Elisa Brighenti

The balance between rRNA and ribosomal protein synthesis up- and downregulates the tumour suppressor p53 in mammalian cells

Data on the relationship between ribosome biogenesis and p53 function indicate that the tumour suppressor can be activated by either nucleolar disruption or ribosomal protein defects. However, there is increasing evidence that the induction of p53 does not always require these severe cellular changes, and data are still lacking on a possible role of ribosome biogenesis in the downregulation of p53. Here, we studied the effect of the up- and downregulation of the rRNA transcription rate on p53 induction in mammalian cells. We found that a downregulation of rRNA synthesis, induced by silencing the POLR1A gene coding for the RNA polymerase I catalytic subunit, stabilised p53 without altering the nucleolar integrity in human cancer cells. p53 stabilisation was due to the inactivation of the MDM2-mediated p53 degradation by the binding of ribosomal proteins no longer used for ribosome building. p53 stabilisation did not occur when rRNA synthesis downregulation was associated with a contemporary reduction of protein synthesis. Furthermore, we demonstrated that in three different experimental models characterised by an upregulation of rRNA synthesis, cancer cells treated with insulin or exposed to the insulin-like growth factor 1, rat liver stimulated by cortisol and regenerating rat liver after partial hepatectomy, the p53 protein level was reduced due to a lowered ribosomal protein availability for MDM2 binding. It is worth noting that the upregulation of rRNA synthesis was responsible for a decreased p53-mediated response to cytotoxic stresses. These findings demonstrated that the balance between rRNA and ribosomal protein synthesis controls the function of p53 in mammalian cells, that p53 can be induced without the occurrence of severe changes of the cellular components controlling ribosome biogenesis, and that conditions characterised by an upregulated rRNA synthesis are associated with a reduced p53 response.

High prevalence of retinoblastoma protein loss in triple-negative breast cancers and its association with a good prognosis in patients treated with adjuvant chemotherapy

BACKGROUND Triple-negative breast cancer (TNBC) is an aggressive disease, nevertheless exhibiting a high response rate to chemotherapy. Since the retinoblastoma protein (pRb) loss confers a high sensitivity to chemotherapy regimens, we evaluated the prevalence of pRb loss in TNBCs and its relevance on the clinical outcome of patients treated with adjuvant chemotherapy. PATIENTS AND METHODS pRb status was prospectively evaluated by immunocytochemistry in 518 consecutive patients with complete receptor information. The predictive value of pRb status in TNBCs was determined according to the adjuvant therapeutic treatments. RESULTS Fifty-three tumors were identified as TNBCs. The prevalence of pRb loss was significantly higher in TNBCs than in the other cancer subtypes. All patients with TNBCs lacking pRb and treated with systemic chemotherapy (cyclophosphamide, methotrexate and 5-fluorouracil) were disease free at a medium follow-up time of 109 months, whereas the clinical outcome of those expressing pRb was significantly poorer (P = 0.008). Analysis of disease-free survival including the established anatomo-clinical prognostic parameters indicated pRb loss as the only significant predictive factor. CONCLUSIONS pRb loss is much more frequent in TNBCs than in the other breast cancer subtypes. Patients with TNBCs lacking pRb had a very favorable clinical outcome if treated with conventional adjuvant chemotherapy.

Interleukin 6 downregulates p53 expression and activity by stimulating ribosome biogenesis: a new pathway connecting inflammation to cancer

Chronic inflammation is an established risk factor for the onset of cancer, and the inflammatory cytokine IL-6 has a role in tumorigenesis by enhancing proliferation and hindering apoptosis. As factors stimulating proliferation also downregulate p53 expression by enhancing ribosome biogenesis, we hypothesized that IL-6 may cause similar changes in inflamed tissues, thus activating a mechanism that favors neoplastic transformation. Here, we showed that IL-6 downregulated the expression and activity of p53 in transformed and untransformed human cell lines. This was the consequence of IL-6-dependent stimulation of c-MYC mRNA translation, which was responsible for the upregulation of rRNA transcription. The enhanced rRNA transcription stimulated the MDM2-mediated proteasomal degradation of p53, by reducing the availability of ribosome proteins for MDM2 binding. The p53 downregulation induced the acquisition of cellular phenotypic changes characteristic of epithelial–mesenchymal transition, such as a reduced level of E-cadherin expression, increased cell invasiveness and a decreased response to cytotoxic stresses. We found that these changes also occurred in colon epithelial cells of patients with ulcerative colitis, a very representative example of chronic inflammation at high risk for tumor development. Histochemical and immunohistochemical analysis of colon biopsy samples showed an upregulation of ribosome biogenesis, a reduced expression of p53, together with a focal reduction or absence of E-cadherin expression in chronic colitis in comparison with normal mucosa samples. These changes disappeared after treatment with anti-inflammatory drugs. Taken together, the present results highlight a new mechanism that may link chronic inflammation to cancer, based on p53 downregulation, which is activated by the enhancement of rRNA transcription upon IL-6 exposure.

