mRNA Copy Number Research Articles

Hypoxia up-regulates VEGF ligand and downregulates VEGF soluble receptor mRNA expression in bovine granulosa cells in vitro

Oxygen concentration (02) in antral ovarian follicles is below that found in most tissues, which is important for adequate granulosa cell function. The VEGF system is linked to angiogenesis and responds to changing 02 by stimulating neovascularization when levels are low. However, in the avascular granulosa cell layer of the follicle, VEGF action is directed to stimulating cell viability and steroidogenesis. The aim of this study was to examine the effect of 02 concentration on granulosa cell expression of the VEGF-system components. Bovine granulosa cells were isolated from medium-sized follicles (4–7 mm in diameter), placed in McCoy 5a medium supplemented with 10 ng/mL of insulin, 1 ng/mL of IGF-I, and 1 ng/mL of FSH, and cultured in four well plates (500 thousand cells per well), on three separate occasions. Culture plates were placed in gas-impermeable jars with a gas mixture containing either 2%, or 5% of O2, or under atmospheric air condition inside an incubator (20% of 02). Media was replaced at 48 h of culture and cells from the plate in each oxygen concentration were pooled for RNA extraction after 96 h. The number of mRNA copies for the VEGF-system components - including ligands (VEGF120, VEGF120b, VEGF165 and VEGF165b), enzymes (cyclin-dependent like kinases-1, CLK1 and serine–arginine protein kinase 1, SRPK1), splicing factors (serine–arginine-rich splicing factors, SRSF1 and SRSF6), and the membrane-bound (VEGFR1, VEGFR2) and soluble forms of the receptors (sVEGFR1 and sVEGFR2) were quantified by qPCR. Granulosa cells cultured with low 02 (2%) had a higher expression of VEGF ligands (P < 0.05) when compared to cells cultured at 20% 02. VEGF164b mRNA was absent in granulosa cells from all culture conditions. The 2 and 5% 02 levels, which coincide with physiological concentrations, in the ovarian follicle, induced higher SRSF6 expression than atmospheric 02 concentrations (20%, P < 0.05). In contrast, mRNA copies for SRPK1, CLK1, SRSF1, VEGFR1 or VEGFR2 did not differ between 02 culture conditions. (P > 0.05). Nonetheless, mRNA copies for the soluble receptors, sVEGFR1 and sVEGFR2, linearly increased (P < 0.05) with 02 concentration. These results suggest that when cultured under hypoxic conditions, granulosa cells may develop an autocrine milieu that favors VEGF’s biological effects on their survival and function.

Abstract PS5-26: Pd-l1 mRNA expression in plasma-derived exosomes predicts prognosis and response to anti-pd-l1 antibodies in unresectable locally advanced triple negative breast cancer (TNBC) treated with atezolizumab-paclitaxel

