Hazel Welch

Efficacy of the Specific Endothelin A Receptor Antagonist Zibotentan (ZD4054) in Colorectal Cancer: A Preclinical Study

Endothelin 1 (ET-1) is overexpressed in cancer, contributing to disease progression. We previously showed that ET-1 stimulated proliferative, migratory, and contractile tumorigenic effects via the ETA receptor. Here, for the first time, we evaluate zibotentan, a specific ETA receptor antagonist, in the setting of colorectal cancer, in cellular models. Pharmacologic characteristics were further determined in patient tissues. Colorectal cancer lines (n = 4) and fibroblast strains (n = 6), isolated from uninvolved areas of colorectal cancer specimens, were exposed to ET-1 and/or ETA/B receptor antagonists. Proliferation (methylene blue), migration (scratch wounds), and contraction (gel lattices) were assessed. Receptor distribution and binding characteristics (Kd, Bmax) were determined using autoradiography on tissue sections and homogenates and cytospun cells, supported by immunohistochemistry. Proliferation was inhibited by ETA (zibotentan > BQ123; P < 0.05), migration by ETB > ETA, and contraction by combined ETA and ETB antagonism. Intense ET-1 stromal binding correlated with fibroblasts and endothelial cells. Colorectal cancer lines and fibroblasts revealed high density and affinity ET-1 binding (Bmax = 2.435 fmol/1 × 106 cells, Kd = 367.7 pmol/L; Bmax = 3.03 fmol/1 × 106 cells, Kd = 213.6 pmol/L). In cancer tissues, ETA receptor antagonists (zibotentan; BQ123) reduced ET-1 binding more effectively (IC50: 0.1–10 μmol/L) than ETB receptor antagonist BQ788 (∼IC50, 1 mmol/L). ET-1 stimulated cancer-contributory processes. Its localization to tumor stroma, with greatest binding/affinity to fibroblasts, implicates these cells in tumor progression. ETA receptor upregulation in cancer tissues and its role in tumorigenic processes show the receptors importance in therapeutic targeting. Zibotentan, the most specific ETA receptor antagonist available, showed the greatest inhibition of ET-1 binding. With its known safety profile, we provide evidence for zibotentans potential role as adjuvant therapy in colorectal cancer. Mol Cancer Ther; 12(8); 1556–67. ©2013 AACR.

Cellular glycosylation affects Herceptin binding and sensitivity of breast cancer cells to doxorubicin and growth factors

Alterations in protein glycosylation are a key feature of oncogenesis and have been shown to affect cancer cell behaviour perturbing cell adhesion, favouring cell migration and metastasis. This study investigated the effect of N-linked glycosylation on the binding of Herceptin to HER2 protein in breast cancer and on the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin (DXR) and growth factors (EGF and IGF-1). The interaction between Herceptin and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using a quartz crystal microbalance biosensor revealing an increase in the accessibility of HER2 to Herceptin following deglycosylation of cell membrane proteins (deglycosylated cells Bmax: 6.83 Hz; glycosylated cells Bmax: 7.35 Hz). The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1 μM DXR P < 0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (P < 0.001). This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and highlights the potential of glycosylation inhibitors as future combination treatments for breast cancer.

The Polymerase Chain Reaction

The polymerase chain reaction (PCR) has had a significant impact on all aspects of the molecular biosciences, from cancer research to forensic science. The sensitivity and specificity inherent in the technique allow minute quantities of genetic material to be detected while the unique properties of thermostable DNA polymerase ensure that abundant copies are reliably reproduced to levels that can be visualized and/or used for further applications. This chapter describes applications of PCR and PCR-RT to investigate primary cancer and metastatic disease at both the DNA and mRNA expression levels.

Biologics against cancer-specific receptors - challenges to personalised medicine from early trial results.

Understanding molecular mechanisms of tumourigenesis underlies new therapeutic strategies that specifically target tumours. This has led to the evolution of personalised therapy that was first used in breast cancer when hormone receptor status was determined. More recently in colorectal cancer treatment the Epidermal Growth Factor receptor and its tumourigenic role has led to its targeting by using Cetuximab and Panitumumab. Addition of these drugs to existing drug regimes (FOLFOX and FOLFIRI) showed improved respectability rates in patients with liver metastasis. Most recently the Endothelin receptor has been implicated in multiple tumourigenic processes. Interest has grown in using Endothelin A receptor antagonists as adjuvant or combination therapy as suggested by the FOLFERA and FOLFIRI trials currently on-going.

