Impact of Janus Kinase Inhibition with Tofacitinib on Fundamental Processes of Bone Healing

Both inflammatory diseases like rheumatoid arthritis (RA) and anti-inflammatory treatment of RA with glucocorticoids (GCs) or non-steroidal anti-inflammatory drugs (NSAIDs) negatively influence bone metabolism and fracture healing. Janus kinase (JAK) inhibition with tofacitinib has been demonstrated to act as a potent anti-inflammatory therapeutic agent in the treatment of RA, but its impact on the fundamental processes of bone regeneration is currently controversially discussed and at least in part elusive. Therefore, in this study, we aimed to examine the effects of tofacitinib on processes of bone healing focusing on recruitment of human mesenchymal stromal cells (hMSCs) into the inflammatory microenvironment of the fracture gap, chondrogenesis, osteogenesis and osteoclastogenesis. We performed our analyses under conditions of reduced oxygen availability in order to mimic the in vivo situation of the fracture gap most optimal. We demonstrate that tofacitinib dose-dependently promotes the recruitment of hMSCs under hypoxia but inhibits recruitment of hMSCs under normoxia. With regard to the chondrogenic differentiation of hMSCs, we demonstrate that tofacitinib does not inhibit survival at therapeutically relevant doses of 10-100 nM. Moreover, tofacitinib dose-dependently enhances osteogenic differentiation of hMSCs and reduces osteoclast differentiation and activity. We conclude from our data that tofacitinib may influence bone healing by promotion of hMSC recruitment into the hypoxic microenvironment of the fracture gap but does not interfere with the cartilaginous phase of the soft callus phase of fracture healing process. We assume that tofacitinib may promote bone formation and reduce bone resorption, which could in part explain the positive impact of tofacitinib on bone erosions in RA. Thus, we hypothesize that it will be unnecessary to stop this medication in case of fracture and suggest that positive effects on osteoporosis are likely

Expression of the MIR-1 molecule in patients with uterine leiomyosarcoma

Department of Surgery, University Medicine Greifswald, Ferdinand-Sauerbruch-Strasse, 17475 Greifswald, Germany, The 6th International Medical Congress for Students and Young DoctorsBackground: The uterine leiomyosarcoma represents the most frequent malignant gynecologic mesenchymal tumor that often develops distant metastases. The diagnosis of these tumors is nowadays still a challenge and the direct implication of the small non-coding RNAs (MicroRNAs ) in gene expression, tumor initiation and tumor progression has already been revealed in scientific studies. Because the aberrant microRNA (miRNA) expression patterns show a diagnostic value as tumor markers, we aimed to identify the gene expression level of miRNA-1 (miR-1) and the protein targets in uterine leiomyosarcoma. Methods: Using the specific cell line - SK-UT-1 with similar biological characteristics of the uterine leiomyosarcoma tissue, in comparison to ovarian carcinoma cell lines: OVCAR-3, TOV-21 and SK-OV-3, and cell lines of mouse heart-muscle (HL-1), we were able to perform real time PCRs and RNA-Isolation arrays, transient and stabile transfection programs with lipofectamine reagents. Tissue samples of uterine leiomyosarcoma and healthy uterus were again analyzed by means of transfection and isolation arrays. The electrophoresis using protein targets of the miR-1 (p38 and ERK 1/2 widely expressed protein kinase intracellular signaling molecules and involved in functions including the regulation of meiosis, mitosis, und postmitotic functions) was also integrated. Results: The analysis of the SK-UT-1 cell line have shown significant differences in comparison to the other studied cell lines, respectively a reduced expression of the miR-1 molecules. The same results were observed in the process of transfection and electrophoresis of the human tissues, where the lowest expression of the miR-1 was evidenced in the uterine leiomyosarcomas. The specific protein targets of miR-1 have shown positive Western Blot signals. Conclusions: The miR-1 non coding molecules may improve our understanding of disease development, progression and gene expression of the uterine leiomyosarcoma. Further prospective translational studies in order to evaluate miR-1 as a prognostic factor are needed. Key words: MIR-1, leiomyosarcoma, Western Blot

Heat Shock Protein HSP27 Secretion by Ovarian Cancer Cells Is Linked to Intracellular Expression Levels, Occurs Independently of the Endoplasmic Reticulum Pathway and HSP27’s Phosphorylation Status, and Is Mediated by Exosome Liberation

The heat shock protein HSP27 has been correlated in ovarian cancer (OC) patients with aggressiveness and chemoresistance and, therefore, represents a promising potential biomarker for OC diagnosis, prognosis, and treatment response. Notably, secretion of soluble HSP27 has been described by a few cell types and may take place as well in OC cells. Therefore, we studied HSP27 secretion mechanisms under diverse cellular conditions in an OC cell model system. Secretion of HSP27 was characterized after overexpression of HSP27 by transfected plasmids and after heat shock. Intra- and extracellular HSP27 amounts were assessed by Western blotting and ELISA. Protein secretion was blocked by brefeldin A and the impact of the HSP27 phosphorylation status was analyzed overexpressing HSP27 phosphomutants. The present study demonstrated that HSP27 secretion by OVCAR-3 and SK-OV-3 cells depends on intracellular HSP27 concentrations. Moreover, HSP27 secretion is independent of the endoplasmic reticulum secretory pathway and HSP27 phosphorylation. Notably, analysis of OC cell-born exosomes not only confirmed the concentration-dependent correlation of HSP27 expression and secretion but also demonstrated a concentration-dependent incorporation of HSP27 protein into exosomes. Thus, secreted HSP27 may become more important as an extracellular factor which controls the tumor microenvironment and might be a noninvasive biomarker

