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Does resveratrol exert a potent inhibitory effect on the development of endometriosis by interfering with some pivotal processes?In-vitro cultures of primary endometriotic stromal cells, immortalized endometrial stromal (St-T1b) and endometriotic epithelial (12Z) cells were used to assess the effects of resveratrol on endometrial cell mechanisms. The effects of resveratrol on 12Z and St-T1b cell viability were assessed by MTT assay, apoptosis by FITC Annexin V assay and cleaved caspase-3 levels and cell migration by wound healing assay. The effect of resveratrol on the expression of genes related to cell migration, angiogenesis and cell stemness was evaluated by qRT-PCR.Resveratrol significantly decreased cell viability (P= 0.0065 to P = 0.0180), cell migration (P0.001 to P = 0.0225) and increased the number of apoptotic cells (P = 0.0031 to P = 0.0432) in both cell lines. In cell lines and primary culture, the treatment reduced MMP-2/TIMP-1 (P0.001 to P = 0.0180), VEGF (P = 0.0052 to P = 0.0243) and Ang-1 mRNA (P0.001 to P = 0.0382) expression. Among the stem cell phenotype markers, resveratrol 100 µM increased mRNA expression levels of Notch-1 (P0.001 to P = 0.0018), KLF-4 (P = 0.0011 to P = 0.0137), SOX-2 (P0.001 to P = 0.0070) and TERT (P0.001 to P = 0.0193) in both cell lines and primary cultures. The mRNA expression level of Snail-1 increased in the cell lines (P0.001 to P = 0.0087), whereas OCT-4 mRNA expression increased in St-T1b (P = 0.0396) and primary cultures (P = 0.0148). Vimentin mRNA expression showed a significant upregulation in primary cultures (P0.001). The expression of Msi-1 (P = 0.0145) and NANOG (P = 0.0080) decreased only in St-T1b cells.Resveratrol showed inhibitory effects on cell behaviour related to the development of endometriosis by differentially affecting growth, apoptosis, migration and stem cell phenotype of endometrial and endometriotic cells in vitro.

Purpose MicroRNA-218 (miR-218) is a key regulator of numerous processes relevant to tumor progression. In the present study, we aimed to characterize the relationship between miR-218 and the Epidermal Growth Factor Receptor (EGFR) as well as to understand downstream effects in triple-negative breast cancer (TNBC). Methods We assessed miR-218 and EGFR expression in cell lines and publicly available primary breast cancer gene expression data. We then overexpressed miR-218 in two TNBC cell lines and investigated effects on EGFR and downstream mitogen-activated protein (MAP) kinase signaling. Luciferase reporter assay was used to characterize a direct binding interaction between miR-218 and EGFR mRNA. Digital holographic microscopy helped investigate cell migration and dry mass after miR-218 overexpression. Cell division and invasion were assessed microscopically, while radiation response after miR-218 overexpression alone or combined with additional EGFR knockdown was investigated via clonogenic assays. Results We found an inverse correlation between EGFR expression and miR-218 levels in cell lines and primary breast cancer tissues. MiR-218 overexpression resulted in a downregulation of EGFR via direct binding of the mRNA. Activation of EGFR and downstream p44/42 MAPK signaling were reduced after pre-miR-218 transfection. Cell proliferation, motility and invasiveness were inhibited whereas cell death and mitotic catastrophe were upregulated in miR-218 overexpressing cells compared to controls. MiR-218 overexpressing and EGFR siRNA-treated cells were sensitized to irradiation, more than miR-218 overexpressing cells alone. Conclusion This study characterizes the antagonistic relationship between miR-218 and EGFR. It also demonstrates downstream functional effects of miR-218 overexpression, leading to anti-tumorigenic cellular changes.

Purpose While the stem cell marker Musashi-1 (MSI-1) has been identified as a key player in a wide array of malignancies, few findings exist on its prognostic relevance and relevance for cancer cell death and therapy resistance in breast cancer. Methods First, we determined prognostic relevance of MSI-1 in database analyses regarding multiple survival outcomes. To substantiate findings, MSI-1 was artificially downregulated in MCF-7 breast cancer cells and implications for cancer stem cell markers, cell apoptosis and apoptosis regulator p21, proliferation and radiation response were analyzed via flow cytometry and colony formation. Radiation-induced p21 expression changes were investigated using a dataset containing patient samples obtained before and after irradiation and own in vitro experiments. Results MSI-1 is a negative prognostic marker for disease-free and distant metastasis-free survival in breast cancer and tends to negatively influence overall survival. MSI-1 knockdown downregulated stem cell gene expression and proliferation, but increased p21 levels and apoptosis. Similar to the MSI-1 knockdown effect, p21 expression was strongly increased after irradiation and was expressed at even higher levels in MSI-1 knockdown cells after irradiation. Finally, combined use of MSI-1 silencing and irradiation reduced cancer cell survival. Conclusion MSI-1 is a prognostic marker in breast cancer. MSI-1 silencing downregulates proliferation while increasing apoptosis. The anti-proliferation mediator p21 was upregulated independently after both MSI-1 knockdown and irradiation and even more after both treatments combined, suggesting synergistic potential. Radio-sensitization effects after combining radiation and MSI-1 knockdown underline the potential of MSI-1 as a therapeutic target.