Abstract Background Despite modern therapies have improved outcomes of patients with non-TNBC cancers, patients diagnosed with unresectable locally advanced TNBC usually have poor outcome for its aggressive clinical behaviour. Over time for cancer management, predicting and monitoring response to treatments is crucial, better if through easy non-invasive procedures. Atezolizumab plus nanoparticle albumin-bound (nab)-Paclitaxel prolonged progression-free survival (PFS) and overall survival (OS) among patients with TNBC, as demonstrated by phase III IMpassion130 trial. Despite this, there is a dire need for biomarkers reflecting treatment response. In our study, we correlated plasma PD-L1 mRNA levels with prognosis and response to anti -PD-L1 antibodies in unresectable locally advanced TNBC in the era of Atezolizumab. Methods We assessed 30 consecutive patients with unresectable locally advanced TNBC treated with Atezolizumab plus nab-Paclitaxel. Blood samples were collected at time point 0 (at baseline) and after 2 months and we assessed the association between PD-L1 mRNA copies per ml in plasma-derived exosomes and response to treatment. Exosomal PD-L1 mRNA in plasma was determined using Bio-Rad QX100 digital droplet PCR system and exoRNeasy kit. Objective responses were defined following the RECIST criteria v.1.1 Results At baseline, patients with complete and partial response (CR+PR, n=11) displayed a significantly higher number of PD-L1 mRNA copies per ml compared to ones with stable or progressive disease (SD+PD, n=19) (mean value: 745.6±131.1 vs 124.7±34.4, p&amp;lt;0.001). In patients with CR and PR mean PD-L1 copies/ml were 745.6±131.1 and 175.4 at baseline vs 2 months, respectively (p=0.001).In patients with stable disease the mean ± s.e.m. values were 270±71.1 vs 217.5±17.3 copies per ml (p=0.614) while in progressive disease PD-L1 mRNA levels were 112.1±31.2 vs 454.3±46.2 copies per ml at baseline vs 2 months, respectively (p&amp;lt;0.001).Two months after the start of Atezolizumab plus nab-Paclitaxel decrease of PD-L1 mRNA copies per ml relative to pre-treatment PD-L1 mRNA copies per ml, was positively correlated with an objective response to treatment.Increase of PD-L1 mRNA copies per ml was significantly associated with worse and shorter OS: median OS in patients with increase of PD-L1 mRNA copies/ml was 5 months (range 2-7 months, 95%CI 1.1-6.1); on the other side median OS in patients with decrease of PD-L1 mRNA copies/ml was more than double (range 8-15 months) (p&amp;lt;0.001).Moreover, prolonged survival outcomes (p&amp;lt;0.001) were recorded in cases with low baseline circulating Lox1+ Myeloid-derived suppressor cells (MDSC) (r=0.62, p&amp;lt;0.001) and high Natural Killer cells (NK) (r=0.66, p&amp;lt;0.001): the NK-to-MDSC ratio (NMr) was significantly higher in patients with higher number of PD-L1 mRNA copies per ml at baseline (p=0.002). During PD-L1 blockade, 2 months after starting treatment, NK progressively raised in clinical benefit group while declined in non-responders (p=0.001) Conclusions Despite the study’s limitations, our data suggest exosomal PD-L1 is significantly associated with outcome and response to treatment. Citation Format: Lucrezia Raimondi, Laura Di Benedetto, Victoria Bitca, Gian Paolo Spinelli. Pd-l1 mRNA expression in plasma-derived exosomes predicts prognosis and response to anti-pd-l1 antibodies in unresectable locally advanced triple negative breast cancer (TNBC) treated with atezolizumab-paclitaxel [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-26.

Is it Possible to Predict Non Sentinel Node Positivity on the Basis of mRNA Copy Numbers of CK19 Receptor in Breast Cancer?

AimsTo determine if there is any correlation between the number of positive non-sentinel lymph nodes (NSLN) and the mRNA copy numbers of cytokeratin 19 receptor on one step nucleic acid amplification (OSNA) in the sentinel lymph node (SLN). MethodsAn 8-year retrospective study of consecutive patients who had primary surgery and sentinel node biopsy for breast cancer from January 2011 to December 2018 was carried out. All these patients had intra-operative analysis of sentinel lymph nodes by OSNA. Patients who had neoadjuvant chemotherapy or neoadjuvant endocrine therapy were excluded. ResultsThere were 1159 patients with an age range of 24 to 90 years and a mean age of 63 years in this study. A total of 1324 SLNs were analyzed by OSNA. Macrometastasis was found in 120 patients and they underwent axillary lymph node dissection (ALND). A total of 2405 NSLNs were analyzed. Of the patients who had ALND, 51 (43%) patients had negative NSLNs and 69 (57%) had positive NSLNs. The mean mRNA copy numbers respectively for the 2 groups were 853,665 and 609,855. The difference between the means is not statistically significant (P = 0.82). Also the Receiver Operating Characteristic (ROC) Curve of the total CK-19 mRNA copy number for both groups-negative and positive NSLN were almost identical (Figure 3) indicating mRNA copy numbers cannot be used to discriminate between positive and negative non-sentinel lymph nodes. ConclusionIt is clear from our study that in patients who have ALND due to macromets on OSNA, there is no correlation between the total tumor load as represented by mRNA copy numbers and the likelihood of positive non-sentinel lymph nodes. We therefore cannot rely solely on the mRNA copy numbers to decide on ALND.

