

Purpose

A 128-gene signature has been proposed to predict outcome in patients with stages II and III colorectal cancers. In the present study, we aimed to reproduce and validate the 128-gene signature in external and independent material.

Methods

Gene expression data from the original material were retrieved from the Gene Expression Omnibus (GEO) (n?=?111) in addition to a Danish data set (n?=?37). All patients had stages II and III colon cancers. A Prediction Analysis of Microarray classifier, based on the 128-gene signature and the original training set of stage I (n?=?65) and stage IV (n?=?76) colon cancers, was reproduced. The stages II and III colon cancers were subsequently classified as either stage I-like (good prognosis) or stage IV-like (poor prognosis) and assessed by the 36?months cumulative incidence of relapse.

Results

In the GEO data set, results were reproducible in stage III, as patients predicted to be stage I-like had a significant lower risk of relapse than patients predicted as stage IV-like (P?=?0.04, Gray test). Results were not reproducible in stage II patients (P?>?0.05, Gray test). In the Danish data set, two of four stage III patients with relapse were correctly predicted as stage IV-like, and the remaining patients were predicted as stage I-like and unclassifiable, respectively. Stage II patients could not be stratified.

Conclusions

The 128-gene signature showed reproducibility in stage III colon cancer, but could not predict recurrence in stage II. Individual patient predictions in an independent Danish material were unsatisfactory. Additional validation in larger cohorts is warranted.     

An E-box-containing region is involved in the tissue-specific expression of the human MC2R gene

Expression of the melanocortin receptor (MC2R) gene is limited to adrenocortical cells and the aim of this study was to determine the factors responsible for this tissue specificity. We used different fragments of the human (h) MC2R gene promoter, inserted in a vector upstream of the luciferase reporter gene, to transiently transfect either bovine adrenocortical (BAC) cells or granulosa cells from bovine ovaries (B-Gran). Similar promoter activities were obtained in both cell types using constructs containing fragments up to 1017 bp of the hMC2R gene promoter. On the contrary, a 2-fold decrease was obtained after transfection of the B-Gran cells with vectors containing 1069 bp and more of the promoter. Results obtained here using BAC cells confirmed our previous data on human cells showing that steroidogenic factor 1 is the major transactivating factor involved in the basal expression of the hMC2R gene in adrenal cells. However, we showed that this factor did not permit, by itself, the expression of the hMC2R gene in B-Gran cells despite its expression in these cells. This study demonstrated for the first time that an E-box (located at -1020 bp) is involved in the repression of hMC2R gene expression in granulosa cells through interactions with several factors, such as activator protein 4, as suggested by electrophoretic mobility shift assay analyses.     

The expression and clinical significance of CD44v in human gastric cancers

INTRODUCTION CD44 was originally implicated as a "homing"receptor directing the migration of recirculating lymphocytes[1]. CD44 expression has been confirmed not only in lymphocytes but also in a wide variety of epithelial tissues. It is considered as an important cell adhesion molecule for cell to cell interactions[2].