Inhibiting Eph kinase activity may not be “Eph”ective for cancer treatment

Several Eph receptor tyrosine kinases (RTKs) are commonly over-expressed in epithelial and mesenchymal cancers and are recognized as promising therapeutic targets. Although normal interaction between Eph receptors and their ephrin ligands stimulates kinase activity and is generally tumor suppressive, significant Eph over-expression allows activation of ligand- and/or kinase-independent signaling pathways that promote oncogenesis. Single-agent kinase inhibitors are widely used to target RTK-driven tumors but acquired and de novo resistance to such agents is a major limitation to effective clinical use. Accumulating evidence suggests that Ephs can be inhibited by “leaky” or low-specificity kinase inhibitors targeted at other RTKs. Such off-target effects may therefore inadvertently promote ligand- and/or kinase-independent oncogenic Eph signaling, thereby providing a new mechanism by which resistance to the RTK inhibitors can emerge. We propose that combining specific, non-leaky kinase inhibitors with tumor-suppressive stimulators of Eph signaling may provide more effective treatment options for overcoming treatment-induced resistance and clinical failure.

EphB4 localises to the nucleus of prostate cancer cells

The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of both NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor.

Murine, but not human, ephrin-B2 can be efficiently cleaved by the serine protease kallikrein-4: Implications for xenograft models of human prostate cancer

Background Ephrin-B2 is the sole physiologically-relevant ligand of the receptor tyrosine kinase EphB4, which is over-expressed in many epithelial cancers, including 66% of prostate cancers, and contributes to cancer cell survival, invasion and migration. Crucially, however, the cancer-promoting EphB4 signalling pathways are independent of interaction with its ligand ephrin-B2, as activation of ligand-dependent signalling causes tumour suppression. Ephrin-B2, however, is often found on the surface of endothelial cells of the tumour vasculature, where it can regulate angiogenesis to support tumour growth. Proteolytic cleavage of endothelial cell ephrin-B2 has previously been suggested as one mechanism whereby the interaction between tumour cell-expressed EphB4 and endothelial cell ephrin-B2 is regulated to support both cancer promotion and angiogenesis. Methods An in silico approach was used to search accessible surfaces of 3D protein models for cleavage sites for the key prostate cancer serine protease, KLK4, and this identified murine ephrin-B2 as a potential KLK4 substrate. Mouse ephrin-B2 was then confirmed as a KLK4 substrate by in vitro incubation of recombinant mouse ephrin-B2 with active recombinant human KLK4. Cleavage products were visualised by SDS-PAGE, silver staining and Western blot and confirmed by N-terminal sequencing. Results At low molar ratios, KLK4 cleaved murine ephrin-B2 but other prostate-specific KLK family members (KLK2 and KLK3/PSA) were less efficient, suggesting cleavage was KLK4-selective. The primary KLK4 cleavage site in murine ephrin-B2 was verified and shown to correspond to one of the in silico predicted sites between extracellular domain residues arginine 178 and asparagine 179. Surprisingly, the highly homologous human ephrin-B2 was poorly cleaved by KLK4 at these low molar ratios, likely due to the 3 amino acid differences at this primary cleavage site. Conclusion These data suggest that in in vivo mouse xenograft models, endogenous mouse ephrin-B2, but not human tumour ephrin-B2, may be a downstream target of cancer cell secreted human KLK4. This is a critical consideration when interpreting data from murine explants of human EphB4+/KLK4+ cancer cells, such as prostate cancer cells, where differential effects may be seen in mouse models as opposed to human clinical situations.

