Expression Of Mutant P53 Protein Research Articles

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.

COMPARISON OF MUTANT P53 PROTEIN EXPRESSION IN PTERYGIUM GROUP WITH AND WITHOUT TRIAMCINOLONE ACETONITED INJECTION

Background: Mutant p53 protein is produced by mutant p53 gene, this protein takes part in tissue hyperplasia process in pterygium. Triamcinolone acetonide in often use to reduce reccurency. Besides decreasing the inflamation triamcinolone acetonide is assumed to be able to repair the function of p53 gene. Thus, it reactivates cell apoptosis system and prevent the tissue hyperplasia process in pterygium.To compare Mutant p53 Protein Expression in Pterygium Group With and Without Triamcinolone Acetonide InjectionMethods: This research is a randomized clinical trial study consists of 31 subject who suffered primary pterygium. All subject divided into 2 grup with and without triamcinolone acetonide injection. After excision the pterygium and the tissue were examined through histopatology and immunohistochemical with Hematcoycline Eosin and reagent Clone DO7 staining to see the mutant p53 protein expression.Results: Mutant p53 protein expression in triamcinolone acetonite injected group is 0% and non triamcinolone acetonite injected group is 33,33%Conclusion: Mutant p53 protein expression in triamconolone acetonite injected group is lower than non injected grou.
 Keywords: Mutant p53 protein, primary pterygium, triamcinolone acetonide immunohistochemicial

COMPARISON OF MUTANT P53 PROTEIN EXPRESSION IN PTERYGIUM GROUP WITH AND WITHOUT TRIAMCINOLONE ACETONITED INJECTION

Background: Mutant p53 protein is produced by mutant p53 gene, this protein takes part in tissue hyperplasia process in pterygium. Triamcinolone acetonide in often use to reduce reccurency. Besides decreasing the inflamation triamcinolone acetonide is assumed to be able to repair the function of p53 gene. Thus, it reactivates cell apoptosis system and prevent the tissue hyperplasia process in pterygium.To compare Mutant p53 Protein Expression in Pterygium Group With and Without Triamcinolone Acetonide InjectionMethods: This research is a randomized clinical trial study consists of 31 subject who suffered primary pterygium. All subject divided into 2 grup with and without triamcinolone acetonide injection. After excision the pterygium and the tissue were examined through histopatology and immunohistochemical with Hematcoycline Eosin and reagent Clone DO7 staining to see the mutant p53 protein expression.Results: Mutant p53 protein expression in triamcinolone acetonite injected group is 0% and non triamcinolone acetonite injected group is 33,33%Conclusion: Mutant p53 protein expression in triamconolone acetonite injected group is lower than non injected grou.
 Keywords: Mutant p53 protein, primary pterygium, triamcinolone acetonide immunohistochemicial

Abstract 5462: Functional characterization of potential gain-of-function p53 mutants in esophageal squamous cell carcinoma

Abstract One of the most frequently mutated genes reported in human cancers is TP53. Esophageal squamous cell carcinoma (ESCC) has an extremely high frequency of p53 mutations of 60-90% in ESCC patients. Evidence of gain-of-function (GOF) p53 mutations that exert wild-type (WT) function-independent oncogenic effects are emerging. We aim to determine the functional role of several candidate GOF p53 mutations in ESCC and explore their clinical utility for prognostication and as potential treatment targets. In our recent study of Hong Kong ESCC specimens, we detected TP53 alterations in over 90% of ESCC tumors, ~36% of which are candidate GOF mutations verified in other cancers. By analyzing published ESCC sequencing data, we showed that patients from the high-risk ESCC region show frequent candidate GOF p53 mutants. We applied the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique to functionally knockout (fKO) mutant p53 protein expression in various ESCC cell lines with different endogenous p53 mutants and subject them to in vivo and in vitro functional assays. Interestingly, only KYSE150 cells with an endogenous p53R248Q mutation show reduced tumor growth in nude mice after p53-fKO, suggesting that there is a specific mutant p53 that confers oncogenic features in ESCC. Consistently, only KYSE150 p53-fKO cells show reduced colony formation in three-dimension in vitro culture. Tyrosine phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) is significantly reduced in KYSE150 p53-fKO cells, suggesting that regulation of the STAT3 pathway contributed to the oncogenic feature of the GOF p53 mutant. p53R248Q exhibits concentrated subcellular localization to the membrane fraction (including ER, mitochondria, etc.) and shows increased resistance to HSP90/HDAC inhibition-induced protein degradation, as compared to other p53 mutants. Together, these results suggest a GOF role of certain mutant p53 proteins with specific characteristics. Targeting such mutants and/or related oncogenic pathways may be of therapeutic benefit. Acknowledgement: We acknowledge the Research Grants Council of Hong Kong Special Administrative Region for General Research Fund support (17109515 to M.L.L). Citation Format: Valen Z. Yu, Maria L. Lung. Functional characterization of potential gain-of-function p53 mutants in esophageal squamous cell carcinoma [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 5462.

