Transcriptional activity of XDR <i>Salmonella</i> Typhi after exposure to subinhibitory antibiotic concentrations

Background: Exposure to subinhibitory antibiotic concentrations can reflect the conditions that bacteria may encounter both in clinical and environmental sample types and could select for antimicrobial resistance. This exposure can induce stress and alter gene expression, promoting both bacterial survival and the emergence of resistance via mutagenic DNA repair. The purpose of this study was to determine the extent to which exposure to subinhibitory antibiotic concentrations alters gene expression in a drug-resistant isolate. Methods: We used an extensively-drug resistant Salmonella Typhi isolate to investigate the effects of treatment with subinhibitory concentrations of ampicillin (AMP), ceftriaxone (CEF), chloramphenicol (CHL), ciprofloxacin (CIP), co-trimoxazole (SXT) and tebipenem-pivoxil (TBP), as well as mitomycin C (MTC), on gene expression. Total RNA was extracted followed by ribosomal RNA depletion and library preparation for sequencing on a NovaSeq platform. Sequenced reads were processed using the nf-core/rnaseq pipeline followed by analysis using DESeq2 and topGO. A q-value of 2 or Results: Treatment with CIP, CHL, MTC and TBP significantly altered gene expression. No genes were significantly down-regulated from treatment with CIP, with the top Gene Ontology (GO) terms for the 25 up-regulated genes associated with SOS response, DNA repair and response to radiation, consistent with previously reported studies. Interestingly, CIP and MTC caused increased expression of both lexA, a main repressor for SOS genes, as well as recA, which is the main inducer of the SOS response pathway. CHL induced a total of 97 up-regulated and 54 down-regulated genes, with the majority involved in transmembrane transport, translation, and response to chemical. LysA (amino acid metabolism) and cpxP (stress response) were the only two genes up-regulated by TBP. No significant expression changes were found for treatment with AMP, CEF or SXT. Conclusion: These initial results replicate findings that subinhibitory concentrations of CIP as well as treatment with MTC can induce significant transcriptional up-regulation of genes in the SOS response pathway. Further investigation into the transcriptional response, specifically genes associated with the SOS pathway, is needed

Identification of candidate genes that may function in ER- breast cancer

Breast cancer is the most common cancer in women worldwide. Most breast cancers are hormone related involving the oestrogen receptor (ER), progesterone receptor (PR) and/or the human epidermal growth factor (HER2). The majority of aggressive (invasive and metastatic) breast cancers are negative for the ER receptor (ER-) including the triple negative and basal subtypes (ER-, PR-, HER2-). Due to this, traditional treatments such as hormonal replacement therapy are not suitable. Using bioinformatics, R scripting, and datasets from 1,485 Caucasian samples (1,437 tumours and 48 normal tissues) with 21,000 genes from the publicly available online GEO database (NCBI), we compared expression levels of ER negative (ER-) tumours to ER positive (ER+) tumours. 7 protein coding genes were identified as candidate functional genes, where their expression was significantly up-regulated in ER- tumours (log2 FC= 1.02-1.24, P=2.98E-31 - 6.47E-19) which correlated with reduced survival using distant metastasis free survival (DMFS) in Kaplan-Meier plotter (Log rank test, P=0.0013-0.033). Next, we will determine the effect of copy number changes, the functions of the genes in triple negative breast cancers and in cellular pathways, and then validate these results in the laboratory to test their potential as novel therapeutic targets in aggressive cancers and as biomarkers to predict prognosis or metastases

The functional Role of CASP8 D302H and Other Apoptosis Gene Variants In Breast Cancer

It is well established that perturbations in high penetrance genes such as BRCA1 and BRCA2 predispose to breast cancer. However, low penetrance genes are still under investigation. We recently reported that a coding single nucleotide polymorphism (SNP) in the caspase 8 gene (CASP8 D302H) is associated with a reduced risk of breast cancer. More recently we identified a CASP8 4-SNP haplotype associated with an increased risk of breast cancer. Our hypothesis is that CASP8 and other apoptotic genes influence breast cancer susceptibility via effects on the apoptotic response. Our objectives are to study the functional effects of CASP8 D302H and the 4-SNP haplotype on apoptosis induction in peripheral blood lymphocytes (PBLs). We have recruited women attending mammography screening and measured the ability of their PBLs to undergo drug-induced apoptosis. Levels of apoptosis and caspase-8 activity were determined by FACS analysis. Based on data from 61 samples, apoptosis levels range from 50% to 92% (median 78%, SD 9.5) and CASP8 protein levels 44% to 94% (median 68%, SD 13.2). We have successfully detected variations in apoptotic/CASP8 response and aim to determine whether these variations correlate with CASP8 genotype, to help us understand the mechanism of the association with breast cancer