Background/aim Heparanase (HPSE) is relevant to therapy resistance in many malignancies yet is largely unstudied in Hodgkin's lymphoma. Here, we investigated links between HPSE, cancer stem cell (CSC) features and radioresistance in KM-H2 and L428 Hodgkin's lymphoma cells. Materials and methods Firstly, HPSE expression in unsorted and sorted CSCs was assessed. Post-irradiation, HPSE and CSC-related gene expression changes were then quantified. Clonogenic ability was investigated with and without artificial changes in HPSE expression pre and post irradiation. Results HPSE was highly expressed in L428 but barely present in KM-H2 cells. HPSE was overexpressed in sorted L428 CSCs. Irradiation induced HPSE and expression of CSC markers. High HPSE-expressing L428 cells showed higher clonogenic ability than low HPSE-expressing KM-H2 cells after irradiation. Down-regulation of HPSE in L428 cells reduced their clonogenic capability post-radiation, whilst overexpression of HPSE in KM-H2 cells increased colony formation. Conclusion HPSE expression is associated with CSC features and contributes to radioresistance in Hodgkin's lymphoma cells.

Syndecan-4, a predicted target of the microRNA miR-140-3p, plays an important role in multiple steps of tumor progression and is the second most abundant heparan sulfate proteoglycan produced by breast carcinoma cell lines. To investigate the potential functional relationship of miR-140-3p and syndecan-4, MDA-MB-231, SKBR3, and MCF-7 breast cancer (BC) cells were transiently transfected with pre-miR-140-3p, syndecan-4 small interfering RNAJ, or control reagents, respectively. Altered cell behavior was monitored by adhesion, migration, and invasion chamber assays. Moreover, the prognostic value of syndecan-4 was assessed by Kaplan-Maier Plotter analysis of gene expression data from tumor samples of 4929 patients. High expression of syndecan-4 was associated with better relapse-free survival in the whole collective of BC patients, but correlated with a worse survival in the subgroup of estrogen receptor negative and estrogen/progesterone-receptor negative patients. miR-140-3p expression was associated with improved survival irrespective of hormone receptor status. miR-140-3p overexpression induced posttranscriptional downregulation of syndecan-4, as demonstrated by quantitative real-time PCR (qPCR), flow cytometry, and luciferase assays, resulting in decreased BC cell migration and matrigel invasiveness. Furthermore, miR-140-3p overexpression and syndecan-4 silencing increased the adhesion of BC to fibronectin and laminin. qPCR analysis demonstrated that syndecan-4 silencing leads to altered gene expression of adhesion-related molecules, such as fibronectin and focal adhesion kinase, as well as in the gene expression of the proinvasive factors matrix metalloproteinase 2 and heparanase (also known as HPSE). We conclude that syndecan-4 is a novel target of miR-140-3p that regulates BC cell invasiveness and cell-matrix interactions in the tumor microenvironment.

STUDY QUESTION What are the effects of plant-derived antioxidant compounds urolithin A (UA) and B (UB) on the growth and pathogenetic properties of an in vitro endometriosis model? SUMMARY ANSWER Both urolithins showed inhibitory effects on cell behavior related to the development of endometriosis by differentially affecting growth, adhesion, motility, and invasion of endometriotic cells in vitro. WHAT IS KNOWN ALREADY Endometriosis is one of the most common benign gynecological diseases in women of reproductive age and is defined by the presence of endometrial tissue outside the uterine cavity. As current pharmacological therapies are associated with side effects interfering with fertility, we aimed at finding alternative therapeutics using natural compounds that can be administered for prolonged periods with a favorable side effects profile. STUDY DESIGN, SIZE, DURATION In vitro cultures of primary endometriotic stromal cells from 6 patients subjected to laparoscopy for benign pathologies with histologically confirmed endometriosis; and immortalized endometrial stromal (St-T1b) and endometriotic epithelial cells (12Z) were utilized to assess the effects of UA and UB on endometriotic cell properties. Results were validated in three-dimensional (3D) in vitro co-culture spheroids of 12Z and primary endometriotic stroma cells of one patient, and organoids from 3 independent donors with endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS The effects on cell growth were measured by non-radioactive colorimetric assay to measure cellular metabolic activity as an indicator of cell viability (MTT assay) and flow cytometric cell cycle assay on primary cultures, St-T1b, and 12Z. Apoptosis analyses, the impact on in vitro adhesion, migration, and invasion were evaluated in the cell lines. Moreover, Real-Time Quantitative Reverse Transcription polymerase chain reaction (RT-qPCR) assays were performed on primary cultures, St- T1b and 12Z to evaluate a plausible mechanistic contribution by factors related to proteolysis (matrix metalloproteinase 2, 3 and 9 -MMP2, MMP3, MMP9-, and tissue inhibitor of metalloproteinases -TIMP-1-), cytoskeletal regulators (Ras-related C3 botulinum toxin substrate 1 -RAC1-, Rho-associated coiled-coil containing protein kinase 2 -ROCK2-), and cell adhesion molecules (Syndecan 1 -SDC1-, Integrin alpha V–ITGAV-). Finally, the urolithins effects were evaluated on spheroids and organoids by formation, viability, and drug screen assays. MAIN RESULTS AND THE ROLE OF CHANCE 40 µM UA and 20 µM UB produced a significant decrease in cell proliferation in the primary endometriotic cell cultures (P LARGE-SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION This study was performed on in vitro endometriosis models. WIDER IMPLICATIONS OF THE FINDINGS These in vitro results provide new insights into the pathogenetic pathways affected by these compounds and mark their use as a potential new therapeutic strategy for the treatment of endometriosis. STUDY FUNDING/COMPETING INTEREST(S) This study was funded EU MSCA-RISE-2015 project MOMENDO (691058). The authors have no conflicts of interest to declare.