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.

Altered epigenetic pathways and cell cycle dysregulation in healthy appearing skin of patients with koebnerized squamous cell carcinomas following skin surgery

Koebnerized non-melanoma skin cancer following skin trauma represents a rare and obscure event. To study molecular pathological parameters in koebnerized squamous cell carcinomas (K-SCCs) occurring after complete tumour removal. We assessed two patients with multiple sclerosis who were on treatment with dimethylfumarate (DMF) preceded by long-term azathioprine therapy. Both patients rapidly developed several K-SCCs following histopathologically proven complete excision of cutaneous SCCs. We performed immunohistochemistry for p53, p16, Ki-67, TET-2, IDH-2, 5-hmc and 5-mc. PCR was carried out for the detection of human papilloma viruses. Mutation analysis was performed for BRAF, K-RAS and EGFR. All lesions investigated were negative for HPV DNA. Mutations were not detected. Healthy appearing skin of both patients showed relatively high Ki-67, p16 and p53 expression which was comparable to the expression observed in primary SCCs as well as K-SCCs. Protein expression of Ki-67, p16 and mutant p53 was barely detected in the specimens of the healthy controls. A decreased protein expression of TET-2 enzyme was seen in all tumours and healthy appearing skin when compared to the skin of healthy controls. We observed two patients with K-SCCs developing under DMF treatment. In healthy appearing skin of patients with K-SCCs, wound healing processes, including induction of proliferation and growth factor release, might promote the growth of preneoplastic keratinocytes and cancer formation on the basis of pre-existing altered epigenetic pathways and cell cycle dysregulation. Although fumarates can reduce TET-2 expression, the role of DMF intake in the development of K-SCCs remains unclear.

Cullin-1 and -2 Protein Expression in Colorectal Cancer: Correlation with Clinicopathological Variables.

The cullin (CUL) family of proteins is involved in the ubiquitin/mediated degradation of proteins, regulating cell proliferation, cell-cycle control, migration, invasion and metastasis in the process of tumor progression. The aim of the present study was to examine if there is any correlation between the immunohistochemical (IHC) expression of Cullin-1 and -2 proteins in colorectal cancer tissue specimens with several clinicopathological variables. Between January 2012 and December 2014, 96 consecutive adenocarcinoma patients were submitted to oncological colectomy, as the first therapeutic option, with a curative intent. CUL-1 and -2 protein expression was examined with IHC on paraffin-embedded tissue sections. CUL-1 and -2 protein positivity, was correlated with patients' age, gender, stage, histological grade, proliferative capacity (Ki-67 labeling index) and mutant p53 protein expression. The positivity for CUL-1, CUL-2, mutant p53 protein and Ki-67 index, was determined by the percentage of their IHC expression in the total number of cancer cells. Choosing as a cut-off point for CUL-1 positivity the 10%, a statistically significant relationship of the expression of the mutant p53 protein (p=0.04) and the co-expression of CUL-2 (p=0.003) were noticed. By setting the cut-off limit for CUL-2 expression to 10%, no statistically significant differences were observed between its expression and the examined clinicopathological variables. However, by increasing the cut-off limit for CUL-2 expression to 30%, a statistically significant correlation of its expression to the mutated p53 protein was noticed (p=0.047). Co-expression of CUL-1 and -2 in more than 10%, significantly correlated to the coexistence of adenomatous polyps along the large bowel (p=0.0329). Multivariate analysis of CUL-1 and -2 co-expression in more than 10% disclosed their expression as an independent factor for adenomatous polyps development in the large bowel (p=0.035, RR=2.1). CUL-1 overexpression may happen early in the process of carcinogenesis mainly affecting the vulnerable p53(+) large bowel cells, arresting them in the G1 phase of cell-cycle, while it may also induce the expression of CUL-2. Co-expression of CUL-1 and CUL-2, in the arrested (in G1 phase) large bowel cells, promotes carcinogenesis up to adenomatous polyp formation. Since no relationship between cullins expression and development of cancer on adenoma was found, the results of the present study may be useful explaining the initiation but not the progression of carcinogenesis in colorectal cancer. Further molecular and clinical studies are needed in order to delineate the clinical importance of these proteins in the management of colorectal cancer patients.

