Background: The AR-V7 splice variant is a cause of castration-resistant prostate cancer (CRPC). However, testing for the presence of AR-V7 by real-time polymerase chain reaction (RT-PCR) shows AR-V7 positivity in healthy individuals. We hypothesized that the positivity reflects contamination by hematopoietic cells. We tried a novel circulating tumor cell (CTC) enrichment instrument, using Celsee, to clear hematopoietic cells. Methods: We tested whole blood or Celsee-enriched samples for AR-V7 by RT-PCR, and included samples from 41 CRPC patients undergoing sequential therapy. We evaluated the associations between AR-V7 status and clinical factors. We evaluated factors affecting AR-V7 positivity. Results: AR-V7 positivity was lower in Celsee-enriched than in whole blood specimens. AR-V7 and clinical factors did not predict the therapy effectiveness. We found no significant differences in the effectiveness of enzalutamide/abiraterone (Enz/Abi) upon AR-V7 evaluation. All AR-V7 positive patients had resistance to Enz/Abi. Docetaxel (DTX), cabazitaxel (CBZ), and Radium223 treatment showed no significant difference in the treatment effectiveness, regardless of AR-V7 presence. AR-V7 was more frequently positive than Extent of disease (EOD) 2 in cases with bone metastases. Conclusion: Celsee CTC enrichment suppresses AR-V7 false positivity. All AR-V7 positive patients presented resistance to Enz/Abi. DTX, CBZ, and Radium223 were effective and remain treatment options. AR-V7 positivity should progressively appear in patients with advanced bone metastases. Androgen receptors (ARs) are encoded by eight exons (exons 2–3 are DNA binding domains and exons 4–8 are ligand binding domains (LBDs)). The AR splice variant 7 (AR-V7) has exon 3 followed by a cryptic exon 3 and omits exons 4 to 8. It lacks the LBD but retains functional, transcriptive element binding domains that mediate intracellular AR signaling in a ligand-independent manner ,2.AR-V7 has attracted attention because of its association with castration-resistant prostate cancer (CRPC). Enzalutamide (Enz), an inhibitor of AR signaling that binds to the LBD of the AR ,4, and aCTC analysis is usually performed in two steps (CTC enrichment and detection), but no standardized method exists. Problems during CTC enrichment include the possibility of contamination by hematopoietic cells and missing CTCs. The PCR detection is directly affected by the accuracy of CTC enrichment, and its results may reflect contaminations by cells other than CTCs . For exaGiven the differing sensitivity and specificity of AR-V7 assessment tests, depending on the test system, we considered the following two points to be important when assessing AR-V7 positivity: one was to exclude hematopoietic cells as much as possible during CTC enrichment, the second one was to target AR-V7 mRNA from CTCs during detection.Celsee  is a microfluidic device that can be used for CTC enrichment. Compared with CellSearch, which is the only system approved by the U.S. Food and Drug Administration (FDA), the capture rate of Celsee for CTCs is high . The basWe hypothesized that Celsee CTC enrichment would suppress AR-V7 false positives. The first aim of this study was to confirm the difference in the detection rate of AR-V7 between the PAXgene systems without CTC enrichment and those with Celsee CTC enrichment. We used samples from patients with CRPC treated with sequential therapy.We assumed that analysis of AR-V7 using Celsee would predict the outcome of CRPC treatment, and our second aim was to evaluate the associations between the current treatment efficacy and variables, such as the presence of AR-V7, and prognostic or clinical factors , lactate dehydrogenase (LDH), albumin (Alb), hemoglobin (Hb), metastatic sites on viscera, bone, or lymph nodes, Gleason score (GS), time-to-CRPC (period from the start of hormone therapy to CRPC), time-from-CRPC (period since becoming CRPC), and the history of therapeutic drug use).AR-V7 is thought to be positive during CRPC treatment. Our third aim was to identify factors affecting AR-V7 positivity by examining the association between prognostic and clinical factors.From January 2018 to September 2019, the study included a total of 41 patients with CRPC, including 38 with metastatic CRPCs (mCRPC) and three with non-metastatic CRPC (nmCRPC). All patients were treated with surgical castration, degarelix acetate, leuprorelin acetate, or goserelin acetate as androgen deprivation therapy in the Showa University hospital and received CRPC treatments with Enz, Abi, DTX, CBZ, Radium223, and traditional hormones . We also included samples from eight healthy controls recruited from volunteers without prostate cancer in the Showa University Urological Department. The Showa university clinical research ethics boards approved the study . All patients provided written informed consents.We measured PSA levels and obtained other laboratory tests for monitoring monthly. Imaging such as computed tomography (CT), magnetic resonance imaging (MRI), and bone scintigraphy were performed at least every 3–6 months.We defined stable disease as that in individuals with a <50% PSA decrease to <25% PSA increase from a PSA nadir, and disease response as that in individuals with a PSA decrease >50% in the absence of radiographic progression. We defined disease progression as radiographic progression or PSA increases ≥25% and ≥2 ng/mL above the nadir or baseline. We used Prostate Cancer Clinical Trials Working Group (PCWG) 3 soft tissue and bone scan criteria to assess radiographic progression . Time pog for 5 min at room temperature, we discarded the supernatants and resuspended the isolated cells in 8 mL of 1% Bovine Serum Albumin (BSA)/ Phosphate-Buffered Saline (PBS). We ran the samples through Celsee and collected the cells on the chip in 1 mL × 8 back flushes using 1% BSA/PBS, and centrifuged them at 326× g for 5 min. We used an RNA isolation NucleoSpin kit , according to the manufacturer’s protocol.We placed 5 mL of blood in anticoagulant, ethylene–diamine–tetra–acetic acid (EDTA) blood tubes. We treated 4 mL blood samples with red blood cell (RBC) lysis buffer (10×)  and let them stand for 20 min on ice. Following centrifugation at 326× We collected 2.5 mL blood samples into Paxgene blood tubes and stored them at −80 °C. We thawed the blood samples prior to extracting RNA from them using the Paxgene blood RNA kit , according to the manufacturer’s protocol.We processed all samples after RNA extraction with a DNase kit  and then ethanol-precipitated the DNA-free RNA samples with Ethachinmate .C system to determine RNA concentrations and absorption at 260 and 280 nm. We only used samples with a 260/280 ratios ≥2.0. We performed reverse transcription (RT) with 1 µg of RNA using a high-capacity complementary DNA (cDNA) Reverse Transcription Kit , according to the manufacturer’s protocol.We used the NanoDrop OneWe used the SYBR Green method for our RT-PCR assays. We made cDNA using the KAPA SYBR FAST qPCR Master Mix  to detect prostate cancer-associated RNA transcripts.We adapted the tests for detection of AR-V7 by RT-PCR, using custom primers specific for AR-V7 . We used primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH)  as a housekeeping gene.We performed RT-PCR under optimized conditions at 95 °C × 30 s, 95 °C × 5 s, and 60 °C × 30 s for 40 cycles, as well as 95 °C × 10 s and 65 °C × 5 s followed by melting curve analysis.We measured product concentrations using the CFX96  instrument and analyzed results with the CFX Maestro software (Bio-Rad). We confirmed that all AR-V7 positive specimens were GAPDH-positive.We collected 4 mL of whole blood samples from eight healthy volunteers into EDTA blood tubes. Subsequently, we performed RNA extraction, RT, and PCR assays on these specimens, and confirmed AR-V7 positivity in six cases .The figure shows the results of PCR tests in AR-V7-positive reactions of five healthy volunteers and Milli-Q water as a negative control (green line). The melting peak of AR-V7 lies around 0.5 degrees of 81.5 degrees.We observed an increase in Threshold Cycle (Ct) value with Milli-Q water, which was used as a negative control, but this probably reflects amplification of primer dimers. We considered AR-V7 positivity only when these conditions applied simultaneously: the melting peak was the same as that for AR-V7, and the Ct value increased.We found a Ct value elevation in the negative control (green line), but the melting peak clearly differed from it. We confirmed AR-V7 positivity in samples from six of eight individuals.We passed 4 mL blood samples from the six healthy volunteers positive for AR-V7 into the whole blood analysis through a Celsee instrument. The RT-PCR results confirmed these samples were negative for AR-V7 .We used Vcap as a positive control (red line) and Milli-Q water as a negative control (green line). The other two lines (blue and brown) are those for the healthy volunteers’ samples.The figure shows that the whole blood of the two healthy volunteers became negative after Celsee enrichment, and the melting peak was detected on the primer dimer side, but not on the AR-V7 graph line. We were able to confirm similar results for all six patients who displayed AR-V7 positivity in their whole blood samples.We spiked 10 and 100 Vcap (ATCC CRL-2876) into 4 mL healthy volunteer blood samples and added Celsee enrichment in the same manner to detect AR-V7 in RT-PCR. We confirmed AR-V7 detection .The red line in We found a melting peak similar to that of AR-V7 in the Vcap (10 and 100) spike group, and confirmed the melting peak only in the primer dimer graph portion in the Vcap 0 spike.By these procedures, we showed that the AR-V7 positivity in the whole blood of healthy volunteers became negative after Celsee enrichment; thereby, we confirmed that tumor-derived AR-V7 can be detected.By these procedures, the AR-V7 positivity in the whole blood samples of healthy volunteers became negative, and we confirmed that tumor-derived AR-V7 cells can be detected.We did not detect AR-V7 after Celsee enrichment in healthy individuals, and thus, we did not set a Ct value threshold for it in this study. We defined AR-V7 positivity as when GAPDH was positive by PCR and the melt peak of AR-V7 was confirmed within 0.5 degrees around 81.5.p < 0.05 as statistically significant.We performed all statistical analyses using the SPSS Software v.21.0 . We applied the Mann–Whitney U test to compare continuous variables , and Fisher’s exact test for categorical variables . We further analyzed factors with a significant difference using a multivariate logistic regression analysis. We considered p = 0.016). □ Hemoglobin (Hb) (SD) was 12.1 g/dl (1.4) for stable disease or response and 10.6 g/dl (1.5) for disease progression (p = 0.005). The time-to-CRPC (SD) was 36.9 months (35.6) for stable disease or response and 16.7 months (13.6) for disease progression (p = 0.005). p = 0.143), Hb , or time-to-CRPC .p = 0.066; We confirmed the effectiveness of Enz and Abi in past and current treatments of patients with or without AR-V7 positivity. We found no statistically significant differences, but none of the AR-V7-positive patients displayed Enz and Abi treatment effectiveness .p = 0.006).Among the patients with bone metastasis, the Celsee AR-V7-positive/negative ratio was 3 (7.3%)/27 (65.9%) in patients with an EOD2 classification or lower, and the Celsee AR-V7 positive/negative ratio was 6 (14.6%)/5 (12.2%) in those with EOD3 or higher (p = 0.058). The difference in the ratio of patients with EOD2 or lower/patients with EOD3 or higher was confirmed .Following our multivariate analysis , we founAR-V7 positivity may vary with the CTC method used to process the samples. False positive results may misguide a patient’s treatment. When interpreting AR-V7 status, the enrichment method used needs to be considered, and AR-V7 should be detected by PCR (reflecting mRNA levels) or by antibody reactions to measure the protein levels.Benign blood cells have been shown to express AR-V7. To further confirm the expression of ARV7, Marín-Aguilera revealed that CD4 and CD8 T-cells, B lymphocytes, T-natural killer cells, and monocytes isolated from PBMC became positive AR-V7 in PCR detection . TakeuchIn the Sciarra review, the mCRPC AR-V7 positive rate was reported at 18.3% (range 17.8–28.8%) , and ourOur AR-V7-positive patients were all resistant to Abi and Enz treatments . Possible causes for the resistance include lineage plasticity and AR indifference, glucocorticoid receptor activation, AR gains or LBD mutations, alternative AR variants and genomic structural rearrangements, AR enhancer amplification, and the presence of additional oncogenic pathways other than AR-V7 . Also, DThe JHU method is known as the AR-V7 PCR detection method after CTC enrichment. CTC enrichment is done with immuno-magnetic beads coated with anti-EpCAM and anti-Her2, and end-product lysates are evaluated on multiplex PCR with AR-V7, AR, PSA, Prostate Specific Membrane Antigen (PSMA), and Epidermal growth factor receptor (EGFR) primers. In the PROPHECY study, Enz and Abi were effective in 6–11% of AR-V7 positive patients [In the Epic Sciences platform, the effectiveness of Enz and Abi were both 0% in AR-V7-positive patients . This plAnother implication of our results is that AR-V7 positive patients have more bone metastases than those with EOD3, and these patients have more CTCs in their blood. A limitation of our system is its inability to assess the CTC counts. Other limitations of our study include the small number of samples studied. We targeted patients undergoing sequential therapy for CRPC and could not assess OS or progression-free survival. However, other reports have shown that AR-V7 negative patients do not usually have AR-V7 re-evaluations, with disease progression after their initial AR-V7 assessment and before the beginning of treatments for CRPC. Nakazawa showed that therapeutic interventions modify the AR-V7 status . StudyinAR-V7 false positives can be suppressed with a Celsee CTC enrichment strategy. All AR-V7-positive patients had resistance to Enz and Abi treatments after Celsee enrichment, and treatment with DTX, CBZ, and Radium223 improved their responses. Our results also showed that some AR-V7-negative patients also presented Enz and Abi resistance, and that AR-V7 is predominantly positive in patients with bone metastases greater than EOD3.


