α5β1 integrin recycling promotes Arp2/3-independent cancer cell invasion via the formin FHOD3

Invasive migration in 3D extracellular matrix (ECM) is crucial to cancer metastasis, yet little is known of the molecular mechanisms that drive reorganization of the cytoskeleton as cancer cells disseminate in vivo. 2D Rac-driven lamellipodial migration is well understood, but how these features apply to 3D migration is not clear. We find that lamellipodia-like protrusions and retrograde actin flow are indeed observed in cells moving in 3D ECM. However, Rab-coupling protein (RCP)-driven endocytic recycling of α5β1 integrin enhances invasive migration of cancer cells into fibronectin-rich 3D ECM, driven by RhoA and filopodial spike-based protrusions, not lamellipodia. Furthermore, we show that actin spike protrusions are Arp2/3-independent. Dynamic actin spike assembly in cells invading in vitro and in vivo is regulated by Formin homology-2 domain containing 3 (FHOD3), which is activated by RhoA/ROCK, establishing a novel mechanism through which the RCP–α5β1 pathway reprograms the actin cytoskeleton to promote invasive migration and local invasion in vivo

Analysis of Chimpanzee History Based on Genome Sequence Alignments

Population geneticists often study small numbers of carefully chosen loci, but it has become possible to obtain orders of magnitude for more data from overlaps of genome sequences. Here, we generate tens of millions of base pairs of multiple sequence alignments from combinations of three western chimpanzees, three central chimpanzees, an eastern chimpanzee, a bonobo, a human, an orangutan, and a macaque. Analysis provides a more precise understanding of demographic history than was previously available. We show that bonobos and common chimpanzees were separated ∼1,290,000 years ago, western and other common chimpanzees ∼510,000 years ago, and eastern and central chimpanzees at least 50,000 years ago. We infer that the central chimpanzee population size increased by at least a factor of 4 since its separation from western chimpanzees, while the western chimpanzee effective population size decreased. Surprisingly, in about one percent of the genome, the genetic relationships between humans, chimpanzees, and bonobos appear to be different from the species relationships. We used PCR-based resequencing to confirm 11 regions where chimpanzees and bonobos are not most closely related. Study of such loci should provide information about the period of time 5–7 million years ago when the ancestors of humans separated from those of the chimpanzees

Clonal replacement and heterogeneity in breast tumors treated with neoadjuvant HER2-targeted therapy.

Genomic changes observed across treatment may result from either clonal evolution or geographically disparate sampling of heterogeneous tumors. Here we use computational modeling based on analysis of fifteen primary breast tumors and find that apparent clonal change between two tumor samples can frequently be explained by pre-treatment heterogeneity, such that at least two regions are necessary to detect treatment-induced clonal shifts. To assess for clonal replacement, we devise a summary statistic based on whole-exome sequencing of a pre-treatment biopsy and multi-region sampling of the post-treatment surgical specimen and apply this measure to five breast tumors treated with neoadjuvant HER2-targeted therapy. Two tumors underwent clonal replacement with treatment, and mathematical modeling indicates these two tumors had resistant subclones prior to treatment and rates of resistance-related genomic changes that were substantially larger than previous estimates. Our results provide a needed framework to incorporate primary tumor heterogeneity in investigating the evolution of resistance

Dynamics of breast-cancer relapse reveal late-recurring ER-positive genomic subgroups.

The rates and routes of lethal systemic spread in breast cancer are poorly understood owing to a lack of molecularly characterized patient cohorts with long-term, detailed follow-up data. Long-term follow-up is especially important for those with oestrogen-receptor (ER)-positive breast cancers, which can recur up to two decades after initial diagnosis1-6. It is therefore essential to identify patients who have a high risk of late relapse7-9. Here we present a statistical framework that models distinct disease stages (locoregional recurrence, distant recurrence, breast-cancer-related death and death from other causes) and competing risks of mortality from breast cancer, while yielding individual risk-of-recurrence predictions. We apply this model to 3,240 patients with breast cancer, including 1,980 for whom molecular data are available, and delineate spatiotemporal patterns of relapse across different categories of molecular information (namely immunohistochemical subtypes; PAM50 subtypes, which are based on gene-expression patterns10,11; and integrative or IntClust subtypes, which are based on patterns of genomic copy-number alterations and gene expression12,13). We identify four late-recurring integrative subtypes, comprising about one quarter (26%) of tumours that are both positive for ER and negative for human epidermal growth factor receptor 2, each with characteristic tumour-driving alterations in genomic copy number and a high risk of recurrence (mean 47-62%) up to 20 years after diagnosis. We also define a subgroup of triple-negative breast cancers in which cancer rarely recurs after five years, and a separate subgroup in which patients remain at risk. Use of the integrative subtypes improves the prediction of late, distant relapse beyond what is possible with clinical covariates (nodal status, tumour size, tumour grade and immunohistochemical subtype). These findings highlight opportunities for improved patient stratification and biomarker-driven clinical trials.Cancer Research UK (CRUK) travel grant (SWAH/047) 282 to visit Prof. Curtis’ Lab. C.R. is supported by award MTM2015-71217-R. Ca.C. is 283 supported by CRUK, ECMC and NIHR. C.C. is supported by the National Institutes 284 of Health through the NIH Director’s Pioneer Award (DP1-CA238296) and the Breast 285 Cancer Research Foundation

Deregulation of Rab and Rab Effector Genes in Bladder Cancer

Growing evidence indicates that Rab GTPases, key regulators of intracellular transport in eukaryotic cells, play an important role in cancer. We analysed the deregulation at the transcriptional level of the genes encoding Rab proteins and Rab-interacting proteins in bladder cancer pathogenesis, distinguishing between the two main progression pathways so far identified in bladder cancer: the Ta pathway characterized by a high frequency of FGFR3 mutation and the carcinoma in situ pathway where no or infrequent FGFR3 mutations have been identified. A systematic literature search identified 61 genes encoding Rab proteins and 223 genes encoding Rab-interacting proteins. Transcriptomic data were obtained for normal urothelium samples and for two independent bladder cancer data sets corresponding to 152 and 75 tumors. Gene deregulation was analysed with the SAM (significant analysis of microarray) test or the binomial test. Overall, 30 genes were down-regulated, and 13 were up-regulated in the tumor samples. Five of these deregulated genes (LEPRE1, MICAL2, RAB23, STXBP1, SYTL1) were specifically deregulated in FGFR3-non-mutated muscle-invasive tumors. No gene encoding a Rab or Rab-interacting protein was found to be specifically deregulated in FGFR3-mutated tumors. Cluster analysis showed that the RAB27 gene cluster (comprising the genes encoding RAB27 and its interacting partners) was deregulated and that this deregulation was associated with both pathways of bladder cancer pathogenesis. Finally, we found that the expression of KIF20A and ZWINT was associated with that of proliferation markers and that the expression of MLPH, MYO5B, RAB11A, RAB11FIP1, RAB20 and SYTL2 was associated with that of urothelial cell differentiation markers. This systematic analysis of Rab and Rab effector gene deregulation in bladder cancer, taking relevant tumor subgroups into account, provides insight into the possible roles of Rab proteins and their effectors in bladder cancer pathogenesis. This approach is applicable to other group of genes and types of cancer

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe