The Ribosome Biogenesis-Cancer Connection.

Multifaceted relations link ribosome biogenesis to cancer. Ribosome biogenesis takes place in the nucleolus. Clarifying the mechanisms involved in this nucleolar function and its relationship with cell proliferation: 1) allowed the understanding of the reasons for the nucleolar changes in cancer cells and their exploitation in tumor pathology, 2) defined the importance of the inhibition of ribosome biogenesis in cancer chemotherapy and 3) focused the attention on alterations of ribosome biogenesis in the pathogenesis of cancer. This review summarizes the research milestones regarding these relevant relationships between ribosome biogenesis and cancer. The structure and function of the nucleolus will also be briefly described.

Ribosome biogenesis and cancer.

There is growing evidence indicating that the human pathological conditions characterized by an up-regulated ribosome biogenesis are at an increased risk of cancer onset. At the basis of this relationship is the close interconnection between the ribosome biogenesis and cell proliferation. Cell proliferation-stimulating factors also stimulate ribosome production, while the ribosome biogenesis rate controls the cell cycle progression. The major tumour suppressor, the p53 protein, plays an important balancing role between the ribosome biogenesis rate and the cell progression through the cell cycle phases. The perturbation of ribosome biogenesis stabilizes and activates p53, with a consequent cell cycle arrest and/or apoptotic cell death, whereas an up-regulated ribosome production down-regulates p53 expression and activity, thus facilitating neoplastic transformation. In the present review we describe the interconnection between ribosome biogenesis and cell proliferation, while highlighting the mechanisms by which quantitative changes in ribosome biogenesis may induce cancer.

The pre-existing population of 5S rRNA effects p53 stabilization during ribosome biogenesis inhibition.

Pre-ribosomal complex RPL5/RPL11/5S rRNA (5S RNP) is considered the central MDM2 inhibitory complex that control p53 stabilization during ribosome biogenesis inhibition. Despite its role is well defined, the dynamic of 5S RNP assembly still requires further characterization. In the present work, we report that MDM2 inhibition is dependent by a pre-existing population of 5S rRNA.

Therapeutic dosages of aspirin counteract the IL-6 induced pro-tumorigenic effects by slowing down the ribosome biogenesis rate.

Chronic inflammation is a risk factor for the onset of cancer and the regular use of aspirin reduces the risk of cancer development. Here we showed that therapeutic dosages of aspirin counteract the pro-tumorigenic effects of the inflammatory cytokine interleukin(IL)-6 in cancer and non-cancer cell lines, and in mouse liver in vivo. We found that therapeutic dosages of aspirin prevented IL-6 from inducing the down-regulation of p53 expression and the acquisition of the epithelial mesenchymal transition (EMT) phenotypic changes in the cell lines. This was the result of a reduction in c-Myc mRNA transcription which was responsible for a down-regulation of the ribosomal protein S6 expression which, in turn, slowed down the rRNA maturation process, thus reducing the ribosome biogenesis rate. The perturbation of ribosome biogenesis hindered the Mdm2-mediated proteasomal degradation of p53, throughout the ribosomal protein-Mdm2-p53 pathway. P53 stabilization hindered the IL-6 induction of the EMT changes. The same effects were observed in livers from mice stimulated with IL-6 and treated with aspirin. It is worth noting that aspirin down-regulated ribosome biogenesis, stabilized p53 and up-regulated E-cadherin expression in unstimulated control cells also. In conclusion, these data showed that therapeutic dosages of aspirin increase the p53-mediated tumor-suppressor activity of the cells thus being in this way able to reduce the risk of cancer onset, either or not linked to chronic inflammatory processes.

Targeted cancer therapy with ribosome biogenesis inhibitors: a real possibility?

