Yes-Associated Protein (YAP) and Transcriptional Co-activator with PDZ-binding Motif (TAZ) have both emerged as important drivers of cancer progression and metastasis. YAP and TAZ are often upregulated or nuclear localized in aggressive human cancers. There is abundant experimental evidence demonstrating that YAP or TAZ activation promotes cancer formation, tumor progression, and metastasis. In this review we summarize the evidence linking YAP/TAZ activation to metastasis, and discuss the roles of YAP and TAZ during each step of the metastatic cascade. Collectively, this evidence strongly suggests that inappropriate YAP or TAZ activity plays a causal role in cancer, and that targeting aberrant YAP/TAZ activation is a promising strategy for the treatment of metastatic disease. To this end, we also discuss several potential strategies for inhibiting YAP/TAZ activation in cancer and the challenges each strategy poses.

Early sorting endosomes are responsible for the trafficking and function of transferrin receptor (TfR) and EGFR. These receptors play important roles in iron uptake and signaling and are critical for breast cancer development. However, the role of morphology, receptor composition, and signaling of early endosomes in breast cancer remains poorly understood. A novel population of enlarged early endosomes was identified in breast cancer cells and tumor xenografts but not in noncancerous MCF10A cells. Quantitative analysis of endosomal morphology, cargo sorting, EGFR activation, and Rab GTPase regulation was performed using superresolution and confocal microscopy followed by 3D rendering. MDA-MB-231 breast cancer cells have fewer, but larger EEA1positive early endosomes compared with MCF10A cells. Live-cell imaging indicated dysregulated cargo sorting, because EGF and Tf traffic together via enlarged endosomes in MDA-MB-231, but not in MCF10A. Large EEA1-positive MDA-MB-231 endosomes exhibited prolonged and increased EGF-induced activation of EGFR upon phosphorylation at tyrosine-1068 (EGFR-p1068). Rab4A overexpression in MCF10A cells produced EEA1-positive enlarged endosomes that displayed prolonged and amplified EGF-induced EGFR-p1068 activation. Knockdown of Rab4A lead to increased endosomal size in MCF10A, but not in MDA-MB-231 cells. Nevertheless, Rab4A knockdown resulted in enhanced EGF-induced activation of EGFR-p1068 in MDA-MB-231 as well as downstream signaling in MCF10A cells. Altogether, this extensive characterization of early endosomes in breast cancer cells has identified a Rab4-modulated enlarged early endosomal compartment as the site of prolonged and increased EGFR activation.Implications: Enlarged early endosomes play a Rab4-modulated role in regulation of EGFR activation in breast cancer cells.

Aim:To assess plasma zinc and copper concentration in individuals with autism and correlate these levels with symptom severity.Subjects and methods:Plasma from 102 autistic individuals, and 18 neurotypical controls, were tested for plasma zinc and copper using inductively-coupled plasma-mass spectrometry. Copper and zinc levels and Cu/Zn were analyzed for possible correlation with severity of 19 symptoms.Results:Autistic individuals had elevated plasma levels of copper and Cu/Zn and lower, but not significantly lower, plasma Zn compared to neurotypical controls. There was a correlation between Cu/Zn and expressive language, receptive language, focus attention, hyperactivity, fine motor skills, gross motor skills and Tip Toeing. There was a negative correlation between plasma zinc concentration and hyperactivity, and fine motor skills severity.Discussion:These results suggest an association between plasma Cu/Zn and severity of symptoms associated with autism.

In the current study, we demonstrate that integrin α3β1 promotes invasive and metastatic traits of triple-negative breast cancer (TNBC) cells through induction of the transcription factor, Brain-2 (Brn-2). We show that RNAi-mediated suppression of α3β1 in MDA-MB-231 cells caused reduced expression of Brn-2 mRNA and protein and reduced activity of the BRN2 gene promoter. In addition, RNAi-targeting of Brn-2 in MDA-MB-231 cells decreased invasion in vitro and lung colonization in vivo, and exogenous Brn-2 expression partially restored invasion to cells in which α3β1 was suppressed. α3β1 promoted phosphorylation of Akt in MDA-MB-231 cells, and treatment of these cells with a pharmacological Akt inhibitor (MK-2206) reduced both Brn-2 expression and cell invasion, indicating that α3β1-Akt signaling contributes to Brn-2 induction. Analysis of RNAseq data from patients with invasive breast carcinoma revealed that high BRN2 expression correlates with poor survival. Moreover, high BRN2 expression positively correlates with high ITGA3 expression in basal-like breast cancer, which is consistent with our experimental findings that α3β1 induces Brn-2 in TNBC cells. Together, our study demonstrates a pro-invasive/pro-metastatic role for Brn-2 in breast cancer cells and identifies a role for integrin α3β1 in regulating Brn-2 expression, thereby revealing a novel mechanism of integrin-dependent breast cancer cell invasion.

