Significant Upregulation Of P21 Research Articles

Co-encapsulation of hydrophilic and hydrophobic drugs into niosomal nanocarrier for enhanced breast cancer therapy: In silico and in vitro studies

Combination therapy has been considered one of the most promising approaches for improving the therapeutic effects of anticancer drugs. This is the first study that uses two different antioxidants in full-characterized niosomal formulation and thoroughly evaluates their synergistic effects on breast cancer cells. In this study, in-silico studies of hydrophilic and hydrophobic drugs (ascorbic acid: Asc and curcumin: Cur) interactions and release were investigated and validated by a set of in vitro experiments to reveal the significant improvement in breast cancer therapy using a co-delivery approach by niosomal nanocarrier. The niosomal nanoparticles containing surfactants (Span 60 and Tween 60) and cholesterol at 2:1 M ratio were prepared through the film hydration method. A systematic evaluation of nanoniosomes was carried out. The release profile demonstrated two phases (initial burst followed by sustained release) and a pH-dependent release schedule over 72 h. The optimized niosomal preparation displayed superior storage stability for up to 2 months at 4 °C, exhibiting extremely minor changes in pharmaceutical encapsulation efficiency and size. Free dual drugs (Asc + Cur) and dual-drug loaded niosomes (Niosomal (Asc + Cur)) enhanced the apoptotic activity and cytotoxicity and inhibited cell migration which confirmed the synergistic effect of co-encapsulated drugs. Also, significant up-regulation of p53 and Bax genes was observed in cells treated with Asc + Cur and Niosomal (Asc + Cur), while the anti-apoptotic Bcl-2 gene was down-regulated. These results were in correlation with the increase in the enzyme activity of SOD, CAT, and caspase, and the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) upon treatment with the mentioned drugs. Furthermore, these anti-cancer effects were higher when using Niosomal (Asc + Cur) than Asc + Cur. Histopathological examination also revealed that Niosomal (Asc + Cur) had a lower mitosis index, invasion, and pleomorphism than Asc + Cur. These findings indicated that niosomal formulation for co-delivery of Asc and Cur would offer a promising delivery system for an effective breast cancer treatment.

The expression level of ARF and p53 in AML patients, and their relation to patients' outcome

BackgroundAcute myeloid leukemia (AML) is a cancer of hematopoietic progenitors characterized by gene mutations. The most popular deregulations are mutation and altered expression in the p53 gene, which is considered the guardian of the genome. Its activity is controlled by regulatory genes, e.g., alternate open reading frame (ARF), whose defects could affect p53 activity.AimTo study the effect of altered expression of p53 and ARF genes in de novo AML patients and correlate the results to the patients’ characteristics and outcomes.MethodsExpression levels of p53 and ARF were assessed in 96 AML adult patients compared to 20 healthy controls using quantitative reverse-transcription PCR (RT-qPCR).ResultsThere was significant up-regulation of p53 [77.6 (3.8–9528.3)] compared to controls [1.031 (0.210–9.051)], p < 0.001]. The expression level of ARF was significantly upregulated [6.2 (0.5–964.0)] compared to controls [0.854 (0.357–2.519), p < 0.001]. All of the low ARF expressers had low p53 overexpression, 61.1% of patients with high ARF expression had high p53 over-expression, and 38.9% with high ARF expression had low p53 over-expression (p < 0.001). ARF expression shows a trend of association with FLT3 mutation, as 89.3% with FLT3 mutation have high ARF expression (p = 0.080). Low p53 over-expression was seen in 77% of APL patients, while high p53 expression was associated with non-APL (p = 0.040). The median DFS of mutant NPM1 patients was higher than wild NPM1 (46.15 vs. 5.89 days, p = 0.045). Patients aged ≤ 50 years had better OS and DFS than those > 50 (p = 0.05, p = 0.035, respectively).There were no significant statistical associations between DFS and p53, ARF, and FLT3 mutations.ConclusionThe p53 and ARF genes are overexpressed in de novo AML patients and they are interrelated. low p53 overexpression is associated with APL phenotype and t(15;17) and patients with t(15;17) had slightly better survival than patients with negative t(15;17) (p = 0.061). AML patients with mutated NPM1 had better DFS than wild NPM1 (p = 0.045). p53 pathway regulation can occur by many alternative ways rather than gene mutation.

Enhanced effects of ferulic acid against the harmful side effects of chemotherapy in colon cancer: docking and in vivo study

BackgroundColorectal cancer is considered a potential causative agent of morbidity and death, making it a particularly dangerous malignancy. The current study aims to assess the efficacy of ferulic acid (FA) to attenuate the harmful side effect of 5-fluorouracil (5FU) in colon cancer tissues induced by 1,2-dimethylhydrazine (DMH).ResultsRegarding the colon tissues of male Wistar-albino rats (Rattus norvegicus), combined FA and 5FU showed the approximately normal structure of mucosa. The treated groups showed a remarkable reduction in Ki67, Ck20, and an elevation in caspase-3 and P53. There was significant upregulation of P53 in both 5FU and combined FA–5FU groups (p < 0.001 and p < 0.00001, respectively).ConclusionsThe present results revealed a potential role of the combined therapy by 5FU and FA in the suppression of colon cancer induced by DMH by upregulation of apoptosis with the clear effect of FA in attenuating the side effects of 5FU on the normal cells.

Abstract 5490: Comparative activation of cellular ISR and cell death induction by p53 pathway restoring small molecules

Abstract Restoration of the p53 pathway has been a long-term goal of the field of cancer research as an approach to treat tumors with mutated p53 and aggressive clinical behavior. In general, p53- pathway-restoring can be achieved by small molecules through p53-dependent or p53-independent ways. For short, hereafter p53-independent restoration of p53-pathway-signaling is also referred as ‘restoration of p53 pathway’. To perform a comparative activation of p53 target genes, novel compounds PG3 and PG3-Oc that activate a subset of p53-target genes in a p53-independent manner, and four known mutant p53-activating compounds were included (ZMC1, APR-246, CP-31398 and Ellipticine). Nutlin-3a was used as a negative control. PG3 and PG3-Oc induced significant upregulation of p21, PUMA (p53 upregulated modulator of apoptosis), and DR5 consistently in five tested cancer cell lines with various p53 status through ATF4 (Activating transcriptional factor 4) via the ISR in a p53-independent manner. However, mutant p53-targeting compounds induced the expression of the three p53 target genes and ATF4 in a highly variable and cell type-dependent manner. PG3 treatment activated ATF4 through the ISR via kinase HRI (Heme-regulated inhibitor). ATF4 mediated the upregulation of PUMA, p21 and NAG-1 (Nonsteroidal anti-inflammatory drug-activated gene 1). We identified PUMA-mediated cell apoptosis through activation of caspase-8 in HT29 cells and possibly caspase-10 in SW480 cells. In summary, we provide a new mechanism that PG3 induced cell death via an HRI/ATF4/PUMA axis that may be helpful in understanding drug efficacy. Citation Format: Xiaobing Tian, Wafik S. El-Deiry. Comparative activation of cellular ISR and cell death induction by p53 pathway restoring small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5490.

Synthetic Design and Biological Evaluation of New p53-MDM2 Interaction Inhibitors Based on Imidazoline Core.

The use of p53-MDM2 inhibitors is a prospective strategy in anti-cancer therapy for tumors expressing wild type p53 protein. In this study, we have applied a simple approach of two-step synthesis of imidazoline-based alkoxyaryl compounds, which are able to efficiently inhibit p53-MDM2 protein–protein interactions, promote accumulation of p53 and p53-inducible proteins in various cancer cell lines. Compounds 2l and 2k cause significant upregulation of p53 and p53-inducible proteins in five human cancer cell lines, one of which possesses overexpression of MDM2.

Identification of CDCA2 as a Diagnostic and Prognostic Marker for Hepatocellular Carcinoma.

ObjectiveHepatocellular carcinoma (HCC) is one of the most common and malignant tumors with an insidious onset, difficult early diagnosis, and limited therapy options, resulting in a poor prognosis. Cell division cycle associated 2 (CDCA2), also known as Repo-Man, plays an important role in regulating mitosis and DNA repair, but the involvement of CDCA2 in HCC remains unclear.MethodsThe differentially expressed genes that were significantly upregulated in multiple RNA sequencing datasets of HCC were screened. Receiver operating characteristic (ROC) curve analysis was performed to identify diagnostic markers for HCC. Least absolute shrinkage and selection operator Cox regression analysis was performed to screen the prognosis-related genes. The screening and analyses identified CDCA2 as the target gene in this study. The expression of CDCA2 was analyzed in public databases and clinical specimens, and CDCA2 involvement in HCC was explored by both bioinformatic analysis and in vitro experiments.ResultsThe level of CDCA2 was enhanced in HCC compared with healthy livers. Overexpression of CDCA2 positively correlated with the pathological grade and TNM stage of the diseases. Furthermore, CDCA2 was found to be an independent prognostic predictor. An excellent prognostic model of HCC was successfully constructed with CDCA2 in combination with TNM stage. Bioinformatic analysis revealed that CDCA2 was closely associated with the cell cycle, apoptosis, and p53 signaling pathway. Silencing CDCA2 in Huh7 cells resulted in significant upregulation of p53 and the downstream PUMA and NOXA and a subsequently increased apoptosis. Inhibition of p53 signaling and apoptosis was found after overexpression of CDCA2 in L02 cells. Strikingly, the proliferation of cells was not affected by CDCA2.ConclusionsCDCA2 was a novel diagnostic marker for HCC, and overexpression of this gene reflected poor pathological grade, stage, and clinical prognosis. CDCA2 promoted the pathogenesis of HCC by suppressing the p53-PUMA/NOXA signaling and the subsequent apoptosis.