Efficacy of zibotentan in colorectal cancer--response.

Panagiotis Vlachostergios highlights the importance of evaluating clinically the targeted therapeutic zibotentan. Despite the wide overexpression of endothelin-1 (ET-1) and endothelin A receptors in the majority of carcinomas ([1][1]), establishing clinical efficacy of receptor antagonism has

PO-487 Cellular glycosylation affects trastuzumab binding and sensitivity of breast cancer cells to doxorubicin and growth factors

Introduction Protein glycosylation is a post-translational event in which oligosaccharides (glycans) are attached to proteins; glycosylation events are mediated by glycosyltransferases and glycosidases. All cancer types studied so far have been shown to exhibit alterations in glycosylation affecting cell-cell adhesion, favouring cell migration and metastasis. The aim of this study was to evaluate whether changes in protein glycosylation affects the responsiveness of cancer cells to1 the chemotherapeutic agent doxorubicin (DXR);2 trastuzumab (Herceptin) interaction with HER2 and3 the responsiveness of cancer cells to growth factors (EGF and IGF-1). Material and methods A range of cancer cell lines were initially investigated and SKBR-3 cells were selected for in-depth analysis by virtue of their HER2 +status. The interaction between trastuzumab and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using the Attana A200 quartz crystal microbalance (QCM) biosensor. The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Results and discussions QCM biosensor analysis revealed that after deglycosylation the HER2 receptor was rendered more accessible to trastuzumab (deglycosylated cells Bmax: 6.83 Hz; glycosylated cells Bmax: 7.35 Hz). Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1 µM DXR p<0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (p<0.001). Conclusion This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and it highlights the potential of glycosylation inhibitors as drugs worth further investigation for the treatment of cancer.

175. MEK inhibitors are effective for specific breast cancer cell sub-types

Background: The MAPK pathway is essential for normal cellular functions; however, it is dysregulated in one third of all cancers and facilitates progression of the disease. MEK 1 & 2 are component kinases responsible for activating specific downstream targets ERK1 and ERK2, which in turn enable cell proliferation, differentiation, motility, apoptosis etc. MEK1/2 are targets for intervention and MEK inhibitors (MEKI) have been approved for treating BRAF-mutated melanoma (Trametinib) and are currently in clinical trials (e.g. metastatic non-small cell lung cancer). The study aim was to examine the effects of different MEKI on breast cancer cells and evaluate therapeutic potential. Materials and Methods: A panel of 10 breast cancer cell lines (including ER+, ER-, HER2 normal, HER2 amplified) were treated with a range of concentrations of the MEKI: AZD6244(AZD), PD98059 (PD) and U0126 (U0). ER+ cells were treated with MEKI 17b-Oestradiol (E2; 10nM). Responses to treatment were examined by (i) metabolic activity via MTT assay and (ii) ability to inhibit phosphorylation of ERK1/2 by Western Blot. Results: Metabolic activity was inhibited significantly in MDA-MB231 cells by all 3 MEKI (AZD, U0, PD p<0.001) while SkBr3 cells were unaffected by the MEKI at all concentrations. U0 inhibited activity significantly(p<0.001) in a further 6 cell lines (5 ER+ & 1 ER-); 3 of the ER+ cells were also inhibited by AZD (p<0.001) while the remaining 2 ER+ were unaffected (ERcell line not tested). Paradoxically: U0 & AZD stimulated activity significantly (p<0.001) in MDA-MB-175 cells while U0 alone stimulated activity in ZR-75-1 cells (p<0.001). None of the MEKI were able to block E2-stimulated activity. Ability to inhibit phosphorylation of ERK1/2 varied considerably across the cell panel, with each inhibitor and their concentration. Conclusions: MEK inhibition may be an effective way of treating triple negative breast cancers that harbour both KRAS and BRAF mutations, as indicated by the response of MDA-MB-231 cells. The lack of consensus across the other cell lines supports the genetic and signalling complexity of breast cancer cells, although there is potential for treating those harbouring KRAS or BRAF mutations (suggested by the responses of Hs578T, SkBr3 and ZR-75-30 cells) with MEKI in combination with other therapeutics; while E2-responsive/ER+ cancers are unlikely to benefit from MEKI alone. No conflict of interest.