Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

The nucleolus is a multifunctional organelle that, in addition to its primary role in ribosome biogenesis, has emerged as a critical stress sensor and coordinator of stress response. However, the molecular nature of how nucleoli sense stress and coordinate downstream cellular consequence remains poorly understood. NF-κB signalling is a critical regulator of stress response. Many cellular stresses that disrupt nucleolar function also stimulate the NF-κB pathway. However, the role of NF-κB as a downstream effector of nucleolar stress has not yet been examined. Aspirin, a known chemopreventative agent, stimulates the NF-κB pathway to mediate apoptosis but the upstream mechanisms are unclear. In this thesis, I identified a novel nucleolar stress response pathway that culminates in activation of NF-κB signalling, and demonstrated the significance of this nucleolar pathway in the anti-tumour effects of aspirin. Using multiple approaches, I made the novel observations that disruption of the Pol I complex activates the cytoplasmic NF-κB signalling pathway. I show that multiple stress stimuli of NF-κB pathway induce degradation of the crucial Pol I complex component, rDNA transcription initiation factor IA (TIF-IA). I identified the tumour suppressor, p14ARF and the Pol I complex component, upstream binding factor (UBF) as mediators of this degradation. I revealed that inhibition of CDK4 activity lies upstream of UBF/p14ARF-facilitated TIF-IA degradation. Furthermore, using different approaches I show that blocking aspirin/CDK4i-mediated degradation of TIF-IA blocks the effects of these agents on nucleolar morphology and NF-κB signalling. Finally, I show this nucleolar stress response pathway, containing a UBF/p14ARF/TIF-IA axis, is utilized by aspirin to kill colon cancer cells. Taken together, this data presented in this thesis advances understanding of nucleolar stress response, and has therapeutic implications with regard to the anti-tumour effects of aspirin.

Contrôle de la signalisation oncogénique du mutant L858R de l'EGFR par la protéine suppresseur de tumeur p14ARF dans les adénocarcinomes pulmonaires. Le récepteur à l'EGF (EGFR) est un oncogène puissant impliqué dans le développement des cancers du poumon. Dans ces cancers, la présence de mutations activatrices de l'EGFR (majoritairement L858R et Del19) est un facteur prédictif de réponse aux agents pharmacologiques qui ciblent spécifiquement ce récepteur (EGFR-TKI). Cependant, l'association entre réponse thérapeutique et mutation est plus complexe que prévue, soulignant la nécessité d'approfondir la compréhension des mécanismes moléculaires impliqués dans développement de ces cancers. Nous avons précédemment montré que la quasi-totalité des tumeurs pulmonaires avec des mutations activatrices de l'EGFR présente une expression faible ou indétectable de la protéine suppressive de tumeur p14ARF. Ces résultats nous ont conduites à émettre l'hypothèse que l'expression de p14ARF était un frein essentiel à l'expansion clonale de ces cellules. Nous décrivons pour la première fois une relation fonctionnelle entre p14ARF et du mutant L858R de l'EGFR dans laquelle p14ARF inhibe la croissance de cellules exprimant ce mutant en induisant leur apoptose. Les effets suppresseurs de tumeur de p14ARF impliquent une fonction pro-apoptotique originale de STAT3 qui conduit à l'inhibition de l'expression de la protéine anti-apoptotique Bcl-2. De plus, nous montrons que les cellules EGFR-L858R maintiennent leur avantage de croissance en inhibant l'expression de p14ARF et la signalisation pro-apoptotique STAT3/Bcl-2 qui en découle. Nos résultats identifient également p14ARF comme une nouvelle cible transcriptionnelle de STAT3, mettant ainsi en évidence une boucle de rétrocontrôle positif entre ces deux protéines qui pourrait entretenir la signalisation pro-apoptotique médiée par STAT3. Sur la base de ces résultats, nous suggérons que la réactivation de la voie p14ARF/STAT3/Bcl-2 pourrait être une nouvelle stratégie thérapeutique dans le traitement de ces cancers.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