Epigallocatechin-3-gallate exhibits antiviral effects against the duck Tembusu virus via blocking virus entry and upregulating type I interferons

The duck Tembusu virus (DTMUV) is a novel mosquito-borne Flavivirus which caused huge economic losses for poultry industries in Southeast Asia and China. Currently, no effective antiviral drugs against this virus have been reported. (-)-Epigallocatechin-3-gallate (EGCG), a polyphenol present in abundance in green tea, has recently been demonstrated to have an antiviral activity for many viruses; however, whether EGCG can inhibit DTMUV infection remains unknown. Here, we tried to explore the anti-DTMUV effects and mechanisms of EGCG both in vitro and in vivo. Several EGCG treatment regimens were used to study the comprehensive antiviral activity of EGCG in DTMUV-infected baby hamster kidney cell line (BHK-21). The DTMUV titers of mock- and EGCG-treated infected cell cultures were determined using the tissue culture infective dose assay and the DTMUV mRNA copy number as determined using quantitative Real Time PCR. Moreover, the therapeutic efficacy of EGCG against DTMUV was assessed in DTMUV-infected ducklings. Our results suggested that EGCG significantly reduced the viral infection in BHK-21 cells in a dose-dependent manner, as reflected by the reduction of virus titers, virus copy number, and the expressions of viral E protein. We also observed that EGCG exhibited direct virucidal abilities against DTMUV. Notably, a significant reduction in virus binding ability was also observed, indicating that EGCG possesses excellent inhibitory effects on the viral adsorption step. In addition, DTMUV replication was also suppressed in BHK-21 cells treated with EGCG after viral entry, likely because of upregulation of the levels of interferon alfa and interferon beta. Finally, we also proved that EGCG exhibited anti-DTMUV efficacy in a duckling infection model because the survival rate was significantly improved. This is the first study to demonstrate the protective effect of EGCG against DTMUV, suggesting its potential use as an antiviral drug for DTMUV infection.

Challenge of Bovine Foot Skin Fibroblasts With Digital Dermatitis Treponemes Identifies Distinct Pathogenic Mechanisms.

Bovine digital dermatitis (BDD) is a common infectious disease of digital skin in cattle and an important cause of lameness worldwide, with limited treatment options. It is of increasing global concern for both animal welfare and food security, imposing a large economic burden on cattle farming industries each year. A polytreponemal etiology has been consistently identified, with three key phylogroups implicated globally: Treponema medium, Treponema phagedenis, and Treponema pedis. Pathogenic mechanisms which might enable targeted treatment/therapeutic development are poorly defined. This study used RNA sequencing to determine global differential mRNA expression in primary bovine foot skin fibroblasts following challenge with three representative BDD treponemes and a commensal treponeme, Treponema ruminis. A pro-inflammatory response was elicited by the BDD treponemes, mediated through IL-8/IL-17 signaling. Unexpectedly, the three BDD treponemes elicited distinct mechanisms of pathogenesis. T. phagedenis and T. pedis increased abundance of mRNA transcripts associated with apoptosis, while T. medium and T. pedis increased transcripts involved in actin rearrangement and loss of cell adhesion, likely promoting tissue invasion. The upregulation of antimicrobial peptide precursor, DEFB123, by T. phagedenis spirochaetes may present a microbial ecological advantage to all treponemes within BDD infected tissue, explaining their dominance within lesions. A commensal, T. ruminis, significantly dysregulated over three times the number of host mRNA transcripts compared to BDD treponemes, implying BDD treponemes, akin to the syphilis pathogen (Treponema pallidum), have evolved as “stealth pathogens” which avoid triggering substantial host immune/inflammatory responses to enable persistence and tissue invasion. Immunohistochemistry demonstrated increased IL-6, IL-8, RND1, and CFB protein expression in BDD lesions, confirming in vitro fibroblast observations and highlighting the system’s value in modeling BDD pathogenesis. Several unique shared gene targets were identified, particularly RGS16, GRO1, MAFF, and ZC3H12A. The three key BDD Treponema phylogroups elicited both distinct and shared pathogenic mechanisms in bovine foot skin; upregulating inflammation whilst simultaneously suppressing adaptive immunity. The novel gene targets identified here should enable future vaccine/therapeutic approaches.

