Mutant P53 Expression Research Articles

Mutant p53 protein is prevalent in Aflatoxin B1 than in Hepatitis B Core antigen associated chronic liver diseases in Southern Nigeria

Background: Aflatoxin B1 (AFB1), hepatitis B virus (HBV) and p53 mutation are major risk factors for liver cancer. However, the presence of AFB1 and expression of mutant p53 protein during active HBV replication (nuclear expression of Hepatitis B Core Antigen; HBcAg) and HBV clearance (nuclear expression of HBcAg) is still under-investigated in Africa. Objectives: This study assessed the prevalence, extent of expression, interaction, and risk associated with single and binary expression of AFB1, HBcAg and mutant p53 (mtp53) in liver diseases in relation to age and sex. Materials and Methods: This retrospective study included 14 and 74 cases of liver cancer (LC) and chronic hepatitis, respectively. Tissues were histochemically and immunohistochemically stained, scored and documented as positive (+) or negative (-) for antigen. Result: In this study, the prevalence of AFB1, HBcAg, and mtp53 were 76.1%, 40.9%, and 61.4%, respectively. Higher prevalence and expression of AFB1 was observed in chronic hepatitis than in LC (p= 0.035 and 0.028, respectively). Higher prevalence of AFB1 and mtp53 and lower prevalence of HBcAg were observed in females than in males (p= 0.186, 0.0003 and 0.062, respectively). Cytoplasmic expression of HBcAg was higher in males (58 than in females were 58.6% (17/29) and 14.3% (1/7), respectively (p= 0.088). AFB1+ and HBcAg+ females were more and less likely to develop LC than males (OR: 3.00 and 0.39, 95%CI: 0.10-4.18 and 0.44-12.20, p= 0.012 and 0.003), respectively. AFB1+mtp53- (23.9%), AFB1+mtp53+ (52.3%), HBcAg+mtp53- (13.6%), HBcAg+mtp53+ (27.3%) and AFB1+HBcAg+ (34.1%) increased liver cancer risk (OR: 6.45, 21.50, 2.80, 0.78, and 5.11; 95%CI: 0.89-29.29, 0.95-43.86, 2.96-156.13, 0.60-13.01, and 0.14-4.25; p= 0.057, 0.002, 0.189, 0.772, and 0.067, respectively). Conclusion: This study suggests that women are at a higher risk of AFB1 exposure and p53 mutation, but are at a lower risk of viral replication than men.

Expression of WDR79 is associated with TP53 mutation and poor prognosis in surgically resected non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) represents a major health burden globally. WD repeat protein 79 (WDR79) is a member of the WD-repeat protein family. WDR79 is a highly conserved and natural antisense transcript to TP53 gene and involved in carcinogenesis of various types of cancer. Whether the alterations of WDR79 protein expression are associated with TP53 mutation and clinicopathological and prognostic implications in the patients with surgically resected NSCLC have not been reported. The purposes of the present study are to investigate the association between the expression of WDR79 and mutant p53 (mtp53) and clinicopathological features in NSCLC by immunohistochemistry. The results showed that positive expression of WDR79 (58.8%, 170/289) and mtp53 (48.1%, 139/289) in NSCLC was significantly higher than that in non-cancerous control lung tissues (5.7%, 3/53 and 1.9%, 1/53, respectively). There was a significantly higher positive percentage of WDR79 expression in NSCLC with lymph node metastasis. The statistically positive correlation between WDR79 and mtp53 expression (r = 0.212, P=0.014) was identified by Spearman's rank correlation analysis. Kaplan-Meier survival curve analysis indicated that positive expression of WDR79 and common positive expression of WDR79 and mtp53 were correlated with poor overall survival rates in NSCLC patients (P = 0.029 and P = 0.041, respectively). Multivariate Cox regression analysis further identified that WDR79 positive expression was an independent unfavorable prognostic factor of NSCLC (P = 0.034). Taken together, positive expression of WDR79 proteins may be related with TP53 mutations and act as valuable independent biomarker to predict poor prognosis of patients with surgically resected NSCLC.

