Accumulation Of Mutant P53 Protein Research Articles

Mutant p53 reactivation restricts the protumorigenic consequences of wild type p53 loss of heterozygosity in Li-Fraumeni syndrome patient-derived fibroblasts

The p53 tumor suppressor, encoded by the TP53 gene, serves as a major barrier against malignant transformation. Patients with Li-Fraumeni syndrome (LFS) inherit a mutated TP53 allele from one parent and a wild-type TP53 allele from the other. Subsequently, the wild-type allele is lost and only the mutant TP53 allele remains. This process, which is termed loss of heterozygosity (LOH), results in only mutant p53 protein expression. We used primary dermal fibroblasts from LFS patients carrying the hotspot p53 gain-of-function pathogenic variant, R248Q to study the LOH process and characterize alterations in various pathways before and after LOH. We previously described the derivation of mutant p53 reactivating peptides, designated pCAPs (p53 Conformation Activating Peptides). In this study, we tested the effect of lead peptide pCAP-250 on LOH and on its associated cellular changes. We report that treatment of LFS fibroblasts with pCAP-250 prevents the accumulation of mutant p53 protein, inhibits LOH, and alleviates its cellular consequences. Furthermore, prolonged treatment with pCAP-250 significantly reduces DNA damage and restores long-term genomic stability. pCAPs may thus be contemplated as a potential preventive treatment to prevent or delay early onset cancer in carriers of mutant p53.

Druggable sites/pockets of thep53-DNAJA1 protein-protein interaction: In silico modeling and in vitro/in vivo validation.

Mutation of p53 is the most common genetic alteration in human cancer. The vast majority of p53 mutations found in cancer are missense mutations, with some single nucleotide point mutations leading to the accumulation of mutant p53 protein with potential gain of oncogenic function. The mechanism for stabilization and accumulation of missense mutant p53 protein in malignant cells is not fully understood. It is thought that DNAJA1 plays a crucial role as a co-chaperone protein by stabilizing mutant p53 and amplifying oncogenic potential. As such, identifying small molecule inhibitors to disrupt the protein-protein interaction between mutant p53 and DNAJA1 may lead to an effective treatment for preventing carcinogenesis. Studying protein-protein interactions and identifying potential druggable hotspots has historically been limited-protein-protein binding sites require more complex characterization than those of single proteins and the crystal structures of many proteins have not been identified. Due to these issues, identifying salient druggable targets in protein-protein interactions through bench research may take years to complete. However, in silico modeling approaches allow for rapid characterization of protein-protein interfaces and the druggable binding sites they contain. In this chapter, we first review the oncogenic potential of mutant p53 and the crucial role of DNAJA1 in stabilizing missense mutant p53. We then detail our methodology for using in silico modeling and molecular biology to identify druggable protein-protein interaction sites/pockets between mutant p53 and DNAJA1. Finally, we discuss screening for and validating the utility of a small molecule inhibitor identified through our in silico framework. Specifically, we describe GY1-22, a unique compound with activity against mutant p53 that demonstrates therapeutic potential to inhibit cancer cell growth both in vivo and in vitro.

P53 partial loss-of-function mutations sensitize to chemotherapy

The tumor suppressive transcription factor p53 is frequently inactivated in cancer cells by missense mutations that cluster in the DNA binding domain. 30% hit mutational hotspot residues, resulting in a complete loss of transcriptional activity and mutant p53-driven chemotherapy resistance. Of the remaining 70% of non-hotspot mutants, many are partial loss-of-function (partial-LOF) mutants with residual transcriptional activity. The therapeutic consequences of a partial-LOF have remained largely elusive. Using a p53 mutation engineered to reduce DNA binding, we demonstrate that partial-LOF is sufficient to enhance oncogene-driven tumorigenesis in mouse models of lung and pancreatic ductal adenocarcinoma and acute myeloid leukemia. Interestingly, mouse and human tumors with partial-LOF mutations showed mutant p53 protein accumulation similar as known for hotspot mutants. Different from the chemotherapy resistance caused by p53-loss, the partial-LOF mutant sensitized to an apoptotic chemotherapy response and led to a survival benefit. Mechanistically, the pro-apoptotic transcriptional activity of mouse and human partial-LOF mutants was rescued at high mutant protein levels, suggesting that accumulation of partial-LOF mutants enables the observed apoptotic chemotherapy response. p53 non-hotspot mutants with partial-LOF, therefore, represent tumorigenic p53 mutations that need to be distinguished from other mutations because of their beneficial impact on survival in a therapy context.

