Compromised P53 Function Research Articles

TP53 mutations in head and neck cancer cells determine the Warburg phenotypic switch creating metabolic vulnerabilities and therapeutic opportunities for stratified therapies.

Patients with mutated TP53 have been identified as having comparatively poor outcomes compared to those retaining wild-type p53 in many cancers, including squamous cell carcinomas of the head and neck (SCCHN). We have examined the role of p53 in regulation of metabolism in SCCHN cells and find that loss of p53 function determines the Warburg effect in these cells. Moreover, this metabolic adaptation to loss of p53 function creates an Achilles’ heel for tumour cells that can be exploited for potential therapeutic benefit. Specifically, cells lacking normal wild-type p53 function, whether through mutation or RNAi-mediated downregulation, display a lack of metabolic flexibility, becoming more dependent on glycolysis and losing the ability to increase energy production from oxidative phosphorylation. Thus, cells that have compromised p53 function can be sensitised to ionizing radiation by pre-treatment with a glycolytic inhibitor. These results demonstrate the deterministic role of p53 in regulating energy metabolism and provide proof of principle evidence for an opportunity for patient stratification based on p53 status that can be exploited therapeutically using current standard of care treatment with ionising radiation.

CSAG1 maintains the integrity of the mitotic centrosome in cells with defective p53.

Centrosomes focus microtubules to promote mitotic spindle bipolarity, a critical requirement for balanced chromosome segregation. Comprehensive understanding of centrosome function and regulation requires a complete inventory of components. While many centrosome components have been identified, others yet remain undiscovered. We have used a bioinformatics approach, based on 'guilt by association' expression to identify novel mitotic components among the large group of predicted human proteins that have yet to be functionally characterized. Here, we identify chondrosarcoma-associated gene 1 protein (CSAG1) in maintaining centrosome integrity during mitosis. Depletion of CSAG1 disrupts centrosomes and leads to multipolar spindles, particularly in cells with compromised p53 function. Thus, CSAG1 may reflect a class of 'mitotic addiction' genes, whose expression is more essential in transformed cells.

The Zn-finger domain of MdmX suppresses cancer progression by promoting genome stability in p53-mutant cells.

The MDMX (MDM4) oncogene is amplified or overexpressed in a significant percentage of human tumors. MDMX is thought to function as an oncoprotein by binding p53 tumor suppressor protein to inhibit p53-mediated transcription, and by complexing with MDM2 oncoprotein to promote MDM2-mediated degradation of p53. However, down-regulation or loss of functional MDMX has also been observed in a variety of human tumors that are mutated for p53, often correlating with more aggressive cancers and a worse patient prognosis. We have previously reported that endogenous levels of MdmX can suppress proliferation and promote pseudo-bipolar mitosis in primary and tumor cells derived from p53-deficient mice, and that MdmX-p53 double deficient mice succumb to spontaneously formed tumors more rapidly than p53-deficient mice. These results suggest that the MdmX oncoprotein may act as a tumor-suppressor in cancers with compromised p53 function. By using orthotopic transplantation and lung colonization assays in mice we now establish a p53-independent anti-oncogenic role for MdmX in tumor progression. We also demonstrate that the roles of MdmX in genome stability and in proliferation are two distinct functions encoded by the separate MdmX protein domains. The central Zn-finger domain suppresses multipolar mitosis and chromosome loss, whereas the carboxy-terminal RING domain suppresses proliferation of p53-deficient cells. Furthermore, we determine that it is the maintenance of genome stability that underlies MdmX role in suppression of tumorigenesis in hyperploid p53 mutant tumors. Our results offer a rationale for the increased metastatic potential of p53 mutant human cancers with aberrant MdmX function and provide a caveat for the application of anti-MdmX treatment of tumors with compromised p53 activity.

Abstract C250: Concurrent targeting Bcl-2/Bcl-xL and MDM2 as a new therapeutic strategy for acute leukemia.

