P53 Point Mutations Research Articles

The influence of p53 mutation status on the anti-cancer effect of cisplatin in oral squamous cell carcinoma cell lines

ObjectivesThe purpose of this study was to evaluate the anti-cancer activity of cisplatin by studying its effects on cell viability and identifying the mechanisms underlying the induction of cell cycle arrest and apoptosis on oral squamous cell carcinoma (OSCC) cell lines with varying p53 mutation status.Materials and MethodsThree OSCC cell lines, YD-8 (p53 point mutation), YD-9 (p53 wild type), and YD-38 (p53 deletion) were used. To determine the cytotoxic effect of cisplatin, MTS assay was performed. The cell cycle alteration and apoptosis were analyzed using flow cytometry. Western blot analysis was used to detect the expression of cell cycle alteration- or apoptosis-related proteins as well as p53.ResultsCisplatin showed a time- and dose-dependent anti-proliferative effect in all cell lines. Cisplatin induced G2/M cell accumulation in the three cell lines after treatment with 0.5 and 1.0 µg/mL of cisplatin for 48 hours. The proportion of annexin V-FITC-stained cells increased following treatment with cisplatin. The apoptotic proportion was lower in the YD-38 cell line than in the YD-9 or YD-8 cell lines. Also, immunoblotting analysis indicated that p53 and p21 were detected only in YD-8 and YD-9 cell lines after cisplatin treatment.ConclusionIn this study, cisplatin showed anti-cancer effects via G2/M phase arrest and apoptosis, with some difference among OSCC cell lines. The mutation status of p53 might have influenced the difference observed among cell lines. Further studies on p53 mutation status are needed to understand the biological behavior and characteristics of OSCCs and to establish appropriate treatment.

Abstract 2707: Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3

Abstract The MDM2-p53 pathway plays a prominent role in various cancer pathogenesis. Nutlin-3, a small-molecule antagonist of MDM2, inhibits proliferation in cancer cells with wild-type p53 by blocking the interaction of MDM2 and p53. However, different cancer cells differ greatly sensitivity of Nutlin-3 because of MDM2 amplification and/or p53 mutation status. Herein, we evaluate the sensitivity of Nutlin-3 in different cancer cell lines including four mesothelioma cell lines, one breast cancer line, and one leiomyosarcoma cell line with nonmutant p53 expression, two liposarcoma cell lines harboring MDM2 amplification and wild-type p53, and one control mesothelioma cell line JMN1B harboring p53 point mutation. Unexpectedly, Nutlin-3 treatment induced cyclin D1 expression accompanied with inhibition of MDM2-p53 interaction in nonmutant cancer cell lines. Additive effects were obtained through a coordinated attack on MDM2-p53 interaction and cyclin D1, as demonstrated by immunoblots, cell viability and cell cycle analyses, showing that lentiviral mediated cyclin D1 knockdown or drug inhibition, and block of MDM2-p53 interaction, in cancer cell lines containing wild-type p53, induced greater anti-proliferative effects, compared to either intervention alone. Further results demonstrated that upregulation of cyclin D1 after Nutlin-3 treatment to some extent was dependent on expression and ubiquitin E3-ligase activity of MDM2. These compelling anti-proliferative responses indicate that combination inhibition of cyclin D1 and MDM2-p53 interaction warrants clinical evaluation as a novel therapeutic strategy in cancer cells harboring wild-type p53. Citation Format: Wenbin Ou, Weicai Chen, Jiaqing Zhu, Grant Eilers, Xuhui Li, Peipei Yang, Hailong Li, Fanguo Meng, Jonathan Fletcher. Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2707. doi:10.1158/1538-7445.AM2015-2707

A high-enrollment course-based undergraduate research experience improves student conceptions of scientific thinking and ability to interpret data.

We present an innovative course-based undergraduate research experience curriculum focused on the characterization of single point mutations in p53, a tumor suppressor gene that is mutated in more than 50% of human cancers. This course is required of all introductory biology students, so all biology majors engage in a research project as part of their training. Using a set of open-ended written prompts, we found that the course shifts student conceptions of what it means to think like a scientist from novice to more expert-like. Students at the end of the course identified experimental repetition, data analysis, and collaboration as important elements of thinking like a scientist. Course exams revealed that students showed gains in their ability to analyze and interpret data. These data indicate that this course-embedded research experience has a positive impact on the development of students' conceptions and practice of scientific thinking.

