Supplemental data files 2 figures including figure S1 related to figure 4 and Figure S2 related to Figure 5 and Figure 6 and detailed supplemental experimental procedures for methods referred in material and method section including RT-PCR analysis and western blot analysis, inmunohistochemistry, co-immunoprecipitation, immunofluorescence and in vitro 3-dimensional proliferation assay and DNA quantification experiments.

Air pollution, especially fine particulate matter, is a major health problem. Several epidemiological studies have demonstrated a significant association between exposure to fine particles and the development of lung and cardiovascular diseases. However, underlying mechanisms of the impact of fine particles on the pathogenesis of lung cancer remain unclear. The aim of this study was to determine whether exposure to fine particles may create a lung microenvironment prone to lung cancer progression. C57BL/6 mice were exposed to diesel exhaust particles (DEP) by intratracheal instillations. After 2 instillations of DEP (200µg/mL), lungs were collected and the immune microenvironment was analyzed by flow cytometry. To evaluate whether the inflammatory microenvironment conditioned by DEP had an impact on tumor progression, Lewis Lung Carcinoma (LLC) tumor cells were orthotopically injected. Cytokines were measured in the bronchoalveolar lavage fluid (BALF) after DEP exposure by cytokine array. Finally, the effect of BALF of naïve or DEP-exposed mice on cancer cells attraction was tested. We demonstrate that lung tumor progression is higher in DEP-instilled mice as compared to control mice. BAL cell counts and FACS analysis have revealed an increase in neutrophils. Moreover, BALF of DEP-instilled mice had a higher chemoattractant effect on tumor cells as compared to control. The expression of 36 cytokines was significantly increased in the BALF of DEP-instilled mice and ongoing analysis will help identifying the key pro-tumor molecules. Exposure to DEP appears to create a supportive lung microenvironment for tumor progression in which recruited neutrophils are probably involved.

The editors are publishing this note to alert readers to concerns about this article (1). In Fig. 2, the actin loading control bands for cyclin D1 and cyclin D2 are identical–the authors clarified that the Western blots for cyclin D1 and cyclin D2 were performed on the same samples, however, this was not indicated in the figure legend. Additionally, in Fig. 6C, the p-EGFR bands in MDA-MB-231 cells showing stimulation by TGFα treatment are identical to the p-EGFR bands showing stimulation by EGF treatment. In the original submission of this manuscript, a correct version of this figure was used to show both TGFα and EGF could stimulate p-EGFR in control vector (CTR)– and MT4-MMP–expressing (MT4) MDA-MB-231 cells, but these panels were mistakenly duplicated in the revised and final versions of the manuscript.

Age-related macular degeneration (AMD) is a worldwide leading cause of blindness affecting individuals over 50 years old.The most aggressive form, wet AMD, is characterized by choroidal neovascularization (CNV) and inflammation involving microglia recruitment.By using a laser-induced CNV mouse model, we provide evidence for a key role played by miR-142-3p during CNV formation.MiR-142-3p was overexpressed in murine CNV lesions and its pharmacological inhibition decreased vascular and microglia densities by 46% and 30%, respectively.Consistently, miR-142-3p overexpression with mimics resulted in an increase of 136% and 126% of blood vessels and microglia recruitment.Interestingly, miR-142-3p expression was linked to the activation state of mouse microglia cells as determined by morphological analysis (cell solidity) through a computational method.In vitro, miR-142-3p overexpression in human microglia cells (HMC3) modulated microglia activation, as shown by CD68 levels.Interestingly, miR142-3p modulation also regulated the production of VEGF-A, the main pro-angiogenic factor.Together, these data strongly support the unprecedented importance of miR-142-3p-dependent vascular-inflammation axis during CNV progression, through microglia activation.