Re-evaluating the clinical significance of serum p53 antibody levels in patients with oral cancer in Japanese clinical practice.

TP53 gene mutations can lead to mutant p53 protein accumulation in cancer cells, thereby inducing the production of serum antip53 antibodies (Ap53Ab) in patients with various types of cancer. The aim of the present study was to re-evaluate the clinicopathological and prognostic significance of Ap53Ab using the Ap53Ab ELISA kit, approved by the Japanese Health Insurance System in 2007. Ap53Ab was measured as a tumor marker in 94 patients with oral squamous cell carcinoma (OSCC), by subjecting paraffin-embedded sections obtained from biopsy specimens to immunohistochemical analysis to confirm p53 expression. The associations among Ap53Ab status, p53 expression and clinical significance in OSCC were examined. A total of 23% of the patients were Ap53Ab-positive. Ap53Ab status was found to be significantly associated with p53 expression status in primary tumors (P=0.027), clinical T-category, pathological N-category and pathological stage (P=0.04, P=0.010 and P=0.013, respectively). Kaplan-Meier curve analysis revealed that Ap53Ab status was significantly associated with poor disease-free survival (DFS; P=0.043), and Cox regression analysis revealed that Ap53Ab status was a significant prognostic factor for DFS in patients with OSCC (hazard ratio=2.807; 95% confidence interval: 1.029-7.160; P=0.044). These results suggested that Ap53Ab measurement may reflect the p53 mutation status and an aggressive malignant phenotype, and it may serve as a useful predictive marker candidate for OSCC in clinical practice.

Targeting mutant p53-expressing tumours with a T cell receptor-like antibody specific for a wild-type antigen

Accumulation of mutant p53 proteins is frequently found in a wide range of cancers. While conventional antibodies fail to target intracellular proteins, proteosomal degradation results in the presentation of p53-derived peptides on the tumour cell surface by class I molecules of the major histocompatibility complex (MHC). Elevated levels of such p53-derived peptide-MHCs on tumour cells potentially differentiate them from healthy tissues. Here, we report the engineering of an affinity-matured human antibody, P1C1TM, specific for the unmutated p53125-134 peptide in complex with the HLA-A24 class I MHC molecule. We show that P1C1TM distinguishes between mutant and wild-type p53 expressing HLA-A24+ cells, and mediates antibody dependent cellular cytotoxicity of mutant p53 expressing cells in vitro. Furthermore, we show that cytotoxic PNU-159682-P1C1TM drug conjugates specifically inhibit growth of mutant p53 expressing cells in vitro and in vivo. Hence, p53-associated peptide-MHCs are attractive targets for the immunotherapy against mutant p53 expressing tumours.

Inhibition of mutant Kras and p53-driven pancreatic carcinogenesis by atorvastatin: Mainly via targeting of the farnesylated DNAJA1 in chaperoning mutant p53.

Recent studies have indicated that using statins to inhibit the mevalonate pathway induces mutant p53 degradation by impairing the interaction of mutant p53 with DnaJ subfamily A member 1 (DNAJA1). However, the role of the C-terminus of DNAJA1 with a CAAX box for farnesylation in the binding, folding, and translocation of client proteins such as mutant p53 is not known. In the present study, we used a genetically engineered mouse model of pancreatic carcinoma and showed that atorvastatin significantly increased animal survival and inhibited pancreatic carcinogenesis. There was a dramatic decrease in mutant p53 protein accumulation in the pancreatic acini, pancreas intraepithelial neoplasia lesions, and adenocarcinoma. Supplementation with farnesyl pyrophosphate, a substrate for protein farnesylation, rescued atorvastatin-induced mutant p53 degradation in pancreatic cancer cells. Tipifarnib, a farnesyltransferase inhibitor, mirrored atorvastatin's effects on mutant p53, degraded mutant p53 in a dose-dependent manner, and converted farnesylated DNAJA1 into unfarnesylated DNAJA1. Farnesyltransferase gene knockdown also significantly promoted mutant p53 degradation. Coimmunoprecipitation either by an anti-DNAJA1 or p53 antibody confirmed the direct interaction of mutant p53 and DNAJA1 and higher doses of atorvastatin treatments converted more farnesylated DNAJA1 into unfarnesylated DNAJA1 with much less mutant p53 pulled down by DNAJA1. Strikingly, C394S mutant DNAJA1, in which the cysteine of the CAAX box was mutated to serine, was no longer able to be farnesylated and lost the ability to maintain mutant p53 stabilization. Our results show that farnesylated DNAJA1 is a crucial chaperone in maintaining mutant p53 stabilization and targeting farnesylated DNAJA1 by atorvastatin will be critical for inhibiting p53 mutant cancer.