Radiation resistance represents a major challenge in the treatment of breast cancer. As heparan sulfate (HS) chains are known to contribute to tumorigenesis, we aimed to investigate the interplay between HS degradation and radiation response in triple-negative breast cancer (TNBC) cells.HS chains were degraded in vitro as TNBC cells MDA-MB-231 and HCC1806 were treated with heparinase I and III. Subsequently, radioresistance was determined via colony formation assay after doses of 2, 4 and 6 Gy. Cell cycle profile, stem cell characteristics, expression of HS, activation of beta integrins, and apoptosis were determined by flow cytometry. Additionally, cell motility was analyzed via wound-healing assays, and expression and activation of FAK, CDK-6, Src, and Erk1/2 were quantified by western blot pre- and post-irradiation. Finally, the expression of cytokines was analyzed using a cytokine array.Radiation promoted cell cycle changes, while heparinase treatment induced apoptosis in both cell lines. Colony formation assays showed significantly increased radio-resistance for both cell lines after degradation of HS. Cell migration was similarly upregulated after degradation of HS compared to controls. This effect was even more prominent after irradiation. Interestingly, FAK, a marker of radioresistance, was significantly activated in the heparinase-treated group. Additionally, we found Src to be dysregulated in MDA-MB-231 cells. Finally, we observed differential secretion of GRO, CXCL1, IGFBP1, IL8, Angiogenin, and Osteoprotegerin after HS degradation and radiotherapy.Our results suggest an influence of HS chains on the development of radioresistance in TNBC.

In colon cancer, downregulation of the transmembrane heparan sulfate proteoglycan syndecan-1 (Sdc-1) is associated with increased invasiveness, metastasis, and dedifferentiation. As Sdc-1 modulates signaling pathways relevant to stem cell function, we tested the hypothesis that it may regulate a tumor-initiating cell phenotype. Sdc-1 small-interfering RNA knockdown in the human colon cancer cell lines Caco2 and HT-29 resulted in an increased side population (SP), enhanced aldehyde dehydrogenase 1 activity, and higher expression of CD133, LGR5, EPCAM, NANOG, SRY (sex-determining region Y)-box 2, KLF2, and TCF4/TCF7L2. Sdc-1 knockdown enhanced sphere formation, cell viability, Matrigel invasiveness, and epithelial-to-mesenchymal transition-related gene expression. Sdc-1-depleted HT-29 xenograft growth was increased compared to controls. Decreased Sdc-1 expression was associated with an increased activation of β1-integrins, focal adhesion kinase (FAK), and wingless-type (Wnt) signaling. Pharmacological FAK and Wnt inhibition blocked the enhanced stem cell phenotype and invasive growth. Sequential flow cytometric SP enrichment substantially enhanced the stem cell phenotype of Sdc-1-depleted cells, which showed increased resistance to doxorubicin chemotherapy and irradiation. In conclusion, Sdc-1 depletion cooperatively enhances activation of integrins and FAK, which then generates signals for increased invasiveness and cancer stem cell properties. Our findings may provide a novel concept to target a stemness-associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence. DATABASES: The GEO accession number of the Affymetrix transcriptomic screening is GSE58751.

Syndecans are transmembrane heparan sulfate proteoglycans that integrate signaling at the cell surface. By interacting with cytokines, signaling receptors, proteases, and extracellular matrix proteins, syndecans regulate cell proliferation, metastasis, angiogenesis, and inflammation. We analyzed public gene expression datasets to evaluate the dysregulation and potential prognostic impact of Syndecan-3 in ovarian cancer. Moreover, we performed functional in vitro analysis in syndecan-3-siRNA-treated SKOV3 and CAOV3 ovarian cancer cells. In silico analysis of public gene array datasets revealed that syndecan-3 mRNA expression was significantly increased 5.8-fold in ovarian cancer tissues (n = 744) and 3.4-fold in metastases (n = 44) compared with control tissue (n = 46), as independently confirmed in an RNAseq dataset on ovarian serous cystadenocarcinoma tissue (n = 374, controls: n = 133, 3.5-fold increase tumor vs. normal). Syndecan-3 siRNA knockdown impaired 3D spheroid growth and colony formation as stemness-related readouts in SKOV3 and CAOV3 cells. In SKOV3, but not in CAOV3 cells, syndecan-3 depletion reduced cell viability both under basal conditions and under chemotherapy with cisplatin, or cisplatin and paclitaxel. While analysis of the SIOVDB database did not reveal differences in Syndecan-3 expression between patients, sensitive, resistant or refractory to chemotherapy, KM Plotter analysis of 1435 ovarian cancer patients revealed that high syndecan-3 expression was associated with reduced survival in patients treated with taxol and platin. At the molecular level, a reduction in Stat3 activation and changes in the expression of Wnt and notch signaling constituents were observed. Our study suggests that up-regulation of syndecan-3 promotes the pathogenesis of ovarian cancer by modulating stemness-associated pathways.

