MDM2 Research Articles

Expression profiling of genes modulated by rosmarinic acid (RA) in MCF-7 breast cancer cells.

Cancer is the second most common cause of death, with breast cancer (BC) as the most frequently diagnosed neoplasm among females. The origin of BC is multifactorial and depends on environmental and genetic factors. The disease presents a significant challenge due to its drug resistance and frequent metastasis. Thus, new effective therapies and metastasis prevention are much needed. Rosmarinic acid (RA) is a natural polyphenol which possesses the ability to inhibit BC cell proliferation and demonstrates cytotoxic properties against those cells. In our study, we examined the effect of RA on the expression of ZEB1, MDM2, ABCB1, PTEN and TWIST1 genes in MCF-7 breast cancer cells. MCF-7 cell cultures were treated with 0.2 μM doxorubicin (DOX) and 1.5, 15 or 50 μM of RA. Real-time PCR reaction was performed to analyze gene expression levels. PCR analysis showed a significant increase of the ZEB1 gene expression, which was about 3-fold for DOX 0.2 μM, 9-fold for 0.2 μM DOX + 1.5 μM RA and 0.2 μM DOX + 15 μM RA (p < 0.05), and about 6.5-fold for 0.2 μM DOX + 50 μM RA (p < 0.05). Furthermore, a decrease of the MDM2 gene expression was observed in all of the examined variants and was about 40-75% (p < 0.05). No influence of DOX and RA combined with DOX on the ABCB1, TWIST1 and PTEN genes was found. The results of our study suggest that RA might be used as an adjuvant therapeutic factor in BC treatment.

Abstract B35: Reactivation of p53 signaling in hepatoblastoma with a stapled peptide dual inhibitor of MDM2 and MDM4

Abstract Introduction: Hepatoblastoma is the most common pediatric liver malignancy. Despite aggressive therapeutic regimens, long-term survival of patients with high-risk, metastatic disease is less than 50%. Most cases of hepatoblastoma do not carry mutations in the TP53 tumor suppressor gene; therefore, we hypothesized that targeting the two major regulators of p53, MDM2 and MDM4, with the stapled peptide dual inhibitor, ATSP-7041, to reactivate p53 signaling would be an effective therapeutic strategy for hepatoblastoma. Methods: Levels of MDM2 and MDM4 gene expression were examined in HB cell lines and in a cohort of 18 primary HB patient samples from stage III (n=12) and IV (n=6) patients in comparison to expression in four adjacent uninvolved liver samples with qPCR experiments. The effects of the dual MDM2/MDM4 inhibitor ATSP-7041 (Aileron Therapeutics) on the same cell lines were analyzed with MTT assays for cytotoxicity and qPCR experiments to look at changes in mRNA expression of p53 targets Bax, Puma, CDKN1A, and MDM2. Results: In 16 of 18 samples, MDM4 expression was higher than average in the uninvolved liver samples; in five samples expression was at least 5-fold higher and in four samples expression was at least 10-fold higher. Of note, all four samples with at least 10-fold higher expression were from stage IV patients. In comparison, MDM2 expression was only elevated in one of the 18 patient samples. All three hepatoblastoma cell lines showed expression of both MDM2 and MDM4. Treatment with ATSP-7041 showed cytotoxic (5.7-8.6 μM) effects in all cell lines tested. All cell lines also showed upregulation of p53 targets Bax (1.4- to 2.4-fold), Puma (2.4- to 3.2-fold), CDKN1A (2.4- to 7.2-fold), and MDM2 (6.1- to 8.3-fold) with treatment. Conclusions: Dual inhibition of MDM2 and MDM4 shows efficacy in preclinical models of hepatoblastoma by upregulating p53 tumor-suppressor signaling in TP53 wild-type cells and is a promising therapeutic strategy for high-risk patients with wild-type TP53. Citation Format: Sarah E. Woodfield, Roma H. Patel, Aryana M. Ibarra, Zhenghu Chen, Sanjeev A. Vasudevan. Reactivation of p53 signaling in hepatoblastoma with a stapled peptide dual inhibitor of MDM2 and MDM4 [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B35.

Advanced Glycation End Products Increase MDM2 Expression via Transcription Factor KLF5.

Type 2 diabetes increases the risk for all-site cancers including colon cancer. Diabetic patients present typical pathophysiological features including an increased level of advanced glycation end products (AGEs), which comes from a series of nonenzymatic reactions between sugars and biological macromolecules, positively associated with the occurrence of diabetic complications. MDM2 is an oncogene implicated in cancer development. The present study investigated whether diabetes promoted MDM2 expression in colon cells and the underlying mechanisms. Our results showed that AGE increased the protein level of MDM2 in a cell model and promoted binding between MDM2 and Rb as well as p53, which led to degradation of Rb and p53. KLF5 was able to bind to the regulatory sequence of the MDM2 gene, and knockdown of the KLF5 protein level inhibited the AGE-triggered MDM2 overexpression, which indicated that KLF5 was the transcription factor for MDM2. In a mouse model of diabetes, we found that AGE level was increased in serum. The protein levels of both KLF5 and MDM2 were increased. KLF5 was able to bind to the regulatory sequence of the MDM2 gene. In conclusion, our results suggest that diabetes increases the level of AGE which enhances the expression of MDM2 via transcription factor KLF5 in colon cells. MDM2 overexpression is a candidate biological link between type 2 diabetes and colon cancer development.

