Breast cancer cells induce osteolytic bone lesions in vivo through a reduction in osteoblast activity in mice

Bone metastases are severely debilitating and have a significant impact on the quality of life of women with metastatic breast cancer. Treatment options are limited and in order to develop more targeted therapies, improved understanding of the complex mechanisms that lead to bone lesion development are warranted. Interestingly, whilst prostate-derived bone metastases are characterised by mixed or osteoblastic lesions, breast-derived bone metastases are characterised by osteolytic lesions, suggesting unique regulatory patterns. This study aimed to measure the changes in bone formation and bone resorption activity at two time-points (18 and 36 days) during development of the bone lesion following intratibial injection of MDA-MB-231 human breast cancer cells into the left tibiae of Severely Combined Immuno-Deficient (SCID) mice. The contralateral tibia was used as a control. Tibiae were extracted and processed for undecalcified histomorphometric analysis. We provide evidence that the early bone loss observed following exposure to MDA-MB-231 cells was due to a significant reduction in mineral apposition rate, rather than increased levels of bone resorption. This suggests that osteoblast activity was impaired in the presence of breast cancer cells, contrary to previous reports of osteoclast-dependent bone loss. Furthermore mRNA expression of Dickkopf Homolog 1 (DKK-1) and Noggin were confirmed in the MDA-MB-231 cell line, both of which antagonise osteoblast regulatory pathways. The observed bone loss following injection of cancer cells was due to an overall thinning of the trabecular bone struts rather than perforation of the bone tissue matrix (as measured by trabecular width and trabecular separation, respectively), suggesting an opportunity to reverse the cancer-induced bone changes. These novel insights into the mechanisms through which osteolytic bone lesions develop may be important in the development of new treatment strategies for metastatic breast cancer patients.

Metastasis of ovarian cancer is mediated by kallikrein related peptidases

Ovarian cancer, in particular epithelial ovarian cancer (EOC), is commonly diagnosed when the tumor has metastasized into the abdominal cavity with an accumulation of ascites fluid. Combining histopathology and genetic variations, EOC can be sub-grouped into Type-I and Type-II tumors, of which the latter are more aggressive and metastatic. Metastasis and chemoresistance are the key events associated with the tumor microenvironment that lead to a poor patient outcome. Kallikrein-related peptidases (KLKs) are aberrantly expressed in EOC, in particular, in the more metastatic Type-II tumors. KLKs are a family of 15 serine proteases that are expressed in diverse human tissues and involved in various patho-physiological processes. As extracellular enzymes, KLKs function in the hydrolysis of growth factors, proteases, cell membrane bound receptors, adhesion proteins, and cytokines initiating intracellular signaling pathways and their downstream events. High KLK levels are differentially associated with the prognosis of ovarian cancer patients, suggesting that they not only have application as biomarkers but also function in disease progression, and therefore are potential therapeutic targets. Recent studies have demonstrated the function of these proteases in promoting and/or suppressing the invasive behavior of ovarian cancer cells in metastasis in vitro and in vivo. Both conventional cell culture methods and three-dimensional platforms have been applied to mimic the ovarian cancer microenvironment of patients, such as the solid stromal matrix and ascites fluid. Here we summarize published studies to provide an overview of our understanding of the role of KLKs in EOC, and to lay the foundation for future research directions.

A bioengineered 3D ovarian cancer model for the assessment of peptidase-mediated enhancement of spheroid growth and intraperitoneal spread

Cancer-associated proteases promote peritoneal dissemination and chemoresistance in malignant progression. In this study, kallikrein-related peptidases 4, 5, 6, and 7 (KLK4-7)-cotransfected OV-MZ-6 ovarian cancer cells were embedded in a bioengineered three-dimensional (3D) microenvironment that contains RGD motifs for integrin engagement to analyze their spheroid growth and survival after chemotreatment. KLK4-7-cotransfected cells formed larger spheroids and proliferated more than controls in 3D, particularly within RGD-functionalized matrices, which was reduced upon integrin inhibition. In contrast, KLK4-7-expressing cell monolayers proliferated less than controls, emphasizing the relevance of the 3D microenvironment and integrin engagement. In a spheroid-based animal model, KLK4-7-overexpression induced tumor growth after 4 weeks and intraperitoneal spread after 8 weeks. Upon paclitaxel administration, KLK4-7-expressing tumors declined in size by 91% (controls: 87%) and showed 90% less metastatic outgrowth (controls: 33%, P<0.001). KLK4-7-expressing spheroids showed 53% survival upon paclitaxel treatment (controls: 51%), accompanied by enhanced chemoresistance-related factors, and their survival was further reduced by combination treatment of paclitaxel with KLK4/5/7 (22%, P=0.007) or MAPK (6%, P=0.006) inhibition. The concomitant presence of KLK4-7 in ovarian cancer cells together with integrin activation drives spheroid formation and proliferation. Combinatorial approaches of paclitaxel and KLK/MAPK inhibition may be more efficient for late-stage disease than chemotherapeutics alone as these inhibitory regimens reduced cancer spheroid growth to a greater extent than paclitaxel alone.

