Centrosome amplification arises before neoplasia and increases upon p53 loss in tumorigenesis.

Centrosome abnormalities are a typical hallmark of human cancers. However, the origin and dynamics of such abnormalities in human cancer are not known. In this study, we examined centrosomes in Barrett's esophagus tumorigenesis, a well-characterized multistep pathway of progression, from the premalignant condition to the metastatic disease. This human cancer model allows the study of sequential steps of progression within the same patient and has representative cell lines from all stages of disease. Remarkably, centrosome amplification was detected as early as the premalignant condition and was significantly expanded in dysplasia. It was then present throughout malignant transformation both in adenocarcinoma and metastasis. The early expansion of centrosome amplification correlated with and was dependent on loss of function of the tumor suppressor p53 both through loss of wild-type expression and hotspot mutations. Our work shows that centrosome amplification in human tumorigenesis can occur before transformation, being repressed by p53. These findings suggest centrosome amplification in humans can contribute to tumor initiation and progression.

Transcription and Signaling Regulators in Developing Neuronal Subtypes of Mouse and Human Enteric Nervous System.

BACKGROUND & AIMS: The enteric nervous system (ENS) regulates gastrointestinal function via different subtypes of neurons, organized into fine-tuned neural circuits. It is not clear how cell diversity is created within the embryonic ENS; information required for development of cell-based therapies and models of enteric neuropathies. We aimed to identify proteins that regulate ENS differentiation and network formation. METHODS: We generated and compared RNA expression profiles of the entire ENS, ENS progenitor cells, and non-ENS gut cells of mice, collected at embryonic days 11.5 and 15.5, when different subtypes of neurons are formed. Gastrointestinal tissues from R26ReYFP reporter mice crossed to Sox10-CreERT2 or Wnt1-Cre mice were dissected and the 6 populations of cells were isolated by flow cytometry. We used histochemistry to map differentially expressed proteins in mouse and human gut tissues at different stages of development, in different regions. We examined enteric neuronal diversity and gastric function in Wnt1-Cre x Sox6fl/fl mice, which do not express the Sox6 gene in the ENS. RESULTS: We identified 147 transcription and signaling factors that varied in spatial and temporal expression during development of the mouse ENS. Of the factors also analyzed in human ENS, most were conserved. We uncovered 16 signaling pathways (such as fibroblast growth factor and Eph/ephrin pathways). Transcription factors were grouped according to their specific expression in enteric progenitor cells (such as MEF2C), enteric neurons (such as SOX4), or neuron subpopulations (such as SATB1 and SOX6). Lack of SOX6 in the ENS reduced the numbers of gastric dopamine neurons and delayed gastric emptying. CONCLUSIONS: Using transcriptome and histochemical analyses of the developing mouse and human ENS, we mapped expression patterns of transcription and signaling factors. Further studies of these candidate determinants might elucidate the mechanisms by which enteric stem cells differentiate into neuronal subtypes and form distinct connectivity patterns during ENS development. We found expression of SOX6 to be required for development of gastric dopamine neurons.

MyT1 Counteracts the Neural Progenitor Program to Promote Vertebrate Neurogenesis.

The generation of neurons from neural stem cells requires large-scale changes in gene expression that are controlled to a large extent by proneural transcription factors, such as Ascl1. While recent studies have characterized the differentiation genes activated by proneural factors, less is known on the mechanisms that suppress progenitor cell identity. Here, we show that Ascl1 induces the transcription factor MyT1 while promoting neuronal differentiation. We combined functional studies of MyT1 during neurogenesis with the characterization of its transcriptional program. MyT1 binding is associated with repression of gene transcription in neural progenitor cells. It promotes neuronal differentiation by counteracting the inhibitory activity of Notch signaling at multiple levels, targeting the Notch1 receptor and many of its downstream targets. These include regulators of the neural progenitor program, such as Hes1, Sox2, Id3, and Olig1. Thus, Ascl1 suppresses Notch signaling cell-autonomously via MyT1, coupling neuronal differentiation with repression of the progenitor fate.

Glial cells in the mouse enteric nervous system can undergo neurogenesis in response to injury.