Abstract 3951: NVP-BEZ235 decreases mutant p53 and affects miR-23a-24a-27a to inhibit metastasis in breast cancer cells

Abstract Nearly half of human cancers harbor TP53 mutations, which are predominantly through missense mutations that result in accumulation of mutant p53 proteins in cancer cells. More and more findings indicate that the mutant p53 proteins acquire gain-of-function abilities to promote carcinogenesis, metastasis, tumor recurrence and chemoresistance. TP53 mutations are the most frequent genetic alterations in breast cancer, observed in 30% of breast carcinomas, especially the triple-negative breast cancer (TNBC), which most easily metastasizes and confers chemoresistance. Therefore, targeting TP53 mutation in breast cancer might be another promising approach of therapeutics. In this study, the AKT-mTOR dual inhibitor, NVP-BEZ235 (called BEZ235 hereafter) was employed to treat two TNBC cell lines, MDA-MB-231 and MDA-MB-468, which serve as TP53 mutation group at different dosages and time points. The results of CCK8, scratch, Transwell chamber and soft agar tests showed that BEZ235 could inhibit cell growth, migration, invasion and transformation abilities of the above TNBC cells. Most importantly, mutant p53 in these TNBC cells decreased significantly with BEZ235 treatment by Western blot. Therefore, we speculated that BEZ235 might inhibit the metastasis of TNBC cells through mutant p53 decline. Furthermore, we observed the autophagy marker LC3I/II conversion elevated obviously by WB, along with the upregulation of LC3 mRNA, which indicated that autophagy was induced after BEZ235 treatment in TNBC cells. To further understand the relationship of autophagy and p53 decrease upon exposure of BEZ235, autophagy inhibitor 3-MA and lentiviral shRNAs embedding ATG5/ATG7 were applied to deplete autophagy, and we found that mutant p53 was still decreased in the cells treated by NVP235 even though the autophagy was inhibited, which indicated that p53 degradation mediated by NVP235 may not relate with autophagy. In order to explore other potential regulatory mechanisms of the p53 downregulation upon BEZ235 exposure, microRNAs were considered to detect in the TNBC cells after treatment. Interestingly, miR-23a-24a-27a cluster was upregulated about 2-fold in TNBC cells after BEZ235 treatment in our study, while tumor growth and metastasis roles were discovered in some previous findings. More studies need to be done to illustrate miR-23a-24a-27a cluster in the TNBC cells treatment with NVP235, as well as the mechanisms with p53 regulation. In conclusion, our results suggest that BEZ235 might inhibit the metastasis abilities of triple-negative breast cancer cells targeting mutant p53 accumulation, which might provide an important direction to further study the breast cancer harboring mutant p53, and also have promising therapeutic benefits for cancer patients carrying p53 mutations. Citation Format: Jiajing Cai, Jingruo Xia, Dan Xiao, Chang Liu, Hebin Liao, Lei Xu, Dongsheng Wang, Xiaolan Guo. NVP-BEZ235 decreases mutant p53 and affects miR-23a-24a-27a to inhibit metastasis in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3951.

Inhibiting the system xC\u2212/glutathione axis selectively targets cancers with mutant-p53 accumulation

TP53, a critical tumour suppressor gene, is mutated in over half of all cancers resulting in mutant-p53 protein accumulation and poor patient survival. Therapeutic strategies to target mutant-p53 cancers are urgently needed. We show that accumulated mutant-p53 protein suppresses the expression of SLC7A11, a component of the cystine/glutamate antiporter, system xC−, through binding to the master antioxidant transcription factor NRF2. This diminishes glutathione synthesis, rendering mutant-p53 tumours susceptible to oxidative damage. System xC− inhibitors specifically exploit this vulnerability to preferentially kill cancer cells with stabilized mutant-p53 protein. Moreover, we demonstrate that SLC7A11 expression is a novel and robust predictive biomarker for APR-246, a first-in-class mutant-p53 reactivator that also binds and depletes glutathione in tumours, triggering lipid peroxidative cell death. Importantly, system xC− antagonism strongly synergizes with APR-246 to induce apoptosis in mutant-p53 tumours. We propose a new paradigm for targeting cancers that accumulate mutant-p53 protein by inhibiting the SLC7A11–glutathione axis.

