Knockdown Of Wild-type P53 Research Articles

Wee1 inhibition by MK1775 potentiates gemcitabine through accumulated replication stress leading to apoptosis in biliary tract cancer

Patients with advanced biliary tract cancer (BTC) have a poor prognosis, and novel treatments are needed. Gemcitabine, the standard of care for BTC, induces DNA damage; however, the ability of cancer cells to repair DNA dampens its effects. To improve the efficacy of gemcitabine, we combined it with MK1775, a Wee1 inhibitor that prevents activation of the G2/M checkpoint. BTC cell lines were treated with gemcitabine only or in combination with MK1775 to determine the therapeutic potential of BTC. Gemcitabine inhibited the growth and induced the apoptosis of four BTC cell lines to a greater extent when added with MK1775 than when added alone. The effects of the combination treatment were observed in both p53 wild-type and p53 mutant cell lines and were unaffected by knockdown of wild-type p53. The combination treatment increased the percentage of apoptotic cells and decreased the percentage of cells synthesizing DNA, suggesting that it caused DNA-damaged cells to accumulate and possibly die in S phase. It did not induce apoptosis when cells were arrested in mitosis using nocodazole. In a xenograft mouse model, gemcitabine plus MK1775 (but not either alone) inhibited the growth of tumors generated from inoculated BTC cells. Our results show that MK1775 highly enhances gemcitabine cytotoxicity in BTC regardless of p53 status. We suggest that the combination treatment elicits a DNA damage response and consequent apoptosis. Our preclinical study provides a basis for future clinical trials of gemcitabine plus MK1775 in patients with BTC.

Upregulation of fibronectin following loss of p53 function is a poor prognostic factor in ovarian carcinoma with a unique immunophenotype

BackgroundWe previously demonstrated that ovarian high grade serous carcinomas (OHGSeCa) and ovarian clear cell carcinomas (OCCCa) with an HNF-1β+/p53+/ARID1A+ immunophenotype were associated with the worst unfavorable prognosis. To clarify the molecular mechanisms underlying this finding, we focused on alterations in the p53 signaling pathway in these tumors.MethodsChanges in cell phenotype and function following knockdown of wild-type p53 (p53-KD) were assessed using OCCCa cells expressing endogenous HNF-1β and ARID1A. The prognostic significance of molecules that were deregulated following p53-KD was also examined using 129 OCCCa/OHGSeCa cases.Resultsp53-KD cells had increased expression of Snail, phospho-Akt (pAkt), and pGSK3β, and decreased E-cadherin expression, leading to epithelial-mesenchymal transition (EMT)/cancer stem cell (CSC) features. The cells also exhibited acceleration of cell motility and inhibition of cell proliferation and apoptosis. Next generation sequencing revealed that fibronectin (FN) expression was significantly increased in the p53 KD-cells, in line with our observation that wild-type p53 (but not mutant p53) repressed FN1 promoter activity. In addition, treatment of OCCCa cells with FN significantly increased cell migration capacity and decreased cell proliferation rate, independent of induction of EMT features. In clinical samples, FN/p53 scores were significantly higher in OCCCa/OHGSeCa with the HNF-1β+/p53+/ARID1A+ immunophenotype when compared to others. Moreover, high FN/high p53 expression was associated with the worst overall survival and progression-free survival in OCCCa/OHGSeCa patients.ConclusionThese findings suggest that upregulation of FN following loss of p53 function may impact the biological behavior of OCCCa/OHGSeCa, particularly in tumors with an HNF-1β+/p53+/ARID1A+ immunophenotype, through alterations in cell mobility and cell proliferation. The accompanying induction of EMT/CSC properties and inhibition of apoptosis due to p53 abnormalities also contribute to the establishment and maintenance of tumor phenotypic characteristics.4HYHfp8oPTLQGnKo7co1TGVideo

Dual Inhibition of MDM2 and XPO1 Synergizes to Induce Apoptosis in Acute Myeloid Leukemia Progenitor Cells with Wild-Type TP53 through Nuclear Accumulation of p53 and Suppression of c-Myc

Dual Inhibition of MDM2 and XPO1 Synergizes to Induce Apoptosis in Acute Myeloid Leukemia Progenitor Cells with Wild-Type TP53 through Nuclear Accumulation of p53 and Suppression of c-Myc

Abstract 2903: A novel compound induces synthetic lethality for p53 mutations in osteosarcoma cells