Neovascular age-related macular degeneration (nAMD) is the leading cause of blindness in aging populations. Here, we applied metabolomics to human sera of patients with nAMD during an active (exudative) phase of the pathology and found higher lactate levels and a shift in the lipoprotein profile (increased VLDL-LDL/HDL ratio). Similar metabolomics changes were detected in the sera of mice subjected to laser-induced choroidal neovascularization (CNV). In this experimental model, we provide evidence for two sites of lactate production: first, a local one in the injured eye, and second a systemic site associated with the recruitment of bone marrow-derived inflammatory cells. Mechanistically, lactate promotes the angiogenic response and M2-like macrophage accumulation in the eyes. The therapeutic potential of our findings is demonstrated by the pharmacological control of lactate levels through pyruvate dehydrogenase kinase (PDK) inhibition by dichloroacetic acid (DCA). Mice treated with DCA exhibited normalized lactate levels and lipoprotein profiles, and inhibited CNV formation. Collectively, our findings implicate the key role of the PDK/lactate axis in AMD pathogenesis and reveal that the regulation of PDK activity has potential therapeutic value in this ocular disease. The results indicate that the lipoprotein profile is a traceable pattern that is worth considering for patient follow-up. KEY MESSAGES: Lactate and lipoprotein profile are associated with the active phase of AMD and CNV development. Lactate is a relevant and functional metabolite correlated with AMD progression. Modulating lactate through pyruvate dehydrogenase kinase led to a decrease of CNV progression. Pyruvate dehydrogenase kinase is a new therapeutic target for neovascular AMD.

MT4-MMP (or MMP17) belongs to the Membrane-Type Matrix Metalloproteinase (MT-MMP) family. This family of proteases contributes to extracellular matrix remodeling during several physiological processes, including embryogenesis, organogenesis, tissue regeneration, angiogenesis, wound healing, and inflammation. MT4-MMP (MMP17) presents unique characteristics compared to other members of the family in terms of sequence homology, substrate specificity, and internalization mode, suggesting distinct physiological and pathological functions. While the physiological functions of MT4-MMP are poorly understood, it has been involved in different pathological processes such as arthritis, cardiovascular disease, and cancer progression. The mt4-mmp transcript has been detected in a large diversity of cancers. The contribution of MT4-MMP to tumor development has been further investigated in gastric cancer, colon cancer, head and neck cancer, and more deeply in breast cancer. Given its contribution to different pathologies, particularly cancers, MT4-MMP represents an interesting therapeutic target. In this review, we examine its biological and structural properties, and we propose an overview of its physiological and pathological functions.

Abstract Purpose: Here, we investigated the clinical relevance of an unprecedented combination of three biomarkers in triple-negative breast cancer (TNBC), both in human samples and in patient-derived xenografts of TNBC (PDX-TNBC): EGFR, its recently identified partner (MT4-MMP), and retinoblastoma protein (RB). Experimental Design: IHC analyses were conducted on human and PDX-TNBC samples to evaluate the production of the three biomarkers. The sensitivity of cancer cells expressing or not MT4-MMP to anti-EGFR (erlotinib) or anti-CDK4/6 inhibitor (palbociclib) was evaluated in vitro in 2D and 3D proliferation assays and in vivo using xenografts and PDX-TNBC displaying different RB, MT4-MMP, and EGFR status after single (erlotinib or palbociclib) or combined (erlotinib + palbociclib) treatments. Results: EGFR and MT4-MMP were coexpressed in >70% of TNBC samples and PDX-TNBC, among which approximately 60% maintained RB expression. Notably, approximately 50% of all TNBC and PDX-TNBC expressed the three biomarkers. Single erlotinib and palbociclib treatments drastically reduced the in vitro proliferation of cells expressing EGFR and MT4-MMP when compared with control cells. Both TNBC xenografts and PDX expressing MT4-MMP, EGFR, and RB, but not PDX-TNBC with RB loss, were sensitive to erlotinib and palbociclib with an additive effect of combination therapy. Moreover, this combination was efficient in another PDX-TNBC expressing the three biomarkers and resistant to erlotinib alone. Conclusions: We defined a new association of three biomarkers (MT4-MMP/EGFR/RB) expressed together in 50% of TNBC and demonstrated its usefulness to predict the TNBC response to anti-EGFR and anti-CDK4/6 drugs used in single or combined therapy.

