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.

Soluble heat-shock protein 27 in blood serum is a non-invasive prognostic biomarker for ovarian cancer.

OBJECTIVES: Ovarian cancer (OC) is the leading cause of death in gynecological oncology, primarily caused by limited prognostic and therapeutic options. The heat shock protein 27 (HSP27) is recognized as a prominent factor in OC, playing a pivotal role in cancer progression machinery such as treatment resistance. Thus, HSP27 may represent an appropriate biomarker for OC diagnosis, prognosis, and therapy response. MATERIALS & METHODS: Extracellular HSP27 levels were measured by enzyme-linked immunosorbent assay (ELISA) in serum samples of OC patients (n = 242) and compared to a non-malignant control group without any history of cancer (n = 200). Correlations between serum levels of HSP27 and clinical pathological parameters were analyzed by bivariate analysis. Survival analyses were carried out by Kaplan-Meier test. RESULTS: This study demonstrated that protein levels of HSP27 are comparable in the blood serum of healthy women and OC patients. However, HSP27 levels are significantly correlated with the volume of ascites, residual tumor mass, and age at first diagnosis in OC patients. Notably, elevated levels of HSP27 demonstrate significantly higher overall survival. CONCLUSION: Taken together, our findings demonstrate that high levels of circulating HSP27 in serum are associated with improved overall survival of OC patients. Even though functionality of secreted HSP27 is still unclear, serum levels of HSP27 represent a putative non-invasive prognostic biomarker candidate for OC progression.

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.

Functionality of the Tumor Suppressor microRNA-1 in Malignant Tissue and Cell Line Cells of Uterine Leiomyosarcoma.

BACKGROUND/AIM: Uterine leiomyosarcoma (uLMS) is a very rare mesenchymal tumor showing an aggressive clinical course and poor prognosis for patients. Due to the low incidence, little is known about molecular tumor biology and biomarkers of uLMS. Micro-RNA-1 (miR-1) has been identified as a pivotal tumor suppressor in numerous entities being suited as a molecular marker for tumor progression. MATERIALS AND METHODS: uLMS patient samples were analyzed regarding their miR-1 expression levels. Furthermore, miR-1 growth inhibitory and target regulatory properties were examined in transfected uLMS cells SK-UT-1. RESULTS: miR-1 was strongly suppressed in uLMS tumor tissue compared to adjacent healthy tissue. In vitro studies, however, failed to detect growth inhibitory properties of miR-1 in SK-UT-1 cells. The expression of the cell survival and MAP kinases Erk-1/2 and p38 was not targeted by miR-1. CONCLUSION: Tumor suppressive mechanisms of miR-1, seem to be inhibited in uLMS SK-UT-1 cells, maybe as part of the malignant transformation process. Regardless of the microRNA's cellular functionality, miR-1 may represent a promising biomarker of diagnosis in uLMS therapy.

Cold Atmospheric Plasma (CAP) and CAP-Stimulated Cell Culture Media Suppress Ovarian Cancer Cell Growth - A Putative Treatment Option in Ovarian Cancer Therapy.

BACKGROUND/AIM: Ovarian cancer (OC) is a gynecologic tumor with poor prognosis. Despite radical cytoreductive surgery and platinum-based adjuvant systemic treatment, OC will relapse in the majority of the cases. Thus, cold atmospheric plasma (CAP), a highly reactive physical state bearing diverse biological activities being suited for anticancer therapy, may be a promising option in OC therapy. MATERIALS AND METHODS: OC cell lines were exposed either directly to the CAP or to cell culture medium previously exposed to CAP. Cell proliferation and cell motility was measured. RESULTS: The data demonstrated, that even a single application of a short-term CAP treatment led to an attenuation of OC cell growth and motility. Moreover, incubation with CAP-treated cell culture medium gave similar effects. Results were consistent in four OC cell lines. CONCLUSION: In summary, the CAP application in oncological surgery leads to strong anti-proliferative effects and opens up novel opportunities for the OC treatment.

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.

The Tumor Suppressor MicroRNA-1 Exhibits Restricted Inhibition of Proliferation of Ovarian Cancer Cells.

BACKGROUND: MicroRNAs are able to control vital tumor biological processes, such as proliferation, tissue transformation and cell migration, as well as apoptosis. One of the micro RNAs, namely miR-1, has been classified as a tumor suppressor, however, preliminary data did not confirm this finding in ovarian cancer (OC) cells. This study examined the impact of miR-1 on OC cell growth. MATERIALS AND METHODS: Recombinant miR-1 was overexpressed in human OC cell lines OVCAR-3, SK-OV-3, TOV-112D, and TOV-21G. Subsequently, cell growth was analyzed. RESULTS: After transfection, 11- to 487-fold overexpression of miR-1 was detectable in the OC cells. However, no significant differences in proliferation compared to control cells were detected, neither in transiently nor in stably transfected cells. CONCLUSION: In numerous cancer entities miR-1 is defined as an antiproliferative tumor suppressor. Notably, the present study demonstrated a loss of growth-inhibitory functionality of miR-1 by so far unknown mechanisms, suggesting dysregulated miR-1 signaling or effector cascades in OC cells.

Drug-induced Modulation of Heat Shock Protein HSPB1 in an Ovarian Cancer Cell Model.

BACKGROUND: Heat-shock protein HSPB1 (alternative name HSP27) plays a pivotal role in cell survival pathways, apoptosis, metastasis and has been frequently linked to treatment resistance in ovarian cancer (OC) and other malignancies. Characteristic HSPB1 induction in different solid tumors is often caused by cytotoxic agents. MATERIALS AND METHODS: An in vitro OC cell model system was established to characterize resistance mechanisms during chemotherapy. Human OC cell lines OVCAR-3, SK-OV-3 and TOV-21G were treated with paclitaxel or carboplatin. Cellular growth was analyzed by cell counting. Intra- and extracellular HSPB1 concentrations were assessed by western blot and enzyme-linked immunosorbent assays. RESULTS: Incubation with paclitaxel, and with carboplatin significantly reduced cell growth without a definitive increase of intracellular HSPB1 expression. HSPB1 demonstrated drug-inducible secretion into the extracellular compartment. CONCLUSION: Despite its current lack of analysis in patient samples, serum soluble HSPB1 may function as a specific biomarker for monitoring response to chemotherapy in patients with OC.