MP36-16 THE ROLE OF P63 AND P53MUTANT IN THE REGULATION OF DNA REPAIR, MUTATOR AND INVASIVE PHENOTYPE OF BLADDER CANCER CELLS

It is well established that the two major phenotypic variants of human bladder cancers, the low-grade non-invasive papillary bladder carcinoma (N-Inv-BC) and the high-grade invasive BC (Inv-BC), have different molecular features. For example, compared to 10% in N-Inv-BC, p53 point mutations are found in 70% of Inv-BC. p63 expression is suppressed in aggressive BC. In contrast, more than 70% of N-Inv-BC have FGFR3 mutations compared to the 10% found in Inv-BC. Using well-established human BC cell lines, we found that while Inv-BC cells have a mutator phenotype, N-Inv-BC cells do not. N-Inv-BC cells are proficient in nucleotide excision repair and base excision repair, whereas Inv-BC are deficient in both, and the expression of several DNA repair genes is suppressed in Inv-BC cells. This study aims to understand the role of p63, mutated p53, DNA repair function and cell mutation susceptibility to BC development. Background

Abstract 881: Identification of autoantibody biomarkers to wild-type and mutant p53 in pancreatic and ovarian cancer

Abstract Introduction: Mutations in TP53 are common in early pancreatic and ovarian cancer development, and are detected in the majority of pancreatic cancers (75%) and serous ovarian cancers (97%). Mutations in TP53 induce autoantibody (AAb) immune responses, which may be biomarkers for the early detection of these cancers. The most common mutations in p53 occur in the DNA binding domain at residues that directly contact the DNA (Arg175, 248, and 273). In contrast, preliminary studies suggest that mutations in the N- and C- termini of p53 alter the thermodynamic stability of p53, leading to increased accumulation in tumors. It is not known if the location of the mutations impacts immunogenicity, or if p53-AAb are specific to mutant p53 proteins. Methods: To evaluate AAb to mutant p53 proteins, we generated custom programmable protein microarrays displaying 52 common p53 point mutations. cDNAs encoding full-length p53 with c-terminal GST tags were printed on arrays with anti-GST antibody, and transcribed and translated in situ using HeLa expression lysate. The microarrays were screened with sera from pancreatic cancer cases (Stage I-III, n=60), healthy and benign disease controls (n=63), ovarian cancer cases (n=19) and controls (n=17). Bound IgG was detected by fluorescence. For validation, four immunogenic mutant p53 proteins Arg248Met, Pro278Ser, Arg213Leu, Gly187Ser and wild-type p53 were individually displayed in 96-well format using a programmable ELISA. The ELISAs were re-screened with a subset of the sera from pancreatic cancer cases (n=49) and controls (n=47) and all of the ovarian cancer cases and controls. Cutoff values were established to determine if a patient is positive for all wild-type and mutant p53 AAb by measuring the mean antigen florescence intensity +2 standard deviations of the controls. Results: AAb were detected to all four mutant and wild-type p53 proteins in sera from 8/49 (16%) of pancreatic cases and 2/47 (4.2%) of pancreatic controls, but the results were not statistically significant. In comparison, AAb to all four mutant and wild-type p53 proteins were identified in sera from 8/19 (42%) of ovarian cases and 0/17 (0%) of healthy control women (p ≤ 0.005). All sera with AAb to mutant p53 also had measurable AAb to wild-type p53 protein. All patients with immune responses had evidence of broad-reactivity to at least at least two mutant p53 proteins, as well as to wild-type p53. Conclusion: Our results indicate that patients with serum AAb to wild type p53 protein also have AAb to multiple mutant p53 antigens, suggesting degeneracy of the epitope specificity. No patients have been identified with AAb specific to mutant-only p53. We observed inter-patient variation in the patterns of immune reactivity to p53 mutations that is of unclear significance. Correlation with tumor p53 mutation status is ongoing. Citation Format: Benjamin A. Katchman, Rizwan Alam, Garrick Wallstrom, Joshua LaBaer, Michael A. Hollingsworth, Daniel W. Cramer, Karen S. Anderson. Identification of autoantibody biomarkers to wild-type and mutant p53 in pancreatic and ovarian cancer. [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 881. doi:10.1158/1538-7445.AM2014-881

Mammary gland-specific ablation of focal adhesion kinase reduces the incidence of p53-mediated mammary tumour formation.

