Davide Treré

Nucleolus, ribosomes, and cancer.

The complex aspects linking the nucleolus and ribosome biogenesis to cancer are reviewed here. The available evidence indicates that the morphological and functional changes in the nucleolus, widely observed in cancer tissues, are a consequence of both the increased demand for ribosome biogenesis, which characterizes proliferating cells, and the changes in the mechanisms controlling cell proliferation. In fact, the loss or functional changes in the two major tumor suppressor proteins pRB and p53 cause an up-regulation of ribosome biogenesis in cancer tissues. In this context, the association in human carcinomas of nucleolar hypertrophy with bad prognoses is worthy of note. Further, an increasing amount of data coming from studies on both hepatitis virus-induced chronic liver diseases and a subset of rare inherited disorders, including X-linked dyskeratosis congenita, suggests an active role of the nucleolus in tumorigenesis. Both an up-regulation of ribosome production and changes in the ribosome structure might causally contribute to neoplastic transformation, by affecting the balance of protein translation, thus altering the synthesis of proteins that play an important role in the genesis of cancer.

Nucleolar size indicates the rapidity of cell proliferation in cancer tissues.

In order to define the importance of the nucleolus in tumour pathology, the relationship between nucleolar size and function and tumour mass growth rate was studied in vivo. Ten established human cancer cell lines from colon carcinomas and neuroblastomas were inoculated subcutaneously in athymic mice and the doubling time (DT) of the xenograft tumour mass was calculated. The tumour DTs ranged from 3.2 to 15.7 days. Nucleolar size was evaluated in sections from formalin‐fixed and paraffin‐embedded tumour samples after silver staining for AgNOR proteins, using a specific image analysis system. The nucleolar area values were inversely related to the xenograft tumour mass DTs (r=−0.90; p<0.001). Nucleolar functional activity was also evaluated using rapid, intermediate, and slow growing tumours (one each). The values of RNA polymerase I activity measured in vitro were strongly related to the corresponding tumour DTs (r=−0.99; p=0.03). The labelling indices (LIs) of three proliferation markers, MIB1, PCNA, and bromodeoxyuridine (BrdU), were also evaluated. As revealed by the MIB1 and PCNA LIs, almost all the cells of the xenograft tumours were cycling (86.6±5.6 SD and 95.5±2.0 SD, respectively). Neither the MIB1, PCNA or BrdU LIs were related to the xenograft tumour mass DT, showing that the different growth rates of tumour xenografts were not due to different growth fractions, but were mainly related to different cell proliferation rates. The present data demonstrate that the size and function of the nucleolus are related to the cell proliferation rate of cancer tissue. Evaluation of nucleolar size after silver staining of AgNOR proteins represents a unique parameter for the histological assessment of rapidity of cell proliferation in tumour lesions. Copyright

The basal-like breast carcinoma phenotype is regulated by SLUG gene expression.

Basal‐like breast carcinoma is an aggressive form of breast cancer, characterized by the absence of oestrogen receptor and HER2 expression, the presence of cytokeratin 5 and epidermal growth factor receptor expression, and by the up‐regulation of stem cell regulatory genes. We show here that tumour tissues expressing high levels of SLUG mRNA show a basal‐like breast carcinoma phenotype and that such tumours also express high levels of stem cell‐regulatory genes, ie CD133, Bmi1. Further, we show that stem/progenitor cells, isolated from ductal breast carcinoma and from normal mammary gland as mammospheres, express SLUG, CD133, and Bmi1 mRNA and show a phenotype similar to that of basal‐like breast carcinoma. We also report that SLUG expression in tumour tissues correlates with that of the hypoxia survival gene carbonic anhydrase IX. In this regard, we report that the exposure of SLUG‐negative/luminal‐like MCF‐7 cells to a hypoxic environment promotes the onset of the basal‐like breast carcinoma phenotype, together with up‐regulation of the SLUG gene, which in turn blunts oestrogen receptor‐α and boosts carbonic anhydrase IX gene expression. Finally, we show that SLUG expression promotes the invasiveness of MCF‐7 cells exposed to hypoxia and sustains the in vivo aggressiveness of hypoxia‐selected, MCF‐7‐derived cells in xenografts. These data indicate that SLUG gene expression is part of a hypoxia‐induced genetic programme which sets up a basal/stem cell‐like, aggressive phenotype in breast cancer cells. Copyright

