Alex J. Eustace

The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, simvastatin, lovastatin and mevastatin inhibit proliferation and invasion of melanoma cells

BackgroundA number of recent studies have suggested that cancer incidence rates may be lower in patients receiving statin treatment for hypercholesterolemia. We examined the effects of statin drugs on in vitro proliferation, migration and invasion of melanoma cells.MethodsThe ability of lovastatin, mevastatin and simvastatin to inhibit the melanoma cell proliferation was examined using cytotoxicity and apoptosis assays. Effects on cell migration and invasion were assessed using transwell invasion and migration chambers. Hypothesis testing was performed using 1-way ANOVA, and Students t-test.ResultsLovastatin, mevastatin and simvastatin inhibited the growth, cell migration and invasion of HT144, M14 and SK-MEL-28 melanoma cells. The concentrations required to inhibit proliferation of melanoma cells (0.8–2.1 μM) have previously been achieved in a phase I clinical trial of lovastatin in patients with solid tumours, (45 mg/kg/day resulted in peak plasma concentrations of approximately 3.9 μM).ConclusionOur results suggest that statin treatment is unlikely to prevent melanoma development at standard doses. However, higher doses of statins may have a role to play in adjuvant therapy by inhibiting growth and invasion of melanoma cells.

Preclinical evaluation of dasatinib, a potent Src kinase inhibitor, in melanoma cell lines

BackgroundMetastatic melanoma is a highly chemotherapy resistant tumour. The use of newer targeted therapies alone and in combination with chemotherapy may offer new hope of improving response to treatment. Dasatinib, a multi-target kinase inhibitor, is currently approved for the treatment of chronic myeloid leukaemia and has shown promising results in preclinical studies in a number of solid tumours.MethodsWe examined the effects of dasatinib on proliferation, chemo-sensitivity, cell cycle arrest, apoptosis, migration and invasion in human melanoma cell lines. Expression and activation of Src kinase, FAK and EphA2 were also examined in the melanoma cells.ResultsDasatinib inhibited growth of three of the five melanoma cell lines. Comparison with sorafenib showed that in these three cell lines dasatinib inhibited growth at lower concentrations than sorafenib. Dasatinib in combination with the chemotherapy drug temozolomide showed greater efficacy than either drug alone. Dasatinib induced cell cycle arrest and apoptosis and significantly inhibited cell migration and invasion of melanoma cells. Dasatinib inhibition of proliferation was associated with reduced phosphorylation of Src kinase, while decreased phosphorylation of FAK was implicated in dasatinib-mediated inhibition of migration and invasion in melanoma cells.ConclusionDasatinib has both anti-proliferative and anti-invasive effects in melanoma cells and combined with chemotherapy may have clinical benefit in the treatment of malignant melanoma.

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.

Metabolomic studies of human lung carcinoma cell lines using in vitro1H NMR of whole cells and cellular extracts

We report principal component analysis (PCA) of 1H NMR spectra recorded for a group of human lung carcinoma cell lines in culture and 1H NMR analysis of extracts from the same samples. The samples studied were cells of lung tumour origin with different chemotherapy drug resistance patterns. For whole cells, it was found that the statistically significant causes of spectral variation were an increase in the choline and a decrease in the methylene mobile lipid 1H resonance intensities, which correlate with our knowledge of the level of resistance displayed by the different cells. Similarly, in the 1H NMR spectra of the aqueous and lipophilic extracts, significant quantitative differences in the metabolite distributions were apparent, which are consistent with the PCA results. Copyright

Membrane transport proteins in human melanoma: associations with tumour aggressiveness and metastasis.

Background:Malignant melanoma, generally described as incurable, is notoriously refractory to chemotherapy. The mechanisms contributing to this have not yet been defined and the contributions of drug efflux pumps, implicated in chemo-resistance of many other cancer types, have not been extensively investigated in melanoma.Methods:In this study, expression of multi-drug resistant (MDR1/P-gp and MRP-1) proteins was examined, by immunohistochemistry, in archival specimens from 134 melanoma patients. This included 92 primary tumours and 42 metastases.Results:On assessing all specimens, MRP-1 and MDR1/P-gp expression was found to be common, with the majority (81%) of melanomas expressing at least one of these efflux pumps. Although there is significant association between expression of these pumps (P=0.007), MRP-1 was found to be the predominant (67% of cases) form detected. χ2 analysis showed significant associations between expression of MRP-1 and/or MDR1/P-gp and the aggressive nature of this disease specifically increased Breslows depth, Clarks level and spread to lymph nodes. This association with aggressiveness and spread is further supported by the observation that a significantly higher percentage of metastases, than primary tumours, express MRP-1 (91% vs 57%; P<0.0001) and MDR1/P-gp (74% vs 50%; P=0.010).Conclusion:The predominant expression of these pumps and, in particular, MRP-1 suggests that they may be important contributors to the inherent aggressive and resistant nature of malignant melanoma.

