Estrogen receptor-? signaling and localization regulates autophagy and unfolded protein response activation in ER+ breast cancer

Antiestrogen therapy is commonly used to treat estrogen receptor (ER)+ breast cancers but acquired and de novo resistance limits their overall curative potential. An endoplasmic reticulum stress pathway, the unfolded protein response, and autophagy are both implicated in the development of antiestrogen therapy resistance in estrogen receptor-? (ER) positive breast cancer. Thus, we recently investigated how ER? can regulate autophagy and the unfolded protein response (Cook et al., FASEBJ, 2014). We showed that inhibiting ER? signaling stimulates autophagosome formation and flux. Moreover, we showed that ER? knockdown inhibited the unfolded protein response (UPR) signaling components. Here we support and extend this recent report showing additional data on ER? localization and provide a schematic of the overall signaling implicated by our results. Differential activation of UPR and autophagy highlight the pivotal role of ER? in regulating pro-survival signaling in breast cancer through UPR and autophagy. Furthermore, these data suggest new approaches to successful targeting ER? and preventing the regulation of key pro-survival signaling that confers resistance to endocrine therapies.&nbsp

Improved Test Planning and Analysis Through the Use of Advanced Statistical Methods

The goal of this work is, through computational simulations, to provide statistically-based evidence to convince the testing community that a distributed testing approach is superior to a clustered testing approach for most situations. For clustered testing, numerous, repeated test points are acquired at a limited number of test conditions. For distributed testing, only one or a few test points are requested at many different conditions. The statistical techniques of Analysis of Variance (ANOVA), Design of Experiments (DOE) and Response Surface Methods (RSM) are applied to enable distributed test planning, data analysis and test augmentation. The D-Optimal class of DOE is used to plan an optimally efficient single- and multi-factor test. The resulting simulated test data are analyzed via ANOVA and a parametric model is constructed using RSM. Finally, ANOVA can be used to plan a second round of testing to augment the existing data set with new data points. The use of these techniques is demonstrated through several illustrative examples. To date, many thousands of comparisons have been performed and the results strongly support the conclusion that the distributed testing approach outperforms the clustered testing approach

Mitochondria directly donate their membrane to form autophagosomes during a novel mechanism of parkin-associated mitophagy

BACKGROUND: Autophagy (macroautophagy), a cellular process of “self-eating”, segregates damaged/aged organelles into vesicles, fuses with lysosomes, and enables recycling of the digested materials. The precise origin(s) of the autophagosome membrane is unclear and remains a critical but unanswered question. Endoplasmic reticulum, mitochondria, Golgi complex, and the plasma membrane have been proposed as the source of autophagosomal membranes. FINDINGS: Using electron microscopy, immunogold labeling techniques, confocal microscopy, and flow cytometry we show that mitochondria can directly donate their membrane material to form autophagosomes. We expand upon earlier studies to show that mitochondria donate their membranes to form autophagosomes during basal and drug-induced autophagy. Moreover, electron microscopy and immunogold labeling studies show the first physical evidence of mitochondria forming continuous structures with LC3-labeled autophagosomes. The mitochondria forming these structures also stain positive for parkin, indicating that these mitochondrial-formed autophagosomes represent a novel mechanism of parkin-associated mitophagy. CONCLUSIONS: With the on-going debate regarding autophagosomal membrane origin, this report demonstrates that mitochondria can donate membrane materials to form autophagosomes. These structures may also represent a novel form of mitophagy where the mitochondria contribute to the formation of autophagosomes. This novel form of parkin-associated mitophagy may be a more efficient bio-energetic process compared with de novo biosynthesis of a new membrane, particularly if the membrane is obtained, at least partly, from the organelle being targeted for later degradation in the mature autolysosome

T cell repertoire to citrullinated self-peptides in healthy humans is not confined to the HLA-DR SE alleles; targeting of citrullinated self-peptides presented by HLA-DP4 for tumour therapy

Post-translational modifications are induced in stressed cells which cause them to be recognised by the immune system. One such modification is citrullination where the positive charged arginine is modified to a neutral citrulline. We demonstrate most healthy donors show an oligoclonal CD4 response in vitro to at least one citrullinated vimentin or enolase peptide. Unlike rheumatoid arthritis patients, these T cell responses were not restricted by HLA-DRB1 shared epitope (SE) alleles, suggesting they could be presented by other MHC-II alleles. As HLA-DP is less polymorphic than HLA-DR, we investigated whether the common allele, HLA-DP4 could present citrullinated epitopes. The modification of arginine to citrulline enhanced binding of the peptides to HLA-DP4 and induced high frequency CD4 responses in HLA-DP4 transgenic mouse models. Our previous studies have shown that tumours present citrullinated peptides restricted through HLA-DR4 which are good targets for anti-tumour immunity. In this study we show that citrullinated vimentin and enolase peptides also induced strong anti-tumour immunity (100% survival,