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<p>Figure S1. Comparison of colocalization analysis coefficients, to support the colocalization approach and use of Pearson's coefficient as detailed in Supplementary Methods section entitled Colocalization analysis. Figure S2. Human mammary epithelial cells (HMEC) were internalized with AF555-Tf (shown as red) at 50 μg/mL continuously for 1 h, fixed and subjected to immunostaining with anti-EEA1 (shown as green). Figure S3. MCF10A, MDAMB231 and T47D cells were internalized with fluorescently labeled Tf for 1 h at 37oC to identify early and recycling endocytic pathway. Figure S4. Rab5-mRFP expression in MDAMB231 results in heterogenous EEA1 staining, not unlike the endogenous EEA1. Figure S5. dSTORM microscopy analysis of Tf and EEA1 endocytic compartments. Figure S6. Whole-cell morphometric quantification of early endosomes using 3D rendering by Imaris Cell Modules. Figure S7. Cells grown on glass-bottom dishes and pre-cleared with imaging medium for 30 minutes were washed, fixed, permeabilized, immunostained with anti-TfR and anti-EEA1, and labeled with DAPI. Figure S8. Immunoblotting analysis of TfR, LDLR and EGFR in whole-cell lysates of MCF10A, MDAMB231 and T47D. Figure S9. Additional time-point specific data corresponding to Figure 3D-E. Figure S10. (A-D) MCF10A and MDAMB231 cells were stimulated with unlabeled EGF for 0 or 5 min and chased for different periods of time. Figure S11. Example of quantitative analysis and visualization assays of EGF-induced EGFR-p1068 activation according to endosomal size. Figure S12. (A) EGF and DAPI merged panels associated with Figure 4D-E. Figure S13. RT-qPCR of Rab4A and Rab4B mRNA in MCF10A and MDAMB231 cells. Figure S14. MCF10A (A-D) and MDAMB231 (E-G) cells were subjected to shRNA Rab4A (shRab4A-KD) or empty vector (EV) with GFP reporter lentiviral infection. Figure S15. MCF10A over-expressing empty vector (EV) or Rab4A-myc constructs were stimulated with unlabeled EGF for 0 or 5 min, and either fixed immediately or chased for different periods of time. Figure S16. (A) EGF and DAPI merged panels associated with Figure 6B-C. Figure S17. Alterations in Rab4 expression can result in enlargement of early endosomes (EEs). Table S1. Solutions and Kits Table S2. Plasmids Table S3. List of primary and secondary antibodies Table S4. Statistical analysis for the quantification of Tf, EEA1 and LC3 colocalization from Figure 1E. Table S5. Statistical analysis (T-test, two-tail: p-value) corresponding to Figure 1G-1 (Rab5 and Rab7), Figure5AC (Rab4 and Rab11). Table S6. Statistical analysis (T-test, two-tailed) of vesicle diameter from super-resolution STORM imaging of Figure 2A, panel q. Table S7. Statistical analysis for the quantification of 3D whole-cell endocytic morphology in Figure 2B-F and Figure S7. Table S8. Statistical analysis for colocalization of EGF vs. EEA1 in Figure 3B. Table S9. Statistical analysis for Tf recycling assay in Figure 3C. t-test, two-tailed. Table S10. Statistical analysis (T-test, two-tail: p-value) corresponding to FigureS8A and S8C. Table S11. Statistical analysis for Figure 3D, for each ligand population with EGF 3D objects (Figure 3D-E and Figure S9A), LDL 3D objects (Figure S9B) and Tf 3D objects (Figure S9C-D). Table S12. Statistical analysis of quantitative immunoblotting analysis from Figure 4B-D. Table S13. Statistical analysis (t-test two-tail: p-value) of data in Figure 4E(panel i-iii, and iv). Table S14. Statistical analysis of endocytic volume data in Figure 5F and Figure 5H. Table S15. Statistical analysis of colocalization analysis in Figure 5G and Figure 5I. Table S16. Statistical analysis of endocytic volume data in Figure 5M and Figure 5O. Table S17. Statistical analysis of quantitative immunoblotting analysis in Figure 6A and Figure S15. Table S18. Statistical analysis of quantitative immunoblotting analysis from Figure 6 and Figure S14. Table S19. Statistical analysis of data in Figure 6B-C.</p>

<p>Video S2. Corresponds to Figure 2A. Created for visualization of MDAMB231 enlarged endosome with EEA1 (red) and Tf (green).</p>

<p>Video S2. Corresponds to Figure 2A. Created for visualization of MDAMB231 enlarged endosome with EEA1 (red) and Tf (green).</p>