Even though aging and cellular senescence appear to be linked, the biological mechanisms interconnecting these two processes remain to be unravelled. Therefore, microRNA (miRNA/miR) profiles were analyzed ex vivo by means of gene array in fibroblasts isolated from young and old human donors. Expression of several miRNAs was positively correlated with donor age. Among them, miR-23a-3p was shown to target hyaluronan synthase 2 (HAS2). HA is a polysaccharide of the extracellular matrix that critically regulates the phenotype of fibroblasts. Indeed, both aged and senescent fibroblasts showed increased miR-23a-3p expression and secreted significantly lower amounts of HA compared with young and non-senescent fibroblasts. Ectopic overexpression of miR-23a-3p in non-senescent fibroblasts led to decreased HAS2-mediated HA synthesis, upregulation of senescence-associated markers, and decreased proliferation. In addition, siRNA-mediated downregulation of HAS2 and pharmacological inhibition of HA synthesis by 4-methylumbelliferone mimicked the effects of miR-23a-3p. In vivo, miR-23a-3p was upregulated and HAS2 was downregulated in the skin of old mice compared with young mice. Inhibition of HA synthesis by 4-methylumbelliferone in mice reduced dermal hydration and viscoelasticity, thereby mimicking an aged skin phenotype. Taken together, these findings appear to link miR-23a-3p and the HA microenvironment as effector mechanisms in both dermal aging and senescence.

While radiotherapy is a mainstay for cancer therapy, pneumonitis and fibrosis constitute dose-limiting side effects of thorax and whole body irradiation. So far, the contribution of immune cells to disease progression is largely unknown. Here we studied the role of ecto-5'-nucelotidase (CD73)/adenosine-induced changes in the myeloid compartment in radiation-induced lung fibrosis. C57BL/6 wild-type or CD73 mice received a single dose of whole thorax irradiation (WTI, 15 Gy). Myeloid cells were characterized in flow cytometric, histologic, and immunohistochemical analyses as well as RNA analyses. WTI induced a pronounced reduction of alveolar macrophages in both strains that recovered within 6 wk. Fibrosis development in wild-type mice was associated with a time-dependent deposition of hyaluronic acid (HA) and increased expression of markers for alternative activation on alveolar macrophages. These include the antiinflammatory macrophage mannose receptor and arginase-1. Further, macrophages accumulated in organized clusters and expressed profibrotic mediators at ≥25 wk after irradiation (fibrotic phase). Irradiated CD73 mice showed an altered regulation of components of the HA system and no clusters of alternatively activated macrophages. We speculate that accumulation of alternatively activated macrophages in organized clusters represents the origins of fibrotic foci after WTI and is promoted by a cross-talk between HA, CD73/adenosine signaling, and other profibrotic mediators.-De Leve, S., Wirsdörfer, F., Cappuccini, F., Schütze, A., Meyer, A. V., Röck, K., Thompson, L. F., Fischer, J. W., Stuschke, M., Jendrossek, V. Loss of CD73 prevents accumulation of alternatively activated macrophages and the formation of prefibrotic macrophage clusters in irradiated lungs.

Biomarkers for prognosis in radiotherapy-treated breast cancer patients are urgently needed and important to stratify patients for adjuvant therapies. Recently, a role of the receptor of hyaluronan-mediated motility (RHAMM) has been suggested for tumor progression. Our aim was (i) to investigate the prognostic value of RHAMM in breast cancer and (ii) to unravel its potential function in the radiosusceptibility of breast cancer cells. We demonstrate that RHAMM mRNA expression in breast cancer biopsies is inversely correlated with tumor grade and overall survival. Radiosusceptibility in vitro was evaluated by sub-G1 analysis (apoptosis) and determination of the proliferation rate. The potential role of RHAMM was addressed by short interfering RNAs against RHAMM and its splice variants. High expression of RHAMMv1/v2 in p53 wild type cells (MCF-7) induced cellular apoptosis in response to ionizing radiation. In comparison, in p53 mutated cells (MDA-MB-231) RHAMMv1/v2 was expressed sparsely resulting in resistance towards irradiation induced apoptosis. Proliferation capacity was not altered by ionizing radiation in both cell lines. Importantly, pharmacological inhibition of the major ligand of RHAMM, hyaluronan, sensitized both cell lines towards radiation induced cell death. Based on the present data, we conclude that the detection of RHAMM splice variants in correlation with the p53 mutation status could help to predict the susceptibility of breast cancer cells to radiotherapy. Additionally, our studies raise the possibility that the response to radiotherapy in selected cohorts may be improved by pharmaceutical strategies against RHAMM and its ligand hyaluronan.