Establishment of a rat model of dimethylbenzanthracene-induced vulvar squamous intraepithelial lesions

To establish a SD rat model of vulvar squamous intraepithelial lesions. Seventy female SD rats were randomized into 4 groups, namely the blank control group (n=10), mechanical irritation group (n=10), acetone solution group (n=10), and mechanical irritation with DMBA acetone solution group (n=40, model group), and the corresponding treatments were administered 3 times a week for 14 weeks. The changes of the vulvar skin of the rats were observed regularly until the 18th week. The expression of mutant p53 (mtp53) and vascular endothelial growth factor (VEGF) proteins were detected using immunohistochemistry and Western blotting, and the expressions of mtp53 and VEGF mRNA were detected with qRT- PCR in the blank control group and model group. No significant differences were found in the morphological or histopathological changes of the skin among the blank control group, mechanical irritation group and acetone solution group. In the model group, low-grade squamous intraepithelial lesions (LSIL) occurred in 28 rats (70%) and high-grade squamous intraepithelial lesions (HSIL) in 11 rats (27.5%) at 14 weeks, with a success rate of 97.5% in inducing vulvar squamous intraepithelial lesions. Compared with the blank control group, the rats in the model group showed significantly increased expressions of mtp53 and VEGF at both the protein level (P < 0.05) and the mRNA level (P < 0.05). DMBA in acetone solution combined with mechanical irritation can induce vulvar squamous intraepithelial lesions in female SD rats.

MDM2, MDM2-C, and mutant p53 expression influence breast cancer survival in a multiethnic population.

The purpose of the study was to examine the association between expression of mutant p53 (mtp53), full-length MDM2 (MDM2), and MDM2 isoform C (MDM2-C) and survival in multiethnic breast cancer patients. A total of 787 invasive breast tumors included in a clinically annotated multiethnic population-based tissue microarray (TMA) were screened utilizing commercially available antibodies to p53 and MDM2, and a newly developed monoclonal antibody recognizing MDM2-C. Mutant p53 (mtp53) was more common in younger (< 50years) breast cancer patients. Among the 787 cases included in the study, mtp53, MDM2, and MDM2-C expression were not significantly associated with risk of overall or breast cancer-specific mortality. However when associations within individual racial/ethnic groups (White, Japanese, and Native Hawaiian) were examined, expression of MDM2-C was found to be associated with lower risk of breast cancer-specific mortality exclusively for White patients HR 0.32, 95% CI 0.15-0.69 and mtp53 expression was associated with higher overall mortality in Japanese patients (HR 1.63, 95% CI 1.02-2.59). Also, Japanese patients positive for the joint expression of MDM2-C and mtp53 had a greater than twofold risk of overall mortality (HR 2.15, 95% CI 1.04-4.48); and White patients with positive MDM2-C and wild-type p53 expression (HR 0.28, 95% CI 0.08-0.96) were at lower risk of mortality when compared to patients withnegative MDM2-C and wild-type p53 expression in their respective racial/ethnic group. Racial/ethnic differences in expression profiles of mtp53, MDM2, and MDM2-C and associations with breast cancer-specific and overall mortality. MDM2-C may have a positive or negative role in breast tumorigenesis depending on mtp53 expression.

Ten-year results of the PORTEC-2 trial for high-intermediate risk endometrial carcinoma: improving patient selection for adjuvant therapy

BackgroundPORTEC-2 was a randomised trial for women with high-intermediate risk (HIR) endometrial cancer, comparing pelvic external beam radiotherapy (EBRT) with vaginal brachytherapy (VBT). We evaluated long-term outcomes combined with the results of pathology review and molecular analysis.Methods427 women with HIR endometrial cancer were randomised between 2002–2006 to VBT or EBRT. Primary endpoint was vaginal recurrence (VR). Pathology review was done in 97.4%, combined with molecular analysis.ResultsMedian follow-up was 116 months; 10-year VR was 3.4% versus 2.4% for VBT vs. EBRT (p = 0.55). Ten-year pelvic recurrence (PR) was more frequent in the VBT group (6.3% vs. 0.9%, p = 0.004), mostly combined with distant metastases (DM). Ten-year isolated PR was 2.5% vs. 0.5%, p = 0.10, and DM 10.4 vs. 8.9% (p = 0.45). Overall survival for VBT vs. EBRT was 69.5% vs. 67.6% at 10 years (p = 0.72). L1CAM and p53-mutant expression and substantial lymph-vascular space invasion were risk factors for PR and DM. EBRT reduced PR in cases with these risk factors.ConclusionLong-term results of the PORTEC-2 trial confirm VBT as standard adjuvant treatment for HIR endometrial cancer. Molecular risk assessment has the potential to guide adjuvant therapy. EBRT provided better pelvic control in patients with unfavourable risk factors.