Anti-p53 autoantibody as early diagnostic and prognostic marker of bronchogenic carcinoma in smokers

Background Mutations in the tumor protein 53 (TP53) gene can lead to expression of mutant p53 proteins that accumulate in cancer cells and can induce circulating p53 antibodies in patients with cancer. Neoplastic transformation leads to elevated plasma sialic acid concentration through shedding or secreting of sialic acid from tumor cell surfaces. Our work aims to evaluate the presence and prognostic role of these antibodies and sialic acid in patients with lung cancer.Patients and methods A total of 30 patients with lung cancer (five patients were presented with stage I, 10 patients with stage II, 10 patients with stage III, and five patients with stage IV) and 60 with nonmalignant disorders (30 smokers and 30 apparently healthy individuals) were evaluated for p53 antibodies by enzyme-linked immunosorbent assay and sialic acid by colorimetric methods.Results Anti-p53 antibodies and sialic acids levels in patients with bronchogenic carcinoma were significantly higher than smokers, and both levels were significantly higher than healthy controls (P<0.001). There was no statistically significant difference between stages III and IV of bronchogenic carcinoma of serum anti-p53 antibodies and sialic acid, but there was a statistically significant difference between stages I, II, and III, where the levels in stage III of bronchogenic carcinoma were significantly higher than stage II, and both levels were significantly higher than in stage I. There was a positive correlation between serum levels of anti P53 autoantibodies and the smoking index of the same patients. There was a positive correlation between serum levels of sialic acid and anti-p53 antibodies in the same patients with bronchogenic carcinoma.Conclusion Anti-p53 antibodies and sialic acid can be considered as sensitive for the biochemical changes associated with smoking and lung cancer. They may be used as markers for early diagnosis of lung cancer, and presence of them in smokers with primary lung cancer correlates with the severity and bad prognostic outcome of the disease.

Sulfation can enhance antitumor activities of Artemisia sphaerocephala polysaccharide in vitro and vivo

In this study, a sulfated Artemisia sphaerocephala polysaccharide (ASPs) was prepared and its antitumor activity was evaluated in tumor cells and Hepatoma 22 (H22) tumor-bearing mice. In vitro experiments, ASPs significantly inhibited the growth of HepG2 and Hela cells with the IC50 values of 172.03 and 161.42μg/mL, respectively. Moreover, no direct cytotoxicity against mouse fibroblast L929 normal cells was observed in vitro. After oral administration for 12days, the tumor growth was significantly suppressed by ASPs at the doses of 200mg/kg (inhibition rate of 60.85%). Results of tumor histological morphology and cell cycle analysis showed that ASPs could arrest H22 cells at S phase and promote cell apoptosis. Additionally, immunohistochemical analysis demonstrated that ASPs caused the down-regulation of mutant p53 protein expression in a dose-dependent manner. Therefore, these findings proposed new insight into antitumor properties of sulfated polysaccharide as a promising agent in cancer treatment.

Correlation between osteosarcoma and the expression of WWOX and p53.

The objective of this study was to analyze the effect of the expression of WWOX and p53 on the growth of MG-63 osteosarcoma cells and to explore the correlation between osteosarcoma and the expression of WWOX and p53. WWOX and p53-overexpressing MG-63 osteosarcoma cell lines were established by transfection and named the MW and MP cell lines, respectively. Untransfected MG-63 cells (blank control) were used as control. Quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect the expression of WWOX and wild-type p53 mRNA and protein, respectively. The effects of WWOX and p53 (wild-type) on the activity of MG-63 cells were determined by MTT assay and flow cytometry. The expression of mutant p53 protein in 65 cases of osteosarcoma was detected by immunohistochemistry to analyze the correlation between p53 and the development of osteosarcoma. qPCR showed that WWOX and p53 mRNA was overexpressed in MW and MP cells, respectively. Western blot analysis showed that the levels of WWOX and p53 protein in MW and MP cells were higher than in the blank control group. MTT assay showed that the cell proliferation ability of MW and MP cells was significantly lower than in the blank control group. Flow cytometry showed that 78.49% of MW and 66.76% of MP cells were arrested in the G0/G1 phase. Immunohistochemistry showed that mutant p53 was highly expressed in osteosarcoma, with a positive expression rate of 47.7%. The expression rate was positively correlated with the pathological grade of cancer. In conclusion, WWOX can affect the cell cycle of MG-63 osteosarcoma cells to inhibit cell proliferation, which provides new insights into gene therapy for osteosarcoma. The two types of the p53 gene have different functions in the development of osteosarcoma. Wild-type p53 acts as a tumor suppressor, while mutant p53, which is overexpressed in malignant osteosarcoma, has a carcinogenic effect associated with the degree of osteosarcoma.

