Mark B. Watson

The analysis of doxorubicin resistance in human breast cancer cells using antibody microarrays

Doxorubicin is considered to be the most effective agent in the treatment of breast cancer patients. Unfortunately, resistance to this agent is common, representing a major obstacle to successful treatment. The identification of novel biomarkers that are able to predict treatment response may allow therapy to be tailored to individual patients. Antibody microarrays provide a powerful new technique, enabling the global comparative analysis of many proteins simultaneously. This technology may identify a panel of proteins to discriminate between drug-resistant and drug-sensitive samples. The Panorama Cell Signaling Antibody Microarray was exploited to analyze the MDA-MB-231 breast cancer cell line and a novel derivative, which displays significant resistance to doxorubicin at clinically relevant concentrations. The microarray comprised 224 antibodies selected from a variety of pathways, including apoptotic and cell signaling pathways. A standard ≥2.0-fold cutoff value was used to determine differentially expressed proteins. A decrease in the expression of mitogen-activated protein kinase–activated monophosphotyrosine (phosphorylated extracellular signal-regulated kinase; 2.8-fold decrease), cyclin D2 (2.5-fold decrease), cytokeratin 18 (2.5-fold decrease), cyclin B1 (2.4-fold decrease), and heterogeneous nuclear ribonucleoprotein m3-m4 (2.0-fold decrease) was associated with doxorubicin resistance. Western blotting was exploited to confirm results from the antibody microarray experiment. These results suggest that antibody microarrays can be used to identify novel biomarkers and further validation may reveal mechanisms of chemotherapy resistance and identify potential therapeutic targets. [Mol Cancer Ther 2006;5(8):2115–20]

The proteomic analysis of cisplatin resistance in breast cancer cells.

Resistance to cisplatin represents a major obstacle in the effective management of many cancers, including metastatic breast cancer. We aimed to gain further understanding of the mechanisms underlying development of cisplatin resistance using an in vitro cell line model. The MCF-7 breast cancer cell line and a novel derivative displaying significant resistance to cisplatin were analyzed using two-dimensional gel electrophoresis. The protein profiles were compared and 15 differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The downregulation of beta-tubulin type 3, cytokeratin 17, tropomyosin 1-alpha, peroxiredoxin 4, heat shock 27-kDa protein 1, glutathione-S-transferase mu 3, ribosomal protein P0, isocitrate dehydrogenase 3, and peptidyl-prolyl isomerase A isoform 1 was associated with cisplatin-resistant cells. In contrast, the expression of hydroxyprostaglandin dehydrogenase 15-(NAD), matrix metalloproteinase 9, heterogeneous nuclear ribonucleoprotein A3, proteasome beta 1 subunit, electron transfer flavoprotein beta-polypeptide isoform 1, and peptidyl-propyl isomerase B precursor was upregulated in cisplatin-resistant cells. The downregulation (at least twofold) of glutathione-S-transferase mu 3, cytokeratin 17, and peroxiredoxin 4 was confirmed by Western blotting. We have identified alterations in the expression levels of several proteins that may be associated with cisplatin resistance and are candidates for further validation in clinical samples.

Establishment of in-vitro models of chemotherapy resistance.

Chemotherapy resistance is one of the most prevalent obstacles to the treatment of cancer, resulting in increased mortality and prolonged exposure to cytotoxic agents with no treatment benefit. One of the tools utilized in the study of mechanisms of chemotherapy resistance are established cell lines derived from human neoplasms. These cell lines can be challenged in vitro with controlled chemotherapy doses to produce chemotherapy-resistant variants. Analysis of these novel chemotherapy-resistant cell lines may then identify genetic and proteomic changes which are associated with the resistant phenotype. Two very important mediators of chemotherapy resistance (P-glycoprotein and multidrug resistance protein-1) were initially identified in chemotherapy-resistant cell lines. To make these in-vitro studies clinically relevant it is, however, necessary to duplicate as far as possible the treatment conditions used in vivo. Considerations should include clinically relevant drug concentrations, such as those derived from peak plasma values, and the type of treatment schedule to be employed.

Expression microarray analysis reveals genes associated with in vitro resistance to cisplatin in a cell line model

We aimed to investigate the mechanisms of cisplatin resistance using an in vitro cancer model. A derivative breast cancer cell line (MCF-7CR) was established which demonstrated significant resistance to cisplatin at clinically relevant low concentrations compared to the MCF-7 parental cell line. Expression microarray analysis was used to identify targets from a 3k cancer-related oligonucleotide platform which were differentially expressed between the derivative and parental cell lines. Real-time quantitative PCR was used to confirm the difference in expression of a subset of genes which demonstrated significant up- or down-regulation. Using expression microarray analysis a total of 28 genes were identified to be differentially expressed (by at least 2-fold) between the MCF-7 and MCF-7CR cells. Real-time quantitative PCR expression analysis confirmed the differential expression of a selection of these genes (ACTG2, ARHD, CTSL, GSTM3, GSTM4 and EHF) between the two cell lines. An in vitro model of cisplatin resistance has been established and expression microarray analysis revealed 28 genes which may be associated with cisplatin resistance.