Effects of liquid ionic silver concentration on caspase-3 and p53mt expressions in the oral mucosal epithelium of Wistar rats

Background: Silver, especially oxidized silver, has been used as a medicine considered to have bactericidal properties. In the present day, ionic silver (Ag+) is also used in the manufacture of cosmetics, socks, food containers, detergents, sprays and other products to prevent the spread of germs. Unfortunately, ionic silver is assumed to be toxic not only to bacteria, but also to humans and the environment. Therefore, it is essential to identify the optimum dosage of ionic silver considered safe by investigating the effects of ionic silver concentration on cell death through activation of mutant p-53 expression by caspase 3 in the oral epithelium. Purpose: This research aimed to analyze the effects of concentrated liquid ionic silver (Ag+) on caspase-3 and mutant p53 expression in the oral mucosal epithelium. Methods: This research constituted a laboratory-based experimental study with posttest-only design. The research subjects consisted of 28 Wistar rats, divided into four treatment groups, namely; KK (with Aquadest), KP 1 (with 5% liquid ionic silver), KP 2 (with 10% liquid ionic silver) and KP 3 (with 15% liquid ionic silver). Each rat was then treated orally with 0.5ml of liquid ionic silver at fixed concentrations twice a day for seven days. The Wistar rats were then terminated and their tissue samples processed by means of histopathological and immunohistochemical staining examination. The monoclonal Caspase-3 and mutant p53 expressions in each group were evaluated with the data being tabulated and analyzed statistically. Results: Mutant p53 expression was also found in the control group. Moreover, the higher the concentration of liquid ionic silver, the greater the elevated Caspase-3 and mutant p53 expressions. Conclusion: The concentration of liquid ionic silver plays an important role in elevating Caspase-3 and mutant p53 expressions.

Differential anticancer activities of arsenic trioxide on head and neck cancer cells with different human papillomavirus status

AimsApproximately 20% of head and neck squamous cell carcinomas (HNSCCs) are caused by human papillomavirus (HPV) infection. The effect of arsenic trioxide (ATO) on HPV oncogene expression of HNSCC cells remains unknown. In this study, we investigated the anti-cancer activity and possible molecular pathways of ATO on the six HNSCC cell lines (three HPV-positive and three HPV-negative). MethodsThe effects of ATO on the cell proliferation, apoptosis, cell cycle of HNSCC cells were analyzed using CCK-8 assay, colony formation and flow cytometry. Transwell assay was used to examine the effect of ATO on cell migration. The transcriptional and protein expression of key genes were determined by real-time PCR and Western blot, respectively. Using a xenograft model, we assessed the effects of ATO on HNSCC cells in vivo. Key findingsHPV-positive and -negative HNSCC cells had different expression of key genes. ATO inhibited HNSCC cell proliferation and migration and induced apoptosis and these effects were more significant in HPV-positive HNSCC cells than in HPV-negative HNSCC cells. ATO treatment reduced the expression of HPV16-E6/E7 and cyclin D1 proteins and enhanced the expression of p16, pRb, and p53 in HPV-positive HNSCC cells. By contrast, ATO treatment reduced the expression of epidermal growth factor receptor, cyclin D1 and mutant p53 and enhanced the expression of pRb in HPV-negative HNSCC cells. Anti-cancer effect of ATO on HNSCCs was confirmed by inhibiting xenograft growth in vivo. SignificanceOur data suggest that ATO is a potential therapeutic drug for HNSCCs, especially HPV-positive HNSCCs.

Genomic instability in mutant p53 cancer cells upon entotic engulfment

Cell-in-cell (CIC) structures are commonly seen in tumours. Their biological significance remains unclear, although they have been associated with more aggressive tumours. Here we report that mutant p53 promotes CIC via live cell engulfment. Engulfed cells physically interfere in cell divisions of host cells and for cells without p53 this leads to host cell death. In contrast, mutant p53 host cells survive, display aberrant divisions, multinucleation and tripolar mitoses. In xenograft studies, CIC-rich p53 mutant/null co-cultures show enhanced tumour growth. Furthermore, our results show that CIC is common within lung adenocarcinomas, is an independent predictor of poor outcome and disease recurrence, is associated with mutant p53 expression and correlated to measures of heterogeneity and genomic instability. These findings suggest that pro-tumorigenic entotic engulfment activity is associated with mutant p53 expression, and the two combined are a key factor in genomic instability.

