Kourtidis, Antonis

Inflammatory breast cancer is a highly aggressive form of breast cancer that forms clusters of tumor emboli in dermal lymphatics and readily metastasizes. These cancers express high levels of E-cadherin, the major mediator of adherens junctions, which enhances formation of tumor emboli. Previous studies suggest that E-cadherin promotes cancer when the balance between apical and basolateral cadherin complexes is disrupted. Here, we used immunohistochemistry of inflammatory breast cancer patient samples and analysis of cell lines to determine the expression of PLEKHA7, an apical adherens junction protein. We used viral transduction to re-express PLEKHA7 in inflammatory breast cancer cells and examined their aggressiveness in 2D and 3D cultures and in vivo. We determined that PLEKHA7 was deregulated in inflammatory breast cancer, demonstrating improper localization or lost expression in most patient samples and very low expression in cell lines. Re-expressing PLEKHA7 suppressed proliferation, anchorage independent growth, spheroid viability, and tumor growth in vivo. The data indicate that PLEKHA7 is frequently deregulated and acts to suppress inflammatory breast cancer. The data also promote the need for future inquiry into the imbalance between apical and basolateral cadherin complexes as driving forces in inflammatory breast cancer.

Abstract Background: Inflammatory breast cancer is a highly aggressive form of breast cancer that robustly forms clusters of tumor emboli in dermal lymphatics and readily metastasizes. Inflammatory breast cancers express high levels of E-cadherin, the major protein of adherens junctions, which may enhance the ability of tumor cells to form such clusters and contribute to metastasis. Seemingly contradictory, E-cadherin has both tumor-suppressing and tumor-promoting roles in cancer; previous studies suggest that this depends on the balance between apical and basolateral cadherin-catenin complexes. Methods: In the present study, we use immunohistochemistry of inflammatory breast cancer patient samples and biochemical analysis of cell lines to determine the expression of PLEKHA7, an apical adherens junction protein. We use viral transduction to ectopically express PLEKHA7 in the SUM149 inflammatory breast cancer cell line. The effect of PLEKHA7 on the aggressiveness of inflammatory breast cancer in 2D, 3D and in-vivo were examined. Results: We determined that PLEKHA7 was deregulated in inflammatory breast cancer, demonstrating improper localization or lost expression in a strong majority of patient samples and very low expression in cell line models. We found that re-expressing PLEKHA7 is sufficient to suppress proliferation, anchorage independent growth, spheroid viability, and tumor growth in-vivo. We also observed a negative-selection pressure within the xenograft tumors to lose PLEKHA7 function or expression.Conclusions: The data indicate that PLEKHA7 is frequently deregulated and acts as a suppressor of inflammatory breast cancer. They also suggest that the resulting imbalance between apical and basolateral cadherin-catenin complexes contributes to growth, survival and emboli-forming capacities of inflammatory breast cancer.

Cumulative evidence demonstrates that most RNAs exhibit specific subcellular distribution. However, the mechanisms regulating this phenomenon and its functional consequences are still under investigation. Here, we reveal that cadherin complexes at the apical zonula adherens (ZA) of epithelial adherens junctions recruit the core components of the RNA-induced silencing complex (RISC) Ago2, GW182, and PABPC1, as well as a set of 522 messenger RNAs (mRNAs) and 28 mature microRNAs (miRNAs or miRs), via PLEKHA7. Top canonical pathways represented by these mRNAs include Wnt/β-catenin, TGF-β, and stem cell signaling. We specifically demonstrate the presence and silencing of MYC, JUN, and SOX2 mRNAs by miR-24 and miR-200c at the ZA. PLEKHA7 knockdown dissociates RISC from the ZA, decreases loading of the ZA-associated mRNAs and miRNAs to Ago2, and results in a corresponding increase of MYC, JUN, and SOX2 protein expression. The present work reveals a mechanism that directly links junction integrity to the silencing of a set of mRNAs that critically affect epithelial homeostasis.