The enteric nervous system (ENS) in mammals forms from neural crest cells during embryogenesis and early postnatal life. Nevertheless, multipotent progenitors of the ENS can be identified in the adult intestine using clonal cultures and in vivo transplantation assays. The identity of these neurogenic precursors in the adult gut and their relationship to the embryonic progenitors of the ENS are currently unknown. Using genetic fate mapping, we here demonstrate that mouse neural crest cells marked by SRY box-containing gene 10 (Sox10) generate the neuronal and glial lineages of enteric ganglia. Most neurons originated from progenitors residing in the gut during mid-gestation. Afterward, enteric neurogenesis was reduced, and it ceased between 1 and 3 months of postnatal life. Sox10-expressing cells present in the myenteric plexus of adult mice expressed glial markers, and we found no evidence that these cells participated in neurogenesis under steady-state conditions. However, they retained neurogenic potential, as they were capable of generating neurons with characteristics of enteric neurons in culture. Furthermore, enteric glia gave rise to neurons in vivo in response to chemical injury to the enteric ganglia. Our results indicate that despite the absence of constitutive neurogenesis in the adult gut, enteric glia maintain limited neurogenic potential, which can be activated by tissue dissociation or injury.

Enteric nervous system development: Recent progress and future challenges.

The enteric nervous system is the largest subdivision of the peripheral nervous system that plays a critical role in digestive functions. Despite considerable progress over the last 15 years in understanding the molecular and cellular mechanisms that control the development of the enteric nervous system, several questions remain unanswered. The present review will focus on recent progress on understanding the development of the mammalian enteric nervous system and highlight interesting directions of future research.

Male and female breast cancer--differences in DNA ploidy, p21 and p53 expression reinforce the possibility of distinct pathways of oncogenesis.

AIM: The purpose of this study was to compare the immunohistochemical profile of cell cycle inhibitors of G1/S phase transition (p21, p53 and pRb), Ki-67 proliferation marker and DNA ploidy in male (MBC) and female breast cancer (FBC). MATERIAL AND METHODS: One hundred patients (50 non-consecutive cases of FBC and an equal number of MBC) were selected according to homogeneous features regarding age, histological type, tumour grading, nodal status and absence of neoadjuvant therapy. The expression of p21, p53, pRb and Ki-67 was assessed by immunohistochemistry, and DNA ploidy was analysed by flow cytometry. Correlations between variables were evaluated using the chi(2) test. RESULTS: The incidence of DNA aneuploid, p21-positive and p53-negative tumours was significantly higher in MBC than in FBC; pRb and Ki-67 revealed no statistically significant differences between the two entities. In MBC, high tumour grade correlated with aneuploidy, Ki-67 and pRb positivity; ploidy and p53 were also associated. In FBC, only ploidy and grade showed a strong correlation. CONCLUSION: The significant dissimilarities regarding DNA ploidy, p21 and p53 in these quite homogeneous groups of FBC and MBC point to different genomic instability and to differences in cell cycle proliferative control, reinforcing the view of somewhat distinct tumour oncogenesis.

Familial breast/ovarian cancer and BRCA1/2 genetic screening: the role of immunohistochemistry as an additional method in the selection of patients.

Only 20-25% of families screened for BRCA1/2 mutations are found positive. Because only a positive result is informative, we studied the role of BRCA1/2 immunohistochemistry as an additional method for patient selection. From 53 high-risk-affected probands, 18 (34%) had available paraffin blocks of their tumors and were selected for this study. Mutation screening was done by conformation-sensitive gel electrophoresis and multiplex ligation-dependent probe amplification. For immunohistochemistry, 21 neoplastic specimens (15 breast carcinomas, 5 ovary neoplasms, and 1 rectal adenocarcinoma) were analyzed with BRCA1 (monoclonal antibody, Ab-1, oncogene) and BRCA2 (polyclonal antibody, Ab-2, oncogene) antibodies. Absence of the BRCA1 protein was confirmed in negative tumors by Western blotting. Seven patients were positive for BRCA1/2 mutations: 5 for BRCA1 and 2 for BRCA2. Four out of five positive patients had tumors negative for BRCA1 immunostaining, and the remaining 13 BRCA1-negative patients had positive BRCA1 immunostaining in all tumor samples. Sensitivity to predict for BRCA1 mutation carriers was 80%, and specificity was 100%, with a positive predictive value of 100% and a negative predictive value of 93%. This correlation was statistically significant (p=0.001). No correlation was observed for BRCA2. If larger studies confirm these results, high-risk patients with BRCA1-negative tumors should be screened first for this gene.

Chromosomal analysis of Barrett's cells: demonstration of instability and detection of the metaplastic lineage involved.