The stem cell marker and RNA-binding protein Musashi-1 is overexpressed in endometriosis. Musashi-1-siRNA knockdown in Ishikawa cells altered the expression of stem cell related genes, such as OCT-4. To investigate the role of both human Musashi homologues (MSI-1 and MSI-2) in the pathogenesis of endometriosis, immortalized endometriotic 12-Z cells and primary endometriotic stroma cells were treated with Musashi-1- and Musashi-2-siRNA. Subsequently, the impact on cell proliferation, cell apoptosis, cell necrosis, spheroid formation, stem cell phenotype and the Notch signaling pathway was studied in vitro. Using the ENDOMET Turku Endometriosis database, the gene expression of stem cell markers and Notch signaling pathway constituents were analyzed according to localization of the endometriosis lesions. The database analysis demonstrated that expression of Musashi and Notch pathway-related genes are dysregulated in patients with endometriosis. Musashi-1/2-double-knockdown increased apoptosis and necrosis and reduced stem cell gene expression, cell proliferation, and the formation of spheroids. Musashi silencing increased the expression of the anti-proliferation mediator p21. Our findings suggest the therapeutic potential of targeting the Musashi–Notch axis. We conclude that the Musashi genes have an impact on Notch signaling and the pathogenesis of endometriosis through the downregulation of proliferation, stemness characteristics and the upregulation of apoptosis, necrosis and of the cell cycle regulator p21.

Background: Truncated tissue factor (tTF) retargeted by NGR-peptides to aminopeptidase N (CD13) in tumor vasculature is effective in experimental tumor therapy. tTF-NGR induces tumor growth inhibition in a variety of human tumor xenografts of different histology. To improve on the therapeutic efficacy we have combined tTF-NGR with radiotherapy. Methods: Serum-stimulated human umbilical vein endothelial cells (HUVEC) and human HT1080 sarcoma cells were irradiated in vitro, and upregulated early-apoptotic phosphatidylserine (PS) on the cell surface was measured by standard flow cytometry. Increase of cellular procoagulant function in relation to irradiation and PS cell surface concentration was measured in a tTF-NGR-dependent Factor X activation assay. In vivo experiments with CD-1 athymic mice bearing human HT1080 sarcoma xenotransplants were performed to test the systemic therapeutic effects of tTF-NGR on tumor growth alone or in combination with regional tumor ionizing radiotherapy. Results: As shown by flow cytometry with HUVEC and HT1080 sarcoma cells in vitro, irradiation with 4 and 6 Gy in the process of apoptosis induced upregulation of PS presence on the outer surface of both cell types. Proapoptotic HUVEC and HT1080 cells both showed significantly higher procoagulant efficacy on the basis of equimolar concentrations of tTF-NGR as measured by FX activation. This effect can be reverted by masking of PS with Annexin V. HT1080 human sarcoma xenografted tumors showed shrinkage induced by combined regional radiotherapy and systemic tTF-NGR as compared to growth inhibition achieved by either of the treatment modalities alone. Conclusions: Irradiation renders tumor and tumor vascular cells procoagulant by PS upregulation on their outer surface and radiotherapy can significantly improve the therapeutic antitumor efficacy of tTF-NGR in the xenograft model used. This synergistic effect will influence design of future clinical combination studies.

In a prospective observational pilot study, we evaluated label-free quantitative phase imaging (QPI) by digital holographic microscopy (DHM) as tool to describe perioperative changes in biophysical cell properties of lymphocytes and monocytes after cardiac surgery.

Breast cancer is the third most common type of cancer diagnosed. Cell cycle is a complex but highly organized and controlled process, in which normal cells sense mitogenic growth signals that instruct them to enter and progress through their cell cycle. This process culminates in cell division generating two daughter cells with identical amounts of genetic material. Uncontrolled proliferation is one of the hallmarks of cancer. In this study, we analyzed the expression of the cell cycle-related genes receptor for hyaluronan (HA)-mediated motility (RHAMM), AURKA, TPX2, PLK1, and PLK4 and correlated them with the prognosis in a collective of 3952 breast cancer patients. A high messenger RNA expression of all studied genes correlated with a poor prognosis. Stratifying the patients according to the expression of hormonal receptors, we found that in patients with estrogen and progesterone receptor-positive and human epithelial growth factor receptor 2-negative tumors, and Luminal A and Luminal B tumors, the expression of the five analyzed genes correlates with worse survival. qPCR analysis of a panel of breast cancer cell lines representative of major molecular subtypes indicated a predominant expression in the luminal subtype. In vitro experiments showed that radiation influences the expression of the five analyzed genes both in luminal and triple-negative model cell lines. Functional analysis of MDA-MB-231 cells showed that small interfering RNA knockdown of PLK4 and TPX2 and pharmacological inhibition of PLK1 had an impact on the cell cycle and colony formation. Looking for a potential upstream regulation by microRNAs, we observed a differential expression of RHAMM, AURKA, TPX2, PLK1, and PLK4 after transfecting the MDA-MB-231 cells with three different microRNAs. Survival analysis of miR-34c-5p, miR-375, and miR-142-3p showed a different impact on the prognosis of breast cancer patients. Our study suggests that RHAMM, AURKA, TPX2, PLK1, and PLK4 can be used as potential targets for treatment or as a prognostic value in breast cancer patients.