Abstract A26: PTEN and colonization of the ovary in metastasis of fallopian tube-derived ovarian cancer

Abstract It is now clear that high-grade serous ovarian cancer (HGSOC) can originate in the fallopian tube epithelium (FTE), making the role of the ovary in these tumors unclear. Therefore, the objectives of this research were to 1) determine if colonization of the ovary was an important step in the spread of FTE-derived tumors to the peritoneum and 2) explore the role of ovarian rupture, independent of hormonal changes, in tumor cell colonization of the ovary. First, 50,000 murine oviductal epithelial (MOE) cells stably expressing PTENshRNA+KRASG12V were allografted into the left ovarian bursa (IB) or peritoneal space (PS). IB-grafted mice developed tumors, with all mice requiring sacrifice within 90 days. Tumors were detected throughout the peritoneum and in the contralateral ovary. In contrast, PS-grafted mice failed to develop tumors for the duration of the experiment (150 days). To determine if rupture of the ovary was important in colonization of the ovary, an ex vivo attachment assay was developed in which ovaries was left intact or cut with a scalpel to rupture the ovarian surface (ovulation mimetic) without associated hormonal changes. Ovulation mimetic ovaries had significantly more MOE GFP and OVCAR8 RFP cells attached, particularly in the wounded areas. To confirm that increased attachment was due to exposure of the ovarian stroma, MOE GFP cells were plated on monolayers of murine ovarian surface epithelium (MOSE), monolayers of murine ovarian stroma (MOST), or three-dimensional (3D) type I collagen gels (the primary extracellular matrix protein in the ovarian stroma). MOE GFP cells attached to MOST more than MOSE and to 3D collagen than MOST, supporting the contention that FTE cells adhere to the extracellular matrix (ECM), such as collagen, exposed during ovulation. We next sought to test the ability of cells to survive in an environment rich in 3D collagen. 3D collagen reduced the viability of normal epithelial cells (MOSE, MOE, and IOSE80) but had no effect on HGSOC cells (OVCAR3, OVCAR4, OVCAR5, OVAR8, and OVSAHO), indicating that transformation of FTE is important for survival in the collagen-rich ovarian stroma. RNA-seq showed that 3D collagen decreases genes associated with focal adhesion, regulation of actin cytoskeleton, and ECM-receptor interaction, supporting the idea that normal epithelial cells respond poorly to a microenvironment rich in 3D collagen. In contrast, genes associated with p53 signaling and suppression of MAPK/AKT were increased when cells were grown on 3D collagen. However, plating cells on 3D collagen did not lead to stabilization of p53 and expression of mutant p53 had no effect on viability of MOE cells on 3D collagen. KRASG12V tended to increase viability, but only PTENshRNA completely rescued viability on 3D collagen as compared to 2D. PTENshRNA had no effect on adhesion to 3D collagen, but it did increase invasion through collagen. Cells expressing PTENshRNA showed increased attachment to 3D ovaries that were wounded to mimic ovulation; however, the AKT inhibitor MK2206 did not reduce this adhesion. Interestingly, after 7 days of culture on 3D collagen, MOE PTENshRNA cells formed spheroids while controls did not, leading to the hypothesis that spheroids may mediate metastasis from the fallopian tube to the ovary. In support of this contention, spheroids were observed in the fallopian tube of PAX8+/cre PTENflox/flox mice, which develop fallopian tube-derived ovarian cancer. Furthermore, OVCAR8 RFP spheroids preferentially attached to wounded sites mimicking ovulation relative to intact ovaries. In conclusion, FTE cells attached to exposed ECM of the ovary and colonization of the ovary enhances further spread of FTE-derived tumors beyond the reproductive tract. Inhibition of ovarian colonization may be a new approach to inhibit peritoneal spread of cancer in women at high risk of developing HGSOC. Citation Format: Matthew Dean, Vivian Jin, Angela Russo, Joanna E. Burdette. PTEN and colonization of the ovary in metastasis of fallopian tube-derived ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A26.

Abstract A31: Mutant p53 increases integrin-ECM interactions in early HGSOC

Abstract Until recently, it was thought that high-grade serous ovarian cancer (HGSOC) arises from the ovarian surface epithelium (OSE). Now, it has become clear that the epithelium of the fallopian tube fimbriae is the starting point of many HGSOCs. Serous tubal intraepithelial carcinomas (STICs) are suggested to be the earliest form of HGSOC and show mutations in the p53 gene. Part of the neoplastic transformation into invasive HGSOC may be the initial adhesion of STIC cells to the surface of the ovary as a first site of metastasis. To examine the role of mutant p53 immortalized fallopian tube secretory epithelial (FTSE) cells in the early onset of HGSOC, we used mechanically tunable hydrogels and developed a protocol to obtain decellularized human ovary scaffolds for 3D cell cultures. Stable expression of mutant p53 in FTSE cells promoted adhesion to hydrogel matrices and invasion into decellularized ovary scaffolds. Next, we sought to analyze the extracellular matrix (ECM) landscape of human fallopian tube and ovary tissues and identified proteins with a high affinity to integrin αvβ3. Mutant p53 FTSE cells had increased levels of integrin αvβ3 and decreased adhesive potential upon integrin αvβ3 blockage compared to wild-type FTSE cells. The results indicate that FTSE cells expressing mutant p53 upregulate integrin expression, which in turn alters their tissue-specific ECM interactions. Our findings give new insights in the early onset of HGSOC and may help to understand the role of p53 in the cells of origin of HGSOC. Citation Format: Laura SM Lecker, Caterina Trevisan, Robin Delaine-Smith, Eleni Maniati, Oliver Pearce, Christoph Meinert, Marcin Iwanicki, Ronny Drapkin, Daniela Loessner, Frances Balkwill. Mutant p53 increases integrin-ECM interactions in early HGSOC. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A31.