In the liver, the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) regulates bile secretion and other functions at the apical membrane of biliary epithelial cells (i.e., cholangiocytes). CF‐related liver disease is a major cause of death in patients with CF. CFTR dysfunction affects innate immune pathways, generating a para‐inflammatory status in the liver and other epithelia. This study investigates the mechanisms linking CFTR to toll‐like receptor 4 activity. We found that CFTR is associated with a multiprotein complex at the apical membrane of normal mouse cholangiocytes, with proteins that negatively control Rous sarcoma oncogene cellular homolog (Src) activity. In CFTR‐defective cholangiocytes, Src tyrosine kinase self‐activates and phosphorylates toll‐like receptor 4, resulting in activation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells and increased proinflammatory cytokine production in response to endotoxins. This Src/nuclear factor kappa‐light‐chain‐enhancer of activated B cells‐dependent inflammatory process attracts inflammatory cells but also generates changes in the apical junctional complex and loss of epithelial barrier function. Inhibition of Src decreased the inflammatory response of CF cholangiocytes to lipopolysaccharide, rescued the junctional defect in vitro, and significantly attenuated endotoxin‐induced biliary damage and inflammation in vivo (Cftr knockout mice). Conclusion: These findings reveal a novel function of CFTR as a regulator of toll‐like receptor 4 responses and cell polarity in biliary epithelial cells; this mechanism is pathogenetic, as shown by the protective effects of Src inhibition in vivo, and may be a novel therapeutic target in CF‐related liver disease and other inflammatory cholangiopathies. (Hepatology 2016;64:2118‐2134).

Abstract 15 - 30 % of all breast cancer cases are classified as HER2/neu-positive, a very aggressive form of breast cancer that correlates with a poor prognosis, reduced survival time and increased incidence of metastasis. Kourtidis et al. (2010) have recently shown that the HER2-positive breast cancer cell line BT474 relies on a unique Warburg-like metabolism for survival and aggressive behavior. These cells are dependent on fatty acid synthesis, presumably as a means to regenerate NAD+ for aerobic glycolysis. The nuclear receptors NR1D1 and PPARγ enable these cells to upregulate de novo fatty acid synthesis while avoiding palmitate-induced lipotoxicity by storing neutral fats (TAGs). The cells show markedly increased levels of stored fats compared to other breast cancer cell lines and disruption of the synthetic process results in apoptosis of BT474 cells. Metabolomic analysis of NR1D1 overexpressing cells compared to normal cells reveals profound differences in their metabolite profiles. This metabolic phenotype is operating at its limits in HER2-positive cells, as addition of exogenous palmitate results in cell death, whereas HER2-normal cells are not affected by this treatment. Microarray analysis suggests that palmitate induces an ER-stress response in the HER2-positive cells, ultimately resulting in cell death. Preliminary results suggest that this response is CHOP-dependent. Combination treatments with palmitate and HER2-targeted therapeutic compounds show synergistic effects and markedly increase the cytotoxic effects of either compound alone. The uptake of exogenous fatty acids as well as the de novo fatty acid synthesis are dependent on the essential co-factor coenzyme A (CoA), which is generated from the precursor pantothenate (vitamin B5). Based on our genomic analysis we predicted that HER2-positive breast cancer cells are more sensitive to changes in intracellular CoA levels. Our results show that HER2-positive breast cancer cells are sensitive to shRNA-mediated knockdown of coenzyme A pathway enzymes whereas MCF-7 cells are not. We observe the same pattern of sensitivity using the competitive CoA pathway inhibitor homopantothenate. Interestingly, limiting the supply of CoA in HER2-positive cells reduces the toxicity of exogenous palmitate. Our data suggest that HER2-positive breast cancer cells are metabolically different from other breast cancer cell lines and that these differences offer new possibilities for therapeutic applications. Citation Format: Jan M. Baumann, Antonis Kourtidis, Douglas S. Conklin. The lipogenic phenotype of HER2/neu-positive breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1894. doi:10.1158/1538-7445.AM2013-1894

Vascular endothelial growth factor (VEGF) and Ang1 (Angiopoietin-1) have opposing effects on vascular permeability, but the molecular basis of these effects is not fully known. We report in this paper that VEGF and Ang1 regulate endothelial cell (EC) junctions by determining the localization of the RhoA-specific guanine nucleotide exchange factor Syx. Syx was recruited to junctions by members of the Crumbs polarity complex and promoted junction integrity by activating Diaphanous. VEGF caused translocation of Syx from cell junctions, promoting junction disassembly, whereas Ang1 maintained Syx at the junctions, inducing junction stabilization. The VEGF-induced translocation of Syx from EC junctions was caused by PKD1 (protein kinase D1)-mediated phosphorylation of Syx at Ser806, which reduced Syx association to its junctional anchors. In support of the pivotal role of Syx in regulating EC junctions, syx−/− mice had defective junctions, resulting in vascular leakiness, edema, and impaired heart function.