Background:Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneous mammary tumour development caused by loss of tumour suppressor genes such as p53 is lacking. Alterations in the tumour suppressor gene p53 have been implicated in over 50% of human breast cancers. Given that elevated FAK expression highly correlates with p53 mutation status in human breast cancer, we set out to investigate the importance of FAK in p53-mediated spontaneous mammary tumour development.Methods:To directly assess the role of FAK, we generated mice with conditional inactivation of FAK and p53. We generated female p53lox/lox/FAK+/+/WapCre, p53lox/lox/FAKflox/+/WapCre and p53lox/lox/FAKflox/−/WapCre mice, and mice with WapCre-mediated conditional expression of p53R270H, the mouse equivalent of human p53R273H hot spot mutation, together with conditional deletion of FAK, P53R270H/+/FAKlox/+/WapCre and p53R270H/+/FAKflox/−/WapCre mice. All mice were subjected to one pregnancy to induce WapCre-mediated deletion of p53 or expression of p53 R270H, and Fak genes flanked by two loxP sites, and subsequently followed the development of mammary tumours.Results:Using this approach, we show that FAK is important for p53-induced mammary tumour development. In addition, mice with the mammary gland-specific conditional expression of p53 point mutation R270H, the mouse equivalent to human R273H, in combination with conditional deletion of Fak showed reduced incidence of p53R270H-induced mammary tumours. In both models these effects of FAK were related to reduced proliferation in preneoplastic lesions in the mammary gland ductal structures.Conclusions:Mammary gland-specific ablation of FAK hampers p53-regulated spontaneous mammary tumour formation. Focal adhesion kinase deletion reduced proliferative capacity of p53 null and p53R270H mammary epithelial cells but did not lead to increased apoptosis in vivo. Our data identify FAK as an important regulator in mammary epithelial cell proliferation in p53-mediated and p53R270H-induced mammary tumour development.

Cooperation of p53 mutations with other oncogenic alterations in cancer.

Following the initial findings suggesting a pro-oncogenic role for p53 point mutants, more than 30 years of research have unveiled the critical role exerted by these mutants in human cancer. A growing body of evidence, including mouse models and clinical data, has clearly demonstrated a connection between mutant p53 and the development of aggressive and metastatic tumors. Even if the molecular mechanisms underlying mutant p53 activities are still the object of intense scrutiny, it seems evident that full activation of its oncogenic role requires the functional interaction with other oncogenic alterations. p53 point mutants, with their pleiotropic effects, simultaneously activating several mechanisms of aggressiveness, are engaged in multiple cross-talk with a variety of other cancer-related processes, thus depicting a complex molecular landscape for the mutant p53 network. In this chapter revealing evidence illustrating different ways through which this cooperation may be achieved will be discussed. Considering the proposed role for mutant p53 as a driver of cancer aggressiveness, disarming mutant p53 function by uncoupling the cooperation with other oncogenic alterations, stands out as an exciting possibility for the development of novel anti-cancer therapies.

Pharmacological targeting of p53 through RITA is an effective antitumoral strategy for malignant pleural mesothelioma

