PP2A inhibition overcomes acquired resistance to HER2 targeted therapy

Background: HER2 targeted therapies including trastuzumab and more recently lapatinib have significantly improved the prognosis for HER2 positive breast cancer patients. However, resistance to these agents is a significant clinical problem. Although several mechanisms have been proposed for resistance to trastuzumab, the mechanisms of lapatinib resistance remain largely unknown. In this study we generated new models of acquired resistance to HER2 targeted therapy and investigated mechanisms of resistance using phospho-proteomic profiling. Results: Long-term continuous exposure of SKBR3 cells to low dose lapatinib established a cell line, SKBR3-L, which is resistant to both lapatinib and trastuzumab. Phospho-proteomic profiling and immunoblotting revealed significant alterations in phospho-proteins involved in key signaling pathways and molecular events. In particular, phosphorylation of eukaryotic elongation factor 2 (eEF2), which inactivates eEF2, was significantly decreased in SKBR3-L cells compared to the parental SKBR3 cells. SKBR3-L cells exhibited significantly increased activity of protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates eEF2. SKBR3-L cells showed increased sensitivity to PP2A inhibition, with okadaic acid, compared to SKBR3 cells. PP2A inhibition significantly enhanced response to lapatinib in both the SKBR3 and SKBR3-L cells. Furthermore, treatment of SKBR3 parental cells with the PP2A activator, FTY720, decreased sensitivity to lapatinib. The alteration in eEF2 phosphorylation, PP2A activity and sensitivity to okadaic acid were also observed in a second HER2 positive cell line model of acquired lapatinib resistance, HCC1954-L. Conclusions: Our data suggests that decreased eEF2 phosphorylation, mediated by increased PP2A activity, contributes to resistance to HER2 inhibition and may provide novel targets for therapeutic intervention in HER2 positive breast cancer which is resistant to HER2 targeted therapies

Prevalence of cryptosporidiosis in dairy cattle, cattle-keeping families, their non-cattle-keeping neighbours and HIV-positive individuals in Dagoretti Division, Nairobi, Kenya

This paper is part of a special supplement on assessing and managing urban zoonoses and food-borne disease in two African cities (Nairobi, Kenya and Ibadan, Nigeria).This paper reports a study estimating the prevalence of cryptosporidiosis, an emerging zoonosis, in people and cattle in Dagoretti, Nairobi. A repeated cross-sectional survey was carried out among randomly selected cattle keepers in Dagoretti, their dairy cattle and their non-cattle-keeping neighbours in the dry and wet seasons of 2006. A survey was also carried out among a group of people living with human immunodeficiency virus (HIV). Faecal samples were examined for Cryptosporidium oocysts using the modified Ziehl–Neelsen method; 16 % of the samples were also examined using immunofluorescence antibody (IFA) technique. Quality control consisted of blind reviews of slides, examining split samples and confirming slide results with IFA. We found that members of dairy households had a dry season cryptosporidiosis prevalence of 4 % and wet season prevalence of 0.3 %, and non-dairy households, a prevalence of 5 and 0 %, respectively. The cattle dry season prevalence was 15 %, and the wet season prevalence, 11 %. The prevalence in people living with HIV was 5 %. The laboratory quality control system showed some inconsistency within and between different tests, indicating challenges in obtaining consistent results under difficult field and working conditions. In conclusion, this is the first reported study to simultaneously survey livestock, livestock keepers and their neighbours for cryptosporidiosis. We failed to find evidence that zoonotic cryptosporidiosis is important overall in this community. This study also draws attention to the importance of quality control and its reporting in surveys in developing countries

Development and delivery of evidence-based messages to reduce the risk of zoonoses in Nairobi, Kenya

This paper is part of a special supplement on assessing and managing urban zoonoses and food-borne disease in two African cities (Nairobi, Kenya and Ibadan, Nigeria).This paper describes a trans-disciplinary process of co-generating and disseminating evidence-based messages for reducing the risk from cryptosporidiosis and other zoonoses in an urban community in Nairobi. Research findings about disease prevalence, risk factors and observed risky and risk-mitigating practices were analysed by a team comprising researchers, community members and local policy- and decision-makers. Using participatory planning, multiple strategies were developed for disseminating key information. We identified five vulnerable groups at higher risk of exposure to cryptosporidiosis and other cattle zoonoses with similar transmission pathways (women, children, elderly people, immunosuppressed people and male farm workers). For each group, targeted messages were developed. Good practices already in use, as were also practices as practices to improve environmental conditions. These messages were disseminated through printed material, in a workshop, through community campaigners and also an edutainment soap opera episode broadcast on Kenyan television. In conclusion, a participatory and trans-disciplinary process can help transform the findings of research into messages that are targeted, attractive and understandable

Social and gender determinants of risk of cryptosporidiosis, an emerging zoonosis, in Dagoretti, Nairobi, Kenya