Ascorbic Acid Chemosensitizes Colorectal Cancer Cells and Synergistically Inhibits Tumor Growth.

Colorectal cancer (CRC) is continuously classified as one of the most incidental and mortal types of cancer worldwide. The positive outcomes of the conventional chemotherapy are frequently associated with high toxicity, which often leads to the suspension of the treatment. Growing evidences consider the use of pharmacological concentrations of ascorbic acid (AA), better known as vitamin C, in the treatment of cancer. The use of AA in a clinical context is essentially related to the adoption of new therapeutic strategies based on combination regimens, where AA plays a chemosensitizing role. The reduced sensitivity of some tumors to chemotherapy and the highly associated adverse effects continue to be some of the major obstacles in the effective treatment of CRC. So, this paper aimed to study the potential of a new therapeutic approach against this neoplasia with diminished side effects for the patient. This approach was based on the study of the combination of high concentrations of AA with reduced concentrations of drugs conventionally used in CRC patients and eligible for first and second line chemotherapeutic regimens, namely 5-fluorouracilo (5-FU), oxaliplatin (Oxa) or irinotecan (Iri). The evaluation of the potential synergy between the compounds was first assessed in vitro in three CRC cell lines with different genetic background and later in vivo using one xenograft animal model of CRC. AA and 5-FU act synergistically in vitro just for longer incubation times, however, in vivo showed no benefit compared to 5-FU alone. In contrast to the lack of synergy seen in in vitro studies with the combination of AA with irinotecan, the animal model revealed the therapeutic potential of this combination. AA also potentiated the effect of Oxa, since a synergistic effect was demonstrated, in almost all conditions and in the three cell lines. Moreover, this combined therapy (CT) caused a stagnation of the tumor growth rate, being the most promising tested combination. Pharmacological concentrations of AA increased the efficacy of Iri and Oxa against CRC, with promising results in cell lines with more aggressive phenotypes, namely, tumors with mutant or null P53 expression and tumors resistant to chemotherapy.

Abstract 2988: Loss of Rbm38 cooperates with mutant p53 to promote lymphomagenesis through downregulation of Pten

Abstract The tumor suppressor TP53 is the most commonly mutated gene in human cancers and mutant p53 has a gain of function by promoting tumor progression, metastasis, and resistance to anticancer therapy. Interestingly, the RNA binding protein Rbm38, a critical regulator of mutant p53 translation, is frequently lost in tumors with mutant p53. To determine the role of the Rbm38-mutant p53 axis in tumorigenesis, we showed that loss of Rbm38 significantly alters the cancer susceptibility of mutant p53 knockin mice by shortening the lifespan, altering the tumor incidence, and promoting T-cell lymphomagenesis. To understand how Rbm38 deficiency promotes mutant p53 gain of function, we showed that loss of Rbm38 not only enhances mutant p53 expression but also decreases expression of tumor suppressor Pten, a critical regulator of T-cell development. Furthermore, we showed that Rbm38 is required for Pten expression by stabilizing Pten mRNA through an AU-rich element in its 3'UTR. Thus, we have uncovered a novel mechanism by which Pten is regulated by Rbm38 via mRNA stability. Our data also suggest that Rbm38 controls T-cell lymphomagenesis by concomitantly modulating mutant p53 and Pten, which may be explored as a therapeutic strategy for the treatment of T-cell malignancies. Citation Format: Jin Zhang, Heejung yang, Yanhong Zhang, Mingyi Chen, Xinbin Chen. Loss of Rbm38 cooperates with mutant p53 to promote lymphomagenesis through downregulation of Pten [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 2988.

