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Supplemental material, JHC772798_Supplemental_Material for Immunolocalization of Advanced Glycation End Products, Mitogen Activated Protein Kinases, and Transforming Growth Factor-β/Smads in Pelvic Organ Prolapse by Antonella Vetuschi, Simona Pompili, Anna Gallone, Angela D’Alfonso, Maria Gabriella Carbone, Gaspare Carta, Claudio Festuccia, Eugenio Gaudio, Alessandro Colapietro and Roberta Sferra in Journal of Histochemistry & Cytochemistry

Table S1. Additional hazard ratio analyses in xenograt models. Table S2 Additional hazard ratio analyses in orthotopic U251models. Table S3 Additional hazard ratio analyses in orthotopic CSCs-5model. (DOCX 23 kb)

Glioblastoma (GBM) is the most common and aggressive tumor of the Central Nervous System (CNS). Unfortunately, patients afflicted with this disease have a very poor prognosis, due to high level of invasiveness and resistance to standard therapies (1). Although the molecular profile of GBM has been extensively investigated, the events responsible for its pathogenesis and progression remain largely unknown. Reports have indicated that HDAC (Histone deacetylases) dependent epigenetic modifications (2) and the Tgfβ/Smad pathway (3) play roles in GBM tumorigenesis. The aim of this study was to evaluate the involvement and the possible interaction between these two molecular cascades in the pathogenesis, therapeutic responsiveness and prognosis of GBM. Immunohistochemestry (IHC) was performed on microdissected GBM samples, collected from 14 patients (n.8 men and n.6 women) ranging in age from 43 to 74 years. The patients were previously divided, on the basis of their overall survival (OS), into three groups: low, intermediate and high OS. Patients with poor prognosis showed hyperexpression of HDAC4 and HDAC6, an activation of the Tgfβ/Smad pathway, with high levels of IL-13, Smad2, PDGF and MMP3 expression, compared to the intermediate and high OS groups, whereas the expression of Smad7 was reduced. The high OS group also exhibits an increase in p21 immunostaining, which represents a common target of the two cascades. The IHC data was confirmed by Immunoblotting. Our results suggest that both HDAC4 and HDAC6 together with the Tgfβ/Smad pathway are involved in progression of GBM and could be a useful prognostic markers and may predict responsive to therapy., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

// Giovanni Luca Gravina 1, 2 , Andrea Mancini 1 , Alessandro Colapietro 1 , Francesco Marampon 1 , Roberta Sferra 3 , Simona Pompili 3 , Leda Assunta Biordi 4 , Roberto Iorio 5 , Vincenzo Flati 4 , Christian Argueta 6 , Yosef Landesman 6 , Michael Kauffman 6 , Sharon Shacham 6 and Claudio Festuccia 1 1 Department of Biotechnological and Applied Clinical Sciences, Laboratory of Radiobiology, University of L’Aquila, L’Aquila, Italy 2 Department of Biotechnological and Applied Clinical Sciences, Division of Radiotherapy, University of L’Aquila, L’Aquila, Italy 3 Department of Biotechnological and Applied Clinical Sciences, Division of Human Anatomy, University of L’Aquila, L’Aquila, Italy 4 Department of Biotechnological and Applied Clinical Sciences, Division of Molecular Pathology, University of L’Aquila, L’Aquila, Italy 5 Department of Biotechnological and Applied Clinical Sciences, Division of Applied Biology, University of L’Aquila, L’Aquila, Italy 6 Karyopharm Therapeutics, Newton, MA, USA Correspondence to: Claudio Festuccia, email: claudio.festuccia@univaq.it Keywords: castration resistant prostate cancer (CrPCa); chromosome region maintenance (CRM-1); exportin-1 (XPO-1); selective inhibitors of nuclear export (SINE); docetaxel (DTX) Received: June 29, 2017 Accepted: November 13, 2017 Published: November 30, 2017 ABSTRACT Background and aims: Docetaxel (DTX) modestly increases patient survival of metastatic castration-resistant prostate cancer (mCRPC) due to insurgence of pharmacological resistance. Deregulation of Chromosome Region Maintenance (CRM-1)/ exportin-1 (XPO-1)-mediated nuclear export may play a crucial role in this phenomenon. Material and methods: Here, we evaluated the effects of two Selective Inhibitor of Nuclear Export (SINE) compounds, selinexor (KPT-330) and KPT-251, in association with DTX by using 22rv1, PC3 and DU145 cell lines with their. DTX resistant derivatives. Results and conclusions: We show that DTX resistance may involve overexpression of β-III tubulin (TUBB3) and P-glycoprotein as well as increased cytoplasmic accumulation of Foxo3a. Increased levels of XPO-1 were also observed in DTX resistant cells suggesting that SINE compounds may modulate DTX effectiveness in sensitive cells as well as restore the sensitivity to DTX in resistant ones. Pretreatment with SINE compounds, indeed, sensitized to DTX through increased tumor shrinkage and apoptosis by preventing DTX-induced cell cycle arrest. Basally SINE compounds induce FOXO3a activation and nuclear accumulation increasing the expression of FOXO-responsive genes including p21, p27 and Bim causing cell cycle arrest. SINE compounds-catenin and survivin supporting apoptosis. βdown-regulated Cyclin D1, c-myc, Nuclear sequestration of p-Foxo3a was able to reduce ABCB1 and TUBB3 H2AX levels, prolonged γ expression. Selinexor treatment increased DTX-mediated double strand breaks (DSB), and reduced the levels of DNA repairing proteins including DNA PKc and Topo2A. Our results provide supportive evidence for the therapeutic use of SINE compounds in combination with DTX suggesting their clinical use in mCRPC patients.