Malignant mesothelioma, a very aggressive tumor associated to asbestos exposure, is expected to increase in incidence, and unfortunately, no curative modality exists. Reactivation of p53 is a new attractive antitumoral strategy. p53 is rarely mutated in mesothelioma, but it is inactivated in most tumors by the lack of p14ARF. Here, we evaluated the feasibility of this approach in pleural mesothelioma by testing RITA and nutlin-3, two molecules able to restore p53 function through a different mechanism, on a panel of mesothelioma cell lines representing the epithelioid (NCI-H28, NCI-H2452, IST-MES 2), biphasic (MSTO-211H), and sarcomatoid (NCI-H2052) histotypes compared with the normal mesothelial HMC-hTERT. RITA triggered robust caspase-dependent apoptosis specifically in epithelioid and biphasic mesothelioma cell lines, both through wild-type and mutant p53, concomitant to p21 downregulation. Conversely, nutlin-3 induced a p21-dependent growth arrest, rather than apoptosis, and was slightly toxic on HMC-hTERT. Interestingly, we identified a previously undetected point mutation of p53 (p.Arg249Ser) in IST-MES 2, and showed that RITA is also able to reactivate this p53 mutant protein and its apoptotic function. RITA reduced tumor growth in a MSTO-211H-derived xenograft model of mesothelioma and synergized with cisplatin, which is the mainstay of treatment for this tumor. Our data indicate that reactivation of p53 and concomitant p21 downregulation effectively induce cell death in mesothelioma, a tumor characterized by a high intrinsic resistance to apoptosis. Altogether, our findings provide the preclinical framework supporting the use of p53-reactivating agents alone, or in combination regimens, to improve the outcome of patients with mesothelioma.

Disarming mutant p53 oncogenic function

In the last decade intensive research has confirmed the long standing hypothesis that some p53 point mutants acquire novel activities able to cooperate with oncogenic mechanisms. Particular attention has attracted the ability of several such mutants to actively promote the development of aggressive and metastatic tumors in vivo. This knowledge opens a new dimension on rational therapy design, suggesting novel strategies based on pharmacological manipulation of those neomorphic activities. P53 point mutants have several characteristics that make them attractive targets for anti-cancer therapies. Remarkably, mutant p53 has been found predominantly in tumor cells and may act pleiotropically by interfering with a variety of cellular processes. Therefore, drugs targeting mutant p53 may selectively affect tumor cells, inactivating simultaneously several mechanisms of tumor promotion. Moreover, the high frequency of missense mutations on the p53 gene suggests that interfering with mutant p53 function may provide a valuable approach for the development of efficient therapies able to target a wide range of tumor types.

Characteristics and survival of patients with advanced cancer and TP53 mutations in phase I clinical trials.

2545 Background: The classical hot spot mutations within the central sequence-specific DNA-binding region of p53 protein (R175H in exon 5 and G245S, R248Q, R249S, R273H and R282W in exon 8) disrupt its ability to bind to DNA. These inactivating point mutations of p53 may result in loss of its function and/or acquisition of a gain of function, which has been associated with the development of invasive and metastatic tumors in mouse models. We assessed the frequency and survival of p53 mutations in patients with advanced cancer. Methods: P53 mutation in patients’ tumor was identified in a CLIA-certified laboratory using PCR, sequenom analysis or next generation sequencing. Results: P53 mutations were identified in 212 patients. Median survival by tumor type and distribution of p53 mutations by exon are shown in the Table. The median survival (from time of referral to Phase I clinic) by mutations in “hot spot” amino acids was as follows: R175H, 9.1 months (n=18); R248Q, not reached/5 deaths (n=22); R273H, 12.6 months (n=11); other mutations, 11.9 months (n=160). The median survival of patients with a tumor mutation in exons 5 or 8 appeared to be shorter than that of patients with mutations in other exons (9.4 vs. 14.6 months) although the difference was not statistically significant (p=.12) owing to the small number of patients. Conclusions: Our results demonstrate that the distribution and clinical significance of various p53 mutations differs by tumor type and suggest that mutations in R248Q may be associated with longer survival. [Table: see text]

Drug Resistance to Inhibitors of the Human Double Minute-2 E3 Ligase Is Mediated by Point Mutations of p53, but Can Be Overcome with the p53 Targeting Agent RITA