Abstract 269: Reactivation of mutant p53 in human prostate cancer cells as a critical mechanism for inhibition of tumor growth by phenethyl isothiocyanate

Abstract Mutations in the p53 gene occur in a wide variety of human cancers at remarkably high frequencies. Prostate cancer (PCa), the most commonly diagnosed cancer and the second leading cause of cancer-related death in men in the United States, show a significantly high frequency of p53 mutations (~30%-70%). However, the role of mutant p53 as a target for the treatment or prevention of PCa remained to be investigated. In this study, we showed that cruciferous vegetable-derived phenethyl isothiocyanate (PEITC) can reactivate p53 mutants, irrespective of its site of mutation, in PCa cells in vitro. Importantly, this study showed that dietary supplementation of PEITC can significantly inhibit DU145 p53 mutant xenograft tumor in a nu/nu Balb/c mice. Interestingly, the animals in the PEITC-treated group showed a significant decrease in the mutant p53 expression levels as compared to the animals in the control group. DU145 cells harboring p53V274F/P223L mutant display maximum sensitivity to PEITC-induced growth inhibition as compared to the wild-type p53 LNCaP and p53 null PC-3 cells. PEITC treatment also caused a significant decrease in the p53 expression level in DU145 cells. No significant inhibition of cell proliferation was detected for the normal prostate epithelial RWPE1 cells treated with PEITC. Mechanistic studies revealed that PEITC induces a conformation change and restores the transactivation functions to the p53 mutant. Accordingly, in PEITC-treated cells the reactivated p53 mutant(s) induces apoptosis by activating canonical wild-type p53 targets, inducing a G1 and S phase delay and by phosphorylating ATM. PEITC treatment of isogenic PC-3 p53 null cells revealed that PC-3 cells expressing p53P223L mutant exhibit maximum inhibition of cell proliferation, suggesting that PEITC acts in a p53P223L mutant-dependent manner. Furthermore, PEITC treatment restores the transactivation functions to p53P223L mutant as shown by the activation of p53 regulated downstream target genes. Interestingly p73, a member of the p53 gene family that shares high functional homology and regulate cell-growth/ cell-death pathways in a manner similar to p53, does not play a role in p53-mediated pathway activation in PEITC treated cells. We further showed that PEITC inhibits cell proliferation and induces apoptosis in prostate cancer cells with hotspot mutants R248W (VCaP) and R175H (LAPC-4). Importantly, PEITC can induce a conformation change, reactivates p53 mutants and induces G1 and S-phase delays in these cells. These results demonstrate that PEITC can reactivate mutant p53, irrespective of the site of p53 mutation, in human prostate cancer cells. These studies provide the first example of targeting mutant p53 in prostate cancer cells by a dietary-related compound that may have impact on the prevention and treatment strategies of this disease. Citation Format: Monika Aggarwal, Rahul Saxena, Nasir Asif, Elizabeth Sinclair, Judy Tan, Idalia Cruz, Deborah Berry, Bhaskar Kallakury, Quynhchi Pham, Thomas Ty Wang, Fung-Lung Chung. Reactivation of mutant p53 in human prostate cancer cells as a critical mechanism for inhibition of tumor growth by phenethyl isothiocyanate [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 269.

PO-330 Prevention of worsening in glioma relapse

IntroductionGliomas are primary tumours, which have aggressively and resiliency characteristics. Even in the last years having proposals of new therapies, the results continue frustrating for this tumour, so developing biocompounds with view to glioma adjuvant will bring very useful to increase therapeutic response to the recurrences. Hydrolysed Rutin (HR), biocompound modified from the de-glycolization of rutin, already demonstrated antiproliferative capacity in vitro for this tumour type. This study intends to confirm the HR antiproliferative action and antitumor activity on human glioblastoma/U251 and to investigate the mechanism related to its action as a potential agent modifying the tumour aggressiveness, with a view to its application in the primary tumour, also as on recurrences.Material and methodsAfter the obtainment of HR, it was used on antiproliferative assays and flow cytometry to verify the cell cycle and apoptosis induction on human glioblastoma/U251 in vitro. On animal glioblastoma model, divided in groups treated (prophylactic and therapy) and untreated, was measured lipid peroxidation, genotoxicity and verified HR antitumoral and the modification of aggressively pattern by histopathological and immunohistoquemistry analysis. The data was analysed adopting p-value lower than 5% (p<0.05).Results and discussionsTGI was 3.6 µg/mL. G1/S delay was observed in treated groups. Large proliferation of undifferentiated cells, with anaplastic degree and high index of atypical mitoses, forming solid neoplastic blocks was found. Tumour growth inhibition can be observed both in prophylactic (p=0.00) and in the therapeutic use of HR (p=0.03). Three cellular patterns were observed that were randomly distributed among groups, which characterises the multiple histological glioblastoma presentations. The HR decrease mitosis and anaplasia. There was a decrease in mitoses, as well as the expression of MDA, without induction of genotoxicity in the tumours submitted to HR treatment when compared to the untreated. In the control group 20% of necrosis was observed, whereas in the prophylactic and treatment groups no necrosis was observed. The number of mitoses was lower in the prophylactic groups (p=0.00) than in the control group. Mutated p53 expression remained the same in all groups, treated or not.ConclusionThe de-glycolization of rutin has potential to be used not only as adjuvant, but also as modifier of tumour aggressively, especially important as a new therapeutic weapon in glioma relapse.