This paper is part of a special supplement on assessing and managing urban zoonoses and food-borne disease in two African cities (Nairobi, Kenya and Ibadan, Nigeria).The aim of the study was to investigate the social and gender determinants of the risk of exposure to Cryptosporidium fromurban dairying in Dagoretti, Nairobi. Focus group discussions were held in six locations to obtain qualitative information on risk of exposure. A repeated cross-sectional descriptive study included participatory assessment and household questionnaires (300 randomly selected urban dairy farming households and 100 non-dairying neighbours). Onehundred dairy households randomly selected from the 300 dairy households participated in an additional economic survey along with 40 neighbouring non-dairy households. We found that exposure to Cryptosporidium was influenced by gender, age and role in the household. Farm workers and people aged 50 to 65 years had most contact with cattle, and women had greater contact with raw milk. However, children had relatively higher consumption of raw milk than other age groups. Adult women had more daily contact with cattle faeces than adult men, and older women had more contact than older men. Employees had greater contact with cattle than other groups and cattle faeces, and most (77 %) were male. Women took more care of sick people and were more at risk from exposure by this route. Poverty did not affect the level of exposure to cattle but did decrease consumption of milk. There was no significant difference between men and women as regards levels of knowledge on symptoms of cryptosporidiosis infections or other zoonotic diseases associated with dairy farming. Awareness of cryptosporidiosis and its transmission increased significantly with rising levels of education. Members of nondairy households and children under the age of 12 years had significantly higher odds of reporting diarrhoea: gender, season and contact with cattle or cattle dung were not significantly linked with diarrhoea. In conclusion, social and gender factors are important determinants of exposure to zoonotic disease in Nairobi

Trans-disciplinary study on the health risks of cryptosporidiosis from dairy systems in Dagoretti, Nairobi, Kenya : study background and farming system characteristics

This paper is part of a special supplement on assessing and managing urban zoonoses and food-borne disease in two African cities (Nairobi, Kenya and Ibadan, Nigeria).This paper characterises the dairy farming system in Dagoretti, Nairobi. Characterisation was part of a broader ecohealth project to estimate the prevalence and risk of cryptosporidiosis and develop risk mitigation strategies. In the project a trans-disciplinary team addressed epidemiological, socioeconomic, environmental and policy aspects of cryptosporidiosis, an emerging zoonosis. This paper also provides background and describes sampling methods for the wider project. Three hundred dairy households were probabilistically sampled from a sampling frame of all dairy households in five of the six locations of Dagoretti, one of the eight districts of Nairobi Province. Randomly selected households identified 100 non-dairy-keeping households who also took part in the study. A household questionnaire was developed, pre-tested and administered in the dry and wet seasons of 2006. An additional study on livelihood and economic benefits of dairying took place with 100 dairy farmers randomly selected from the 300 farms (as well as 40 non-dairy neighbours as a control group), and a risk targeted survey of environmental contamination with Cryptosporidium was conducted with 20 farmers randomly selected from the 29 farmers in the wider survey who were considered at high risk because of farming system. We found that around 1 in 80 urban households kept dairy cattle with an average of three cattle per household. Cross-breeds of exotic and local cattle predominate. Heads of dairykeeping households were significantly less educated than the heads of non-dairy neighbours, had lived in Dagoretti for significantly longer and had significantly larger households. There was a high turnover of 10 % of the cattle population in the 3-month period of the study. Cattle were zero grazed, but productivity parameters were sub-optimal as were hygiene and husbandry practices. In conclusion, dairy keeping is a minor activity in urban Nairobi but important to households involved and their community. Ecohealth approaches are well suited to tackling the complex problem of assessing and managing emerging zoonoses in urban settings

HER2-Targeted Tyrosine Kinase Inhibitors Cause Therapy-Induced-Senescence in Breast Cancer Cells

Prolonged treatment of HER2 positive breast cancer cells with tyrosine kinase inhibitors (TKIs) leads to the emergence of acquired resistance. However, the effects of continuous TKI exposure on cell fate, and the steps leading to the acquisition of a resistant phenotype are poorly understood. To explore this, we exposed five HER2 positive cells lines to HER2 targeted therapies for periods of up to 4 weeks and examined senescence associated β-galactosidase (SA-β-gal) activity together with additional markers of senescence. We found that lapatinib treatment resulted in phenotypic alterations consistent with a senescent phenotype and strong SA-β-gal activity in HER2-positive cell lines. Lapatinib-induced senescence was associated with elevated levels of p15 and p27 but was not dependent on the expression of p16 or p21. Restoring wild type p53 activity either by transfection or by treatment with APR-246, a molecule which reactivates mutant p53, blocked lapatinib-induced senescence and caused increased cell death. In contrast to lapatinib, SA-β-gal activity was not induced by exposing the cells to trastuzumab as a single agent but co-administration of lapatinib and trastuzumab induced senescence, as did treatment of the cells with the irreversible HER2 TKIs neratinib and afatinib. Neratinib- and afatinib-induced senescence was not reversed by removing the drug whereas lapatinib-induced senescence was reversible. In summary, therapy-induced senescence represents a novel mechanism of action of HER2 targeting agents and may be a potential pathway for the emergence of resistance