Pharmacological modulation and manipulation of cancer drug resistance

The aim of this project was to investigate pharmacological methods of overcoming resistance in cancer. Novel compounds, targeted therapies and non-steroidal anti-inflammatory drugs (NSAIDs) were examined for their potential to modulate and manipulate specific forms of drug resistance. Sixty one novel compounds were tested in combination with chemotherapeutic drugs in proliferation assays for their ability to overcome MRP1 and P-gp-mediated drug resistance. Two compounds were successful P-gp modulators in the DLKP-A cell line; the ditrifluoroacetyl resveratrol derivative, RBM15, and the macrocycle derivative, KG104. A panel of nine therapeutic agents were evaluated for their potential to down-regulate multidrug resistant protein expression and thus, overcome P-gp, MRP1 or BCRP-mediated drug resistance, at and below pharmacologically-relevant concentrations. Two of these agents (indomethacin and 17-AAG) partially down-regulated the expression of P-gp in the A549-Taxol cell line but did not overcome P-gp-mediated resistance when combined with docetaxel simultaneously or in pre-treated proliferation assays. Three agents (lapatinib, sulindac sulphide and 17-AAG) reduced the expression of MRP1 in the A549 cell line. Only sulindac sulphide overcame MRP1-mediated resistance in the combination proliferations assays; however, this was due to the inhibitory mechanism of sulindac sulphide and not due to the down-regulation of the MRP1 protein. Five agents (17-AAG, lapatinib, indomethacin, elacridar and gefitinib) down-regulated the expression of BCRP in the DLKP-SQ/mitox cell line. Lapatinib, gefitinib, elacridar and 17-AAG overcame BCRP-mediated resistance in both the combination and pre-treatment proliferation assays. The data indicates that the amount of down-regulation resulting from treatment with these drugs was insufficient to overcome drug resistance. Up-regulation of the three MDR transporter proteins was observed with a variety of agents tested. This suggests that, long-term treatment with such agents could lead to the development and amplification of multidrug resistance, and therefore, reduce the effectiveness of substrate chemotherapies in patients. Targeted therapies, including tyrosine kinase inhibitors (TKIs, such as lapatinib), are the latest significant development in the treatment of cancer. Lapatinib sensitised HER2-expressing cell lines to chemotherapeutic agents in the presence or absence of EGFR expression. This agent was also found to be a more active sensitiser in P-gp-expressing cell lines, while erlotinib was more active in BCRP-expressing cell lines. Gefitinib was the least active of three TKIs at modulating P-gp, MRP1 or BCRP. Following a 48 hours treatment, lapatinib up-regulated the expression and function of COX-2. It also stimulated COX-2 activity directly. This lapatinib-mediated COX-2 induction was independent of its TKI action on EGFR, and HER2 and could have serious therapeutic effects as COX-2 is known to increase cell growth, inhibit apoptosis, and enhance metastasis and angiogenesis. A COX-2-specific inhibitor, celecoxib, overcame P-gp, MRP1 and BCRP-mediated resistance. At pharmacologically relevant concentrations, celecoxib significantly overcame MRP1 and BCRP-mediated resistance. And to a much lesser extent celecoxib overcame P-gp mediated resistance above pharmacologically relevant concentrations. The combination of lapatinib with celecoxib could be of therapeutic benefit, as the combination of these agents could collectively inhibit COX-2, P-gp, MRP1, BCRP, HER2 and EGFR activity in tumours expressing multiple oncoproteins resistant pathways and enhanced signalling pathways. It is hoped that a novel treatment regimens, using these agents and TKI drugs with traditional chemotherapeutic agents, could improve current treatment strategies resulting in increased survival rates and decreased mortality

Phenotypic consequences of somatic mutations in the ataxia-telangiectasia mutated gene in non-small cell lung cancer

Mutations in the Ataxia-telangiectasia mutated (ATM) gene are frequently found in human cancers, including non-small cell lung cancer (NSCLC). Loss of ATM function confers sensitivity to ionising radiation (IR) and topoisomerase inhibitors and may thus define a subset of cancer patients that could get increased benefit from these therapies. In this study, we evaluated the phenotypic consequences of ATM missense changes reported in seven NSCLC cell lines with regard to radiosensitivity and functionality of ATM signalling. Our data demonstrate that only 2/7 NSCLC cell lines (H1395 and H23) harbouring ATM missense mutations show a functional impairment of ATM signalling following IR-exposure. In these two cell lines, the missense mutations caused a significant reduction in ATM protein levels, impairment of ATM signalling and marked radiosensitivity. Of note, only cell lines with homozygous mutations in the ATM gene showed significant impairment of ATM function. Based on these observations, we developed an immunohistochemistry-based assay to identify patients with loss or reduction of ATM protein expression in a clinical setting. In a set of 137 NSCLC and 154 colorectal cancer specimens we identified tumoral loss of ATM protein expression in 9.5% and 3.9% of cases, respectively, demonstrating the potential utility of this method