The effects of many chemotherapeutic drugs on ribosome biogenesis have been underestimated for a long time. Indeed, many drugs currently used for cancer treatment--and which are known to either damage DNA or hinder DNA synthesis--have been shown to exert their toxic action mainly by inhibiting rRNA synthesis or maturation. Moreover, there are new drugs that have been proposed recently for cancer chemotherapy, which only hinder ribosome biogenesis without any genotoxic activity. Even though ribosome biogenesis occurs in both normal and cancer cells, whether resting or proliferating, there is evidence that the selective inhibition of ribosome biogenesis may, in some instances, result in a selective damage to neoplastic cells. The higher sensitivity of cancer cells to inhibitors of rRNA synthesis appears to be the consequence of either the loss of the mechanisms controlling the cell cycle progression or the acquisition of activating oncogene and inactivating tumor suppressor gene mutations that up-regulate the ribosome biogenesis rate. This article reviews those cancer cell characteristics on which the selective cancer cell cytotoxicity induced by the inhibitors of ribosome biogenesis is based.

The emerging role of RNA polymerase I transcription machinery in human malignancy: a clinical perspective.

Ribosome biogenesis - the complex and highly coordinated cellular process leading to the production of ribosomes - is strictly dependent on the activity of RNA polymerase I (Pol I) transcriptional machinery. Pol I activity is continually increased in proliferating cells to sustain the increased demand for ribosome production and protein synthesis, which are necessary for appropriate cell growth and division. The integrity of the process of ribosome biogenesis represents an important sensor of cellular stress: when this process is altered, a tumor suppressor response is triggered, which leads to proliferative arrest. The present review focuses on the possible implications of Pol I targeting in the treatment of human malignancies.

An innovative hyperbaric hypothermic machine perfusion protects the liver from experimental preservation injury.

PURPOSE: Hypothermic machine perfusion systems seem more effective than the current static storage to prevent cold ischemic liver injury. Thus, we test an innovative hyperbaric hypothermic machine perfusion (HHMP), which combines hyperbaric oxygenation of the preservation solution and continuous perfusion of the graft. METHODS: Rat livers were preserved with Celsior solution according to 4 different modalities: normobaric static preservation; hyperbaric static preservation at 2 atmosphere absolute (ATA); normobaric dynamic preservation, with continuous perfusion; hyperbaric dynamic preservation, with continuous perfusion at 2 ATA. After 24 h cold preservation, we assessed different parameters. RESULTS: Compared to baseline, livers preserved with the current static storage showed severe ultrastructural damage, glycogen depletion and an increased oxidative stress. Normobaric perfused livers showed improved hepatocyte ultrastructure and ameliorated glycogen stores, but they still suffered a significant oxidative damage. The addition of hyperbaric oxygen produces an extra benefit by improving oxidative injury and by inducing endothelial NO synthase (eNOS) gene expression. CONCLUSIONS: Preservation by means of the present innovative HHMP reduced the liver injury occurring after the current static cold storage by lowering glycogen depletion and oxidative damage. Interestingly, only the use of hyperbaric oxygen was associated to a blunted oxidative stress and an increased eNOS gene expression.

Ribosome biogenesis and control of cell proliferation: p53 is not alone.

Cell growth is a prerequisite for cell proliferation, and ribosome biogenesis is a limiting factor for cell growth. In mammalian cells, the tumor suppressor p53 has been shown to induce cell-cycle arrest in response to impaired ribosome biogenesis. Recently, p53-independent mechanisms of cell-cycle arrest in response to alterations of ribosome biogenesis have been described. These findings provide a rational basis for the use of drugs that specifically impact ribosome biogenesis for the treatment of cancers lacking active p53 and extend the scenario of mechanisms involved in the relationship between cell growth and cell proliferation.

Changes in ribosome biogenesis may induce cancer by down-regulating the cell tumor suppressor potential.

Many human pathological conditions, not linked to genetic alterations of oncogenes or tumor suppressors, are nevertheless associated with an increased risk of developing cancer, and some of them are characterized by quantitative and/or qualitative changes in ribosome biogenesis. Indeed, there is evidence that both an up-regulation of ribosome biogenesis, such as that occurring during the abnormal stimulation of cell growth, and intrinsic dysfunctions of ribosomes, such as those characterizing a series of inherited disorders, show an increased incidence of tumor onset. Here we discuss some recent insights into the mechanisms by which these alterations in ribosome biogenesis may facilitate tumorigenesis.