Abstract 4357: Harnessing system xCT- to target mutant p53 cancer cells

Abstract p53, a critical tumour suppressor is mutated in over half of all human cancers. The loss of wild-type p53 activity together with oncogenic gain-of-function, secondary to aberrant accumulation of mutant p53 protein frequently results in aggressive tumour phenotype, resistance to conventional therapies and poor survival. Therefore, effective therapeutic strategies to target mutant p53 cancer cells remain an urgent and unmet medical need. Here we show that mutant p53 accumulation across multiple tumour types represses the transcription of SLC7A11, a key component of system xCT-, resulting in reduced cystine uptake, lowering endogenous glutathione stores and predisposing cells to oxidative damage. Notably, genetic knockdown or pharmacological inhibition (erastin and sulfasalazine) of system xCT- preferentially induces apoptosis in cancer cells with mutant p53 accumulation. Moreover, we found that APR-246 (PRIMA-1met), a first-in-class reactivator of mutant p53 currently in early clinical trials, depletes cellular glutathione and induces significantly higher amounts of reactive oxygen species in mutant p53 cancer cells compared with normal cells. This leads to lipid peroxidation of mitochondrial membranes and the release of matrix contents, culminating in apoptotic cell death. Conversely, APR-246-induced cytotoxicity could be rescued by cysteine or glutathione replacement, or with lipophilic antioxidants. In extension, we identified and functionally validated SLC7A11 expression as a specific predictive biomarker for APR-246. Importantly, we demonstrate that antagonising system xCT- activity in combination with APR-246 selectively and synergistically inhibit mutant p53 cancer cells. Together, our findings propose that accumulation of mutant p53 protein in cancer cells, through its repressive effects on SLC7A11 expression, creates an ‘Achilles heel’ that can be targeted by further perturbations of the glutathione pathway. Citation Format: David SH Liu, Matthew Read, Klas G. Wiman, Sue Haupt, Cuong P. Duong, Lars Abrahmsen, Ygal Haupt, Nicholas J. Clemons, Wayne A. Phillips. Harnessing system xCT- to target mutant p53 cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4357.

New perspective on targeting the tumor suppressor p53 pathway in the tumor microenvironment to enhance the efficacy of immunotherapy.

About 50% of human cancers harbor somatic mutations of the tumor suppressor p53 (p53 or Trp53) gene. Many of those mutations result in the inactivation of the p53 pathway and are often associated with the stabilization and accumulation of mutant p53 proteins. Therefore, increased p53 expression in tumors is frequently used as a surrogate marker for p53 mutation and inactivation. Moreover, this elevated p53 expression also makes it an ideal tumor associated antigen (TAA) for cancer vaccines. Recent advances in our understanding of p53 as a crucial transcription factor reveal that p53 is an important sensor of cellular stress under genotoxic, chemotoxic, pathological, and even normal physiological conditions. Experimental and clinical observations by our laboratory and others have demonstrated that p53 also participates in immune regulation as p53 dysfunction skews host immune responses towards pro-inflammation, which further promotes tumor progression. Furthermore, recent studies using a genetic approach revealed that p53-restoration or re-activation led to tumor regression and clearance, which were at least partially caused by the activation of innate antitumor immunity. Since many of the currently used cancer therapeutics, including radiotherapy and chemotherapy, disrupt tumor growth by inducing DNA damage via genotoxic or chemotoxic stress, which activates the p53 pathway in the tumor microenvironment, we postulate that some of those observed therapeutic benefits might also be partially mediated through their immune stimulatory effects. Here, we briefly review our current understanding of the potential cellular and molecular mechanisms by which p53 participates in immune regulation and, subsequently, extend our discussion to the immunostimulatory potential of existing and new approaches of targeting the p53-pathway to alter the immunological landscape of tumors for maximizing immunotherapy outcome.