An integrated inspection of the somatic mutations in a lung squamous cell carcinoma using next-generation sequencing

Squamous cell carcinoma (SCC) of the lung kills over 350,000 people annually worldwide, and is the main lung cancer histotype with no targeted treatments. High-coverage whole-genome sequencing of the other main subtypes, small-cell and adenocarcinoma, gave insights into carcinogenic mechanisms and disease etiology. The genomic complexity within the lung SCC subtype, as revealed by The Cancer Genome Atlas, means this subtype is likely to benefit from a more integrated approach in which the transcriptional consequences of somatic mutations are simultaneously inspected. Here we present such an approach: the integrated analysis of deep sequencing data from both the whole genome and whole transcriptome (coding and non-coding) of LUDLU-1, a SCC lung cell line. Our results show that LUDLU-1 lacks the mutational signature that has been previously associated with tobacco exposure in other lung cancer subtypes, and suggests that DNA-repair efficiency is adversely affected; LUDLU-1 contains somatic mutations in TP53 and BRCA2, allelic imbalance in the expression of two cancer-associated BRCA1 germline polymorphisms and reduced transcription of a potentially endogenous PARP2 inhibitor. Functional assays were performed and compared with a control lung cancer cell line. LUDLU-1 did not exhibit radiosensitisation or an increase in sensitivity to PARP inhibitors. However, LUDLU-1 did exhibit small but significant differences with respect to cisplatin sensitivity. Our research shows how integrated analyses of high-throughput data can generate hypotheses to be tested in the lab

Characterisation and manipulation of docetaxel resistant prostate cancer cell lines

Abstract Background There is no effective treatment strategy for advanced castration-resistant prostate cancer. Although Docetaxel (Taxotere®) represents the most active chemotherapeutic agent it only gives a modest survival advantage with most patients eventually progressing because of inherent or acquired drug resistance. The aims of this study were to further investigate the mechanisms of resistance to Docetaxel. Three Docetaxel resistant sub-lines were generated and confirmed to be resistant to the apoptotic and anti-proliferative effects of increasing concentrations of Docetaxel. Results The resistant DU-145 R and 22RV1 R had expression of P-glycoprotein and its inhibition with Elacridar partially and totally reversed the resistant phenotype in the two cell lines respectively, which was not seen in the PC-3 resistant sublines. Resistance was also not mediated in the PC-3 cells by cellular senescence or autophagy but multiple changes in pro- and anti-apoptotic genes and proteins were demonstrated. Even though there were lower basal levels of NF-κB activity in the PC-3 D12 cells compared to the Parental PC-3, docetaxel induced higher NF-κB activity and IκB phosphorylation at 3 and 6 hours with only minor changes in the DU-145 cells. Inhibition of NF-κB with the BAY 11-7082 inhibitor reversed the resistance to Docetaxel. Conclusion This study confirms that multiple mechanisms contribute to Docetaxel resistance and the central transcription factor NF-κB plays an immensely important role in determining docetaxel-resistance which may represent an appropriate therapeutic target.</p

Structural somatic variants identified in LUDLU-1.

<p>Structural somatic variants identified in LUDLU-1.</p

The consequence of LUDLU-1 somatic substitutions.

<p>The consequence of LUDLU-1 somatic substitutions.</p

LUDLU-1 non-synonymous variants that are either somatic (first row) or germline but in cancer-associated genes (last 3 rows).

<p>LUDLU-1 non-synonymous variants that are either somatic (first row) or germline but in cancer-associated genes (last 3 rows).</p

Molecular functionality testing of LUDLU-1.

<p>a) The effect of increasing dose of cisplatin on the cell proliferation of LUDLU-1 and A549; b) The survival fraction of LUDLU-1 and A549 when irradiated. Proliferation data is representative of duplicate independent experiments, with significance <i>p</i> values of less than 0.001. Radiation sensitivity data is representative of triplicate independent experiments.</p