Evaluation of epithelial dysplasia adjacent to lip squamous cell carcinoma indicates that the degree of dysplasia is not associated with the occurrence of invasive carcinoma in this site.

We analyzed the different grades of dysplasia in the epithelium adjacent to lip squamous cell carcinoma (LSCC), as a parallel to actinic cheilitis (AC) that suffered malignant transformation. Forty samples of epithelium adjacent to LSCC were histologically graded according to the World Health Organization (WHO) and the binary systems. The expression of mutated p53 was evaluated through immunohistochemistry. According to WHO system, 37.5% of the cases were graded as mild, 45% as moderate and 17.5% as severe dysplasia (P = 0.09). Considering the binary system, 90% of the cases were classified as low-risk and 10% as high-risk lesions. Mutated p53 was present in 73.3% of mild, 88.8% of moderate and 71.4% of severe dysplasia cases. Considering the binary system, 80.5% of the low-risk and 75% of high-risk lesions were immunopositive; 62.5% expressed the protein in both tumor cells and adjacent epithelium; 17.5% in adjacent epithelium only, and 7.5% in LSCC islands only (P = 0.03). We observed heterogeneous grades of epithelial dysplasia in the epithelium adjacent to LSCC, which indicates that the analysis of AC morphological features is insufficient to predict patient's prognosis and to determine a treatment decision. Positive expression of mutant p53 in mild dysplasia reinforces this idea.

Mutant p53-Expressing Cells Undergo Necroptosis via Cell Competition with the Neighboring Normal Epithelial Cells

p53 is a tumor suppressor protein, and its missense mutations are frequently found in human cancers. During the multi-step progression of cancer, p53 mutations generally accumulate at the mid or late stage, but not in the early stage, and the underlying mechanism is still unclear. In this study, using mammalian cell culture and mouse exvivo systems,we demonstrate that when p53R273H- or p53R175H-expressing cells are surrounded by normal epithelial cells, mutant p53 cells undergo necroptosis and are basally extruded from the epithelial monolayer. When mutant p53 cells alone are present, cell death does not occur, indicating that necroptosis results from cell competition with the surrounding normal cells. Furthermore, when p53R273H mutation occurs within RasV12-transformed epithelia, cell death is strongly suppressed and most of the p53R273H-expressing cells remain intact. These results suggest that the order of oncogenic mutations in cancer development could be dictated by cell competition.

Andrographolide induces degradation of mutant p53 via activation of Hsp70

The tumor suppressor gene p53 encodes a transcription factor that regulates various cellular functions, including DNA repair, apoptosis and cell cycle progression. Approximately half of all human cancers carry mutations in p53 that lead to loss of tumor suppressor function or gain of functions that promote the cancer phenotype. Thus, targeting mutant p53 as an anticancer therapy has attracted considerable attention. In the current study, a small-molecule screen identified andrographlide (ANDRO) as a mutant p53 suppressor. The effects of ANDRO, a small molecule isolated from the Chinese herb Andrographis paniculata, on tumor cells carrying wild-type or mutant p53 were examined. ANDRO suppressed expression of mutant p53, induced expression of the cyclin-dependent kinase inhibitor p21 and pro-apoptotic proteins genes, and inhibited the growth of cancer cells harboring mutant p53. ANDRO also induced expression of the heat-shock protein(Hsp70) and increased binding between Hsp70 and mutant p53 protein, thus promoting proteasomal degradation of p53. These results provide novel insights into the mechanisms regulating the function of mutant p53 and suggest that activation of Hsp70 may be a new strategy for the treatment of cancers harboring mutant p53.

Association of Protein Expression p53 Mutants with Regional Lymph Gland Status on type III Carcinoma Nasofaring Patients.