The tumour-promoting receptor tyrosine kinase, EphB4, regulates expression of Integrin-β8 in prostate cancer cells

Background The EphB4 receptor tyrosine kinase is overexpressed in many cancers including prostate cancer. The molecular mechanisms by which this ephrin receptor influences cancer progression are complex as there are tumor-promoting ligand-independent mechanisms in place as well as ligand-dependent tumor suppressive pathways. Methods We employed transient knockdown of EPHB4 in prostate cancer cells, coupled with gene microarray analysis, to identify genes that were regulated by EPHB4 and may represent linked tumor-promoting factors. We validated target genes using qRT-PCR and employed functional assays to determine their role in prostate cancer migration and invasion. Results We discovered that over 500 genes were deregulated upon EPHB4 siRNA knockdown, with integrin β8 (ITGB8) being the top hit (29-fold down-regulated compared to negative non-silencing siRNA). Gene ontology analysis found that the process of cell adhesion was highly deregulated and two other integrin genes, ITGA3 and ITGA10, were also differentially expressed. In parallel, we also discovered that over-expression of EPHB4 led to a concomitant increase in ITGB8 expression. In silico analysis of a prostate cancer progression microarray publically available in the Oncomine database showed that both EPHB4 and ITGB8 are highly expressed in prostatic intraepithelial neoplasia, the precursor to prostate cancer. Knockdown of ITGB8 in PC-3 and 22Rv1 prostate cancer cells in vitro resulted in significant reduction of cell migration and invasion. Conclusions These results reveal that EphB4 regulates integrin β8 expression and that integrin β8 plays a hitherto unrecognized role in the motility of prostate cancer cells and thus targeting integrin β8 may be a new treatment strategy for prostate cancer.

Colonoscopy requirements of population screening for colorectal cancer in New Zealand

Aim: To estimate the colonoscopy burden of introducing population screening for colorectal cancer in New Zealand. Methods: Screening for colorectal cancer using biennial immunochemical faecal occult blood tests offered to people aged 50-74 years of age was modelled using population estimates from Statistics New Zealand for 2011-2031. Modelling to determine colonoscopy requirements was based on participation and test positivity rates from published results of screening programmes. Estimates of the number of procedures required for ongoing adenoma surveillance were calculated using screening literature results of adenoma yield, and New Zealand Guidelines for Adenoma Surveillance. Sensitivity analysis was undertaken on key parameters. Results: For a test positivity of 6.4%, biennial screening using immunochemical faecal occult blood testing with a 60% participation rate, would require 18,000 colonoscopies nationally, increasing to 28,000 by 2031. The majority of procedures are direct referrals from a positive FOBT, with surveillance colonoscopy numbers building over time. Conclusion: Colonoscopy requirements for immunochemical faecal occult blood based population screening for colorectal cancer are high. Significant expansion of services is required and careful management of surveillance procedures to ensure timely delivery of initial colonoscopies whilst maintaining symptomatic services. A model re-run informed by data from the screening pilot will allow improved estimates for the New Zealand setting.

Assessment of fundamental movement skills in childhood cancer patients

Background The improved treatment protocols and subsequent improved survival rates amongst childhood cancer patients has shifted the focus towards the long-term consequences arising from cancer treatment. Children who have completed cancer treatment are at a greater risk of delayed development, diminished functioning, disability, compromised fundamental movement skill (FMS) attainment and long term chronic health conditions. The aim of the study was to compare FMS of childhood cancer patients with an aged matched healthy reference group. Methods Pediatric cancer patients aged 5-8 years of age (n=26; median age 6.91 years), who completed cancer treatment (<5 years) at the Sydney Children’s Hospital were assessed performing 7 key FMS; sprint, side-gallop, vertical-jump, catch, over-arm throw, kick and leap. Results were compared to the reference group (n=430; 6.56 years). Results Childhood cancer patients scored significantly lower on 3 out of 7 FMS tests when compared to the reference group. These results equated to a significantly lower overall score for FMS. Conclusion This study highlighted the significant deficits in FMS within pediatric patients having completed cancer treatment. In order to reduce the occurrence of significant FMS deficits in this population, FMS interventions maybe warranted to assist in recovery from childhood cancer, prevent late effects and improve the quality of life in survivors of childhood cancer.