Consistent Amplification of FRS2 and MDM2 in Low-grade Osteosarcoma: A Genetic Study of 22 Cases With Clinicopathologic Analysis.

Low-grade osteosarcoma (LGOS) encompasses low-grade central osteosarcoma (LGCOS) and parosteal osteosarcoma (POS). LGOSs are characterized by a supernumerary ring and giant rod chromosomes containing the 12q13-15 amplicon. The fibroblast growth factor receptor substrate 2 (FRS2) gene is located close to MDM2 and CDK4. Recent studies identified consistent amplification of FRS2 gene in atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma. The aim of this study was to evaluate the frequency of FRS2 amplification and its relationship with the clinicopathologic features of LGOSs. The amplification of FRS2 and MDM2 genes were analyzed by fluorescence in situ hybridization using 22 LGOSs (3 LGCOSs, 14 classic POSs, and 5 dedifferentiated POSs) and 85 control samples of bone and soft tissue. The clinicopathologic features of the 22 LGOSs were described. Amplification of FRS2 was detected in 21/22 (95%) of the LGOSs, including 3 (100%) LGCOSs and 18 (95%) POSs. All 22 LGOSs showed MDM2 amplification (100%). The only MDM2/FRS2 LGOS was dedifferentiated POS (the dedifferentiated component was conventional osteosarcoma). In the control group, all of the atypical lipomatous tumor/well-differentiated liposarcoma/dedifferentiated liposarcomas (DDLs) (10/10, 100%) were FRS2-amplified, whereas the remaining 75 control cases were FRS2-nonamplified. These findings indicate that the FRS2 gene is consistently amplified in classic and dedifferentiated LGOSs but not in their histologic mimics. These results offer another avenue for investigating the biology of LGOSs. Whether FRS2-nonamplified tumors exhibit unusual clinicopathologic features needs further investigation. Some so-called "high-grade osteosarcomas harboring 12q13-15 amplification" may be unrecognized dedifferentiated LGOSs.

Транскрипционная активность генов TP53 и MDM2 в отдаленный период у лиц, подвергшихся хроническому радиационному воздействию

Purpose: To study the levels of transcriptional activity of TP53 and MDM2 genes in the residents of the Techa riverside villages chronically exposed at a wide dose range. Material and methods: transcriptional activity of TP53 and MDM2 genes was assessed in 95 persons. The main study group included 80 people exposed to combined external and internal radiation (peripheral blood samples were taken 60–70 years after the beginning of chronic radiation exposure), mean accumulated dose to red bone marrow was 0.86 ± 0.08 Gy (doses varied in the range 0.1–3.65 Gy). The control group consisted of 15 people living in similar socio-economic conditions in the Southern Urals; the accumulated doses to red bone marrow did not exceed 0.07 Gy. Gene transcription activity profile was studied with real-time PCR assay. The data were analyzed using a comparative CT method with normalization to the “housekeeping” gene transcription in each sample. Statistical analysis was performed using the software PAST. Results and conclusion: In the course of the analysis we did not receive statistically significant differences between the study groups, but there was a tendency to a decrease in gene transcription in the group of exposed persons. The correlation analysis showed a weak negative dependence for TP53 and MDM2 genes, and this dependence was characterized not only by the accumulated dose value but was also associated with the age of the individuals under study. A tendency to a decrease in the transcription activity of the genes under study was noted when studying the effect of the dose. Statistically significant differences were shown for MDM2 gene in the group of individuals whose accumulated doses exceeded 2 Gy (p = 0.044). The analysis of age-peculiarities on gene transcription revealed a statistically significant decrease in TP53 gene transcription with increasing age of patients (p = 0.02). Non-radiation factors including smoking were also studied. The levels of gene transcription were compared between men and women of 2 main ethnicities (Bashkirs/Tartars and Slavs). Results of the study showed that neither sex nor ethnicity had any effect on the levels of TP53 and MDM2 gene transcription in the study groups. The effect of smoking on the activity of the genes under study was negligible.

