BACKGROUND: It has been well established that circular RNAs (circRNAs) play an important regulatory role during tumor progression. Recent studies have indicated that even though circRNAs generally regulate gene expression through miRNA sponges, they may encode small peptides in tumor pathogenesis. However, it remains largely unexplored whether circRNAs are involved in the tumorigenesis of colon cancer (CC).
METHODS: The expression profiles of circRNAs in CC tissues were assessed by circRNA microarray. Quantitative real-time PCR, RNase R digestion assay and tissue microarray were used to confirm the existence and expression pattern of circPPP1R12A. The subcellular distribution of circPPP1R12A was analyzed by nuclear mass separation assay and fluorescence in situ hybridization (FISH). SDS-PAGE and LC/MS were employed to evaluate the protein-coding ability of circPPP1R12A. CC cells were stably transfected with lentivirus approach, and cell proliferation, migration and invasion, as well as tumorigenesis and metastasis in nude mice were assessed to clarify the functional roles of circPPP1R12A and its encoded protein circPPP1R12A-73aa. RNA-sequencing and Western blotting analysis were furthered employed to identify the critical signaling pathway regulated by circPPP1R12A-73aa.
RESULTS: We firstly screened the expression profiles of human circRNAs in CC tissues and found that the expression of hsa_circ_0000423 (termed as circPPP1R12A) was significantly increased in CC tissues. We also found that circPPP1R12A was mostly localized in the cytoplasm of CC cells. Kaplan-Meier analysis showed that patients with higher levels of circPPP1R12A had a significantly shorter overall survival. By gain- and loss-of-function approaches, the results suggested that circPPP1R12A played a critical role in proliferation, migration and invasion of CC cells. Furthermore, we showed that circPPP1R12A carried an open reading frame (ORF), which encoded a functional protein (termed as circPPP1R12A-73aa). Next, we found that PPP1R12A-C, not circPPP1R12A, promoted the proliferation, migration and invasion abilities of CC in vitro and in vivo. Finally, we identified that circPPP1R12A-73aa promoted the growth and metastasis of CC via activating Hippo-YAP signaling pathway. In addition, the YAP specific inhibitor Peptide 17 dramatically alleviated the promotive effect of circPPP1R12A-73aa on CC cells.
CONCLUSIONS: In the present study, we illustrated the coding-potential of circRNA circPPP1R12A in the progression of CC. Moreover, we identified that circPPP1R12A-73aa promoted the tumor pathogenesis and metastasis of CC via activating Hippo-YAP signaling pathway. Our findings might provide valuable insights into the development of novel potential therapeutic targets for CC.

Epigenetic changes have major role in the normal development and programming of gene expression. Aberrant methylation results in carcinogenesis. The primary objective of our study is to determine whether primary tumor tissue and cultured tumor cells in 2D and 3D tissue culture systems have the same methylation signature for

The problems including narrow indications, low drug loading, and difficulty in intervention severely affect the clinical efficacy of anti-tumor embolization. Here, we designed a novel tTF-EG3287 protein consisting of the truncated tissue factor (tTF) fused with the bicyclic polypeptide which was encoded by exons 7 and 8 for accurate localization in the tumor vascular endothelial cells (EG3287). This study aims to explore its anti-cancer effect. Gene sequencing was used to verify the fusion gene and SDS-PAGE gel to confirm the optimal induction time and concentration of tTF-EG3287. Nickel affinity chromatography column was used to purify the fusion protein. Confocal microscopy was used to assess the target activity of tTF-EG3287 on colon cancer cells in vitro. Thrombelastography assay was used to identify the pro-coagulant activity of tTF-EG3287. In in vivo experiments, the specific localization of tTF-EG3287 in tumor tissues and the effect of tTF-EG3287 on tumor thrombosis were further detected by in vivo imaging and HE staining, respectively. The tTF-EG3287 fusion protein was efficiently purified by nickel-affinity chromatography column. Moreover, tTF-EG3287 fusion protein showed strong coagulation a ctivity and specific binding ability to the cell surface of colon cancer. In vivo, tTF-EG3287 stably and persistently accumulated in tumor tissues, and specifically induced mixed thrombus formation in tumor vessels, and then impaired tumor growth (tumor inhibition rate=79.2%, p<0.01). Our data prove that the fusion protein tTF-EG3287 could be used as a novel and promising anti-cancer strategy and has great potential value for clinical applications.