Citrullinated α-enolase is an effective target for anti-cancer immunity

Targeting post-translationally modified epitopes may provide a new strategy for generating tumor specific immune responses. Citrullination is the post-translational modification of arginine to citrulline catalyzed by peptidylarginine deaminase (PAD) enzymes. Presentation of citrullinated peptides on MHC-II has been associated with autophagy. Tumors upregulate autophagy and present citrullinated peptides in response to stresses including nutrient deprivation, oxygen deprivation, redox stress and DNA damage, making them good targets for immune attack. The ubiquitous glycolytic enzyme α-enolase (ENO1) is often citrullinated and degraded during autophagy. Immunization of mice with two citrullinated ENO1 peptides (ENO1 241-260cit253 or 11-25cit15) induced strong Th1 responses that recognize the post-translationally modified, but not the wild type unmodified epitope. ENO1 11-25cit15 induced tumor therapy of melanoma cells in C57Bl/6 (B16F1 50% survival p = 0.0026) and ENO1 241-260cit253 in HLA-DR4 transgenic mice (B16-DR4 50% survival p = 0.0048). In addition, ENO1 241-260cit253 induced therapy of pancreatic (Pan02-DR4 50% survival p = 0.0076) and lung (LLC/2-DR4 40% survival p = 0.0142) tumors in HLA-DR4 transgenic mice. The unmodified epitope induced no anti-tumor response. Minimal regression of class II negative B16 or LLC/2 tumor was seen, confirming direct recognition of MHC-II was required. Most tumors only express MHC-II in the presence of IFNγ; an IFNγ inducible model showed strong responses, with rejection of tumors in up to 90% of animals (p = 0.0001). In humans, a repertoire to ENO1 241-260cit253 was observed in healthy donors. This response was CD4 mediated and seen in people with a variety of HLA types suggesting a broad application for this vaccine in human cancer therapy

Citrullinated vimentin presented on MHC-II in tumor cells is a target for CD4+ T-cell-mediated antitumor immunity

Stressful conditions in the harsh tumor microenvironment induce autophagy in cancer cells as a mechanism to promote their survival. However, autophagy also causes post-translational modification of proteins that are recognized by the immune system. In particular, modified self-antigens can trigger CD4(+) T-cell responses that might be exploited to boost antitumor immune defenses. In this study, we investigated the ability of CD4 cells to target tumor-specific self-antigens modified by citrullination, which converts arginine residues in proteins to citrulline. Focusing on the intermediate filament protein vimentin, which is frequently citrullinated in cells during epithelial-to-mesenchymal transition of metastasizing epithelial tumors, we generated citrullinated vimentin peptides for immunization experiments in mice. Immunization with these peptides induced IFN?- and granzyme B-secreting CD4 T cells in response to autophagic tumor targets. Remarkably, a single immunization with modified peptide, up to 14 days after tumor implant, resulted in long-term survival in 60% to 90% of animals with no associated toxicity. This antitumor response was dependent on CD4 cells and not CD8(+) T cells. These results show how CD4 cells can mediate potent antitumor responses against modified self-epitopes presented on tumor cells, and they illustrate for the first time how the citrullinated peptides may offer especially attractive vaccine targets for cancer therapy

Establishment of wMel Wolbachia in Aedes aegypti mosquitoes and reduction of local dengue transmission in Cairns and surrounding locations in northern Queensland, Australia.

Background: The wMel strain of Wolbachia has been successfully introduced into Aedes aegypti mosquitoes and subsequently shown in laboratory studies to reduce transmission of a range of viruses including dengue, Zika, chikungunya, yellow fever, and Mayaro viruses that cause human disease. Here we report the entomological and epidemiological outcomes of staged deployment of Wolbachia across nearly all significant dengue transmission risk areas in Australia. Methods: The  wMel strain of  Wolbachia was backcrossed into the local  Aedes aegypti genotype (Cairns and Townsville backgrounds) and mosquitoes were released in the field by staff or via community assisted methods. Mosquito monitoring was undertaken and mosquitoes were screened for the presence of  Wolbachia. Dengue case notifications were used to track dengue incidence in each location before and after releases. Results: Empirical analyses of the Wolbachia mosquito releases, including data on the density, frequency and duration of Wolbachia mosquito releases, indicate that Wolbachia can be readily established in local mosquito populations, using a variety of deployment options and over short release durations (mean release period 11 weeks, range 2-22 weeks). Importantly, Wolbachia frequencies have remained stable in mosquito populations since releases for up to 8 years. Analysis of dengue case notifications data demonstrates near-elimination of local dengue transmission for the past five years in locations where Wolbachia has been established. The regression model estimate of Wolbachia intervention effect from interrupted time series analyses of case notifications data prior to and after releases, indicated a 96% reduction in dengue incidence in Wolbachia treated populations (95% confidence interval: 84 - 99%). Conclusion: Deployment of the wMel strain of Wolbachia into local Ae. aegypti populations across the Australian regional cities of Cairns and most smaller regional communities with a past history of dengue has resulted in the reduction of local dengue transmission across all deployment areas