Secretome and degradome profiling shows that Kallikrein-related peptidases 4, 5, 6, and 7 induce TGFβ-1 signaling in ovarian cancer cells

Kallikrein-related peptidases, in particular KLK4, 5, 6 and 7 (4-7), often have elevated expression levels in ovarian cancer. In OV-MZ-6 ovarian cancer cells, combined expression of KLK4-7 reduces cell adhesion and increases cell invasion and resistance to paclitaxel. The present work investigates how KLK4-7 shape the secreted proteome ("secretome") and proteolytic profile ("degradome") of ovarian cancer cells. The secretome comparison consistently identified >900 proteins in three replicate analyses. Expression of KLK4-7 predominantly affected the abundance of proteins involved in cell-cell communication. Among others, this includes increased levels of transforming growth factor β-1 (TGFβ-1). KLK4-7 co-transfected OV-MZ-6 cells share prominent features of elevated TGFβ-1 signaling, including increased abundance of neural cell adhesion molecule L1 (L1CAM). Augmented levels of TGFβ-1 and L1CAM upon expression of KLK4-7 were corroborated in vivo by an ovarian cancer xenograft model. The degradomic analysis showed that KLK4-7 expression mostly affected cleavage sites C-terminal to arginine, corresponding to the preference of kallikreins 4, 5 and 6. Putative kallikrein substrates include chemokines, such as growth differentiation factor 15 (GDF 15) and macrophage migration inhibitory factor (MIF). Proteolytic maturation of TGFβ-1 was also elevated. KLK4-7 have a pronounced, yet non-degrading impact on the secreted proteome, with a strong association between these proteases and TGFβ-1 signaling in tumor biology. © 2013 Federation of European Biochemical Societies.

PITX2 and non-canonical Wnt pathway interaction in metastatic prostate cancer

The non-canonical Wnt pathway, a regulator of cellular motility and morphology, is increasingly implicated in cancer metastasis. In a quantitative PCR array analysis of 84 Wnt pathway associated genes, both non-canonical and canonical pathways were activated in primary and metastatic tumors relative to normal prostate. Expression of the Wnt target gene PITX2 in a prostate cancer (PCa) bone metastasis was strikingly elevated over normal prostate (over 2,000-fold) and primary prostate cancer (over 200-fold). The elevation of PITX2 protein was also evident on tissue microarrays, with strong PITX2 immunostaining in PCa skeletal and, to a lesser degree, soft tissue metastases. PITX2 is associated with cell migration during normal tissue morphogenesis. In our studies, overexpression of individual PITX2A/B/C isoforms stimulated PC-3 PCa cell motility, with the PITX2A isoform imparting a specific motility advantage in the presence of non-canonical Wnt5a stimulation. Furthermore, PITX2 specific shRNA inhibited PC-3 cell migration toward bone cell derived chemoattractant. These experimental results support a pivotal role of PITX2A and non-canonical Wnt signaling in enhancement of PCa cell motility, suggest PITX2 involvement in homing of PCa to the skeleton, and are consistent with a role for PITX2 in PCa metastasis to soft and bone tissues. Our findings, which significantly expand previous evidence that PITX2 is associated with risk of PCa biochemical recurrence, indicate that variation in PITX2 expression accompanies and may promote prostate tumor progression and metastasis.