We report here numerous novel genes and multiple new signatures which robustly predict prostate cancer (PC) recurrence. We extracted 696 differentially expressed genes relative to a reported PC signature from the TCGA dataset (n = 492) and built a 15-gene signature (SigMuc1NW) using Elastic-net with 10-fold cross-validation through analyzing their expressions at 1.5 standard deviation/SD below and 2 SD above a population mean. SigMuc1NW predicts biochemical recurrence (BCR) following surgery with 56.4% sensitivity, 72.6% specificity, and 63.24 median months disease free (MMDF) (P = 1.12e-12). The prediction accuracy is improved with the use of SigMuc1NW's cutpoint (P = 3e-15) and is further enhanced (sensitivity 67%, specificity 75.7%, MMDF 45.2, P = 0) when all 15 genes were analyzed through their cutpoints instead of their SDs. These genes individually associate with BCR using either SD or cutpoint as the cutoff points. Eight of 15 genes are individual risk factors after adjusting for age at diagnosis, Gleason score, surgical margin, and tumor stage. Eleven of 15 genes are novel to PC. SigMuc1NW discriminates BCR with time-dependent AUC (tAUC) values of 76.6% at 11.5 months (76.6%-11.5 m), 73.8%-22.3 m, 78.5%-32.1 m, and 76.4%-48.4 m. SigMuc1NW is correlated with adverse features of PC, high Gleason scores (odds ratio/OR 1.48, P < 2e-16), and advanced tumor stages (OR 1.33, P = 4.37e-13). SigMuc1NW remains an independent risk factor of BCR (HR 2.44, 95% CI 1.53-3.87, P = 1.62e-4) after adjusting for age at diagnosis, Gleason score, surgical margin, and tumor stage. In an independent PC (MSKCC) cohort (n = 140), these 15 genes were altered in PC vs normal tissue, metastatic PCs vs primary PCs, and recurrent PCs vs nonrecurrent PCs. Importantly, a 10-gene subsignature SigMuc1NW1 predicts BCR in MSKCC (P = 3.11e-15) and TCGA (P = 3.13e-12); SigMuc1NW1 discriminates BCR at 18.4 m with tAUC as 82.5%. Collectively, our analyses support SigMuc1NW as a novel and robust signature in predicting BCR of PC.

BACKGROUND: Glioblastoma is one of the most malignant brain cancer, thus, establishing an effective therapy is paramount. Our previous results indicate that dendritic cell-derived factor (DCF1) is an attractive candidate for therapy against Glioblastoma, since its overexpression in Glioblastoma U251 cells leads to apoptosis. However, the delivery approach limits its clinical application, in this paper, we expressed TAT-DCF1 fusion protein in E.coli in order to surmount its current delivery problems.
METHODS: The coding sequences of the different domains of DCF1 (full length, cytoplasmic, extracellular, 19-amino acid), together with the N-terminal transactivator of transcription (TAT) sequence, were amplified and subcloned into the bacterial expression vector pET30a(+) in order to produce (His)
RESULTS: Immunofluorescence and flow cytometry show that U251 cells were efficiently transduced with the fusion proteins. Cell viability, proliferation, and migration assays suggest that the complete TAT-DCF1 fusion protein significantly decreased U251 proliferation and migration. Flow cytometry further reveals that TAT-DCF1 triggered cellular apoptosis.
CONCLUSIONS: In conclusion, these findings suggest that the TAT-DCF1 fusion protein was efficiently transduced into Glioblastoma U251 cells and induced the antitumor effect and support further investigation into specific targeting and side effects of TAT-DCF1 during drug delivery.

Splice variants are known to be important in the pathophysiology of tumors, including the brain cancers. We applied a proteogenomics pipeline to identify splice variants in glioblastoma (GBM, grade IV glioma), a highly malignant brain tumor, using in-house generated mass spectrometric proteomic data and public domain RNASeq dataset. Our analysis led to the identification of a novel exon that maps to the long isoform of Neural cell adhesion molecule 1 (NCAM1), expressed on the surface of glial cells and neurons, important for cell adhesion and cell signaling. The presence of the novel exon is supported with the identification of five peptides spanning it. Additional peptides were also detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel separated proteins from GBM patient tissue, underscoring the presence of the novel peptides in the intact brain protein. The novel exon was detected in the RNASeq dataset in 18 of 25 GBM samples and separately validated in additional 10 GBM tumor tissues using quantitative real-time-polymerase chain reaction (qRT-PCR). Both transcriptomic and proteomic data indicate downregulation of NCAM1, including the novel variant, in GBM. Domain analysis of the novel NCAM1 sequence indicates that the insertion of the novel exon contributes extra low-complexity region in the protein that may be important for protein-protein interactions and hence for cell signaling associated with tumor development. Taken together, the novel NCAM1 variant reported in this study exemplifies the importance of future multiomics research and systems biology applications in GBM.

Parasporins, a class of non-insecticidal crystal proteins of Bacillus thuringiensis (Bt) are being explored as promising anticancer agents due to their specific toxicity to cancer cells. The present study has identified 25 Bt isolates harbouring parasporin genes from Western Ghats region, the hotspot of biodiversity in India. Among these, the isolate, KAU 41 (Kerala Agricultural University isolate 41) contained non-hemolytic homogenous crystals showing specific cytotoxicity towards cancer cells. SDS-PAGE analysis of this crystal, isolated by aqueous biphasic separation, revealed a 31 kDa sized peptide. The N-terminal sequence deciphered in BLAST analysis showed homology to a hypothetical Bt protein. Upon proteolysis, a 29 kDa active peptide was generated which exhibited heterogenic cytotoxic spectrum on various cancer cells. HeLa cells were highly susceptible to this peptide with IC 50 1 lg/mL and showed characteristics of apoptosis. RT-qPCR analysis revealed the overexpression of APAF1, caspase 3 and 9 by 14.9, 8 and 7.4 fold, respectively which indicates the activation of intrinsic pathway of apoptosis. However, at higher concentrations of peptide (greater than 3 lg/mL), necrotic death was prominent. The results suggest that the 31 kDa protein from Bt isolate, KAU 41 is a parasporin that may have high therapeutic potential.