MDM4 contributes to the increased risk of glioma susceptibility in Han Chinese population

Recently, MDM4 gene has been reported to be a susceptibility gene for glioma in Europeans, but the molecular mechanism of glioma pathogenesis remains unknown. The aim of this study was to investigate whether common variants of MDM4 contribute to the risk of glioma in Han Chinese individuals. A total of 24 single-nucleotide polymorphisms (SNPs) of the MDM4 gene were assessed in a dataset of 562 glioma patients (non-glioblastoma) and 1,192 cancer-free controls. The SNP rs4252707 was found to be strongly associated with the risk of non-GBM (P = 0.000101, adjusted odds ratio (OR) = 1.34, 95% confidence interval (CI) = 1.16–1.55). Further analyses indicated that there was a significant association between A allele of rs4252707 associated with the increased non-GBM risk. Haplotype analysis also confirmed a result similar to that of the single-SNP analysis. Using stratification analyses, we found the association of rs4252707 with an increased non-GBM risk in adults (≥18 years, P = 0.0016) and individuals without IR exposure history (P = 0.0013). Our results provide strong evidence that the MDM4 gene is tightly linked to genetic susceptibility for non-GBM risk in Han Chinese population, indicating a important role for MDM4 gene in the etiology of glioma.

Potent effect of the MDM2 inhibitor AMG232 on suppression of glioblastoma stem cells

Testing new ways to identify untapped opportunities for glioblastoma therapies remains highly significant. Amplification and overexpression of MDM2 gene is frequent in glioblastoma and disrupting the MDM2−p53 interaction is a promising strategy to treat the cancer. RG7112 is the first-in class inhibitor and recently discovered AMG232 is the most potent MDM2 inhibitor known to date. Here, we compared the effects of these two clinical MDM2 inhibitors in six glioblastoma cell lines and ten patient-derived glioblastoma stem cells. Targeted sequencing of the TP53, MDM2 genes and whole transcriptome analysis were conducted to verify genetic status associated with sensitivity and resistance to the drugs. Although TP53 wild-type glioblastoma cell lines are similarly sensitive to AMG232 and RG7112, we found that four TP53 wild-type out of ten patient-derived glioblastoma cells are much more sensitive to AMG232 than RG7112 (average IC50 of 76 nM vs. 720 nM). Among these, 464T stem cells containing MDM2 gene amplification were most sensitive to AMG232 with IC50 of 5.3 nM. Moreover, AMG232 exhibited higher selectivity against p53 wild-type cells over p53 mutant stem cells compared to RG7112 (average selectivity of 512-fold vs. 16.5-fold). Importantly, we also found that AMG232 is highly efficacious in three-dimensional (3D) tumor spheroids growth and effectively inhibits the stemness-related factors, Nestin and ZEB1. Our data provide new evidence that glioblastoma stem cells have high susceptibility to AMG232 suggesting the potential clinical implications of MDM2 inhibition for glioblastoma treatment. These will facilitate additional preclinical and clinical studies evaluating MDM2 inhibitors in glioblastoma and direct further efforts towards developing better MDM2-targeted therapeutics.

The proto‐oncogene function of Mdm2 in bone

Mouse double minute 2 (Mdm2) is a multifaceted oncoprotein that is highly regulated with distinct domains capable of cellular transformation. Loss of Mdm2 is embryonically lethal, making it difficult to study in a mouse model without additional genetic alterations. Global overexpression through increased Mdm2 gene copy number (Mdm2Tg ) results in the development of hematopoietic neoplasms and sarcomas in adult animals. In these mice, we found an increase in osteoblastogenesis, differentiation, and a high bone mass phenotype. Since it was difficult to discern the cell lineage that generated this phenotype, we generated osteoblast-specific Mdm2 overexpressing (Mdm2TgOb ) mice in 2 different strains, C57BL/6 and DBA. These mice did not develop malignancies; however, these animals and the MG63 human osteosarcoma cell line with high levels of Mdm2 showed an increase in bone mineralization. Importantly, overexpression of Mdm2 corrected age-related bone loss in mice, providing a role for the proto-oncogenic activity of Mdm2 in bone health of adult animals.

PO-450 MEK inhibition synergizes with MDM2 inhibitors through DUSP6 suppression

IntroductionMAPK and ARF-MDM2-p53 pathways are critical in cutaneous melanomas. This study examined the combination treatment of MEK inhibitor, trametinib, with MDM2 inhibitors, nutlin-3/RG7388/HDM201, in BRAFV600E mutant and p53 wild-type (p53WT) melanoma cell lines and explored the mechanistic basis of these responses.Material and methodsTwo BRAFV600E and p53WT melanoma cell lines, A375 and WM35, were treated with either trametinib or MDM2 inhibitors, or combinations.Results and discussionsThe combination treatment in both A375 and WM35 showed additive to synergistic effects by Chou-Talalay median effect analysis and combination index scores. The combination treatments induced higher levels of p53 target gene transcripts (CDKN1A(p21), MDM2, BAX, BBC3(PUMA), TP53I3(PIG-3)) and protein products (p21, MDM2), resulting in increased cell cycle arrest and apoptosis compared with MDM2 inhibitors alone, suggesting trametinib synergized with MDM2 inhibitors via upregulation of p53-dependent pathways. In addition, DUSP6 phosphatase mRNA and protein were down-regulated following pERK reduction by trametinib, but did not change after p53 activation by MDM2 inhibitors. Furthermore, suppression of DUSP6 by siRNA, or inhibition with the small molecule inhibitor, BCI, potentiated the effect of MDM2 inhibitors through increased ATM-dependent p53 phosphorylation, as demonstrated by complete reversal with the ATM inhibitor, KU55933.ConclusionTrametinib synergizes with MDM2 inhibitors through a DUSP6 mechanism in BRAFV600E and p53WT melanoma cells. DUSP6 regulation of p53 phosphorylation via ATM, could be a therapeutic target for combination with treatments involving activation of the ATM/p53 pathway.