EFFECTS OF CO-CHEMOTHERAPY ETHYL ACETATE FRACTION OF EURYCOMA LONGIFOLIA JACK ROOTS AND DOXORUBICIN AGAINST APOPTOSIS THROUGH EXPRESSION P53 MUTANT AND BCL-2

Background : It was found mutations of p53 gene in Breast cancer. Mutant p53 protein caused a decrease in cell apoptosis mechanisms through increased expression of Bcl-2. Breast cancer therapy is commonly used chemotherapy using Doxorubicin. However, effectiveness of the use of this chemotherapeutic agent is limited due to the emergence of side effects and toxic to normal cells. Therefore, it is necessary to develop new drugs for combination of chemotherapy. Eurycoma longifolia Jack roots has the potential as co-chemotherapy of breast cancer and it is not toxic to normal cells. Method : Rats were divided into 5 groups. Each group consisted of four female white Sprague Dawley rats. Group 1 (Normal), group 2 (DMBA 20 mg/kgB.W), group 3 (DMBA +Doxorubicin 1.12 mg/kgB.W), group 4 (DMBA +fractions 100 mg/kgB.W), group 5 (DMBA+Doxorubicin +fractions). All the rats were sacrificed at weeks 16 and to be taken their breast tissue. Immunohistochemistry was performed using a mouse monoclonal antibody mutant (BioGenex) and Bcl-2 (BIOSS). Results : expression of mutant p53 percentage obtained for group I (9.35 ± 0.32)%, II (21.65 ± 1.60)%, III (10.72 ± 2.52)%, IV (11.63 ± 3.39)%, V (12.72 ± 3.44)%, While the percentage of Bcl-2 expression obtained for I (20.62 ± 10.09)%, II (52.83 ± 3.61)%, III (24.38 ± 3.54)%, IV (38.01 ± 6.25)%, V (27.99 ± 4.27)%. The data was statistically tested by Kruskal Wallis test (p< 0.005). Conclussion : Co-chemotherapy of E. longifolia Jack roots and Doxorubicin can stimulate apoptosis through decreased in the expression of mutant p53 protein and Bcl-2 in breast tissue of rats induced by DMBA.

Molecular genetic and morphological features of malignant neoplasms of the stomach and colon with tissue eosinophilia

Aim. To study molecular genetic and morphological features of neoplasms in stomach and colon cancer associated with tissue eosinophilia. &#x0D; Methods. Study material was the tissue samples of malignant neoplasms of the stomach and colon obtained from the surgical procedures. Study groups were identified depending on the presence or absence of eosinophilic infiltration of the tumour tissue as well as on neoplasm localization. Medical records and outpatient patient cards were analyzed, presence of matastases in regional lymph nodes and grade of differentiation of malignant cells were evaluated. In tumour tissue expression of p53 and p21 proteins was evaluated by means of immunohistochemical technique. To study the distribution of polymorphic variants of р53 (G215C) and р21 (A1026G) genes, deoxyribonucleic acid extraction was performed from formalin-fixed and paraffin-embedded tissue samples obtained from the resection margins. Polymorphism genotyping of deoxyribonucleic acid samples was performed by restriction fragment length polymorphism analysis of amplification products with the use of polymerase chain reaction of specific genome regions with further visualization with ultraviolet light. &#x0D; Results. In patients with colon cancer associated with tissue eosinophilia tumour cells were shown to have higher grade of differentiation than in patients without eosinophilia. In stomach cancer associated with eosinophilic infiltration low expression of mutant p53 protein was revealed significantly more frequently as opposed to cancer without eosinophilia. Also in patients with stomach and colon cancer, association of tissue eosinophilia with carriage of G allele of р53 (G215C) gene polymorphism was found. &#x0D; Conclusion. Tissue eosinophilia in stomach and colon cancer is associated with favourable morphological cancer characteristics, low expression of mutant p53 protein and carriage of G allele of р53 (G215C) polymorphism.

