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Mammary gland development starts during embryogenesis, and the process continues after birth. During development, the mammary gland undergoes massive morphological and physiological alterations including growth, invasion, and branching morphogenesis providing an ideal model for stem cell and cancer biology studies. Great efforts have been made in understanding mammary gland development during puberty and adulthood; however, the process during embryogenesis is still elusive. One reason is that the tools to study tissue dynamics during development are limited, which is partially due to the lack of an ex vivo culture method. Here we describe an updated organ culture protocol of the murine embryonic mammary gland. This powerful tool allows monitoring of growth and branching morphogenesis of mammary gland ex vivo by live imaging. In addition, we introduce a novel method for culturing intact, stroma-free mammary rudiments from late gestation mouse embryos in 3D in Matrigel. This approach can be used to identify the direct stromal cues for branching morphogenesis.

Selective catalytic reduction of nitrogen oxides is an efficient technique for emission abatement in heavy-duty vehicles. Cu-SSZ-13 SCR catalysts are more active than vanadium-based catalysts at low temperatures, but are more sensitive to deactivation by sulfur. Consequently, there is a need to study poisoning by sulfur for this catalyst material. This experimental investigation focuses on the effect of sulfur on the low-temperature performance of Cu-SSZ-13 SCR catalysts. The effect of sulfur exposure temperature, and the influence of the NO2/NOx ratio, are considered and two different regeneration temperatures are compared. In addition, catalyst samples from an engine-aged catalyst are evaluated. The SO2 exposure temperature is shown to have an important impact on the deactivation of the Cu-SSZ-13 catalyst. The lowest sulfur exposure temperature (220 °C) results in the most severe deactivation, while the highest temperature during sulfur exposure (400 °C) results in the lowest degree of deactivation. This was found to be related to the amount of sulfur on the catalyst. Additionally, SO2 exposure was shown to decrease the N2O selectivity. The engine-aged catalyst has a decreased performance in terms of both decreased activity and increased N2O selectivity. For this catalyst, impurities from fuel and engine-oil can play a role in the deactivation. Different deactivation mechanisms are seen for the laband engine-aged catalysts.

Funding Information: Acknowledgements We thank the Core Services and Advanced Technologies at the Cancer Research UK Beatson Institute (C596/A17196 and A31287), and particularly the Biological Services Unit, Histology Service and Molecular Technologies; members of the Sansom and Katajisto laboratories for discussions of the data and manuscript; and BRC Oxford for supplying patient material. O.J.S. and his laboratory members were supported by Cancer Research UK (A28223, A21139, A12481 and A17196). D.J.F. and M.C.H. were supported by the UK Medical Research Council (MR/R017247/1 and MR/J50032X/1, respectively). SpecifiCancer CRUK Grand Challenge (C7932/A29055) is funded by Cancer Research UK and the Mark Foundation for Cancer Research. P.K. and his laboratory members were supported by the Academy of Finland Centre of Excellence MetaStem (266869, 304591 and 320185), the ERC Starting Grant 677809, the Swedish Research Council 2018-03078, the Cancerfonden 190634, the Jane and Aatos Erkko Foundation and the Cancer Foundation Finland. N.P. was supported by the Finnish Cultural Foundation, the Biomedicum Helsinki Foundation, the Orion Research Foundation sr and The Paulo Foundation. P.V.F. was supported by Alzheimer’s Research UK and The Francis Crick Institute. The ARUK UCL Drug Discovery Institute receives its core funding from Alzheimer’s Research UK (520909). The Francis Crick Institute receives its core funding from Cancer Research UK (FC001002), the UK Medical Research Council (FC001002) and the Wellcome Trust (FC001002). Publisher Copyright: © 2021, The Author(s), under exclusive licence to Springer Nature Limited. The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling(1), but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation)(2). Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.

Transforming growth factor‐beta (TGFβ) is a multifunctional cytokine with a well‐established role in mammary gland development and both oncogenic and tumor‐suppressive functions. The extracellular matrix (ECM) indirectly regulates TGFβ activity by acting as a storage compartment of latent‐TGFβ, but how TGFβ is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGFβ signaling through the release of latent‐TGFβ from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFβ signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent‐TGFβ1, while overexpression of hepsin in mammary tumors increased TGFβ signaling. Cell‐free and cell‐based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent‐TGFβ and, importantly, that the ability of hepsin to activate TGFβ signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFβ pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin., TGFβ is released from the ECM compartments of the mammary glands by hepsin mediated proteolytic cleavage of the ECM component fibronectin.

Two different SCR catalysts, V2O5-WO3/TiO2 and Cu-SSZ-13, were exposed to biodiesel exhausts generated by a diesel burner. The effect of phosphorus and sulfur on the SCR performance of these cataly ...