Collagen and MMPs play a pivotal role in the pathophysiology of the Pelvic Organ Prolapse (1). In POP samples a switch between type I and type III collagen together with a simultaneous activation of MMPs have been observed and the main consequence of these changes is the loss of mechanical support in the vaginal wall (2). Aim of this study was to prove that AGEs induces the activation of MMPs through ERK1/2 and synchronically stimulates changes in collagen composition directly through Smads. The case group consisted of 20 patients suffering from stage III genital prolapse undergoing colpohysterectomy and anterior and posterior plastic vaginal surgery and 10 control patients treated with laparohysterectomy for uterine fibromatosis. Histological and Immunohistochemical analysis using AGE, RAGE, ERK 1/2, Smads 2-3, Smad 7, MMP-3 and collagen I-III were performed. AGE and ERK 1/2 were also evaluated using Western-Blot analysis. POP samples from anterior vaginal wall showed disorganization and a distortion of the normal muscularis architecture. In POP samples AGE, ERK 1/2, Smad 2-3, MMP-3 and collagen III were upregulated in muscularis whereas in controls Smad 7 and collagen I were increased in the same layer. RAGE was mild or absent both in controls and prolapse. In summary we suggest the possible role of these new markers in the pathogenesis of POP but further studies are required to elucidate if the change of these molecules is the reason or the result of POP disease., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

