Proliferative diabetic retinopathy (PDR) is a sight-threatening diabetic complication in urgent need of new therapies. In this study we identify potential molecular mechanisms and target candidates ...in the pathogenesis of PDR fibrovascular tissue formation. We performed mRNA sequencing of RNA isolated from eleven excised fibrovascular membranes of type 1 diabetic PDR patients and two non-diabetic patients with rhegmatogenous retinal detachment with proliferative vitreoretinopathy. We determined differentially expressed genes between these groups and performed pathway and gene ontology term enrichment analyses to identify potential underlying mechanisms, pathways, and regulators. Multiple pro-angiogenic processes, including VEGFA-dependent and -independent pathways, as well as processes related to lymphatic development, epithelial to mesenchymal transition (EMT), wound healing, inflammation, fibrosis, and extracellular matrix (ECM) composition, were overrepresented in PDR. Overrepresentation of different angiogenic processes may help to explain the transient nature of the benefits that many patients receive from current intravitreal anti-angiogenic therapies, highlighting the importance of combinatorial treatments. Enrichment of genes and pathways related to lymphatic development indicates that targeting lymphatic involvement in PDR progression could have therapeutic relevance. Together with overrepresentation of EMT and fibrosis as well as differential ECM composition, these findings demonstrate the complexity of PDR fibrovascular tissue formation and provide avenues for the development of novel treatments.
Cancer cells balance with the equilibrium of cell death and growth to expand and metastasize. The activity of mammalian sterile20-like kinases (MST1/2) has been linked to apoptosis and tumor ...suppression via YAP/Hippo pathway-independent and -dependent mechanisms. Using a kinase substrate screen, we identified here MST1 and MST2 among the top substrates for fibroblast growth factor receptor 4 (FGFR4). In COS-1 cells, MST1 was phosphorylated at Y433 residue in an FGFR4 kinase activity-dependent manner, as assessed by mass spectrometry. Blockade of this phosphorylation by Y433F mutation induced MST1 activation, as indicated by increased threonine phosphorylation of MST1/2, and the downstream substrate MOB1, in FGFR4-overexpressing T47D and MDA-MB-231 breast cancer cells. Importantly, the specific knockdown or short-term inhibition of FGFR4 in endogenous models of human HER2
breast cancer cells likewise led to increased MST1/2 activation, in conjunction with enhanced MST1 nuclear localization and generation of N-terminal cleaved and autophosphorylated MST1. Unexpectedly, MST2 was also essential for this MST1/N activation and coincident apoptosis induction, although these two kinases, as well as YAP, were differentially regulated in the breast cancer models analyzed. Moreover, pharmacological FGFR4 inhibition specifically sensitized the HER2
MDA-MB-453 breast cancer cells, not only to HER2/EGFR and AKT/mTOR inhibitors, but also to clinically relevant apoptosis modulators. In TCGA cohort, FGFR4 overexpression correlated with abysmal HER2
breast carcinoma patient outcome. Therefore, our results uncover a clinically relevant, targetable mechanism of FGFR4 oncogenic activity via suppression of the stress-associated MST1/2-induced apoptosis machinery in tumor cells with prominent HER/ERBB and FGFR4 signaling-driven proliferation.
Initial triggers for diabetic retinopathy (DR) are hyperglycemia-induced oxidative stress and advanced glycation end-products. The most pathological structural changes occur in retinal ...microvasculature, but the overall development of DR is multifactorial, with a complex interplay of microvascular, neurodegenerative, genetic/epigenetic, immunological, and secondary inflammation-related factors. Although several individual factors and pathways have been associated with retinopathy, a systems level understanding of the disease is lacking. To address this, we performed mass spectrometry based label-free quantitative proteomics analysis of 138 vitreous humor samples from patients with nonproliferative DR or the more severe proliferative form of the disease. Additionally, we analyzed samples from anti-VEGF (vascular endothelial growth factor) (bevacizumab)-treated patients from both groups. In our study, we identified 2482 and quantified the abundancy of 1351 vitreous proteins. Of these, the abundancy of 230 proteins was significantly higher in proliferative retinopathy compared with nonproliferative retinopathy. This specific subset of proteins was linked to inflammation, complement, and coagulation cascade proteins, protease inhibitors, apolipoproteins, immunoglobulins, and cellular adhesion molecules, reflecting the multifactorial nature of the disease. The identification of the key molecules of the disease is critical for the development of new therapeutic molecules and for the new use of existing drugs.