Total Tumor Load of mRNA Cytokeratin 19 in the Sentinel Lymph Node as a Predictive Value of Axillary Lymphadenectomy in Patients with Neoadjuvant Breast Cancer.

Although sentinel lymph node biopsy (SLNB) has proved to be able to diagnose axillary lymph node status safely and reliably, there is still not enough evidence to suggest that it can be used in patients who have undergone neoadjuvant chemotherapy (NAC) for lymph node-sparing surgery. The present study used molecular approaches to determine whether SLNB can be reliably used in patients who have been treated with NAC before SLN surgery, and whether the total tumor load of the SLN can be used as a predictive factor in axillary lymphadenectomy (ALD). We used one-step nucleic acid amplification (OSNA) to analyze a total of 111 consecutive patients who presented operable invasive breast carcinomas and who had been treated with NAC. SLN was positive in 55 patients and the identification rate was 100%. In 9 of these 55 patients, ALD showed that other lymph nodes were also involved. In all of the other 46 patients, the only lymph node to be identified as positive was SLN. Metastasis was not found in any of the axillary lymph nodes in the isolated tumor cell group. The total tumor load, defined as the amount of cytokeratin 19 mRNA copy numbers in all positives SLN (copies/µL), showed three risk groups related to the possibility of positive non-sentinel nodes. OSNA is a diagnostic technique that is highly sensitive, specific, and reproducible and it can be used to analyze sentinel lymph nodes after NAC. Total tumor load may be able to help predict additional metastases in axillary lymphadenectomy.

MicroRNA Based Feedforward Control of Intrinsic Gene Expression Noise.

Intrinsic noise, which arises in gene expression at low copy numbers, can be controlled by diverse regulatory motifs, including feedforward loops. Here, we study an example of a feedforward control system based on the interaction between an mRNA molecule and an antagonistic microRNA molecule encoded by the same gene, aiming to quantify the variability (or noise) in molecular copy numbers. Using linear noise approximation, we show that the mRNA noise is sub-Poissonian in case of non-bursty transcription, and exhibits a nonmonotonic response both to the species natural lifetime ratio and to the strength of the antagonistic interaction. Additionally, we use the Chemical Reaction Network Theory to prove that the mRNA copy number distribution is Poissonian in the absence of spontaneous mRNA decay channel. In case of transcriptional bursts, we show that feedforward control can attenuate the super-Poissonian gene-expression noise that is due to bursting, and that the effect is more considerable at the protein than at the mRNA level. Our results indicate that the strong coupling between mRNA and microRNA in the sense of burst stoichiometry and also of timing of production events renders the microRNA based feedforward motif an effective mechanism for the control of gene expression noise.