Glioblastoma is the most common and lethal primary malignant brain tumor. Although standard treatments have been improving, the clinical outcome remains unacceptably poor. Several genetic alterations are supposed to be involved in the eti- ology of different grades of astrocytoma, including epimutations. Histone deacety- lases (HDACs) are involved in the post-translational modification on the lysines of histone tails. For this reason HDACs are recognized as promising targets for cancer treatment (1). In the past several years, HDAC inhibitors (HDACis) have been used as radiosensitizers in glioblastoma treatment. However, no study has demonstrated the status of global HDAC expression in gliomas and its possible correlation to the use of HDACis (2). Aim of our study was to evaluate with an immunohistochemi- cal and immunoblotting analyses the expression of different classes of HDACs (Class I: HDAC 1-2-3-8; class II: HDAC 4-6) in microdissected glioblastoma. Tumor sam- ples were taken from 14 patients (n.8 men and n.6 women) ranging in age from 43 to 74 years. HDAC1 and HDAC3 expression was not significantly different between the two proteins and was predominantely located at cytoplasmic level of cancer cells with different intensity of immureaction from mild to moderate whereas HDAC2 staining was localized to the nucleous of neoplastic cells. The pattern of HDAC4 immureactivity was always cytoplasmatic and showed a marked and diffuse increase of immunostaining in neoplastic areas. HDAC8 was always absent in cancer cells and the only positivity was located in the endothelial cells of the vessels. HDAC6 was often absent and, if present, showed a very low cytoplasmic immunopositivity in cancer cells. HDAC1, HDAC2 and HDAC3 levels were not significantly different in immunoblotting results; HDAC4 showed a marked increase while HDAC6 and HDAC8 expression was poor, confirming the IHC data. These previous results dem- onstrate a different pattern of HDAC expression and could suggest a more addressed therapeutical use of HDACis in glioblastoma., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Intestinal fibrosis is a progressive process characterized by de novo synthesis and uncontrolled deposition of extracellular matrix components (ECM) following a tissue chronic inflammation mainly regulated by Transforming Growth Factor (TGF)β/ Smads pathway. Frequently associated to Inflammatory Bowel Disease (IBD), intestinal fibrosis may lead to stenosis and obstructions that require surgery up to 75% of patients as drugs currently used in IBD are unable to improve fibrostenosis lesions (1). Peroxisome proliferator-activated receptor (PPAR)-γ is able to antagonize (TGF) β/Smads and could be an crucial target to develop novel antifibrotic therapeutic strategies (2). Aim of this study is to evaluate the antifibrotic action of a novel PPARγ agonist, GED 0507-34 levo, in colonic fibrosis in mice. Immunohistochemistry and immunoblotting evaluations, TGFβ1, CTGF, Collagen types I-III, Smad3, α-SMA, were performed in in three groups of C57BL/6 mice: Dextran Sulphate Sodium (DSS) colitis group, DSS+GED group and controls. Evident macroscopic and microscopic lesions in the most of colons of DSS treated mice were observed compared to DSS+GED mice and controls. The tissue levels of the main markers of fibrosis resulted significantly increased in DSS mice and restored by administration of GED. GED seems to prevents ECM colonic deposition and to improve the intestinal fibrotic lesions by its ability in controlling TGFβ/Smads pathway signalling activation., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Inflammatory Bowel Diseases (IBD) are chronic and progressive inflammatory disorders that may result in intestinal fibrosis, characterized by excessive and uncon- trolled deposition of extracellular matrix components (ECM). Other than Smad- dependent the profibrotic effects of TGFβ signaling that leads to a phenotype switch of intestinal mesenchymal cells, resulting in proliferation and collagene deposition (1), source of fibrotic tissue comes from the process called Epithelial-to-Mesenchymal Transition (EMT), in which also epithelial cells acquire fibroblastic phenotype and related function (2). The purpose of our study was to evaluate the expression of pro- tein markers of EMT in a mouse model of intestinal fibrosis and their changes after the administration of the experimental drug GED- 0507-34 Levo, that is able to inhibit and revert the fibrotic phenotypic switch. Immunohistochemistry (IHC) and immunofluorescence (IF) evalutations for E-cadherin and β-catenin were performed in three groups of C57BL/6 mice: Dextran Sulphate Sodium (DSS) colitis group, DSS+GED group and controls. In the same groups E-cadherin, β-catenin, p-GSK3-beta and GSK3-beta were evaluated by immunoblotting. In IHC and IF analysis, was observed a significant decrease of E-cadherin and β-catenin expression in DSS group compared to control. GED administration was able to revert the DSS effect by increasing the expression of both proteins to levels similar to the control. Comparable results were obtained by immunoblotting. Furthermore the immunoblotting assay showed that the known EMT inhibitor GSK3-beta is activated in the DSS+GED group suggesting that GSK3-beta activity is required for the restoration of the epithelial phenotype exerted by GED. Taken together, our results indicates that the EMT is another cause of colla- gen deposition in our mouse model and thus it could be a valid target for the devel- opment of a treatment for intestinal fibrosis., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Intestinal fibrosis is a common complication of inflammatory bowel disease (IBD) in which chronic inflammation leads to abnormal deposition of extracellular matrix components (ECM) causing obstruction and loss of function of the intestinal tract involved (1). Fibrogenesis is mainly regulates by trasforming growth factor (TGF) β/Smad pathway and a key antagonist of this signaling is represented by peroxisome proliferator-activated receptor (PPAR)-γ. As the anti-inflammatory drugs currently used in IBD are unable to improve intestinal fibrosis the exploration of new therapeutical approaches has now became crucial (2). Aim of this study is to evaluate the antifibrotic action of a novel PPARγ agonist, GED-0507-34-LEVO (GED), in colon fibrosis in mice. Chronic colitis and fibrosis were induced in C57BL/6 mice by administration of 2,5% (w/v) dextrane sulphate sodium (DSS) in drinking water for 5 days followed by 7 days of water for 3 cycles. Mice were divided into 3 groups: DSS, DSS+GED and control. 30mg/Kg/mice of GED was daily administrated by oral gavage starting from the second DSS cycle. Samples from colon were excised and processed to assess macroscopic lesions, histological and morphometrical aspects and immunohistochemical and immunoblotting analysis for TGFβ1, CTGF, collagen types I-III, Smad3,α-SMA. Evident shortening and dilation in the most of colons of DSS treated mice were observed. Macroscopic and microscopic findings were significantly improved in DSS mice+GED compared with control mice. The tissue levels of collagen and α-SMA, specific markers of fibrosis, resulted significantly increased in mice receving DSS compared to control mice, as well as the expression of TGFβ1, CTGF, Smad3. DSS+GED group showed reduced expression of all markers involved. GED significantly improves the intestinal fibrotic lesions in DSS chronic colitis murine model and controls the pivotal molecular events leading to fibrosis., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

Italian Journal of Anatomy and Embryology, Vol 115, No 1/2 (Supplement) 2010