Artificial Neural Networks for Classification in Metabolomic Studies of Whole Cells Using 1H Nuclear Magnetic Resonance

We report the successful classification, by artificial neural networks (ANNs), of 1H NMR spectroscopic data recorded on whole-cell culture samples of four different lung carcinoma cell lines, which display different drug resistance patterns. The robustness of the approach was demonstrated by its ability to classify the cell line correctly in 100% of cases, despite the demonstrated presence of operator-induced sources of variation, and irrespective of which spectra are used for training and for validation. The study demonstrates the potential of ANN for lung carcinoma classification in realistic situations.

Growth factor receptor/steroid receptor cross talk in trastuzumab-treated breast cancer

Treatment with tyrosine kinase inhibitors (TKIs) including trastuzumab has revolutionized the management of HER2-positive breast cancer. Recent evaluation of clinical trial data suggests that a subset of HER2/ER double-positive cancers may not receive significant benefit from the TKI therapy. Here we investigate the cross talk between HER2 and ER in breast cancer and monitor the effect of trastuzumab on the tyrosine kinase effector transcription factor Myc. In HER2-positive breast cancer patients treated with neoadjuvant trastuzumab, steroid receptor-negative status (ER and PR negative) of pre-treatment biopsies predicted pathological complete response (pCR) (n=31 patients, P=0.0486), whereas elevated Myc protein inversely associated with pCR (P=0.0446). Liquid chromatography mass spectrometry identified the corepressor SMRT as a novel Myc-interacting protein. Trastuzumab treatment enhanced Myc–SMRT interactions in HER2-overexpressing breast cancer cells (LCC1) and inhibited expression of the Myc target gene survivin. In HER2-low, ER-positive steroid-dominant cells (MCF7), trastuzumab therapy repressed Myc–SMRT interactions and upregulated survivin expression. Trastuzumab treatment induced ER–CBP interactions, enhanced ER transcriptional activity and upregulated expression of the ER target gene pS2. The absence of pS2 expression in pre-treatment biopsies predicted pCR to neoadjuvant trastuzumab in breast cancer patients (n=25, P=0.0089) and pS2 expression associated with residual cancer burden (P=0.0196). Furthermore, metastatic tissues from patients who had failed trastuzumab therapy were pS2 positive. In HER2-overexpressing cells, trastuzumab treatment can repress Myc transcriptional activity and clinical response is favorable. However, with co-expression of the steroid pathway, this inhibition is lost and response to treatment is often poor.

2D-DIGE analysis of phospho-enriched fractions from dasatinib-treated melanoma cell lines.

Current therapeutic regimes for metastatic melanoma have failed to provide robust clinical responses. Dasatinib has shown anti-proliferative and anti-invasive effects in vitro; however, not all melanoma cells tested were sensitive to dasatinib. We used 2D-DIGE analysis of phospho-enriched fractions to identify phosphoproteins involved in regulating response to dasatinib in an isogenic pair of melanoma cell lines, one sensitive to dasatinib (WM-115) and the other resistant (WM-266-4). In WM-115 cells treated with dasatinib, 18 unique protein species with altered phosphorylation levels were detected. Dasatinib treatment of WM-266-4 cells resulted in phosphoprotein alterations to four unique protein species. Four phosphorylated forms of Annexin-A2 (ANXA2) were increased in WM-115 cells treated with dasatinib, whilst dasatinib treatment did not alter ANXA2 phosphoprotein levels in WM-266-4 cells. Immunoblotting confirmed that phosphorylation of ANXA2, on tyrosine residues, was increased in WM-115 cells treated with dasatinib. Subsequent knockdown of ANXA2 by siRNA significantly inhibited proliferation of WM-115 cells but did not significantly reduce proliferation of WM-266-4 cells. Therefore, ANXA2 plays a role in regulating proliferation in dasatinib-sensitive WM-115 cells and could potentially play a role in sensitivity to dasatinib in melanoma cells.

Impact of somatic PI3K pathway and ERBB family mutations on pathological complete response (pCR) in HER2-positive breast cancer patients who received neoadjuvant HER2-targeted therapies.