Septic shock as a trigger of arterial stress-induced premature senescence: A new pathway involved in the post sepsis long-term cardiovascular complications

BackgroundMajor adverse cardiovascular events among sepsis survivors is an emerging health issue. Because endothelial senescence leads to vascular dysfunction and atherothrombosis, sepsis could be associated to vascular stress-induced premature senescence and thus with long-term cardiovascular events. Materials & methodsAdult Wistar male rats were submitted to cecal ligation and puncture, or a SHAM operation. Markers of inflammation, oxidative stress and endothelial senescence were assessed at 3, 7 and 90 days (D), and vascular reactivity was assessed in conductance and resistance vessels at D90. Expression of proteins involved in senescence and inflammation was assessed by Western blot analysis and confocal microscopy, oxidative stress by dihydroethidium probing. ResultsPro-inflammatory endothelial ICAM-1 and VCAM-1 were up-regulated by three-fold in CLP vs. SHAM at D7 and remained elevated at D90. Oxidative stress followed a similar pattern but was detected in the whole vascular wall. Sepsis accelerated premature senescence in aorta vascular tissue as shown by the significant up-regulation of p53 and down-stream p21 and p16 senescent markers at D7, values peaking at D90 whereas the absence of significant variation in activated caspase-3 confirmed p53 as a prime inducer of senescence. In addition, p53 was mainly expressed in the endothelium. Sepsis-induced long-term vascular dysfunction was confirmed in aorta and main mesenteric artery, with a major alteration of the endothelial-dependent nitric oxide pathway. ConclusionsSeptic shock-induced long-term vascular dysfunction is associated with endothelial and vascular senescence. Our model could prove useful for investigating senotherapies aiming at reducing long-term cardiovascular consequences of septic shock.

Chemopreventive Effect of 5-Flurouracil Polymeric Hybrid PLGA-Lecithin Nanoparticles against Colon Dysplasia Model in Mice and Impact on p53 Apoptosis.

The use of 5-fluorouracil (5FU) is associated with multifaceted challenges and poor pharmacokinetics. Poly(lactic-co-glycolic acid)-lipid hybrid nanoparticles (PLNs)-based therapy has received attention as efficient carriers for a diversity of drugs. This study evaluated the in vivo chemotherapeutic and anti-proliferative efficacy of 5FU-loaded PLNs against 1,2-dimethylhydrazine (Di-MH) prompted colon dysplasia in mice compared to free 5FU. 5FU PLNs were prepared. Male Swiss albino mice were distributed to six experimental groups. Group 1: Saline group. All the other groups were injected weekly with Di-MH [20 mg/kg, s.c.]. Group 2: Di-MH induced colon dysplasia control group. Groups 3 and 4: Di-MH + free 5FU treated group [2.5 and 5 mg/kg]. Groups 5 and 6: Di-MH + 5FU-PLNs treated group [2.5 and 5 mg/kg]. Free 5FU and 5FU-PLNs doses were administered orally, twice weekly. Treatment with 5FU-PLNs induced a higher cytoprotective effect compared to free 5FU as indicated by lower mucosal histopathologic score and reduction in number of Ki-67 immunpositive proliferating nuclei. Additionally, there was significant upregulation of p53 and caspase 3 genes in colon specimens. Our results support the validity of utilizing the PLNs technique to improve the chemopreventive action of 5FU in treating colon cancer.

Protoflavones in melanoma therapy: Prooxidant and pro-senescence effect of protoapigenone and its synthetic alkyl derivative in A375 cells

AimsIn our study, the anticancer effects of a semisynthetic p-quinol, protoapigenone 1′-O-butyl ether (PABut), were tested in human melanoma A375 cells also in comparison with natural congener, protoapigenone (PA). Main methodsThe cytotoxic effect of PABut and PA was determined using MTT assay. Flow cytometry analysis was used to evaluate the influence of the compounds tested on ROS generation and cell cycle distribution in A375 cells. Moreover, apoptosis was evaluated by AO/EB dual staining as well as by flow cytometry. Markers of senescence were quantified by spectrofluorimetry and by Western blot analysis. Key findingsBoth PABut and PA showed significant cytotoxicity against melanoma A375 cells at sub-micromolar concentrations. Both protoflavones induced comparable cell cycle arrest in G2/M phase. However, a more profound upregulation of intracellular ROS levels was found following PABut treatment. An increased apoptosis in the cells following 48 h treatment with both protoflavones tested was also confirmed. Both compounds tested remarkably upregulated p21 protein levels in A375 cells. Unlike PA, PABut significantly decreased protein levels of NAD+-dependent deacetylase SirT1 and β-actin accompanied by mild significant upregulation of mitochondrial SOD2 and senescence markers, p16 protein and SA-β-Gal activity. However, a significant upregulation of p53 only following PA treatment was found. SignificanceThese results suggest that PABut and PA confer high chemotherapeutic potential in melanoma cells and are suitable for further testing. Furthermore, modification of protoapigenone with 1′-O-butyl ether moiety can be associated with improved senescence-inducing effect and, thus, enhanced chemotherapeutic potency of PABut compared to the unmodified natural protoflavone.

