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Supplementary Figure 4 from The Promyelocytic Leukemia Zinc Finger–MicroRNA-221/-222 Pathway Controls Melanoma Progression through Multiple Oncogenic Mechanisms

Supplementary Methods, Figure Legends 1-3, Table 1 from The Promyelocytic Leukemia Zinc Finger–MicroRNA-221/-222 Pathway Controls Melanoma Progression through Multiple Oncogenic Mechanisms

Supplementary Figure 2 from The Promyelocytic Leukemia Zinc Finger–MicroRNA-221/-222 Pathway Controls Melanoma Progression through Multiple Oncogenic Mechanisms

Supplementary Figure 1 from The Promyelocytic Leukemia Zinc Finger–MicroRNA-221/-222 Pathway Controls Melanoma Progression through Multiple Oncogenic Mechanisms

The incidence of cutaneous melanoma is steadily increasing. Although several molecular abnormalities have been associated with melanoma progression, the mechanisms underlying the differential gene expression are still largely unknown and targeted therapies are not yet available. Noncoding small RNAs, termed microRNAs (miR), have been recently reported to play important roles in major cellular processes, including those involved in cancer development and progression. We have identified the promyelocytic leukemia zinc finger (PLZF) transcription factor as a repressor of miR-221 and miR-222 by direct binding to their putative regulatory region. Specifically, PLZF silencing in melanomas unblocks miR-221 and miR-222, which in turn controls the progression of the neoplasia through down-modulation of p27Kip1/CDKN1B and c-KIT receptor, leading to enhanced proliferation and differentiation blockade of the melanoma cells, respectively. In vitro and in vivo functional studies, including the use of antisense “antagomir” oligonucleotides, confirmed the key role of miR-221/-222 in regulating the progression of human melanoma; this suggests that targeted therapies suppressing miR-221/-222 may prove beneficial in advanced melanoma. [Cancer Res 2008;68(8):2745–10]

Supplementary Figure 3 from The Promyelocytic Leukemia Zinc Finger–MicroRNA-221/-222 Pathway Controls Melanoma Progression through Multiple Oncogenic Mechanisms

ACTA1 gene encodes the skeletal muscle alpha-actin, the core of thin filaments of the sarcomere. ACTA1 mutations are responsible of several muscle disorders including nemaline, cores, actin aggregate myopathies and fiber-type disproportion. We report clinical, muscle imaging, histopatological and genetic data of an Italian family carrying a novel ACTA1 mutation. All affected members showed a late-presenting, diffuse muscle weakness with sternocleidomastoideus and temporalis atrophy. Mild dysmorphic features were also detected. The most affected muscles by muscle MRI were rectus abdominis, gluteus minimus, vastus intermedius and both gastrocnemii. Muscle biopsy showed the presence of nemaline bodies with several unusual dark areas at Gomori Trichrome, corresponding to unstructured cores with abundant electrodense material by electron microscopy. The molecular analysis revealed missense variant c.148G>A; p.(Gly50Ser) in the exon 3 of ACTA1, segregating with affected members in the family. We performed a functional essay of fibre contractility showing a higher pCa50 (a measure of the calcium sensitivity of force) of type 1 fibers compared to control subjects’ type 1 muscle fibers. Our findings expand the clinico-pathological spectrum of ACTA1-related congenital myopathies and the genetic spectrum of core-rod myopathies.

"Cast nephropathy" (CN) is a pathological feature of myeloma kidney, also seen to a lesser extent in the context of severe nephrotic syndrome from non-haematological diseases. The name relates to obstruction of distal tubules by "casts" of luminal proteins concentrated by intensive water reabsorption resulting from dehydration or high-dose diuretics. Filtered proteins form complexes with endogenous tubular Tamm-Horsfall glycoprotein. The resulting gel further slows or stops luminal flow upon complete obstruction of distal convoluted tubules and collecting ducts. Thus, a tubular obstructive form of acute kidney injury (AKI) is a common consequence of CN. The pathogenesis of CN will be reviewed in light of recent advances in the understanding of monoclonal disorders of B lymphocytes, leading to the release of immunoglobulin components (free light chains, FLC) into the bloodstream and their filtration across the glomerular basement membrane. Treatment aiming at reduction of the circulating burden of FLC may help recovery of renal function in a fraction of these patients, besides filling the void between the onset of AKI, histopathological diagnosis, and full response to pharmacologic treatment.