We have investigated how the exhaust gases from a heavy-duty Euro VI engine, powered with biogas impact a vanadium-based selective catalytic reduction (SCR) catalyst in terms of performance. A full Euro VI emission control system was used and the accumulation of catalyst poisons from the combustion was investigated for the up-stream particulate filter as well as the SCR catalyst. The NO x reduction performance in terms of standard, fast and NO 2 -rich SCR was evaluated before and after exposure to exhaust from a biogas-powered engine for 900 h. The SCR catalyst retains a significant part of its activity towards NO x reduction after exposure to biogas exhaust, likely due to capture of catalyst poisons on the up-stream components where the deactivation of the oxidation catalyst is especially profound. At lower temperatures some deactivation of the first part of the SCR catalyst was observed which could be explained by a considerably higher surface V 4 + /V 5 + ratio for this sample compared to the other samples. The higher value indicates that the reoxidation of V 4 + to V 5 + is partially hindered, blocking the redox cycle for parts of the active sites.

The reduction of anthropogenic greenhouse gas emissions is crucial to avoid further warming of the planet. We investigated how effluent gases from a biogas powered Euro VI heavy-duty engine impact the performance of a bimetallic (palladium and platinum) oxidation catalyst. Using synthetic gas mixtures, the oxidation of NO, CO, and CH4 before and after exposure to biogas exhaust for 900 h was studied. The catalyst lost most of its activity for methane oxidation, and the activity loss was most severe for the inlet part of the aged catalyst. Here, a clear sintering of Pt and Pd was observed, and higher concentrations of catalyst poisons such as sulfur and phosphorus were detected. The sintering and poisoning resulted in less available active sites and hence lower activity for methane oxidation.

Invasive cancer growth occurs when epithelial tumor cells individually or collectively detach from the primary tumor and migrate into the adjacent healthy tissue. This process starts a gradual tumor progression that may lead to life-threatening metastatic disease. Hepsin is a type II transmembrane serine protease that is frequently overexpressed in breast, prostate, and other epithelial cancers. It promotes tumor progression through activating growth factors and disrupting cell-cell and cell-basement membrane (BM) junctions. Although hepsin participates in pericellular cancer degradome, the oncogenic upstream mechanisms that activate hepsin in cancer are not well known. We found that oncogenic Ras proteins by activating MAPK pathway signaling alter the ratio of hepsin and HAI-1 to increase the proteolytic activity of hepsin. In 3D epithelial culture models of Ras-driven cancer, depletion of hepsin by shRNA restored desmosomes, BM defects, and glandular morphology but not the apical polarity. Mechanistically, Ras-MAPK pathway affected hepsin/HAI-1 via two distinct downstream pathways: Hepsin protein was stabilized via Ras-induced and heat shock transcription factor-1-mediated stress pathway, whereas HAI-1 was down-regulated by Ras via tumor suppressor LKB1 and PI3K-dependent pathways. Altogether, the results suggest that abnormal activation of Ras-MAPK pathway elevates hepsin activity to execute early steps of breast cancer invasion. Citation Format: Shishir Mani Pant, Topi Tervonen, Denis Belitskin, Johanna Englund, Emmy Verschuren, Panu Kovanen, Juha Klefstrom. Oncogenic Ras signaling requires serine protease hepsin to induce invasive breast cancer phenotype [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-204.

SO2-poisoning results in deactivation of Cu-CHA SCR under standard SCR conditions; however regeneration at 700 °C completely restores the SCR performance. To understand the nature of these effects, Cu-species in the fresh and poisoned catalysts were characterized by in-situ temperature-dependent time-resolved Cu K-edge X-ray absorption spectroscopy using the multivariate curve resolution alternating least squares (MCR-ALS) approach and continuous Cauchy wavelet transforms. The extracted chemically-meaningful reference spectra of Cu-species were analyzed by DFT-assisted XANES calculations. Cu-bisulfates were found as the most energetically favorable poisoned Cu-species. The response of Cu-species to a reducing environment differs in the fresh and SO2-poisoned catalysts. Differences in reducibility are related to the formation of quasi-linear Cu-complexes in the SO2-poisoned catalyst formed during heating in H2/He. Heating in H2/He leads to partial desulfurization of the poisoned catalyst. Cooling in H2/He after heating results in more facile formation of Cu-metal clusters in fresh catalyst than in SO2-poisoned.

The European Network for Breast Development and Cancer (ENBDC), a worldwide network ( http://www.enbdc.org/ ), celebrated its tenth anniversary with a fantastic meeting last March 15–17, 2018 in Weggis with 76 attendees.