Polymeric nanocapsular baicalin: Chemometric optimization, physicochemical characterization and mechanistic anticancer approaches on breast cancer cell lines

Baicalin is a multi-purpose flavonoid known for its anticancer properties, but its application is hindered by its low water solubility and bioavailability. Polymeric nanocapsules were proposed in this work as a promising system for enhancing baicalin delivery, and potentiating its anticancer properties. The characterization of nanocapsules was augmented with chemometric analysis, and the selected formulations were tested on two breast cancer cell lines (MCF-7 and MDA-MB-231), with mechanistic anticancer elucidation using MTT assay, confocal microscopy uptake, flow cytometry, mechanism of cell death, reactive oxygen species production, caspase 3/7 activity and death biomarker expression using quantitative real time PCR. Results showed that baicalin nanocapsules displayed favorable pharmaceutical properties; with the formulation variables affecting their properties elucidated using chemometric factorial analysis. Nanocapsules enhanced the anticancer activity of baicalin up to 216 times for MCF-7 cells and 31 times for MDA-MB-231 after 24 hr incubation. Cellular internalization of the fluorescently labeled nanocapsules was confirmed after 4 hr incubation for both cell lines. Apoptosis was the dominant cell death mechanism, with significant up-regulation of P53 in baicalin nanocapsules treated cells. Data here presented drive to further preclinical studies to investigate the delivery of baicalin polymeric nanocapsules and their anti-cancer activity.

Cells of adult T-cell leukemia evade HTLV-1 Tax/NF-κB hyperactivation–induced senescence

AbstractHuman T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATL). The HTLV-1 viral trans-activator/oncoprotein Tax is a major driver of ATL, yet it induces rapid p21Cip1/Waf1 (p21)- and p27Kip1-mediated cellular senescence through constitutive activation (hyperactivation) of NF-κB. Although constitutive NF-κB activation is a common feature of T/B-cell leukemia/lymphoma, including ATL, it is not known how ATL cells maintain chronic NF-κB activation without undergoing senescence. Here, we demonstrate that, in contrast to HTLV-1− T-cell lines, ATL cell lines no longer undergo Tax-induced senescence. Although Tax+ and Tax− ATL cell lines showed signatures of constitutive NF-κB activation, their ability to progress through the cell cycle was unaffected. In some cases, ATL cell lines continued to proliferate despite significant upregulation of p21; additionally, many cell lines displayed altered expression of G1 and G1/S cyclins, particularly overexpression of cyclin D2. We propose that, during the course of ATL development, leukemia cells acquire genetic/epigenetic changes that can mitigate the senescence response triggered by NF-κB hyperactivation. Restoring the NF-κB–induced senescence response would likely help to control the development and progression of ATL and similar lymphoid malignancies.

Design, Synthesis and Screening of 4,6-Diaryl Pyridine and Pyrimidine Derivatives as Potential Cytotoxic Molecules.

A new series of pyridine and pyrimidine derivatives is designed and synthesized as potential antitumor molecules. The tested compounds show promising in vitro cytotoxic activity against HL-60 cell line as eight compounds: 4, 6, 11, 13, 14, 15, 18 and 21 exhibit potent cytotoxic activity in sub-micromolar concentration higher than the combretastatin A4 (CA-4). Compound 21 shows a cytotoxic activity 5-fold more potent than CA-4 on HL-60 cells. DNA-Flow cytometry cell cycle analysis and annexin-V assay on HL-60 cells show that compounds 4, 18 and 21 exhibit potent cell growth inhibition, cell cycle arrest at G2/M phase and pro-apoptotic inducing activities. The percentage inhibition assay of β-tubulin polymerization on HL-60 cells shows that the antitumor activity of the tested compounds appears to correlate well with its ability to inhibit β-tubulin polymerization. In addition, enzyme-linked immunosorbene assay (ELlSA) measurement for compound 21 shows apoptotic inducing activities through significant up regulation of p53, Bax/Bcl-2 ratio and caspase-3 proteins parallel to down regulation of the level of survivin proteins.

Abstract 2339: Targeting ICMT in pancreatic cancer

Abstract Introduction: Pancreatic Cancer remains one of the most difficult to treat human cancers with the 5-year survival as low as 5%. No targeted therapy yet developed against Pancreatic cancer. Here we want to target Isoprenylcysteine carboxylmethyltransferase (ICMT), an enzyme that post translationally modifies a group of proteins including several small GTPases against Pancreatic Cancer. Method: ICMT inhibition was achieved using lentivirus expressing ICMT shRNA and a pharmacological inhibitor named Cysmethynil. We evaluated a panel of human pancreatic cancer cell lines and identified those that are sensitive to ICMT inhibition by cell viability assay. This responsiveness to ICMT inhibition was confirmed in in vivo xenograft tumor mouse models using both Cysmethynil and shRNA-targeting ICMT. Flow cytometry analysis after DAPI or PI staining is used to detect cell cycle arrest and apoptosis. We used western blot analysis to study autophagy, apoptosis and cell cycle markers. ShRNA for P21 is used for p21 knock down studies. SiRNA for p53 is used to study whether p21 induction is p53 dependent. BNIP3, ULK1 and ATG5 knock down was done using specific shRNAs to study their role in ICMT inhibition induced autophagy and apoptosis. BCL-xL is over expressed in the cells using retroviral vectors to study the role of apoptosis in ICMT inhibition induced cell death. Cell and mitochondrial respiration was determined by oxygen consumption rate analysis using Seahorse XF24 analyzer. Summary: In sensitive pancreatic cancer cells, ICMT inhibition induces mitochondrial respiratory deficiency and cellular energy depletion, leading to significant upregulation of p21 and p21 dependent induction of BNIP3 leading to autophagy and apoptosis. But unlike in prostate and liver cancer, ICMT inhibition induced apoptosis not the autophagy responsible for inhibition of cell viability in pancreatic cancer. Conclusion: These findings suggest ICMT is a potential therapeutic target against pancreatic cancers and atleast in pancreatic cancer, ICMT inhibition induced apoptosis but not autophagy responsible for its therapeutic efficacy. Citation Format: Kanjoormana A. Manu, Tin F. Chai, Jing T. Teh, Wan Zhu, Patrick J. Casey, Mei Wang. Targeting ICMT in pancreatic cancer [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 2339.