BH3-only Proteine, eine pro-apoptotische Untergruppe der Bcl-2 Proteinfamilie, sind zentrale Mediatoren von apoptotischen Signalen durch die Regulierung intrinsischer Apoptose-signalwege. Unsere Arbeitsgruppe hat vor kurzem gezeigt, dass Apoptose, die durch den p14ARF Tumorsuppressor induziert wird über die p53-abhängige Aktivierung des BH3-only Proteins Puma/Bbc3 vermittelt wird. Interessanterweise induziert p14ARF aber auch in p53 defizienten Zellen Zellzyklusarrest und Apoptose. Die dahinterliegenden Signalwege sind jedoch nicht bekannt. In dieser Arbeit berichten wir, dass das BH3-only Protein Bmf (Bcl-2 modifying factor) beim p14ARF-induzierten Zelltod in p53 defizienten Zellen eine wichtige Rolle spielt. Expression von p14ARF führt zu einer Induktion der PERK Kinase, daran anschließender Phosphorylierung von eIF2α sowie Aktivierung der stromabwärts liegenden Transkriptionsfaktoren ATF4 und CHOP. Diese Signalkaskade ist normalerweise Teil einer zellulären Antwort auf fehl- oder ungefaltete Proteine im Endoplasmatischen Retikulum (ER), der sogenannten ‘unfolded protein response’ (UPR), die zum einen durch verminderte Translationsinitiation und Hochregulierung von Chaperonen die Menge der fehlgefalteten Proteine reduzieren soll. Allerdings induziert p14ARF keinen ER Stress, sondern den PERK‒CHOP Signalweg. Die Transkriptionsfaktoren ATF4 und CHOP binden direkt in der Promotorregion von bmf und sind für dessen transkriptionelle Regulation verantwortlich. Unsere Daten zeigen, dass der PERK‒eIF2α‒ATF4‒CHOP Signalweg eine wesentliche Rolle bei der Induktion von Apoptose durch p14ARF spielt. Dieser Weg könnte ein Sicherungsmechanismus sein, der es den Zellen auch nach Verlust von p53 erlaubt Apoptose einzuleiten, nachdem p14ARF durch Onkogene hochreguliert wurde. / BH3-only proteins, a pro-apoptotic subgroup of the Bcl-2 family of proteins, are central mediators of apoptosis signals by regulating the intrinsic apoptosis pathway. We have recently shown, that apoptosis triggered by the p14ARF tumour suppressor protein is mediated by the p53-dependent activation of the BH3-only protein Puma/Bbc3. Nevertheless, expression of p14ARF in p53-family deficient cells is capable of inducing both cell cycle arrest and apoptosis, but the signalling pathways initiated remain elusive. Here, we report that the BH3-only protein Bmf (Bcl-2 modifying factor) is involved in cell death in p53-deficient cells triggered by p14ARF. Expression of p14ARF leads to the induction of the PERK kinase, subsequent phosphorylation of eIF2α and activation of transcription factors ATF4 and CHOP. This signalling cascade is usually part of the ‘unfolded protein response’ (UPR), which is activated upon ER stress to reduce the amount of misfolded proteins by reduction of global protein translation initiation and upregulation of chaperones. Of note, p14ARF does not induce ER stress but activates the PERK‒CHOP pathway. ATF4 and CHOP transcription factors directly bind to the promotor region of bmf and induce its transcription. These data suggest that the PERK‒eIF2α‒ATF4‒CHOP signalling pathway may play a substantial role in mediating p14ARF-triggered apoptosis. This pathway could play the role of a ‘fail-safe’ mechanism that allows cells, even after loss of p53, to undergo apoptosis induced by upregulation of p14ARF by oncogenes.

ARF, a potent tumor suppressor, positively regulates p53 by antagonizing MDM2, a negative regulator of p53, which in turn, results in either apoptosis or cell cycle arrest. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF-null or p53-null mice, results in a broadened tumor spectrum and decreased tumor latency. This evidence suggests that ARF exerts both p53-dependent and p53-independent tumor suppressor activity. However, the molecular pathway and mechanism of ARF’s p53-independent tumor suppressor activity is not understood. The antiapoptotic, metabolically regulated, transcriptional corepressor C-terminal binding protein 2 (CtBP2) has been identified as a specific target of ARF’s p53-independent tumor suppression. CtBPs are phosphoproteins with PLDLS-binding motif and NADH-binding central dehydrogenase domains. ARF interacts with CtBP1 and CtBP2 both in vitro and in vivo, and induces their proteasome-mediated degradation, resulting in p53-independent apoptosis in colon cancer cells. ARF’s ability to target CtBP2 for degradation, and its induction of p53-independent apoptosis requires an intact interaction with CtBP2, and phosphorylation at S428 of CtBP2. As targets for inhibition by ARF, CtBPs are candidate oncogenes, and their expression is elevated in a majority of human colorectal adenocarcinomas specimens in comparison to normal adjacent tissue. Relevant to its targeting by ARF, there is an inverse correlation between ARF and CtBP expression, and CtBP2 is completely absent in a subset of colorectal adenocarcinomas that retains high levels of ARF protein. CtBPs are activated under conditions of metabolic stress, such as hypoxia, and they repress epithelial and proapoptotic genes. BH3-only genes such as Bik, Bim and Bmf have been identified as mediators of ARF-induced, CtBP2-mediated p53-indpendent apoptosis. CtBP2 repressed BH3-only genes in a tissue specific manner through BKLF (Basic kruppel like factor)-binding elements. ARF regulation of BH3-only genes also required intact interaction with CtBP2. ARF antagonism of CtBP repression of Bik and other BH3-only genes may play a critical role in ARF-induced p53-independent apoptosis, and in turn, tumor suppression. To study the physiologic effect of ARF/CtBP2 interaction at the organismal level, the p19ArfL46D knock-in mice, in which the Arf/CtBP2 interaction was abrogated, was generated. Analysis of the primary cells derived from these mice, revealed that the Arf/CtBP2 interaction contributes to regulation of cell growth and cell migration. Overexpression of CtBP in human tumors, and ARF antagonism of CtBP repression of BH3-only gene expression and CtBP-mediated cell migration may therefore play a critical role in the p53-independent tumor suppressor function/s of ARF.