Abstract Acquired resistance of tumor cells to anticancer drugs is a major cause of cancer therapy failure in the clinic. In this study, we have investigated the acquired resistance mechanisms in vitro and in vivo for two novel anticancer drugs currently in clinical trials, a Bcl-2/Bcl-xL inhibitor ABT-263 and a MDM2-p53 interaction inhibitor MI-77301/SAR405838. In adult relapsed ALL (RS4;11) xenograft model, both ABT-263 and SAR405838 induced rapid and complete tumor regression but tumors regrew shortly after treatment cessation for both drugs. In pediatric AML (MV4;11) xenograft model, ABT-263 induced tumor growth inhibition and SAR405838 induced rapid and complete tumor regression but tumors also regrew after treatment stopped. Analysis of RS4;11 and MV4;11 resistant tumors to both drugs show that tumor cells evade apoptosis induction by the MDM2 inhibitor by mutation of p53 gene or compromised p53 function, whereas in RS4;11 tumor cells lose their sensitivity to the Bcl-2/Bcl-xL inhibitor through down-regulation of pro-apoptotic Bcl-2 family member BAX. Interestingly, RS4;11 tumors which became resistant to SAR405838, retained sensitivity to ABT-263 and rapid tumor regression was observed upon treatment with ABT-263. Similarly, in both RS4;11 and MV4;11 models, tumor cells which became highly resistant to ABT-263 were still very sensitive to SAR405838 and tumors resistant to the treatment of ABT-263 underwent rapid and complete tumor regression upon treatment with SAR405838. Furthermore, in both leukemia xenograft mouse models, sequential treatment with ABT-263 followed by SAR405838 achieved rapid and complete tumor regression with extended tumor free survival compared to the single agents. Additionally, the combination of SAR405838 and ABT-263 achieved a higher degree of long-term tumor regression than either agent alone. Our study provides new insights into the resistant mechanisms for both Bcl-2/Bcl-xL and MDM2 inhibitors in acute leukemia. Importantly, our preclinical data suggest that the combination and sequential treatment of these two distinct classes of novel apoptosis-inducing agents should be explored as an innovative treatment strategy for acute leukemia in the clinic with the goal to overcome acquired drug resistance and to improve the long-term clinical outcome for acute leukemia patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C250. Citation Format: C. Gianna Hoffman-Luca, Donna McEachern, Daniel Ziazadeh, Yu-Jun Zhao, Wei Sun, Laurent Debussche, Shaomeng Wang. Concurrent targeting Bcl-2/Bcl-xL and MDM2 as a new therapeutic strategy for acute leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C250.

A role for p53 in the response of bystander cells to receipt of medium borne signals from irradiated cells

Purpose: A number of contradictory studies have reported a role or not for p53 (protein 53) in the production of radiation-induced bystander effects. Most of these studies looked at a range of cell lines with normal or compromised p53 function.Methods: In this study, Human Colon Tumour line 116 (HCT 116) cells with confirmed wild type p53 function and a corresponding p53 null HCT 116 line were used to test for bystander signal production and response to bystander signals in a mix/match protocol using the medium transfer technique.Results: The results showed that both the null cells and the wild type cells produced bystander signals. However, only the p53 wild type cells responded to signals from either cell line. The Human Papilloma Virus transfected keratinocyte line G (HPV-G) reporter cell line used routinely in our laboratory was used to confirm that the null cells were producing signals.Conclusions: We conclude that in this system the p53 pathway is involved in response of cells to bystander signals but that signals can be produced by cells which do not have functional p53. If these results apply in vivo, they could be important in radiotherapy where tumours may have compromised p53 function but surrounding (and distant) normal tissue may have wild type functional p53.

UV-inducible base excision repair of oxidative damaged DNA in human cells

Methylene blue (MB) acts as a photosensitizer and after excitation by visible light (VL) produces reactive oxygen species that result in oxidatively damaged DNA. (MB + VL) produces predominantly 8-hydroxyguanine as well as other single base modifications in DNA that are repaired by base excision repair (BER). We have used a recombinant non-replicating human adenovirus, Ad5HCMVlacZ, which expresses the beta-galactosidase (beta-gal) reporter gene, to examine the role of the p53 tumor suppressor in constitutive and inducible BER of MB + VL-damaged DNA in human cells. Host cell reactivation (HCR) of beta-gal activity for MB + VL-treated Ad5HCMVlacZ was examined in normal human fibroblasts and several transformed and tumor cell lines with compromised p53 function using both non-treated cells and cells pretreated with ultraviolet light of 200-280 nm wavelength (UVC). Constitutive HCR of the MB + VL-treated reporter gene in untreated cells did not correlate with wild-type p53 expression levels, suggesting that factors other than p53 expression levels can influence constitutive BER of the reporter gene. UVC pre-treatment of the normal fibroblast strains resulted in an enhanced HCR of the MB + VL-treated reporter gene and a concomitant increase in the expression of p53, suggesting that p53 may be involved in UV-inducible BER in normal human fibroblasts. In contrast, p53 expression did not correlate with HCR values for the p53-compromised cells in UVC-pre-treated cells. In particular, the SKOV-3, LFS 087 and NF-E6 cells showed no up-regulation of p53 expression following UVC, and yet these cells showed significant enhancement of HCR following UVC pre-treatment. These results indicate that BER of MB + VL-damaged DNA is inducible in human cells by pre-UVC treatment and that the enhancement in BER may result from both p53-dependent and p53-independent mechanisms.

Induction of Apoptosis by Vinblastine via c-Jun Autoamplification and p53-Independent Down-Regulation of p21WAF1/CIP1

