Towards Identifying Pharmacodynamic Blood Biomarkers of MAP3K12 Inhibition

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons in the brain and spinal cord, for which there is no cure. Activation of the stress-induced c-Jun N-terminal kinase (JNK) signaling pathway, a critical mediator of neuronal apoptosis and axon degeneration, is evident in ALS and is therefore considered a potential target to prevent neurodegeneration. Pharmacological inhibition of an upstream activator of the JNK pathway, the mitogen-activated protein kinase kinase kinase (MAP3K12), has demonstrated neuroprotection in vitro as well as suppression of pathway activation in vivo. These observations, along with the largely neuronal-specific expression of MAP3K12, makes inhibition of this kinase a promising therapeutic target for ALS. Furthermore, a pharmacodynamic (PD) blood biomarker would be beneficial to help assess efficacy of MAP3K12 inhibition. A crucial step to interpreting the PD effects of MAP3K12 inhibition using blood biomarkers is to acquire evidence of MAP3K12 protein expression in blood, which was the aim of this study. First, we found Map3k12 transcript levels to be highly reduced in blood from Map3k12 conditional knockout (cKO) mice, demonstrating that these are excellent negative controls for studying MAP3K12 expression in blood. Next, three antibodies were identified that recognized mouse and human MAP3K12. Utilizing these antibodies, an intracellular flow cytometry method was developed that successfully detected MAP3K12 protein in human peripheral blood mononuclear cells (PBMCs). Collectively, our data provide evidence of MAP3K12 expression in human blood and thus pave the way for identifying PD blood biomarkers of MAP3K12 inhibition.Blood Biomarkers – a Window into the Brain? Little is known about the underlying cause of neurodegenerative diseases such amyotrophic lateral sclerosis (ALS). The protein MAP3K12 was recently identified as an important player in neurodegeneration and a potential therapeutic target for ALS. Our study found evidence of MAP3K12 in blood, which brings us closer towards identifying blood biomarkers to evaluate the biological effects of ALS drugs. ALS is a rare but terrifying neurodegenerative disease that kills half of the patients within 3 years from onset. The disease attacks the cells that are in charge of muscle movement, so called motor neurons. As the disease worsens, ALS patients lose their ability to move, speak, eat and ultimately breathe. After decades of research, there is still no cure to ALS. The cause of motor neuron death still puzzles scientists, but more clues are being revealed about the underlying disease mechanisms. Recent studies have uncovered a molecular pathway leading to degeneration of motor neurons in ALS. With increased attention to this pathway, a protein called MAP3K12 was found to be one of its key components. This new piece to the puzzle makes MAP3K12 a promising therapeutic target to prevent the death of motor neurons in people with ALS. These findings have led to development of drugs, so called inhibitors that block the activation of MAP3K12. However, the impossibility of collecting brain samples makes it difficult to measure the effect of these inhibitors within the brain. A way to overcome this obstacle is to use biomarkers. Biomarkers are measurable indicators that provide a window into health and disease. A classic example of a biomarker is the cholesterol levels in blood that serve as a risk indicator for heart disease. Another use of biomarkers is to assess the biological effects produced by a drug. Finding such biomarkers of MAP3K12 inhibitors would open up ways to monitor their effectiveness to help find the right dose for ALS patients. Ultimately this could speed up drug trials for ALS, which is of great importance for a disease of such urgent unmet medical need. Although biomarkers can be found throughout the body, we set out to find a biomarker in blood. Why, you might ask? Well, just imagine being able to predict the inhibitors effects in the brain with only a few drops of blood. The main challenge with finding such biomarkers is that MAP3K12 protein mainly exists in the brain and has not previously been found in blood. Without the protein, the drug would have little to no effect in blood, which would make the use of a blood biomarker pointless. Promisingly, the gene encoding the protein is indeed present in blood, which motivated us to search for protein expression in the blood. First off, we used mice in which the gene encoding MAP3K12 had been genetically removed, so called knockout mice, as well as cells that we genetically engineered to express the protein. Using these two sources where we clearly knew the presence or absence of the protein, we tested the ability of different antibodies to recognize MAP3K12 and found several good candidates to use for protein detection in blood. By using our newfound antibodies in flow cytometry, a laser-based technique useful for studying protein expression, we developed a novel method for detecting the protein in human blood cells. Armed with laser and antibodies, we found evidence of MAP3K12 protein expression in human blood, which brings us one step closer to finding a blood biomarker of this protein. In the future, a quick blood sample from an ALS patient could allow us to measure levels of MAP3K12 activity in the blood and predict the effects of MAP3K12 inhibition in the brain

Discovery of dachshund 2 protein as a novel biomarker of poor prognosis in epithelial ovarian cancer