Codelivery of chemo-sensitizers with chemotherapeutics using combo nanomedicine is a promising platform for overcoming chemoresistance in breast cancer. However, tumor accumulation of nano-carriers based on enhanced permeability and retention (EPR) effect is confounded by heterogeneity in tumor microenvironment. Adsorption of protein corona on surface of nanoparticle boost up clearance by reticulo-endothelial system. In this study, a surface functionalized magnetic nanocomposite (NC) for codelivery of doxorubicin (DOX) and curcumin (CUR) is developed. NCs were coated with hydroxyapatite and were also cross linked with β-cyclodextrin. NCs efficiently encapsulated DOX and CUR. Release of CUR and DOX were in a sustained pH-depended pattern. β-cyclodextrin functionalization reduced protein corona according sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. As shown by flowcytometric and confocal microscopy analyses, NCs internalized efficiently by human breast carcinoma cells MCF-7 and adriamycin resistant MCF-7 (MCF-7/adr) cells. 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test demonstrated superior cytotoxicity of DOX-CUR loaded NCs. Anti-tumor efficacy analyses confirmed reduction in relative tumor volume size (RTV%) compared to control group. Western blot analyses demonstrated marginal CUR mediated P-glycoprotein (P-gp) down regulation. DOX-CUR loaded NCs efficiently accumulated into the tumor via external magnet guidance. Nevertheless, the increased tumor accumulation did not correlate with pharmacologic responses such as RTV% and significant superiority over free DOX was not observed.

The concept that abnormal protein aggregates show prion-like propagation between cells has been considered to explain the onset and progression of many neurodegenerative diseases. Indeed, both synthetic amyloid-like fibrils and pathogenic proteins extracted from patients' brains induce self-templated amplification and cell-to-cell transmission in vitro and in vivo. However, it is unclear whether exposure to exogenous prion-like proteins can potentially cause these diseases in humans. Here, we investigated in detail the prion-like seeding activities of several kinds of pathogenic α-synuclein (α-syn), including synthetic fibrils and detergent-insoluble fractions extracted from brains of patients with α-synucleinopathies. Exposure to synthetic α-syn fibrils at concentrations above 100 pg/mL caused seeded aggregation of α-syn in SH-SY5Y cells, and seeded aggregation was also observed in C57BL/6 J mice after intracerebral inoculation of at least 0.1 μg/animal. α-Syn aggregates extracted from brains of multiple system atrophy (MSA) patients showed higher seeding activity than those extracted from patients with dementia with Lewy bodies (DLB), and their potency was similar to that of synthetic α-syn fibrils. We also examined the effects of various methods that have been reported to inactivate abnormal prion proteins (PrP

The

BACKGROUND: Interleukin-6 (IL-6) is a potent inflammatory cytokine that appears to play a key role in cancer growth and metastasis. In the present study, the effects of IL-6 receptor (IL-6R) on breast cancer aggressiveness and bone metastases were investigated.
METHODS: MDA-MB-231 (MDA-231) cells were treated in the presence or absence of anti-human IL-6 receptor (IL-6R) monoclonal antibody and examined with respect to cell survival. The expressions of signal transducer and activator of transcription 3 (Stat3), vascular endothelial growth factor (VEGF), and receptor activator of NF-κB (RANK) were analyzed by SDS-PAGE and immunoblotting. MDA-231 cells were injected into the left ventricle of mice, and then anti-human IL-6R monoclonal antibody or saline was administered intraperitoneally for 28 days. After 28 days, the incidence of bone metastases was evaluated in the hind limbs by radiography and histology.
RESULTS: Anti-human IL-6R monoclonal antibody reduced bone metastases in an animal model injected with MDA-231 cells on radiological and histomorphometric analyses. The mechanism of bone metastasis inhibition involved inhibited cell proliferation and decreased expressions of phospho-Stat3, VEGF, and RANK in MDA-231 cells.
CONCLUSIONS: The results of the present study suggest that inhibition of IL-6 signaling may become a preventive therapeutic option for breast cancer and bone metastases.

Maslinic acid (MA), a natural pentacyclictriterpene, displays cytotoxic activity on various types of cancer cells. However, its underlying mechanism is unclear. In this study, we assessed the effect of MA on autophagy of human pancreatic cancer cells, and the potential autophagic pathway was presented. MA inhibited the proliferation and induced autophagy of Panc-28 cells by altering the expressions of autophagy related proteins. SDS-PAGE analysis revealed that one protein band was significantly down-regulated in cells treated with MA, and the band was identified as heat shock protein HSPA8 as analyzed using Western blot and MS, MS/MS approaches. HSPA8 knockdown could significantly inhibit cell viability and enhance the cytotoxic effects of MA, whereas HSPA8 overexpression was able to enhance cell viability, diminishing the effects of MA. Western blot analysis indicated that the effect of MA on the expression of autophagy related genes was increased significantly in cells treated with HSPA8 inhibitor VER-155008, whereas HSPA8 inducer geranylgeranylacetone antagonized the effects of MA. Our study provides evidence that MA is able to induce of autophagy via down-regulation of HSPA8 in Panc-28 cells.