Erythropoietin producing hepatocellular (Eph) receptors and their membrane-bound ligands ephrins are variably expressed in epithelial cancers, with context-dependent implications to both ...tumor-promoting and -suppressive processes in ways that remain incompletely understood. Using ovarian cancer tissue microarrays and longitudinally collected patient cells, we show here that ephrinA5/EFNA5 is specifically overexpressed in the most aggressive high-grade serous carcinoma (HGSC) subtype, and increased in the HGSC cells upon disease progression. Among all the eight ephrin genes, high EFNA5 expression was most strongly associated with poor overall survival in HGSC patients from multiple independent datasets. In contrast, high EFNA3 predicted improved overall and progression-free survival in The Cancer Genome Atlas HGSC dataset, as expected for a canonical inducer of tumor-suppressive Eph receptor tyrosine kinase signaling. While depletion of either EFNA5 or the more extensively studied, canonically acting EFNA1 in HGSC cells increased the oncogenic EphA2-S897 phosphorylation, EFNA5 depletion left unaltered, or even increased the ligand-dependent EphA2-Y588 phosphorylation. Moreover, treatment with recombinant ephrinA5 led to limited EphA2 tyrosine phosphorylation, internalization and degradation compared to ephrinA1. Altogether, our results suggest a unique function for ephrinA5 in Eph-ephrin signaling and highlight the clinical potential of ephrinA5 as a cell surface biomarker in the most aggressive HGSCs.
Diabetic retinopathy (DR) is the most common diabetic microvascular complication and major cause of blindness in working-age adults. According to the level of microvascular degeneration and ischemic ...damage, DR is classified into non-proliferative DR (NPDR), and end-stage, proliferative DR (PDR). Despite advances in the disease etiology and pathogenesis, molecular understanding of end-stage PDR, characterized by ischemia- and inflammation-associated neovascularization and fibrosis, remains incomplete due to the limited availability of ideal clinical samples and experimental research models. Since a great portion of patients do not benefit from current treatments, improved therapies are essential. DR is known to be a complex and multifactorial disease featuring the interplay of microvascular, neurodegenerative, metabolic, genetic/epigenetic, immunological, and inflammation-related factors. Particularly, deeper knowledge on the mechanisms and pathophysiology of most advanced PDR is critical. Lymphatic-like vessel formation coupled with abnormal endothelial differentiation and progenitor cell involvement in the neovascularization associated with PDR are novel recent findings which hold potential for improved DR treatment. Understanding the underlying mechanisms of PDR pathogenesis is therefore crucial. To this goal, multidisciplinary approaches and new ex vivo models have been developed for a more comprehensive molecular, cellular and tissue-level understanding of the disease. This is the first step to gain the needed information on how PDR can be better evaluated, stratified, and treated.
Changes in EphA2 signaling can affect cancer cell-cell communication and motility through effects on actomyosin contractility. However, the underlying cell-surface interactions and molecular ...mechanisms of how EphA2 mediates these effects have remained unclear. We demonstrate here that EphA2 and membrane-anchored membrane type-1 matrix metalloproteinase (MT1-MMP) were selectively up-regulated and coexpressed in invasive breast carcinoma cells, where, upon physical interaction in same cell-surface complexes, MT1-MMP cleaved EphA2 at its Fibronectin type-III domain 1. This cleavage, coupled with EphA2-dependent Src activation, triggered intracellular EphA2 translocation, as well as an increase in RhoA activity and cell junction disassembly, which suggests an overall repulsive effect between cells. Consistent with this, cleavage-prone EphA2-D359I mutant shifted breast carcinoma cell invasion from collective to rounded single-cell invasion within collagen and in vivo. Up-regulated MT1-MMP also codistributed with intracellular EphA2 in invasive cells within human breast carcinomas. These results reveal a new proteolytic regulatory mechanism of cell-cell signaling in cancer invasion.