The safety and efficacy of light emitting diodes-based ultraviolet A1 phototherapy in bleomycin-induced scleroderma in mice

PurposeTo define the efficacy and safety of narrowband ultraviolet A1 (UVA1) for the treatment of dermal fibrosis in bleomycin-induced mouse model of scleroderma. Materials and methods42 DBA/2 strain mice were included in the study: healthy mice and mice with established scleroderma, treated with high or medium dose of UVA1. Non-treated groups served as control. The equipment emitting 365±5nm UVA1 radiation was used in the study. The average cumulative doses were 1200J/cm2 for high and 600J/cm2 for medium dose course. Histological analysis was performed for the evaluation of the dermal thickness and mast cells density. The expressions of p53 and Ki-67 proteins were assessed by immunohistochemical analyses. ResultsSkin thickness of mice with scleroderma, treated with high and medium dose of UVA1, were lower (272.9±113.2μm and 394±125.9μm, respectively) in comparison to the dermal thickness of non-treated animals (599±55.7μm). The dermal mast cells count in mice with scleroderma was reduced after high and medium dose treatment to 11±1.7 and 13±2.2, respectively, as compared to that in non-treated mice (23±3.0). No significant upregulation of p53 nor Ki-67 proteins was observed in the skin of healthy mice and mice with scleroderma after high- and medium-dose of UVA1. ConclusionsThe results of this study indicate that 365nm UVA1 with the cumulative doses of 1200J/cm2 and 600J/cm2 is safe and effective for the dermal fibrosis treatment.

Abstract P1-09-04: Proliferation and p21 refine risk of relapse in residual disease after HER2-directed therapies

Abstract Background: Patients (pts) with residual disease (RD) after neoadjuvant therapy are at higher risk of relapse. We investigated whether biomarkers assessed at surgery in patients pts with RD in the NeoSphere study were informative for risk of distant event free survival (DEFS) Methods: In NeoSphere 417 HER2+ pts were randomized to neoadjuvant TD, TPD, TP or PD (T=trastuzumab, P=pertuzumab, D=docetaxel), and received FAC/FEC and trastuzumab after surgery. 296 pts had RD. Affymetrix derived gene expression profiles (GEPs) were available at surgery for 201 pts (67.9%). 176 pts (60.1%) had paired samples before and after treatment with available GEPs. We investigated the prognostic value of proliferation evaluated by Mitosis Kinase Score (MKS) (Bianchini G Cancer Res 2010), and performed a gene discovery for association between gene expression at surgery and DEFS. Results: MKS as continuous marker was associated with significantly higher risk of relapse when assessed at surgery (HR 1.80 [1.23-2.65]; p=0.002), but not before treatment (HR 1.50 [0.80-2.78]; p=0.20). In paired samples, there was an average decrease (p=9.2E-11) of MKS after treatment, which was prominent in ER+ and chemotherapy-containing arms. In ER- and TP arm there were cases of increase and of decrease of MKS. In ER+ the 5 years DEFS was 94.3% in the Low/Int MKS tertiles group (pooled) vs 70.5% in the High MKS tertile group (HR 5.41 [1.87-15.6]; p=0.002). In ER-, the 5 years DEFS was 85.0% in the Low/Int vs 64.1% in the High group (HR 2.89 [1.08-7.76]; p=0.035). Notably, MKS at surgery after the two monoclonal alone was also prognostic. In the gene discovery approach only the expression of CDKN1A (p21)at surgery was associated with DEFS after correction for false discovery rate (FDR=0.01). Pre-treatment p21 was not associated with DEFS. Paired comparison showed significant upregulation of p21 in all patients, treatment arms and ER groups. The Int/High p21 tertiles group (pooled) had lower risk of recurrence than the low tertile in ER+ (HR 4.31 [1.60-11.6]; p=0.004) and in ER- (HR 5.81 [1.87-18.1]; p=0.002) groups. p21 in TP arm was also prognostic. MKS and p21 expression provided independent prognostic information and remained significant after correction for clinico-pathological variables (nodes and T stage) and tumor-infiltrating lymphocytes. Combining the two markers, there was a group at very low risk (Low/Int MKS and Int/High p21) and one at high risk (High MKS and Low p21). The other tertiles combinations had intermediate risk. In ER+, the 5 yrs DEFS was 94.9% in the low risk group and 52.9% in the high risk (p=1.9E-05). In ER-, the 5 yrs DEFS was 96.5% in the low and 45.5% in the high risk group (p=0.001).The markers' combination was also prognostic in the two monoclonal only arm. Conclusions: Proliferation (MKS) and p21 expression are modulated by trastuzumab and/or pertuzumab regimens. Tumors with high MKS and low p21 in RD after neoadjuvant therapy defined a group at very high risk of relapse. Tumors with low/int proliferation and int/high p21 had low risk of recurrence similar to that of patients achieving pCR. Whether the pharmacodynamic modulation of p21 could be used as surrogate marker of long term benefit in patients with RD deserves additional investigation. Citation Format: Bianchini G, Pienkowski T, Im Y-H, Bianchi GV, Tseng L-M, Liu M-C, Lluch A, de la Haba-Rodríguez J, Semiglazov V, Oh D-Y, Poirier B, Pedrini JL, Valagussa P, Gianni L. Proliferation and p21 refine risk of relapse in residual disease after HER2-directed therapies [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-09-04.