Bacillus caldovelox arginase mutant (BCA-M) for human cervical cancer treatment. Our studies demonstrated that BCA-M significantly inhibited the growth of human cervical cancer cells in vitro regardless of argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) expression. Drug susceptibilities correlate well with the expressions of major urea cycle genes and completeness of L-arginine regeneration pathways. With the expressions of ASS and ASL genes conferring resistance to L-arginine deiminase (ADI) which is undergoing Phase III clinical trial, BCA-M offers the advantage of a broader spectrum of susceptible cancer cells. Mechanistic studies showed that BCA-M inhibited the growth of human cervical cancer cells by inducing apoptosis and cell cycle arrest at S and/or G2/M phases. Our results also displayed that autophagy served as a protective mechanism, while the growth inhibitory effects of BCA-M could be enhanced synergistically by its combination to the autophagy inhibitor, chloroquine (CQ), on human cervical cancer cells.With our recent success in developing a recombinant human arginase drug against broad-spectrum cancer cell lines, we have explored the potential of a recombinant  Bacillus caldovelox arginase mutant (BCA-M) [Human cervical cancer is a disease that arises from the malignant growth of cells originating from the uterine cervix. Its development is strongly associated with the infection of high-risk type Human Papillomavirus (HPV) while the formation of invasive cancer requires the further accumulation of mutations and epigenetic alterations in certain tumor suppressor genes and oncogenes . Despite (BCA-M)  in the tBefore introducing BCA-M, we may first begin with the urea cycle . Apart fFor mechanistic studies, L-arginine depletion has been reported to induce cell cycle arrest and apoptosis in various susceptible cancer cells ,5,16,22.50 values calculated from the non-linear regression model ranged from the lowest of 0.19 U/mL in C-33A cells to the highest of 1.42 U/mL in ME-180 cells. Both IC50 values and max. % indicated a similar trend of drug susceptibility in the order of C-33A > SiHa > HeLa > CC3 ~ ME-180. On the other hand, ADI only showed effective inhibition on the growth of SiHa and C-33A cells but were highly resisted by the remaining cell lines even at a 10-fold higher maximum concentration tested than the two sensitive cell lines  using flow cytometry with propidium iodide (PI) staining and RNase digestion . Owing tTo further understand the mechanism of BCA-M-induced cell cycle arrest, the transcriptional regulations of cyclin genes were analyzed using quantitative real-time PCR . The expThe potential of apoptosis induction by BCA-M treatment in human cervical cancer cells was explored in C-33A, SiHa, HeLa and ME-180 cells using flow cytometric analysis with annexin V-FITC and propidium iodide staining. Owing to the different drug susceptibilities of these cancer cells and to optimize the detection of cellular responses, the cancer cells were treated with BCA-M at different doses and times chosen specifically for them. Our results showed that BCA-M significantly induced apoptosis in all the tested cell lines that have different drug susceptibilities and urea cycle gene expression profiles . For theOur results showed that both HeLa and SiHa cells treated with 0.4–1.6 U/mL BCA-M for 1 h displayed early signs of autophagy . In orde50 values, the combination indexes (C.I.) for BCA-M and CQ combinations were 0.78 ± 0.06 and 0.70 ± 0.21 for SiHa and HeLa cells, respectively  and ICle genes . All fivpathways . No one pathways . In this50 value was calculable. The max. % within the tested dose range for these resistant cells was below 33% and seemed to reach the lowest plateau  with positive expression of ASL and ASS genes exhibit a contrasting difference in susceptibility to ADI when compared to cell lines (SiHa and C-33A) that do not express the ASS gene . The thr plateau . In facter cells , we expe2/M and S phase while it arrested SiHa cells at the S phase alone and arrested ME-180 cells at G2/M phase only, with statistical significance in each case (0/1 phase via inactivation of the retinoblastoma protein (Rb) and p53 [1 phase progression but, unlike other cyclins, it is expressed for as long as mitogenic stimulation persists [1 phase, the cell is committed to progress through the cell cycle [1-S transition period, starting from the mid-G1 phase, peak at late G1 phase or the G1-S transition and then degraded sharply upon entering the S phase [1-S transition and serves a unique role in the initiation of DNA replication [1-S transition somewhat later than the initiation of cyclin E expression and its expression increases thereafter, reaching a peak in the S phase and persisting at a high level until its degradation just after the mitotic entry of the cell into prophase [2 phase progression via interactions with different CDKs [2/M phase and reaches its peak at G2/M transition. Then, it will be degraded in mitosis at the metaphase–anaphase transition [2/M phases . MTT -2,5-diphenyltetrazolium bromide) reagent, and all cell culture media, supplement and sera, were purchased from Invitrogen Life Technologies, Inc. . Human cervical cancer cell lines HeLa, ME-180, C-33A and SiHa  were obtained from American Type Culture Collection . Another human cervical cancer cell line CC-3 was obtained from the Chinese University of Hong Kong.Bacillus caldovelox arginase mutant (BCA-M) was prepared from E. coli harboring the expression vector for the his-tagged Bacillus caldovelox arginase and purified using nickel affinity column chromatography [Recombinant tography ,48. Recotography . Activittography ,51, withv/v) mixture of DMEM and Roswell park memorial institute 1640 medium (RPMI1640). All the culture media were supplemented with 10% fetal calf serum (FCS) and 100 units/mL penicillin/streptomycin (PS) and the cultures incubated at 37 °C in a humidified 5% CO2 incubator.HeLa, ME-180, C-33A and SiHa cells were maintained in Dulbecco’s modified eagle’s medium (DMEM). Another human cervical cancer cell line CC-3 was cultured in a 1:1 . The absorbance of untreated cells was considered 100% and the drug doses needed to produce 50% growth inhibition (IC50) were calculated using GraphPad Prism 5  with non-linear regression sigmoidal dose–response curve model. Maximum percentage (Max. %) mean suppressions were determined within the tested dose ranges of the respective drugs. Combination index (C.I.) was calculated using the Chou-Talalay method [50 values of drugs in the respective treatments with the formulaFive thousand cells from log-phase cultures were seeded in 100 μL per well of a 96-well plate  separately and incubated at 37 °C in a humidified 5% COy method  using ICTotal RNA was extracted using the RNeasy mini kit  according to manufacturer’s instruction from cultured cells. For RT-PCR, cDNA was reverse-transcribed from 1 μg total RNA in a 20 μL reaction at 42 °C for 30 min using iScript™ cDNA synthesis kit  according to manufacturer’s instruction. Then, 1 μL of the cDNA was used as a template for PCR amplification using GoTaqGreen master mix  in a 20 μL reaction according to manufacturer’s instruction in a MyCycler thermocycler (Bio-Rad Laboratories). Primers used for OTC  and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH)  were as described by [Log-phase cultured cervical cancer cells without drug treatment were used for protein expression profiling and cells with or without treatment of BCA-M or the combination of BCA-M and CQ was used for LC3 protein detection. Total protein from these cells was extracted from cultured cells using lysis buffer  supplemented with 1× complete mini protease inhibitor cocktail . For confirmation of OTC expression in OTC rescue study, transduced cells were lysed with Radio-immunoprecipitation assay (RIPA) lysis buffer . To prepare rat liver for OTC positive control, a small piece of rat liver was resuspended in RIPA lysis buffer and passed through a 25 G needle several times and filtered. In each case, cell debris was removed by centrifugation at 12,000 rpm at 4 °C. The extracted protein was resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto Polyvinylidene fluoride (PVDF) Immobilon-P membrane . The membranes were blocked with Tris-buffered saline  containing 5% blotting-grade blocker (Bio-Rad Laboratories) and the specific protein detected by hybridization with primary antibodies and visualized with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies using SuperSignal West Pico Chemiluminescent Substrate . For primary antibodies, mouse anti-ASS , rabbit anti-OTC , rabbit anti-LC3  and rabbit anti-GAPDH  were used to detect the respective proteins. HRP-conjugated goat anti-mouse secondary antibody  was used to detect anti-ASS. HRP-conjugated goat anti-rabbit secondary antibody  was used to detect anti-LC3 antibody. Another HRP-conjugated goat anti-rabbit secondary antibody  was used to detect anti-OTC antibody  and anti-GAPDH antibody . Protein signal intensities were determined using the ImageJ software .Cervical cancer cells were treated with BCA-M at various concentrations for 48 or 72 h and then harvested by trypsinization, fixed in 60% ethanol at 4 °C, washed in PBS, filtered via 60 μm nylon mesh , resuspended in PI/RNase staining buffer , and stained at 4 °C overnight. Finally, FACS analyses were performed on the stained cells using the FACSAria flow cytometer . For each sample, data from 10,000 cells were collected and cell cycle phase distribution analyses were performed using ModFit LT 3.1 .Cervical cancer cells were seeded and treated as in cell cycle analysis. Total RNA was extracted, and cDNA was synthesized as described above. Then, quantitative real-time PCR was performed with the cDNA using ABI Power Syber Green PCR master mix  on an MyiQ and iQ5 real-time PCR Detection System (Bio-Rad Laboratories). Reactions for each gene of each sample were performed in triplicate and the gene expression levels were calculated using ΔΔCT method with normalization to GAPDH expression and relative to the normalized expression levels of the respective genes in the drug treatment control groups. Primers used for the cyclin A1 (CCNA1) , cyclin B1 (CCNB1) , cyclin D1 (CCND1)  and cyclin E1 (CCNE1)  were as described by .2), and stained with Annexin V-FITC