The empire of cancer: Gene patents and cancer voices

In his book, The Emperor of All Maladies, Siddhartha Mukherjee writes a history of cancer — "It is a chronicle of an ancient disease — once a clandestine, 'whispered-about' illness — that has metamorphosed into a lethal shape-shifting entity imbued with such penetrating metaphorical, medical, scientific, and political potency that cancer is often described as the defining plague of our generation." Increasingly, an important theme in the history of cancer is the role of law, particularly in the field of intellectual property law. It is striking that a number of contemporary policy debates over intellectual property and public health have concerned cancer research, diagnosis, and treatment. In the area of access to essential medicines, there has been much debate over Novartis’ patent application in respect of Glivec, a treatment for leukaemia. India’s Supreme Court held that the Swiss company’s patent application violated a safeguard provision in India’s patent law designed to stop evergreening. In the field of tobacco control, the Australian Government introduced plain packaging for tobacco products in order to address the health burdens associated with the tobacco epidemic. This regime was successfully defended in the High Court of Australia. In the area of intellectual property and biotechnology, there have been significant disputes over the Utah biotechnology company Myriad Genetics and its patents in respect of genetic testing for BRCA1 and BRCA2, which are related to breast cancer and ovarian cancer. The Federal Court of Australia handed down a decision on the validity of Myriad Genetics’ patent in respect of genetic testing for BRCA1 in February 2013. The Supreme Court of the United States heard a challenge to the validity of Myriad Genetics’ patents in this area in April 2013, and handed down a judgment in July 2013. Such disputes have involved tensions between intellectual property rights, and public health. This article focuses upon one of these important test cases involving intellectual property, public health, and cancer research. In June 2010, Cancer Voices Australia and Yvonne D’Arcy brought an action in the Federal Court of Australia against the validity of a BRCA1 patent — held by Myriad Genetics Inc, the Centre de Recherche du Chul, the Cancer Institute of Japan and Genetic Technologies Limited. Yvonne D’Arcy — a Brisbane woman who has had treatment for breast cancer — maintained: "I believe that what they are doing is morally and ethically corrupt and that big companies should not control any parts of the human body." She observed: "For my daughter, I've had her have [sic] mammograms, etc, because of me but I would still like her to be able to have the test to see if the mutation gene is in there from me." The applicants made the following arguments: "Genes and the information represented by human gene sequences are products of nature universally present in each individual, and the information content of a human gene sequence is fixed. Genetic variations or mutations are products of nature. The isolation of the BRCA1 gene mutation from the human body constitutes no more than a medical or scientific discovery of a naturally occurring phenomenon and does not give rise to a patentable invention." The applicants also argued that "the alleged invention is not a patentable invention in that, so far as claimed in claims 1–3, it is not a manner of manufacture within the meaning of s 6 of the Statute of Monopolies". The applicants suggested that "the alleged invention is a mere discovery". Moreover, the applicants contended that "the alleged invention of each of claims 1-3 is not a patentable invention because they are claims for biological processes for the generation of human beings". The applicants, though, later dropped the argument that the patent claims related to biological processes for the generation of human beings. In February 2013, Nicholas J of the Federal Court of Australia considered the case brought by Cancer Voices Australia and Yvonne D’Arcy against Myriad Genetics. The judge presented the issues in the case, as follows: "The issue that arises in this case is of considerable importance. It relates to the patentability of genes, or gene sequences, and the practice of 'gene patenting'. Briefly stated, the issue to be decided is whether under the Patents Act 1990 (Cth) a valid patent may be granted for a claim that covers naturally occurring nucleic acid — either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) — that has been 'isolated'". In this context, the word "isolated" implies that naturally occurring nucleic acid found in the cells of the human body, whether it be DNA or RNA, has been removed from the cellular environment in which it naturally exists and separated from other cellular components also found there. The genes found in the human body are made of nucleic acid. The particular gene with which the patent in suit is concerned (BRCA1) is a human breast and ovarian cancer disposing gene. Various mutations that may be present in this gene have been linked to various forms of cancer including breast cancer and ovarian cancer.' The judge held in this particular case that Myriad Genetics’ patent claims were a "manner of manufacture" under s 6 of the Statute of Monopolies and s 18(1)(a) of the Patents Act 1990 (Cth). The matter is currently under appeal in the Full Court of the Federal Court of Australia. This article interprets the dispute over Myriad Genetics in light of the scholarly work of Nobel Laureate Professor Joseph Stiglitz on inequality. Such work has significant explanatory power in the context of intellectual property and biotechnology. First, Stiglitz has contended that "societal inequality was a result not just of the laws of economics, but also of how we shape the economy — through politics, including through almost every aspect of our legal system". Stiglitz is concerned that "our intellectual property regime … contributes needlessly to the gravest form of inequality." He maintains: "The right to life should not be contingent on the ability to pay." Second, Stiglitz worries that "some of the most iniquitous aspects of inequality creation within our economic system are a result of 'rent-seeking': profits, and inequality, generated by manipulating social or political conditions to get a larger share of the economic pie, rather than increasing the size of that pie". He observes that "the most iniquitous aspect of this wealth appropriation arises when the wealth that goes to the top comes at the expense of the bottom." Third, Stiglitz comments: "When the legal regime governing intellectual property rights is designed poorly, it facilitates rent-seeking" and "the result is that there is actually less innovation and more inequality." He is concerned that intellectual property regimes "create monopoly rents that impede access to health both create inequality and hamper growth more generally." Finally, Stiglitz has recommended: "Government-financed research, foundations, and the prize system … are alternatives, with major advantages, and without the inequality-increasing disadvantages of the current intellectual property rights system.’" This article provides a critical analysis of the Australian litigation and debate surrounding Myriad Genetics’ patents in respect of genetic testing for BRCA1. First, it considers the ruling of Nicholas J in the Federal Court of Australia that Myriad Genetics’ patent was a manner of manufacture as it related to an artificially created state of affairs, and not mere products of nature. Second, it examines the policy debate over gene patents in Australia, and its relevance to the litigation involving Myriad Genetics. Third, it examines comparative law, and contrasts the ruling by Nicholas J in the Federal Court of Australia with developments in the United States, Canada, and the European Union. Fourth, this piece considers the reaction to the decision of Nicholas at first instance in Australia. Fifth, the article assesses the prospects of an appeal to the Full Federal Court of Australia over the Myriad Genetics’ patents. Finally, this article observes that, whatever happens in respect of litigation against Myriad Genetics, there remains controversy over Genetic Technologies Limited. The Melbourne firm has been aggressively licensing and enforcing its related patents on non-coding DNA and genomic mapping.