Application of OSNA Nomogram in Patients With Macrometastatic Sentinel Lymph Node: A Retrospective Assessment of Accuracy

Introduction:Almost 50% to 70% of patients who undergo axillary lymph node dissection (ALND) because of a single metastatic sentinel lymph node (SLN) have no further metastatic nodes at the axillary histology. On these grounds, the one-step nucleic acid amplification (OSNA) nomogram was designed and validated. As a mathematical model, calculated through tumor size (expressed in millimeters) and CK19 mRNA copy number, it is thought to predict nonsentinel lymph node (NSLN) status. The aim of the study is to verify the diagnostic accuracy of the OSNA nomogram in a group of patients with macrometastatic SLN, with a retrospective analysis.Methods:The OSNA nomogram was retrospectively applied to a group of 66 patients with macrometastatic SLN who underwent ALND. The result of the final histology of the axillary cavity was compared to the nomogram prediction. We calculated the prevalence of NSLN metastasis in patients who underwent ALND, sensitivity and specificity, negative and positive predictive value of the nomogram.Results:In patients with macrometastasis in SLN, the prevalence of patients with metastatic NSLN was 45%. The sensitivity of the nomogram was excellent (90%). The specificity was low (36%). Positive predictive value amounted to 54%, while negative predictive value was good (81%).Conclusions:These results suggest that the OSNA nomogram is a valid instrument that can help choose the best surgical strategy for the treatment of axillary cavity. The mathematical model is useful to avoid surgery in a selected group of patients because it accurately predicts NSLN status.

Development and Validation of the Trans-Omics Model for Pancreatic Adenocarcinoma

Aim: To develop a trans-omics-based molecular clinicopathological algorithm for predicting pancreatic adenocarcinoma prognosis, we performed a comprehensive analysis of the expression levels of mRNA, DNA methylation and DNA copy number in The Cancer Genome Atlas dataset. Materials & methods: Based on the least absolute shrinkage and selection operator method - COX regression analysis, a trans-omics-based classifier was established to predict overall survival. Nomogram was constructed by combining the classifier band clinical pathological characterization. Results: Based on trans-omics, we developed a 10-gene-based classifier and a molecular-clinicopathologic nomogram for predicting overall survival with satisfactory accuracy. Conclusion: Trans-omics-based classifier and molecule-clinicopathological nomogram based on the classifier can accurately predict the prognosis of pancreatic adenocarcinoma patients.

Is it possible to predict non sentinel node positivity on the basis of mRNA copy numbers of CK19 receptor in breast cancer?

Background: One step nucleic acid amplification (OSNA) has found wide acceptance as a tool for intraoperative assessment of the sentinel lymph node (SLN). This is a semiquantitative assay based on quantification of mRNA copy numbers of cytokeratin 19 (CK19) receptor. The study aimed to determine if there is any correlation between the number of positive non sentinel lymph nodes (NSLN) and the mRNA copy numbers of CK19 receptor on OSNA in the SLN.

VSG mRNA levels are regulated by the production of functional VSG protein

The bloodstream form of Trypanosoma brucei persists in mammalian hosts through a population survival strategy depending on antigenic variation of a cell surface coat composed of the variant surface glycoprotein (VSG). The integrity of the VSG coat is essential and blocking its synthesis results in a cell division cycle arrest just prior to cytokinesis. This observation indicates that VSG levels are monitored and that the cell has mechanisms to respond to a disruption of synthesis. Here, the regulation of VSG mRNA levels has been investigated by first measuring VSG mRNA copy number, and second using ectopic expression of VSG transgenes containing premature termination codons. The findings are that (i) VSG mRNA copy number varies with the identity of the VSG and (ii) a pathway detects synthesis of non-functional VSG protein and results in an increase in VSG mRNA levels.

Transcription factors CP2 and YY1 as prognostic markers in head and neck squamous cell carcinoma: analysis of The Cancer Genome Atlas and a second independent cohort