and PI for over 15 min at room temperature in the dark. Finally, the cells were applied to FACS analysis using FACSAria flow cytometer  within 1 h after staining.Cancer cells were treated with BCA-M at various concentrations and then harvested by trypsinization and washed in PBS. Detection of apoptosis was performed using the Annexin V-FITC Apoptosis Detection Kit  according to manufacturer’s instruction. Briefly, the washed live cells were resuspended in binding buffer (10 mmol/L HEPES/NaOH (pH 7.4), 140 mmol/L NaCl, 2.5 mmol/L CaCl® Autophagy detection kit was used to monitor autophagy in live cancer cells by Leica TCS SPE confocal microscope following the manufacturer’s instructions .CYTO-IDNotI and EcoRV of a transfer vector pShuttle-CMV using the forward primer 5′-AAGGAAAAAAGCGGCCGCATGCTGTTTAATCTGAGGATCCTGTTAAA-3′ and reverse primer 5′-CCGGATATCTCAAAATTTAGGCTTCTGGAGCTGAGGTGAGT-3′. The EGFP gene, isolated from vector pEGFP-C1 (Clontech Laboratories), was inserted into KpnI and NotI separately as control using the forward primer 5′-CCGGGTACCATGGTGAGCAAGGGCGAGGAGCTGTT-3′ and reverse primer 5′-AAGGAAAAAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAGAGT-3′. The resulting plasmid was then linearized with PmeI and cotransformed with plasmid pADEasy-1 into E. coli strain BJ5183 for homologous recombination. After screening for successful recombination and amplification using XL10 gold ultracompetent cells for greater DNA yield, the recombinant adenovirus construct was digested with PacI and transfected into AD293 cells by FugeneHD  for viral particle production. The effect of OTC expression was tested with cell proliferation assay in cancer cells transduced at 1:50 MOI with the recombinant adenovirus 24 h before addition of drug. The expression of OTC in transduced cells was also confirmed by Western blotting.The recombinant adenoviruses used for OTC expression were generated using AdEasy Vector System . Briefly, the OTC gene, amplified from human liver marathon-ready cDNA , was inserted between the restriction sites p values < 0.05 were considered statistically significant.Statistical analysis was performed with one-way ANOVA with post hoc Dunnett’s test and 2/M phases. Thus, BCA-M resulted in multiple suppression mechanisms towards all tested cancers, meaning that BCA-M may be a promising candidate for the treatment of cervical cancers.BCA-M significantly inhibited the growth of human cervical cancer cells in vitro regardless of the expression of ASS, while the ASS expression is strongly associated with the susceptibility of cancer cells towards ADI. BCA-M, therefore, offers the advantage of a broader spectrum of susceptible cancer cells. Drug susceptibilities correlate well with the expressions of major urea cycle genes and completeness of L-arginine regeneration pathways. Mechanistic studies showed that autophagy served as a protective mechanism and the growth inhibitory effects of BCA-M could be enhanced synergistically by its combination with the autophagy inhibitor, CQ, regardless of ASS expression. On the other hand, the anticancer mechanisms of BCA-M involved efficient induction of apoptosis and cell cycle arrest at S and/or G


Escherichia coli lysates and verified this approach as detecting 41 of the 54 r-proteins separately synthesized in E. coli S30 extracts. The SRM methods will enable us to utilize qMS as a highly specific analytical tool in the research of E. coli ribosomes, and this methodology have potential to accelerate the understanding of ribosome biogenesis, function, and the development of engineered ribosomes with additional functions.Ribosomes are the sophisticated machinery that is responsible for protein synthesis in a cell. Recently, quantitative mass spectrometry (qMS) have been successfully applied for understanding the dynamics of protein complexes. Here, we developed a highly specific and reproducible method to quantify all ribosomal proteins (r-proteins) by combining selected reaction monitoring (SRM) and isotope labeling. We optimized the SRM methods using purified ribosomes and  Escherichia coli ribosome is a multimeric protein complex composed of 30S and 50S subunits. The 30S subunit consists of 21 ribosomal proteins (r-proteins) and 16S ribosomal ribonucleic acid (rRNA), whereas the 50S subunit consists of 33 r-proteins, 5S rRNA, and 23S rRNA. The biogenesis of ribosomes is very complex, and several assembly-associated factors are involved in the process [E. coli 30S subunit was first reconstituted by Traub & Nomura in 1968 [E. coli 50S subunit was not achieved until 1974 by Nierhaus & Dohme [Ribosomes are the sophisticated machinery that synthesize proteins in all organisms. The in vitro integrated synthesis, assembly, and translation (iSAT) method that enables the co-activation of rRNA transcription, and the assembly of the transcribed rRNA with r-proteins into functional ribosomes in E. coli lysates. In contrast to the iSAT-based ribosome reconstruction, which uses a mixture of r-proteins, previous studies have also reported that individually purified ribosomal components can be reconstructed into 30S subunits [These early researchers revealed essential components of today’s knowledge about the assembly of ribosomes, which provides the basis of ribosome reconstruction and engineering. Jewett and coworkers –8 develosubunits –12. Thessubunits , which csubunits . Moreovesubunits .in vitro evolution, and a liposome sorting technique [In parallel to these efforts on ribosome reconstruction, researchers have tried to redesign the translational machinery. Orthogonal translation systems based on modified Shine Dalgarno (SD)/anti-SD sequence pairs enable the translation of orthogonal mRNAs independent of the cellular translation system –19. Thisechnique .E. coli r-proteins.It is valuable to analyze the dynamics of each component to understand the process of ribosome reconstruction. For example, tracking expression levels or incorporation rates of each r-protein into ribosome assembly intermediates provides crucial information toward understanding the assembly process. Previous studies used quantitative mass spectrometry (qMS) to reveal the composition of r-proteins and associated factors of ribosome assembly intermediates , 32, 33 E. coli r-proteins by combining targeted proteomics using SRM with isotope labeling of nascent r-proteins. We determined optimized transitions for the quantification of 54 r-proteins using protein digests of purified ribosomes, E. coli lysate, and r-protein-overexpressed E. coli lysates. Further, we verified the SRM methods as a high-throughput analytical tool by detecting 41 of the 54 r-proteins separately synthesized in E. coli S30 extracts. This method will enable the utilization of the SRM methods as a highly specific analytical tool in the field of ribosome research, and it has the potential to accelerate the understanding of ribosome biogenesis and development of functionally modified ribosomes  library [® HD Cloning Kit . Full sequences of the plasmids used in this study are provided in Genes encoding ribosomal proteins were amplified from the complete set of  library , and a vlacZ gene of BL21 Star™ (DE3)  was replaced with the kanamycin-resistance gene (kmr) using Red-mediated recombination [E. coli cells, and the transformants were cultured in 50 mL SOC medium with 100 μg/ml ampicillin  and 10 mM l-(+)-arabinose . The fragment of the kmr gene was amplified using primers from pKD13: the H1P1 forward primer, 5’- GAAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTGTGTAGGCTGGAGCTGCTTC-3', and the P4H2 reverse primer, 5’-TTACGCGAAATACGGGCAGACATGGCCTGCCCGGTTATTAATTCCGGGGATCCGTCGACC-3'. Then, it was purified using a MinElute® PCR Purification Kit . Electroporation was performed using a Gene Pulser® II  with a Nepa Electroporation Cuvette with a 1 mm gap  at 1350 V, 10 μF, and 600 Ohm. Next, the electroporated cells were incubated overnight at room temperature in SOC medium. After incubation, the cells were cultured on an LB agar plate containing 50 μg/ml kanamycin monosulfate  to select KmR transformants. The deletion of the lacZ gene was verified using blue-white selection on an LB agar plate containing 0.1 mM isopropyl-β-d-thiogalactopyranoside  and 4.0 × 10−3% 5-bromo-4-chloro-3-indolyl-β-d-galactoside (Takara Bio).The chromosomal bination . The Red™ Plasmid Mini  to prepare plasmids for cell-free protein synthesis (CFPS). Then, the extracted plasmids were purified further using a QIAquick® PCR Purification Kit (QIAGEN).The Miraprep method  was perf™ (DE3) lacZ::kmr cells were grown in 1 L of 2×YPTG medium. Then, the cells were disrupted using an EmulsiFlex-C5 homogenizer  with a single pass at a pressure of 20,000 psi. The lysate was centrifuged twice at 30,000 g for 30 min at 4 °C. Then, the supernatant was dialyzed using an iSAT buffer according to previous reports [S30 extract was prepared as previously reported –7. BL21  reports . The prod-thiogalactopyranosides. CFPS solutions (15 μL) were incubated at 37 °C for 3 h in a black-walled, polystyrene 96-well plate with a solid bottom and half volume . The expression of sfGFP and LacZ was monitored using a Fluoroskan Ascent FL™ 96-well-plate reader (Thermo Fisher Scientific) at λex = 485 nm and λem = 510 nm. To quantify the activity of lacZ, 3.3 nM of 5-chloromethylfluoresecein di-beta-d-galactopyranoside (CMFDG)  was added to the reaction solution. For isotope labeling, we used 20 amino acids that contained 2 mM of 13C615N2l-lysine (Thermo Fisher Scientific) and 13C615N4l-arginine (Thermo Fisher Scientific) instead of 12C614N2 l-lysine and 12C614N4l-arginine . The synthesized proteins were quantified using a liquid chromatograph-triple quadrupole mass spectrometer LCMS-8060 , as described below.Cell-free protein synthesis was conducted according to previous reports with slight modifications , 8. The E. coli BL21 (DE3) lacZ::kmr. The cells were grown at 37 °C to OD600 = 0.5–0.6 in 5 mL LB medium containing 100 μg/ml ampicillin , incubated at 37 °C for 3 h with 0.1 or 1 mM IPTG , and then centrifuged at 3,000 rpm for 5 min. Then, the pellets of the cells were suspended in 200 μL of lysis buffer of 2 M urea, 50 mM ammonium bicarbonate, and with a pH of 8.0 and transferred into 1.5 mL microtubes in an ice-water bath. The cells were disrupted using a sonicator  for 10 min at 4 °C . Then, the