Increased Angiogenesis and Decreased Programmed Cell Death Increases the Risk of Uterine Cervical Cancer

Cervical cancer is one of the most common female malignancies and leading cause for high mortality rate. In the present study we made an attempt to determine the extent of angiogenesis, apoptosis, accumulation of mutant p53 protein, cell proliferation rate in the uterine cervical cancer tissues. Cervical cancer samples were obtained from patients and they were subjected to PCR analysis and immunocytochemistry. A total of 30 cervical cancer tissue samples were analyzed, by PCR, we found 25 collected cervical cancer samples showed HPV-16 and E6 positive. Further, we observed the increased CD34 expression was associated with HPV-16 and E6 positive cancer tissues when compared to the corresponding control tissues. This elevated level of CD34 confirms the increased extent of angiogenesis in cervical cancer tissues. Further by immunocytochemistry we have demonstrated that the rate of apoptosis is reduced, over expression of bcl-2, Ki 67 and thus increases rate of cell proliferation. Therefore, our data suggest that development of new anticancer or antiviral drugs could efficiently compromise the HPV-16 mediated angiogenesis and reduced apoptosis in cervical cancer and thus will improve the survival rate of patients.

Abstract 584: NVP-BEZ235 decreases mutant p53 and downregulates miR-23a-24-27a to inhibit metastasis in breast cancer cells

Abstract Nearly half of human cancers harbor TP53 mutations, which are predominantly through missense mutations that result in accumulation of mutant p53 proteins in cancer cells. More and more findings indicate that the mutant p53 proteins acquire gain-of-function abilities to promote carcinogenesis, metastasis, tumor recurrence and chemoresistance. TP53 mutations are the most frequent genetic alterations in breast cancer, observed in 30% of breast carcinomas, especially the triple negative breast cancer (TNBC),which most easily metastasizes and confers chemoresistance. Therefore targeting TP53 mutation in breast cancer might be another promising approach of therapeutics. Our previous investigation showed that the AKT-mTOR dual inhibitor, NVP-BEZ235(called BEZ235 thereafter), could inhibit AKT-mTOR pathway activation, most interestingly mutant p53 was significantly reduced with BEZ235 treatment(unpublished data). In this study, three TNBC cell lines MDA-MB-231, MDA-MB-436, MDA-MB-468 as TP53 mutation group and MCF-7 as control were employed with BEZ235 treatment at different dosages and time points. We found that AKT-mTOR signaling pathway were highly activated in these TNBC cells but were inhibited obviously after BEZ235 treatment; most importantly, mutant p53 in these TNBC cells decreased significantly, but rare changes were detected in MCF-7 cells with BEZ235 treatment. Interestingly, we found miR-23a-24-27a cluster was about 2 fold upregulation in TNBC cells after BEZ235 treatment while exploring the decline of p53 by detecting microRNAs expression in those cell lines. To address whether BEZ235 could affect the metastasis ability of TNBC cells, cell invasion assay Transwell was used to measure the invasiveness of TNBC cells with BEZ235, and we observed that BEZ235 could inhibit the metastasis of TNBC cells, on the other hand miR-23a-24-27a cluster mimics could also induce the inhibition of metastasis in TNBC cells. Our results suggest that BEZ235 might target mutant p53 accumulation and downregulate miR-23a-24-27a cluster to inhibit the metastasis abilities of triple negative breast cancer cells, which might provide an important direction to further study the breast cancer harboring mutant p53, and also have promising therapeutic benefits for cancer patients carrying such p53 mutations. Citation Format: Dongsheng Wang, Qin Du, Binfeng He, Yibin Deng, Xiaolan Guo. NVP-BEZ235 decreases mutant p53 and downregulates miR-23a-24-27a to inhibit metastasis in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 584. doi:10.1158/1538-7445.AM2014-584

Abstract 4049: The gain-of-function properties of mutant p53 rescues cells from telomere dysfunction-induced crisis and promotes tumorigenesis.