In a prospective observational pilot study on patients undergoing elective cardiac surgery with cardiopulmonary bypass, we evaluated label-free quantitative phase imaging (QPI) with digital holographic microscopy (DHM) to describe perioperative inflammation by changes in biophysical cell properties of lymphocytes and monocytes. Blood samples from 25 patients were investigated prior to cardiac surgery and postoperatively at day 1, 3 and 6. Biophysical and morphological cell parameters accessible with DHM, such as cell volume, refractive index, dry mass, and cell shape related form factor, were acquired and compared to common flow cytometric blood cell markers of inflammation and selected routine laboratory parameters. In all examined patients, cardiac surgery induced an acute inflammatory response as indicated by changes in routine laboratory parameters and flow cytometric cell markers. DHM results were associated with routine laboratory and flow cytometric data and correlated with complications in the postoperative course. In a subgroup analysis, patients were classified according to the inflammation related C-reactive protein (CRP) level, treatment with epinephrine and the occurrence of postoperative complications. Patients with regular courses, without epinephrine treatment and with low CRP values showed a postoperative lymphocyte volume increase. In contrast, the group of patients with increased CRP levels indicated an even further enlarged lymphocyte volume, while for the groups of epinephrine treated patients and patients with complicative courses, no postoperative lymphocyte volume changes were detected. In summary, the study demonstrates the capability of DHM to describe biophysical cell parameters of perioperative lymphocytes and monocytes changes in cardiac surgery patients. The pattern of correlations between biophysical DHM data and laboratory parameters, flow cytometric cell markers, and the postoperative course exemplify DHM as a promising diagnostic tool for a characterization of inflammatory processes and course of disease.

Ductal carcinoma in situ (DCIS) is a form of breast cancer that is restricted to the lactiferous ducts and has not yet invaded the surrounding breast tissue. Dysregulation of the transmembrane heparan sulphate proteoglycan Syndecan-1 (Sdc-1) plays a role in tumour progression of invasive breast cancer (IBC). In DCIS, Sdc-1, c-Met and E-cadherin are part of a proangiogenic expression signature. In this study, we employed a siRNA knockdown approach in the DCIS model cell line MCF10A DCIS.com to investigate a potential connection between Sdc-1 and epithelial mesenchymal transition (EMT), proteolysis and the Rho kinase pathway. Analysis of gene expression data of the TNMplot.com database revealed that Sdc-1 expression was higher in primary breast tumours compared to metastases. The impact of Sdc-1-depletion on the cellular phenotype was investigated in a Matrigel-based three-dimensional cell culture model. Sdc-1 depletion resulted in the formation of larger spheroids and the formation of invasive protrusions. Application of matrix metalloproteinase (MMP) and Rho kinase inhibitors could block the Sdc-1-induced phenotype. qPCR analysis of Sdc-1-depleted cells in two-dimensional culture revealed upregulated expression of the EMT-markers CDH1, FN-1, CLDN1, the proteolysis markers MMP3, and MMP9, and HPSE, while MMP2, VIM and ROCK-2 were downregulated. Immunocytochemistry confirmed upregulation of MMP9 and fibronectin, the latter being particular prominent after ROCK inhibition. STRING analysis confirmed an interaction of the investigated gene products at the protein level. Our results suggest that diminished Sdc-1 expression plays a role in DCIS progression to IBC through deregulation of proteolytic factors and a partial EMT.

Endometrial carcinoma is the most common gynecological cancer in Europe. Musashi-1 is known to be a key regulator of endometrial cancer stem cells and a negative prognostic marker. In the present study, we aimed to understand growth and gene expression patterns in endometrial carcinoma after Musashi-1 knockdown in vitro and in vivo. Changes in therapeutic resistance were also assessed.First, we performed analyses to understand Musashi-1 expression patterns using The Cancer Genome Atlas database. We then proceeded to assess effects of small interfering RNA-based Musashi-1 targeting in two endometrial carcinoma cell lines, Ishikawa and KLE. After quantifying baseline changes in cell metabolism, we used MTT tests to assess chemotherapy effects and colony formation assays to understand changes in radioresistance. For mechanistic study, we used quantitative polymerase chain reaction (qPCR) and western blotting of key Musashi-1 target genes and compared results to primary tissue database studies. Finally, xenograft experiments in a mouse model helped understand in vivo effects of Musashi-1 knockdown.Musashi-1 is aberrantly expressed in primary tumor tissues. In vitro, silencing of Musashi-1 resulted in a strong decline in cell proliferation and radioresistance, while chemoresistance remained unchanged. Loss of Musashi-1 led to downregulation of telomerase, DNA-dependent protein kinase, the Notch pathway and overexpression of cyclin-dependent kinase inhibitor p21, the latter of which we identified as a key mediator of Msi-1 knockdown-related anti-proliferative signaling. In vivo, the anti-proliferative effect was confirmed, with Msi-1 knockdown tumors being about 40% reduced in size.Musashi-1 knockdown resulted in a strong decrease in endometrial cancer proliferation and a loss of radioresistance, suggesting therapeutic potential.