PurposeThe transcription factors YY1 and CP2 have been associated with tumor promotion and suppression in various cancers. Recently, simultaneous expression of both markers was correlated with negative prognosis in cancer. The aim of this study was to explore the expression of YY1 and CP2 in head and neck squamous cell carcinoma (HNSCC) patients and their association with survival.MethodsFirst, we analyzed mRNA expression and copy number variations (CNVs) of YY1 and CP2 using “The Cancer Genome Atlas” (TCGA) with 510 HNSCC patients. Secondly, protein expression was investigated via immunohistochemistry in 102 patients, who were treated in the Vienna General Hospital, utilizing a tissue microarray.ResultsThe median follow-up was 2.9 years (1.8–4.6) for the TCGA cohort and 10.3 years (6.5–12.8) for the inhouse tissue micro-array (TMA) cohort. The median overall survival of the TCGA cohort was decreased for patients with a high YY1 mRNA expression (4.0 vs. 5.7 years, p = 0.030, corr. p = 0.180) and high YY1-CNV (3.53 vs. 5.4 years, p = 0.0355, corr. p = 0.213). Furthermore, patients with a combined high expression of YY1 and CP2 mRNA showed a worse survival (3.5 vs. 5.4 years, p = 0.003, corr. p = 0.018). The mortality rate of patients with co-expression of YY1 and CP2 mRNA was twice as high compared to patients with low expression of one or both (HR 1.99, 95% CI 1.11–3.58, p = 0.021). Protein expression of nuclear YY1 and CP2 showed no association with disease outcome in our inhouse cohort.ConclusionOur data indicate that simultaneous expression of YY1 and CP2 mRNA is associated with shorter overall survival. Thus, combined high mRNA expression might be a suitable prognostic marker for risk stratification in HNSCC patients. However, since we could not validate this finding at genomic or protein level, we hypothesize that unknown underlying mechanisms which regulate mRNA transcription of YY1 and CP2 are the actual culprits leading to a worse survival.

The value of HPV E6/E7 mRNA quantitative analysis in distinguishing high-grade cervical squamous intraepithelial lesions from low-grade cervical squamous intraepithelial lesions

A quantitative assay for HPV E6/E7 mRNA may be a valuable tool for cervical cancer screening. The purpose of this study is to compare the expression levels of HPV E6/E7 mRNA in high-grade cervical squamous intraepithelial lesion (HSIL) and low-grade squamous intraepithelial lesions (LSIL) and to determine a new method that can be used to distinguish cervical squamous intraepithelial lesions. Routine cytology, HR-HPV E6/E7 mRNA, histology, and p16 immunohistochemistry were performed in tissues from 142 patients with cervical squamous intraepithelial lesions. Significant differences were observed between the E6/E7 mRNA copy number values between the LSIL and HSIL cases (Mann–Whitney U‐test, P < 0.001). The optimal cut-off value (≥9,222.00 copies/mL) was determined using the receiver operating characteristic curve to predict diagnostic performance. Out of the 161 samples tested in this study, four cases were classified cytologically as HSIL but had normal histology. The E6/E7 copy numbers in these cases were all higher than 9,222 copies/mL. Therefore, a quantitative assay for HPV E6/E7 mRNA may be a valuable tool that can be used to distinguish HSIL and LSIL, especially for those with HSIL, for which samples are not obtained by biopsy, or when HSIL is difficult to distinguish by morphology and p16 immunohistochemistry.

Abstract B05: mEAK-7 supports self-renewal and radioresistance in metastatic cancer

