αvβ3 and α5β1 integrin recycling pathways dictate downstream Rho kinase signaling to regulate persistent cell migration

Accumulating evidence suggests that integrin recycling regulates cell migration. However, the lack of reagents to selectively target the trafficking of individual heterodimers, as opposed to endocytic transport as a whole, has made it difficult to define the contribution made by particular recycling pathways to directional cell movement. We show that autophosphorylation of protein kinase D1 (PKD1) at Ser916 is necessary for its association with αvβ3 integrin. Expression of PKD1916A or the use of mutants of β3 that do not bind to PKD1 selectively inhibits short-loop, Rab4-dependent recycling of αvβ3, and this suppresses the persistence of fibroblast migration. However, we report that short-loop recycling does not directly contribute to fibroblast migration by moving αvβ3 to the cell front, but by antagonizing α5β1 recycling, which, in turn, influences the cell's decision to migrate with persistence or to move randomly

Tensin links energy metabolism to extracellular matrix assembly.

The regulation of integrin function is key to fundamental cellular processes, including cell migration and extracellular matrix (ECM) assembly. In this issue, Georgiadou et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201609066) report that the metabolic sensor adenosine monophosphate-activated protein kinase influences tensin production to regulate α5β1-integrin and fibrillar adhesion assembly and thus reveal an important connection between energy metabolism and ECM assembly

A proteomic approach to identify endosomal cargoes controlling cancer invasiveness

We have previously shown that Rab17 - a small GTPase associated with epithelial polarity - is specifically suppressed by ERK2 signalling to promote an invasive phenotype. However, the mechanisms through which Rab17 loss permits invasiveness, and the endosomal cargoes that are responsible for mediating this are not known. Using quantitative mass spectrometry-based proteomics, we have found that knockdown of Rab17 leads to highly selective reduction in the cellular levels of a v-SNARE (Vamp8). Moreover, proteomics and immunofluorescence indicate that Vamp-8 is associated with Rab17 at late endosomes. Reduced levels of Vamp8 promote transition between ductal carcinoma in situ (DCIS) and a more invasive phenotype. We developed an unbiased proteomic approach to elucidate the complement of receptors that redistributes between endosomes and the plasma membrane, and have pin-pointed neuropilin-2 (NRP2) as a key pro-invasive cargo of Rab17/Vamp8-regulated trafficking. Indeed, reduced Rab17 or Vamp8 levels lead to increased mobilisation of NRP2-containing late endosomes and upregulated cell surface expression of NRP2. Finally, we show that NRP2 is required for the basement membrane disruption which accompanies transition between DCIS and a more invasive phenotype

The initiator methionine tRNA drives cell migration and invasion leading to increased metastatic potential in melanoma

The cell's repertoire of transfer RNAs (tRNAs) has been linked to cancer. Recently, levels of the initiator methionine tRNA (tRNAiMet) in stromal fibroblasts have been shown to influence extracellular matrix (ECM) secretion to drive tumour growth and angiogenesis. Here we show that increased tRNAiMet within cancer cells does not influence tumour growth, but drives cell migration and invasion via a mechanism that is independent from ECM synthesis and dependent on α5β1 integrin and levels of the translation initiation ternary complex. In vivo and ex vivo migration (but not proliferation) of melanoblasts is significantly enhanced in transgenic mice which express additional copies of the tRNAiMet gene. We show that increased tRNAiMet in melanoma drives migratory, invasive behaviour and metastatic potential without affecting cell proliferation and primary tumour growth, and that expression of RNA polymerase III-associated genes (which drive tRNA expression) are elevated in metastases by comparison with primary tumours. Thus specific alterations to the cancer cell tRNA repertoire drive a migration/invasion programme that may lead to metastasis

Effect of Supplemental Protein and Glucogenic Precursors on Digestibility and Energy Metabolism

A metabolism study was conducted to evaluate the impact of increasing levels of glucogenic precursors on diet digestibility and acetate clearance. Four supplementation strategies containing 0, 30, 40, and 70 g of supplemental glucogenic potential were supplied to a basal diet of bromegrass hay. Addition of glucogenic potential in the form of rumen undegradable protein improved dry matter, organic matter, and acid detergent fiber digestibility efficiency of acetate utilization in growing lambs fed moderate- quality hay. However, no additive effect of supplementing propionate salts and rumen undegradable protein were observed in this study. Th is would suggest that rumen undegradable protein requirements must be met to observe effects from increasing levels of glucogenic potential

Rab-coupling protein coordinates recycling of α5β1 integrin and EGFR1 to promote cell migration in 3D microenvironments