The silver-stained proteins of interphasic nucleolar organizer regions as a parameter of cell duplication rate

The relationship between interphasic silver-stained proteins of the nucleolar organizer regions (Ag-NOR proteins) and cell replication rate has been studied in 13 established neuroblastoma cell lines. The quantity of Ag-NOR proteins was measured in silver-stained cells by means of an automated image analyzer. The results indicated that the amount of Ag-NOR proteins is strictly proportional to the proliferative activity of the cells. Cell lines with a difference of only 4 h in doubling time were characterized by a statistically significant difference in Ag-NOR protein amount. The Ag-NOR protein quantity can therefore be used as a parameter for evaluating the cell proliferation rate.

What the nucleolus says to a tumour pathologist

The importance of nucleolar changes in cancer cells is underestimated in tumour pathology. There is evidence that the nucleolus is the mirror of a series of metabolic changes that characterize cancer cells. Cell entry into the cell cycle is always associated with up‐regulation of the nucleolar function and increased nucleolar size, which are also directly dependent on the rapidity of cell cycle progression. Furthermore, alterations of the major tumour suppressor retinoblastoma (Rb) and p53 pathways also contribute to the stimulation of nucleolar function and to nucleolar enlargement. High cell growth fraction, high cell growth rate and disruption of the Rb and p53 pathways are responsible for greater aggressiveness of cancer tissues. Therefore, the evaluation of nucleolar size allows one to obtain reliable information on the clinical outcome of the cancer: the greater the nucleolar size, the worse the tumour prognosis. Indeed, a series of studies carried out on numerous human tumours has shown that nucleolar hypertrophy (prominent nucleolus) was an independent predictive and prognostic parameter of a fatal clinical outcome.

Importance of interphase nucleolar organizer regions in tumor pathology

SummaryThe importance of the distribution of silverstained nucleolar organizer regions (Ag-NORs) in interphase nuclei for diagnostic and prognostic purposes in tumor pathology has been reviewed. The available data demonstrated that interphase Ag-NOR evaluation may be of help in distinguishing malignant from hyperplastic or normal cells. On the other hand, there is increasing evidence that a relationship exists between the quantity of interphase Ag-NORs and the prognosis of malignant tumors: the greater the number of interphase Ag-NORs, the worse is the prognosis. This can be explained by the observation that the interphase Ag-NOR quantity is strictly related to the cell proliferation rate. The procedures used for the measurement of the interphase Ag-NOR quantity are also critically discussed.

The asialoglycoprotein receptor in human hepatocellular carcinomas: its expression on proliferating cells

SummaryThe expression of the asialoglycoprotein receptor (ASGP-R) on human hepatocellular carcinoma (HCC) cells might be exploited to reduce the extrahepatic toxicity of DNA synthesis inhibitors by their conjugation with galactosyl-terminating peptides. In the present study we first assessed the frequency of ASGP-R expression in 60 HCCs. Secondly, we investigated whether the receptor was maintained on the plasma membranes of DNA synthesizing cancer cells. Needle biopsies of HCC were evaluated. Diagnosis and grading of HCC were performed on routine haematoxylin and eosin-stained sections according to Edmondson and Steiner (1953). Thirty-five tumours were grade I and II and were classified as well differentiated, while 25 tumours were grade III and IV and were classified as poorly differentiated. Sections from formalin-fixed, paraffin-embedded samples were incubated, after antigen retrieval, with an anti-ASGP-R monoclonal antibody revealed by secondary biotinylated antibody and streptavidin–biotin–peroxidase–diaminobenzidine reaction. A clear immunolabelling of plasma membranes of HCC cells was observed in 28 out of 35 (80%) well differentiated (grade I and II) and in five out of 25 (20%) poorly differentiated (grade III and IV) HCCs. The presence of the ASGP-R on the surface of DNA synthesizing cancer cells was also investigated after in vitro bromodeoxyuridine (BrdU) labelling of HCC samples by immunohistochemical visualization of both the ASGP-R and incorporated BrdU on the same section. The results obtained clearly demonstrated that DNA synthesizing cancer cells expressed the ASGP-R on their surface. The presence of ASGP-R on cell plasma membrane in the majority of differentiated HCCs and its maintenance on proliferating cells encourages studies in order to restrict the action of the inhibitors of DNA synthesis of HCC cells by their conjugation with galactosyl-terminating carriers internalized through this receptor.