BackgroundThe Cancer Genome Atlas analysis revealed that somatic EGFR, receptor tyrosine-protein kinase erbB-2 (ERBB2), Erb-B2 receptor tyrosine kinase 3 (ERBB3) and Erb-B2 receptor tyrosine kinase 4 (ERBB4) gene mutations (ERBB family mutations) occur alone or co-occur with somatic mutations in the gene encoding the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (PIK3CA) in 19% of human epidermal growth factor receptor 2 (HER2)-positive breast cancers. Because ERBB family mutations can activate the PI3K/AKT pathway and likely have similar canonical signalling effects to PI3K pathway mutations, we investigated their combined impact on response to neoadjuvant HER2-targeted therapies.MethodsBaseline tumour biopsies were available from 74 patients with HER2-positive breast cancer who were enrolled in the phase II TCHL neoadjuvant study (ICORG 10-05) assessing TCH (docetaxel, carboplatin, trastuzumab) (n = 38) versus TCL (docetaxel, carboplatin, lapatinib) (n = 10) versus TCHL (docetaxel, carboplatin, trastuzumab, lapatinib) (n = 40), each for six cycles. Activating mutations in PIK3CA and ERBB family genes were identified using mass spectrometry-based genotyping. Phosphatase and tensin homolog (PTEN) expression was assessed by immunohistochemistry.ResultsPIK3CA and/or ERBB family mutations were detected in 23 (31.1%) tumour samples tested, whereas PTEN expression was low in 31.1% of cases tested. Mutation frequency was similar in each treatment arm (31.3% in TCH arm, 30% in TCL arm and 31.3% in TCHL arm) and was not influenced by oestrogen receptor (ER) status (27.6% in ER-negative patients, 33.3% in ER-positive patients) or progesterone receptor (PR) status (32.6% in PR-negative patients, 29% in PR-positive patients). There was no significant difference in pathological complete response (pCR) rates between 47 patients with wild-type (WT) tumours and 22 patients whose tumours carried mutations (in either PIK3CA or ERBB family genes) (42.5% vs. 54.5%; p = 0.439). Similarly, there was no significant difference in pCR rates between patients with PIK3CA/ERBB family mutated/PTEN-low (i.e., PI3K-activated) tumours and patients without PI3K activation (50% vs. 44%; p = 0.769). However, in the TCHL (but not the TCH) group, the pCR rate was higher for 9 patients with PIK3CA/ERBB family mutated tumours than for 20 patients with PIK3CA/ERBB family WT tumours (77.8% vs. 35%; p = 0.05).ConclusionsOur results indicate that patients who receive neoadjuvant TCHL and have PIK3CA/ERBB family mutated tumours may be more likely to have a pCR than patients with WT tumours.Trial registrationClinicalTrials.gov, NCT01485926. Registered on 2 December 2011.

Kinase inhibitor screening identifies CDK4 as a potential therapeutic target for melanoma

Despite recent advances in targeted therapies and immunotherapies metastatic melanoma remains only rarely curable. The objective of the present study was to identify novel therapeutic targets for metastatic melanoma. A library of 160 well-characterised and potent protein kinase inhibitors was screened in the BRAF mutant cell line Sk-Mel-28, and the NRAS mutant Sk-Mel-2, using proliferation assays. Of the 160 inhibitors tested, 20 achieved >50% growth inhibition in both cell lines. Six of the 20 were cyclin dependent kinase (CDK) inhibitors, including two CDK4 inhibitors. Fascaplysin, a synthetic CDK4 inhibitor, was further tested in 8 melanoma cell lines. The concentration of fascaplysin required to inhibit growth by 50% (IC50 value) ranged from 0.03 to 0.22 μM. Fascaplysin also inhibited clonogenic growth and induced apoptosis. Sensitivity to PD0332991, a therapeutic CDK4/6 inhibitor was also evaluated in the melanoma cell lines. PD0332991 IC50 values ranged from 0.13 to 2.29 μM. Similar to fascaplysin, PD0332991 inhibited clonogenic growth of melanoma cells and induced apoptosis. Higher levels of CDK4 protein correlated with lower sensitivity to PD0332991 in the cell lines. Combined treatment with PD0332991 and the BRAF inhibitor PLX4032, showed additive anti-proliferative effects in the BRAF mutant cell line Malme-3M. In summary, targeting CDK4 inhibits growth and induces apoptosis in melanoma cells in vitro, suggesting that CDK4 may be a rational therapeutic target for metastatic melanoma.