Implication of the BRCA2 and Putative “BRCA3” Genes in Dukes’ Stage C, Replication Error–Negative Colon Cancer

BackgroundAlthough BRCA genes have been implicated in certain tumors, particularly breast tumors, their role in colon tumorigenesis has not been fully explored. We aimed to investigate the association of the BRCA2 and putative “BRCA3” genes in a homogeneous series of right-sided colon cancer specimens.MethodsTwenty-three Dukes’ stage C, replication error–negative carcinomas were selected from patients with right-sided colon cancer. After histological examination and microdissection, DNA was extracted from normal colon and carcinoma from each patient. Five microsatellite markers spanning the region of BRCA2 and BRCA3 on chromosome 13 (D13S218, D13S219, D13S165, D13S156, and D13S160) and two markers intragenic to BRCA2 and BRCA3 (D13S171 and D13S1308, respectively) were used. Polymerase chain reaction products were analyzed by using a fluorescent allele imbalance assay.ResultsMarkers demonstrating the highest allelic imbalance were D13S1308 (53%), D13S171 (33%), and D13S160 (37%).ConclusionsThe intragenic markers D13S1308 (BRCA3) and D13S171 (BRCA2) on chromosome 13 demonstrated a high frequency of allelic imbalance in primary colon carcinoma. This suggests an involvement of BRCA2 and putative BRCA3 in colon tumorigenesis in right-sided, replication error–negative, Dukes’ stage C cancers. Further studies are needed to confirm the precise role of these genes, and any prognostic significance, in colon cancer.

Quantitative proteomic analysis of radioresistant breast cancer cell lines.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #5074 Background: Resistance to radiotherapy may be a significant factor in the development of local recurrence following surgical resection and radiotherapy. We aimed to develop a novel in vitro model of radioresistance using a breast cancer cell line and to subsequently identify molecular biomarkers which may be associated with the radioresistant phenotype. We utilised a quantitative proteomics technique (iTRAQ) based on MALDI-TOF/TOF mass spectrometry (MS) to identify differentially expressed proteins. Material and Methods: We established 3 novel breast cancer cell sublines which were significantly resistant to radiotherapy when compared with the parental cells. The radioresistant sublines were created by irradiating cells in fractionated doses of 2Gy up to a total dose of 40Gy. Sufficient time was allowed for the cells to recover between subsequent irradiations. A dose response curve was assessed at the end of treatment to demonstrate a statistically significant increase in radioresistance for each novel cell subline when compared with parental cells. One radioresistant/parental cell pair was first analysed using in-solution digestion and liquid chromatographic separation with protein identification by MALDI-TOF/TOF (LC-MALDI analysis) on an Applied Biosystems 4800 Plus instrument. Quantitative iTRAQ (Applied Biosystems) was then performed on the same instrument for all 3 radioresistant/parental cell pairs. Results: A total of 586 and 652 proteins were identified in T47D and T47D RR cells respectively by LC-MALDI. Those proteins identified in both cell lines and any redundant entries were removed to reveal those proteins which were unique to each cell line. In total 244 unique proteins were identified in T47D cells and 311 unique proteins were identified in T47D RR cells. Comparison of the 3 pairs of radioresistant/parental cell samples by iTRAQ revealed a number of differentially expressed proteins. Using a standard ≥2-fold change in expression, these iTRAQ analyses revealed significant changes in the expression of 51 proteins in one or more of the radio-resistant derivatives. Further confirmation by immunoblotting is underway. Currently the decrease in expression of 26S proteasome associated subunits has been confirmed by this method. Conclusion: LC-MALDI and iTRAQ analysis has revealed a large number of candidate proteins which may be associated with a radioresistant phenotype. These now require further confirmatory studies. These MS-based techniques offer a powerful proteomic approach to identify candidate biomarkers which may be involved in radioresistance. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5074.

Antibody microarray analysis identifies biomarkers associated with radioresistant breast cancer cell lines.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #5072 Background: Resistance to radiotherapy may be a significant factor in the development of local recurrence following surgical resection and radiotherapy. In addition, if patients with radioresistant breast cancers can be identified, harmful side effects from exposure to unnecessary ionizing radiation could be prevented. We aimed to develop a novel in vitro model of radioresistance using breast cancer cell lines and to subsequently identify molecular biomarkers which may be associated with the radioresistant phenotype. Antibody microarrays offer a complementary approach for proteomic analysis in conjunction with standard screening methods such as two dimensional gel electrophoresis/ mass spectrometry and other quantitative proteomic techniques. We previously utilised the Panorama Cell Signalling Antibody Microarray Kit (Sigma-Aldrich) consisting of 224 antibodies (Smith et al Mol Cancer Ther 5:2115-20, 2006). In this study we utilised a novel high density 725 antibody microarray to screen for proteins associated with radioresistance. Material and Methods: We established novel breast cancer cell sublines which were significantly resistant to radiotherapy when compared with the parental cells (T47D; MCF-7). The radioresistant sublines were created by irradiating cells in fractionated doses of 2Gy up to a total dose of 40Gy. Sufficient time was allowed for the cells to recover between subsequent irradiations. A dose response curve was assessed at the end of treatment to demonstrate a statistically significant increase in radioresistance for the novel cell subline when compared with parental cells. The radioresistant/parental cell pairs were analysed using the Panorama Antibody Microarray XPRESS Profiler725 Kit (Sigma-Aldrich). The microarray comprised 725 different antibodies on nitrocellulose coated microscope slides. The antibodies were selected from a wide variety of pathways, including apoptotic and cell signalling pathways. Results: Utilising a Cy3/Cy5 labelling strategy the antibody microarray approach yielded a number of a total of 28 targets for further study. Of these, three proteins were identified independently from both of the radioresistant cell lines. These were GFI1 (Growth Factor Independence-1), DR4 (Death Receptor 4) and Importin a1. Immunoblotting and other proteomic approaches have confirmed the identities and differential expression of some candidate protein targets. Conclusion: High density antibody microarrays potentially offer a powerful new proteomic technique to allow the global analysis of many proteins simultaneously. This analysis has produced both complementary and confirmatory data in our proteomic screening for putative biomarkers associated with radiotherapy resistance. We successfully identified a number of protein targets which may be associated with a radioresistant phenotype. Further confirmatory and validation studies are ongoing. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5072.