Cell proliferation of esophageal squamous epithelium in erosive and non-erosive reflux disease.

AIM: To elucidate cell proliferation in erosive reflux disease (ERD) and non-erosive reflux disease (NERD), we evaluated markers in squamous epithelial cells. METHODS: Thirty-four consecutive patients with gastroesophageal-reflux-disease-related symptoms (21 NERD and 13 ERD) were evaluated for the enrolment into the study. All patients underwent 24-h pH monitoring, standard endoscopy, and biopsy for histological evaluation. The expression of cyclins D and A was evaluated by real-time reverse transcription polymerase chain reaction (RT-PCR) from isolated epithelial cells. In all samples, analysis of the isolated cell population revealed the presence of epithelial cells only. RESULTS: Real-time RT-PCR showed that, in patients with ERD, the relative expression of cyclin D1 mRNA in esophageal epithelium was strongly decreased in comparison with NERD patients. The mean value of relative expression of cyclin D1 mRNA in NERD patients was 3.44 ± 1.9, whereas in ERD patients, it was 1.32 ± 0.87 (P = 0.011). Real-time RT-PCR showed that, in patients with ERD, relative expression of cyclin A mRNA in esophageal epithelium was decreased in comparison with that in NERD patients (2.31 ± 2.87 vs 0.66 ± 1.11). The mean bromodeoxyuridine labeling index in the NERD patients was 5.42% ± 1.68%, whereas in ERD patients, it was 4.3% ± 1.59%. CONCLUSION: We confirmed reduced epithelial proliferation in ERD compared with NERD patients, and that individuals who develop ERD are characterized by weaker epithelial cell proliferation.

Selective inhibition of rRNA transcription downregulates E2F-1: a new p53-independent mechanism linking cell growth to cell proliferation.

The tumour suppressor p53 negatively controls cell cycle progression in response to perturbed ribosome biogenesis in mammalian cells, thus coordinating growth with proliferation. Unlike mammalian cells, p53 is not involved in the growth control of proliferation in yeasts and flies. We investigated whether a p53-independent mechanism of response to inadequate ribosome biogenesis rate is also present in mammalian cells. We studied the effect of specific inhibition of rRNA synthesis on cell cycle progression in human cancer cell lines using the small-interfering RNA procedure to silence the POLR1A gene, which encodes the catalytic subunit of RNA polymerase I. We found that interference of POLR1A inhibited the synthesis of rRNA and hindered cell cycle progression in cells with inactivated p53, as a consequence of downregulation of the transcription factor E2F-1. Downregulation of E2F-1 was due to release of the ribosomal protein L11, which inactivated the E2F-1-stabilising function of the E3 ubiquitin protein ligase MDM2. These results demonstrated the existence of a p53-independent mechanism that links cell growth to cell proliferation in mammalian cells, and suggested that selective targeting of the RNA polymerase I transcription machinery might be advisable to hinder proliferation of p53-deficient cancer cells.

Location of rRNA transcription to the nucleolar components: disappearance of the fibrillar centers in nucleoli of regenerating rat hepatocytes.

The precise location of rDNA transcription to the components of mammalian cell nucleolus is still debated. This was due to the fact that all the molecules necessary for rRNA synthesis are located in two of the three components, the fibrillar centers (FCs) and the dense fibrillar component (DFC), which together with the granular component (GC) are considered to be constantly present in mammalian cell nucleoli. In the present study we demonstrated that in nucleoli of many regenerating rat hepatocytes at 15 h after partial hepatectomy the FCs were no longer present, only the DFC and the GC being detected. At this time of regeneration the rRNA transcriptional activity was three fold that of resting hepatocytes, while the synthesis of DNA was not yet significantly increased, indicating that these nucleolar changes were due to the rRNA synthesis up-regulation. The DFC appeared to be organized in numerous, small, roundish tufts of fibrils. The silver staining procedure for AgNOR proteins, which are associated with the ribosomal genes, selectively and homogeneously stained these fibrillar tufts. Immuno-gold visualization of the Upstream Binding Factor (UBF), which is associated with the promoter region and the transcribed portion of the rRNA 45S gene, demonstrated that UBF was selectively located in the fibrillar tufts. We concluded that in proliferating rat hepatocytes the increased synthesis of rRNA induced an activation of the rRNA transcription machinery located in the fibrillar centers which, by becoming associated with the ribonucleoprotein transcripts, assumed the morphological pattern of the DFC.