Novel Dyskerin-Mediated Mechanism of p53 Inactivation through Defective mRNA Translation

In up to 60% of human cancers, p53 gene mutations are responsible for direct inactivation of the tumor suppressor function of p53. Alternative mechanisms of p53 inactivation described thus far mainly affect its posttranslational regulation. In X-linked dyskeratosis congenita, a multisystemic syndrome characterized by increased cancer susceptibility, mutations of the DKC1 gene encoding dyskerin cause a selective defect in the translation of a subgroup of internal ribosome entry site (IRES)-containing cellular mRNAs. In this study, we show that impairment of dyskerin function can cause p53 inactivation due to a defect in p53 mRNA translation. siRNA-mediated reduction of dyskerin levels caused a decrease of p53 mRNA translation, protein levels, and functional activity, both in human breast cancer cells and in primary mammary epithelial progenitor cells. These effects seemed to be independent of the known role of dyskerin in telomerase function, and they were associated with a specific impairment of translation initiation mediated by IRES elements present in p53 mRNA. In a series of human primary breast cancers retaining wild-type p53, we found that low levels of dyskerin expression were associated with reduced expression of p53-positive target genes. Our findings suggest that a dyskerin-mediated mechanism of p53 inactivation may occur in a subset of human tumors.

Nucleolar Size and Activity Are Related to pRb and p53 Status in Human Breast Cancer

Cell proliferation is tightly coordinated with cell growth. The oncosuppressor proteins pRb and p53 may exert a key role in coupling growth and proliferation by controlling both ribosome biogenesis and cell cycle progression. In the present study we evaluated the relationship between the pRb and p53 status and rRNA transcriptional activity in histological sections of 343 human primary breast carcinomas. Ribosomal biogenesis was quantified by morphometric analysis of silver-stained interphase nucleolar organizer regions (AgNORs). pRb and p53 status was assessed by immunohistochemistry. Twenty-four tumors were considered to be pRb deleted, 260 tumors showed a phosphorylated-pRb labeling index (LI) up to 25%, and 55 tumors an LI >25%. Tumors with deleted pRb or phosphory-lated-pRb-LI ×25% were characterized by significantly greater mean AgNOR area values than those with unaltered pRb (p<0.001). In the 71 tumors with mutated p53 the NOR area mean value was greater than in the 272 tumors with normal p53 (p<0.001). Our results demonstrate, for the first time in vivo, that pRb and p53 status is related to the ribosome biogenesis rate and suggest that in tumors with altered pRb and p53 function the up-regulation of rRNA synthesis may always assure an adequate growth to cancer cells with uncontrolled cell cycle progression.

c-erbB-2 over-expression in amplified and non-amplified breast carcinoma samples

We investigated c‐erbB‐2 oncogene amplification and over‐expression in 79 invasive breast carcinoma samples using fluorescence in situ hybridization (FISH) and immuno‐histochemistry, with the aim of studying relationships between neoplasms over‐expressing c‐erbB‐2 with or without amplification and bio‐pathological parameters used in clinical breast cancer. Nineteen samples showed amplification, and all of these were positive by immuno‐histochemistry. Moderate or intense immunostaining was present in a further 22 samples without c‐erbB‐2 amplification and was not related to any increased number of c‐erbB‐2 signals: 15 samples exhibited chromosome 17 polysomy, 3 monosomy and 4 no FISH abnormalities. Thirty‐eight samples were immunonegative: 18 exhibited chromosome 17 polysomy, 9 monosomy and 11 no alterations. Samples having c‐erbB‐2 over‐expression associated with amplification showed DNA aneuploidy and hormonal receptor loss to a greater extent than those expressing c‐erbB‐2 without amplification or immunonegative samples (χ2 test, p = 0.007, 0.008 and 0.008, respectively). The proliferation rate, detected by Ag‐NOR staining, was highest in amplified samples (Kruskal Wallis test, p = 0.009). Our results indicate that tumours showing both c‐erbB‐2 over‐expression and amplification exhibit more aggressive biological characteristics than those with only over‐expression or immunonegative tumours. Since both c‐erbB‐2 amplification and over‐expression have been related to negative responses to chemotherapy and poor prognosis, these differences might have clinical implications. The combination of FISH and immuno‐histochemistry may be helpful to achieve this aim. Int. J. Cancer (Pred. Oncol.) 84:273–277, 1999.