Placement Trajectory : mapping the journeys of children and young people in out-of-home care

Significant problems confront our child protection out-of-home care system including: high costs; increasing numbers of children and young people entering and remaining in care longer; high frequency of placement movement; and, negative whole-of-life outcomes for children and young people who have exited care. National policy and research agendas recognise the importance of enhancing the evidence base in out-of-home care to inform the development of policy, programs and practice, and improve longitudinal outcomes of children and young people. The authors discuss the concept of placement trajectory as a framework for research and systems analysis in the out-of-home context. While not without limitations, the concept of placement trajectory is particularly useful in understanding the factors influencing placement movement and stability. Increasing the evidence base in this area can serve to enhance improved outcomes across the lifespan for children and young people in the out-of-home care system.

A comparison of children's needs models in the Australian and Chinese context

The diverse needs of children have been drawing global attention from both academic and practitioner communities. Based on semi-structured interviews with 23 kin caregivers and five school personnel in the Shijiapu Town of Jilin Province, China, this paper presents a needs model for rural school-age children left behind by their migrant parents. This Chinese model is compared to the needs identification mechanism developed by the Australian Research Alliance for Children and youth. The paper outlines the common needs of children in different contexts, and also highlights the needs that are not explicit in the Australian Research Alliance for Children and Youth framework, such as empowerment and agency or perhaps given insufficient weight, such as education. In discussing relationships among different needs, aspects that are missing in the framework it is argued that culture should be more explicitly recognised when defining need.

Informal kinship care in rural China : the influence of Confucianism and attachment

This article examines the role of informal kinship care in addressing the emotional needs and mental health, along with relationships, of school-age children left behind in rural China. Rural–urban migration in China has caused many rural children to be left behind in their local communities. Based on semi-structured interview data, this article explores Confucianism’s impact on Chinese kin caregivers’ understandings of children’s needs and their childrearing practices to address these needs. Through the lens of attachment theory, this study identified a close affective bond between children left behind and their kin caregivers. This relationship is underpinned by kin caregivers’ high commitment and love for children, and the Confucian concept of ‘benevolence’. It not only provides children left behind with a sense of belonging, it also alleviates their trauma/grief due to separation from their parents

A better future for young people leaving care

The Australian National Homelessness Research Agenda funded this study which explored post-care homelessness experiences of young people, asked them what they considered helpful, and identified practice and policy implications. Participants were 48 care leavers aged 19 to 23 years in Queensland and Victoria. Of these 17 young people were interviewed twice over a four month period. In addition focus groups were held with service providers and current postcare service arrangements in the two states mapped. The study found there is a need for policy and practice to more effectively orient towards a holistic and future oriented view of outcomes for young people transitioning from care.

Using cost-effective multimedia to create engaging learning experiences : Resources Manual

A guide to utilising multi-media for teaching and learning.

Future-proofing the pharmacy profession in a hypercompetitive market

This paper highlights the hypercompetitive nature of the current pharmacy landscape in Australia and to suggest either a superior level of differentiation strategy or a focused differentiation strategy targeting a niche market as two viable, alternative business models to cost leadership for small, independent community pharmacies. A description of the Australian health care system is provided as well as background information on the current community pharmacy environment in Australia. The authors propose a differentiation or focused differentiation strategy based on cognitive professional services (CPS) which must be executed well and of a superior quality to competitors' services. Market research to determine the services valued by target customers and that they are willing to pay for is vital. To achieve the superior level of quality that will engender high patient satisfaction levels and loyalty, pharmacy owners and managers need to develop, maintain and clearly communicate service quality specifications to the staff delivering these services. Otherwise, there will be a proliferation of pharmacies offering the same professional services with no evident service differential. However, to sustain competitive advantage over the long-term, these smaller, independent community pharmacies will need to exploit a broad core competency base in order to be able to continuously introduce new sources of competitive advantage. With the right expertise, the authors argue that smaller, independent community pharmacies can successfully deliver CPS and sustain profitability in a hypercompetitive market.

The impact on clinical practice of a postgraduate clinical pharmacy programme, incorporating competency-based performance evaluation

Background A goal of the postgraduate clinical pharmacy programme (PGCPP) at the University of Queensland is to enhance clinical practice. Aims To evaluate student perceptions of the impact of the PGCPP on practice and the inclusion of a competency-based performance evaluation as a formative component of the curriculum. Method In 2010, students completed a questionnaire to evaluate the impact of the PGCPP. In 2011, formative competency-based performance evaluations were conducted as a component of the course and the questionnaire was repeated. Responses, competency ratings and evaluation feedback were collated. Data were analysed using descriptive statistics. Results 51/57 (89%) of students completed the questionnaire in 2010 and 2011. Over 90% of students agreed or strongly agreed that the PGCPP enhanced practice, knowledge, confidence and contribution to patient care. Responses were similarly positive after the inclusion of the performance evaluation. Conclusion This study demonstrated that the PGCPP is achieving the goal of enhancing the practice of pharmacists.