Correlative Studies Unravelling the Possible Mechanism of Cell Death in Tideglusib-Treated Human Ovarian Teratocarcinoma-Derived PA-1 Cells.

This study aims to unravel the use of GSK-3 inhibitors as viable apoptotic inducers for teratocarcinoma-derived ovarian PA-1 cells. MTT assay was carried out to assess inhibitory concentrations of LiCl and TDG. AO/EB staining and Hoechst 33258 staining were employed to assess the damage. Mitochondrial membrane potential (ΔΨm) and ROS generation were assessed with IC50 concentrations of LiCl and TDG. Tumor-related genes (p53, p21, IL-8, TNF-α, MMP-2, Fas-L, Cox-2, and caspase-3) were assessed with 1/4 IC50, 1/2 IC50, IC50 concentrations by semi-quantitative RT- PCR. Cell cycle analysis was performed with IC50 concentration of LiCl and TDG. Western blot analysis was performed for caspase-3, caspase-7, caspase-9, PARP to estimate the possible damage induced by GSK-3 inhibitors and regulation of GSK-3β, pGSK-3β, Cox-2. GSK-3 inhibitors demonstrated a concentration and time-dependent reduction in cell viability, exhibiting significant ROS generation and reduced ΔΨm at their IC50 values. Substantial concentration-dependent gene expression changes with significant upregulation of P21, Cox-2, TNF-α, caspase-3, Fas-L were observed. Protein expression of caspase-3 caspase-7, caspase-9, PARP exhibited significant cleavage in LiCl and TDG-treated cells. Protein expression of Cox-2 was significantly increased in IC50 concentration of TDG. Cell cycle analysis showed significant accumulation of cells at sub-G0-G1.

Enantioselective bioaccumulation of hexaconazole and its toxic effects in adult zebrafish (Danio rerio)

Little is known about the bioaccumulation and toxicity of hexaconazole (HEX) in spite of the fact that they are indispensable parts for a comprehensive assessment of its environmental behavior and toxic effects in organisms of freshwater ecosystems. In this study, adult zebrafish were used to study the enantioselective bioaccumulation of HEX and its effect endpoints in liver, including oxidative stress and the regulation of apoptosis-related gene expression. Significant enantioselective bioaccumulation was demonstrated when exposed to HEX of 100 and 200μgL−1, finding that the (−)-enantiomer tended to accumulate in zebrafish more easily than (+)-enantiomer. Activities of antioxidant enzymes (SOD, CAT and GPx) and GSH content were all significantly decreased when zebrafish were exposed to 50 and 200μgL−1 HEX for 21d. A series of genes of the apoptosis pathway were examined in groups treated with 50 and 200μgL−1 HEX for 21d using real-time PCR. Significant up-regulation of p53, Puma, Apaf-1, caspase-3 and caspase-9 expression and down-regulation of Bcl-2/Bax expression ratio were proved. The overall results indicated that waterborne HEX was able to produce oxidative stress and induce apoptosis through the involvement of caspases in adult zebrafish. The above information will play a vital role in the integrated environmental risk assessment of HEX and make its toxic mechanism in fish clear.