Animals have evolved complicated and overlapping mechanisms to guard against the development of cancer and infection by pathogenic organisms. ARF, a potent tumor suppressor, positively regulates p53 by antagonizing p53’s negative regulator, MDM2, which in turn results in either apoptosis or cell cycle arrest. ARF also has p53-independent tumor suppressor activity. The CtBP transcriptional co-repressors promote cancer cell survival and migration/invasion. CtBP senses cellular metabolism via a regulatory dehydrogenase domain, and is a target for negative regulation by ARF. ARF targets CtBP to the proteasome for degradation, which results in the up regulation of proapoptotic BH3-only proteins, and p53-independent apoptosis. CtBP inhibition by ARF also up regulates PTEN, reducing cancer cell motility, making CtBP a potential therapeutic target in human cancer. The CtBP dehydrogenase substrate 4-methylthio-2-oxobutyric acid (MTOB) can act as a CtBP inhibitor at high concentrations, and is cytotoxic to cancer cells from a wide variety of tissues. MTOB induced apoptosis was independent of p53, and correlated with the de-repression of the pro-apoptotic CtBP repression target Bik. CtBP over-expression, or Bik silencing, rescued MTOB-induced cell death. MTOB did not induce apoptosis in mouse embryonic fibroblasts (MEFs), but was increasingly cytotoxic to immortalized and transformed MEFs, suggesting that CtBP inhibition may provide a suitable therapeutic index for cancer therapy. In human colon cancer cell peritoneal xenografts, MTOB treatment decreased tumor burden, and induced tumor cell apoptosis. To verify the potential utility of CtBP as a therapeutic target in human cancer the expression of CtBP and its negative regulator ARF was studied in a series of resected human colon adenocarcinomas. CtBP and ARF levels were inversely-correlated, with elevated CtBP levels (compared with adjacent normal tissue) observed in greater than 60% of specimens, with ARF absent in nearly all specimens exhibiting elevated CtBP levels. Targeting CtBP with a small molecule like MTOB may thus represent a useful and widely applicable therapeutic strategy in human malignancies. ARF has long been known to respond to virally encoded oncogenes. Recently, p14ARF was linked to the innate immune response to non-transforming viruses in mice. Therefore a wider role for the ARF pathway in viral infection was considered. Previous studies linking p53 to multiple points of the Human Immunodeficiency Virus-1 (HIV-1) life cycle suggested that ARF may also play a role in the HIV life cycle. In this study the interdependency of ARF and HIV infection was investigated. ARF expression was determined for a variety of cell types upon HIV infection. In every case, ARF levels exhibited dynamic changes upon HIV infection-in most cases ARF levels were reduced in infected cells. The impact of ARF over-expression or silencing by RNAi on HIV infection was also examined. Consistently, p24 levels were increased with ARF overexpression, and decreased when ARF was silenced. Thus ARF and HIV modulate each other, and ARF may paradoxically play a positive role in the HIV life cycle.

BACKGROUND: The tumor suppressors p14(ARF) (ARF) and p16(INK4A) (p16) are encoded by overlapping reading frames at the CDKN2A/INK4A locus on chromosome 9p21. In human melanoma, the accumulated evidence has suggested that the predominant tumor suppressor at 9p21 is p16, not ARF. However, recent observations from melanoma-prone families and murine melanoma models suggest a p16-independent tumor suppressor role for ARF. We analyzed a group of melanoma metastases and cell lines to investigate directly whether somatic alterations to the ARF gene support its role as a p16-independent tumor suppressor in human melanoma, assuming that two alterations (genetic and/or epigenetic) would be required to inactivate a gene. METHODS: We examined the p16/ARF locus in 60 melanoma metastases from 58 patients and in 9 human melanoma cell lines using multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction (PCR) to detect deletions, methylation-specific PCR to detect promoter methylation, direct sequencing to detect mutations affecting ARF and p16, and, in a subset of 20 tumors, immunohistochemistry to determine the effect of these alterations on p16 protein expression. All statistical tests were two-sided. RESULTS: We observed two or more alterations to the ARF gene in 26/60 (43%) metastases. The p16 gene sustained two or more alterations in 13/60 (22%) metastases (P = .03). Inactivation of ARF in the presence of wild-type p16 was seen in 18/60 (30%) metastases. CONCLUSION: Genetic and epigenetic analyses of the human 9p21 locus indicate that modifications of ARF occur independently of p16 inactivation in human melanoma and suggest that ARF is more frequently inactivated than p16.