Abstract 3056: Lung tumor spheres as in vitro platform for testing new therapeutic strategies against cancer stem cells

Abstract Background: Treatment resistance is related to cancer stem cells (CSCs), a highly tumorigenic subpopulation of cells capable of growing and forming tumor spheres under non-adherent conditions. This study aimed to isolate and characterise CSCs from resected non-small cell lung cancer (NSCLC) patients' tumor-tissue and cell lines like tumor spheres and to use them as an in vitro platform for drug screening. Methods: The study was performed on tumour cells from 8 resected NSCLC patients and 12 NSCLC cell lines grown in monolayer and as spheres. The expression of 60 genes, including CSC-markers, pluripotency inducers, cell cycle regulators and components of the Notch, Wnt and Hedgehog pathways was analysed by RTqPCR. Protein levels of relevant markers were assessed by WB and IF. Based on CSCs' characterization, two new-synthetized compounds, called compound A and compound B, were tested in the 12 cell lines and on tumour cells from 4 resected NSCLC patients, in 8 different concentrations. Cell viability was measured after 48 hours using MTS assay normalized to the respective mock-treated control cells and presented as a percentage of the control. Statistical analyses were considered significant at p&amp;lt;0.05. Results: Patients´ median age was 65 years [56-76], 73.3% were male and 45% were adenocarcinomas. Lung tumor spheres had a significantly higher expression of CSC-related genes CD44, ALDH1A1, CD90, CDKN1A, JUNB, NANOG, KLF4 and MDM2 than their paired-adherent cells. Invasion promoters SNAI1, ITGA6, and MMP9 were overexpressed in tumor spheres. Notch pathway ligands, JAG1 and DLL4, and receptors, NOTCH1 and NOTCH3, showed increased expression in spheroids than in differentiated cells. In Wnt, higher expression levels of CTNNB1 and GSK3B were found in spheres; when comparing the expression for both conditions. WB and IF revealed that EpCAM, CD44, ALDH1A1, Sox2 and β-catenin were overexpressed in spheroids whereas E-cadherin was overexpressed in the adherent cells. Drug screening showed that classical anticancer drugs had mild cytotoxic effects on both conditions. Stemness pathways inhibitors IWP2, XAV939, Salinomycin and Vismodegib had stronger cytotoxic effects on spheroids than on the cells grown in monolayers. Cytotoxic effect was stronger in the tumor spheres of the lines and patients treated with compounds A and B. Currently, these compounds are being tested in vivo, in tumors induced in xenografts with cells of patients tested in vitro, to determine the mechanism of action and cytotoxic effect on cancer stem cells in the future. Conclusions: Our data suggest that lung tumor spheres provide a useful short-term culture platform for the simple and cost-effective characterization of CSCs. We found molecules that could be used as novel therapeutic approach in NSCLC. Supported by grants RD12/0036/0025 from RTICC-FEDER, and PI12-02838 and PI15-00753 from ISCIII. Citation Format: Alejandro Herreros Pomares, Cristóbal Aguilar-Gallardo, Hector Amado, Eva Escorihuela, Ana Blasco, Silvia Calabuig-Fariñas, Juan Murga-Clausell, Eloísa Jantus-Lewintre, Carlos Camps. Lung tumor spheres as in vitro platform for testing new therapeutic strategies against cancer stem 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 3056.

Abstract 1652: Development of a potent class of small molecule inhibitors of the MDM2-p53 protein-protein interaction