Correlation of P53 Expression with Various Clinicopathological Parameters of Gastric Carcinoma and Its Relationship with Survival

AIM: Gastric cancer is the most aggressive type of cancer. The immunohistochemical protein expression of mutant p53 has been proposed as a potential tool to evaluate the biological behavior of gastric cancer. Predictive value of p53 for survival is debatable; hence this study was formulated to know the survival of patients with p53 expression in gastric cancer. METHODS: It is prospective study from September 2014 - July 2015, included 58 consecutive patients of gastric cancer. Biopsy specimens were treated immuno-histochemically and expression of p53 gene was analyzed by Immunoreactive Score (IRS). These findings were then compared with clinico-pathological parameters like age, gender, tumor location, tumor size, Laurens classification and TNM staging according to American joint committee for cancer guidelines, using CT scan of abdomen, and histopathological grading and types according to WHO classification. RESULTS: Mp53 expression was observed in 90% of gastric cancer patients among which 37 (63.8%) patients showed high and 21 (36.2%) patients showed low p53 expression. Level of p53 expression was found significantly associated with age, tumor site, tumor size, histological grade, T stage, M stage and Clinical stage. Multivariate analysis shows that high p53 expression is an independent predictor of survival. On Kaplan-Meier survival analysis, patients with p53 high expression had significantly shorter overall survival than those patients with low p53 expression. CONCLUSION: Expression of p53 correlates with the survival and is a simple, effective and reproducible modality to determine the prognosis and survival in various grades & stages of gastric cancer.

Yield of p53 expression in esophageal squamous cell cancer and its relationship with survival.

Background/Aims:Esophageal squamous cell carcinoma (ESCC) is the most aggressive type of cancer. Mutation of tumor suppressor gene p53 is observed in many gastrointestinal malignancies including ESCC. The immunohistochemical protein expression of mutant p53 has been proposed as a potential tool to evaluate the biological behavior of ESCC. Predictive value of p53 for survival is debatable, hence this study was formulated to know the survival of patients with p53 expression in ESCC.Patients and Methods:We prospectively included 91 consecutive patients of ESCC from August 2014 to August 2016. Biopsy specimens were treated immunohistochemically and expression of p53 gene was analyzed by Immunoreactive Score (IRS). These findings were then compared with clinicopathological parameters such as age, gender, histological grades, and TNM stages. All patients received treatment and were kept under regular follow-up.Results:M: F ratio was 2.03:1. p53 expression analyzed by IRS showed low expression (score ≤6) in 35 patients (38.46%) and high expression (>6) in 56 patients (61.54%). Level of p53 expression increased significantly with increasing histological grades of ESCC and TNM stage (P ≤ 0.001). Multivariate analysis shows p53 expression as independent predictor of survival. After 1 year of follow up, survival in the p53 high-expression group was 67.86% [standard error (SE) = 0.0473, confidence interval (CI) = 0.75–0.97) and in low p53 expression group was 91.43% (SE = 0.06, CI = 0.53–0.78) with statistically significant difference P = 0.0001 when analyzed with Kaplan–Meier method.Conclusion:Expression of p53 correlates with the survival and is a simple, effective and reproducible modality to determine the prognosis and survival in ESCC.

116P - Correlation of mutant P53 protein expression and Ki67 index with tumor response to concurrent chemoradiation in locally advanced head and neck cancer

116P - Correlation of mutant P53 protein expression and Ki67 index with tumor response to concurrent chemoradiation in locally advanced head and neck cancer

Abstract 672: Differential effects of p53 mutations on cancer invasion and metastasis in a mouse model of colon cancer