Cervical cancer ranks fourth among the most commonly diagnosed malignant tumors in women worldwide. Previously published evidence suggested a possible connection between the expression of the membrane-bound heparan sulfate proteoglycan syndecan-1 (Sdc-1) and the development of cervical carcinoma. Sdc-1 serves as a matrix receptor and coreceptor for receptor tyrosine kinases and additional signaling pathways. It influences cell proliferation, adhesion, and migration and is seen as a modulator of the tumor microenvironment. Following proteolytic cleavage of its extracellular domain in a process called shedding, Sdc-1 can act as a paracrine effector. The loss of Sdc-1 expression is associated with low differentiation of cervical carcinoma and with an increased rate of lymph node metastases. Here, we analyzed the clinical impact of Sdc-1 expression by analysis of public gene expression datasets and studied the effect of an overexpression of Sdc-1 and its membrane-bound and soluble forms on the malignant properties of the human cervical carcinoma cell line HeLa through functional analysis. For this purpose, the HeLa cells were stably transfected with the control plasmid pcDNA3.1 and three different Sdc-1-DNA constructs,encoding wild-type, permanently membrane-bound, and constitutively soluble Sdc-1. In clinical specimens, Sdc-1 mRNA was more highly expressed in local tumor tissues than in normal and metastatic cervical cancer tissues. Moreover, high Sdc-1 expression correlated with a poor prognosis in Kaplan-Meier survival analysis, suggesting the important role of Sdc-1 in the progression of this type of cancer. In vitro, we found that the soluble, as well as the permanently membrane-bound forms of Sdc-1 modulated the proliferation and the cell cycle, while membrane-bound Sdc1 regulated HeLa cell apoptosis. The overexpression of Sdc-1 and its soluble form increased invasiveness. In vitro scratch/wound healing assay, showed reduced Sdc-1-dependent cell motility which was linked to the Rho-GTPase signaling pathway. In conclusion, in cervical cancer Sdc-1 modulates pathogenetically relevant processes, which depend on the membrane-association of Sdc-1.

Total skin electron beam therapy (TSEBT) has proved to be a safe and effective treatment for cutaneous T‑cell lymphomas. Here, we examined the impact of this treatment on patient quality of life and outcome. Forty-four patients with mycosis fungoides (MF) or Sezary syndrome (SS) received 48 TSEBT courses with a median dose of 12 Gy within the past 8 years at our institute. Patient and treatment characteristics for these cases as well as the impact of TSEBT on quality of life and duration of response were retrospectively analyzed and compared. The median modified Severity-Weighted Assessment Tool score before the start of TSEBT was 44. The overall response rate was 88%, with a complete response (CR) rate of 33%. The median follow-up period was 13 months. The median duration of response (DOR) and progression-free survival (PFS) for the entire cohort were 10 months and 9 months, respectively. Patient-reported symptom burden was measured with the Dermatological Life Quality Index and Skindex-29 questionnaires. The mean symptom reductions were 6 ± 8 (P = 0.005) and 21 ± 24 (P = 0.002), respectively. In the Functional Assessment of Cancer Therapy-General Assessment, significant improvements in the emotional (P = 0.03) domains were observed after TSEBT. Patients who received maintenance or adjuvant treatments had a longer PFS (P = 0.01). TSEBT improved disease symptoms and significantly improved emotional domains of patients’ quality of life in patients with MF or SS. In addition, our results indicate that maintenance or adjuvant therapy after TSEBT may improve the PFS.

Purpose While the stem cell marker Musashi-1 (MSI-1) has been identified as a key player in a wide array of malignancies, few findings exist on its prognostic relevance and relevance for cancer cell death and therapy resistance in breast cancer. Methods First, we determined prognostic relevance of MSI-1 in database analyses regarding multiple survival outcomes. To substantiate findings, MSI-1 was artificially downregulated in MCF-7 breast cancer cells and implications for cancer stem cell markers, cell apoptosis and apoptosis regulator p21, proliferation and radiation response were analyzed via flow cytometry and colony formation. Radiation-induced p21 expression changes were investigated using a dataset containing patient samples obtained before and after irradiation and own in vitro experiments. Results MSI-1 is a negative prognostic marker for disease-free and distant metastasis-free survival in breast cancer and tends to negatively influence overall survival. MSI-1 knockdown downregulated stem cell gene expression and proliferation, but increased p21 levels and apoptosis. Similar to the MSI-1 knockdown effect, p21 expression was strongly increased after irradiation and was expressed at even higher levels in MSI-1 knockdown cells after irradiation. Finally, combined use of MSI-1 silencing and irradiation reduced cancer cell survival. Conclusion MSI-1 is a prognostic marker in breast cancer. MSI-1 silencing downregulates proliferation while increasing apoptosis. The anti-proliferation mediator p21 was upregulated independently after both MSI-1 knockdown and irradiation and even more after both treatments combined, suggesting synergistic potential. Radio-sensitization effects after combining radiation and MSI-1 knockdown underline the potential of MSI-1 as a therapeutic target. Supplementary Information The online version contains supplementary material available at 10.1007/s00432-021-03743-y.