Abstract Mammalian EAK-7, or MTOR associated protein, eak-7 homolog, (mEAK-7) functions as a crucial regulator of mTOR signaling. Dysregulation of mTOR signaling contributes to the pathogenicity of human carcinomas, so we sought to clarify the role of mEAK-7 in tumorigenesis and metastasis. Bioinformatics analysis revealed that MEAK7 mRNA or gene copy number amplification was observed in a multitude of human carcinomas, including non-small cell lung carcinoma (NSCLC). Tissue microarrays containing sections of primary NSCLC tumors, noncancerous adjacent tissue, and metastatic lymph tissue were used to quantify mEAK-7 and mTOR signaling. Our analyses revealed high mEAK-7 expression and mTOR signaling in metastasized lymph nodes. NSCLC cell lines H1299 and H1975 were used for all in vitro experiments. To examine the role mEAK-7 may play in cancer stem cell (CSC) self-renewal, NSCLC cells were FACS sorted into two groups: CD44+/CD90+ CSCs and non-CSCs. CSCs expressing CD44+/CD90+ demonstrated higher mEAK-7 expression and mTOR activation than non-CSCs, suggesting a role for mEAK-7 in cancer self-renewal and radiation resistance. Following X irradiation-induced DNA damage, 2D clonogenicity and 3D spheroid assays were used to further examine mEAK-7's role in clonogenic potential and self-renewal, respectively. These assays revealed that mEAK-7 is essential for the clonogenic potential and self-renewal of cancer cells in response to DNA damage. Comet assays were used to quantify the DNA damage response, and immunoblot and immunoprecipitation analysis confirmed that mEAK-7 is required for promoting radiation stress. Similarly, siRNA-mediated knockdown of mEAK-7 resulted in reduced DNA damage repair and enhanced cellular apoptosis following genotoxic stress. To do so, mEAK-7 may be interacting with components of the DNA damage response, such as DNA-PK. Immunoprecipitation and mass spectrometry analyses identified that mEAK-7 interacts with DNA-PK, and this interaction is strengthened following treatment with genotoxic stress. Further, DNA-PK interacts with mEAK-7 to regulate S6K2, a crucial target of the mTOR. This suggests crosstalk between the DNA-PK and PI3K pathways, since mEAK-7 interacts with DNA-PK to impact the DNA damage response in human cancer cells. Therefore, DNA-PK inhibitors may be utilized to regulate mEAK-7 and mTOR signaling in specific human carcinomas. Thus, mEAK-7 is a novel marker for NSCLC metastasis; overexpressed in both NSCLC and CSCs; and required for clonogenic potential, spheroid formation, and radiation resistance. Citation Format: Joe T. Nguyen, Fatima S. Haidar, Alexandra L. Fox, Connor Ray, Daniela B. Mendonça, Jin K. Kim, Paul H. Krebsbach. mEAK-7 supports self-renewal and radioresistance in metastatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B05.

High expression of forkhead box M1 (FOXM1) is a poor prognostic biomarker in lung adenocarcinoma.

BackgroundForkhead box M1 (FOXM1) is closely related to the formation and development of cancer. Because of differences in cellular origin, lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SCC) usually exhibit different signatures. Therefore, it is essential to investigate the abnormalities of FOXM1 in the two subtypes separately.MethodsThrough the Oncomine and TCGA databases, we investigated the expression of FOXM1 mRNA, its prognostic value and possible mechanisms leading to its dysregulation. Furthermore, networks involving FOXM1 and its significantly altered neighboring genes were identified using the cBioPortal database. GO and KEGG enrichment analyses were performed using DAVID.ResultsExpression of FOXM1 mRNA was higher in lung tumor tissues than in normal tissues, and higher in SCC tissues than in ADC tissues. FOXM1 mRNA expression was correlated with N stage, TNM stage, age, sex and smoking history in ADC, but only correlated with N stage, age and sex in SCC. Survival analysis indicated that high expression of FOXM1 mRNA resulted to poor overall survival (OS) for ADC patients, but not for SCC patients. Cox regression analysis confirmed that FOXM1 mRNA expression was an independent prognostic indicator for ADC patients, and regression analysis identified a moderately positive correlation between FOXM1 mRNA levels and copy number alterations (CNAs), but a weakly negative association with DNA methylation. FOXM1 was mainly involved in cell cycle regulation, G2/M transition, G1/S transition and p53, PI3K-Akt and TGF-beta signaling pathway.ConclusionsHigh expression of FOXM1 mRNA might be an independent biomarker of poor OS in ADC patients.

Polyunsaturated Fatty Acids of Both the Omega-3 and the Omega-6 Family Abrogate the Cytokine-Induced Upregulation of miR-29a-3p by Endothelial Cells.

