Thorsten Stiewe

Role of the p53-homologue p73 in E2F1-induced apoptosis

Most human cancers harbour aberrations of cell-cycle control, which result in deregulated activity of the E2F transcription factors with concomitant enhanced cell-cycle progression. Oncogenic signalling by E2F1 has recently been linked to stabilization and activation of the tumour suppressor p53 (refs 1,3,4). The p73 protein shares substantial sequence homology and functional similarity with p53 (refs 5–7 ). Hence, several previously considered p53-independent cellular activities may be attributable to p73. Here we provide evidence that E2F1 directly activates transcription of TP73, leading to activation of p53-responsive target genes and apoptosis. Disruption of p73 function by a tumour-derived p53 mutant reduced E2F1-mediated apoptosis. Thus, p73 activation by deregulated E2F1 activity might constitute a p53-independent, anti-tumorigenic safeguard mechanism.

The p53 family in differentiation and tumorigenesis

The role of p53 as a tumour suppressor is generally attributed to its ability to stop the proliferation of precancerous cells by inducing cell-cycle arrest or apoptosis. The relatives and evolutionary predecessors of p53 — p63 and p73 — share the tumour-suppressor activity of p53 to some extent, but also have essential functions in embryonic development and differentiation control. Recent evidence indicates that these ancestral functions in differentiation control contribute to the tumour-suppressor activity that the p53 family is famous for.

Role of p73 in malignancy: tumor suppressor or oncogene?

The recently identified p53 family member, p73, shows substantial structural and functional homology with p53. However, despite the established role of p53 as a proto-type tumor suppressor, a similar function of p73 in malignancy is questionable. Overexpression of p73 can activate typical p53-responsive genes, and activation of p73 has been implicated in apoptotic cell death induced by aberrant cell proliferation and some forms of DNA-damage. These data together with the localization of TP73 on chromosome 1p36, a region frequently deleted in a variety of human tumors, led to the hypothesis that p73 has tumor suppressor activity just like p53. However, unlike p53−/− mice, p73 knockout mice do not develop tumors. Extensive studies on primary tumor tissues have revealed overexpression of wild-type p73 in the absence of p73 mutations instead, suggesting that p73 may augment, rather than inhibit tumor development. In contrast to p53, differential splicing of the TP73 gene locus gives rise to a complex pattern of interacting p73 isoforms with antagonistic functions. In fact, induction of apoptosis by increased levels of p73 can be blocked by both p53 mutants and the N-terminally truncated p73 isoforms, which were recently shown to possess oncogenic potential. In the light of these new findings the contradictory role of p73 in malignancy will be discussed.

A Senescence-Inflammatory Switch from Cancer-Inhibitory to Cancer-Promoting Mechanism

Senescence, perceived as a cancer barrier, is paradoxically associated with inflammation, which promotes tumorigenesis. Here, we characterize a distinct low-grade inflammatory process in stressed epithelium that is related to para-inflammation; this process either represses or promotes tumorigenesis, depending on p53 activity. Csnk1a1 (CKIα) downregulation induces a senescence-associated inflammatory response (SIR) with growth arrest in colorectal tumors, which loses its growth control capacity in the absence of p53 and instead, accelerates growth and invasiveness. Corresponding processes occur in CKIα-deleted intestinal organoids, assuming tumorigenic transformation properties ex vivo, upon p53 loss. Treatment of organoids and mice with anti-inflammatory agents suppresses the SIR and prevents p53-deficient organoid transformation and mouse carcinogenesis. SIR/para-inflammation suppression may therefore constitute a key mechanism in the anticarcinogenic effects of nonsteroidal anti-inflammatory drugs.

Adaptation of cancer cells from different entities to the MDM2 inhibitor nutlin-3 results in the emergence of p53-mutated multi-drug-resistant cancer cells