Collaborating to develop clinical pharmacy teaching in Sri Lanka

Background Bachelor of Pharmacy programs were introduced in 2006 into two Sri Lankan universities - University of Peradeniya and University of Sri Jayewardenepura. Due to minimal clinical pharmacy experience in the country, these universities invited international colleagues to develop and teach the clinical pharmacy course. Aims To describe development, delivery and evaluation of both a clinical pharmacy undergraduate course and a "Train-thetrainer”program provided to local academics delivering undergraduate pharmacy programs. Method In 2009, Australian pharmacist academics developed and piloted an undergraduate clinical pharmacy course at University of Peradeniya. In 2010, this was refined and delivered at University of Sri Jayewardenepura, along with a “train-thetrainer”program for local academics. These were evaluated using surveys. Results Most students considered lecture delivery speed and use of audio visual aids appropriate, and lecture content relevant.Most academics found the “Train-the-Trainer” program increased their knowledge and improved their teaching skills. Conclusion Experienced pharmacist academics can improve the quality of clinical pharmacy teaching in developing countries such as Sri Lanka.

Species-specific homing mechanisms of human prostate cancer metastasis in tissue engineered bone

The development of effective therapeutic strategies against prostate cancer bone metastases has been impeded by the lack of adequate animal models that are able to recapitulate the biology of the disease in humans. Bioengineered approaches allow researchers to create sophisticated experimentally and physiologically relevant in vivo models to study interactions between cancer cells and their microenvironment under reproducible conditions. The aim of this study was to engineer a morphologically and functionally intact humanized organ bone which can serve as a homing site for human prostate cancer cells. Transplantation of biodegradable tubular composite scaffolds seeded with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone construct including a large number of human mesenchymal cells which were shown to be metabolically active and capable of producing extracellular matrix components. Micro-CT analysis demonstrated that the newly formed ossicle recapitulated the morphological features of a physiological organ bone with a trabecular network surrounded by a cortex-like outer structure. This microenvironment was supportive of the lodgement and maintenance of murine haematopoietic cell clusters, thus mimicking a functional organ bone. Bioluminescence imaging demonstrated that luciferase-transduced human PC3 cells reproducibly homed to the humanized tissue engineered bone constructs, proliferated, and developed macro-metastases. This model allows the analysis of interactions between human prostate cancer cells and a functional humanized bone organ within an immuno-incompetent murine host. The system can serve as a reproducible platform to study effects of therapeutics against prostate cancer bone metastases within a humanized microenvironment.

Hydrogel microwell arrays allow the assessment of protease-associated enhancement of cancer cell aggregation and survival

Current routine cell culture techniques are only poorly suited to capture the physiological complexity of tumor microenvironments, wherein tumor cell function is affected by intricate three-dimensional (3D), integrin-dependent cell-cell and cell-extracellular matrix (ECM) interactions. 3D cell cultures allow the investigation of cancer-associated proteases like kallikreins as they degrade ECM proteins and alter integrin signaling, promoting malignant cell behaviors. Here, we employed a hydrogel microwell array platform to probe using a high-throughput mode how ovarian cancer cell aggregates of defined size form and survive in response to the expression of kallikreins and treatment with paclitaxel, by performing microscopic, quantitative image, gene and protein analyses dependent on the varying microwell and aggregate sizes. Paclitaxel treatment increased aggregate formation and survival of kallikrein-expressing cancer cells and levels of integrins and integrin-related factors. Cancer cell aggregate formation was improved with increasing aggregate size, thereby reducing cell death and enhancing integrin expression upon paclitaxel treatment. Therefore, hydrogel microwell arrays are a powerful tool to screen the viability of cancer cell aggregates upon modulation of protease expression, integrin engagement and anti-cancer treatment providing a micro-scaled yet high-throughput technique to assess malignant progression and drug-resistance.