lysates were centrifuged at 14,000 g for 10 min at 4 °C, and the supernatants were collected. The ribosomal proteins were quantified using the LCMS-8060  as described below.The plasmids encoding each ribosomal protein were purified using the ASKA library . Then, tE. coli lysate, E. coli lysates containing each overexpressed r-protein, r-proteins produced in S30 extract, and LacZ produced in S30 extract were mixed with 120 μL of methanol, 30 μL of chloroform, and 90 μL of ultrapure water, which were added to 30 μL of each protein sample and vortexed thoroughly. Then, the mixtures were centrifuged at 13,000 g for 5 min. The top aqueous layer was pipetted off, and 90 μL of methanol was added to the solution. Then, the solution was mixed thoroughly and centrifuged at 13,000 g for 5 min. The aqueous layer was pipetted off and the pellet was air-dried for 5 min. Then, the pellet was dissolved in a lysis buffer of 2 M urea and 50 mM ammonium bicarbonate with a pH of 8.0. After alkylation and reduction steps [® C18 column  and dried using vacuum centrifugation. Then, the dried samples were reconstituted to 5 μg/μL using 10% formic acid. Next, the peptide samples were filtered through 0.45 μm filters . For the digestion of rplF, rplT, rplY, and rpmC that were synthesized in S30 extracts, samples were digested using Lys-C for 4 h, and then they were digested using trypsin for 24 h. The same enzyme-to-substrate ratio (75:1) was used when digesting with both Lys-C and trypsin.The protein samples of purified ribosome (New England BioLabs), on steps , proteol13C615N2l-lysine and 13C615N4l-arginine. Doubly and triply charged states of 6–25 mer peptides were selected to predict precursor ion candidates. Peptides, including methionine, were excluded from the candidates because they are susceptible to oxidation and unsuitable for quantitative analysis. Carbamidomethyl cysteine was set as a fixed modification. For fragment ions, singly charged or doubly charged b- or y-series ions with 50–1500 m/z were predicted. All selected peptides were evaluated for the uniqueness of their sequences using an E. coli background, and the SRM methods were scheduled based on the obtained retention time with a dwell time of 5 ms.The digested proteins were analyzed using an UltiMate 3000 RSLCnano (Thermo Fisher Scientific) and an LCMS-8060  triple quadrupole mass spectrometer. A 500 mm monolithic column with a100 μm ID  was connt-test.We analyzed protein digests of purified ribosome (New England BioLabs) and purified LacZ  and drew calibration curves to quantify the r-proteins and LacZ synthesized in S30 extracts. Up to three quantitative transitions were selected for r-proteins and six transitions were selected for LacZ. As for the quality control metrics, r-proteins with S/N ratio over 5 was defined as detected. The relative amount of each r-protein was quantified using a single transition with the highest S/N ratio among the selected transitions. We calculated the ratio of peak areas from isotope-labeled ‘heavy’ r-proteins and ‘light’ endogenous r-proteins to compare the abundance ratio of r-proteins synthesized in S30 extracts to the endogenous counterparts. The names of r-proteins were written according to the universal nomenclature for r-proteins . The datE. coli lysate and show the same retention times observed at (1), and (3) peptides that show stronger intensities when prepared using r-protein-overexpressed E. coli lysates compared with that of (2). We attempted to select three peptides for each r-protein and 3–4 fragment ions for each peptide to enable precise quantification.For the SRM analysis, we selected peptides that met the following criteria: (1) peptides that show intense peaks when prepared using purified ribosomes, (2) peptides that show intense peaks when prepared using E. coli lysate or r-protein-overexpressed E. coli lysates, as described in the Materials and methods section  lacZ::kmr strain. First, we developed SRM methods for LacZ with high specificity and accuracy  for LacZ quantification. The protein yield was deduced based on the peak area of the transitions. A non-isotope-labeled control was used to measure background signals. Data are shown as mean ± standard error. The mean values were calculated based on triplicate analysis. Statistical significance was determined using a two-tailed Student’s t-test with significance at *p <0.05, **p <0.01. The blue bar indicates heavy proteins and the red bar indicates light proteins.Quantification of LacZ synthesized with or without 2 mM of 13C615N2l-lysine and 13C615N4l-arginine instead of 12C614N2 l-lysine and 12C614N4l-arginine. The S30 extracts were digested and analyzed as described in the Materials and methods section, and the ratios of nascent r-proteins (‘heavy’) to endogenous r-proteins (‘light’) were calculated (Heavy/Light ratio) to investigate the abundance ratio of nascent proteins to endogenous counterparts. The r-proteins, which had an S/N ratio >5 were defined as detected proteins . In previous studies, r-proteins are known to be differentially modified in response to different stimuli , 59. In E. coli r-proteins by combining SRM with isotope labeling of nascent r-proteins. The SRM methods for r-proteins established in this study enable the profiling of ribosome biogenesis by quantifying each r-protein in a highly quantitative manner, and they enable us to utilize qMS as a high-throughput hypothesis testing tool in the field of ribosome research. This study provides a powerful method for understanding ribosome biogenesis, and it accelerates research on the reconstruction or engineering of E. coli ribosomes.In conclusion, we developed a highly specific qMS methods for 54 S1 FigE. coli lysate, and r-protein-overexpressed E. coli lysates using a liquid chromatograph-triple quadrupole mass spectrometer.Candidate peptides for selected reaction monitoring analysis were selected using Skyline software. We quantified the prepared r-proteins using purified ribosomes, (PDF)Click here for additional data file.S2 FigFor each peptide, several transitions with intense peaks were selected based on the quantification of r-proteins from purified ribosomes. The calibration curves of all transitions are described in (PDF)Click here for additional data file.S3 Figex = 485 nm and λem = 510 nm. Data are shown as the mean ± standard error of the mean (N = 3).sfGFP was expressed in S30 extracts prepared using an EmulsiFlex-C5 homogenizer. The expression of sfGFP was monitored using a 96-well-plate reader at λ(TIF)Click here for additional data file.S4 Fig(A) An ion chromatogram corresponding to the transitions associated with peptides from LacZ. Tryptic digests of LacZ standard were analyzed by a liquid chromatograph-triple quadrupole mass spectrometer. (B) Ion chromatograms corresponding to the selected transitions associated with peptides from LacZ. Tryptic digests of LacZ standard were quantified using triple quadrupole LC-MS/MS, and transitions with intense peak were selected. The whole peaks of all transition were provided in (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Although senescent cells display various morphological changes including vacuole formation, it is still unclear how these processes are regulated. We have recently identified the gene, lymphocyte antigen 6 complex, locus D (LY6D), to be upregulated specifically in senescent cells. LY6D is a glycosylphosphatidylinositol-anchored cell-surface protein whose function remains unknown. Here, we analyzed the functional relationship between LY6D and the senescence processes. We found that overexpression of LY6D induced vacuole formation and knockdown of LY6D suppressed the senescence-associated vacuole formation. The LY6D-induced vacuoles were derived from macropinocytosis, a distinct form of endocytosis. Furthermore, Src family kinases and Ras were found to be recruited to membrane lipid rafts in an LY6D-dependent manner, and inhibition of their activity impaired the LY6D-induced macropinocytosis. Finally, reduction of senescent-cell survival induced by glutamine deprivation was recovered by albumin supplementation to the culture media in an LY6D-dependent manner. Because macropinocytosis acts as an amino acid supply route, these results suggest that LY6D-mediated macropinocytosis contributes to senescent-cell survival through the incorporation of extracellular nutrients. Cellular senescence is defined as an irreversible cell proliferation arrest induced by various stresses, such as telomere erosion, activated oncogenes, oxidative stress, and DNA damage , 2. It hLymphocyte antigen 6 complex, locus D (LY6D) is a membrane-bound protein attached to the cell surface via a C-terminal glycosylphosphatidylinositol (GPI) anchor . AlthougGPI-anchored proteins are known to show dynamic behaviors in terms of their diffusion, organization, and interactions with other membrane proteins, and at the same time, a certain portion of GPI-anchored proteins is enriched in specialized domains of the plasma membrane to efficiently relay the extracellular signals to intracellular effector molecules , 10. TheMacropinocytosis is a clathrin- and dynamin-independent endocytic process by which substantial amounts of extracellular fluid and macromolecules are internalized into cells through large irregular vacuoles called macropinosomes . Unlike In the present study, we explored the relationship between LY6D and the senescence program. We have found that LY6D is needed for the senescence-associated vacuole formation, which occurs through the induction of Ras-mediated macropinocytosis. SFK and Ras were accumulated in the membrane lipid rafts upon senescence in an LY6D-dependent manner. Furthermore, LY6D-induced macropinocytosis can promote survival of senescent cells by the internalization of extracellular fluid under poor nutritional conditions.A; Δ1-20 LY6D mutant lacking N-terminal 20 amino acids was also used, as discussed later). Unexpectedly, we observed the cells showing extensive cytoplasmic vacuole formation after overexpression of LY6D (B). The vacuole formation is known as a typical feature of senescence (C and D). Furthermore, the vacuole formation associated with etoposide-induced senescence was also observed in normal human diploid fibroblasts Hs68 cells (E and F) in which the LY6D gene was previously reported to be upregulated during