Here we show that blocking the adhesive function of αvβ3 integrin with soluble RGD ligands, such as osteopontin or cilengitide, promoted association of Rab-coupling protein (RCP) with α5β1 integrin and drove RCP-dependent recycling of α5β1 to the plasma membrane and its mobilization to dynamic ruffling protrusions at the cell front. These RCP-driven changes in α5β1 trafficking led to acquisition of rapid/random movement on two-dimensional substrates and to a marked increase in fibronectin-dependent migration of tumor cells into three-dimensional matrices. Recycling of α5β1 integrin did not affect its regulation or ability to form adhesive bonds with substrate fibronectin. Instead, α5β1 controlled the association of EGFR1 with RCP to promote the coordinate recycling of these two receptors. This modified signaling downstream of EGFR1 to increase its autophosphorylation and activation of the proinvasive kinase PKB/Akt. We conclude that RCP provides a scaffold that promotes the physical association and coordinate trafficking of α5β1 and EGFR1 and that this drives migration of tumor cells into three-dimensional matrices

Universal Third Trimester Ultrasonic Screening Using Fetal Macrosomia in the Prediction of Adverse Perinatal Outcome, a Systematic Review and Meta-analysis of Diagnostic Test Accuracy.

Background: The effectiveness of screening for macrosomia is not well established. One of the critical elements of an effective screening program is the diagnostic accuracy of a test at predicting the condition. The objective of this study is to investigate the diagnostic effectiveness of universal ultrasonic fetal biometry in predicting the delivery of a macrosomic infant, shoulder dystocia, and associated neonatal morbidity in low- and mixed-risk populations. Methods and findings: We conducted a predefined literature search in Medline, Excerpta Medica database (EMBASE), the Cochrane library and ClinicalTrials.gov from inception to May 2020. No language restrictions were applied. We included studies where the ultrasound was performed as part of universal screening and those that included low- and mixed-risk pregnancies and excluded studies confined to high risk pregnancies. We used the estimated fetal weight (EFW) (multiple formulas and thresholds) and the abdominal circumference (AC) to define suspected large for gestational age (LGA). Adverse perinatal outcomes included macrosomia (multiple thresholds), shoulder dystocia, and other markers of neonatal morbidity. The risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. Meta-analysis was carried out using the hierarchical summary receiver operating characteristic (ROC) and the bivariate logit-normal (Reitsma) models. We identified 41 studies that met our inclusion criteria involving 112,034 patients in total. These included 11 prospective cohort studies (N = 9986), one randomized controlled trial (RCT) (N = 367), and 29 retrospective cohort studies (N = 101,681). The quality of the studies was variable, and only three studies blinded the ultrasound findings to the clinicians. Both EFW >4,000 g (or 90th centile for the gestational age) and AC >36 cm (or 90th centile) had >50% sensitivity for predicting macrosomia (birthweight above 4,000 g or 90th centile) at birth with positive likelihood ratios (LRs) of 8.74 (95% confidence interval [CI] 6.84–11.17) and 7.56 (95% CI 5.85–9.77), respectively. There was significant heterogeneity at predicting macrosomia, which could reflect the different study designs, the characteristics of the included populations, and differences in the formulas used. An EFW >4,000 g (or 90th centile) had 22% sensitivity at predicting shoulder dystocia with a positive likelihood ratio of 2.12 (95% CI 1.34–3.35). There was insufficient data to analyze other markers of neonatal morbidity. Conclusions: In this study, we found that suspected LGA is strongly predictive of the risk of delivering a large infant in low- and mixed-risk populations. However, it is only weakly (albeit statistically significantly) predictive of the risk of shoulder dystocia. There was insufficient data to analyze other markers of neonatal morbidity

Differential uptake, kinetics and mechanisms of intracellular trafficking of next-generation antisense oligonucleotides across human cancer cell lines.

Antisense oligonucleotides (ASOs) modulate cellular target gene expression through direct binding to complementary RNA. Advances in ASO chemistry have led to the development of phosphorothioate (PS) ASOs with constrained-ethyl modifications (cEt). These next-generation cEt-ASOs can enter cells without transfection reagents. Factors involved in intracellular uptake and trafficking of cEt-ASOs leading to successful target knockdown are highly complex and not yet fully understood. AZD4785 is a potent and selective therapeutic KRAS cEt-ASO currently under clinical development for the treatment of cancer. Therefore, we used this to investigate mechanisms of cEt-ASO trafficking across a panel of cancer cells. We found that the extent of ASO-mediated KRAS mRNA knockdown varied significantly between cells and that this did not correlate with bulk levels of intracellular accumulation. We showed that in cells with good productive uptake, distribution of ASO was perinuclear and in those with poor productive uptake distribution was peripheral. Furthermore, ASO rapidly trafficked to the late endosome/lysosome in poor productive uptake cells compared to those with more robust knockdown. An siRNA screen identified several factors mechanistically involved in productive ASO uptake, including the endosomal GTPase Rab5C. This work provides novel insights into the trafficking of cEt-ASOs and mechanisms that may determine their cellular fate