Laura Breen

Integrated miRNA, mRNA and protein expression analysis reveals the role of post-transcriptional regulation in controlling CHO cell growth rate

BackgroundTo study the role of microRNA (miRNA) in the regulation of Chinese hamster ovary (CHO) cell growth, qPCR, microarray and quantitative LC-MS/MS analysis were utilised for simultaneous expression profiling of miRNA, mRNA and protein. The sample set under investigation consisted of clones with variable cellular growth rates derived from the same population. In addition to providing a systems level perspective on cell growth, the integration of multiple profiling datasets can facilitate the identification of non-seed miRNA targets, complement computational prediction tools and reduce false positive and false negative rates.Results51 miRNAs were associated with increased growth rate (35 miRNAs upregulated and 16 miRNAs downregulated). Gene ontology (GO) analysis of genes (n=432) and proteins (n=285) found to be differentially expressed (DE) identified biological processes driving proliferation including mRNA processing and translation. To investigate the influence of miRNA on these processes we combined the proteomic and transcriptomic data into two groups. The first set contained candidates where evidence of translational repression was observed (n=158). The second group was a mixture of proteins and mRNAs where evidence of translational repression was less clear (n=515). The TargetScan algorithm was utilised to predict potential targets within these two groups for anti-correlated DE miRNAs.ConclusionsThe evidence presented in this study indicates that biological processes such as mRNA processing and protein synthesis are correlated with growth rate in CHO cells. Through the integration of expression data from multiple levels of the biological system a number of proteins central to these processes including several hnRNPs and components of the ribosome were found to be post-transcriptionally regulated. We utilised the expression data in conjunction with in-silico tools to identify potential miRNA-mediated regulation of mRNA/proteins involved in CHO cell growth rate. These data have allowed us to prioritise candidates for cell engineering and/or biomarkers relevant to industrial cell culture. We also expect the knowledge gained from this study to be applicable to other fields investigating the role of miRNAs in mammalian cell growth.

In vitro Development of Chemotherapy and Targeted Therapy Drug-Resistant Cancer Cell Lines: A Practical Guide with Case Studies

The development of a drug-resistant cell line can take from 3 to 18 months. However, little is published on the methodology of this development process. This article will discuss key decisions to be made prior to starting resistant cell line development; the choice of parent cell line, dose of selecting agent, treatment interval, and optimizing the dose of drug for the parent cell line. Clinically relevant drug-resistant cell lines are developed by mimicking the conditions cancer patients experience during chemotherapy and cell lines display between two- and eight-fold resistance compared to their parental cell line. Doses of drug administered are low, and a pulsed treatment strategy is often used where the cells recover in drug-free media. High-level laboratory models are developed with the aim of understanding potential mechanisms of resistance to chemotherapy agents. Doses of drug are higher and escalated over time. It is common to have difficulty developing stable clinically relevant drug-resistant cell lines. A comparative selection strategy of multiple cell lines or multiple chemotherapeutic agents mitigates this risk and gives insight into which agents or type of cell line develops resistance easily. Successful selection strategies from our research are presented. Pulsed-selection produced platinum or taxane-resistant large cell lung cancer (H1299 and H460) and temozolomide-resistant melanoma (Malme-3M and HT144) cell lines. Continuous selection produced a lapatinib-resistant breast cancer cell line (HCC1954). Techniques for maintaining drug-resistant cell lines are outlined including; maintaining cells with chemotherapy, pulse treating with chemotherapy, or returning to master drug-resistant stocks. The heterogeneity of drug-resistant models produced from the same parent cell line with the same chemotherapy agent is explored with reference to P-glycoprotein. Heterogeneity in drug-resistant cell lines reflects the heterogeneity that can occur in clinical drug resistance.

Re-programming CHO cell metabolism using miR-23 tips the balance towards a highly productive phenotype

microRNA engineering of CHO cells has already proved successful in enhancing various industrially relevant phenotypes and producing various recombinant products. A single miRNAs ability to interact with multiple mRNA targets allows their regulatory capacity to extend to processes such as cellular metabolism. Various metabolic states have previously been associated with particular CHO cell phenotypes such as glycolytic or oxidative metabolism accommodating growth and productivity, respectively. miR-23 has previously been demonstrated to play a role in glutamate metabolism resulting in enhanced oxidative phosphorylation through the TCA cycle. Re-programming cellular bioenergetics through miR-23 could tip the balance, forcing mammalian production cells to be more productive by favoring metabolic channelling into oxidative metabolism. CHO clones depleted of miR-23 using a miR-sponge decoy demonstrated an average ∼three-fold enhanced specific productivity with no impact on cell growth. Using a cell respirometer, mitochondrial activity was found to be enhanced by ∼30% at Complex I and II of the electron transport system. Additionally, label-free proteomic analysis uncovered various potential novel targets of miR-23 including LE1 and IDH1, both implicated in oxidative metabolism and mitochondrial activity. These results demonstrate miRNA-based engineering as a route to re-programming cellular metabolism resulting in increased productivity, without affecting growth.