Identifying the molecular mediators of VN and the IGF:VN complex-stimulated breast cancer cell survival

This project has identified a molecular signature involved in functions critical to breast cancer progression and metastasis mediated by vitronectin, an abundant protein in human plasma and victornectin:insulin-like growth factor complexes. This may have significant implications in designing future therapeutic targets for patient with tumours overexpressing vitronectin and/or the components of the insulin-like growth factor system:vitronectin axis. In particular, the findings from this project have identified Cyr61 and CTGF as key mediators involved in vitroncetin- and insulin-like growth factor I: Insulin-like growth factor-binding protein:vitronectin-induced breast cancer cell survival and migration.

Understanding the function and mechanisms of intestinal cell kinase in the growth and survival of prostate cancer cells

This project was a step forward in discovering the potential role of intestinal cell kinase in prostate cancer development. Intestinal cell kinase was shown to be upregulated in prostate cancer cells and altered expression led to changes in key cell survival proteins. This study used in vitro experiments to monitor changes in cell growth, protein and RNA expression.

The Piper Fatigue Scale-Revised: Translation and psychometric evaluation in Spanish-speaking breast cancer survivors

Background Cancer-related fatigue (CRF) is the most common and distressing symptom reported by breast cancer survivors. The primary aim of this study was to translate and evaluate psychometrically for the first time a Spanish version of the Piper Fatigue Scale-Revised (S-PFS-R). Methods One hundred and eleven women with stage I–IIIA breast cancer who had completed their primary cancer therapy in the previous 6 months with the exception of hormone therapy completed the S-PFS-R, the Profile of Mood States (POMS) Fatigue (POMS-F) and Vigor subscales (POMS-V), and bilateral force handgrip testing. Data analysis included test–retest reliability, construct validity, criterion-related validity, and exploratory factor analyses. Results Test–retest reliability was satisfactory (r > 0.86), and all subscales showed moderate to high construct validity estimates [corrected item-subscale correlations (Pearson r = ≥ 0.65)]. The exploratory factor analysis revealed four dimensions with 75.5 % of the common variance explained. The S-PFS-R total score positively correlated with the POMS-F subscale (r = 0.50–0.78) and negatively with the POMS-V subscale (r = −0.13 to −0.44) confirming criterion-related validity. Negative correlations among force handgrip testing, subscales, and total scores were weak (r = −0.26 to −0.29). Conclusions The Spanish version of PFS-R shows satisfactory psychometric properties in a sample of breast cancer survivors. This is the first study to translate the PFS-R into Spanish and further testing is warranted.