Cellular processes fundamentally depend on protein expression control. At this, protein expression is regulated on the transcriptional and the post-transcriptional level. PUFAs are already known to affect gene transcription. The present study was conducted to answer the question whether PUFAs are also able to impact on the miRNA-mediated post-transcriptional fine-tuning of mRNA copy numbers. To this end, cellular miRNA profiles were screened by means of next-generation sequencing and NanoString analysis to compare PUFA-enriched to unsupplemented endothelial cells exposed to an inflammatory milieu. Validation took place by droplet digital PCR, allowing for an absolute quantification of RNA copy numbers. The analyses revealed that the stimulation-induced upregulation of miR-29a-3p is blocked by PUFA enrichment of endothelial cells. What is more, mRNA copy numbers of miR-29a-3p targets, namely the coagulation factors PAI-1, TF, and vWF, as well as the proinflammatory cytokines IL-1β, IL-6, and IL-8, were reduced in PUFA-enriched endothelial cells compared to unsupplemented cells, counteracting the stimulatory effect of an inflammatory environment. These data hint toward a new mechanism of action by which PUFAs modulate the functionality of endothelial cells. Apparently, the inflammation-modulating properties of PUFAs are also mediated at the post-transcriptional level.

180P Development of prognosis prediction model using cytokeratin 19 mRNA copy number of sentinel lymph node metastasis in breast cancer: A multicenter study in Japan

The one-step nucleic acid amplification (OSNATM) assay (Sysmex, Kobe, Japan) is an intraoperative diagnostic procedure performed for the detection of lymph node metastases using molecular techniques to amplify and detect cytokeratin 19 (CK19) mRNA. This multicenter study aimed to construct stratification of prognosis by CK19 mRNA copy number and development of a prognosis prediction model in breast cancer patients.

Probing mRNA Kinetics in Space and Time in &lt;em&gt;Escherichia coli&lt;/em&gt; using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization.

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

CD44v8-10 mRNA contained in serum exosomes as a diagnostic marker for docetaxel resistance in prostate cancer patients

BackgroundDocetaxel is first-line chemotherapy for castration-resistant prostate cancer (CRPC), but most patients acquire docetaxel resistance. CD44 has been shown to be involved in drug resistance of cancers including prostate cancer. We hypothesized that CD44 in serum exosomes could be a diagnostic marker for docetaxel resistance in CRPC patients. In this study, we examined CD44 protein and mRNA expression in cell lysates and exosomes isolated from prostate cancer cells, evaluated the effect of CD44v8-10 knockdown on docetaxel sensitivity and measured CD44 mRNA copy numbers contained in serum exosomes in prostate cancer patients. Materials and methodsDocetaxel-sensitive PC-3 prostate cancer cells and docetaxel-resistant PC-3R cells established previously from parental PC-3 cells were used. CD44v8-10 knockdown was performed by siRNA transfection. Blood was collected from 50 docetaxel-naïve and 10 docetaxel-resistant patients and 15 control males. CD44 protein expression was evaluated by Western blotting. CD44 mRNA expression was measured by RT-digital PCR. ResultsThe levels of CD44v8-10 protein and mRNA in cell lysates and exosomes were higher in PC-3R cells than in PC-3 cells. CD44v8-10 knockdown significantly increased docetaxel sensitivity of PC-3R cells. The CD44v8-10 mRNA copy numbers in serum exosomes were higher in docetaxel-resistant patients than in docetaxel-naïve patients and control males (median 46, 12 and 17 copies/mL serum, respectively, P = 0.032). In contrast, the serum exosomal mRNA copy numbers of CD44 standard isoform (CD44s) were not different among 3 groups (median 25, 14 and 13 copies/mL serum, respectively, P = 0.150). ConclusionsCD44v8-10 may be involved in docetaxel resistance in prostate cancer and serum exosomal CD44v8-10 mRNA could be a diagnostic marker for docetaxel-resistant CRPC.