Abstract In response to cellular stress, the p53 tumor suppressor is activated to modulate cell cycle progression, DNA repair, and cell death. The activity of p53 is tightly regulated by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. Inhibition of the MDM2-p53 interaction in tumors carrying wild-type p53 can therefore reactivate p53 and elicit an anti-cancer effect. Small molecule inhibitors of the MDM2-p53 interaction remains a promising strategy for cancer therapy and a number of these compounds are in clinical development. An isoindolinone series, identified by the Northern Institute for Cancer Research (NICR), has been used as a starting point for the development of potent MDM2-p53 inhibitors. Structure based drug design was applied during lead optimisation to gain potency whilst also focusing on stabilizing the main metabolically labile position and reducing lipophilicity. This approach led to potent compounds with EC50 &amp;lt;1 nM against MDM2 in cell-free ELISA assays and EC50 &amp;lt;30 nM for p53 induction in SJSA-1 osteosarcoma cells. Further analyses of the compounds demonstrated an increase in the levels of p53 and p53 transcriptional targets as a result of inhibiting the MDM2-p53 interaction. Using three pairs of isogenic cell lines, the compounds were shown to be specific for cell lines with wild-type p53. Key compounds were also characterized in pharmacokinetic and pharmacodynamic studies in mice bearing the SJSA-1 tumor xenograft where they displayed strong induction of p53, 3 hours post oral administration, together with an increase in the expression of p53 target genes p21 and MDM2. These potent MDM2-p53 inhibitors have also shown significant in vivo efficacy in the SJSA-1 xenograft model at well tolerated oral doses. Thus, promising lead compounds were identified, meriting further optimization of the series. Citation Format: Lynsey Fazal, Maria Ahn, Luke Bevan, Ildiko Buck, Juan Castro, Gianni Chessari, Ben Cons, Keisha Hearn, Steven Howard, Chris Johnson, Judith Reeks, Emiliano Tamanini, Neil Thompson, Hugh Walton, Pamela Williams, Ruth H. Bawn, Tim J. Blackburn, Celine Cano, Sarah J. Cully, Bernard Golding, Roger Griffin, Karen Haggerty, Ian Hardcastle, Herbie Newell, Martin Noble, Huw Thomas, Elaine Willmore, Yan Zhao, Steve Wedge. Development of a potent class of small molecule inhibitors of the MDM2-p53 protein-protein interaction [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 1652.

Abstract 3529: Modulation of MDM2 alters the metabolomic programming of dedifferentiated liposarcoma and its sensitivity to cholesterol inhibition

Abstract BACKGROUND: Dedifferentiated liposarcoma (DDLPS) is a highly morbid, mesenchymal tumor characterized by amplification of the 12q chromosomal loci. Although DDLPS are lipid tumors, they produce little lipid. Life expectancy is typically less than 15 months in the advanced setting and standard treatments remain highly toxic. Superior treatment options are clearly needed. Amplification of the MDM2 oncogene that resides in these loci is observed in 100% of all DDLPS; however, to variable levels. Through a negative feedback loop, MDM2 inhibits the tumor suppressive functions of p53 that halts cell growth after cellular stress. Previously, we have reported that MDM2 levels correspond with cellular growth and drug metabolism, but little is known about the effects of MDM2 alterations on global metabolomic profiles. These profiles will help us pinpoint dysregulated pathways that explain, at least partially, the functional effects associated with MDM2 amplification. METHODS: Six DDLPS cell lines were brought directly into culture from patients. MDM2 levels were determined via Western blot and RNA-sequencing. Metabolomics data was generated using the Metabolon platform. Cell viability assays were performed in ZOOM IncuCyte or measured by XTT. Atorvastatin was used to inhibit cholesterol synthesis. MDM2 levels were altered using SAR405838 to raise MDM2 levels and MI-192 to lower MDM2 levels. Synergy was calculated via Chou-Talalay method to determine the combination index (CI) using Compusyn software. RESULTS: MDM2 levels are inversely correlated with metabolites in the lipid and cholesterol pathway (Fisher's, p &amp;lt; 0.001). The lipid metabolism pathway was also the top deregulated pathway (pathway enrichment p-value = 0.03) in transcriptionally profiled DDLPS cell lines treated with MDM2 elevating agent SAR405838. MDM2 low DDLPS cell lines were exquisitely sensitive to HMGA-CoA reductase inhibitors in the low micromolar range. Lipid metabolite profiling of MDM2 low versus high cell lines treated with atorvastatin demonstrated that twice as many lipid metabolites were altered in MDM2 low versus high cells (Chi-square, p &amp;lt; 0.001). MDM2 levels by RNA-seq demonstrated significant correlation between MDM2 gene expression and atorvastatin IC50 doses (r=0.963). MDM2 modulation by use of SAR405838 and MI-192 respectively in combination with atorvastatin displayed antagonism (average CI = 1.5) and synergy (average CI = 0.63), respectively. CONCLUSION: Modulation of MDM2 alters cholesterol metabolism in DDLPS and may serve as druggable target. Citation Format: Bryce Demoret, Andrew Patt, John Hays, Ewy Mathé, James L. Chen. Modulation of MDM2 alters the metabolomic programming of dedifferentiated liposarcoma and its sensitivity to cholesterol inhibition [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 3529.

The MDM2 rs937283 A>G variant significantly increases the risk of lung and gastric cancer in Chinese population.