Abstract Inactivation of the APC (adenomatous polyposis coli) tumor suppressor gene plays an important role in initiating most adenomas and colorectal cancers (CRCs). Somatic mutations in the TP53 tumor suppressor gene and KRAS oncogene are found in roughly 60% and 40% of CRCs, respectively, and contribute to tumor progression. In the vast majority of human CRCs, TP53 missense mutations lead to high levels of mutant p53 protein expression and the loss of the other wild type TP53 allele. To evaluate the role of TP53 missense mutations in CRC progression, we generated a genetically engineered mouse model of colorectal carcinoma, via combined targeting of Apc, Kras, and Trp53 alleles in mouse colon epithelium, focusing on comparing phenotypic effects of the murine equivalent of the human R273H mutation (i.e., murine R270H mutation) or a large deletion mutation of mouse Trp53. Inactivation of one allele Apc and activation of an oncogenic Kras allele in colon epithelium generated serrated and hyperplastic morphologic epithelium and adenomas. The addition of either the R270H missense mutation or the Trp53 null mutation to the Apc and Kras mutations led to markedly shortened survival of the mice, due to the development of multiple colon tumors in each mouse ranging from adenomas to late stage adenocarcinomas. Evidence of invasion into the smooth muscle and serosa area was found in both compound mice with missense or deletion mutations in Trp53, with the Trp53R270H mutant mice displaying an increased prevalence of deeply invasive tumors relative to the mice with deletion of Trp53. Furthermore, we found that the missense mutant Trp53R270H allele in combination with Apc and Kras mutations, but not the null-mutant Trp53 allele, was strongly linked to metastases to lymph nodes and lung. We have developed a useful mouse model of metastatic colon cancer that recapitulates the role of TP53 mutations in cooperating with APC and KRAS mutations in human CRC development and progression. In addition, our findings strongly suggest a powerful role for missense mutant p53 proteins compared to TP53 null mutations in promoting invasion and metastasis in CRC progression. Citation Format: Ying Feng, Naoya Sakamoto, Maranne Green, Megan Greenn, Kathleen R. Cho, Eric R. Fearon. Differential effects of p53 mutations on cancer invasion and metastasis in a mouse model of colon cancer. [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 672.

Platycodin D, a metabolite of Platycodin grandiflorum, inhibits highly metastatic MDA-MB-231 breast cancer growth in vitro and in vivo by targeting the MDM2 oncogene

The objective of the present study was to explore the invitro and invivo anticancer effects of PlatycodinD (PD), derived from Platycodin grandiflorum, on highly metastatic MDA-MB-231 breast cancer cells. Using the MTT assay, we found that PD inhibited MDA-MB-231 cell growth in a concentration-dependent manner, with an IC50 value of 7.77±1.86µM. Further studies showed that PD had anti-proliferative effects and induced cell cycle arrest in the G0/G1 phase. To explore the detailed mechanism(s) by which PD suppressed MDA-MB-231 cell growth, western blot analyses were used to detect the expression levels of proteins related to cell proliferation and survival. The data showed that PD decreased the expression of proteins related to the G0/G1 phases, downregulated the protein expression of MDM2, MDMX, and mutant p53, and increased the expression levels of p21 and p27 invitro. We verified the effects of PD on the expression of MDM2, MDMX, mutant p53, p21 and p27 using a pcDNA3-Flag-MDM2 plasmid and MDM2 siRNA transfection, and found that PD inhibited MDA-MB-231 cell viability by targeting MDM2 and mutant p53. Compared with the corresponding parental cells, the cells with siRNA-MDM2 transfection had a greater decrease in cell viability and proliferation, while those with pcDNA3-MDM2 plasmid transfection did not show any increase in the effects of PD. We also established a MDA-MB-231 xenograft model in BALB/c nude mice, and found that PD significantly inhibited the growth of MDA-MB-231 xenograft tumors in these mice. The expression levels of various proteins in the tumor tissue exhibited changes similar to those observed invitro. These findings indicate that PD exerted invitro and invivo anticancer effects against MDA-MB-231 breast cancer cells, that PD is a potential MDM2/MDMX inhibitor, and that the anticancer effects of PD were likely associated with its inhibition of these proteins. Our observations help to identify a mechanism by which PD functions as an anti-breast cancer agent.

Regulation of Mutant p53 Protein Expression.

For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation.