Abstract Osteosarcoma is the second-highest cause of cancer-related death in children and adolescents. Despite advances in chemotherapy and surgery, the survival rate for metastatic osteosarcoma remains below 30% for the last three decades. Discovery of new chemotherapy agents would be crucial for improving outcomes of osteosarcoma patients. Here, using high-throughput screening, we have found a new compound, called 2-{2-[(3,5-dimethoxybenzyl)sulfanyl]-1,3-thiazol-4-yl}-N-(2-thienylmethyl)acetamide (referred to as KU0171032), as an inducer of apoptosis in various canine and human osteosarcoma cells. This compound shows minimal effects on non-transformed osteoblast and fibroblast cells. Intriguingly, KU0171032-induced apoptosis is more robust in osteosarcoma cells having mutant p53 or null for p53, as compared to cells with wild-type p53. Knockdown of wild-type p53 in U2OS and SJSA-1 cells significantly enhances sensitivity to KU0171032 with increase in DNA damage and caspase-3 cleavage. Moreover, KU0171032 significantly reduces in vivo tumor growth of osteosarcoma cells with p53 knockdown or carrying mutant p53. Our results strongly suggest that KU0171032 shows synthetic lethality with p53 mutations in osteosarcoma cells. Given that loss of p53 activity is frequent event in many cancer types including osteosarcoma while normal cells usually retain wild-type p53, this compound could be used to develop novel therapeutic strategies that capitalize on vulnerabilities in osteosarcoma and other types of cancer. Citation Format: Tomoo Iwakuma, Alejandro Parrales, Peter McDonald, Anuradha Roy, Mitchell W. Braun, Frank J. Schoenen, Jenna Wang, Steve Rogers, Melinda Broward, Tyce Bruns, Shrikant Anant, Dan A. Dixon, Fred Meyer, Katherine M. Chastain, Douglas H. Thamm, Scott J. Weir, Kathleen Neville, Joy M. Fulbright. A novel compound induces synthetic lethality for p53 mutations in osteosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2903.

Abstract 2532: Wild-type p53 represses MELK expression by blocking the recruitment of FOXM1 to MELK promoter

Abstract Background: Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer with poor prognosis due to frequent distant metastasis and lack of successful targeted therapies. To identify potential druggable targets, previously we performed a gene expression analysis and identified protein kinases that are highly expressed in TNBCs. In this study, we investigated the molecular mechanism through which wild-type p53 represses MELK expression in TNBC cells. Through these studies, we discovered that the expression of Maternal Embryonic Leucine zipper Kinase (MELK) is highly elevated in TNBC when wild-type p53 is lost or mutated. We also show that overexpression of wild-type p53 represses MELK expression without binding to MELK promoter. Experimental Design and Methods: To identify the p53-responsive region in MELK promoter, we have made multiple promoter deletions using PCR deletion mutagenesis. The p53-responsive region was determined by measuring the activity of these promoter constructs in the presence of wild-type p53 using luciferase assays. TransFac software was used to identify potential transcription factors in the p53-responsive region. The effect of overexpression of p53 and FOXM1 on MELK expression was examined through Western blotting analysis. Using co-immuno precipitation (Co-IP) studies, we determined the protein-protein interactions between wild-type p53 and FOXM1. The effect of wild-type on FOXM1 recruitment was determined using chromatin immuno precipitation (ChIP) assays. Results: Through promoter deletion analysis, we identified that deletion of the region between -1.69kb to -0.69kb in MELK promoter significantly reduced p53-dependent suppressive effect on MELK promoter activity. Screening this p53-responsive region using TransFac software, we identified a binding site for FOXM1, a previously reported MELK transcription factor. Similar to the regulation of MELK by wild-type p53, overexpression of wild-type p53 repressed FOXM1 and knockdown of wild-type p53 increased FOXM1 expression. Overexpression of FOXM1 increased MELK expression in p53-null and p53-mutant cells but not in p53 wild-type cells. Co-IP studies revealed that wild-type p53 interacts with FOXM1. ChIP studies revealed that overexpression of wild-type p53 significantly reduced the recruitment of FOXM1 to MELK promoter in TNBC cells. Conclusion: These studies elucidated the regulation of MELK expression by wild-type p53 and demonstrated that wild-type p53 represses MELK expression by inhibiting expression and recruitment of FOXM1 to the MELK promoter. In this study, we identified a novel mechanism for upregulation of MELK expression in TNBC, which is associated with poor prognosis of breast cancer patients. This work was supported by Susan G Komen Promise Grant (PB, SH, GM), SAB Komen grant (PB) and Young Foundation grant (PB). Citation Format: Lakshmi Reddy Bollu, Jonathan Shepherd, Dekuang Zhao, Yanxia Ma, Abhijit Mazumdar, Powel H. Brown. Wild-type p53 represses MELK expression by blocking the recruitment of FOXM1 to MELK promoter [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 2532.