Nasopharyngeal carcinoma (NPC) is a malignant disease originating from the nasopharyngeal epithelial cells. The molecular mechanism of cancer occurrence is a change in the oncogene and tumor suppressor genes. One of the tumor suppressor genes that mutate in cancer cells is the mutant p53 gene. One of nasopharyngeal carcinoma progression is determined by the status of regional lymph gland. The enormous regional lymph node has a poor prognosis. To analyze the expression of the mutant p53 protein in Nasopharyngeal carcinoma (NPC) that correlated with regional lymph gland status (N) as a clinical manifestation. Expression of mutant p53 protein from NPC tissue paraffin block with immunohistochemical cracking technique was using monoclonal rabbit Anti Human p53 clone 318-6-11 (Dako, North America, Inc., 6392 Via Real Carpinteria, CA 93013 USA), microscope light binoculars was assessed visually by an Anatomical Pathology Consultant. Positive expression of p53 mutants was obtained 57.58% from all the sample in N0 by 0 subjects, N1 was 6 subjects, N2 was 7 subjects, and N3 was 7 subjects. The results of Mann-Whitney U test was p = 0.706, then there was no significant (p > 0.05) correlation between positive expression of p53 protein in type III WHO NPC with the regional lymph gland were N0, N1, N2, and N3. There was no significant between expression protein p53 mutants' regional and lymph gland in type III WHO NPC.

Abstract A28: Understanding the role of the tumor suppressor p53 in pancreatic cancer development

Abstract In the next decade, pancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer-related death. This high rate of mortality is attributed to late-stage diagnosis and lack of effective therapeutic strategies. The tumor-suppressor gene TP53 is mutated in ~75% of PDAC patients. The most frequently observed TP53 mutations in PDAC occur within the DNA binding domain at codons 175 and 273. TP53 mutations may induce loss of wild-type p53 function and gain of novel, oncogenic functions. We aim to use different mouse models of PDAC to identify the molecular pathways deregulated when wild-type p53 is lost and mutant p53 is expressed, to elucidate the molecular mechanisms driving PDAC development. While previous studies have suggested that point mutant p53 drives gain-of-function phenotypes, our studies of aging Pdx1-Cre;KrasLSL-G12D/+;Rosa26LSL-tdTomato/LSL-tdTomato mice with conditional p53 knockout (p53Flox/Flox or p53Flox/Null) or p53 mutation (p53LSL-R172H/Flox or p53LSL-R172H/Null) did not demonstrate a clear mutant p53 gain-of-function phenotype in tumor latency. We hypothesize that tumor latency is too rapid in these models to manifest a clear gain-of-function phenotype, requiring the establishment of different model systems. Indeed, our preliminary studies using tumor-derived p53R172H cell lines and isogenic p53 null cell lines suggest a role for mutant p53 gain of function in later-stage tumorigenesis, specifically in metastatic seeding. Furthermore, we are evaluating the role of the putative PDAC cells of origin, pancreatic acinar and ductal cells, in PDAC development. Using both tumor-derived mouse cell lines and pancreatic lineage-specific genetically engineered mouse models of PDAC, we can evaluate the consequences of loss of wild-type p53 expression and gain of mutant p53 expression in different pancreatic compartments. Ultimately, using these systems we aim to elucidate the target genes and molecular pathways controlled by wild-type p53 or by mutant p53 to regulate PDAC initiation. Understanding the molecular mechanisms driving PDAC initiation is critical for the development of new therapeutic strategies for early detection and effective intervention in PDAC patients. Citation Format: Brittany M. Flowers, Patty B. Garcia, Barbara M. Grüner, Monte M. Winslow, Laura D. Attardi. Understanding the role of the tumor suppressor p53 in pancreatic cancer development [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A28.

Endogenous Leu332Gln mutation in p53 disrupts the tetramerization ability in a canine mammary gland tumor cell line.

Mutations in the p53 gene are associated with more than half of all human cancers. These mutations often cause a disruption of the tumor-suppressor function of p53 and induce genomic instabilities. Wild‑type p53 requires tetramerization to function as an initiator of cell cycle arrest and apoptosis. Although alterations in p53 tetramerization caused by mutation have been well studied, there are few cell lines containing an endogenous mutation in the tetramerization domain of p53. Here, we report the discovery of a canine mammary gland tumor cell line CTB‑m2, which contains the Leu332Gln (L332Q) mutation corresponding to Leu344 in the tetramerization domain of human p53. Although CTB‑m2 cells are genetically heterozygous for the Leu332Gln mutation, the mutant mRNA was almost exclusively expressed. CTB‑m2 cells showed enhanced cell proliferation compared to wild‑type p53-expressing CTB‑m cells of the same lineage. A p53 tetramerization reporter assay showed that the ability of the p53 mutant to form tetramers was significantly lower than that of wild‑type p53. An immunoblot analysis of cross-linked p53 oligomerized forms demonstrated that the L332Q mutant lacked the ability to form tetramers but retained the ability to form dimers. These data suggest that the p53 mutant cell line CTB‑m2 could be a useful tool for analyzing the precise tetramerization mechanisms of p53 and verifying the effects of therapeutic agents against tumors expressing p53 mutants that lack the ability to tetramerize.