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.

The p53-mediated sensitivity of cancer cells to chemotherapeutic agents is conditioned by the status of the retinoblastoma protein.

Despite the well-established function of p53 in determining cell cycle arrest and/or apoptosis in response to cytostatic/cytotoxic stresses, the role of the p53 status in the response to chemotherapeutic agents in human cancers has been not clearly defined. We wondered whether this was due to the fact that the p53-mediated response to chemotherapy drugs might be conditioned by the status of the retinoblastoma protein (pRb), a downstream factor of the pathway activated by p53 stabilization, which is frequently disrupted in cancer. The dependence of p53-mediated chemosensitivity on pRb status was first investigated in a prospective study on the prognostic relevance of p53 in breast cancer patients treated with adjuvant chemotherapy (5-fluorouracil, methotrexate and cyclophosphamide). Univariate analysis of disease-free survival (DFS) indicated that the p53 status, immunohistochemically evaluated, had no predictive value if considered independently of the pRb status. However, in patients with cancer with pRb neither lost nor hyperphosphorylated, p53 was significantly associated with the prognosis and, in a multivariate analysis of DFS including the established clinical and histopathological prognostic parameters, was found to be the only factor predicting the progression of the disease. We then studied the role of pRb status in the p53-mediated response to 5-fluorouracil and methotrexate or doxorubicin treatment in three human cancer cell lines. We found that in these cells the chemosensitivity was strictly dependent on the p53 status. However, either RB1 silencing or pRb hyperphosphorylation, caused by p16(INK4a) silencing, strongly reduced the p53-mediated response to chemotherapeutic agents. These results demonstrated that: (a) the p53-mediated response to chemotherapeutic agents induces a cytostatic/cytotoxic effect only in cancers with unaltered pRb pathway; and (b) the p53 status can actually predict the clinical outcome in this group of cancer patients.

Esophageal cell proliferation in gastroesophageal reflux disease: clinical-morphological data before and after pantoprazole.

AIM: To evaluate esophageal mucosal defense mechanisms at an epithelial level to establish if pantoprazole treatment can induce ultrastructural healing and improvement in the proliferation activity of the esophageal epithelium in gastroesophageal reflux disease (GERD). METHODS: This was a single-blinded study for pH-monitoring, and histological, ultrastructural and MIB1 immunostaining evaluation. Fifty eight patients with GERD were enrolled and underwent 24 h pH-monitoring and endoscopy. Patients were treated for 12 and 24 mo with pantoprazole. Esophageal specimens were taken for histological and ultrastructural evaluation, before and after the treatment. RESULTS: With transmission electron microscopy, all patients with GERD showed ultrastructural signs of damage with dilation of intercellular spaces (DIS). After 3 mo of therapy the mean DIS values showed a significant reduction and the mean MIB1-LI values of GERD showed an increase in cell proliferation. A further 3 mo of therapy significantly increased cell proliferation only in the erosive esophagitis (ERD) group. CONCLUSION: Three months of pantoprazole therapy induced ultrastructural healing of mucosal damage in 89% and 93% of ERD and non-erosion patients, respectively. Moreover, long-term pantoprazole treatment may be helpful in increasing the capability for esophageal cell proliferation in GERD, particularly in ERD patients.

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.

Relationship between dyskerin expression and telomerase activity in human breast cancer.