<p>Abstract</p> <p>Background</p> <p>The Dachshund homolog 2 (<it>DACH2</it>) gene has been implicated in development of the female genital tract in mouse models and premature ovarian failure syndrome, but to date, its expression in human normal and cancerous tissue remains unexplored. Using the Human Protein Atlas as a tool for cancer biomarker discovery, DACH2 protein was found to be differentially expressed in epithelial ovarian cancer (EOC). Here, the expression and prognostic significance of DACH2 was further evaluated in ovarian cancer cell lines and human EOC samples.</p> <p>Methods</p> <p>Immunohistochemical expression of DACH2 was examined in tissue microarrays with 143 incident EOC cases from two prospective, population-based cohorts, including a subset of benign-appearing fallopian tubes (n = 32). A nuclear score (NS), i.e. multiplier of staining fraction and intensity, was calculated. For survival analyses, cases were dichotomized into low (NS < = 3) and high (NS > 3) using classification and regression tree analysis. Kaplan Meier analysis and Cox proportional hazards modelling were used to assess the impact of DACH2 expression on survival. DACH2 expression was analysed in the cisplatin sensitive ovarian cancer cell line A2780 and its cisplatin resistant derivative A2780-Cp70. The specificity of the DACH2 antibody was tested using siRNA-mediated silencing of DACH2 in A2780-Cp70 cells.</p> <p>Results</p> <p>DACH2 expression was considerably higher in the cisplatin resistant A2780-Cp70 cells compared to the cisplatin-sensitive A2780 cells. While present in all sampled fallopian tubes, DACH2 expression ranged from negative to strong in EOC. In EOC, DACH2 expression correlated with several proteins involved in DNA integrity and repair, and proliferation. DACH2 expression was significantly higher in carcinoma of the serous subtype compared to non-serous carcinoma. In the full cohort, high DACH2 expression was significantly associated with poor prognosis in univariable analysis, and in carcinoma of the serous subtype, DACH2 remained an independent factor of poor prognosis.</p> <p>Conclusions</p> <p>This study provides a first demonstration of DACH2 protein being expressed in human fallopian tubes and EOC, with the highest expression in serous carcinoma where DACH2 was found to be an independent biomarker of poor prognosis. Future research should expand on the role of DACH2 in ovarian carcinogenesis and chemotherapy resistance.</p

Expression of the global regulator SATB1 is an independent factor of poor prognosis in high grade epithelial ovarian cancer

Background: The global gene regulator Special AT-rich sequence-binding protein1 (SATB1) has been reported to reprogramme tumour cells into a more malignant phenotype and associate with poor clinical outcome in several cancer forms. In this study, we investigated the molecular correlates and prognostic impact of SATB1 expression in human epithelial ovarian cancer (EOC). Findings: Immunohistochemical expression of SATB1 was examined in tissue microarrays with tumours from 151 incident EOC cases from two prospective, population-based cohorts. Benign-appearing fallopian tube epithelium from 32 cases was also analyzed. A multiplier of nuclear fraction and staining intensity of SATB1 was calculated. While barely expressed in tubal epithelium, nuclear SATB1 expression was denoted in 35/151 (23.2%) EOC cases. Spearman's Rho test revealed an inverse correlation between SATB1 expression and histological grade (R = -0.22, p = 0.006) and a positive correlation with expression of dachshund 2 protein (R = 0.28, p = 0.001), phosphorylated Chek1 (R = 0.26, p = 0.002) and minichromosome maintenance protein 3 (R = 0.17, p = 0.042). Univariable Cox regression analysis revealed that SATB1 expression, while not prognostic in the full cohort, was associated with a reduced ovarian cancer-specific survival and 5-year overall survival in high grade tumours (n = 105) (HR = 2.14 and HR = 1.96, respectively). This association remained significant in multivariable analysis, adjusted for age and clinical stage (HR = 2.20 and HR = 2.06, respectively). Conclusions: These results demonstrate that SATB1 expression is an independent factor of poor prognosis in high grade EOC and correlates in vivo with cellular processes involved in the maintenance of DNA integrity. The functional basis for these observations merits further investigation

African farm trajectories and the sub-continental food crisis

This is a study of farm dynamics in eight African countries, drawing on a sample of more than 3000 farm households. It deals mainly with food crops and in detail with maize and makes a longitudinal analysis by systematically comparing current conditions with those obtaining when the farm was set up under its present management. From the study emerges an overall picture of inadequately exploited production potentials where farmers’ commercial energies are driven towards other food crops than grains, especially vegetables for urban markets. Commercial incentives in food grain production favour small groups of well-placed and usually male farmers, while, the lack of seed-fertiliser technology and commercial incentives means that smallholders devote their energies to other crops or to non-farm sources of income

Drills and Diets, Consumption and Conservation– the Role of Primate Meat in Local Diets in and Around Cross River National Park, Nigeria