Vinblastine treatment in all cell lines examined causes a robust increase in c-Jun protein expression and phosphorylation and a corresponding increase in activator protein-1 (AP-1) transcriptional activity. We show in KB-3 carcinoma cells that this is due to a strong autoamplification loop involving the proximal AP-1 site in the c-Jun promoter, resulting in highly increased c-Jun mRNA and c-Jun protein. Inhibitors of RNA transcription and protein translation blocked both vinblastine-induced c-Jun expression and apoptotic cell death, suggesting that apoptosis is dependent, at least in part, on transcription/translation. Small interfering RNA (siRNA) to c-Jun was used to interrupt the amplification cycle and was found to be highly effective, reducing vinblastine-induced c-Jun expression at both the mRNA and protein levels by 90%. Apoptosis and caspase-3 activation were significantly inhibited in c-Jun siRNA-treated cells. To uncover potential mechanisms of c-Jun-mediated cell death and protection by c-Jun siRNA, candidate target genes were examined. Chromatin immunoprecipitation revealed preferential association of c-Jun with the p21 (cyclin-dependent kinase inhibitor) gene promoter after vinblastine treatment. In KB-3 cells, which have compromised p53 function, and in p53-null cells but not in p53 wild-type cells, vinblastine caused down-regulation of p21 expression concomitant with increased c-Jun expression, suggesting a role for c-Jun in negative regulation of the p21 promoter independent of p53. These results provide strong evidence that c-Jun induction in response to vinblastine plays a proapoptotic role in part via down-regulation of p21, promoting cycling and subsequent cell death of mitotically impaired cells.

Genotoxic stress leads to centrosome amplification in breast cancer cell lines that have an inactive G1/S cell cycle checkpoint.

Centrosome amplification plays a key role in the origin of chromosomal instability during cancer development and progression. In this study, breast cancer cell lines with different p53 backgrounds were used to investigate the relationship between genotoxic stress, G(1)/S cell cycle checkpoint integrity, and the development of centrosome amplification. Introduction of DNA damage in the MCF-7 cell line by treatment with hydroxyurea (HU) or daunorubicin (DR) resulted in the arrest of both G(1)/S cell cycle progression and centriole duplication. In these cells, which carry functional p53, HU treatment also led to nuclear accumulation of p53 and p21(WAF1), retinoblastoma hypophosphorylation, and downregulation of cyclin A. MCF-7 cells carrying a recombinant dominant-negative p53 mutant (vMCF-7(DNp53)) exhibited a shortened G(1) phase of the cell cycle and retained a normal centrosome phenotype. However, these cells developed amplified centrosomes following HU treatment. The MDA-MB 231 cell line, which carries mutant p53 at both alleles, showed amplified centrosomes at the outset, and developed a hyperamplified centrosome phenotype following HU treatment. In cells carrying defective p53, the development of centrosome amplification also occurred following treatment with another DNA damaging agent, DR. Taken together, these findings demonstrate that loss of p53 function alone is not sufficient to drive centrosome amplification, but plays a critical role in this process following DNA damage through abrogation of the G(1)/S cell cycle checkpoint. Furthermore, these studies have important clinical implications because they suggest that breast cancers with compromised p53 function may develop centrosome amplification and consequent chromosomal instability following treatment with genotoxic anticancer drugs.

Immunohistochemical determination of p53 protein does not predict clinical response in advanced colorectal cancer with low thymidylate synthase expression receiving a bolus 5-fluorouracil—leucovorin combination

We assessed the hypothesis that a compromised p53 function could account for the non response of colon cancer patients with low thymidylate synthase (TS) expression receiving a bolus 5-fluorouracil (5-FU) leucovorin (LV) combination. The study population consisted of 41 patients with unresectable metastatic colon cancer, homogeneously, treated with bolus 5-FU and LV. Twenty-seven patients (66%) showed high levels of TS expression. The difference in the proportion of objective responses between patients with low (CR + PR: 7 of 14, 50%) and high (CR + PR: 0 of 27) TS levels was statistically significant (P = 0.0001, chi-square test). p53 nuclear over-expression was found in 27 of 41 patients (66%). No differences were observed in p53 overexpression in patients with high (66%) or low (66%) TS expression. p53 status was not found to be associated with response even in patients with low TS expression. p53 status measured by immunohistochemistry does not seem to be useful to identify unresponsive patients with low TS expression.

Regulation of a senescence checkpoint response by the E2F1 transcription factor and p14(ARF) tumor suppressor.

Normal cells do not divide indefinitely due to a process known as replicative senescence. Human cells arrest growth with a senescent phenotype when they acquire one or more critically short telomeres as a consequence of cell division. Recent evidence suggests that certain types of DNA damage, chromatin remodeling, and oncogenic forms of Ras or Raf can also elicit a senescence response. We show here that E2F1, a multifunctional transcription factor that binds the retinoblastoma (pRb) tumor suppressor and that can either promote or suppress tumorigenesis, induces a senescent phenotype when overexpressed in normal human fibroblasts. Normal human cells stably arrested proliferation and expressed several markers of replicative senescence in response to E2F1. This activity of E2F1 was independent of its pRb binding activity but dependent on its ability to stimulate gene expression. The E2F1 target gene critical for the senescence response appeared to be the p14(ARF) tumor suppressor. Replicatively senescent human fibroblasts overexpressed p14(ARF), and ectopic expression of p14(ARF) in presenescent cells induced a phenotype similar to that induced by E2F1. Consistent with a critical role for p14(ARF), cells with compromised p53 function were immune to senescence induction by E2F1, as were cells deficient in p14(ARF). Our findings support the idea that the senescence response is a critical tumor-suppressive mechanism, provide an explanation for the apparently paradoxical roles of E2F1 in oncogenesis, and identify p14(ARF) as a potentially important mediator of the senescent phenotype.