Introduction Triple negative breast cancer (TNBC) comprises heterogeneous agressive tumours. The standard treatment relies entirely on chemotherapy. While EGFR is expressed in 50%–75% of TNBC, clinical trials with erlotinib or cetuximab fail to show clinical benefit. Recently, we demonstrated that EGFR interacts with MT4-MMP, the membrane-type 4 matrix metalloprotease in TNBC. Here, we investigated the clinical significance of the MT4-MMP/EGFR axis and inactivation of protein of retinoblastoma (Rb) in human TNBC samples and patient-derived xenografts (PDX). We tested the efficacy of targeting this pathway in vivo in xenografts and PDX with a distinct expression profile of MT4-MMP, EGFR and Rb. Material and methods Immunohistochemistry (IHC) analysis for MT4-MMP, EGFR, Rb and Ki67 expression were performed on human TNBC samples (n=78) and Patient-Derived Xenograft (PDX-TNBC) (n=38). Single and combined treatments with erlotinib and palbociclib were applied in vitro and in vivo. In vitro 2D and 3D proliferation assays were performed on TNBC cells naturally producing EGFR and transfected or not with MT4-MMP cDNA. For the in vivo study, xenografts and PDX were engrafted with Rag1-/- mice and nude mice, respectively. Mice bearing tumours producing or not MT4-MMP, EGFR and Rb were treated with vehicle, erlotinib (50 mg/kg/day), palbociclib (75 mg/kg/day) or the combination. Results and discussions By IHC analysis we reveal that Rb is maintained functional in 68% of human samples and 59% of PDX-TNBC. Co-expression of MT4-MMP, EGFR and Rb is obtained in 49% of human samples and 43% in PDX-TNBC. In a 2D culture model, response to erlotinib and palbociclib is enhanced in cells expressing both MT4-MMP and EGFR. In 3D culture, their combination is synergistic for the inhibition of proliferation of MT4-MMP and EGFR expressing cells. In vivo, tumours expressing MT4-MMP and EGFR show a better response to erlotinib and palbociclib than tumours negative for MT4-MMP with a striking effect of the combination on MT4-MMP tumours. Moreover, PDX-TNBC expressing only EGFR and Rb show no response to erlotinib, a partial response to palbociclib and a strong response to the combination. Importantly, co-expression of EGFR and MT4-MMP strongly sensitises Rb positive PDX-TNBC to both erlotinib and palbociclib with a striking effect of the combination, whereas PDX-TNBC with Rb loss didn’t respond to any single or combination therapy. Conclusion Our data highlight MT4-MMP, EGFR and Rb as predictive biomarkers for response to erlotinib-palbociclib combination.

Background: Triple-negative breast cancers (TNBC) are heterogeneous cancers with poor prognosis. We aimed to determine the clinical relevance of membrane type-4 matrix metalloproteinase (MT4-MMP), a membrane type matrix metalloproteinase that interacts with epidermal growth factor receptor (EGFR) overexpressed in >50% of TNBC.Methods: We conducted a retrospective immunohistochemical analysis on human TNBC samples (n = 81) and validated our findings in in vitro and in vivo assays.Results: Membrane type-4 matrix metalloproteinase and EGFR are produced in 72.5% of TNBC samples, whereas those proteins are faintly produced by healthy tissues. Unexpectedly, tumour relapse after chemotherapy was reduced in samples highly positive for MT4-MMP. Mechanistically, this is ascribed to a higher sensitivity of MT4-MMP-producing cells to alkylating or intercalating chemotherapeutic agents, as assessed in vitro. In sharp contrast, MT4-MMP expression did not affect tumour cell sensitivity to paclitaxel that interferes with protease trafficking. Importantly, MT4-MMP expression sensitised cancer cells to erlotinib, a tyrosine kinase EGFR inhibitor. In a pre-clinical model, the growth of MT4-MMP overexpressing xenografts, but not of control ones, was reduced by epirubicin or erlotinib. The combination of suboptimal drug doses blocked drastically the growth of MT4-MMP-producing tumours.Conclusions: We demonstrate that MT4-MMP defines a sub-population of TNBC sensitive to a combination of DNA-targeting chemotherapeutic agents and anti-EGFR drugs.

Background This study was to examine the insular cortical functional connectivity in drug naïve patients with first episode schizophrenia and to explore the relationship between the connectivity and the severity of clinical symptoms. Methods Thirty-seven drug naïve patients with schizophrenia and 25 healthy controls were enrolled in this study. A seed-based approach was used to analyze the resting-state functional imaging data. Insular cortical connectivity maps were bilaterally extracted for group comparison and validated by voxel-based morphometry (VBM) analysis. Clinical symptoms were measured using the Positive and Negative Syndrome Scale (PANSS).

Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c.

their association with nucleo-cytoplasmic proteins associated with mRNP granules. Eugénie Ansseau, Jocelyn O. Eidahl, Céline Lancelot, Alexandra Tassin, Christel Matteotti, Cassandre Yip, Jian Liu, Baptiste Leroy, Céline Hubeau, Cécile Gerbaux, Samuel Cloet, Armelle Wauters, Sabrina Zorbo, Pierre Meyer, Isabelle Pirson, Dalila Laoudj-Chenivesse, Ruddy Wattiez, Scott Q. Harper, Alexandra Belayew and Frédérique Coppée. Laboratory of Molecular Biology, Research Institute for Health Sciences and Technology, University of Mons, Mons, Belgium 2 Center for Gene Therapy, Research Institute at Nationwide Children's Hospital, Columbus, OH, USA Laboratory of Proteomic and Microbiology, Research Institute for Biosciences, University of Mons, Mons, Belgium Pediatric Department, CHRU Montpellier, Montpellier, France I.R.I.B.H.M., Free University of Brussels, Belgium Laboratory of Physiology and Experimental Medicine, INSERM U1046, Montpellier, France Department of Pediatrics, Ohio State University College of Medicine, Columbus, OH, USA These authors contributed equally as second authors. & These authors contributed equally as senior authors.

Membrane type 4 matrix metalloproteinase (MT4‐MMP) [matrix metalloproteinase (MMP) 17] is a GPI‐anchored membrane‐type MMP expressed on the cell surface of human breast cancer cells. In triple‐negative breast cancer cells, MT4‐MMP promotes primary tumour growth and lung metastases. Although the trafficking and internalization of the transmembrane membrane type 1 MMP have been extensively investigated, little is known about the regulatory mechanisms of the GPI‐anchored MT4‐MMP. Here, we investigated the fate and cellular trafficking of MT4‐MMP by analysing its homophilic complex interactions, internalization and recycling dynamics as compared with an inert form, MT4‐MMP‐E249A. Oligomeric and dimeric complexes were analysed by cotransfection of cells with FLAG‐tagged or Myc‐tagged MT4‐MMP in reducing and nonreducing immunoblotting and coimmunoprecipitation experiments. The trafficking of MT4‐MMP was studied with an antibody feeding assay and confocal microscopy analysis or cell surface protein biotinylation and western blot analysis. We demonstrate that MT4‐MMP forms homophilic complexes at the cell surface, and internalizes in early endosomes, and that some of the enzyme is either autodegraded or recycled to the cell surface. Our data indicate that MT4‐MMP is internalized by the clathrin‐independent carriers/GPI‐enriched early endosomal compartments pathway, a mechanism that differs from that responsible for the internalization of other membrane‐type MMP members. Although MT4‐MMP localizes with caveolin‐1, MT4‐MMP internalization was not affected by inhibitors of caveolin‐1 or clathrin endocytosis pathways, but was reduced by CDC42 or RhoA silencing with small interfering RNA. We provide a new mechanistic insight into the regulatory mechanisms of MT4‐MMP, which may have implications for the design of novel therapeutic strategies for metastatic breast cancer.

Abstract MT4-MMP (MMP-17) is a glycosylphosphatidyl inositol–anchored matrix metalloprotease expressed on the surface of cancer cells that promotes tumor growth and metastasis. In this report, we identify MT4-MMP as an important driver of cancer cell proliferation through CDK4 activation and retinoblastoma protein inactivation. We also determine a functional link between MT4-MMP and the growth factor receptor EGFR. Mechanistic experiments revealed direct association of MT4-MMP and its positive effects on EGFR phosphorylation in response to TGFα and EGF in cancer cells. Notably, the effects of MT4-MMP on proliferation and EGFR activation did not rely on metalloprotease activity. Clinically, MT4-MMP and EGFR expressions were correlated in human triple-negative breast cancer specimens. Altogether, our results identify MT4-MMP as a positive modifier of EGFR outside-in signaling that acts to cooperatively drive cancer cell proliferation. Cancer Res; 74(23); 6758–70. ©2014 AACR.