senescence (A). Knockdown of LY6D markedly suppressed the vacuole formation during etoposide-induced senescence . Similar results were obtained in Hs68 cells (D–F and D and E). As confirmed by immunoblotting analysis, the siRNA against LY6D inhibited the etoposide-induced upregulation of LY6D in Hs68 cells . These results suggest that LY6D is required for the development of senescence-associated vacuoles but not for the induction of senescence itself.To elucidate the possible function of LY6D in the senescence program, an expression vector containing N-terminal HA-tagged LY6D was constructed and introduced into human osteosarcoma U2OS cells . As shown in Fig. 2G, WT-LY6D was preferentially localized at the cell surface, consistent with the reported localization of LY6D , which leads us to speculate that the membrane-localized LY6D induces the vacuole formation. Because GPI-anchored proteins are known to be distributed to cell membrane lipid rafts/microdomains , as determined by using caveolin 1 as a raft marker. Furthermore, LY6D was barely detected even in the whole-cell lysate of untreated cells (fraction L), suggesting that LY6D was upregulated in response to DNA damage and hence localized to the raft areas. To examine the functional consequences of LY6D raft localization on vacuole formation, the cells overexpressed with LY6D were treated with methyl-β-cyclodextrin (MβCD), a cyclic oligosaccharide that disrupts the lipid rafts by depletion of membrane cholesterol . Most importantly, overexpression of integrinα4 and integrinβ1 failed to induce vacuole formation . These results suggest that the cell-surface localization of LY6D, more particularly in the raft areas, contributes to vacuole formation.Next, we generated an LY6D mutant (Δ1-20 LY6D) harboring a deletion of N-terminal 20 amino acids corresponding to the signal sequence A. As sho of LY6D , whereasodomains , 15, we odomains . As showlesterol , and vacA). Moreover, treatment with 3-methyladenine (3-MA), an inhibitor of class III PI3K widely used to inhibit autophagy, failed to suppress the LY6D-induced vacuole formation (B). To further confirm this, we tested the effect of knockdown of ATG5, a key autophagy gene, on the vacuole formation. The ATG5 expression was efficiently suppressed by the transfection of siRNA (C). Furthermore, when GFP-LC3 was introduced as an autophagy marker, knockdown of ATG5 inhibited GFP-LC3 puncta formation under normal growth conditions and serum starvation, indicating the successful suppression of autophagy by ATG5 knockdown (D). Most importantly, depletion of ATG5 failed to inhibit the LY6D-induced vacuole formation (E). Altogether, we concluded that the LY6D-induced vacuoles were not derived from autophagy.We next tried to determine the origin of LY6D-induced vacuoles. Given the significant association between senescence and autophagy, a catabolic process that consists of the isolation of cytoplasmic proteins and organelles by double-membrane cytoplasmic vacuoles (autophagosomes) and their degradation through fusion with lysosomes (forming autolysosomes) , we firsormation B. To furof siRNA C. Furthenockdown D. Most iormation E. AltogeA), raising the possibility that LY6D-induced vacuoles are derived from Ras-mediated macropinocytosis. To confirm this possibility, we incubated the LY6D-overexpressing cells with high-molecular-mass dextran, a fluid-phase macropinocytic marker . When combined with immunostaining for HA-LY6D, the signals of dextran and LY6D were barely overlapping with each other, suggesting that LY6D appears not to be internalized together with the newly formed vacuoles . For further confirmation, we tested the effect of ethylisopropylamiloride (EIPA), a potent inhibitor of Na+/H+ exchangers widely used to block macropinocytosis . Moreover, etoposide-induced vacuole formation was also diminished by EIPA in both U2OS and Hs68 cells (E and F). Finally, cytochalasin D, a potent inhibitor of actin polymerization required for macropinocytosis , collectively indicating that the LY6D-induced vacuole formation was mediated by macropinocytosis.It has been reported that oncogenic Ras stimulates cytoplasmic vacuole formation  and thatFig. 4c marker . We obsevacuoles . In addiocytosis , on the ocytosis , abolishA). As a result, only when both of the tagged LY6D overexpressed, the coprecipitated 20HA-LY6D was evidently detected by immunoblot analysis, suggesting that LY6D forms multimers in the cells like other GPI-anchor proteins. LY6D has a well-conserved LY6/uPAR domain that is characterized by five disulfide bonds and three flexible loops extended from the cysteine-rich core. This three-fingered structural motif is typically involved in the protein–protein interaction, although the binding partners of LY6D are currently unidentified. To elucidate whether the formation of LY6D multimers leads to the vacuole formation or not, we generated two LY6D mutants in which the second disulfide bond (Cys26-Cys32) and fifth disulfide bond (Cys87-Cys92) were disrupted by replacing Cys26/32 and Cys87/92 with serine (C26/32S and C87/92S), respectively, because the disruption of the second or fifth disulfide bonds in GPIHBP1, a member of the LY6 family, was shown to effectively inhibit the binding of GPIHBP1 to its binding partner, lipoprotein lipase , suggesting that these disulfide bonds (Cys26-Cys32 and Cys87-Cys92) play a vital role in the LY6D multimerization. Furthermore, vacuole formation was hardly induced by overexpression of the C26/32S mutant, whereas overexpression of the C87/92S mutant resulted in a slight decrease in the percentage of vacuole-forming cells than WT . Moreover, the C26/32S mutation reduced the raft localization of LY6D . These results suggest that the multimerization of LY6D is required for the induction of vacuole formation.We next sought to delineate the molecular mechanism(s) underlying the LY6D-induced vacuole formation. Because several GPI-anchored proteins are known to form homomultimers or heteromultimers at the raft areas , 23, we Fig. 5n lipase , 25. ImmE and Fig. S6C). These results suggest that a cluster of signaling proteins including SFK and Ras is formed in the lipid raft during senescence in an LY6D-dependent manner.Because it has been shown that the clusters of GPI-anchored proteins can act as signaling platforms for intracellular signaling proteins such as SFK , 13, 14,Fig. 5A), and the Ras-induced vacuole formation was remarkably inhibited by the treatment with FTS , all of which indicates that macropinocytosis induced by Ras is dependent on Ras activity itself as previously reported (A). As a result, both Wt-Ras and active G12V-Ras did not influence the LY6D-induced macropinocytosis, but dominant-negative S17N-Ras impaired it, suggesting that LY6D and Ras function in the same signaling pathway and Ras acts downstream of LY6D to induce macropinocytosis. Moreover, PP2, a specific SFK inhibitor, suppressed the LY6D-induced macropinocytosis but not the Ras-induced one (B). These results collectively indicate that the LY6D-SFK-Ras axis functions to induce macropinocytosis.Next, to investigate the functional relationships between the accumulation of signaling factors and macropinocytosis, we tested the impact of various Ras mutants on macropinocytosis , 27. OveFig. S5reported . To examreported , A. As auced one B. These C), suggesting that the MAP kinase pathway is dispensable for the induction of macropinocytosis. Therefore, to determine which pathway(s) is responsible for the LY6D-induced vacuole formation, we constructed three Ras mutants, T35S and E37G, and Y40C, that only interact with each of the effectors, Raf (MAP kinase), Ral-GEF , and PI3K (PI3K pathway), respectively , which raises the possibility that the PI3K pathway induced macropinocytosis. Consistent with the previous report that the Y40C mutant interacts with and activates PI3K, immunoblot analysis revealed that overexpression of the Y40C mutant activated the PI3K pathway, as judged by an increase in the phosphorylation level of AKT, a downstream effector of PI3K . In addition, class I PI3K inhibition by inhibitors, Wortmannin and LY294002, effectively suppressed both LY6D-induced and Ras-induced macropinocytosis (C). These results suggest that the LY6D-SFK-Ras axis induces macropinocytosis through the activation of the PI3K pathway.We next set out to elucidate the signaling pathway that acts downstream of LY6D-SFK-Ras to induce macropinocytosis. Ras can activate several different downstream pathways such as MAP kinase, Ral, and PI3K pathways , 29. We Fig. S5ectively , and tesocytosis C. These et al have shown that a glutamine deprivation–induced decline in survival of Ras-transformed cells is recovered by extracellular supplementation with bovine serum albumin (BSA) in a macropinocytosis-dependent manner, leading to the conclusion that Ras-induced macropinocytosis contributes to cancer cell survival through the incorporation of extracellular fluid (A). We found that cell viability was remarkably decreased when cultured in the low-glutamine medium  as previously reported  but not by either BSA or LY6D alone . To clearly show the effects of BSA supplementation on cell survival, the data of Fig. 7A, left panel were re-represented in Fig. 7A, right panel, in which viability rates of cells with to without BSA supplementation at each condition are shown. These results support the hypothesis that LY6D-induced macropinocytosis contributes to cell survival through the uptake of the extracellular fluid. To expand this observation in nonsenescent cells into the senescence context, we examined whether the BSA supplementation restores cell viability in etoposide-induced senescent cells . U2OS cells were treated with etoposide for 7 days and subsequently cultured in the high- or low-glutamine medium with/without BSA for additional 7 days. Nonsenescent cells were not sensitive to the low-glutamine treatment, whereas viability of senescent cells markedly decreased by glutamine deprivation , which was partially restored by the BSA supplementation . Because the BSA addition by itself did not improve viability of senescent cells under the high glutamine condition, these results suggest that senescence-associated macropinocytosis promote senescent cell survival through the incorporation of extracellular BSA. To confirm this using a biochemical approach, cell