Abstract 2844: Association of genetic variants in TNFRSF10B and breast cancer

Susceptibility to non-Mendelian forms of breast cancer likely involves many low penetrance variants from multiple genes. Recent associations between genetic variants in CASP8 and breast cancer suggest that CASP8 is one such gene contributing to breast cancer risk (Cox et al. 2007; Shephard et al. 2009). Here we investigate TNFRSF10B for associations with breast cancer. TNFRSF10B is a death receptor gene whose protein signals via caspase-8 in the extrinsic apoptosis pathway. We selected 11 tagging-SNPs (tSNPs) to represent the majority of the common genetic variation in the TNFTRSF10B gene. These tSNPs were genotyped on 1,992 independent cases and controls from Sheffield, UK. We performed 11 single marker association analyses and a haplotype-mining analysis using hapConstructor (Abo et al. 2008). HapConstructor implements a stepwise forward-backward procedure to search for haplotypes associated with disease. The algorithm allows for automatic consideration of non-contiguous SNP sets and the construction of numerous different genetic models that consider haplotypes, composite genotypes, and both monotype (chromosome-based) and diplotype (individual-based) tests. Briefly, all single locus tests are performed, then loci that surpass predefined significance thresholds are considered in all two-locus combinations, and so on. For the single marker association testing, dominant, recessive, and trend tests were performed. For the hapConstructor analysis, monotype haplotype tests and diplotype dominant and recessive tests were considered. HapConstructor is implemented within a Monte Carlo testing framework, with all p-values estimated empirically from 100,000 simulations under the null. Seven SNPs reached nominal significance in at least one of the association tests performed in the single SNP tests. Five of these were based on a recessive model (most significant single SNP yielded p = 0.002). Using hapConstructor, the most significant finding was a 4-SNP haplotype that was associated with an increase of risk (p=0.0006; OR=1.75). Four haplotypes were also identified that were associated with a decrease in risk and each obtained the same level of significance and risk (p=0.00096; OR=0.35). These results for reduced risk were obtained using the diplotype recessive model, similar to the single SNP findings. These results provide initial evidence that further common genetic variants in genes in the extrinsic apoptosis pathway, beyond CASP8, are involved in breast cancer susceptibility

Identification of a long non-coding RNA that mediates response to therapy in castration-resistant prostate cancer

Prostate cancer (PCa) is the second most commonly diagnosed neoplasm and the sixth leading cause of cancer related death in males, worldwide. It is commonly treated using androgen deprivation therapy, but around 25% of PCas develop resistance to this treatment and are therefore called Castration-Resistant Prostate Cancers (CRPCs). Effective therapies for CRPC include: the second-generation anti-androgen enzalutamide; the taxane cabazitaxel; carboplatin, which is active in androgen receptor negative CRPCs. Nevertheless, response to these therapies is often short-lived making CRPC incurable. Recent evidence suggests that long non-coding RNAs (lncRNAs) can play a role in drug resistance. Using RNA sequencing and qPCR validation, we analysed the expression of lncRNAs in a panel of patient-derived PCa xenografts (PDXs) with opposing sensitivity to castration. Our data showed that HORAS5 was the most consistently up-regulated lncRNA among the CRPC vs. hormone-sensitive PDXs. Moreover, HORAS5 protected CRPC cells from androgen-deprivation induced apoptosis. We next investigated whether HORAS5 had any effect on PCa cells` response to therapies. We analysed the expression and sub-cellular localization of HORAS5 in CRPC cell lines with endogenous or lentiviral-induced expression of HORAS5. CRPC cells were treated with different concentrations of cabazitaxel, carboplatin and enzalutamide. Upon treatment, the expression of HORAS5 was tested via RT-qPCR. Our data showed a significant, dose-dependent increase in HORAS5 expression in cells exposed to cabazitaxel (p<0.01; maximum fold change: 95.07918) and carboplatin (p<0.01; maximum fold change: 71.324). We did not register any significant increase in HORAS5 expression after treatment with enzalutamide. Hence, our results revealed that the expression of HORAS5 can be modulated by two chemotherapeutics in a dose-dependent manner. We are currently evaluating whether HORAS5 silencing affects CRPC cells` response to these drugs. Our data demonstrated that HORAS5 is located mainly in the cytoplasm of PCa cells, and we have predicted that HORAS5 may bind several microRNAs. We therefore hypothesized the involvement of HORAS5 in RNA-induced silencing, where it could act like a competitive endogenous RNA. Our findings suggest that HORAS5 could have a role in CRPC cells` response to therapy. We will further investigate the molecular mechanisms by which HORAS5 drives this phenomenon