Breast cancer imposes a considerable amount of cancer-related mortality and morbidity among women worldwide. Many efforts are in progress to reduce the disease burden and amongst the bacterial-based products received considerable attention as potential anti-cancer drugs. In the present study, the effect of recombinant pro-inflammatory outer membrane protein (HopH) of Helicobacter pylori on the angiogenic factor and tumor development in metastatic breast cancer model was evaluated. The HopH gene was cloned into Pet28a vector, induced by IPTG and expressed and purified by Ni-NTA affinity chromatography. The expressed protein was confirmed by SDS-page. The breast cancer tumor induction was performed using Breast cancer cell line (4T1). The mice were divided into different groups and underwent treatment by recombinant HopH and Herceptin, subsequently. The treatment effectiveness on tumor size was followed, and the expression level of vascular endothelial growth factor was evaluation by real time PCR. The SDS-PAGE analysis confirmed the expression of HopH protein with an approximate 34KD weight. Based on our results, the expression level of VEGF was significantly reduced in HopH-treated mice group comparing to the control and Herceptin group. Our results have shown that the recombinant HopH protein can effectively reduce VEGF expression in breast cancer tumor which was associated with reduction of tumor size. The HopH protein can be considered as a potential anti-cancer agent for future cancer therapeutic studies.

Human epidermal growth factor receptor 2 (HER2) is a powerful target for cancer immune therapy. The development of anti-HER2 monoclonal antibodies targeting different domains of HER2 is quite effective. However, the selection and production of multivalent antibodies are complicated. In this study, a mimivirus-based designed ankyrin repeat protein (DARPin) targeting HER2 was selected from an artificial library by bacteria surface display. The selection was performed on HER2-positive B16BL6/E2 melanoma cells and HER2-nagative cells. DARPin selected from the library could be expressed in soluble form with a yield of 70 mg/L. After purified by two continuous and easy steps, the purity of DARPin was 90% as established by SDS-PAGE and RP-HPLC. Selected DARPin showed significant HER2-targeting ability with an affinity of 1.05 ± 0.47 µM. MTT assay demonstrated that at the concentration of 640 nM, the selected DARPin dimer could inhibit the SK-BR-3 growth at a rate of 36.63 and 46.34% in 48 and 72 hr incubation separately, which was similar to trastuzumab (43.12 and 49.14% separately). These findings suggested that it was an effective method to select antibody mimetic DARPin by bacteria surface display combined with live cells sorting and provided a drug candidate for cancer therapy.

Cancer metastasis risk increases in older individuals, but the mechanisms for this risk increase are unclear. Many peritoneal cancers, including ovarian cancer, preferentially metastasize to peritoneal fat depots. However, there is a dearth of studies exploring aged peritoneal adipose tissue in the context of cancer. Because adipose tissue produces signals which influence several diseases including cancer, proteomics of adipose tissue in aged and young mice may provide insight into metastatic mechanisms. We analyzed mesenteric, omental, and uterine adipose tissue groups from the peritoneal cavities of young and aged C57BL/6J mouse cohorts with a low-fraction SDS-PAGE gelLC-MS/MS method. We identified 2308 protein groups and quantified 2167 groups, among which several protein groups showed twofold or greater abundance differences between the aged and young cohorts. Cancer-related gene products previously identified as significant in another age-related study were found altered in this study. Several gene products known to suppress proliferation and cellular invasion were found downregulated in the aged cohort, including R-Ras, Arid1a, and heat shock protein β1. In addition, multiple protein groups were identified within single cohorts, including the proteins Cd11a, Stat3, and Ptk2b. These data suggest that adipose tissue is a strong candidate for analysis to identify possible contributors to cancer metastasis in older subjects. The results of this study, the first of its kind using uterine adipose tissue, contribute to the understanding of the role of adipose tissue in age-related alteration of oncogenic pathways, which may help elucidate the mechanisms of increased metastatic tumor burden in the aged. Graphical abstract We analyzed mesenteric, omental, and uterine adipose tissue groups from the peritoneal cavities of young and aged C57BL/6J mouse cohorts with a low-fraction SDS-PAGE gelLC-MS/MS method. These fat depots are preferential sites for many peritoneal cancers. The results of this study, the first of its kind using uterine adipose tissue, contribute to the understanding of the role of adipose tissue in age-related alteration of oncogenic pathways, which may help elucidate the mechanisms of increased metastatic tumor burden in the aged.