The human double minute (HDM)-2 E3 ubiquitin ligase plays a key role in p53 turnover and has been validated preclinically as a target in multiple myeloma (MM) and mantle cell lymphoma (MCL). HDM-2 inhibitors are entering clinical trials, and we therefore sought to understand potential mechanisms of resistance in lymphoid models. Wild-type p53 H929 MM and Granta-519 MCL cells resistant to MI-63 or Nutlin were generated by exposing them to increasing drug concentrations. MI-63-resistant H929 and Granta-519 cells were resistant to Nutlin, whereas Nutlin-resistant cells displayed cross-resistance to MI-63. These cells also showed cross-resistance to bortezomib, doxorubicin, cisplatin, and melphalan, but remained sensitive to the small molecule inhibitor RITA (reactivation of p53 and induction of tumor cell apoptosis). HDM-2 inhibitor-resistant cells harbored increased p53 levels, but neither genotoxic nor nongenotoxic approaches to activate p53 induced HDM-2 or p21. Resequencing revealed wild-type HDM-2, but mutations were found in the p53 DNA binding and dimerization domains. In resistant cells, RITA induced a G(2)-M arrest, upregulation of p53 targets HDM-2, PUMA, and NOXA, and PARP cleavage. Combination regimens with RITA and MI-63 resulted in enhanced cell death compared with RITA alone. These findings support the possibility that p53 mutation could be a primary mechanism of acquired resistance to HDM-2 inhibitors in MCL and MM. Furthermore, they suggest that simultaneous restoration of p53 function and HDM-2 inhibition is a rational strategy for clinical translation.

P53-Mediated Senescence Impairs the Apoptotic Response to Chemotherapy and Clinical Outcome in Breast Cancer

Studies on the role of TP53 mutation in breast cancer response to chemotherapy are conflicting. Here, we show that, contrary to dogma, MMTV-Wnt1 mammary tumors with mutant p53 exhibited a superior clinical response compared to tumors with wild-type p53. Doxorubicin-treated p53 mutant tumors failed to arrest proliferation, leading to abnormal mitoses and cell death, whereas p53 wild-type tumors arrested, avoiding mitotic catastrophe. Senescent tumor cells persisted, secreting senescence-associated cytokines exhibiting autocrine/paracrine activity and mitogenic potential. Wild-type p53 still mediated arrest and inhibited drug response even in the context of heterozygous p53 point mutations or absence of p21. Thus, we show that wild-type p53 activity hinders chemotherapy response and demonstrate the need to reassess the paradigm for p53 in cancer therapy.

Abstract 1158: Clinical significance of p53 codon 72 polymorphism in hepatocellular carcinomas

Abstract Purpose: We recently demonstrated that, in colorectal carcinomas, the presence of a single nucleotide polymorphism (SNP) at codon 72 of the p53 gene was associated with a poor outcome for African American (AA) patients. Since such studies have not been performed for hepatocellular carcinomas (HCCs), we determined, for AAs and non-Hispanic Caucasians (NHCAs), the prevalence of p53 SNPs in codon 72 of HCCs and its association with adverse clinical and pathological features. Methods: DNA samples extracted from 48 archived, formalin-fixed, paraffin-embedded HCC tissues and from matching, uninvolved liver were analyzed for the mutational status of the p53 gene by sequencing exon 4 through exon 9. The results obtained were correlated with the demographic and clinicopathological features of the HCCs and with patient survival. Results: Overall, the frequency of missense point mutations in p53 of HCCs was low (6 of 48, 12%). Missense point mutations, however, were common in NHCAs (4 of 6). Genotyping of codon 72 of p53 demonstrated that the Arg/Arg phenotype had the highest frequency (27 of 48; 56%), followed by Arg/Pro (16 of 48; 33%) and Pro/Pro (5 of 48; 11%). Four of five patients with the Pro/Pro phenotype were AAs (χ2 p = 0.028). Of these, three of the four tumors had high proliferative (ki67) indices; two of the four were poorly differentiated. In contrast, poorly differentiated tumors were few in Arg/Arg (1 of 27, 4%) and Arg/Pro (1 of 16; 6%) (χ2p = 0.016) phenotype groups. For all patients, the Pro/Pro phenotype were associated with hepatitis C virus (HCV) infection (χ2 p = 0.048). Furthermore, AAs with the Pro/Pro phenotype had lower median survival (15.5 months) compared to those with the Arg/Arg and Arg/Pro (32 months) phenotypes. NHCA patients with missense point mutations had shorter median survival (22.5 months) relative to NHCAs with wild-type p53 (34 months).Conclusion: These preliminary findings suggest that, in AAs, the Pro/Pro phenotype at codon 72 of the p53 gene is associated with aggressive tumor behavior, shorter median survival, and HCV infection. These pilot studies were supported jointly by a Charles Barkley Award through the UAB Minority Health Disparities Research Center (MHRC) and the UAB Center for Clinical and Translational Science (5UL1 RR025777-04). 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 1158. doi:1538-7445.AM2012-1158