Reduced Protein Synthesis and p53 Activation in Late-Stage Erythroblasts Mediate the Erythroid Differentiation Defect in Mice with Ribosomal Protein S14 Haploinsufficiency

The 5q-syndrome is a subtype of myelodysplastic syndrome (MDS) with a defined clinical phenotype associated with heterozygous deletion of Chromosome 5q. The RPS14 gene was identified as a critical gene for the erythroid phenotype of the 5q- syndrome using an RNA interference screen. We generated a murine model for conditional, heterozygous inactivation of Rps14 in the bone marrow to investigate the biological basis of del(5q) MDS.To explore the role of Rps14 on hematopoietic stem cell (HSC) function and erythroid differentiation, we generated a mouse model in which Rps14 exons 2-4 are flanked by loxP sites. Following crosses to Mx1Cre transgenic mice, we induced Rps14 excision in hematopoietic cells by poly(I:C). Two weeks after induction of the gene excision, mice developed significantly reduced hemoglobin and red blood cell counts with a significantly higher MCV compared to Mx1Cre+ controls. Bone marrow analysis confirmed an erythroid differentiation defect specifically at the transition from the CD71+Ter119+ (RII, basophilic erythroblasts/early polychromatophilic erythroblasts) to the CD71intermediate/lowTer119+ (RIII/IV, poly/orthochromatophilic erythroblasts, enucleated erythrocytes) population, accompanied by significant up-regulation of p21 in the RIII population. Histopathology of spleens demonstrated compensatory extramedullar erythropoiesis. The bone marrow was normocellular with a significant increase in hypolobulated megakaryocytes correlating with high platelet counts and dysplastic platelets in the peripheral blood, reflecting the pathognomonic megakaryocyte phenotype in del(5q) MDS. At 50 weeks of age, Rps14-/+Mx1Cre+ developed a more severe macrocytic anemia in the peripheral blood.We thus tested whether a more severe erythroid phenotype could be induced by treatment with the hemolytic agent phenylhydrazine (PH; 2x single dose 25mg/kg body weight, s.c.). In response to PH treatment, Rps14 haploinsufficient mice developed a more severe anemia than control mice, had a delayed reticulocyte response, and a differentiation defect again specifically at the transition from RII to RIII/IV accompanied by a compensatory increase in the RI (CD71+Ter119-) pro-erythroblast/early basophilic population. Megakaryocyte-erythrocyte progenitors were increased, and pre-CFU-E and CFU-E numbers were normal. To further elucidate the mechanisms by which haploinsufficiency of Rps14 causes ineffective erythropoiesis, we analyzed cell cycle, p53 and apoptosis. We found a significant induction of p53 specifically in the RI population and a dramatic increase of apoptotic cells in RIII, suggesting that increased apoptosis accounts for the erythroid failure. We therefore tested if genetic inactivation of p53 rescues the erythroid phenotype. In Rps14-/+p53-/+Mx1Cre+ mice the erythroid differentiation defect was restored and mice had a comparable response to PH as Mx1Cre+ controls. In PH dose escalation experiments (35mg/kg), Rps14 haploinsufficient mice died from the severe anemia and delayed reticulocyte response, while compound p53 loss rescued the erythroid failure. Accordingly, forced erythroid differentiation in vitro was significantly impaired in Rps14 haploinsufficient hematopoietic stem and progenitor cells.We measured protein synthesis by O-propargyl (OP)-puromycin incorporation. Rps14 haploinsufficient cells had a significantly reduced protein synthesis in CD71low cells (late erythroblasts) that could not be fully restored by p53 inactivation although the protein synthesis in p53 heterozygote cells alone was significantly higher. The decreased protein synthesis in Rps14-/+p53-/-Mx1Cre+ compound cells might also account for the decreased stem cell expansion in repopulation assays compared to cells with only p53 loss.Our data provide in vivo evidence that Rps14 haploinsufficiency contributes to the erythroid differentiation defect in del(5q) MDS by reduced protein synthesis and p53 induction in late-stage erythroblasts. This murine model recapitulates the erythroid und megakaryocytic phenotype of the 5q-sydrome and provides a model for understanding the underlying mechanisms in the pathogenesis of ribosomal-mediated erythroid failure in del(5q) MDS. DisclosuresNo relevant conflicts of interest to declare.

Decitabine Priming Enhances The Antileukemic Effects Of The Selective Inhibitor Of Nuclear Export (SINE) CRM1/XPO1 Antagonist (Selinexor) In Acute Myeloid Leukemia (AML)