Currently, the MDM2 promoter rs937283 A > G variant that is able to alter MDM2 gene expression has been widely studied to explore the association of MDM2 with cancer risk. In this report, we investigate the association of MDM2 rs937283 A > G variant with risk of lung cancer (LC) and gastric cancer(GC) in a Chinese population of Hubei province, which was followed by a meta-analysis. The genotyping of rs937283 was performed by polymerase chain reaction-restriction fragment length polymorphism and confirmed by sequencing. The results of the present study showed that rs937283 was significantly associated with the risk of LC, and the factors of age, gender, smoking status and drinking status would affect such association. However, rs937283 was only associated with the risk of GC in male, smoking and drinking subgroups. The following meta-analysis demonstrated that rs937283 was associated with the overall cancer risk particularly in Chinese population, which reinforced our present finding. Moreover, the meta-analysis according to cancer types revealed that rs937283 was associated with retinoblastoma risk, but not squamous cell carcinoma risk. Collectively, the MDM2 rs937283 A > G variant may be a valuable risk factor or diagnostic biomarker for Chinese cancer patients.

Establishment of a biodosimeter model based on multiple gene expressions in human peripheral blood cells after 60Co γ-rays irradiation

Objective To investigate a potential radiation biodosimetry based on multiple gene expressions. Methods Human peripheral blood were exposed to 60Co γ-rays at doses from 0 to 8 Gy. The mRNA expression levels of 10 selected genes were detected 6 and 12 h after irradiation by RT-PCR. Individual variation was also examined. An optimal mathematical model of the dose response of these gene expression levels at each time point was obtained by the stepwise regression method. A blind test was conducted to validate the statistical accuracy of dose estimation. Results The 10 selected genes expression levels at each time point were significantly increased along with dose from 0.5 to 8 Gy (R2=0.61-0.97, P<0.05). Individual variations were evident in the gene expressions of TNFSF4, PHPT1 and FDXR. The gene expression levels of PCNA, CCNG1, TNFSF4, PHPT1, GADD45A and FDXR were incorporated into the model at 6 h after exposure (R2=0.88, F=54.8, P<0.001); the gene expression levels of PCNA, CCNG1, TNFSF4, MDM2, GDF15 and TNFRSF10B were included in the model at 12 h after irradiation (R2=0.82, F=42.767, P<0.001). These two statistical models can be utilized for the dose estimation accurately. Conclusions The multiple gene expressions have a potential as a radiation biodosimetry. Key words: Gene expressions; Radiation biodosimetry; Ionizing radiation

Pharmacogenetics of platinum-based chemotherapy: impact of DNA repair and folate metabolism gene polymorphisms on prognosis of non-small cell lung cancer patients.

Chemotherapy based on platinum compounds is the standard treatment for NSCLC patients with EGFR wild type, and is also used as second line in mutated EGFR patients. Nevertheless, this therapy presents poor clinical outcomes. ERCC1, ERCC2, XRCC1, MDM2, MTHFR, MTR, and SLC19A1 gene polymorphisms may contribute to individual variation in response and survival to platinum-based chemotherapy. The aim of this study was to investigate the influence of these polymorphisms on response and survival of NSCLC patients treated with platinum-based chemotherapy. A retrospective-prospective cohorts study was conducted, including 141 NSCLC patients. Polymorphisms were analyzed by PCR real-time with Taqman® probes. Patients with ERCC1 rs3212986-GG (p = 0.0268; OR = 2.50; CI95% = 1.12-5.69) and XRCC1 rs25487-GG (p = 0.0161; OR = 2.99; CI95% = 1.26-7.62) genotype showed significantly better ORR. Cox survival analysis revealed that patients carrying the MDM2 rs1690924-GG genotype (p = 0.0345; HR = 1.99; CI95% = 1.05-3.80) presented higher risk of death. Furthermore, carriers of MTR rs1805087-A alleles (p = 0.0060; HR = 8.91; CI95% = 1.87-42.42) and SLC19A1 rs1051266-AA genotype (p = 0.0130; HR = 1.74; CI95% = 1.12-2.68) showed greater risk of progression. No influence of ERCC1 rs11615, ERCC2 rs13181, ERCC2 rs1799793, XRCC1 rs1799782, MDM2 rs1470383, MTHFR rs1801131, and MTHFR rs1801133 on platinum-based chemotherapy clinical outcomes was found. In conclusion, our results suggest that ERCC1 rs3212986, XRCC1 rs25487, MDM2 rs1690924, MTR rs1805087, and SLC19A1 rs1051266 gene polymorphisms may significantly act as predictive factors in NSCLC patients treated with platinum-based chemotherapy.