Discovery of candidate hub genes in breast cancer

The functions of clinically relevant genes in breast cancer and their molecular role in cell pathways has not been completely discovered yet. Here we aimed to identify candidate hub genes in breast cancer that reduce survival and are confirmed in ovarian and prostate cancers, so we could select the most impactful genes. Using R scripting with Empirical Bayes Modified t-test we compared the gene expression levels of 436 normal tissues to 3,612 tumours with up to 13,000 genes from Caucasian microarray datasets in the Gene Expression Omnibus (NCBI). Seven novel candidate protein coding hub genes were identified. They were amongst the most significant (adjusted Benjamini-Hochberg FDR, P=1.20-3-8.76-11) and upregulated genes (log2 fold change=0.29-2.86), and correlated with overall survival (HR=2.12-4.95, P=0.01-0.04) in SurvExpress (omicX). We also constructed weighted gene co-expression network images using Cytoscape 3.7.1 (NIGMS) and identified corresponding cell pathways using The Gene Ontology knowledgebase and literature searches. So far, three of the candidate hub genes were linked in their cell pathways; mainly operating in mitotic spindle formation and the G2/M phase cell cycle check point, with one gene that may act to bypass the cell cycle checkpoint and apoptosis, and thereby allow uncontrolled proliferation. Next, we will analyse the functions of the remaining four candidate hub genes and propose a mechanism of action and validate these findings using siRNA assays. The discovery of novel candidate hub genes could be used to develop novel biomarkers and treatments for breast cancer that may also be applicable to ovarian and prostate cancers

Discovery of candidate long non-coding RNAs as biomarkers and treatment targets for prostate cancer

The role of long non-coding RNAs (lncRNAs) in prostate cancer is largely unknown, and discovery of consistent biomarkers for cancer subtype diagnosis and novel treatment targets is needed, particularly for more aggressive tumours such as neuroendocrine prostate cancers (NEPCs). To identify lncRNA signatures in Gleason group subtypes, we used R scripting with Empirical Bayes Modified t-test to compare lncRNA expression levels of 98 normal samples to 262 prostate cancer tissue samples with up to 3,750 lncRNAs from 3 Caucasian microarray datasets in the Gene Expression Omnibus (NCBI). Across the Gleason group subtypes we identified 61-287 significantly (adjusted Benjamini-Hochberg FDR, P=1.09E-13-0.049) upregulated (log2 fold change=0.19-0.78) lncRNAs. The most significant and highest upregulated lnRNAs were identified as candidate biomarker/novel treatment target signatures. Consistently across all subtypes, one of the lncRNAs (log2 fold change=0.44-0.77) was significantly correlated with reduced overall survival (combined HR=10.44, P=0.026) using SurvExpress (omicX) and was identified as a candidate generic biomarker/treatment target for prostate cancer regardless of subtype. In Gleason groups 2-5, eight of the lncRNAs (log2 fold change=0.29-0.99) were correlated with reduced relapse free survival (HR=2.09-14.29, P=0.002-0.05). We also constructed weighted gene co-expression network images using Cytoscape 3.7.1 (NIGMS) and identified corresponding cell pathways using The Gene Ontology knowledgebase. We determined that across all subtypes of prostate cancer, the lncRNAs (with co-expressed protein coding genes) were operating mainly in immune response pathways. Next, we will analyse the functions of the candidate lncRNAs and propose a mechanism of action. We also hope to validate these findings using siRNA assays. The discovery of these lncRNAs together with our candidate protein coding genes could be developed as biomarkers (like the PAM50 genes in breast cancer) for the first time in prostate cancer and may be potential novel treatment targets

T-type calcium channels drive the proliferation of androgen-receptor negative prostate cancer cells

Background: Androgen deprivation therapy (ADT) is the treatment of choice for metastatic prostate cancer (PCa). After an initial response to ADT, PCa cells can generate castration resistant (CRPC) or neuroendocrine (NEPC) malignancies, which are incurable. T-type calcium channels (TTCCs) are emerging as promising therapeutic targets for several cancers, but their role in PCa progression has never been investigated. Methods: To examine the role of TTCCs in PCa, we analyzed their expression level, copy number variants (CNV) and prognostic significance using clinical datasets (Oncomine and cBioPortal). We then evaluated TTCC expression in a panel of PCa cell lines and measured the effect of their inhibition on cell proliferation and survival using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and caspase assays. Results: TTCCs were upregulated in PCas harboring androgen receptor (AR) mutations; CNV rate was positively associated with PCa progression. Higher expression of one TTCC isoform (CACNA1G) predicted poorer postoperative prognosis in early stage PCa samples. Pharmacological or small interfering RNA (siRNA)-based inhibition of TTCCs caused a decrease in PC-3 cell survival and proliferation. Conclusions: Our results show that TTCCs are overexpressed in advanced forms of PCa and correlate with a poorer prognosis. TTCC inhibition reduces cell proliferation and survival, suggesting that there may be possible value in the therapeutic targeting of TTCCs in advanced PCa