Abstract SY14-02: p53-mediated senescence impairs the apoptotic response to chemotherapy in breast cancer

Abstract Studies on the role of p53 mutation in human breast cancer response to chemotherapy are controversial. Using MMTV-Wnt1 murine breast tumor model, we show that tumors with mutant p53 exhibited a superior clinical response compared to tumors with wild-type p53. Doxorubicin treated p53-mutant tumors failed to arrest proliferation leading to abnormal mitoses and cell death, while p53 wild-type tumors underwent arrest and senescence with expression of senescence-associated cytokines that led to autocrine/paracrine activity and mitogenic potential. Wild-type p53 could still mediate arrest and inhibit drug response even in the context of a heterozygous p53 point mutation or absence of p21. Thus, we show wild-type p53 activity hinders chemotherapy response and demonstrate the need to reassess the paradigm for p53 in cancer therapy. 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 SY14-02. doi:1538-7445.AM2012-SY14-02

Drug Resistance to the HDM-2 Inhibitors MI-63 and Nutlin Is Mediated by Point Mutations of p53, but Can Be Overcome with the p53 Targeting Agent RITA

Abstract 2726 Background:The ubiquitin-proteasome pathway has been validated as a target for multiple myeloma (MM) and mantle cell lymphoma (MCL) by the demonstration of the effectiveness of the proteasome inhibitor bortezomib (BZB) in these malignancies. E3 ubiquitin ligases are involved in ubiquitination of a small subset of cellular proteins and play an integral part in regulation of turnover of cellular proteins. HDM-2 is the E3 ubiquitin ligase responsible for degradation and regulation of the p53 tumor suppressor, and HDM-2 inhibitors such as nutlin (NUT) and MI-63 stabilize p53 and enhance its pro-apoptotic effects in the absence of DNA damage. Methods:To evaluate the mechanisms through which wild type p53 MCL and MM cells could evade the therapeutic effects of HDM-2 inhibitors, we generated cell lines resistant to MI-63 and Nutlin. Resistant lines were generated by growing cells over a period of 3 months in increasing concentrations of either MI-63 or NUT until the cells were resistant to 10 mM of either agent. Results:H929 MM and Granta-519 MCL cells resistant to MI-63 (MI63R) were cross resistant to NUT, and NUT resistant (NUTR) cells displayed resistance to MI-63. Indeed, treatment of resistant cells with either MI-63 or NUT resulted in enhanced proliferation of these cells. MI-63R and NUTR cells showed enhanced resistance to both BZB and doxorubicin, whilst unexpectedly maintaining sensitivity to RITA (reactivation of p53 and induction of tumor cell apoptosis). Immunoblot analysis indicated increased levels of p53 in resistant cells at baseline and upon treatment with MI-63, NUT, or doxorubicin, but these treatments failed to induce p21 and HDM-2. Sequencing of genomic DNA from these cells for both HDM-2 and p53 genes indicated no alteration in the sequence of HDM-2. However, substitution mutations were found within p53 encompassing exons 4 through 8 for both NUTR and MI63R cells. When RITA was used in the resistant cells, an SG2 cell cycle arrest was seen at 48 hours post treatment. A time course analysis indicated that resistant cells treated with RITA displayed up-regulation of HDM-2, PUMA and NOXA from around 12 hours, with a corresponding induction of PARP cleavage from 48 to 72 hours. These data suggested RITA was able to restore wtp53 function resulting in induction of p53 transcriptional targets and thereby overcoming resistance to NUT and MI-63. The combination of either MI-63 or NUT with RITA demonstrated that simultaneous addition of the agents or pretreatment with RITA resulted in an enhancement of cell death over RITA alone. This result indicates that RITA restored the function of mutp53 within MI-63 and NUT resistant cells and thereby resensitized the cells to the effects of MI-63 or NUT. Conclusions:Chronic exposure of MM and MCL cells to HDM-2 inhibitors resulted in cross resistance to other inhibitors in the same class through mutation of p53 as the primary mechanism of resistance. HDM-2 inhibitor resistant cells did remain sensitive to the effects of RITA through restoration of p53 function, and restoring the sensitivity of such cells to agents that promote p53's pro apoptotic effects may be a rational strategy for translation to the clinic. Disclosures:No relevant conflicts of interest to declare.