Six p53 wild-type cancer cell lines from infrequently p53-mutated entities (neuroblastoma, rhabdomyosarcoma, and melanoma) were continuously exposed to increasing concentrations of the murine double minute 2 inhibitor nutlin-3, resulting in the emergence of nutlin-3-resistant, p53-mutated sublines displaying a multi-drug resistance phenotype. Only 2 out of 28 sublines adapted to various cytotoxic drugs harboured p53 mutations. Nutlin-3-adapted UKF-NB-3 cells (UKF-NB-3rNutlin10 μM, harbouring a G245C mutation) were also radiation resistant. Analysis of UKF-NB-3 and UKF-NB-3rNutlin10 μM cells by RNA interference experiments and lentiviral transduction of wild-type p53 into p53-mutated UKF-NB-3rNutlin10 μM cells revealed that the loss of p53 function contributes to the multi-drug resistance of UKF-NB-3rNutlin10 μM cells. Bioinformatics PANTHER pathway analysis based on microarray measurements of mRNA abundance indicated a substantial overlap in the signalling pathways differentially regulated between UKF-NB-3rNutlin10 μM and UKF-NB-3 and between UKF-NB-3 and its cisplatin-, doxorubicin-, or vincristine-resistant sublines. Repeated nutlin-3 adaptation of neuroblastoma cells resulted in sublines harbouring various p53 mutations with high frequency. A p53 wild-type single cell-derived UKF-NB-3 clone was adapted to nutlin-3 in independent experiments. Eight out of ten resulting sublines were p53-mutated harbouring six different p53 mutations. This indicates that nutlin-3 induces de novo p53 mutations not initially present in the original cell population. Therefore, nutlin-3-treated cancer patients should be carefully monitored for the emergence of p53-mutated, multi-drug-resistant cells.

Quantitative TP73 Transcript Analysis in Hepatocellular Carcinomas

Purpose: The p53 family member p73 displays significant homology to p53, but data from primary tumors demonstrating increased expression levels of p73 in the absence of any gene mutations argue against a classical tumor suppressor function. A detailed analysis of the p73 protein in tumor tissues has revealed expression of two classes of p73 isoforms. Whereas the proapoptotic, full-length, transactivation-competent p73 protein (TA-p73) has a putative tumor suppressor activity similar to p53, the antiapoptotic, NH2-terminally truncated, transactivation-deficient p73 protein (ΔTA-p73) has been shown to possess oncogenic activity. The oncogenic proteins can be generated by the following two different mechanisms: (a) aberrant splicing (p73Δex2, p73Δex2/3, ΔN′-p73) and (b) alternative promoter usage of a second intronic promoter (ΔN-p73). The purpose of our study was to elucidate the origin of ΔTA-p73 isoforms in hepatocellular carcinomas. Experimental Design: We analyzed the underlying mechanisms of p73 overexpression in cancer cells by quantification of p73 transcripts from 10 hepatocellular carcinoma patients using isoform-specific real-time reverse transcription-PCR. Results: Our data demonstrate that only aberrantly spliced ΔTA-p73 transcripts from the TA promoter show significantly increased expression levels in the tumor whereas the ΔN-p73 transcript generated from the second promoter is not significantly up-regulated. Conclusions: Although we only analyzed 10 patient samples the results strongly suggest that the elevated activity of the first promoter (TA promoter) accounts for high-level expression of both full-length TA-p73 and aberrantly spliced ΔTA-p73 isoforms in hepatocellular carcinoma tissues.

E1A is sufficient by itself to induce apoptosis independent of p53 and other adenoviral gene products.

Induction of apoptosis seems to be a key function in maintaining normal cell growth by exerting negative controls on cell proliferation and suppressing tumorigenesis. The adenovirus E1A oncogene shows both cell cycle progression and apoptotic functions. To understand the mechanism of E1A-induced apoptosis, the apoptotic function of E1A 13S was investigated in p53-null cells. We show here that E1A is sufficient by itself to induce substantial apoptosis independent of p53 and other adenoviral genes. The apoptotic function of E1A is accompanied by processing of caspase-3 and cleavage of poly(ADP-ribose)-polymerase. Cell death is significantly blocked by the caspase inhibitor zVAD-fmk and when coexpressed with E1B19K, Bcl-2 or the retinoblastoma protein (RB). Analyses of E1A mutants indicated that the apoptotic activity of E1A correlates closely with the ability to bind the key regulators of E2F1-induced apoptosis, p300 and RB. Finally, in vivo relevance of down-modulation of p53-independent apoptosis for efficient transformation is demonstrated.

DNA Binding Cooperativity of p53 Modulates the Decision between Cell-Cycle Arrest and Apoptosis

p53 limits the proliferation of precancerous cells by inducing cell-cycle arrest or apoptosis. How the decision between survival and death is made at the level of p53 binding to target promoters remains unclear. Using cancer cell lines, we show that the cooperative nature of DNA binding extends the binding spectrum of p53 to degenerate response elements in proapoptotic genes. Mutational inactivation of cooperativity therefore does not compromise the cell-cycle arrest response but strongly reduces binding of p53 to multiple proapoptotic gene promoters (BAX, PUMA, NOXA, CASP1). Vice versa, engineered mutants with increased cooperativity show enhanced binding to proapoptotic genes, which shifts the cellular response to cell death. Furthermore, the cooperativity of DNA binding determines the extent of apoptosis in response to DNA damage. Because mutations, which impair cooperativity, are genetically linked to cancer susceptibility in patients, DNA binding cooperativity contributes to p53s tumor suppressor activity.