Abstract 1785: Chemical inhibition or transient knockdown of wild-type p53 induced phosphatase 1 (WIP1/PPM1D) potentiates the response to MDM2 inhibitors in a p53-dependent manner

Abstract The mechanisms that determine p53 cell fate decision after its non-genotoxic activation by MDM2 inhibitors are poorly understood. Wild-type p53-inducible phosphatase-1 (WIP1/PPM1D) forms an autoregulatory loop with p53 and is involved in the homeostasis of the p53 stress response network. WIP1 is activated, gained/amplified and overexpressed in a range of p53 wild-type malignancies. We investigated cellular growth/proliferation of p53 wild-type (wt) and matched p53 mutant (mut)/null cell line pairs, differing in WIP1 genetic status, in response to Nutlin-3 + GSK2830371 (WIP1i)/WIP1 siRNA-mediated knockdown. We also assessed the effects of WIP1i or knockdown in combination with Nutlin-3 on p53 post-translational modifications, p53 downstream transcriptional targets and apoptosis. Phosphorylation of p53S15 (pp53S15) was used as a marker of WIP1 catalytic activity. WIP1-amplified MCF-7p53wt cells were very sensitive to the WIP1i, with a 50% growth inhibitory concentration (GI50) of 2.65μM + 0.54 (SEM). The three p53 wild-type and mutant/null cell line pairs showed relatively little sensitivity to single agent WIP1i (GI50>10μM). However the WIP1i, at a non-growth inhibitory dose (2.5μM) in vitro, potentiated their response to Nutlin-3 in a p53-dependent manner. HCT116p53+/+; WIP1L450fs*1/+ (L450fs*1 is a WIP1 activating mutation), NGPp53wt and SJSA-1p53wt cell lines showed a 2.4-fold (p = 0.007), 2.1-fold (p = 0.04) and 1.3-fold (p = 0.02) decrease in their Nutlin-3 GI50 values respectively in the presence of WIP1i at 2.5μM. In contrast Nutlin-3 GI50 did not change in HCT116p53-/−, N20R1p53Mut and SN40R2p53Mut, NGP and SJSA-1 Nutlin-3 resistant daughter cell lines respectively. WIP1 transient siRNA knockdown also resulted in potentiation of cells to Nutlin-3. In WIP1-non-amplified (WNA) cells treated with either Nutlin-3 at the GI50 dose or 2.5μM WIP1i, pp53S15 was not detected, however in combination, these drugs resulted in dramatic levels of pp53S15. Interestingly protein expression of p53 targets MDM2 and p21 were not affected in WNA cells by the increased p53S15 phosphorylation; whereas BAX expression was elevated. The Nutlin-3 + WIP1i/siRNA knockdown combination also resulted in a significant increase in cell death markers between 24-48Hrs post-treatment in a p53-dependent manner. Although the strongest WIP1i potentiation of Nutlin-3 was observed in HCT116p53+/+; WIP1L450fs*1/+ cells which possess an activating WIP1 mutation, the NGPp53wt and SJSA-1p53wt cell lines with wild-type WIP1 also showed potentiation. These findings highlight the mechanistic importance of the p53-WIP1 autoregulatory loop in cell fate determination after non-genotoxic p53 activation by Nutlin-3. Potent inhibitors of WIP1 may potentiate the response to other therapeutics aiming to activate p53 mediated tumour suppression. Citation Format: Arman Esfandiari, Nicola J. Curtin, John Lunec. Chemical inhibition or transient knockdown of wild-type p53 induced phosphatase 1 (WIP1/PPM1D) potentiates the response to MDM2 inhibitors in a p53-dependent manner. [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 1785. doi:10.1158/1538-7445.AM2015-1785

Wild-type and mutant p53 differentially regulate NADPH oxidase 4 in TGF-β-mediated migration of human lung and breast epithelial cells.