Abstract Telomeres are nucleoprotein complexes that cap the ends of eukaryotic chromosomes and maintain chromosomal stability. Telomeres are protected by shelterin, a complex of six core proteins (TRF1, TRF2, RAP1, TIN2, TPP1 and POT1) that prevents the chromosome ends from being mistakenly recognized as damaged DNA. Telomeres that can no longer exert end-protective functions are said to be dysfunctional. Telomere dysfunction can be caused by progressive telomere attrition (telomere shortening) or through disruption of the shelterin complex (telomere uncapping). To study whether chromosomal instability caused by telomere uncapping leads to tumorigenesis, we used the Cre-lox system to delete the telomere binding protein TRF2 in mouse embryonic fibroblasts (MEFs). In the presence of tumor suppressor protein p53, loss of TRF2 inhibited cell proliferation via induction of cellular senescence. In contrast, the absence of p53 allowed the TRF2-deficient cells to bypass senescence as these cells were negative for SA-β-galactosidase staining. However, these p53-deficient cells stopped proliferating ten days after TRF2 depletion and eventually entered telomere uncapping-induced crisis (indicated by the significant appearance of chromosomal end to end fusions, mitotic failure and induction of apoptosis). Our mechanistic studies revealed that these p53-deficient cells underwent a novel ATM-caspase 2-dependent apoptosis upon loss of TRF2. Accordingly, knocking out caspase 2 rescued these cells from apoptosis, and thus increased long-term survival of the cell colonies as well as promoted cell transformation. Mutations of p53 are predominantly missense mutations that result in accumulation of mutant p53 proteins with loss of wild-type p53 tumor-suppressive functions and gain of oncogenic activities. To study whether the gain-of-function properties of mutant p53 can rescue cells undergoing telomere crisis from cell death, we generated MEFs expressing mutant p53R172H that corresponds to the p53R175H “hot spot” mutation in human cancers. We observed that mutant p53R172H did not prevent TRF2 depletion-induced chromosomal instability as indicated by extensive chromosome fusions. However, expression of mutant p53R172H significantly rescued cells from TRF2 loss-initiated cell apoptosis and promoted cell transformation as determined by anchorage-independent soft agar assay. More importantly, mutant p53R172H promoted tumor development of TRF2-deficient MEFs in immunodeficient mice. Collectively, our findings provide compelling genetic evidence that telomere uncapping-driven chromosomal instability activates ATM-caspase 2-dependent apoptosis to suppress tumorigenesis. However, the gain-of-function properties of mutant p53 can rescue the cells undergoing telomere crisis from cell death and thus promote tumorigenesis. Citation Format: Li-Ju Chang, Fengxia Wu, Xiaolan Guo, Yibin Deng. The gain-of-function properties of mutant p53 rescues cells from telomere dysfunction-induced crisis and promotes tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4049. doi:10.1158/1538-7445.AM2013-4049

Ionizing radiation induces a dramatic persistence of p53 protein accumulation and DNA damage signaling in mutant p53 zebrafish

Mutant p53 proteins accumulate to high levels in human tumors and in preneoplastic lesions in the skin and fallopian tube. However examination of tissues from mice and fish that are homozygous for mutant p53 surprisingly showed that the protein was present only at low levels except in the tumors that arose in these animals. The mutant protein did accumulate, however, following treatment with ionizing radiation in the same tissues in which the wild-type protein is induced. Here we study in detail the accumulation of mutant and wild-type p53 proteins following ionizing radiation in zebrafish embryos. We found that the mutant protein was induced by lower levels of radiation and reached higher levels than the wild-type protein. Morpholino knockdown of the zebrafish homologs of Mdm2 and Mdm4 caused dramatic accumulation of mutant p53 protein. The most remarkable results were observed by examining p53 protein levels over an extended time course. Mutant p53 protein increased and persisted for days after irradiation and this was accompanied by persistent elevation of phosphorylated H2AX (γH2AX), implying that the resolution of DNA damage signaling in these embryos is severely compromised by mutations in p53. Thus mutation in p53 results in an exaggerated and persistent damage response, which could in turn drive the process of cancer development as high levels of mutant p53 can act as an oncoprotein to drive invasion and metastasis.

Abstract 1173: AKT-mTOR pathway mediates mutant p53 gain-of-function by inhibiting autophagy