The study uses household level data from four villages inand around Cross River National Park (CRNP), Nigeria to assess therole of primate meat in local livelihoods and diets. Okwangwo is anenclave community within the national park, Butatong houses theCRNP headquarters. Kanyang1 and Abo Ebam are located fartheraway from the park. 149 respondents were surveyed. Sale ofbushmeat contributed 4 percent of total cash income on average, butis important as a source of protein in the context of poorly developedlivestock systems. 98 percent of the households ate bushmeat duringthe past year and 74 percent hunted for consumption. 77 percent atemeat from primates, although this varied from 53 percent in Butatongto 97 percent in Okwangwo. Differences emerge among the villageswith less reliance on bushmeat, less hunting and a dietary shifttowards poultry in Butatong. There is no correlation between incomelevels and consumption of primate meat. The overwhelming motivefor eating primate meat was taste preferences. Solutions tounsustainable extraction of primate meat must be sourced in relationto local consumption. Improving access to animal source foods,through widening the livestock basis of local agraria

Analysing diet composition and food insecurity by socio-economic status in secondary African cities

This chapter takes as its starting point theorizing around nutrition and food system transitions thought to be increasingly occurring in urban Africa, and how this may be linked to a growing non-communicable disease burden. We focus specifically on the secondary city context by analysing household survey data gathered from six cities across Ghana, Kenya and Uganda during 2013–2015. We asked how diet composition and diversity, food sources and food security varied by socio-economic status, using expenditure and demographic data to create a proxy for household well-being. In this way, we investigate one of the claimed keystones affecting urban food systems and dietary health in sub-Saharan Africa—that of obesogenic urban food environments. Our findings indicate that the socio-economic status of a household was the most important factor influencing household dietary diversity and food security status, i.e. better-off households were more likely to feel food secure and eat from a greater variety of food groups. In addition, the number of income sources was additionally associated with higher dietary diversity. We also found that a household’s involvement in agriculture had only a small positive effect on food security in one city and was associated with a reduction in dietary diversity scores. Our findings emphasize the importance of supporting aggregated national and international statistics on agricultural production and trade with detailed local analyses that focus on actual household food access and consumption. We also see reasons to be cautious about making causal claims regarding consumption change and obesogenic urban environments as the major contributor to a rising obesity and non-communicable disease burden in Africa

Cyclin D1 expression in colorectal cancer is a favorable prognostic factor in men but not in women in a prospective, population-based cohort study

<p>Abstract</p> <p>Background</p> <p>Although colorectal cancer (CRC) is generally not considered to be a hormone-dependent malignancy, several sex-related differences in incidence, molecular characteristics and survival have been reported. Epidemiological studies have consistently shown that increased exposure to female sex hormones is associated with a lower risk of CRC in women, and cyclin D1, an important downstream effector in estrogen-mediated signaling, is commonly activated in CRC. In this study, we analyzed the prognostic significance of cyclin D1 expression in CRC, with particular reference to sex-related differences, in tumors from a large, prospective, population-based cohort.</p> <p>Methods</p> <p>Using tissue microarrays and immunohistochemistry, the fraction and intensity of cyclin D1 expression was evaluated in 527 incident CRC cases from the Malmö Diet and Cancer Study. The χ²and Spearman's rho (ρ) tests were used for comparison of cyclin D1 expression and relevant clinicopathological characteristics. Kaplan-Meier analysis and Cox proportional hazards modeling were used to assess the effect of cyclin D1 expression on cancer-specific survival (CSS) in univariate and multivariate analysis, adjusted for established prognostic factors.</p> <p>Results</p> <p>Cyclin D1 intensity was significantly lower in male compared with female CRC (<it>P </it>= 0.018). In the full cohort, cyclin D1 expression was associated with a significantly prolonged CSS (hazard ratio (HR) = 0.69; 95% CI 0.49 to 0.96, <it>P </it>= 0.026) but subgroup analysis according to gender revealed a strongly accentuated prognostic effect of cyclin D1 in male CRC (HR = 0.48; 95% CI 0.31 to 0.74, <it>P </it>< 0.001), which was in contrast to female CRC, where cyclin D1 was not prognostic (HR = 1.05; 95% CI 0.62 to 1.78, <it>P </it>= 0.864) (<it>P</it>_interaction= 0.024). The prognostic value of cyclin D1 was not retained in multivariate analysis, either in the full cohort or in male CRC.</p> <p>Conclusions</p> <p>Cyclin D1 expression is strongly associated with prolonged survival in male CRC. These findings not only support an important role for cyclin D1 in colorectal carcinogenesis, but also add further weight to the accumulating evidence that CRC is indeed a hormone-dependent malignancy, for which prognostic and treatment-predictive molecular biomarkers should be evaluated differently in women and men.</p