PURPOSE: The molecular mechanism of breast and/or ovarian cancer susceptibility remains unclear in the majority of patients. While germline mutations in the regulatory non-coding regions of BRCA1 and BRCA2 genes have been described, screening has generally been limited to coding regions. The aim of this study was to evaluate the contribution of BRCA1/2 non-coding variants.
METHODS: Four BRCA1/2 non-coding regions were screened using high-resolution melting analysis/Sanger sequencing or next-generation sequencing on DNA extracted from index cases with breast and ovarian cancer predisposition (3926 for BRCA1 and 3910 for BRCA2). The impact of a set of variants on BRCA1/2 gene regulation was evaluated by site-directed mutagenesis, transfection, followed by Luciferase gene reporter assay.
RESULTS: We identified a total of 117 variants and tested twelve BRCA1 and 8 BRCA2 variants mapping to promoter and intronic regions. We highlighted two neighboring BRCA1 promoter variants (c.-130del; c.-125C > T) and one BRCA2 promoter variants (c.-296C > T) inhibiting significantly the promoter activity. In the functional assays, a regulating region within the intron 12 was found with the same enhancing impact as within the intron 2. Furthermore, the variants c.81-3980A > G and c.4186-2022C > T suppress the positive effect of the introns 2 and 12, respectively, on the BRCA1 promoter activity. We also found some variants inducing the promoter activities.
CONCLUSION: In this study, we highlighted some variants among many, modulating negatively the promoter activity of BRCA1 or 2 and thus having a potential impact on the risk of developing cancer. This selection makes it possible to conduct future validation studies on a limited number of variants.

Human epidermal growth factor receptor 2 (HER2) is an important biomarker for detection and treatment of different types of cancers such as breast, ovarian, stomach cancer. In this study, we developed a monoclonal antibody against the extracellular domain (ECD) of HER2 biomarker of breast cancer. For this purpose, the ECD-HER2 gene was amplified and cloned into an expression vector. Gene was generated in Escherichia coli BL21 (DE3) strain for expression of recombinant protein. The expressed protein was separated by SDS-PAGE and detected by anti-his monoclonal antibody in immunoblotting. Hybridoma cells were obtained by fusing myeloma cells with mouse spleen cells injected with recombinant ECD-HER2 and screened by ELISA for the production of monoclonal antibody. The results indicate that out of three candidate hybridoma cells one clone (1E7) was producing the highest titer and antibody specificity was envisioned in ELISA results. In vivo scaling up culture of hybridoma cells in BALB/C mice lead to significant increase in the monoclonal antibody concentration up to 16 mg/ml. Immunochemical methods demonstrated the specificity of developed antibody against ECD-HER2 protein.

Few susceptibility genes for gastric cancer have been identified. We sought to identify germline susceptibility genes from participants with gastric cancer from an international hereditary cancer research network. Adults with gastric cancer of any histology, and with a germline DNA sample (n = 51), were retrospectively selected. For those without previously identified germline mutations (n = 43), sequencing was performed for 706 candidate genes. Twenty pathogenic or likely pathogenic variants were identified among 18 participants. Eight of the 18 participants had previous positive clinical testing, including six with CDH1 pathogenic or likely pathogenic variants, and two with pathogenic MSH2 and TP53 variants. Of the remaining 10, six were in BRCA1 DNA damage response pathway genes (ATM, ATR, BRCA2, BRIP1, FANCC, TP53), other variants were identified in CTNNA1, FLCN, SBDS, and GNAS. Participants identified with pathogenic or likely pathogenic variants were younger at gastric cancer diagnosis than those without, 39.1 versus 48.0 years, and over 50% had a close family member with gastric cancer (p-values < 0.0001). In conclusion, many participants were identified with mutations in clinically-actionable genes. Age of onset and family history of gastric cancer were mutation status predictors. Our findings support multigene panels in identifying gastric cancer predisposition.

The main intention of this study was the investigation of impact of natural biologically active ligands of nuclear retinoid/retinoid X receptors (all-trans and 9-cis retinoic acid) on proteomic pattern in human estrogen receptor negative breast cancer cell line MDA-MB-231. For this purpose, proteomic strategies based on bottom-up method were applied. The total cell proteins were extracted utilizing a commercially Radio-Immunoprecipitation Assay (RIPA) buffer and separated on 2D sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE). The proteins were subsequently digested in-gel by trypsin and their characterization was achieved by MALDI-TOF/TOF. By employing PDQuest™ software, we identified more than 50 proteins affected by retinoic acid isomers. For more information, 9 proteins which are associated with tumor process were selected. We determined that derivatives of retinoic acid led to significantly reduced level of proteins belonging to metabolic pathway (e.g. glyceraldehyde-3-phosphate dehydrogenase or pyruvate kinase 2) or to other cellular processes as apoptosis, regulation of transcription process or epithelial-mesenchymal transition (e.g. annexins, nucleoside diphosphate kinase B, vimentin). On the other hand all-trans retinoic acid treatment indicates up-regulated effect for heterogeneous nuclear ribonucleoprotein A2/B1.