Spindle cell lipoma and pleomorphic lipoma: a clinicopathologic analysis of 65 cases

Objective: To investigate the clinical and pathological features of spindle cell lipoma (SCL) and pleomorphic lipoma (PL) with emphasis on differential diagnosis. Methods: Sixty-five cases of SCL/PL from the archive of Department of Pathology, Fudan University Shanghai Cancer Center, from August of 2006 to June of 2017, were included in the study. Immunohistochemistry by EnVision method, MDM2 gene amplifycation and DDIT3 gene translocation by fluorescence in situ hybridization(FISH) were detected and the literature was reviewed. Results: There were 53 males and 12 females with age ranging from 26 to 82 years (mean, 57 years; median, 59 years). The majority of the lesions occurred in the neck/posterior neck/nuchal region, upper back and shoulder. A small percentage of lesions developed in the extremities, face and trunk, and rarely in the epiglottis, mediastinum, labium majus and perineum. Clinically, the tumor usually manifested as a slowly growing mass or nodule. It was often encapsulated, ranging from 1 to 13 cm (mean, 4.1 cm; median, 3.5 cm) in size. Microscopically, SCL was composed of mature adipose tissue, bland spindle cells and bright eosinophilic "ropey" collagen fibers that varied in proportion. Of 45 SCL, 28 cases (62.2%) were classical, 6 cases (13.3%) were myxoid, 5 cases (11.1%) were fat-poor, 3 cases (6.7%) were fat-free, and 3 cases (6.5%) were pseudoangiomatoid. PL showed similar features as SCL, but was characterized by the presence of scattered floret giant cells. Of 20 PL, 11 cases (55.0%) were classical, 6 cases (30.0%) were mixed SCL/PL, 3 cases (15.0%) were fat-free. Both SCL and PL contained scattered mast cells in the stroma. By immunohistochemistry, the spindle cells in SCL and the floret giant cells in PL showed strong positivity for CD34 (52/52, 100.0%), bcl-2 (24/26, 92.3%) and CD99 (6/6), whereas they were negative for S-100 protein and STAT6. FISH analysis of MDM2 (17 cases) and DDIT3 (4 cases) was negative in all cases. Follow-up information was available in 32 patients (3 to 96 months), with local recurrence in only one patient. Conclusions: SCL and PL belong to the same entity but may exhibit morphological disparities. Of note, the fat-poor and fat-free variants are easily mistaken for other mesenchymal tumors. Attention should be paid in their differential diagnosis.

Long‐term Ouabain treatment promotes ATP1A1‐dependent IL‐6 expression in a fetal human lens epithelial cell line (FHL124)

The cardiac glycoside ouabain (OUA) selectively inhibits Na+/K+‐ATPase (NKA) governing the steady state maintenance of the electro‐chemical gradient of Na+ and K+ across the cell membranes. OUA, following binding to the NAK, activates several growth‐related signaling pathways: the EGFR/Src–Ras–ERK pathway and the PI3K1A–PDK–Akt pathway (Xie and Askari, Eur J Biochem 269(10):2434–2439, 2002). OUA also influences pro‐ and anti‐inflammation (Cavalcante‐Silva et al., Frontiers in Physiology, 8:895, 2017) and cell proliferation depending on OUA concentration and incubation time, and cell type (Kulikov et al., Biochim et Biophys Acta 1768: 1691–1702, 2007 and Nguyen et al. JASN 18 (1): 46–57, 2007). Our group proposed and showed the direct interaction between NKA and BcL2 family proteins, which promote or inhibit apoptosis in human fetal lens epithelial cell line (FHL124) (Lauf et al., Cell Physiol Biochem 31:257–276, 2013; Lauf et al., Am J Physiol. Cell Physiol. 308, C51‐60, 2015). Here, we aimed to study the time dependence of OUA action at a concentration of 10−7M on cell viability and transcriptional gene regulation of various pro‐apoptotic and pro‐inflammatory related genes in FHL124 cells. Cells were incubated with either 1–100 μM OUA in 7 μM DMSO or 7 μM DMSO alone (solvent control) relative to an untreated control for 1, 24 and 48 h. The OUA‐treated cells cultured for 24 h revealed a slightly decreased cell viability, however, 48 h incubation dramatically decreased the number of cells by 75–80% while DMSO alone reduced cell viability by 40–50% compared to untreated cells. Like all epithelial cells, FHL124 cells possess α1 (atp1a1) isoform of NAK. The 5′‐untranslated region (5′UTR) of the atp1a1 gene contains two p53 and three NF‐kB (transcription factors nuclear factor‐kappa‐B) response elements. To test for the functional expression of the p53 and NFKB target genes, we selected mdm2, which negatively regulates the p53 gene and IL‐6 (interleukin‐6), which is induced by NF‐KB. Quantitative polymerase chain reaction (qPCR) method, using TaqMan probes, was employed to measure comparative gene expression of the atp1a1, mdm2, and IL‐6 genes. Cells OUA‐treated for 48 h promoted an increase of the IL‐6 expression by ~eight fold and the atp1a1 expression by two fold, but not the mdm2 expression. These findings suggest that OUA, in addition to utilizing existing signaling pathways mentioned above, may act either in complex with cytosolically internalized NKA (see Stricker et al., this FASEB meeting 2018) or derived therefrom through to be determined gene regulation factors proximal to the NF‐kB response elements. Further studies are aimed to confirm NKA‐dependent IL‐6 expression after long‐treatment with OUA.Support or Funding InformationSupported by the P&amp;T Graduate program and WSU Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comprehensive analysis of cancers of unknown primary for the biomarkers of response to immune checkpoint blockade therapy