Development of taxane resistance in a panel of human lung cancer cell lines

Using a selection process designed to reflect clinically relevant conditions, a panel of taxane-selected variants were developed to study further the mechanisms of resistance in lung cancer. Unlike continuous or pulse exposure to high concentrations of chemotherapeutic drugs which yield high resistance and often cross resistance, most variants developed here displayed low level resistance to the selecting drug with slight cross-resistance. Pulsing with taxol resulted in more highly resistant clones (up to 51.4-fold). Analysis of taxol and taxotere in the four major lung cancer cell types showed the taxanes to be more effective against NSCLC (with the exception of SKMES-taxane selected variants) than against the SCLC. Comparison of taxol and taxotere shows that taxol induces higher levels of resistance than taxotere. Further, in taxotere-selected cell lines, the cells are more resistant to taxol than taxotere, suggesting that taxotere may be a superior taxane from a clinical view. Taxol treatment resulted in increased cross-resistance to 5-FU in all classes of lung cancer except DMS-53. The high levels of Pgp in the DMS-53 and selected variant suggests this mechanism is not related to Pgp expression. Analysis of the Pgp and MRP-1 status by combination inhibitory assays and Western blotting showed no consistent relationship between expression of the membrane pumps Pgp or MRP-1 and resistance. However, where high level resistance was seen, the parent cell line expressed Pgp or MRP-1 and was accompanied by increased levels in the variants. Overall we found that the clinically relevant models used here are useful for investigating mechanisms of taxane resistance.

Modulation of P-gp expression by lapatinib

SummaryChemotherapy drug resistance is a major obstacle in the treatment of cancer. It can result from an increase in levels of cellular drug efflux pumps, such as P-glycoprotein (P-gp). Lapatinib, a growth factor receptor tyrosine kinase inhibitor, is currently in clinical trials for treatment of breast cancer. We examined the impact of co-incubation of chemotherapy drugs in combination with lapatinib in P-gp over-expressing drug resistant cells. Unexpectedly, lapatinib treatment, at clinically relevant concentrations, increased levels of the P-gp drug transporter in a dose- and time-responsive manner. Conversely, exposure to the epidermal growth factor (EGF), an endogenous growth factor receptor ligand, resulted in a decrease in P-gp expression. Despite the lapatinib-induced alteration in P-gp expression, use of accumulation, efflux and toxicity assays demonstrated that the induced alteration in P-gp expression by lapatinib had little direct impact on drug resistance.

Generation of Lung Cancer Cell Line Variants by Drug Selection or Cloning

Clonal variants or subpopulations have been isolated from every major histological type of cancer, and cellular heterogeneity in lung cancer is a common occurrence. These subpopulations may exhibit differences in drug resistance and invasive potential. One therefore needs to consider the subpopulations as well as the tumour to overcome the barriers of drug resistance and metastasis for successful treatment. Isogenic variants of cancer cell lines can be very valuable in providing controlled human experimental systems to study clinically relevant parameters such as drug resistance and invasiveness. These variants can be established by selection based on a characteristic of the subpopulation or by isolating clonal subpopulations from a heterogeneous population. Drug-resistant variants can be generated by pulse selection, which usually generates low-level resistance, which may as well be clinically relevant, or by continuous exposure, which can be used to obtain high-level resistant variants. Clonal subpopulations may also be isolated based on morphological differences using simple cell-culture-based techniques.

The novel toluidine sulphonamide EL102 shows pre-clinical in vitro and in vivo activity against prostate cancer and circumvents MDR1 resistance

Background:Taxanes are routinely used for the treatment of prostate cancer, however the majority of patients eventually develop resistance. We investigated the potential efficacy of EL102, a novel toluidine sulphonamide, in pre-clinical models of prostate cancer.Methods:The effect of EL102 and/or docetaxel on PC-3, DU145, 22Rv1 and CWR22 prostate cancer cells was assessed using cell viability, cell cycle analysis and PARP cleavage assays. Tubulin polymerisation and immunofluorescence assays were used to assess tubulin dynamics. CWR22 xenograft murine model was used to assess effects on tumour proliferation. Multidrug-resistant lung cancer DLKPA was used to assess EL102 in a MDR1-mediated drug resistance background.Results:EL102 has in vitro activity against prostate cancer, characterised by accumulation in G2/M, induction of apoptosis, inhibition of Hif1α, and inhibition of tubulin polymerisation and decreased microtubule stability. In vivo, a combination of EL102 and docetaxel exhibits superior tumour inhibition. The DLKP cell line and multidrug-resistant DLKPA variant (which exhibits 205 to 691-fold greater resistance to docetaxel, paclitaxel, vincristine and doxorubicin) are equally sensitive to EL102.Conclusion:EL102 shows potential as both a single agent and within combination regimens for the treatment of prostate cancer, particularly in the chemoresistance setting.