Most human epithelial tumors harbor numerous alterations, making it difficult to predict which genes are required for tumor survival. To systematically identify cancer dependencies, we analyzed 501 genome-scale loss-of-function screens performed in diverse human cancer cell lines. We developed DEMETER, an analytical framework that segregates on- from off-target effects of RNAi. 769 genes were differentially required in subsets of these cell lines at a threshold of six SDs from the mean. We found predictive models for 426 dependencies (55%) by nonlinear regression modeling considering 66,646 molecular features. Many dependencies fall into a limited number of classes, and unexpectedly, in 82% of models, the top biomarkers were expression based. We demonstrated the basis behind one such predictive model linking hypermethylation of the UBB ubiquitin gene to a dependency on UBC. Together, these observations provide a foundation for a cancer dependency map that facilitates the prioritization of therapeutic targets.

Programmed cell death 1 ligand 1 (PD-L1) is a trans-membrane protein highly expressed on the membrane of cancer cell, which binds inhibitory receptor of PD-1 on the T cells and attenuates anti-tumor immune response.The strategy of blocking PD1 and PD-L1 interaction has been widely used for anti-cancer drug development. The DNA encoding extracellular domain of PD-L1 was cloned and expressed with the pET30(+) and Escherichia coli BL21(DE3) system. Cloning of PD-L1 extracellular domain was confirmed by PCR and enzymatic digestion. Sequence analysis of cloned targeted genes showed 100% homology of original sequence. The recombinant protein was expressed using 1mM/mL IPTG and purified by affinity chromatography on a column of Ni-NTA and confirmed by SDS-PAGE and western blot analysis. Results showed that our constructed pET30(+)/PDL1-ECD system efficiently produces desired recombinant protein with molecular weight of 38.1kDa. The prokaryotic expression system provides an easy method to express PD-L1 extracellular domain that further facilitate the role of PD-1/PD-L1 binding inhibition and helps in valuable drug and antibodies production.

BACKGROUND: Melanoma-associated antigen-A3 (MAGE-A3) is a tumor specific antigen and a potential candidate for cancer immunotherapy. We had screened three immunodominant multiepitopes of MAGE-A3, and identified these multiepitope peptides had significantly higher reactivity to serum samples from gastric cancer patients. However, the immune responses of three multiepitope peptides carried by HBcAg in mice have not been investigated.
OBJECTIVES: The main objective of this study was to analyze the humoral and cellular immune responses in mice induced by these three multiepitope vaccines of MAGE-A3.
METHODS: Three multiepitopes of MAGE-A3 (MAGE-A3(EPI-1, or -2, or -3)) were respectively inserted at HBcAg major immunodominant region (HBcAg(MIR)) of the pET21a(+)/HBcAg(MIR) recombinant plasmid. These recombinant chimeras were identified by PCR, and transfected respectively into E. Coli Ressotta strain. The expression products of rHBcAg(MIR)/MAGE-A3(EPI-1, or -2, or -3) were purified respectively by Ni2+ chelated affinity column, and then confirmed by SDS-PAGE and Western-blot analysis.Purified three rHBcAg(MIR)/MAGE-A3 multiepitopes were administrated respectively into BALB/c (H-2Kd) mice by intradermal injection. The production of rHBcAg(MIR)/MAGE-A3(EPI-1, or -2, or -3) specific IgG in serum from immunized mice were measured by ELISA. Spleen cells from all immunized mice were harvested after one week of last immunization for lymphocyte proliferation assay and cytotoxic T-lymphocyte assay.
RESULTS: PCR and Sequencing analysis showed the presence of the required gene fragment in pET21a(+)/ HBcAg(MIR)/MAGE-A3(EPI-1, or -2, or -3) recombinant plasmid. Purified rHBcAg(MIR)/MAGE-A3(EPI-1, or -2, or -3) could be probed specifically by McAb of 6×his-tag. ELISA analysis indicated that serum from immunized mice with rHBcAg(MIR)/MAGE-A3(EPI-1, -2, or -3) proteins could be discerned specifically by complete MAGE-A3 protein, and high level of antibodies in immune serum were obtained, and all antibody titers could reach above 1:1600. The splenocytes from groups of rHBcAg(MIR)/MAGE-A3(EPI-1,-2, or -3), stimulated respectively with corresponding peptides showed the higher proliferative responses comparing with control groups of HBcAg(MIR) or PBS (p<0.05, respectively). Splenocytes from mice immunized with rHBcAg(MIR)/MAGE-A3 (EPI-1, or -2, or -3) could killed target cells effectively, and there were significant difference of CTL activities compared with control groups of HBcAg(MIR), or PBS (p<0.05, respectively) at any ratio of effector : target.
CONCLUSION: Our results indicated MIR in HBcAg presenting platform could present MAGE-A3 multiepitopes efficiently and induced significant humoral or cellular immunity. The immune strategy based on multiepitopeimmunization could have potential for preventing or controlling MAGE-A3 associated malignant disease.