P53 Dependent Centrosome Clustering Prevents Multipolar Mitosis in Tetraploid Cells

Backgroundp53 abnormality and aneuploidy often coexist in human tumors, and tetraploidy is considered as an intermediate between normal diploidy and aneuploidy. The purpose of this study was to investigate whether and how p53 influences the transformation from tetraploidy to aneuploidy.Principal FindingsLive cell imaging was performed to determine the fates and mitotic behaviors of several human and mouse tetraploid cells with different p53 status, and centrosome and spindle immunostaining was used to investigate centrosome behaviors. We found that p53 dominant-negative mutation, point mutation, or knockout led to a 2∼ 33-fold increase of multipolar mitosis in N/TERT1, 3T3 and mouse embryonic fibroblasts (MEFs), while mitotic entry and cell death were not significantly affected. In p53-/- tetraploid MEFs, the ability of centrosome clustering was compromised, while centrosome inactivation was not affected. Suppression of RhoA/ROCK activity by specific inhibitors in p53-/- tetraploid MEFs enhanced centrosome clustering, decreased multipolar mitosis from 38% to 20% and 16% for RhoA and ROCK, respectively, while expression of constitutively active RhoA in p53+/+ tetraploid 3T3 cells increased the frequency of multipolar mitosis from 15% to 35%.Conclusionsp53 could not prevent tetraploid cells entering mitosis or induce tetraploid cell death. However, p53 abnormality impaired centrosome clustering and lead to multipolar mitosis in tetraploid cells by modulating the RhoA/ROCK signaling pathway.

Recent advances in mutant p53 and novel personalized strategies for cancer therapy

Protein p53 is the most intensively studied tumor suppressor protein. Recent studies keep revealing its new function in metabolism and reproduction. At the same time, it is also found that varieties of p53 mutant gained new function in promoting tumorigenesis. These studies provide the basis for understanding the personalized gain of function of p53 mu-tants and help us searching for the new strategies for reactivation of wild-type p53 and correction of the function of p53 mutants. The personalized treatment targeting different p53 mutants will be the focus for cancer treatment. Here, we re-viewed the discovered gain of function of some p53 mutants and the molecular strategies for reactivating wild type p53 function: by use of small molecules or polypeptides to reactivate the wild type function of p53 mutants in tumor cells; by exogenous expression of wild type p53 carried by recombinant adenovirus in tumor cells; and by inhibition the interaction between p53 and mdm2 to stabilize wild type p53 proteins. Further study of variety of p53 point mutations farcilitates de-signing more effectively personalized strategies in the cancer therapy.

Abstract 2262: The prognostic value of p53 abnormalities in colorectal cancers varies based on detection methods

Abstract Background: Although abnormalities in p53 (gene mutations or nuclear accumulation) are considered, by most, but not all, investigators, as prognostic markers for colorectal cancers (CRCs), their clinical utility remains controversial. The conflicting findings may be attributed, in part, to technical variations in detecting p53 abnormalities. Therefore, this study examined the effect of detection methods on the prognostic value of p53 abnormalities in CRCs. Methods: We evaluated 107 CRCs and their corresponding normal snap-frozen and formalin-fixed paraffin-embedded (FFPE) tissues for p53 abnormalities. To detect p53 mutations, DNA extracted from frozen and FFPE tissues were analyzed by single-strand conformation polymorphisms (SSCP) and by direct sequencing using exon-specific primers, respectively. Also, the FFPE tissue sections were evaluated for p53 nuclear accumulation (p53nac) by immunohistochemistry (IHC), using an anti-human p53 monoclonal antibody (Clone BP53.12.1, BioGenex, San Ramon, CA), with and without antigen retrieval (AR). A correlation coefficient test was performed to determine the concordance between the molecular and IHC methods with different cutoff-values (>0%, 10%, 20%, and 50% positive cells) to categorize tumors as positive for p53nac. Data derived by the molecular and IHC methods were correlated independently with patient survival by Kaplan-Meier analyses. Subsequently, with an expanded CRC cohort of 339 tissues, we validated the findings obtained from the exon-wise direct DNA sequencing and IHC without AR. Results The highest concordance (79.5%, Kappa co-efficient 0.612; p<0.0001) was between p53nac by IHC without AR, at a cut-off value of ≥10% positive cells and mutations detected by molecular methods. Also, this IHC method detected 95% of the p53 point mutations. Although statistically not significant, Kaplan-Meier analyses demonstrated short CRC-specific survival for patients whose tumors exhibited p53nac, detected by IHC at ≥ 10% cut-off value without AR, (log-rank p value=0.0590); for patients with mutated p53, survival rates were similar to those derived by SSCP (log-rank p value=0.0592) and by direct DNA sequencing (log-rank p value=0.096). The validation studies demonstrated that abnormal p53, detected by IHC (log-rank p value=0.042) and direct sequencing methods (log-rank p value<0.0001), was associated with shorter patient survival. Conclusions: These findings demonstrate that IHC without AR is an effective approach to detect clinically useful p53 status and can be routinely used as a prognostic marker for colorectal cancer. These studies were supported by grants 2U54-CA118948-03, R01-CA98932, and R03-CA139629 from the NCI/NIH. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2262. doi:10.1158/1538-7445.AM2011-2262