Differentiated epithelial structure communicates with individual constituent epithelial cells to suppress their proliferation activity. However, the pathways linking epithelial structure to cessation of the cell proliferation machinery or to unscheduled proliferation in the context of tumorigenesis are not well defined. Here we demonstrate the strong impact of compromised epithelial integrity on normal and oncogenic Myc-driven proliferation in three-dimensional mammary epithelial organoid culture. Systematic silencing of 34 human homologs of Drosophila genes, with previously established functions in control of epithelial integrity, demonstrates a role for human genes of apico-basal polarity, Wnt and Hippo pathways and actin dynamics in regulation of the size, integrity and cell proliferation in organoids. Perturbation of these pathways leads to diverse functional interactions with Myc: manifested as a RhoA-dependent synthetic lethality and Par6-dependent effects on the cell cycle. Furthermore, we show a role for Par6G as a negative regulator of the phosphatidylinositol 3'-kinase/phosphoinositide-dependent protein kinase 1/Akt pathway and epithelial cell proliferation and evidence for frequent inactivation of Par6G gene in epithelial cancers. The findings demonstrate that determinants of epithelial structure regulate the cell proliferation activity via conserved and cancer-relevant regulatory circuitries, which are important for epithelial cell cycle restriction and may provide new targets for therapeutic intervention.

Type II transmembrane serine protease hepsin is overexpressed and redistributed in clinical breast cancer samples, although the HPN gene is not a frequent target of cancer-specific genetic alterations. Here, we sought to identify cancer relevant upstream factors responsible for oncogenic deregulation of hepsin. We explored the effects of ectopic expression of major oncogenes and tumor suppressors on hepsin protein expression levels in non-malignant mammary epithelial cells, finding that HrasV12, KrasV12, ectopic wild-type Kras, endogenous mutant KrasD12 and E2F-1 induce the expression of active form of hepsin. Oncogenic Ras proteins also reduced expression levels of cognate hepsin inhibitor, HAI-1. Concomitantly, oncogenic Ras expression led to disappearance of desmoplakin and desmoglein 2 from desmosomal junctions, which correlated with mislocalization of hepsin from its predominant localization in desmosomes to cytosol. To explore the effects of mutated Ras expressed at endogenous levels we used lung tumors derived from lox-stop-lox (LSL)-KrasD12; p53-/- and LSL-KrasV12 mammary epithelial structures in ex vivo 3D cultures. In both cases, hepsin was highly expressed and predominantly localized to cytosol. Knockdown of hepsin by shRNA rescued HrasV12-induced epithelial integrity defects in mammary epithelial 3D culture and furthermore, MEK and ERK inhibitors prevented HrasV12-induced hepsin deregulation as well as desmosomal and basement membrane defects. Critical pathways downstream of MAPK pathway are being studied and will be discussed. These findings suggest a critical role for hepsin in mediating Ras-MAPK-mediated disruption of epithelial integrity during tumorigenesis. Citation Format: Topi A. Tervonen, Shishir M. Pant, Denis Belitskin, Johanna Englund, Katja Närhi, Emmy Verschuren, Panu Kovanen, Juha Klefström. Ras recruits oncogenic serine protease hepsin to disrupt mammary epithelial integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3484.

Hepsin belongs to a family of cell-surface serine proteases, which have sparked interest as therapeutic targets because of the accessibility of extracellular protease domain for inhibitors. Hepsin is frequently amplified and/or overexpressed in epithelial cancers, but it is not clear how enhanced hepsin expression confers a potential for oncogenicity. We show that hepsin is consistently overexpressed in more than 40% of examined breast cancers, including all major biological subtypes. The effects of doxycycline-induced hepsin overexpression were examined in mammary epithelial organoids, and we found that induced hepsin acutely downmodulates its cognate inhibitor, hepatocyte growth factor (HGF) activator inhibitor type 1 (HAI-1). Hepsin-induced depletion of cellular HAI-1 led to a sharp increase in pericellular serine protease activity. The derepressed hepsin proteolytically activated downstream serine proteases, augmented HGF/MET signalling and caused deterioration of desmosomes and hemidesmosomes; structures important for cell cohesion and cell-basement membrane interaction. Moreover, chronic induction of hepsin considerably shortened the latency of Myc-dependent tumourigenesis in the mouse mammary gland. The serine protease and uPA system inhibitor WX-UK1, identified as a micromolar range hepsin inhibitor, prevented hepsin from augmenting HGF/MET signalling and disrupting desmosomes and hemidesmosomes. The findings suggest that the oncogenic activity of hepsin arises not only from elevated expression level but also from depletion of HAI-1, events which together trigger gain-of-function activity impacting HGF/MET signalling and epithelial cohesion. Thus, hepsin overexpression is a major oncogenic conferrer to a serine protease activity involved in breast cancer dissemination.