Lynn Cawkwell

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]

Do β-tubulin mutations have a role in resistance to chemotherapy?

Summary β-tubulin is the target of various antitubulin agents used in the treatment of cancer. After β tubulin was shown to confer resistance to antitubulin agents in established cell lines, several studies have investigated the DNA sequence of this compound in clinical samples. However, these findings are highly controversial, since sequencing experiments showed that the original clinical observation of mutations in the gene resulted from inclusion of non-functional β-tubulin pseudogenes. At least nine such pseudogenes are known, and all share substantial sequence homology with the functional gene. Subsequent studies have concluded that β-tubulin mutations in clinical samples are rare, and unlikely to contribute to drug resistance. Here, we overview the β-tubulin gene family and summarise the results of studies done comparing β-tubulin mutations with antitubulin drug resistance.

Bcl-2 expression predicts radiotherapy failure in laryngeal cancer.

Early stage laryngeal cancer can be effectively cured by radiotherapy or conservative laryngeal surgery. In the UK, radiotherapy is the preferred first line treatment. However, up to 25% of patients with T2 tumours will demonstrate locally persistent or recurrent disease at the original site, requiring salvage surgery to achieve a definitive cure. Patients experiencing treatment failure have a relatively poor prognosis. A retrospective analysis was conducted consisting of 124 patients with early stage (T1–T2, N0) laryngeal squamous cell carcinoma. In total, 62 patients who failed radiotherapy were matched for T stage, laryngeal subsite and smoking history to a group of 62 patients successfully cured by radiotherapy. Using immunohistochemistry the groups were compared for expression of apoptotic proteins: bcl-2, bcl-XL, bax, bak and survivin. Radioresistant laryngeal cancer was associated with bcl-2 (P<0.001) and bcl-XL (P=0.005) expression and loss of bax expression (P=0.012) in pretreatment biopsies. Bcl-2 has an accuracy of 71% in predicting radiotherapy outcome. The association between expression of bcl-2, bcl-XL and bax with radioresistant cancer suggests a potential mechanism by which cancer cells avoid the destructive effects of radiotherapy. Predicting radioresistance, using bcl-2, would allow the clinician to recommend conservative laryngeal surgery as an alternative first line treatment to radiotherapy.

Overexpression of Bcl‐2 in squamous cell carcinoma of the larynx: A marker of radioresistance

Squamous cell carcinoma of the larynx can be treated using radiotherapy or surgery, either alone or in combination. Radiotherapy is preferred for early‐stage tumours, as it spares the larynx and therefore preserves speech and swallowing. Unfortunately, approximately 15% of tumours treated this way will prove to be radioresistant, as manifest by tumour recurrence within the original radiotherapy field over the ensuing 12 months. By causing extensive DNA damage, radiotherapy aims to induce apoptosis and tumour regression. Our hypothesis was that defects in the mechanisms that recognise DNA damage, induce cell cycle arrest or control apoptosis, either alone or in combination, may be responsible for radioresistance. We therefore undertook an immunohistochemic analysis of pretreatment biopsies of radioresistant (n = 8) and radiosensitive (n = 13) laryngeal tumours. To minimise the impact of confounding factors, strict inclusion criteria were observed; all tumours were of the glottic subsite and all recurrences developed within 12 months of radiotherapy at the site of the original tumour. The expression of key proteins involved in DNA damage recognition (p53), cell cycle arrest (ATM, p16 and p21/WAF1) and apoptosis (Bcl‐2 and BAX) were studied. Ki‐67 was also assessed as a marker of cell proliferation to exclude low mitotic rate as a cause of radioresistance. A statistically significant correlation was observed between overexpression of Bcl‐2 and radioresistance (p = 0.003, Fishers exact test). We hypothesise that overexpression of the anti‐apoptotic protein Bcl‐2 allows tumour cells with extensive radiation‐induced DNA damage to continue proliferating; the absence of an appropriate apoptotic response manifests clinically as radioresistance.

Prognostic value of genomic alterations in head and neck squamous cell carcinoma detected by comparative genomic hybridisation

A total of 45 primary head and neck squamous cell carcinomas were analysed by comparative genomic hybridisation to identify regions of chromosomal deletion and gain. Multiple regions of copy number aberration were identified including gains affecting chromosomes 3q, 8q, 5p, 7q, 12p and 11q and deletion of material from chromosomes 3p, 11q, 4p, 5q, 8p, 10q, 13q and 21. Kaplan–Meier survival analysis revealed significant correlations between gain of 3q25–27 and deletion of 22q with reduced disease-specific survival. In addition, gain of 17q and 20q, deletion of 19p and 22q and amplification of 11q13 were significantly associated with reduced disease-free survival. A Cox proportional hazards regression model identified deletion of 22q as an independent prognostic marker. The data presented here provide further evidence that the creation of a genetically based tumour classification system will soon be possible, complementing current histopathological characterisation.