Parallel mRNA, proteomics and miRNA expression analysis in cell line models of the intestine

AIM To identify miRNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine. METHODS Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for mRNA, miRNA and proteomic profiling. mRNA microarray profiling analysis was carried out using the Affymetrix GeneChip Human Gene 1.0 ST array. miRNA microarray profiling analysis was carried out using the Affymetrix Genechip miRNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear ion trap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets (miRNA, proteomics, mRNA) was conducted in the R software environment using the Euclidean distance measure and Ward’s clustering algorithm. The prediction of miRNA and oppositely correlated protein/mRNA interactions was performed using TargetScan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE miRNA, protein and mRNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database. RESULTS Differential expression (DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 miRNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the miRNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology (GO) analysis of the DE mRNA identified cell adhesion, migration and ECM organization, cellular lipid and cholesterol metabolic processes, small molecule transport and a range of responses to external stimuli, while similar analysis of the DE protein list identified gene expression/transcription, epigenetic mechanisms, DNA replication, differentiation and translation ontology categories. The DE protein and gene lists were found to share 15 biological processes including for example epithelial cell differentiation [P value ≤ 1.81613E-08 (protein list); P ≤ 0.000434311 (gene list)] and actin filament bundle assembly [P value ≤ 0.001582797 (protein list); P ≤ 0.002733714 (gene list)]. Analysis was conducted on the three data streams acquired in parallel to identify targets undergoing potential miRNA translational repression identified 34 proteins, whose respective mRNAs were detected but no change in expression was observed. Of these 34 proteins, 27 proteins downregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 19 unique anti-correlated/upregulated microRNAs and 7 proteins upregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 15 unique anti-correlated/downregulated microRNAs. CONCLUSION This first study providing “tri-omics” analysis of the principal intestinal cell line models Caco-2 and HT-29 has identified 34 proteins potentially undergoing miRNA translational repression.

KH-type splicing regulatory protein controls colorectal cancer cell growth and modulates the tumor microenvironment

Background & Aims KH-type splicing regulatory protein (KHSRP) is a multifunctional nucleic acid binding protein. KHSRP regulates transcription, mRNA decay and translation, and miRNA biogenesis. These distinct functions are associated with key aspects of cancer cell biology: inflammation and cell-fate determination. However, the role KHSRP plays in colorectal cancer (CRC) tumorigenesis remains largely unknown. Methods We investigated the oncogenic role of KHSRP in CRC using a combination of in silico analysis of large datasets, ex vivo analysis of protein expression in patient samples, and mechanistic studies using in vitro models of CRC. Results KHSRP was expressed in the epithelial and stromal compartments of both primary and metastatic tumors. Elevated KHSRP expression was found in tumor versus matched normal tissue in a cohort of 62 patients. This finding was validated in larger independent cohorts in silico. KHSRP expression was a prognostic indicator of worse overall survival (HR=3.74, 95% CI = 1.43-22.97, p=0.0138). Mechanistic data in CRC cell line models supported a role of KHSRP in driving epithelial cell proliferation in both a primary and metastatic setting, through control of the G1/S transition. Additionally, epithelial KHSRP was involved in promoting a pro-angiogenic extracellular environment, as well as regulating the secretion of oncogenic proteins involved in diverse cellular processes such as migration and response to cellular stress. Conclusion Our study has uncovered novel mechanistic-based data on the tumor-promoting effects of KHSRP in CRC. Synopsis Here we describe a pro-tumorigenic role for KHSRP in colorectal cancer. Higher KHSRP expression is associated with shorter overall survival. KHSRP regulates cancer cell proliferation and expression of proteins that govern processes including angiogenesis, migration and response to cellular stress.

Acute exposure to organic and inorganic sources of copper: Differential response in intestinal cell lines

Abstract Scope Copper supplementation in nutrition has evolved from using inorganic mineral salts to organically chelated minerals but with limited knowledge of the impact at the cellular level. Methods Here, the impact of inorganic and organic nutrient forms (glycinate, organic acid, and proteinate) of copper on the cellular level is investigated on intestinal cell lines, HT29 and Caco‐2, after a 2‐hr acute exposure to copper compounds and following a 10‐hr recovery. Results Following the 10‐hr recovery, increases were observed in proteins involved in metal binding (metallothioneins) and antioxidant response (sulfiredoxin 1 and heme oxygenase 1), and global proteomic analysis suggested recruitment of the unfolded protein response and proteosomal overloading. Copper organic acid chelate, the only treatment to show striking and sustained reactive oxygen species generation, had the greatest impact on ubiquitinated proteins, reduced autophagy, and increased aggresome formation, reducing growth in both cell lines. The least effect was noted in copper proteinate with negligible impact on aggresome formation or extended growth for either cell line. Conclusion The type and source of copper can impact significantly at the cellular level.