The inherited marrow failure syndromes (IBMFS) are a heterogeneous group of diseases characterized by failure in the production of one or more blood lineage. The clinical manifestations of the IBMFS vary according to the type and number of blood cell lines involved, including different combinations of anemia, leukopenia, and thrombocytopenia. In some IBMFS, systemic non-hematologic manifestations, including congenital malformations, mucocutaneous abnormalities, developmental delay, and other medical complications, may be present. Fanconi anemia (FA), caused by germline pathogenic variants in the DNA repair genes comprising the FA/BRCA pathway is associated with congenital anomalies, bone marrow failure, and increased risk of myelodysplastic syndrome (MDS), acute myelogenous leukemia (AML), and solid tumors. Dyskeratosis congenita (DC) is a telomere biology disorder (TBD) caused by aberrations in key telomere biology genes. In addition to mucocutaneous manifestations, patients with DC are at increased risk of marrow failure, MDS, AML, pulmonary fibrosis, and other complications. Ribosomal biology defects are the primary causes of Diamond Blackfan anemia (DBA) and Shwachman Diamond syndrome (SDS). In addition to pure red blood cell aplasia, DBA is associated with elevated risk of solid tumors, AML, and MDS. Patients with SDS have pancreatic insufficiency, neutropenia, as well as MDS and AML risks. Patients with severe congenital neutropenia (SCN), caused by pathogenic variants in genes essential in myeloid development, have profound neutropenia and high risk of MDS and AML. Herein we review the genetic causes, clinical features, diagnostic modalities, predisposition to malignancies with focus on leukemogenic markers whenever available, and approaches to treatments of the classical IBMFS: FA, DC, SDS, DBA, and SCN.

INTRODUCTION: Prostate-specific membrane antigen (PSMA) is an extensively studied antigen for imaging prostate cancer. We prepared a single-chain variable fragment (scFv) of J591, a monoclonal antibody that recognises an external epitope of PSMA, incorporating a His-tag for labelling with Tc tricarbonyl, and evaluated its binding using human PCa cell lines.
METHODS: J591(scFv) was expressed in HEK-293T cells and purified by metal ion affinity chromatography, followed by size exclusion chromatography. Stability and monomer/dimer ratios of purified scFv under different storage conditions were analysed by SDS-PAGE and analytical size exclusion chromatography. J591(scFv) was labelled with (Equation is included in full-text article.)at 37°C for 60 min. The stability of Tc-scFv in human serum was analysed by SDS-PAGE with autoradiography. Cell-binding studies were carried out using PC3LN3 (PSMA negative) and PC3LN3-PSMA (a variant engineered to express PSMA) cell lines.
RESULTS: J591(scFv) was most stable to dimerisation on storage at -80°C compared with -20 and 4°C. Radiochemical yields of 85-90% were obtained with the final radiochemical purity of more than 99% after purification by gel filtration. In these small-scale studies, the maximum specific activity achieved was 7 MBq/μg. Liquid chromatography-mass spectrometry showed the formation of Tc-J591(scFv), which was radiochemically stable in serum, with no dissociation of Tc over 24 h. Cell-binding assays showed specific binding to PSMA-positive cells.
CONCLUSION: J591(scFv) can be radiolabelled with (Equation is included in full-text article.)conveniently and efficiently. The labelled product was stable in serum. It showed selective binding to PSMA-positive cells compared with PSMA-negative cells. This potential radiotracer warrants evaluation in PCa xenograft models.

AIM: Development of EGF and VEGF vaccines with high antigenicity for combined immunotherapy of EGF-EGFR signaling-dependent epithelial tumors such as breast cancer.
METHOD: EGF genes from mouse, human and chicken were randomly assembled to chimeric genes by DNA shuffling, then a chimeric EGF was selected out by PCR, SDS-PAGE and immunization for combined immunotherapy of breast cancer with a previously constructed chimeric VEGF vaccine from shuffling.
RESULTS: Combined vaccination with chimeric EGF and VEGF from shuffling could induce high titer of antibodies against EGF and VEGF to inhibit tumor growth and angiogenesis, and improve the survival rate of mice with breast cancer.
CONCLUSION: Combined vaccination with EGF and VEGF from shuffling showed better immunotherapy on EGF-EGFR signaling-dependent epithelial tumors such as breast cancer than the single-agent EGF vaccination.

BACKGROUND: Accurate determination of the predictive markers human epidermal growth factor receptor 2 (HER2/ERBB2), estrogen receptor (ER/ESR1), progesterone receptor (PgR/PGR), and marker of proliferation Ki67 (MKI67) is indispensable for therapeutic decision making in early breast cancer. In this multicenter prospective study, we addressed the issue of inter- and intrasite reproducibility using the recently developed reverse transcription-quantitative real-time polymerase chain reaction-based MammaTyper® test.
METHODS: Ten international pathology institutions participated in this study and determined messenger RNA expression levels of ERBB2, ESR1, PGR, and MKI67 in both centrally and locally extracted RNA from formalin-fixed, paraffin-embedded breast cancer specimens with the MammaTyper® test. Samples were measured repeatedly on different days within the local laboratories, and reproducibility was assessed by means of variance component analysis, Fleiss' kappa statistics, and interclass correlation coefficients (ICCs).
RESULTS: Total variations in measurements of centrally and locally prepared RNA extracts were comparable; therefore, statistical analyses were performed on the complete dataset. Intersite reproducibility showed total SDs between 0.21 and 0.44 for the quantitative single-marker assessments, resulting in ICC values of 0.980-0.998, demonstrating excellent agreement of quantitative measurements. Also, the reproducibility of binary single-marker results (positive/negative), as well as the molecular subtype agreement, was almost perfect with kappa values ranging from 0.90 to 1.00.
CONCLUSIONS: On the basis of these data, the MammaTyper® has the potential to substantially improve the current standards of breast cancer diagnostics by providing a highly precise and reproducible quantitative assessment of the established breast cancer biomarkers and molecular subtypes in a decentralized workup.