Abstract# 2556: An investigation of the role of the caspase-8 gene in prostate and colon cancer susceptibility using a SNP-tagging approach

We showed recently in a large collaborative study that the rare allele of the caspase-8 (CASP8) gene polymorphism, D302H, is associated with a reduced risk of breast cancer (Cox et al 2007). Subsequently the same SNP has been associated with an increased risk of glioma (Bethke et al, 2008). Further studies based on a panel of SNPs that tag the common variation in CASP8 have indicated that CASP8 variants other than D302H may also be important in cancer susceptibility (Shephard et al, submitted). It is not yet known whether CASP8 variation affects susceptibility to other cancers. In order to test the hypothesis that CASP8 is associated with colon or prostate cancer, we have adopted a SNP-tagging approach. Fifteen CASP tagging-SNPs (tSNPs) were selected by principle components analysis based on in-house genotyping data for 33 common SNPs (minor allele frequency >0.05) on 135 healthy individuals. Colon cancer cases and controls were identified from 4 studies, based in Sheffield and Leeds, UK and Utah, (1261 cases and 1723 controls in total). Prostate cancer cases and controls were derived from the UK ProtecT trial, and comprised 1009 cases, and 987 controls with normal serum PSA ( 0.15). In the prostate cancer cohort, 3 SNPs in CASP8 yielded significant results when cases were compared to low PSA controls; rs3769826, rs3769824 and rs6723097, with odds ratios (95% confidence intervals) [OR (95%CI)] of 0.87 (0.76, 0.99), 0.68 (0.49, 0.94), and 0.87 (0.75, 1.00) respectively, Ptrend = 0.034, 0.021, 0.049 respectively. Consistent results were also obtained for these 3 SNPs when cases were compared to the normal PSA controls, with OR (95% CI) of 0.876 (0.75, 0.98), 0.68 (0.50, 0.93), and 0.83 (0.73, 0.96) and Ptrend = 0.027, 0.015, 0.010, respectively. These data suggest that there may be an association between CASP8 and prostate cancer, but require replication. Therefore an independent replication study, based on 1262 cases, 1258 normal PSA controls and 609 low PSA controls from the ProtecT study is in progress. It is of interest to note that the rare alleles of these 3 SNPs are carried on European haplotypes that are associated with an increased risk of breast cancer rather than a decreased risk as shown here. If these results were replicated, this would point to a different mode of action of caspase-8 in breast and prostate cancer

EZH2 as a therapeutic target for aggressive prostate cancer

Aggressive variants of prostate cancer (AVPC) are a subtype of metastatic castration resistant prostate cancer (mCRPC), which express no androgen receptor (AR) and are currently incurable. The main AVPC subtypes are neuroendocrine prostate cancer (NEPC) and anaplastic prostate cancer (AR-, NEPC-). EZH2 is an epigenetic regulator that mediates gene silencing via histone H3 Lys27 trimethylation (H3K27me3). EZH2 acts as an oncogene in several malignancies. Three pharmacologic EZH2 inhibitors (Tazemetostat, GSK-126, and CPI-1205) are in clinical trials. Volition Nu.Q kits enable the detection of histone variants in biological fluids. We hypothesize that EZH2 could be a viable therapeutic target for AVPC. We queried the expression of EZH2 in a database comprising 444 mCRPC clinical samples. Our results show that higher EZH2 expression is positively correlated with NEPC features (p<<0.0001, T test), and negatively correlated with AR activity (p< 4.57e-11). Higher EZH2 expression predicts shorter overall survival (p< 0.0276, log-rank test). The three EZH2 inhibitors induce a dose-dependent inhibition of cell proliferation in two AVPC cell lines (DU-145, PC-3). GSK-126 is the most potent inhibitor. Total histone H3 and H3K27me3 are measurable in the supernatant from AVPC cell lines (Nu.Q assay). Treatment with EZH2 inhibitors induces a measurable reduction in H3K27me3. We are now testing the interaction between GSK-126 and chemotherapy drugs employed for the treatment of AVPC. We are also testing Nu.Q kits in blood samples from patient-derived xenografts. This information will be useful to develop tailored epigenetic therapies for AVPC patients