Background:Transforming growth factor-beta (TGF-β) induces the epithelial-to-mesenchymal transition (EMT) leading to increased cell plasticity at the onset of cancer cell invasion and metastasis. Mechanisms involved in TGF-β-mediated EMT and cell motility are unclear. Recent studies showed that p53 affects TGF-β/SMAD3-mediated signalling, cell migration, and tumorigenesis. We previously demonstrated that Nox4, a Nox family NADPH oxidase, is a TGF-β/SMAD3-inducible source of reactive oxygen species (ROS) affecting cell migration and fibronectin expression, an EMT marker, in normal and metastatic breast epithelial cells. Our present study investigates the involvement of p53 in TGF-β-regulated Nox4 expression and cell migration.Methods:We investigated the effect of wild-type p53 (WT-p53) and mutant p53 proteins on TGF-β-regulated Nox4 expression and cell migration. Nox4 mRNA and protein, ROS production, cell migration, and focal adhesion kinase (FAK) activation were examined in three different cell models based on their p53 mutational status. H1299, a p53-null lung epithelial cell line, was used for heterologous expression of WT-p53 or mutant p53. In contrast, functional studies using siRNA-mediated knockdown of endogenous p53 were conducted in MDA-MB-231 metastatic breast epithelial cells that express p53-R280K and MCF-10A normal breast cells that have WT-p53.Results:We found that WT-p53 is a potent suppressor of TGF-β-induced Nox4, ROS production, and cell migration in p53-null lung epithelial (H1299) cells. In contrast, tumour-associated mutant p53 proteins (R175H or R280K) caused enhanced Nox4 expression and cell migration in both TGF-β-dependent and TGF-β-independent pathways. Moreover, knockdown of endogenous mutant p53 (R280K) in TGF-β-treated MDA-MB-231 metastatic breast epithelial cells resulted in decreased Nox4 protein and reduced phosphorylation of FAK, a key regulator of cell motility. Expression of WT-p53 or dominant-negative Nox4 decreased TGF-β-mediated FAK phosphorylation, whereas mutant p53 (R280K) increased phospho-FAK. Furthermore, knockdown of WT-p53 in MCF-10A normal breast epithelial cells increased basal Nox4 expression, whereas p53-R280K could override endogenous WT-p53 repression of Nox4. Remarkably, immunofluorescence analysis revealed MCF-10A cells expressing p53-R280K mutant showed an upregulation of Nox4 in both confluent and migrating cells.Conclusions:Collectively, our findings define novel opposing functions for WT-p53 and mutant p53 proteins in regulating Nox4-dependent signalling in TGF-β-mediated cell motility.

Mutation or Loss of p53 Differentially Modifies TGFβ Action in Ovarian Cancer

Ovarian cancer is the most lethal gynecological disease affecting women in the US. The Cancer Genome Atlas Network identified p53 mutations in 96% of high-grade serous ovarian carcinomas, demonstrating its critical role. Additionally, the Transforming Growth Factor Beta (TGFβ) pathway is dysfunctional in various malignancies, including ovarian cancer. This study investigated how expression of wild-type, mutant, or the absence of p53 alters ovarian cancer cell response to TGFβ signaling, as well as the response of the ovarian surface epithelium and the fallopian tube epithelium to TGFβ. Only ovarian cancer cells expressing wild-type p53 were growth inhibited by TGFβ, while ovarian cancer cells that were mutant or null p53 were not. TGFβ induced migration in p53 null SKOV3 cells, which was not observed in SKOV3 cells with stable expression of mutant p53 R273H. Knockdown of wild-type p53 in the OVCA 420 ovarian cancer cells enhanced cell migration in response to TGFβ. Increased protein expression of DKK1 and TMEPAI, two pro-invasive genes with enhanced expression in late stage metastatic ovarian cancer, was observed in p53 knockdown and null cells, while cells stably expressing mutant p53 demonstrated lower DKK1 and TMEPAI induction. Expression of mutant p53 or loss of p53 permit continued proliferation of ovarian cancer cell lines in the presence of TGFβ; however, cells expressing mutant p53 exhibit reduced migration and decreased protein levels of DKK1 and TMEPAI.

Mutant p53 Cooperates with Knockdown of Endogenous Wild-Type p53 to Disrupt Tubulogenesis in Madin-Darby Canine Kidney Cells