viability was measured by another method, the WST-1 assay, in which cell viability is monitored by the activity of mitochondrial dehydrogenases . Consistent with the results obtained from crystal violet staining, the WST-1 assay showed that the low glutamine–induced decline in survival of senescent cells was recovered by the BSA supplementation . Furthermore, to determine whether the effect of BSA supplementation on improvement in senescent cell survival is dependent on LY6D, the BSA-mediated recovery of viability was compared between LY6D-depleted and nondepleted cells . siRNA-mediated knockdown of LY6D clearly impaired the survival improvement by the BSA addition . Finally, the same experiment was performed using Hs68 cells . Senescent Hs68 cells were more susceptible to the low-glutamine condition than nonsenescent cells , which was mitigated by the BSA supplementation as is the case in U2OS cells . In addition, the BSA-mediated recovery of senescent cell viability was again dependent on LY6D . These results collectively indicate that senescence-associated macropinocytosis induced by LY6D contributes to survival of senescent cells in a certain low-nutritional status.We next aimed to determine the physiological significance of senescence-associated induction of macropinocytosis. Macropinocytosis has been recently reported to function as an amino acid supply route in Ras-transformed cancer cells , 16. Comar fluid , it is par fluid . Cell viar fluid A. We fouAlthough senescent cells acquire various phenotypic changes such as enlarged and flattened cell morphology, increased granularity, and vacuole formation, the molecular mechanisms underlying each phenotype are currently unclear ,2. We haFurthermore, we revealed by sucrose density-gradient centrifugation that LY6D was accumulated in the lipid raft areas in response to DNA damage. The lipid rafts are platforms for concentrating signaling proteins, such as SFK and Ras, and can influence signal transduction , 10, 30.How the SFK-Ras cascade induces macropinocytosis? Ras can activate multiple downstream pathways, including MAP kinase, Ral, and PI3K pathways , 29. Amoin vivo and whether or not LY6D induces macropinocytosis in malignant cancer cells are the key questions for future research. Regardless of their outcomes, our results clearly show that LY6D is responsible for the senescence-associated vacuole formation through the induction of Ras-dependent macropinocytosis, which may shed new light on the LY6D roles in the survival of senescent cells.Despite the loss of proliferative ability, senescent cells are known to have a high energy requirement . ActuallU2OS  and Hs68   cells weFor the construction of pcDNA3-HA-LY6D-Wt, an expression vector for N-terminal hemagglutinin (HA)-tagged human full-length (1-387 bp) LY6D, the LY6D cDNA was amplified with a pair of primers (a forward primer: 5’-CGTGCTCGGAATTCATGAGGACAGCATTGCTGCT-3’ and a reverse primer: 5’-CGTGCTCGGCGGCCGCTCACAGGCTGGGGGCTAAGA-3’) using a cDNA sample prepared from U2OS cells as a template. The resultant fragments were digested with Eco RI and Not I and cloned into pcDNA3 vector . The fragment of signal sequence–deleted LY6D (Δ1-20 LY6D) was amplified with the same reverse primer and a forward primer 5’-GCTGCTTCGAATTCCTGCGCTGCCACGTGTGCA-3’ and cloned into the pcDNA3 to generate pcDNA3-HA-LY6D-Δ1-20. For the construction of LY6D containing an internal HA tag (20HA-LY6D) or Flag tag (20Flag-LY6D) between amino acid residues 20 and 21, DNA fragments corresponding to the 5’ of LY6D (1-60 bp), the HA (TACCCATACGACGTGCCAGACTACGCC) or Flag tag (GACTACAAAGACGATGACGACAAG) sequence, and the 3’ of LY6D (61-387 bp) were ligated in this order to the pcDNA3 vector by using the In-Fusion cloning system  to generate pcDNA3-20HA-LY6D and pcDNA3-20Flag-LY6D. For the construction of transmembrane domain–containing versions of LY6D, DNA fragments corresponding to the N-terminal half of 20HA-LY6D (1-318 bp) and the transmembrane domain of human integrinα4 (2932-3096 bp) or integrinβ1 (2185-2394 aa) were ligated to the pcDNA3 vector by using the In-Fusion cloning system to construct pcDNA3-20HA-LY6D-integrinα4 and pcDNA3-20HA-LY6D-integrinβ1, respectively. The plasmid encoding GFP-fused human LC3, pBABEpuro GFP-LC3 (plasmid #22405), was obtained from Addgene . For the generation of HA- and Flag-tagged C26/32S-LY6D and C87/92S-LY6D mutants, PCRs were performed using mutagenic primers (a forward primer: TGCGCTGCCACGTGTCAACCAGCTCCAGCAACTCAAAGCATTCTGTGGTC and a reverse primer: TGCGCTGCCACGTGTCAACCAGCTCCAGCAACTCAAAGCATTCTGTGGTC for C26/32S-LY6D and a forward primer: GCTCCACCCAGTGCTCACAGGAGGACCTGTCAAATGAGAAGCTGCAC and a reverse primer: GTGCAGCTTCTCATTTGACAGGTCCTCCTGTGAGCACTGGGTGGAGC for C87/92S-LY6D) using pcDNA3-20HA-LY6D or pcDNA3-20Flag-LY6D as the template to generate pcDNA3-20HA-LY6D-C26/32S, pcDNA3-20HA-LY6D-C87/92S, pcDNA3-20Flag-LY6D-C26/32S, and pcDNA3-20Flag-LY6D-C87/92S. To construct the expression vectors, pcDNA3-HA-Ras-Wt, pcDNA3-HA-Ras-G12V, and pcDNA3-HA-Ras-S17N, that contain full-length human H-Ras-Wt, G12V, and S17N, respectively, the corresponding cDNA fragments were amplified with a pair of primers  using pCMV-HA-Ras-Wt, pCMV-HA-Ras-G12V, and pCMV-HA-Ras-S17N as the templates . The resultant fragments were digested with Eco RI and Not I and cloned into downstream of the HA tag sequence in the pcDNA3-HA vector. For the construction of H-Ras mutants , PCRs were performed using mutagenic primers  with pCMV-G12V-Ras as the template to generate pcDNA3-HA-Ras-G12V/T35S, pcDNA3-HA-Ras-G12V/E37G, and pcDNA3-HA-Ras-G12V/Y40C.Anti-LY6D antibody (sc-373838), HRP-conjugated anti-rat antibody (sc-2032), anti-Pan P-Tyr PY99 antibody (sc-7020), anti-c-Src antibody (sc-19), anti-c-Yes antibody (sc-8403), anti-c-Fgr antibody (sc-130), anti-Lyn antibody (sc-015), anti-Hck antibody (sc-072), and anti-Blk antibody (sc-329) were obtained from Santa Cruz Biotechnology ; anti-ATG5 (#12994), anti-P-Erk (T202/204) antibody (#9101S), anti-Erk antibody (#9102S), anti-P-Akt (S473) antibody (#9271S), and anti-Akt antibody (#9272S) were from Cell Signaling Technology ; anti–γ-tubulin antibody (T6557) and anti–FLAG M2 monoclonal antibody (F3165) were from Sigma Aldrich; anti–P-SFK (Y418) antibody (44--660G) and anti–P-SFK (Y529) antibody (44--662G) were from Thermo Fisher Scientific ; anti-HA 3F10 monoclonal antibody (1867423) and anti-HA 12CA5 monoclonal antibody (11666606) were from Roche ; HRP-conjugated anti-mouse antibody (W4021) was from Promega; anti-Ras antibody (#06-570) was from Merck Millipore; anti-Fyn antibody (PC32) was from Oncogene science ; anti-caveolin 1 antibody was from BD Biosciences .The cells were lysed in the lysis buffer , and the lysates were separated by SDS-PAGE and blotted onto Immobilon polyvinylidene difluoride membrane (Merck Millipore). Each protein was detected using primary antibodies as indicated, HRP-conjugated secondary antibodies, and the ECL detection reagent .2) for 24 h, the cells were examined under fluorescence microscope . Senescent cells were identified as blue-stained cells with phase contrast, and at least 100 cells in randomly selected microscopic fields were counted to determine the percentage of SA-β-Gal–positive cells. For EdU incorporation assay, U2OS cells were labeled with EdU for 3 h before fixation, and then, EdU incorporation was detected using the Click-iT EdU Imaging Kit (Life Technologies) according to the manufacturer's instructions. The stained cells were observed under fluorescence microscope .For detection of SA-β-Gal activity, the senescence β-galactosidase staining kit  was used according to the manufacturer’s instruction. Briefly, the cells were fixed with 2% formaldehyde/0.2% glutaraldehyde for 15 min and washed with PBS. After incubation with SA-β-Gal staining solution  and its control siRNA (D-001810-10) were from GE Healthcare Dharmacon . ATG5 siRNA (sc-41445) was obtained from Santa Cruz Biotechnology. Cells were seeded and transfected with 30-nM siRNA using HiPerFect Transfection Reagent  according to the manufacturer’s instruction.For immunofluorescence analysis, the cells were fixed with 4% paraformaldehyde and permeabilized in 0.5% Triton X-100 and then incubated with primary antibodies in Can Get Signal immunostain Solution B  overnight at 4 °C followed by incubation with the Alexa Fluor 488–conjugated secondary antibody (Life Technologies) for 1 h at room temperature. After staining cell nuclei with Hoechst 33258, the cells were observed under fluorescence microscope .g for 10 min. The supernatants were mixed with equal volumes of ice-cold 85% sucrose. The resulting mixtures (1 ml) were placed in an ultracentrifuge tube. A stepwise sucrose gradient (2.5 ml of 30% and 1.5 ml of 5% sucrose) was layered over the mixtures. The samples were centrifuged at 100,000g for 20 h in an SW55Ti rotor . After the centrifugation, 1-ml aliquots of 5 fractions were collected from the top to the bottom of the tubes. Fraction 2 was pooled as raft fraction, whereas fraction 5 was pooled as nonraft fraction , and the lysates were collected and centrifuged at 10,000fraction .For immunoprecipitation experiments, cells were lysed in Triton X-100–containing lysis buffer. The lysates were incubated with 2 μg of anti-FLAG M2 monoclonal antibody together with Protein G Sepharose  for 2 h at 4 °C with constant rotation. The bound proteins were eluted by addition of the SDS sample buffer . The obtained cell lysates and eluates were separated by SDS-PAGE and immunoblotted with the indicated antibodies.4 in a 12-well plate, cultured for 7 to 14 days, and stained with crystal violet (Wako). For WST-1 assay, U2OS cells were plated at 5 × 103 cells per well in a 96-well plate and cultured for 14 days, followed by the addition of WST-1 reagent to the medium and additional incubation for 20 min. The cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases was measured by absorbance at 450 nm.Cell viability was measured by crystal violet staining or by a cell proliferation reagent WST-1 (Roche). For crystal violet staining, cells were plated at 4 or 7.5 × 10p-values for all data sets.The two-tailed Student’s t-test was used to calculate All data are given in the main manuscript or supporting information.The authors declare that they have no conflicts of interest with the contents of this article.