The nucleolar protein dyskerin is involved in the modification of specific uridine residues to pseudouridine on ribosomal and small nuclear RNAs and in the stabilization of the telomerase RNA component (TERC). In this study we investigated for the first time the relationship between dyskerin expression and telomerase activity in a series of 61 primary breast carcinomas. We found that when dyskerin mRNA values were very low the telomerase activity was markedly reduced, independently of the expression of other important components of the telomerase complex such as telomerase reverse transcriptase (TERT). In vitro experiments showed that reduction of dyskerin expression affect telomerase activity through the reduction of TERC. Only when TERC levels were strongly reduced telomerase activity was hindered. Retroviral mediated over-expression of TERC abolished the telomerase impairment due to dyskerin knock down. In conclusion, our results indicated that, beside its effect on ribosome biogenesis, the levels of dyskerin in cancer cells modulate telomerase activity through the regulation of TERC levels, independently of TERT expression. This should be taken into consideration when utilizing TERT expression as a surrogate indicator of telomerase activity in tumour pathology.

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.

Loss of retinoblastoma tumor suppressor protein makes human breast cancer cells more sensitive to antimetabolite exposure.

PURPOSE: The RB tumor-suppressor activity may influence the therapeutic response in human breast cancers. The effect of adjuvant therapy on clinical outcome of breast cancer patients was analyzed, and the sensitivity to 5-fluorouracil (5-FU) and methotrexate was investigated in MCF-7 and HCT-116 human cancer cells, according to their RB status. EXPERIMENTAL DESIGN: RB protein (pRB) expression was prospectively evaluated by immunocytochemistry in 518 consecutive patients and its predictive value was determined according to the adjuvant therapeutic treatments. MCF-7 and HCT-116 human cancer cells silenced for RB1 expression were treated with 5-FU and methotrexate, at the same concentrations and time exposures as determined in the interstitium of breast cancers of patients treated with adjuvant chemotherapy. RESULTS: Multivariate analysis of disease-free survival, including all the established clinical and histopathologic prognostic variables, indicated that the absence of pRB expression was the only predictive factor of good clinical outcome in patients treated with standard systemic chemotherapy (cyclophosphamide, methotrexate, and 5-FU) but not in patients treated with endocrine therapy alone. 5-FU and methotrexate significantly reduced the growth rate of RB1-silenced but not of control MCF-7 and HCT-116 cells. This was likely due to the absence of a DNA damage checkpoint with accumulation of DNA double-strand breaks in RB1-silenced but not in control cells. CONCLUSIONS: The absence of pRB expression renders human breast cancer cells more sensitive to 5-FU and methotrexate and predicts a good clinical outcome for patients treated with adjuvant chemotherapy. We suggest that patients with RB-negative breast cancers should be treated with systemic chemotherapy.

Cell proliferation in breast cancer is a major determinant of clinical outcome in node-positive but not in node-negative patients.

The growth rate of a tumor cell population depends on two major factors: the percentage of proliferating cells (cell growth fraction) and the rapidity of their duplication (cell proliferation rate). The authors evaluated the prognostic and predictive value of both kinetics parameters in a large series of breast cancer patients (n=504). The cell growth fraction was determined by MIB-1 immunostaining, the cell proliferation rate by AgNOR analysis. Ki-67 LI (labeling index) and AgNOR area were significantly associated with histotype, histologic grade, tumor size, estrogen/progesterone receptor status, patient age, and lymph node involvement (P<0.005). In the entire series of patients, both kinetics variables were significantly and independently associated with the clinical outcome, but their prognostic relevance was quite different when node-negative and node-positive patients were considered separately. Although in node-positive patients Ki-67 LI and AgNOR area were the unique independent predictors of disease-free and overall survival, they were excluded by the multivariate Cox model in node-negative patients, where only tumor size and estrogen receptor status retained a significant P-value. These results show that in breast carcinoma the cell growth fraction and the cell proliferation rate have a different prognostic impact with respect to the lymph node status and are major determinants of clinical outcome in node-positive patients only. Within this subgroup, the rapidity of cell proliferation as assessed by AgNOR analysis also served as a sensitive predictor of the response to adjuvant treatments.