Expression pattern of the microRNA miR-1 in ovarian cancer cell lines and tumor tissue samples implies a loss of miR-1's tumor suppressor properties: DOI: 10.14800/rd.348

Despite ovarian cancer (OC) represents the leading cause of death in gynecology, however, current understanding of the molecular machinery governing initiation, development and recurrence of the tumor is still limited. Particularly, lack of specific biomarkers defining the complex states of OC limits effective diagnosis and prognosis and, subsequently, hinders appropriate therapy strategies for OC patients. MicroRNA (miR) are small non-coding, regulatory RNA molecules which have critical functions in tumor biology, and which have been functionally grouped into tumor suppressive and tumor promoting miR. miR-1 has been described operating as a tumor suppressor which attenuates proliferation and progression in divers solid cancer entities. Currently, there are no data available concerning miR-1 functionality in progression of OC, particularly, in regard to its potentialas a biomarker for OC diagnosis and treatment. Therefore, the present study examined miR-1 expression levels in established OC cell lines as well as in tissue samples from primary and relapsed OC patients to get a first understanding of putative miR-1 properties in OC progression. Notably, we found miR-1 in OC cell lines was linked to higher cell growth rates. Moreover, analysis of patient samples revealed miR-1 levels in relapsed tumors appeared being up-regulated compared to primary tumors. The findings of our preliminary study (1) may suggest a novel role of miR-1 in OC as a promotor of cancer progression or (2) exhibit a dysregulation of miR-1 functions by a so far unknown disruption of miR-1 regulatory cascades

Impact of Janus Kinase Inhibition with Tofacitinib on Fundamental Processes of Bone Healing

Both inflammatory diseases like rheumatoid arthritis (RA) and anti-inflammatory treatment of RA with glucocorticoids (GCs) or non-steroidal anti-inflammatory drugs (NSAIDs) negatively influence bone metabolism and fracture healing. Janus kinase (JAK) inhibition with tofacitinib has been demonstrated to act as a potent anti-inflammatory therapeutic agent in the treatment of RA, but its impact on the fundamental processes of bone regeneration is currently controversially discussed and at least in part elusive. Therefore, in this study, we aimed to examine the effects of tofacitinib on processes of bone healing focusing on recruitment of human mesenchymal stromal cells (hMSCs) into the inflammatory microenvironment of the fracture gap, chondrogenesis, osteogenesis and osteoclastogenesis. We performed our analyses under conditions of reduced oxygen availability in order to mimic the in vivo situation of the fracture gap most optimal. We demonstrate that tofacitinib dose-dependently promotes the recruitment of hMSCs under hypoxia but inhibits recruitment of hMSCs under normoxia. With regard to the chondrogenic differentiation of hMSCs, we demonstrate that tofacitinib does not inhibit survival at therapeutically relevant doses of 10–100 nM. Moreover, tofacitinib dose-dependently enhances osteogenic differentiation of hMSCs and reduces osteoclast differentiation and activity. We conclude from our data that tofacitinib may influence bone healing by promotion of hMSC recruitment into the hypoxic microenvironment of the fracture gap but does not interfere with the cartilaginous phase of the soft callus phase of fracture healing process. We assume that tofacitinib may promote bone formation and reduce bone resorption, which could in part explain the positive impact of tofacitinib on bone erosions in RA. Thus, we hypothesize that it will be unnecessary to stop this medication in case of fracture and suggest that positive effects on osteoporosis are likely

p53 is functionally inhibited in clear cell renal cell carcinoma (ccRCC): a mechanistic and correlative investigation into genetic and molecular characteristics

Purpose!#!Although p53 is rarely mutated in ccRCC, its overexpression has been linked to poor prognosis. The current study sought to elucidate the unique role of p53 in ccRCC with genomic, proteomic, and functional analyses.!##!Materials and methods!#!Data from the Cancer Genome Atlas (TCGA) were evaluated for genomic and proteomic characteristics of p53; a tissue micro array (TMA) study was carried out to evaluate the association of p53 and phosphorylated p53 (pp53) with clinical outcome. Mechanistic in vitro experiments were performed to confirm a pro-apoptotic loss of p53 in ccRCC and p53 isoforms as well as posttranslational modifications of p53 where assessed to provide possible reasons for a functional inhibition of p53 in ccRCC.!##!Results!#!A low somatic mutation rate of p53 could be confirmed. Although mRNA levels were correlated with poor prognosis and clinicopathological features, there was no monotonous association of mRNA levels with survival outcome. Higher p53 protein levels could be confirmed as poor prognostic features. In vitro, irradiation of ccRCC cell lines markedly induced levels of p53 and of activated (phosphorylated) p53. However, irradiated ccRCC cells demonstrated similar proliferation, migration, and p53 transcriptional activity like non-irradiated controls indicating a functional inhibition of p53. p53 isoforms and could not be correlated with clinical outcome of ccRCC patients.!##!Conclusions!#!p53 is rarely mutated but the wildtype p53 is functionally inhibited in ccRCC. To investigate mechanisms that underlie functional inhibition of p53 may provide attractive therapeutic targets in ccRCC