Abstract 2215: Transcriptional co-regulation of Nox4 by p53 and SMAD3

Abstract Recent studies suggest p53 plays an important role in TGF-beta-mediated cell signaling and migration. Previously, we showed that wild-type (WT) and mutant forms of p53 differentially regulate reactive oxygen species (ROS) generation by NADPH oxidase-4 (Nox4). We found that WT-p53 is a potent suppressor of TGF-beta-induced Nox4, ROS production, and cell migration, whereas tumor-associated mutant p53 proteins enhanced Nox4 mRNA and protein expression and cell migration by both TGF-beta-dependent and independent mechanisms (Boudreau et al. Br J Cancer. 2014 May 13;110(10):2569-82). In this study, we examined the basis of human Nox4 promoter regulation by p53 and SMAD3. By constructing a deletion series of Nox4 promoter-reporter constructs, we discovered two regions (-4.76kb to -3.9kb and -1.8kb to - 0.2kb) upstream of the transcription start site that induce promoter activity upon activation of the TGF-beta/SMAD3 pathway. Interestingly, TGF-beta/SMAD3-mediated Nox4 promoter activity was abolished by heterologous expression of p53-WT in p53-null cell lines Hep3B (hepatocytes) and H1299 (lung epithelial). This activity was repressed by wild-type p53 in a dose-dependent manner. In contrast, expression of common tumor-associated mutant-p53 proteins (R175H, R248H, R249Q, R273H) did not repress but enhanced Nox4 promoter activity. We observed this enhancement of Nox4 promoter activity by mutant p53 (R273H) in H1299 cells expressing T204D, a constitutively active TGF-beta receptor. When individual p53 response elements were mutated, however, both untreated and R273H-expressing cells showed 50% reduction in promoter activity. These results indicate that the Nox4 promoter region between -3.97kb and -4.76kb, containing candidate p53 response elements, plays a role in modulating Nox4 expression. Preliminary chromatin immunoprecipitation results suggest that endogenous mutant p53 R249S in PLC/PRF/5 hepatoma cells complexes with the Nox4 promoter region between -3.97kb and -4.76kb. Furthermore, constructs lacking this region show significantly reduced Nox4 promoter activity in response to TGF-beta. Our results provide a basis for Nox4 co-regulation by SMAD3 and mutant p53 that may be involved in tumor progression and metastasis. Citation Format: Howard E. Boudreau, Jonathan J. Park, Thomas L. Leto. Transcriptional co-regulation of Nox4 by p53 and SMAD3. [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 2215. doi:10.1158/1538-7445.AM2015-2215

Abstract 2643: Small molecule identification for the restoration of p53 pathway through p73 and by degradation of mutant p53

Abstract One of the most explored cancer targets has been the tumor suppressor p53. p53 is mutated in more than half of all human tumors and therefore provides an attractive way to selectively target cancer cells that harbor the mutant protein. Mutant p53 protein can acquire a gain-of-function activity, losing its normal tumor suppressor properties and gaining oncogenic characteristics. Various approaches have been taken to restore the p53 pathway in mutant p53 expressing cells including the modulation of mutant p53 conformation to wild-type, and through the upregulation of p53 family members i.e. p73. Unlike p53, p73 is not commonly mutated in cancer cells and thus confers restoration of the p53 pathway through pharmacological stimulation. We characterized a small molecule that restores the p53 pathway through p73. A high-throughput cell-based screen identified small molecule CB002 as a potential candidate for the restoration of the p53 pathway in mutant p53 containing human colorectal cancer cells. Two colorectal cancer cell lines: SW480, DLD-1, and the RXF393 renal cancer cell line, were treated with different concentrations of CB002 for various time points. SW480 cells treated with CB002 for 8 hrs showed a decrease in p53 followed by an increase in DR5 expression at 16 hrs. DLD-1 cells treated with CB002 showed a decrease in p53 expression at 8 hrs. Although no change was observed in proteins involved in cell cycle arrest or cell death that were tested, colony formation was suppressed as compared to control. RXF393 cells treated with CB002 for 48 hrs showed decreased expression of p53 levels and increased levels of p21. These data suggest that CB002 is able to decrease mutant p53 protein expression, increase p53 target genes and promote expression of proteins involved in apoptosis, such as p21 and DR5, respectively. It appears that the CB002 mechanism is different depending on the cell line tested. We hypothesize that CB002 is capable of degrading mutant p53 and restoring the p53 pathway through p73 in colorectal cancer cells. Current undergoing experiments are focused on unraveling the p73 dependence through p73 knockdown and the mechanism of action involved in each cell line. The following project will aid in the understanding of the role of p73 in pharmacological restoration of the p53 pathway in cancer therapy. Citation Format: Liz J. Hernandez Borrero, Shengliang Zhao, David T. Dicker, Wafik El-Deiry. Small molecule identification for the restoration of p53 pathway through p73 and by degradation of mutant p53. [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 2643. doi:10.1158/1538-7445.AM2015-2643