Estrogen receptor α (ESR1) plays a critical role in promoting growth of various cancers. Yet, its role in the development of pancreatic cancer is not well-defined. A less studied region of ESR1 is the hinge region, connecting the ligand binding and DNA domains. rs142712646 is a rare SNP in ESR1, which leads to a substitution of arginine to cysteine at amino acid 269 (R269C). The mutation is positioned in the hinge region of ESR1, hence may affect the receptor structure and function. We aimed to characterize the activity of R269C-ESR1 and study its role in the development of pancreatic cancer.Transcriptional activity was evaluated by E2-response element (ERE) and AP1 –luciferase reporter assays and qRT-PCR. Proliferation and migration were assessed using MTT and wound healing assays. Gene-expression analysis was performed using RNAseq.n = 2800) compared to pancreatic ductal adenocarcinoma (PDAC) as well as other tumor types . Studies in breast and pancreatic cancer cells indicated cell type-dependent activity of ESR1 harboring R269C. Thus, expression of R269C-ESR1 enhanced proliferation and migration of PANC-1 and COLO-357 pancreatic cancer cells but not of MCF-7 breast cancer cells. Moreover, R269C-ESR1 enhanced E2-response elements (ERE) and AP1-dependent transcriptional activity and increased mRNA levels of ERE and AP1-regulated genes in pancreatic cancer cell lines, but had a modest effect on MCF-7 breast cancer cells. Accordingly, whole transcriptome analysis indicated alterations of genes associated with tumorigenicity in pancreatic cancer cells and upregulation of genes associated with cell metabolism and hormone biosynthesis in breast cancer cells.We examined the presence of this SNP in various malignancies, using the entire database of FoundationOne and noted enrichment of it in a subset of pancreatic non-ductal adenocarcinoma (Our study shed new light on the role of the hinge region in regulating transcriptional activity of the ER and indicates cell-type specific activity, namely increased activity in pancreatic cancer cells but reduced activity in breast cancer cells. While rare, the presence of rs142712646 may serve as a novel genetic risk factor, and a possible target for therapy in a subset of non-ductal pancreatic cancers. ESR1, is a member of the steroid/nuclear receptor superfamily and functions as ligand-activated transcription factors [The human estrogen receptor α (ERα), encoded by  factors . A less  factors –6.p = 0.07) [Approximately 75% of all breast cancers express ERα, and targeting ERα signaling, is a key treatment strategy in these tumors. ERα plays a major role in the development of other malignancies, including endometrial and ovarian cancers  gastric  = 0.07) . Yet, no = 0.07) .A documented rare SNP in the ESR1 gene (rs142712646) leads to a substitution of arginine at position 269 of the hinge region to cysteine (R269C). To our knowledge, the activity of this SNP, and its potential role in tumorigenesis, has not been reported yet. We report here on enrichment of this functional variant in non-PDAC pancreatic cancers. In this study, we aimed to characterize the activity of R269C-ESR1 in pancreatic and breast cancer cells and identify its role as a potential driver of proliferation of pancreatic cancer cells. Functional analysis revealed increased AP-1 dependent gene-expression of this variant in pancreatic but not in breast cancer cells, and expression of the R269C variant enhanced proliferation and migration of pancreatic cancer cells. These data indicate unique, cell-type dependent activity, of R269C and its contribution to tumor aggressiveness in a small subset of pancreatic cancers.We searched for mutations in the ESR1 gene, in the entire database of the Foundation One clinical database . The database consists of > 100,000 cases, of them > 4000 PDAC cases and ~ 2800 non-PDAC pancreatic cancer. The test has been described previously  and consThe secondary structure of ERα was predicted using ConSSert , PsiPred2) and crystal violet were obtained from Sigma ; ICI 182,780 from Tocris Bioscience , G418 from Life Technologies ; qScript cDNA SuperMix and PerfeCTa SYBR Green FastMix from Quanta BioSciences . Primers synthesis- IDT .17β-Estradiol , consists of 2 repeats of the upstream region of the vitellogenin ERE promoter. pRL Renilla luciferase control was purchased from Promega . The generation of WT-ER construct (in pcDNA3) was described elsewhere . ArgininHuman kidney cell line HEK293, breast cancer cell line MCF-7 and pancreatic cancer cell lines COLO-357 and PANC-1 were obtained from ATCC . All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (1%) at 37 °C in a humidified 5% CO2 atmosphere. All experiments were conducted with cells under 15 passages. For estrogen studies, cells were cultured in phenol-free media using 10% charcoal-treated serum  for 2 days before treatment. All transfections were conducted with Jet PEI  according to the manufacturer’s instructions.The assays were conducted essentially as described –42. In bTwo days after transfection with the different constructs, total RNA was prepared using the High Pure RNA Isolation Kit Roche (Roche). Total RNA (1 μg) was reverse transcribed using qScript cDNA synthesis kit (Quanta Biosciences). Quantitative RT-PCR (qRT-PCR) was used to determine mRNA level. Primers were designed using Primer Express  and synthesized by IDT . Primers used: GREB1a (human): F 5′-ACGTGTGGTGACTGGAGTAGC, R 5′- CCACGCAAGGTAGAAGGTGA; TGF-α (human): F 5′- CCCGCTGAGTGCAGACC, R 5′-ACGTACCCAGAATGGCAGAC; CyclinD-1 (human): F 5′- TGGAGGTCTGCGAGGAACAG, R 5′- AGCTGCAGGCGGCTCTTT; IGF-1R (human): F 5′- ATGTCCAGGCCAAAACAGGAT, R 5′- CAACCCTCCCACGATCAACA. Equal loading was determined using β-actin–specific primers. Amplification reactions were performed with Platinum qPCR SuperMix in triplicate using StepOne Plus (Applied Biosystems). PCR conditions: 50 °C for 2 min, 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 45 s.Migration was assessed using the wound-healing (“scratch”) assay. COLO-357, PANC-1 and MCF-7 cells were grown to confluency in 6-well plates, with the various constructs  and grown in phenol-free media with 10% charcoal-treated serum for 24 h. Cells’ monolayer was scraped in a straight line with a 200 μL sterile pipette tip for 48 h. The cells were photographed at 0, 24, and 48 h with an inverted phase-contrast microscope . Calculation of cell migration (d) was determined using the equation Cells were washed twice with PBS, snap frozen in liquid nitrogen and stored at − 80 °C until the analysis. Cells were harvested and lysed for total protein extraction in radio-immunoprecipitation assay (RIPA) buffer  together with a protease inhibitor cocktail (Sigma). A total of 50 μg protein extracts were loaded on 10% polyacrylamide gels, separated electrophoretically and blotted from the gel onto nitrocellulose membrane . The membranes were blocked with skim milk 1% in PBS-T  for an hour, and then immunoblotted overnight with the indicated antibodies. β-actin antibody was used as loading control. Membranes were washed 5 times with TBS-T, followed by incubation with horse raddish peroxidase  conjugated antibodies and were detected by using enhanced chemiluminescence (ECL) reaction.3--2,5-diphenyltetrazolium bromide (MTT) assay was conducted essentially as described . In brieMCF-7 or COLO-357 cells were seeded in phenol red depleted medium with charcoal stripped serum. Cells then were transfected with either WT-ER or R269C-ER in triplicates and then treated with vehicle control or E2 (10nM) for 24 h. Total RNA was extracted using the High Pure RNA Isolation Kit . RNAseq and bioinformatics were conducted at the Tel-Aviv University Genomics Research Unit and Bioinformatics Unit . The libraries were prepared using NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® . For sequencing: briefly, 1000 ng of total RNA were fragmented followed by reverse transcription and second strand cDNA synthesis. The double strand cDNA was subjected to end repair, A-base addition, adapter ligation and PCR amplification to create barcoded libraries. Libraries were evaluated by Qubit and TapeStation. Sequencing was conducted with NextSeq 500/550 v2.5 (Illumina) at 75-cycles, Single Read kit. The output was ~ 21 million reads per sample.Homo sapiens GRCm38 reference genome using BWA-MEM [Poly-A/T stretches and Illumina adapters were trimmed from the reads using cutadapt; resulting reads shorter than 30 bp were discarded. FastQ files were uploaded to Partek Flow  for proc BWA-MEM  . This enrichment suggests that the R269C substitution may play a role in the development of these tumors.The substitution of C to T at position 1039, leading to a substitution of arginine at position 269 to cysteine (R269C), was observed in a patient with mucinous pancreatic adenocarcinoma conducting tumor genomic analysis for clinical purposes. This substitution is a known rare SNP (rs142712646)  and its R269 lies within a potential nuclear localization sequences (NLS), known as p-NLS2 , 53. Anap < 0.01). In PANC-1 cells the mutant-ER activity was increased by 44% compared to WT-ER . In MCF-7 cells, R269C-ER increased the activity, with or without E2 treatment, by 61 and 114%, respectively . Finally, in HEK-293 the mutation increased the activity by 93%  and by only 16% with E2 treatment .In order to study the transcriptional activity of R269C-ER, we generated ER harboring the R269C substitution. Expression of R269C-ER in pancreatic cancer cells was similar to that of overexpressed WT-ER , it enhanced GREB and TGF-α mRNA levels in COLO-357 cells in response to E2 . These data indicate complex, cell-type dependent transcriptional activity of both WT-ER and R269C-ER in pancreatic cancer cells.In order to further validate the transcriptional activity of R269C-ER, we examined its ability to induce transcription of the classic estrogen-regulated genes GREB1 and TGF-α, which their promoter contains ERE sequences . Surprisp < 0.05 for all comparisons).The hinge region has been suggested to mediate non-classical transcription through interaction with the AF-1 domain and with transcription factors regulating AP-1 activity . In order to analyze the effects of R269C-ER on AP-1 activity, an AP-1-luciferase reporter was employed . COLO-35p < 0.01). Similarly, in PANC-1 cells we observed an increase of cyclin D1 and IGF-1R mRNA by 64 and 62%, respectively . While statistically significant, the effect of R269C-ER on MCF-7 cells was less pronounced: it decreased the levels of cyclin D1 by 25%, and increased levels of IGF-1 by 25% . Taken together, these data indicate AP-1 mediated transcriptional activity of R269C-ER, which is more profound in pancreatic cancer cells compared to breast cancer cells.Next, the effect of R269C-ER on the transcription of the AP-1-regulated genes was examined. Expression of R269C-ER in COLO-357 cells increased mRNA levels of both cyclin D1 and IGF-1R by 60 and 65%, respectively  with most upregulated genes associated with cell metabolism and hormone biosynthesis Fig. , a gene p < 0.01) and in PANC-1 by 16% . In contrary, it significantly decreased proliferation of MCF-7 cells by 12% .The effects of R269C-ER on proliferation of MCF-7, COLO-357 and PANC-1 cells were evaluated using MTT assay. For the analysis, cells were transfected with WT-ER or R269C-ER, grown in estrogen-depleted medium, and treated with either a vehicle control or E2. While R269C-ER enhanced proliferation of COLO-357 in the basal state by 149% . Similar results were observed with E2-untreated PANC-1 cells, after 24 h the migration was enhanced by 37% and after 48 h by 25% . However, the mutation did not increase migration