Triple-negative breast cancer (TNBC) is defined by aggressive behavior, limited response to chemotherapy and lower overall survival rates. The increased metastatic potential of TNBC is a combined result of extensive extracellular matrix (ECM) remodeling that leads to cytoskeleton rearrangement and activation of epithelial-to-mesenchymal transition (EMT). The overexpression of epidermal growth factor receptor (EGFR) in TNBC tumors has been linked to induced expression of EMT-related molecules. EMT activation has often been associated with increased metastasis and stemness. Recently, we described the crucial role of EGFR/estrogen receptor beta (ERβ) interplay in the regulation of invasion and cell-matrix interactions. In this study, we report on the EGFR-ERβ functional relationship in connection to the aggressiveness and cancer stem cell (CSC)-like characteristics of TNBC cells. ERβ-suppressed and MDA-MB-231 cells were subjected to downstream EGFR inhibition and/or estradiol stimulation to assess alterations in functional parameters as well as in morphological characteristics, studied by scanning electron, atomic force, and immunofluorescence microscopies. Moreover, the expression and localization of key EMT and CSC-related markers were also evaluated by real-time qPCR, immunofluorescence microscopy, and flow cytometry. EGFR inhibition resulted in an overall suppression of aggressive functional characteristics, which occurred in an ERβ-mediated manner. These changes could be attributed to a reduction, at the molecular level, of EMT and stemness-linked markers, most notably reduced expression of Notch signaling constituents and the cell surface proteoglycan, syndecan-1. Collectively, our study highlights the importance of EGFR signaling as a key effector of aggressiveness, EMT, and stemness in an ERβ-dependent way in TNBC.

Syndecan-1 (CD138) is a cell surface heparan sulfate proteoglycan that is frequently misexpressed in cancer. Under physiological and pathological conditions, it is involved in the regulation of diverse processes, such as leukocyte recruitment, wound repair, angiogenesis, exosome formation, and epithelial-to-mesenchymal transition. Apart from its role as an adhesion molecule and a modulator of proteolysis, Syndecan-1 has a pivotal function as a co-receptor for multiple signal transduction pathways, including Wnt, hedgehog, FGF, and NF-kB/IL-6/JAK/STAT3 signaling. Notably, the activity of these pathways plays an important role in determining the functional state of cancer stem cells. This cell population shares several properties with stem cells, and has been linked to cancer recurrence due to its property of increased resistance to chemo- and radiation therapies. In this chapter, we summarize the current knowledge on the role of Syndecan-1 in cancer stem cell function. Due to its simultaneous involvement as an enhancer of multiple stemness-associated signaling pathways, Syndecan-1 emerges as an attractive target for novel therapeutic approaches, which could be utilized to overcome recurrence after an otherwise successful therapy.

Inflammatory breast cancer (IBC) is a rare, but aggressive entity of breast carcinoma with rapid dermal lymphatic invasion in young females. It is either poorly or misdiagnosed as mastitis because of the absence of a distinct lump. Small extracellular vesicles (sEVs) circulating in liquid biopsies are a novel class of minimally invasive diagnostic alternative to invasive tissue biopsies. They modulate cancer progression via shuttling their encapsulated cargo including microRNAs (miRNAs) into recipient cells to either trigger signaling or induce malignant transformation of targeted cells. Plasma sEVs < 200 nm were isolated using a modified cost-effective polyethylene glycol (PEG)-based precipitation method and compared to standard methods, namely ultracentrifugation and a commercial kit, where the successful isolation was verified by different approaches. We evaluated the expression levels of selected sEV-derived miR-181b-5p, miR-222-3p and let-7a-5p using quantitative real PCR (qPCR). Relative to non-IBC, our qPCR data showed that sEV-derived miR-181b-5p and miR-222-3p were significantly upregulated, whereas let-7a-5p was downregulated in IBC patients. Interestingly, receiver operating characteristic (ROC) curves analysis revealed that diagnostic accuracy of let-7a-5p alone was the highest for IBC with an area under curve (AUC) value of 0.9188, and when combined with miR-222-3p the AUC was improved to 0.973. Further, 38 hub genes were identified using bioinformatics analysis. Together, circulating sEV-derived miR-181b-5p, miR-222-3p and let-7a-5p serve as promising non-invasive diagnostic biomarkers for IBC.

Breast cancer continues to be a serious public health problem. The role of the hedgehog pathway in normal development of the mammary gland as well as in carcinogenesis and progression of breast cancer is the subject of intense investigation, revealing functional interactions with cell surface heparan sulfate. Nevertheless, its influence on breast cancer prognosis, and its relation to specific sulfation motifs in heparan sulfate have only been poorly studied in large patient cohorts. Using the public database KMplotter that includes gene expression and survival data of 3951 patients, we found that the higher expression of SHH, HHAT, PTCH1, GLI1, GLI2, and GLI3 positively influences breast cancer prognosis. Stratifying patients according to the expression of hormone receptors, histological grade, lymph node metastasis, and systemic therapy, we observed that GLI1, GLI2, and GLI3 expression, as well as co-expression of SHH and ELP1 were associated with worse relapse-free survival in patients with HER2-positive tumors. Moreover, GLI1 expression in progesterone receptor-negative tumors and GLI3 expression in grade 3 tumors correlated with poor prognosis. SHH, in a panel of cell lines representing different breast cancer subtypes, and HHAT, PTCH1, GLI1, GLI2, and GLI3 were mostly expressed in cell lines classified as HER2-positive and basal-like. Expression of SHH, HHAT, GLI2, and GLI3 was differentially affected by overexpression of the heparan sulfate sulfotransferases HS2ST1 and HS3ST2 in vitro. Although high HS2ST1 expression was associated with poor prognosis in KMplotter analysis, high levels of HS3ST2 were associated with a good prognosis, except for ER-positive breast cancer. We suggest the GLI transcription factors as possible markers for the diagnosis, treatment, and prognosis of breast cancer especially in HER2-positive tumors, but also in progesterone receptor-negative and grade-3 tumors. The pathway interaction and prognostic impact of specific heparan sulfate sulfotransferases provide novel perspectives regarding a therapeutical targeting of the hedgehog pathway in breast cancer.