Direct relationship between the level of p53 stabilization induced by rRNA synthesis-inhibiting drugs and the cell ribosome biogenesis rate.

Many drugs currently used in chemotherapy work by hindering the process of ribosome biogenesis. In tumors with functional p53, the inhibition of ribosome biogenesis may contribute to the efficacy of this treatment by inducing p53 stabilization. As the level of stabilized p53 is critical for the induction of cytotoxic effects, it seems useful to highlight those cancer cell characteristics that can predict the degree of p53 stabilization following the treatment with inhibitors of ribosome biogenesis. In the present study we exposed a series of p53 wild-type human cancer cell lines to drugs such as actinomycin D (ActD), doxorubicin, 5-fluorouracil and CX-5461, which hinder ribosomal RNA (rRNA) synthesis. We found that the amount of stabilized p53 was directly related to the level of ribosome biogenesis in cells before the drug treatment. This was due to different levels of inactivation of the ribosomal proteins–MDM2 pathway of p53 digestion. Inhibition of rRNA synthesis always caused cell cycle arrest, independent of the ribosome biogenesis rate of the cells, whereas apoptosis occurred only in cells with a high rDNA transcription rate. The level of p53 stabilization induced by drugs acting in different ways from the inhibition of ribosome biogenesis, such as hydroxyurea (HU) and nutlin-3, was independent of the level of ribosome biogenesis in cells and always lower than that occurring after the inhibition of rRNA synthesis. Interestingly, in cells with a low ribosome biogenesis rate, the combined treatment with ActD and HU exerted an additive effect on p53 stabilization. These results indicated that (i) drugs inhibiting ribosome biogenesis may be highly effective in p53 wild-type cancers with a high ribosome biogenesis rate, as they induce apoptotic cell death, and (ii) the combination of drugs capable of stabilizing p53 through different mechanisms may be useful for treating cancers with a low ribosome biogenesis rate.

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 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. Copyright

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.

The inhibition of lactate dehydrogenase A hinders the transcription of histone 2B gene independently from the block of aerobic glycolysis

Most cancer cells use aerobic glycolysis to fuel their growth and many efforts are made to selectively block this metabolic pathway in cancer cells by inhibiting lactate dehydrogenase A (LDHA). However, LDHA is a moonlighting protein which exerts functions also in the nucleus as a factor associated to transcriptional complexes. Here we found that two small molecules which inhibit the enzymatic activity of LDHA hinder the transcription of histone 2B gene independently from the block of aerobic glycolysis. Moreover, we observed that silencing this gene reduces cell replication, hence suggesting that the inhibition of LDHA can also affect the proliferation of normal non-glycolysing dividing cells.