UNLABELLED: Barrett's esophagus is lined by columnar and goblets cells with gastric and intestinal characteristics. Despite the association between goblet elements and malignancy, it was not demonstrated that other columnar cells lineages are not related to neoplasia. Chromosomal abnormalities were described in metaplasia adjacent to Barrett's neoplasia, but it is unknown which metaplastic lineages are involved. This work assessed the frequency and the type of chromosomal abnormalities in Barrett's esophagus without neoplasia and performed the identification of the metaplastic cells carrying chromosomal gains. Barrett's esophagus biopsies were collected and processed for short-term cell culture and cytogenetic analysis. Combined immunofluorescence/fluorescence in situ hybridization was performed in cases exhibiting chromosomal gains by using antisera against intestinal (MUC2) and gastric (MUC5AC and MUC6) apomucins and chromosome pericentromeric alpha satellite DNA probes for the chromosomes involved. Each case was scored for the number of spots (0, 1, 2, >2) in 200 nonoverlapping nuclei. Columnar and goblet cells were separately assessed. Short-term cell cultures were achieved in 40/60 cases (67%). There were clonal abnormalities in 27/40 cases (68%) and tetraploid (4n) clones in 10/40 (25%). Structural alterations were detected in 14/40 (35%) with recurrent breakpoints at 1q21, 15q15 and 15q22. Numerical changes (trisomies 7 and 18 and loss of Y) occurred in 16/40 (40%). Gains of chromosomes 7 and 18 were more frequent in columnar than in goblet cells (9.8% vs 0.7% (P<0.05)) and (7.9 vs 1.9% (P<0.05)) respectively. These alterations were detected in cells exhibiting gastric as well as intestinal features and were more frequent in cells without apomucin production. CONCLUSIONS: (1) chromosomal instability is a common finding in Barrett's esophagus without neoplasia. (2) The two metaplastic populations are committed, chromosomal gains being more frequent in columnar nongoblet than in goblet cells. (3) The two metaplastic phenotypes, gastric and intestinal, are equally involved.

Correlations of cell cycle regulators (p53, p21, pRb and mdm2) and c-erbB-2 with biological markers of proliferation and overall survival in breast cancer.

AIM: The biological impact of cell cycle regulatory proteins on breast cancer progression is widely recognised, although mostly unclear. The aim of this preliminary study was to investigate the correlations of several cell cycle modulators (p53, p21, pRb, and mdm2) and c-erbB-2 expression with cell proliferation markers (S-phase fraction [SPF] and Ki-67) and overall survival in breast cancer. METHODS: The series comprised 50 women with stage I-II invasive ductal breast carcinoma (median follow-up 87 months), who were selected for their tumour proliferative characteristics (15 low, 15 high, and 20 intermediate proliferative tumours). Tumour differentiation was assessed following the Nottingham grading criteria. Cell cycle regulators, oestrogen receptor status, and Ki-67 index were analysed by immunohistochemistry on paraffin embedded material (cut-offs 10%). c-erbB-2 was evaluated according to a standardised immunohistochemical assay and borderline cases were confirmed by FISH analysis. Ploidy and SPF were determined by DNA flow cytometry on frozen samples. Chi-square test and Fisher's exact test were applied to analyse the statistical significance of data. RESULTS: Positive immunostaining was observed in nine (18%) p53+, 30 (60%) p21+, 13 (26%) pRb+, and one (2%) mdm2+ cases. c-erbB-2 expression was considered positive in 11 (22%) cases. In the subset of patients dead of the disease, a high incidence of c-erbB-2 over-expression (7/10, 70%) was verified. In general, no significant correlations among cell cycle regulators or between the latter and histopathological or proliferative characteristics were found. Only the p53-/p21+ phenotype significantly correlated with low SPF (p=0.048), and p21 positivity showed a trend to be associated with low SPF (p=0.083). No statistically significant correlations between cell cycle inhibitors and clinical outcome were found. On the contrary, c-erbB-2 over-expression showed significant correlations with DNA aneuploidy (p<0.001), high SPF (p<0.001), high tumour grading (p=0.008), lack of oestrogen receptors (p=0.036), and poor overall survival (p<0.001). CONCLUSIONS: The results seem to indicate the lack of correlations of cell cycle regulatory proteins with cell proliferation markers and overall survival in breast cancer, in contrast to c-erbB-2 over-expression which was found to be associated with increased proliferation rate and worse prognosis.