of MCF-7 cells, but rather slowed migration compared to WT-expressing cells  to cysteine is expected to alter its interactions with both AF-1 and AF-2. In silico study via multiple bioinformatics tools , predicted with high probability that R269C substitution may damage the structure-function of this protein .In agreement with these predictions, our data indicates altered transcriptional activity of the variant compared to the WT receptor, an effect that was even more pronounced toward AF-1 than toward AF-2. These observations point to an important role of the hinge region in maintaining proper transcriptional activity of the ER and may explain the low frequency of alterations observed in this area in the general population and among species. We noted a discrepancy between direct transcriptional activity of R269C-ER, as determined using the ERE reporter, and the effect on mRNA of ERE-regulated genes in MCF-7 cells. This observation suggests a role for additional, possibly non-genomic mechanisms regulating the activity of R269C-ER specifically in these cells. The mutation lies within putative NLS2 and possibly affects the activity of the receptor by modifying its subcellular localization, however nuclear and cytoplasmic localization were not disrupted compared to WT-ER (data is not shown).Surprisingly, the effect of R269C variant was cell type specific. It inhibited gene expression, proliferation and migration of MCF-7 cells. The precise upstream regulatory elements leading to these differential effects are yet to be determined. This observation explains the lack of enrichment of R269C among breast cancer patients. Thus, expression of R269C is probably not associated with increased breast cancer risk. On the other hand, expression of R269C had profound effect on pancreatic cancer cells. It enhanced transcriptional activity of both classical and AF-1-regulated genes, enhanced proliferation and promoted migration of two pancreatic cancer cell lines. These observations may explain the enrichment of R269C among a subset of pancreatic cancers. It also highlights the need to study the effects of this mutation in additional pancreatic cancer subtype models.A global transcriptome analysis, using RNAseq, indicated differential gene expression among breast and pancreatic cancer cell lines expressing the mutated ER. Thus, expression of the R269C-ER was associated with ligand-independent upregulation of genes associated with cell metabolism and hormone biosynthesis, and not with growth or invasion. This observation may explain the lack of association between the presence of this SNP and increase breast cancer risk. On the one hand, alterations of genes associated with tumorogenicity were observed in COLO-357 cells. These included upregulation SERPINB2, a known oncogene –65, and Previous studies, published mostly during the 1980s and 1990s, explored the role of anti-estrogens as a potential treatment for pancreatic cancer. While pre-clinical studies were promising , clinicaOur data suggest a role for R269C functional variant of the ESR1 in the development of a small subset of non-PDAC pancreatic cancers. Moreover, our data suggest that this variant may have a protective effect against breast cancer. The underlying mechanism should be further explored.Additional file 1: Figure S1. Expression of R269C-ER in pancreatic cancer cells. PANC-1 (A) or COLO-357 (B) cells were transfected with either pCDNA3, WT-ER or R269C-ER grown in estrogen-depleted medium and treated with E2 (10nM) or a control vehicle for 24 h. Cells were harvested, lysed and analyzed by Western blotting. The results of (A) and (B) are from a representative experiment of n = 3.Additional file 2: Figure S2. R269C-ER exhibits differential effect following E2 treatment on gene expression in pancreatic cancer cells. A list of genes down and upregulated by R269C-ER compared to WT-ER following E2 treatment in COLO-357 cells.Additional file 3: Figure S3. R269C-ER exhibits differential effect on gene expression in pancreatic cancer cells compared to breast cancer. MCF-7 and COLO-357 cells were seeded in phenol red depleted medium with charcoal stripped serum. Cells then were transfected with either WT-ER or R269C-ER in triplicates and then treated with vehicle control or E2 (10nM) for 24 h. Total RNA was extracted and RNAseq was performed.  A heatmap of differentially expressed genes in MCF-7 cells treated with E2 (A) and COLO-357 cells treated with E2 (B) was generated.


This process was used to synthesise three specifically tailored nitrogen (N)-doped MCM catalysts: N-SAO, N-SAO  (PF) and N-SAO-reduced graphene oxide (rGO). Analysis using Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR) and X-ray diffraction (XRD) analysis confirmed the formation of macropores (100–350 µm). Investigation of ORR efficacy showed that N-SAOPF performed with the highest onset potential of 0.98 V (vs. RHE) and N-SAOrGO showed the highest limiting current density of 7.89 mAcm−2. The macroporous structure and ORR efficacy of the MCM catalysts synthesised using this novel process suggest that this method can be used to streamline MCM production while enabling the formation of composite materials that can be tailored for greater efficiency in many applications.Macroporous carbon materials (MCMs) are used extensively for many electrocatalytic applications, particularly as catalysts for oxygen reduction reactions (ORRs)—for example, in fuel cells. However, complex processes are currently required for synthesis of MCMs. We present a rapid and facile synthetic approach to produce tailored MCMs efficiently via pyrolysis of sulfonated aniline oligomers (SAOs). Thermal decomposition of SAO releases SO A SEM image of the MCM synthesised by the pyrolysis of SAO under inert (argon) conditions (P-SAO) is presented in The TEM image of the SAO presented in 0–300 °C . Therefoe of SO2 G. FigureUsing this unique synthesis process, two composites materials of SAO were synthesised using PF (SAOPF) and rGO (SAOrGO) and the photographic images of SAOPF and SAOrGO are shown in 2 gas from SAO.The composite materials SAO, SAOPE, SAOrGO and rGOPF were pyrolysed with a nitrogen precursor melamine to synthesise N-doped MCM catalyst materials for ORR. The SEM images of N-SAO, N-SAOPE and N-SAOrGO are shown in To investigate the presence of different chemical species present in N-SAO, N-SAOrGO and N-SAOPF, an XPS survey spectrum was conducted on these samples and is presented in The low intensity S 2p peak suggests the existence of small amounts of residual sulphur in the materials due to decomposition of sulphates. However, the atomic concentration of this peak is less than 0.80% in all samples.p electron to the aromatic π system and raises the density of the π state closer to the Fermi level to enhance the electron-donating capability of the ORR activity. The pyridinic nitrogen induces high positive charge density on adjacent carbon atoms and increases the electron donor properties of these carbon atoms to adsorb O2. This diatomic oxygen adsorption on the carbon atom adjacent to pyridinic nitrogen weakens the O–O bond and facilitates the ORR reaction [To probe the different nitrogen species, present in the graphitic frame work, we deconvoluted high resolution N 1s measurements on N-SAO, N-SAOrGO and N-SAOPF and presented in reaction .The comparison of the percentage atomic concentrations of these peaks  reveals This shows that the addition of PF and rGO to SAO has influence the formation of pyridinic nitrogen in the carbon frame work. N-SAOPF showed the highest percentage (%) atomic concentration of pyridinic nitrogen (42.4%) while N-SAOrGO showed 36.69%. Furthermore, the results show that SAO facilitates more graphitic N substitution and less pyridinic N substitution.−1 corresponding to D band and G band of graphitic carbon. The graphitic peaks, G and D, arise due to the E2g vibrational mode of the C–C bond stretching and the disorder peak due the A1g vibrational mode. The intensity ratio of the D and G band (ID/IG) determine the defects associated with the pyrolysed and N-doped samples, where a higher intensity (ID/IG) ratio show more defects [D/IG) showed 1.07, 1.08 and 1.02 for N-SAO, N-SAOrGO and N-SAOPF, respectively. The higher intensity ratio shows that reasonable distortion to the graphitic framework has taken place due to N-doping.XRD and Raman analyses were utilised to analyse the presence of graphitic carbon materials in the product. The XRD patterns E and Ram defects ,52. The 2 saturated 0.10 M KOH solution in the potential range of 0.00 to 1.20 V vs. RHE. The voltammograms show that, for all catalysts, the CV curves displayed well defined oxygen reduction cathodic peaks centred at 0.67, 0.68, 0.76, 0.59 and 0.84 V, for N-SAO, N-SAOrGO, N-SAO, N-rGOPF and Pt/C, respectively, demonstrating catalytic activity for ORR. The ORR cathodic peak positively shifted in the order of Pt/C > N-SAOPF > N-SAOrGO > N-SAO > N-rGOPF. This reveals that the N-doped composite materials of SAO (N-SAOrGO and N-SAOPF) enhanced the catalytic activity of ORR as opposed to that of the N-SAO itself. It is interesting to note that the cathodic peaks shifted positively according to the increasing percentage of pyridinic nitrogen seen in The electrochemical catalytic activity for ORR of N-doped  catalysts were examined by cyclic voltammetry (CV) and presented in 2− generated at the disc electrode using N-doped electrocatalysts in 0.10 M KOH on oxygen saturated solution [To quantify the ORR electron transfer pathway, we employed an RRDE technique to accurately measure the ring and disc currents, electron transfer number and percentage HOsolution   > N-SAO (6.29 mAcm−2) > Pt/C (5.85 mAcm−2) > N-SAOPF (5.18 mAcm−2) > N-rGOPF (3.99 mAcm−2) at 0.00 V (RHE)  . Lai et 2− generated in the potential region 0.00–1.15 V are presented in 2− yield (2− yield (19.02%). According to Liu et al. [p electron to the aromatic π system of the carbon matrix to increase the electron-donating capability of carbons adjacent to nitrogen to weaken the O-O bond strength and facilitate the ORR reaction [2−.The number of electrons transferred and percentage HO2− yield  at 0.5 Vu et al. , pyridinreaction . The hig1/2) of the catalysts positively increased according to N-SAOPF (0.79 V) > N-SAOrGO (0.66 V) > N-SAO (0.65 V) > N-rGOPF (0.44 V), showing that the ORR performed through both mixed diffusion and kinetic regions where current is controlled by both mass transport and kinetics of electron transfer [1/2 of the catalysts shifted more negatively compared to the E1/2 of the standard Pt/C catalyst (0.88 V). However, among all catalysts E1/2 of N-SAOPF shifted more positively in the mixed kinetics and diffusion range region revealing the coverage of high oxygen adsorption. The negative shift of the E1/2 of the catalysts can be attributed to their N-C active sites as reported by Liu et al. [1/2 can be shifted positively by the addition of Fe-N-C or Co-N-C active sites [The half-wave potential , highest electron transfer number (3.32) and lowest percentage HO2− yield (19.02%). Conversely, N-SAOrGO which exhibited more graphitic nitrogen species showed an increased limiting current density (7.89 mVcm−2).In this study, we have demonstrated a unique synthesis method to tailor MCMs by a pyrolysis process of SAO using in situ released SOFurthermore, this method can be a game changing synthesis process to fabricate new-state-of-art MCM N-doped carbon catalysts with selected active sites for ORR. Electrocatalytically active materials such as metallic nanoparticles, carbon nanotubes (CNTs) and single or few-layer graphene can be used to form composites with SAO to create new catalysts.