Mutation of the p53 gene is the most common genetic alteration in human malignances and associated clinically with tumor progression and metastasis. To determine the effect of mutant p53 on epithelial differentiation, we developed three-dimensional culture (3-D) of Madin-Darby canine kidney (MDCK) cells. We found that parental MDCK cells undergo a series of morphological changes and form polarized and growth-arrested cysts with hollow lumen, which resembles branching tubules in vitro. We also found that upon knockdown of endogenous wild-type p53 (p53-KD), MDCK cells still form normal cysts in 3-D culture, indicating that p53-KD alone is not sufficient to disrupt cysts formation. However, we found that ectopic expression of mutant R163H (human equivalent R175H) or R261H (human equivalent R273H) in MDCK cells leads to disruption of cyst polarity and formation of invasive aggregates, which is further compounded by knockdown of endogenous wild-type p53. Consistently, we found that expression of E-cadherin, β-catenin, and epithelial-to-mesenchymal transition (EMT) transcription factors (Snail-1, Slug and Twist) is altered by mutant p53, which is also compounded by knockdown of wild-type p53. Moreover, the expression level of c-Met, the hepatocyte growth factor receptor and a key regulator of kidney cell tubulogenesis, is enhanced by combined knockdown of endogenous wild-type p53 and ectopic expression of mutant R163H or R261H but not by each individually. Together, our data suggest that upon inactivating mutation of the p53 gene, mutant p53 acquires its gain of function by altering morphogenesis and promoting cell migration and invasion in part by upregulating EMT and c-Met.

Abstract LB-114: Genome wide analysis reveals a "liability" in the mutant p53 transcriptome.

Abstract One of the mechanisms by which the oncogene mutant p53 (mtp53) suppresses chemotherapy induced apoptosis is by directly regulating gene expression. We speculated that within the mtp53 transcriptome, there may be transcriptional targets that constitute "liabilities" that can be exploited to kill mtp53 bearing cancer cells. CHIP-Seq analysis of mtp53 revealed that the key enzyme required for activation of nucleoside analogs, deoxycytidine kinase (DCK), is a direct target of mtp53. We demonstrate that mtp53 associates with the promoter of the DCK gene and that knockdown of mtp53 in multiple cancer cell lines results in reduced DCK mRNA and protein levels. Furthermore, we show that mtp53 cooperates with the ETS transcription factors to induce DCK expression. Knockdown of mtp53 or ETS proteins reduces the activity of a DCK promoter-luciferase construct, thus reinforcing that DCK is a transcriptional target of an ETS/mtp53 complex. In contrast, knockdown of wildtype p53 has no effect on the expression of DCK, indicating that the ability of mtp53 to regulate DCK expression is a gain-of-function activity. Based on our findings, we reasoned that by upregulating DCK expression, mtp53 may increase the sensitivity of cancer cells to nucleoside analogs. Indeed, shRNA knockdown of DCK reduces the sensitivity of cancer cells to gemcitabine. Importantly, suppression of mtp53 or ETS proteins also renders cancer cells less sensitive to gemcitabine. Thus, these data support the notion that mtp53 can sensitize cells to nucleoside analogs via DCK. Our data reveal the presence of a transcriptional "liability" in the mtp53 transcriptome that may help predict which patients will have a better therapeutic response to nucleoside analogs. Citation Format: Luis A. Martinez, Madhusudhan Kollareddy, Lakshman Varanasi. Genome wide analysis reveals a "liability" in the mutant p53 transcriptome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-114. doi:10.1158/1538-7445.AM2013-LB-114

97 Cisplatin-induced apoptosis in malignant pleural mesothelioma cells involves p53 but not p14ARF

Background: Malignant pleural mesothelioma (MPM) tumours often contain homozygous deletions in the INK4A locus that encodes p14ARF, a tumour suppressor protein that promotes p53 stability by blocking the p53 inhibitor MDM2. It has been suggested that p53 is inactive in MPM due to the absence of p14ARF expression. Methods and Results: We investigated p53 activation in p14ARF negative MPM cells or primary cultures after cisplatin treatment and found increased levels of phospho-p53 (serine-15), total p53 protein and p53 specific DNAbinding activity in the majority of cell lines. In addition, the expression levels of p53-positively controlled genes p21WAF, Bax, pig3 and MDM2 increased on incubation with cisplatin, whereas levels of the negatively regulated p53 target gene survivin decreased. Cisplatin-induced apoptosis was detected in p14ARF-deficient cells expressing wild type p53 but not in a p53 mutant cell line. Knockdown of wild type p53 with siRNA rendered MPM cells more resistant to cisplatin-induced apoptosis. Over-expression of p14ARF in p53 wild type MPM cells by transient transfection did not increase their sensitivity to cisplatininduced apoptosis, however, pre-incubation with antisense oligonucleotides targeting the inhibitor of apoptosis protein survivin lead to p53 activation and increased apoptosis sensitivity to cisplatin. Conclusions: These results suggest that p53 is functional in MPM in the absence of p14ARF, and that therapies that target the inhibitor of apoptosis protein survivin are worthy of investigation in MPM.