Abstract 4694: The small molecule CHK1/CHK2 inhibitor PF-0477736 (Pfizer) demonstrates single agent activity in diffuse large B-cell lymphoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Few data are available on the role of CHK inhibitors in Diffuse Large B cell Lymphoma (DLBCL). The checkpoint kinases 1 (CHK1) and 2 (CHK2) are serine-threonine kinases involved in the DNA damage response pathway. CHK inhibition enhances the efficacy of DNA damaging agents in a variety of tumors, including p53 deficient cells, that rely on the G2/M checkpoint, having a dysfunctional G1 checkpoint. DLBCL with dysfunctional p53 axis (harboring p53 mutations and/or CDKN2A loss) have been recently shown to have a dismal outcome. In this study we report the activity profile of the CHK1/2 inhibitor PF-0477736 (Pfizer) in a panel of B cell lymphoma cell lines and primary cells, and explore its mechanisms of action. Three Germinal center B cell (GCB) DLBCL derived cell lines (SUDHL-4, SHDHL-6, BJAB), 3 Activated B cell (ABC) DLBCL (HBL-1, U2932, TMD8), 2 mantle cell lymphoma (Mino, SP-53), and the Hodgkin Lymphoma cell line KM-H2 were first screened for p53 and CDKN2A mutations and deletions. P53 mutations were detected in the following cell lines: HBL-1, U2932, SUDHL-6, BJAB, Mino, SP-53. TMD8 was p53 wild-type but with an homozygous deletion of CDKN2A. Of note SUDHL-4 and KM-H2 were p53 wild type, with no deletion of CDKN2A. To assess the effect of PF-0477736 on cell proliferation, cells were incubated with increasing concentrations of PF-0477736 (from 5 to 2000 nM) for 24, 48 and 72 hours (hrs), and cell viability assessed by WST-1 assay (Roche). A significant growth inhibition was evident after 48 hrs, in all cell lines, excluding SUDHL-4 and KM-H2 that were resistant (IC50 8300 and 6800 nM at 48 hrs, respectively). The BJAB cell line showed the highest sensitivity (IC50 10 nM at 24 hrs). The IC50 ranged from 140 to 230 nM at 48 hrs in the other sensitive cell lines. Using Annexin V- propidium iodide staining, we found that PF-0477736 25-500 nM induced cell death by apoptosis in a time and dose dependent manner. Of note PF-0477736 100-1000 nM demonstrated activity also in primary DLBCL cells. We found no correlations between baseline levels of CHK1/2 activation and outcome. In the sensitive cell lines inhibition of the downstream target CDC25c ser216 phosphorylation coupled with a marked increase in levels of the DNA damage marker γH2AX was observed by western blot as soon as after 24 hrs of incubation with concentrations equal to the IC50 (25 - 250 nM). PF-0477736 at the dose of 50-100 nM synergistically enhanced the efficacy of Doxorubicin (0.1 to 1 μM) at 24 hrs. These data suggest that PF-0477736 has single agent activity and synergizes with chemotherapy in DLBCL. The drug shows high single agent activity in the subset of DLBCL with genomic lesions of the p53 pathway, that are resistant to conventional chemotherapy and associated with dismal outcome, providing the rationale for further clinical investigation of PF-0477736 in DLBCL alone or in combination with chemotherapy. PF-0477736 was provided by Pfizer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4694. doi:1538-7445.AM2012-4694

Abstract 1772: Inhibition of DNA repair by the small molecule Chk1/Chk2 inhibitor PF-0477736 (Pfizer) in B-acute lymphoblastic leukemia (ALL)

Checkpoint kinase 1 (Chk1) and 2 (Chk2) are serine/threonine kinases involved in the DNA damage response and in the regulation of cell cycle progression at S-G 2 phase. Based on the potential utility of DNA checkpoint inhibition in enhancing tumor cell death, we investigated the preclinical activity of PF-0477736 (Pfizer), a potent and selective Chk1/2 inhibitor, in B-ALL and we determined potential biomarkers of functional inhibition. BCR-ABL1-positive (BV-173, SUPB-15) and negative cell lines (NALM6, NALM19, REH) were incubated with increasing concentrations of PF-0477736 (0.005-2 μM) for 24, 48 and 72 hours. PF-0477736 inhibition of Chk1 resulted in dose and time-dependent cytotoxicity with IC50 at 24 hours of 0.1-1.5 µM, with BV-173 being the most sensitive, while NALM6 the most resistant (WST-1 assay, Roche). Consistent with the viability results, Annexin V/Propidium Iodide staining analysis showed a significant increase of apoptosis at 24 and 48 hours in all cell lines. Western Blot analysis showed that PF-0477736 decreases the inhibitory phosphorylation of Cdc25c Ser 216 which is inactivated by Chk1 to prevent mitotic entry, and increases phosphorylation of γH2AX Ser 139 , a marker of DNA damage. The efficacy of PF-0477736 was thereafter confirmed in primary blast cells from 11 B-ALL patients. Based on the viability results, three groups of patients were identified: very good responders, 46% (IC50 ranged from 0.1-0.5 µM at 24 hours); good responders, 36% (IC50 ranged from 0.6-1 µM at 24 hours); poor responders, 18% (IC50 > 1 µM at 24 hours). Since multiple studies reported a higher activity of PF-0477736 against p53-defective cancer cells, we performed a mutational screening of all coding exons of p53. All cell lines and primary leukemia blasts lacked mutations, demonstrating that in B-ALL the sensitivity to PF-0477736 is independent of p53 status. Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated cell lines and their untreated counterparts. Consistent with a specific Chk1-mechanism of action, treatment resulted in differential expression (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1772. doi:1538-7445.AM2012-1772