Autophagy and unfolded protein response (UPR) regulate mammary gland involution by restraining apoptosis-driven irreversible changes

The postnatal mammary gland undergoes repeated cycles of proliferation and cell death, most notably when the fully differentiated (lactating) gland dedifferentiates to a prelactation state. Accumulation of milk proteins in the secretory epithelium creates the stress signal that triggers this process (involution). How this stress is perceived, and the cellular processes that are subsequently activated, remain unclear. We now report that Unfolded Protein Response (UPR), autophagy, and apoptosis related genes cluster separately during lactation and involution in the mouse mammary gland. Time-course experiments in rodents show that autophagy and UPR signaling are tightly co-regulated at the transition from reversible to irreversible involution. Inhibition of autophagy by chloroquine or genetic deletion of one ATG7 allele enhanced progression of mammary involution into the irreversible phase, as characterized by an early/precocious induction of apoptosis. These are the first preclinical in vivo data in support of a clinical trial testing an autophagy inhibitor for prevention of intraductal breast malignancy progression to invasive breast cancer. In marked contrast, stimulation of autophagy by low dose tunicamycin treatment reduced apoptosis and extended the reversible phase of involution by sustaining the secretory epithelium. Autophagy stimulators could be used short-term to promote lactation in women experiencing difficulties or irregularities in nursing. Taken together, these data indicate that UPR and autophagy play a key role in regulating the balance between cell survival and apoptosis during normal mammary gland regression.</p

Evaluation of Two Methods to Estimate and Monitor Bird Populations

Background: Effective management depends upon accurately estimating trends in abundance of bird populations over time, and in some cases estimating abundance. Two population estimation methods, double observer (DO) and double sampling (DS), have been advocated for avian population studies and the relative merits and short-comings of these methods remain an area of debate. Methodology/Principal Findings: We used simulations to evaluate the performances of these two population estimation methods under a range of realistic scenarios. For three hypothetical populations with different levels of clustering, we generated DO and DS population size estimates for a range of detection probabilities and survey proportions. Population estimates for both methods were centered on the true population size for all levels of population clustering and survey proportions when detection probabilities were greater than 20%. The DO method underestimated the population at detection probabilities less than 30 % whereas the DS method remained essentially unbiased. The coverage probability of 95 % confidence intervals for population estimates was slightly less than the nominal level for the DS method but was substantially below the nominal level for the DO method at high detection probabilities. Differences in observer detection probabilities did not affect the accuracy and precision of population estimates of the DO method. Population estimates for the DS method remained unbiased as the proportion of units intensively surveyed changed, but the variance of th

A review of research on the intersection between breast cancer and cardiovascular research in the Women’s Health Initiative (WHI)

Both obesity and metabolic syndrome are linked to increased incidence of type 2 diabetes, cardiovascular disease (CVD), and cancers of the breast (post-menopausal), and other obesity-related cancers. Over the past 50 years, the worldwide prevalence of obesity and metabolic syndrome has increased, with a concomitant higher incidence of associated co-morbidities and mortality. The precise mechanism linking metabolic syndrome to increased cancer incidence is incompletely understood, however, individual components of metabolic syndrome have been linked to increased breast cancer incidence and worse survival. There is a bidirectional relationship between the risk of CVD and cancer due to a high burden of shared risk factors and higher rates of CVD among cancer survivors, which may be impacted by the pro-inflammatory microenvironment associated with metabolic syndrome and cancer-directed therapies. The Women’s Health Initiative (WHI) is an excellent resource to study a dual relationship between cancer and CVD (cardio-oncology) with extensive information on risk factors and long-term outcomes. The purpose of this review is to provide an overview of research on cardio-oncology conducted utilizing WHI data with focus on studies evaluating both breast cancer and CVD including shared risk factors and outcomes after cancer. The review also includes results on other obesity related cancers which were included in the analyses of breast cancer, articles looking at cancer after heart disease (reverse cardio-oncology) and the role of Clonal Hematopoiesis of Indeterminate Potential (CHIP) as a shared risk factor between CVD and cancer. A summary of pertinent WHI literature helps to delineate the direction of future research evaluating the relationship between CVD and other cancer sites, and provides information on the opportunity for other novel analyses within the WHI