Cancer genes alterations and HPV infection in oral squamous cell carcinoma

The aim of this study was to gain a better understanding of cancer genes contributing to oral squamous cell (OSCC) development and progression and correlate genetic changes to clinical parameters. Human papilloma virus (HPV) 16 detection is also included in the study. 60 samples of OSCC were analysed for c-erbB2 and c-myc amplification by dPCR, H-ras and p53 point mutations by PCR/SSCP. HPV was detected via amplification of its E1 and E6 genes. c-erbB2 was altered in 45%, c-myc in 35%, H-ras in 22% and p53 in 60% of samples. HPV was detected in 10% of cases. The frequency of p53 gene mutations showed a statistically significant association with tumour stage. Patients with c-erbB2 and H-ras alterations had lower survival than patients without these alterations. The number of detected genetic changes was remarkable but statistical association with tumour natural history was poor, indicating high clonal heterogeneity and multiple pathways of carcinogenesis.

902 Investigating Clonal Competition in Barrett's Associated Tumorigenesis Using Spatial Maps of Genetic Heterogeneity

Introduction Genetic heterogeneity within Barrett9s mucosa is significantly associated with the development of oesophageal adenocarcinoma (OA). Why genetic diversity promotes tumorigenesis is unclear. Pro-tumorigenic interaction between clones is a possible explanation. To look for evidence of clonal interaction within Barrett9s lesions, we have undertaken a high-resolution clonal-ordering analysis to produce phylogenetic trees and spatial maps that illustrate the spread of genetically distinct clones in endoscopic mucosal resection specimens (EMRs). Methods Entire EMR specimens were serially sectioned. Each crypt was classified according to histological grade. Aneuploidy was assessed using image cytometry. More than 50 individual crypts were then laser-capture microdissected from each EMR. The genetic mutation burden of individual crypts was assessed (K-ras, p53 and p16 point mutations and microsatellite loss of heterozygosity (LOH) data for up to16 loci on chromosomes 3p (FHIT), 5q (APC), 9p (p16), 17p (p53), 17q and 18q (SMAD4)). In each EMR, a point mutation in at least one gene was identified. The relative location of each analysed crypt was recorded and used to reconstruct a genetic and histological map of each EMR. Results EMRs were rarely genetically homogeneous. Typically, dysplastic crypts within individual lesions were clonal for a founding p53 or p16 point-mutated clone, whereas the adjacent metaplastic (non-dysplastic) crypts analysed did not contain the point mutation. Mosaic subclones with distinct patterns of LOH (often 18q and/or 3p) were present. In one EMR, the same p53 and p16 point mutations and 5q and 17p LOH were detected across the entire EMR. Conclusion Clonal expansion of mutated cells is frequently restricted to small numbers of crypts. Interactions between genetically distinct clones appears to be predominantly competitive: a crypt acquiring an additional pro-tumorigenic mutation can outcompete, and so clonally expand to the detriment of, the original crypts. Further investigation will reveal whether this progression is driven by mutation and selection or by additional mutations altering the behaviour of neighbouring pre-existing clones.