Telomere dysfunction accurately predicts clinical outcome in chronic lymphocytic leukaemia, even in patients with early stage disease

Defining the prognosis of individual cancer sufferers remains a significant clinical challenge. Here we assessed the ability of high‐resolution single telomere length analysis (STELA), combined with an experimentally derived definition of telomere dysfunction, to predict the clinical outcome of patients with chronic lymphocytic leukaemia (CLL). We defined the upper telomere length threshold at which telomere fusions occur and then used the mean of the telomere ‘fusogenic’ range as a prognostic tool. Patients with telomeres within the fusogenic range had a significantly shorter overall survival (P < 0·0001; Hazard ratio [HR] = 13·2, 95% confidence interval [CI] = 11·6–106·4) and this was preserved in early‐stage disease patients (P < 0·0001, HR=19·3, 95% CI = 17·8–802·5). Indeed, our assay allowed the accurate stratification of Binet stage A patients into those with indolent disease (91% survival at 10 years) and those with poor prognosis (13% survival at 10 years). Furthermore, patients with telomeres above the fusogenic mean showed superior prognosis regardless of their IGHV mutation status or cytogenetic risk group. In keeping with this finding, telomere dysfunction was the dominant variable in multivariate analysis. Taken together, this study provides compelling evidence for the use of high‐resolution telomere length analysis coupled with a definition of telomere dysfunction in the prognostic assessment of CLL.

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.

Expression of Bcl-2 Family Members in Malignant Pleural Mesothelioma

Little is known about the Bcl-2 family members in mesothelioma. These proteins are involved in the control of apoptosis, carrying out both pro- and anti- apoptotic functions. Immunohistochemistry was used to examine the expression of p53 and Bcl-2 family members in 54 archival mesothelioma samples (39 epithelial, 15 sarcomatoid tumours). Overexpression of p53 was observed in 81% (44/54). For anti-apoptotic proteins, overexpression was recorded as follows: Bcl-2 40% (22/54), Bcl-XL 24% (13/54), Mcl-1 92% (50/54). For pro-apoptotic proteins, loss of expression was recorded as follows: Bad 25% (14/54), Bak 24% (13/54), Bax 42% (23/54), Bid 37% (20/54), Bim 18% (10/54). Statistically significant differences between epithelial and sarcomatoid tumours were observed for Bid (p < 0.001), Bad (p = 0.012) and Bcl-XL (p = 0.03). Significant differences in abnormal expression of apoptosis proteins were found between epithelial and sarcomatoid subtypes but histological subtype was the only factor with significant association to patient prognosis.

Chromosomal alterations in small cell lung cancer revealed by multicolour fluorescence in situ hybridization

Small cell lung cancer (SCLC) is a major cause of cancer related morbidity and mortality. Karyotypic studies have revealed numerous chromosomal aberrations in most SCLC however, classical G‐banding analysis is unable to fully characterise complex marker chromosomes. Recent developments in molecular cytogenetics now allow accurate identification of the chromosomal components of complicated rearrangements. We have applied the technique of multicolour fluorescence in situ hybridization (M‐FISH) in combination with comparative genomic hybridization (CGH) to the analysis of 5 SCLC cell lines and 1 primary tumour specimen to characterise the chromosomal abnormalities. CGH analysis identified many similarities between specimens, with frequent DNA copy number decreases on chromosomes 3p, 5q, 10, 16q, 17p and frequent gains on 3q, 1p, 1q and 14q. In contrast, M‐FISH analysis revealed a large number of structural abnormalities, with each specimen demonstrating an individual pattern of chromosomal translocations. Forty different translocations were identified with the vast majority (39) being unbalanced. Chromosome 5 was the most frequently rearranged chromosome (9 translocations) followed by chromosomes 2, 10 and 16 (6 translocations each). Further investigation of these frequently involved chromosomes is warranted to establish whether consistent break points are involved in these translocations, causing dysregulation of specific genes that are crucial for tumour progression and secondly to identify the affected genes.

Cancer proteomics and its application to discovery of therapy response markers in human cancer

The administration of chemotherapy either alone or in combination with radiotherapy is an important factor in reducing the mortality and morbidity of cancer patients. Resistance to both chemotherapy and radiotherapy represents a major obstacle to a successful outcome. The identification of novel biomarkers that can be used to predict treatment response would allow therapy to be tailored on an individual patient basis. Although the mechanisms are unclear, it is accepted that development of therapy resistance is a multifactorial phenomenon involving alterations in several cellular pathways. Proteome analysis methods are powerful tools for identifying factors associated with resistance to anticancer therapy because they facilitate the simultaneous analysis of whole proteomes. The current review describes the plethora of existing proteomic approaches and details the studies that have identified biomarkers that may be useful in the prediction of clinical response to anticancer therapy. Cancer 2006.