BackgroundCancer of unknown primary (CUP) accounts for approximately 3% of all malignancies. Avoiding immune destruction is a major cancer characteristic and therapies aimed at immune checkpoint blockade are in use for several specific cancer types. A comprehensive survey of predictive biomarkers to immune checkpoint blockade in CUP were explored in this study. MethodsAbout 389 cases of CUP were analysed for mutations in 592 genes and 52 gene fusions using a massively parallel DNA sequencing platform (next-generation sequencing [NGS]). Total mutational load (TML) and microsatellite instability (MSI) were calculated from NGS data. PD-L1 expression was explored using immunohistochemistry (with 5% cutoff value). ResultsHigh TML was seen in 11.8% (46/389) of tumours. MSI-high (MSI-H) was detected in 7/384 (1.8%) of tumours. Tumour PD-L1 expression was detected in 80/362 CUP (22%). A small proportion of CUP cases harboured genetic alterations of negative predictive biomarkers to immune checkpoint inhibitors (predictors to hyperprogression) including MDM2 gene amplification (2%) and loss of function JAK2 gene mutations (1%). Amplifications of CD274 (PD-L1) and PDCD1LG2 (PD-L2) genes were also rare (1.4% and 0.8%, respectively). The most frequently mutated genes were TP53 (54%), KRAS (22%), ARID1A (13%), PIK3CA (9%), CDKN2A (8%), SMARCA4 (7%) and PBRM1, STK11, APC, RB1 (5%, respectively). ConclusionsUsing a multiplex testing approach, 28% of CUP carried one or more predictive biomarkers (MSI-H, PD-L1 and/or TML-H) to the immune checkpoint blockade, providing a novel option for treatment in patients with CUP.

Clinicopathologic features of atypical spindle cell lipomatous tumor

Objective: To investigate the clinicopathologic characteristics, immunophenotype, differential and diagnostic features of atypical spindle cell lipomatous tumor (ASLT). Methods: Three cases of ASLT were collected from January 2010 to March 2017 at Zhejiang Provincial People's Hospital. The clinical and imaging features, histomorphology, immunophenotype and prognosis were analyzed. Fluorescence in situ hybridization (FISH) was used to detect MDM2 gene amplification, and relevant literature was reviewed. Results: All three patients were adult males, aged 38, 43 and 54 years, respectively. One tumor originated in the subcutaneous soft tissue in the head and neck, one was located in the left primary bronchus and one in the latissimus dorsi muscle. Grossly, all three tumors were circumscribed and ranged from 4.0 to 5.8 cm in size. Microscopically, all showed a focally infiltrative front. These tumors were composed of variable proportions of spindle-shaped and adipocytic cells in a background of variable fibrous and edematous matrix. Scattered lipoblasts were easily seen. One tumor was composed predominately of spindle tumor cells, one of adipocytic cells, and one of equally mixed cell populations. The spindle tumor cells were generally bland-appearing with focal nuclear enlargement and hyperchromasia noted in one case. Mitosis was not seen in neither the spindle cells nor the adipocytic cells. By immunohistochemistry, diffuse and strong reactivity to CD34 of the spindle cells was noted in all cases, definite loss of Rb expression was noted in one of three cases, and S-100 protein was expressed only in the adipocytic cells. INI-1 was intact and Ki-67 index was 1% to 3%. All other markers including CDK4, MDM2, STAT6, SOX10, CD99, bcl-2, β-catenin, CD117, GFAP, CK, EMA, SMA and desmin were negative. FISH of MDM2 was done in two cases, and both showed no amplification. The ASLT in the head and neck had two recurrences during 17 months of follow-up, whereas the tumor in the latissimus dorsi was free of disease during 33 months of follow-up. Conclusions: ASLT is a rare subtype of low-grade adipocytic neoplasm and is distinctive from atypical lipomatous tumor/well-differentiated liposarcoma. The histomorpholgy of ASLT has significant heterogeneity and forms a continuous spectrum. ASLT needs to be distinguished from a series of benign and malignant soft tissue tumors.

Primary Breast Atypical Lipomatous Tumor/ Well-Differentiated Liposarcoma and Dedifferentiated Liposarcoma.

Atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) and its higher-grade counterpart, dedifferentiated liposarcoma (DDL), are extraordinarily rare tumors in the breast. The main differential diagnostic consideration of primary breast ALT/WDL is malignant phyllodes tumor with liposarcomatous differentiation, and the main differential diagnostic consideration of DDL in the breast is metaplastic breast carcinoma, particularly the spindle cell type, with heterologous sarcomatous differentiation. These differential diagnoses may be particularly challenging when evaluating limited core needle biopsy sampling. MDM2 and/or CDK4 protein overexpression and gene amplification are beneficial ancillary studies that can help establish the diagnosis of primary breast ALT/WDL and DDL, and effectively rule out the diagnoses of malignant phyllodes tumor and metaplastic breast carcinoma.