Predicting the need for adaptive radiotherapy in head and neck cancer

Background and purpose Adaptive radiotherapy (ART) can account for the dosimetric impact of anatomical change in head and neck cancer patients; however it can be resource intensive. Consequently, it is imperative that patients likely to require ART are identified. The purpose of this study was to find predictive factors that identify oropharyngeal squamous cell carcinoma (OPC) and nasopharyngeal carcinoma (NPC) patients more likely to need ART. Materials and methods One hundred and ten patients with OPC or NPC were analysed. Patient demographics and tumour characteristics were compared between patients who were replanned and those that were not. Factors found to be significant were included in logistic regression models. Risk profiles were developed from these models. A dosimetric analysis was performed. Results Nodal disease stage, pre-treatment largest involved node size, diagnosis and initial weight (categorised in 2 groups) were identified as significant for inclusion in the model. Two models were found to be significant (p = 0.001), correctly classifying 98.2% and 96.1% of patients respectively. Three ART risk profiles were developed. Conclusion Predictive factors identifying OPC or NPC patients more likely to require ART were reported. A risk profile approach could facilitate the effective implementation of ART into radiotherapy departments through forward planning and appropriate resource allocation.

Investigation of the relationship between BMI and prostate motion in radiation therapy treatment for prostate cancer

This thesis studied the influence of patient obesity on prostate motion during radiation therapy treatment delivery, an important consideration in the accurate treatment of prostate cancer. The study highlighted the importance of daily image guidance to correct for prostate motion, increasing radiation dose to the prostate while decreasing radiation dose to surrounding healthy tissues, thereby increasing patient quality of life.

Effects of compression on lymphoedema during resistance exercise in women with breast cancer-related lymphoedema: A randomised, cross-over trial

Background The use of compression garments during exercise is recommended for women with breast cancer-related lymphoedema, but the evidence behind this clinical recommendation is unclear. The aim of this randomised, cross-over trial was to compare the acute effects of wearing versus not wearing compression during a single bout of moderate-load resistance exercise on lymphoedema status and its associated symptoms in women with breast cancer-related lymphoedema. Methods Twenty-five women with clinically diagnosed, stable unilateral breast cancer-related lymphoedema completed two resistance exercise sessions, one with compression and one without, in a randomised order separated by a 14 day wash-out period. The resistance exercise session consisted of six upper-body exercises, with each exercise performed for three sets at a moderate-load (10-12 repetition maximum). Primary outcome was lymphoedema, assessed using bioimpedance spectroscopy (L-Dex score). Secondary outcomes were lymphoedema as assessed by arm circumferences (percent inter-limb difference and sum-of-circumferences), and symptom severity for pain, heaviness and tightness, measured using visual analogue scales. Measurements were taken pre-, immediately post- and 24 hours post-exercise. Results There was no difference in lymphoedema status (i.e., L-Dex scores) pre- and post-exercise sessions or between the compression and non-compression condition [Mean (SD) for compression pre-, immediately post- and 24 hours post-exercise: 17.7 (21.5), 12.7 (16.2) and 14.1 (16.7), respectively; no compression: 15.3 (18.3), 15.3 (17.8), and 13.4 (16.1), respectively]. Circumference values and symptom severity were stable across time and treatment condition. Conclusions An acute bout of moderate-load, upper-body resistance exercise performed in the absence of compression does not exacerbate lymphoedema in women with breast cancer-related lymphoedema.