Abstract AML is a clonal hematopoietic disorder characterized by genetic and epigenetic alterations. The prognosis of AML is poor highlighting the urgent need for novel therapeutic approaches. Targeting aberrant DNA hypermethylation by using hypomethylating drugs such as decitabine has been recently investigated in AML. Our group reported a relatively effective (47% complete response) single agent decitabine treatment schedule for older AML patients. These results suggested the opportunity to capitalize on this relatively effective and non-toxic treatment by incorporating this compound into novel molecularly targeted approaches. Recent data indicate that targeting nuclear exporter proteins is a novel therapeutic strategy to overcome cancer. In particular, CRM1/XPO1 is the only nuclear exporter involved in the active transport of the majority of tumor suppressor proteins (TSP) [e.g. p53 and FOX03A] out of the nucleus resulting in their inactivation. We recently reported the anti-leukemic activity of oral SINE CRM1/XPO1 Antagonists in AML. SINEs displayed potent anti-proliferative properties, induced apoptosis, cell-cycle arrest and myeloid differentiation in AML cell lines and patient blasts. In addition, treatment of leukemic mice with oral SINE significantly prolongs their survival. By crossing the lists of genes known to be regulated by DNA methylation in AML with the ones whose nuclear transport is XPO1 dependent, we identified important TSPs such as FOXO3A and p21. Here we hypothesize that treatment of AML cells with decitabine will increase the transcription and expression of a subset of TSPs (including FOX03A and p21) whose nuclear anti-leukemic effects could be enhanced by blocking their XPO1 mediated nuclear export using the clinical stage oral SINE (Selinexor). Thus, we expect that the sequential treatment of decitabine followed by Selinexor will be more effective than each drug alone. To confirm this hypothesis first we treated the AML line OCI-AML3 cells with decitabine (500nM) overnight (ovn) followed by Selinexor (200nM, 2 fold lower than IC50) for an additional 24 hours (hrs) and measured cell proliferation using WST-1 assay. Controls include: 1) DMSO ovn +DMSO 24 hours, 2) Selinexor ovn +selinexor 24 hours (200 nM) and 3) decitabine ovn + decitabine 24 hrs (500nM). AML cells that were treated first with decitabine followed by Selinexor exhibited a higher cytotoxicity (absorbance (Abs) 0.7) than cells treated with either decitabine (Abs 1.36), Selinexor (Abs 1, p=0.006) or DMSO (Abs 1.7). Similar results were observed with the MV4-11 cell line. Next we measured the candidate TSPs (FOX03A and p21) mRNA and protein expression in OCI-AML-3 and MV4-11 cell lines after 24 hrs of decitabine treatment. We found a significant up-regulation of p21 in decitabine versus DMSO treated cells (MV4-11, Fold change (FC) 4.67±1.4; OCI-AML3, FC 3.98±1.19, p&lt;0.05). We also detected a modest up-regulation of FOXO3A in both cell lines treated with decitabine when compared to the DMSO controls (MV4-11, FC 2.56±0.74 and OCI-AML3, FC 1.5±0.23, p&lt;0.05). These results were confirmed also at the protein level by using western blot. Next, we asked whether ectopic up-regulation of p21 (mimicking decitabine effects) in OCI-AML3 cells could re-capitulate the decitabine enhancing antileukemic effects of Selinexor. Overexpression of p21 followed by Selinexor (200nM) for 24 hrs resulted in a larger decrease of cell proliferation (Abs 0.5) with respect to controls (Abs 0.7, p&lt;0.05) using the WST assay. Similar results were observed for the MV4-11 cell line. Finally, we tested the efficacy of the sequential decitabine–Selinexor in vivo using the MV4-11 xenograft model. Treatment began one week after leukemic cell inoculation in 4 different cohorts; 1) Vehicle, 2) decitabine i.p. twice weekly (BIW) (0.4mg/kg); 3) Selinexor BIW (20mg/kg by oral gavage) and 4) decitabine BIW i.p (0.4 mg/kg) followed by Selinexor (10 mg/kg BIW). We found no difference in median survival time (MST) between vehicle and decitabine only treated mice. As expected, Selinexor alone treated mice have significantly improved MST at 36.5 days vs. 28.5 days, vehicle, p=&lt;0.01). Most importantly, the sequential treatment of decitabine followed by Selinexor significantly improved MST compared to Selinexor alone 47 vs 36.5, p=0.008). These pre-clinical results hold great promise for the use of this combination in human clinical trials in AML. Disclosures: Schacham: Karyopharm: Membership on an entity’s Board of Directors or advisory committees. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents &amp; Royalties. Garzon:Karyopharm: Research Funding.

Bmi1 is required for tumorigenesis in a mouse model of intestinal cancer

The epigenetic regulator BMI1 is upregulated progressively in a wide variety of human tumors including colorectal cancer. In this study, we assessed the requirement for Bmi1 in intestinal tumorigenesis using an autochthonous mouse model in which Apc was conditionally ablated in the intestinal epithelium. Germline mutation of Bmi1 significantly reduced both the number and size of small intestinal adenomas arising in this model, and it acted in a dose-dependent manner. Moreover, in contrast to wild-type controls, Bmi1(-/-) mice showed no increase in median tumor size, and a dramatic decrease in tumor number, between 3 and 4 months of age. Thus, Bmi1 is required for both progression and maintenance of small intestinal adenomas. Importantly, Bmi1 deficiency did not disrupt oncogenic events arising from Apc inactivation. Instead, the Arf tumor suppressor, a known target of Bmi1 epigenetic silencing, was upregulated in Bmi1 mutant tumors. This was accompanied by significant upregulation of p53, which was confirmed by sequencing to be wild-type, and also elevated apoptosis within the smallest Bmi1(-/-) adenomas. By crossing Arf into this cancer model, we showed that Arf is required for the induction of both p53 and apoptosis, and it is a key determinant of the ability of Bmi1 deficiency to suppress intestinal tumorigenesis. Finally, a conditional Bmi1 mutant strain was generated and used to determine the consequences of deleting Bmi1 specifically within the intestinal epithelium. Strikingly, intestinal-specific Bmi1 deletion suppressed small intestinal adenomas in a manner that was indistinguishable from germline Bmi1 deletion. Thus, we conclude that Bmi1 deficiency impairs the progression and maintenance of small intestinal tumors in a cell autonomous and highly Arf-dependent manner.