Inflammatory breast cancer (IBC) is a rare yet aggressive breast cancer variant, associated with a poor prognosis. The major challenge for IBC is misdiagnosis due to the lack of molecular biomarkers. We profiled dysregulated expression of microRNAs (miRNAs) in primary samples of IBC and non-IBC tumors using human breast cancer miRNA PCR array. We discovered that 28 miRNAs were dysregulated (10 were upregulated, while 18 were underexpressed) in IBC vs. non-IBC tumors. We identified 128 hub genes, which are putative targets of the differentially expressed miRNAs and modulate important cancer biological processes. Furthermore, our qPCR analysis independently verified a significantly upregulated expression of miR-181b-5p, whereas a significant downregulation of miR-200b-3p, miR-200c-3p, and miR-203a-3p was detected in IBC tumors. Receiver operating characteristic (ROC) curves implied that the four miRNAs individually had a diagnostic accuracy in discriminating patients with IBC from non-IBC and that miR-203a-3p had the highest diagnostic value with an AUC of 0.821. Interestingly, a combination of miR-181b-5p, miR-200b-3p, and miR-200c-3p robustly improved the diagnostic accuracy, with an area under the curve (AUC) of 0.897. Intriguingly, qPCR revealed that the expression of zinc finger E box-binding homeobox 2 (ZEB2) mRNA, the putative target of miR-200b-3p, miR-200c-3p, and miR-203a-3p, was upregulated in IBC tumors. Overall, this study identified a set of miRNAs serving as potential biomarkers with diagnostic relevance for IBC.

Despite the functional role of serglycin as an intracellular proteoglycan, a variety of malignant cells depends on its expression and constitutive secretion to advance their aggressive behavior. Serglycin arose to be a biomarker for glioblastoma, which is the deadliest and most treatment-resistant form of brain tumor, but its role in this disease is not fully elucidated. In our study we suppressed the endogenous levels of serglycin in LN-18 glioblastoma cells to decipher its involvement in their malignant phenotype. Serglycin suppressed LN-18 (LN-18shSRGN) glioblastoma cells underwent astrocytic differentiation characterized by induced expression of GFAP, SPARCL-1 and SNAIL, with simultaneous loss of their stemness capacity. In particular, LN-18shSRGN cells presented decreased expression of glioma stem cell-related genes and ALDH1 activity, accompanied by reduced colony formation ability. Moreover, the suppression of serglycin in LN-18shSRGN cells retarded the proliferative and migratory rate, the invasive potential in vitro and the tumor burden in vivo. The lack of serglycin in LN-18shSRGN cells was followed by G2 arrest, with subsequent reduction of the expression of cell-cycle regulators. LN-18shSRGN cells also exhibited impaired expression and activity of proteolytic enzymes such as MMPs, TIMPs and uPA, both in vitro and in vivo. Moreover, suppression of serglycin in LN-18shSRGN cells eliminated the activation of pro-tumorigenic signal transduction. Of note, LN-18shSRGN cells displayed lower expression and secretion levels of IL-6, IL-8 and CXCR-2. Concomitant, serglycin suppressed LN-18shSRGN cells demonstrated repressed phosphorylation of ERK1/2, p38, SRC and STAT-3, which together with PI3K/AKT and IL-8/CXCR-2 signaling control LN-18 glioblastoma cell aggressiveness. Collectively, the absence of serglycin favors an astrocytic fate switch and a less aggressive phenotype, characterized by loss of pluripotency, block of the cell cycle, reduced ability for ECM proteolysis and pro-tumorigenic signaling attenuation., Highlights • Knockdown (KD) of serglycin results in glioblastoma cell differentiation and decreased stemness. • KD of serglycin diminishes the proteolytic and inflammatory potential of glioblastoma cells. • KD of serglycin represses the glioblastoma aggressiveness via attenuation of pro-tumorigenic signaling cascades.

Endometriosis is a painful gynaecological condition characterized by ectopic growth of endometrial cells outside of the uterus. Little is known about the mechanisms by which endometrial fragments invade tissues. This is partially due to a lack of suitable experimental models. In this study, we show that a spheroid 3D model, but not single cells mimic the collective endometrial fragment-like invasion through the extracellular matrix. This model reveals that collagen I, the main constituent of surgical scars, significantly increases the rate of lesion formation by healthy endometrial stromal cells (St-T1b) in vitro compared to the basement membrane-like matrix Matrigel. Stromal cell invasion of collagen I requires MMPs, whereas collective migration of endometriotic epithelial 12Z cells involves Rac-signalling. We show that inhibiting ROCK signalling responsible for actomyosin contraction increases the lesion-size. Moreover, endometriotic epithelial 12Z cells, but not eutopic stromal cells St-T1b migrate on Matrigel. The rate of this migration is decreased by the microRNA miR-200b and increased by miR-145. Our 3D model offers a facile approach to dissect how endometrial fragments invade tissues and is an important step toward developing new personalized therapeutics for endometriosis. Moreover, our model is a suitable tool to screen small molecule drugs and microRNA-based therapeutics.