Abstract Nearly half of human cancers harbor p53 mutations, which are predominantly through missense mutations that result in accumulation of mutant p53 proteins in cancer cells. Persistent evidence supports that the mutant p53 proteins not only lose tumor suppressor activity, but also acquire gain-of-function abilities to promote carcinogenesis, metastasis, tumor recurrence and chemoresistance. Indeed, knock-in mouse models bearing the mutant p53R172H (equivalent to human R175H mutation) at physiological level in vivo display gain-of-function phenotypes, such as enhanced cell transformation potentials and tumor metastasis. However, the molecular mechanism of mutated p53R172H conferring gain-of-function remains poorly understood. In this study, utilizing mouse embryonic fibroblasts (MEFs) and lung adenocarcinoma cells (93-1) derived from p53R172H knock-in mice, we observed that AKT was preferentially activated in p53R172H mutant cells over p53-deficient cells. This activation of AKT allows p53R172H mutant cells to bypass contact inhibition, grow in an anchorage-independent manner and form tumors after subcutaneous injection into SCID mice. In contrast, expression of dominant negative AKT or shRNA-mediated AKT knockdown in p53R172H mutant cells inhibited colony formation in soft-agar and suppressed tumor growth in mouse xenograft tumor model. To directly dissect the essential role of AKT in p53R172H mutant-initiated tumorigenesis in vivo, we bred AKT1 knockout mice to p53R172H mutant knock-in mice. Loss of AKT1 significantly reduced the incidence of tumor development and extended the life span from around 3 months in AKT1-proficient and p53R172H mutant mice (AKT1+/+; p53R172H/R172H) to about 12 months in AKT1-deficient and p53R172H mutant mice (AKT1−/−; p53R172H/R172H). To understand how the activated AKT conferred mutant p53 gain-of-function, we demonstrated that p53R172H mutant activated AKT-mTOR signaling pathway that led to an inhibition of autophagy both in vitro and in vivo. Furthermore, induction of autophagy in p53R172H mutant lung cancer cells through suppressing AKT-mTOR pathway pharmacologically by the dual kinase inhibitor NVP-BEZ235 treatment or genetically by RNAi-mediated knockdown of mTOR dramatically inhibited p53R172H mutant-induced colony formation in soft agar and tumor growth in SCID mice. Our results support that AKT plays a crucial role in mediating mutant p53 gain-of-function phenotypes in vivo through the inhibition of autophagy by the activated AKT-mTOR signaling pathway. These findings provide the proof-of-principle that targeting the AKT-mTOR-autophagy pathway might have promising therapeutic benefits for cancer patients carrying such p53 mutations. 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 1173. doi:1538-7445.AM2012-1173

Titration of Serum p53 Antibodies Predicts Pathological Response in Women with Operable Early Stage Breast Cancer Receiving Preoperative Sequential FEC and Docetaxel Chemotherapy.

Abstract Background: In breast cancer, emerging dates demonstrate that p53 mutation is associated with response to preoperative chemotherapy, indicating that p53 mutation may be a predictive factor for response to preoperative chemotherapy. The accumulation of mutant p53 protein has been found to induce the formation of serum p53 IgG antibodies in breast cancer patients. A highly specific quantitative enzyme-linked immunosorbent assay (ELISA) kit has been developed to detect p53 antibody titers. However, whether this titration of serum p53 antibody reflects response to preoperative chemotherapy is unknown. The aim of this study was to evaluate the clinical implication of serum p53 antibody titers as a predictive factor for preoperative chemotherapy in breast cancer.Patinets and Methods: In this study, we analyzed 103 women with operable early stage breast cancer treated by preoperative chemotherapy and definitive curative surgery. Patients received four cycles of FEC (fluorouracil 500 mg/m2, epirubicin 100 mg/m2, cyclophosphamide 500 mg/m2 q3w) followed by four cycles of docetaxel (75 mg/m2 q3w). The pretreatment serum samples were used to determine the p53 antibody titers. The test's cut-off value was determined to be 1.3 U/ml based on reference distribution in healthy control individuals.Results: Twenty-three of 103 patients (22.3%) were positive for serum p53 antibody. The frequency of pathological complete response (pCR) was significantly higher in cases with positive for p53 antibody than in patients with negative for p53 antibody (9/23 or 39% vs. 15/80 or 23%; p=0.04). According to intrinsic subtypes, the tendency was most emphasized in cases with HER2-positive cases (6/9 or 67% vs. 8/25 or 32%). Interestingly, among 8 patients with high p53 antibody titers (>100 IU/ml) 7 patients experienced pCR and remaining one patients showed only foci invading areas in primary tumor and complete remission in axillary lymph node metastasis, suggesting that the value of p53 antibody titers may more precisely predict response to preoperative chemotherapy.Conclusion: p53 mutation analysis using titration of serum p53 antibody is useful predictive test for response to preoperative chemotherapy in patients with breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2038.