Lymphatic invasion and accumulation of continuous collagen bundles around tumor cells are associated with poor melanoma prognosis, but the underlying mechanisms and molecular determinants have ...remained unclear. We show here that a copy-number gain or overexpression of the membrane-type matrix metalloproteinase MMP16 (MT3-MMP) is associated with poor clinical outcome, collagen bundle assembly around tumor cell nests, and lymphatic invasion. In cultured WM852 melanoma cells derived from human melanoma metastasis, silencing of MMP16 resulted in cell-surface accumulation of the MMP16 substrate MMP14 (MT1-MMP) as well as L1CAM cell adhesion molecule, identified here as a novel MMP16 substrate. When limiting the activities of these trans-membrane protein substrates toward pericellular collagen degradation, cell junction disassembly, and blood endothelial transmigration, MMP16 supported nodular-type growth of adhesive collagen-surrounded melanoma cell nests, coincidentally steering cell collectives into lymphatic vessels. These results uncover a novel mechanism in melanoma pathogenesis, whereby restricted collagen infiltration and limited mesenchymal invasion are unexpectedly associated with the properties of the most aggressive tumors, revealing MMP16 as a putative indicator of adverse melanoma prognosis.
Basal-like breast carcinomas, characterized by unfavorable prognosis and frequent metastases, are associated with epithelial-to-mesenchymal transition. During this process, cancer cells undergo ...cytoskeletal reorganization and up-regulate membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP14), which functions in actin-based pseudopods to drive invasion by extracellular matrix degradation. However, the mechanisms that couple matrix proteolysis to the actin cytoskeleton in cell invasion have remained unclear. On the basis of a yeast two-hybrid screen for the MT1-MMP cytoplasmic tail-binding proteins, we identify here a novel Src-regulated protein interaction between the dynamic cytoskeletal scaffold protein palladin and MT1-MMP. These proteins were coexpressed in invasive human basal-like breast carcinomas and corresponding cell lines, where they were associated in the same matrix contacting and degrading membrane complexes. The silencing and overexpression of the 90-kDa palladin isoform revealed the functional importance of the interaction with MT1-MMP in pericellular matrix degradation and mesenchymal tumor cell invasion, whereas in MT1-MMP-negative cells, palladin overexpression was insufficient for invasion. Moreover, this invasion was inhibited in a dominant-negative manner by an immunoglobulin domain-containing palladin fragment lacking the dynamic scaffold and Src-binding domains. These results identify a novel protein interaction that links matrix degradation to cytoskeletal dynamics and migration signaling in mesenchymal cell invasion.
Reticulons (RTNs) are a large family of membrane associated proteins with various functions. NOGO-A/RTN4A has a well-known function in limiting neurite outgrowth and restricting the plasticity of the ...mammalian central nervous system. On the other hand, Reticulon 4 proteins were shown to be involved in forming and maintaining endoplasmic reticulum (ER) tubules. Using comparative transcriptome analysis and qPCR, we show here that NOGO-B/RTN4B and NOGO-A/RTN4A are simultaneously expressed in cultured epithelial, fibroblast and neuronal cells. Electron tomography combined with immunolabelling reveal that both isoforms localize preferably to curved membranes on ER tubules and sheet edges. Morphological analysis of cells with manipulated levels of NOGO-B/RTN4B revealed that it is required for maintenance of normal ER shape; over-expression changes the sheet/tubule balance strongly towards tubules and causes the deformation of the cell shape while depletion of the protein induces formation of large peripheral ER sheets.
PDF