KINK-1, a Novel Small-Molecule Inhibitor of IKKβ, and the Susceptibility of Melanoma Cells to Antitumoral Treatment

BACKGROUND Increasing the efficacy of chemotherapeutics by reducing chemoresistance may be a useful strategy in cancer therapy. Constitutive activation of nuclear factor-kappa B (NF-kappaB) is a hallmark of various cancers, including melanoma, which is almost universally resistant to chemotherapy. NF-kappaB is regulated by inhibitory kappaB (IkappaB) proteins, which are in turn phosphorylated by the IkappaB kinase (IKK) complex. METHODS The effect on NF-kappaB activity of a novel small-molecule inhibitor of the beta subunit of IKK (KINK-1; kinase inhibitor of nuclear factor-kappaB-1) was assessed by measuring phosphorylation of the alpha subunit of IkappaB by immunoblotting, DNA binding by electrophoretic mobility shift assays, and nuclear translocation of NF-kappaB using immunofluorescence. Regulation of NF-kappaB-dependent gene expression was determined by microarray analysis, real-time and semiquantitative reverse transcription polymerase chain reaction (RT-PCR), and Western blot analyses. The effects of KINK-1 (alone and in combination with cytostatic agents) on melanoma cells were characterized by assessing proliferation, soft agar colony formation, and markers of apoptosis. The antitumoral efficacy of KINK-1 in combination with the cytostatic agents doxorubicin or camptothecin (all injected intraperitoneally) was tested in vivo by measuring lung weight and counting metastases in C57BL6 mice (groups of six) bearing metastases of melanoma cells. All statistical tests were two-sided. Results KINK-1 strongly suppressed both constitutive and induced NF-kappaB activity in melanoma cells. It reduced the expression of NF-kappaB-dependent gene products that regulate proliferation, cytokine production, and antiapoptotic responses but exhibited little antiproliferative or proapoptotic activity at the cellular level. However, KINK-1 markedly increased the activities of some cytostatic agents in vitro and abrogated doxorubicin-induced NF-kappaB activation. Combined treatment of C57BL6 mice that had been injected with melanoma cells with KINK-1 and doxorubicin or camptothecin reduced metastases and pulmonary tumor mass compared with either treatment alone (mean lung weight 19 days after injection of melanoma cells of mice treated with 3 mg/kg KINK-1 alone, 1 mg/kg doxorubicin alone, and 1 mg/kg doxorubicin plus 3 mg/kg KINK-1 = 260 mg, 95% confidence interval (CI) = 216 to 305 mg; 268 mg, 95% CI = 224 to 313 mg; and 181 mg, 95% CI = 171 to 192 mg, respectively, P < .001 from t tests comparing mean lung weight of double-treated mice to that in mice treated with either compound alone). CONCLUSION Inhibition of constitutive and induced IKKbeta-activity through treatment with KINK-1 might increase tumor susceptibility to chemotherapy.

C-terminal diversity within the p53 family accounts for differences in DNA binding and transcriptional activity

The p53 family is known as a family of transcription factors with functions in tumor suppression and development. Whereas the central DNA-binding domain is highly conserved among the three family members p53, p63 and p73, the C-terminal domains (CTDs) are diverse and subject to alternative splicing and post-translational modification. Here we demonstrate that the CTDs strongly influence DNA binding and transcriptional activity: while p53 and the p73 isoform p73γ have basic CTDs and form weak sequence-specific protein–DNA complexes, the major p73 isoforms have neutral CTDs and bind DNA strongly. A basic CTD has been previously shown to enable sliding along the DNA backbone and to facilitate the search for binding sites in the complex genome. Our experiments, however, reveal that a basic CTD also reduces protein–DNA complex stability, intranuclear mobility, promoter occupancy in vivo, target gene activation and induction of cell cycle arrest or apoptosis. A basic CTD therefore provides both positive and negative regulatory functions presumably to enable rapid switching of protein activity in response to stress. The different DNA-binding characteristics of the p53 family members could therefore reflect their predominant role in the cellular stress response (p53) or developmental processes (p73).