Mit drei Urteilen hat der BFH in jüngerer Zeit die Anforderungen an eine organisatorische Eingliederung im Rahmen einer umsatzsteuerlichen Organschaft präzisiert (BFH, Urt. v. 3.4.2008 -V R 76/05, BStBl. II 2008, 905 = UR 2008 BFH, Urt. v. 14.2.2008 -V R 12/06, V R 13/06, BFH/NV 2008, 1365 BFH, Urt. v. 5.12.2007 -V R 26/06, BStBl. II 2008, 451 = UR 2008. Da zwei der Urteile im Bundessteuerblatt veröffentlicht wurden, sind sie von der Finanzverwaltung über den entschiedenen Einzelfall hinaus anzuwenden. Aus den Leitsätzen dieser Urteile muss auf eine deutlich stärkere Ausprägung der organisatorischen Eingliederung im Rahmen der Organschaftsvoraussetzungen geschlossen werden. Dies hat zu vielen Unsi-cherheiten in den betroffenen Unternehmen geführt. Fraglich ist, ob deren bisherige Einflussnahme auf die Willensbildung der Organgesellschaft auch künftig von der Finanzverwaltung als ausreichend anerkannt wird. Dies gilt umso mehr, als eine rückwirkende ¾nderung der Beziehungen zwischen Organträger und Organgesellschaft nicht möglich ist. Das Risiko einer nachträglichen Aberkennung der Organschaft führt zu erheblichen Umsatzsteuer-Nachforderungen und Nachzahlungszinsen. Dieser Beitrag soll die sich aus der BFH-Rechtsprechung ergebenden Anforderungen kritisch analysieren und deren Konsequenzen für die praktische Umsetzung darstellen. I. Rechtliche VorgabenOrganschaft liegt vor, wenn eine juristische Person nach dem Gesamtbild der tatsächlichen Verhältnisse finanziell, wirtschaftlich und organisatorisch in ein anderes Unternehmen eingegliedert ist ( § 2 Abs. 2 Nr. 2 UStG).Es ist nicht erforderlich, dass sich alle drei Merkmale der Eingliederung gleichermaßen deutlich feststellen lassen. Nach dem Gesamtbild der tatsächlichen Verhältnisse kann die gewerbliche oder berufliche Tätigkeit auch nicht selbständig ausgeübt werden, wenn die Eingliederung auf einem der drei Gebiete nicht vollkommen, jedoch in den anderen beiden Gebieten so eindeutig ist, dass sich die Organschaft aus dem Gesamtbild der tatsächlichen Verhältnisse ergibt. Allerdings reicht es nicht aus, dass eine Eingliederung nur in Bezug auf zwei der drei Merkmale besteht. 1 Dies gilt auch für den Fall einer nicht voll ausgeprägten organisatorischen Eingliederung. 2 II. Neue Rechtsprechungsgrundsätze des BFH 1. Vermutung des § 17 Abs. 2 AktG ist nicht ausreichend In den vergangenen Jahren schien sich -zumindest in der Literatur 3 -die überwiegende Meinung herauszubilden, dass die aktienrechtliche Fiktion des § 17 Abs. 2 AktG auch für die umsatzsteuerliche Organschaft von Bedeutung sei. Da von einem in Mehrheitsbesitz stehenden Unternehmen vermutet wird, dass es von dem an ihm mit Mehrheit beteiligten Unternehmen abhängig ist, spräche hierfür die allgemeine Lebenserfahrung. Das beherrschende Unternehmen

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Despite surgery or chemo radiotherapy the five-year survival rate of esophageal carcinoma (eSCC) patients remains around 15% indicating the need for new therapy regimes. Recently the role of the tumor microenvironment has been recognized as an important determinant of radiation responses. Here we investigate the influence of stromal fibroblasts (hF) on radiation induced tumor cell death. To evaluate functional consequences of ionizing radiation hF and eSCC cells were either cultured separately and irradiated (2Gy) or co-cultured after irradiation to investigate radiation dependent tumor-stroma interactions. Subsequently cells were monitored by time-lapse microscopy and cell death was assessed visually as well as by PARP cleavage, live cell staining with Hoechst33342/propidium iodide and quantification of sub-G1 cell cycle. The rate of cell death was barely altered in monocultures. Of note significantly increased cell death of eSCC was observed in co-culture with irradiated hF compared to co-cultures with mock-irradiated fibroblasts (16.10±4.25 fold of control). Microscopic evaluation of co-cultures revealed that hF and cancer cells are connected via tunnelling nanotubes (TNT). Furthermore, TNT formation was found to be dependent on the extracellular matrix component hyaluronan. eSCC cell death was prevented by pharmacological inhibition of TNT formation by cytochalasin B as well as inhibition of hyaluronan by 4-MU. As TNTs are known to mediate intercellular protein or mitochondrial transfer we investigated which stromal proteins are shuttled into eSCC cells by trans-SILAC experiments. For this purpose the proteome of fibroblasts was labeled with heavy amino acids. After co-culturing hF and eSCC were separated by FACS-sorting and labeled stromal proteins were identified in the cancer cells by mass spectrometry. Four consistently shuttled proteins were identified in the cancer cells, which were exclusively transferred after irradiation of both cell lines. Knock-down experiments of these proteins in hF were performed and SHANK1, which is not expressed in eSCC cells, was found to be responsible for the hF induced cell death of eSCC cells in response to radiation. In conclusion, by usage of a novel trans-SILAC approach, we provide evidence that in response to ionizing radiation stromal hF transfer SHANK1 to eSCC cells, thereby inducing cellular death. SHANK1 might therefore serve as potential new pharmacological target to sensitize cancer cells to radiation therapy. Citation Format: Katharina Röck, Alexandra Schütze, Janna Morawitz, Verena Jendrossek, Klaus Pantel, Jens W. Fischer. Deadly fuel: Fibroblasts mediate cancer cell death through tunneling nanotubes in response to ionizing radiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1649.