BACKGROUND/AIM: Telomerase activity is influenced by hTERT transcriptional regulation, shelterin, and posttranscriptional alternative splicing. Telomerase shelterin such as POT1 is highly correlated with various cancers. However, the profile of POT1 in cervical cancer has not been clearly identified. Therefore, it is important to identify its profile in cervical cancer biopsy tissue and normal cervical smears.
MATERIALS AND METHODS: Biopsy tissue of cervical cancer patients and normal cervical smears were characterized using SDS-PAGE and western blot. Sixteen biopsy tissues of cervical cancer patients and 15 normal cervical smears were measured for POT1 level using ELISA.
RESULTS: The inline band at 70 kDa indicated that all samples had protein that was identified as POT1. Western blot showed that telomerase antibody only recognized POT1 in biopsy tissue of cervical cancer patients. There was a significant difference (P = 0.01) in POT1 level between biopsy tissue of cervical cancer patients and normal cervical smears.
CONCLUSION: POT1 was identified at 70 kDa in biopsy tissue of cervical cancer patients and its level was higher than that in normal cervical smears. The high level of POT1 in the biopsy tissue of cervical cancer patients showed the influence of this shelterin component in cervical carcinogenesis and also cell immortalization.

Natural antisense transcripts (NATs) as one of the most diverse classes of long noncoding RNAs (lncRNAs), have been demonstrated involved in fundamental biological processes in human. Here, we reported that human prohibitin gene pseudogene 1 (PHBP1) was upregulated in ESCC, and increased PHBP1 expression in ESCC was associated with clinical advanced stage. Functional experiments showed that PHBP1 knockdown inhibited ESCC cells proliferation, colony formation and xenograft tumor growth in vitro and in vivo by causing cell-cycle arrest at the G1-G0 phase. Mechanisms analysis revealed that PHBP1 transcript as an antisense transcript of PHB is partially complementary to PHB mRNA and formed an RNA-RNA hybrid with PHB, consequently inducing an increase of PHB expression at both the mRNA and protein levels. Furthermore, PHBP1 expression is strongly correlated with PHB expression in ESCC tissues. Collectively, this study elucidates an important role of PHBP1 in promoting ESCC partly via increasing PHB expression.

Despite affecting up to 70% of HIV-positive patients and being the leading cause of dementia in patients under 40 years, the molecular mechanisms involved in the onset of HIV-associated neurocognitive disorders (HAND) are not well understood. To address this, we performed SILAC-based quantitative proteomic analysis on HIV-Tat treated SH-SY5Y neuroblastoma cells. Isolated protein was fractionated by SDS-PAGE and analyzed by nLC-MS/MS on an Orbitrap Velos. Using MaxQuant, we identified and quantified 3077 unique protein groups, of which 407 were differentially regulated. After applying an additional standard deviation-based cutoff, 29 of these were identified as highly significantly and stably dysregulated. GO term analysis shows dysregulation in both protein translation machinery as well as cytoskeletal regulation that have both been implicated in other dementias. In addition, several key cytoskeletal regulatory proteins such as ARHGEF17, the Rho GTPase, SHROOM3, and CMRP1 are downregulated. Together, these data demonstrate that HIV-Tat can dysregulate neuronal cytoskeletal regulatory proteins that could lead to the major HAND clinical manifestation-synapse loss.

Recurrent non-random chromosomal translocations are hallmark characteristics of leukemogenesis, and however, molecular mechanisms underlying these rearrangements are less explored. The fundamental question is, why and how chromosomes break and reunite so precisely in the genome. Meticulous understanding of mechanism leading to chromosomal rearrangement can be achieved by characterizing breakpoints. To address this hypothesis, a novel multi-parametric computational approach for characterization of major leukemic translocations within and around breakpoint region was performed. To best of our knowledge, this bioinformatic analysis is unique in finding the presence of segmental duplications (SDs) flanking breakpoints of all major leukemic translocation. Breakpoint islands (BpIs) were analyzed for stress-induced duplex destabilization (SIDD) sites along with other complex genomic architecture and physicochemical properties. Our study distinctly emphasizes on the probable correlative role of SDs, SIDD sites and various genomic features in the occurrence of breakpoints. Further, it also highlights potential features which may be playing a crucial role in causing double-strand breaks, leading to translocation.