When mutant p53 plays hide and seek: a new challenge for diagnosis and therapy?

p53 missense mutations observed in human cancers are often associated with an increased level of p53 protein in the tumour. Using mouse models, Terzian et al. recently showed that this accumulation of mutant p53 protein is not associated with specific properties of the protein itself but instead depends on the endogenous genetic background of the tumours and on two important genes, mouse double minute 2 (Mdm2) and the cyclin kinase inhibitor p16INK4a. Mice expressing mutant p53 in the absence of Mdm2 display more aggressive metastatic tumours. In light of these observations, targeting the MDM2-p53 interaction for therapy of human cancer could be more complicated than previously anticipated.

A comparison between p53 accumulation determined by immunohistochemistry and TP53 mutations as prognostic variables in tumours from breast cancer patients

Background. p53 accumulation and TP53 mutations are known prognostic markers for breast cancer. To clarify their interrelationship and the importance of different TP53 mutation types, these markers were investigated in tumours from 630 patients with breast cancer. Materials and methods. Tumour sections were stained for p53 and scored based on staining intensity and percentages of invasive tumour cells with nuclear staining. TP53 mutations were identified by sequencing. Patient cohorts were from the DBCG (Danish Breast Cancer Cooperative Group) protocols DBCG82 and DBCG89. Results. TP53 was mutated in 29% of the patients. The disease-specific survival (DSS) at 15 years of follow-up for patients with missense mutations directly involved in DNA or zinc binding was 21±8%. Patients with the remaining missense mutations within the structural/conserved domains and patients with null mutations had a DSS of 36±6% and 31±17%, respectively. For patients without TP53 mutations and patients with mutations affecting amino acids outside these domains, the 15 year DSS was 51±3% and 71±10%, respectively. p53 accumulation was successfully scored in 567 patients and categorized into three groups. Tumours with no p53 expression had a high frequency of null mutations (37% compared to 10% in the whole cohort), and tumours with high p53 expression contained 82% of the missense mutations inside structural/conserved domains including those directly involved in DNA or zinc binding. Conclusion. The clinical outcome for breast cancer patients is significantly different for different TP53 mutation types, but further functional studies are required to clarify the exact role of these mutation types. Most of the mutations that lead to mutant p53 protein accumulation can be detected by immunohistochemistry but the specificity is low. Samples showing lack of detectable p53 protein should be considered as an indication of a possible null mutation.

P53 alterations in human cancer: more questions than answers

The strongest and undisputed fact about p53 is the high frequency of p53 alterations in human cancer and that mutant p53 proteins constitute a complex family of several hundred proteins with heterogeneous properties. Beyond these observations, the p53 pathway and its regulation in a normal cell is like a desert trail, always moving with the wind of novel findings. The field is full of black boxes that are often ignored for sake of simplicity or because they do not fit with the current dominant view. Mutant p53 protein accumulation in tumours is the best example of a preconceived idea, as there is no experimental evidence to explain this observation. In this review, we will discuss several questions concerning the activity or selection of p53 mutations. The central domain of the p53 protein targeted by 80% of p53 mutations is associated with the DNA-binding activity of the p53 protein, but it is also the binding site for several proteins that play a key role in p53 regulation such as ASPP proteins or BclxL. The role of impaired DNA binding and/or protein interactions in tumour development has not been fully elucidated. Similarly, novel animal models carrying either missense p53 mutations or inducible p53 have provided abundant observations, some of which could challenge our view on p53 function as a tumour suppressor gene. Finally, the possible clinical applications of p53 will be discussed.

APOPTOSIS AND CHEMOTHERAPY FOR BLADDER CANCER

We discuss the role of apoptosis, that is gene directed self-destruction of a cell, in the response of bladder transitional cell carcinoma cells to chemotherapy. A directed MEDLINE literature search of apoptosis, bladder cancer and chemotherapy was performed to extract relevant information for review. The characteristics of apoptotic cells were defined and the methods in common use to detect these traits is described. The role of the key mediators of the apoptotic process in bladder cancer is discussed in the context of chemosensitivity and disease stage. The importance of the apoptosis induction after chemotherapy is highlighted. On stimulus by appropriate external or internal signals a cell may alter the expression of genes encoding for proteins associated with the apoptotic process. The development of apoptosis depends on the balance between pro-apoptotic and anti-apoptotic proteins. Key alterations in genes and proteins related to apoptosis within bladder cancer result in a shift away from the default state of apoptosis toward a cell with increased survival properties that is chemoresistant. Much current research in bladder cancer is aimed at restoring chemosensitivity by shifting the cell toward a pro-apoptotic phenotype. Successful translation of this work into clinical practice may improve survival in patients in whom prognosis is currently poor.