This file includes supplementary figures S1-S9: Figure S1. Immunofluorescence and western blot results showing differences in CXADR expression between EpH4, EpRas and EpXT cells. Figure S2. Western blots showing how incubatino with an AKT inhibitor, or knockdown of CXADR affects EMT markers in EpRas cells. Figure S3. Immunofluorescence images showing co-localization of CXADR with PHLPP2, PTEN and MAGI-1 in EpH4 cells. Figure S4. Results from Co-IP experiments, protein stability assays, and confuency experiments. Figure S5. Immunofluorescence staining of PHLPP2 and PTEN in the intestinal epithelium of CXADR-deficient mice. Figure S6. Kaplan-Meier curves showing how the expression of CXADR and other junction proteins correlates with overall survival in the GOBO database. Figure S7. Kaplan-Meier curves showing how the expression of MAGI1, PTEN and PHLPP2 correlates with overall survival in the GOBO database. Figure S8. Schematic diagram showing the expression of CXADR in 51 human breast cancer cells. Immunofluorescence and western blot results showing how overexpression of CXADR affects EMT markers and signalosome components in breast and lung cancer cells. Figure S9. Scatter plots showing variability in staining of MAGI-1, PTEN, PHLPP2 and E-cadherin in luminal A and basal tumors.

<p>This supplementary information includes 3 supplementary tables. Table S1. List of antibodies. Table S2. List of plasmids. Table S3. List of human tumor samples.</p>

Multiple viruses are implicated in atherosclerosis, but the mechanisms by which they infect cells and contribute to plaque formation in arterial walls are not well understood. Based on reports showing the presence of enterovirus in atherosclerotic plaques we hypothesized that the coxsackievirus and adenovirus receptor (CXADR/CAR), although absent in normal arteries, could be induced during plaque formation. Large-scale microarray and mass spectrometric analyses revealed significant up-regulation of CXADR messenger RNA and protein levels in plaque-invested carotid arteries compared with control arteries. Macrophages were identified as a previously unknown cellular source of CXADR in human plaques and plaques from Ldr-/-Apob100/100 mice. CXADR was specifically associated with M1-polarized macrophages and foam cells and was experimentally induced during macrophage differentiation. Furthermore, it was significantly correlated with receptors for other viruses linked to atherosclerosis. The results show that CXADR is induced in macrophages during plaque formation, suggesting a mechanism by which enterovirus infect cells in atherosclerotic plaques.

Abstract Tight junctions (TJ) act as hubs for intracellular signaling pathways controlling epithelial cell fate and function. Deregulation of TJ is a hallmark of epithelial–mesenchymal transition (EMT), which contributes to carcinoma progression and metastasis. However, the signaling mechanisms linking TJ to the induction of EMT are not understood. Here, we identify a TJ-based signalosome, which controls AKT signaling and EMT in breast cancer. The coxsackie and adenovirus receptor (CXADR), a TJ protein with an essential yet uncharacterized role in organogenesis and tissue homeostasis, was identified as a key component of the signalosome. CXADR regulated the stability and function of the phosphatases and AKT inhibitors PTEN and PHLPP2. Loss of CXADR led to hyperactivation of AKT and sensitized cells to TGFβ1–induced EMT. Conversely, restoration of CXADR stabilized PHLPP2 and PTEN, inhibited AKT, and promoted epithelial differentiation. Loss of CXADR in luminal A breast cancer correlated with loss of PHLPP2 and PTEN and poor prognosis. These results show that CXADR promotes the formation of an AKT-inhibitory signalosome at TJ and regulates epithelial–mesenchymal plasticity in breast cancer cells. Moreover, loss of CXADR might be used as a prognostic marker in luminal breast cancer. Significance: The tight junction protein CXADR controls epithelial-mesenchymal plasticity in breast cancer by stabilizing the AKT regulators PTEN and PHLPP2.