Overcoming the systemic administration of chemotherapy to reduce drug toxicity and the application of personalised medicine are two of the major challenges in the treatment of cancer. To this aim, efforts are focused on finding novel nanomaterials for the targeted administration of drugs and bioactive molecules in the tumor sites. DNA-based hydrogels are promising candidates for these applications. However, while such materials are fairly known from a structural and physical standpoint, their effects on cell cultures are far less investigated. Here, we studied the biological response of three different cell lines (clear cell renal cell carcinoma 786-O, lung adenocarcinoma H1975 and glioblastoma U87MG) to the treatment with DNA-GEL - a DNA-based hydrogel composed of interacting DNA nanostars. Additionally, we investigated the structural modification of DNA-GELs under cell culture conditions. The results we collected show a cell type specificity of the response, with interesting implications for future applications.

Background: Human Dachshund homologue 1 (DACH1) is involved in carcinogenesis with opposite roles reported in different tumor types. Four alternatively spliced transcripts encoding different DACH1 isoforms were described but their specific role in human cancers is still unknown. Prostate cancer (PCa) is a heterogeneous disease with a very wide variability, so there is yet a relevant need to find new diagnostic and therapeutic biomarkers to make a safe clinical evaluation. It is well known that the differential expression of protein isoforms can induce distinct transcriptional programs with opposing effects on tumor progression and therapy. Thus, in this study we aimed to correlate the functional role of DACH1 with its splicing variants expression in PCa.Methods: The expression and functional role of DACH1 splicing variants in PCa were investigated using tumor (PC3) and normal (RWPE-1) cell lines, patient biopsies and TCGA dataset. Flow-cytometry, western blots and RT-qPCR were used for in vitro molecular characterization; invasion, adhesion, clonogenic assays and cell cycle analysis for functional characterization. Immunohystochemistry and western blot were performed on human PCa biopsies.Results: RT-qPCR and Western Blot revealed that DACH1-positive PC3 cells predominantly expressed DACH1 variant 4 (DACH1-v4), whereas RWPE-1 cells mostly expressed DACH1 variant 3. Stable DACH1-v4 overexpression enhanced the transformed phenotype of PC3 cells by inducing proliferation, colony formation, invasion ability, epithelial to mesenchymal transition. Given its intrinsic radioresistance, PCa frequently recurs after radiotherapy. Of note, DACH1-v4-overexpressing PC3 cells displayed higher radioresistant behavior. Overexpression of DACH1-v4 also transformed RWPE-1 cells to oncogenic phenotype, suggesting a pro-oncogenic role for this specific isoform. PCa biopsies analysis showed DACH1 nuclear staining enhanced throughout the increase of the tumor grade. Remarkably, tumor glands were found to express a long DACH1 variant, while normal prostate tissue expressed the short DACH1 isoform, in line with data from TCGA-PRAD analysis and our data in RWPE-1 cells. Conclusions: Our findings highlight the oncogenic role of DACH1-v4 in PCa and suggest that the longer DACH1 variants could be associated to pro-tumor function, while the shortest DACH1 variant would perform tumor suppression. The expression of specific DACH1 isoforms could represent a novel diagnostic/prognostic marker in PCa.

BACKGROUND Radiomic models outperform clinical data for outcome prediction in high-grade gliomas (HGG). Many machine learning (ML) radiomic models have been developed, mostly employing single classifiers with variable results. However, comparative analyses of different ML models for clinically-relevant tasks are lacking in the literature. OBJECTIVE We aimed to compare well-established ML learning classifiers, including single and ensemble learners, to predict clinically-relevant tasks for HGG: overall survival (OS), isocitrate dehydrogenase (IDH) mutation, O-6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation, epidermal growth factor receptor (EGFR) amplification and Ki-67 expression in HGG patients, based on radiomic features from conventional and advanced MRI. Our objective was to identify the best algorithm for each task in terms of accuracy of the prediction performance. METHODS 156 adult patients with pathologic diagnosis of HGG were included. Three tumoral regions were manually segmented: contrast-enhancing tumor, necrosis and non-enhancing tumor. Radiomic features were extracted with a custom version of Pyradiomics, and selected through Boruta algorithm. A Grid Search algorithm was applied when computing 4 times K-fold cross validation (K=10) to get the highest mean and lowest spread of accuracy. Model performance was assessed as Area Under The Curve-Receiver Operating Characteristics (AUC-ROC). RESULTS Ensemble classifiers showed the best performance across tasks. xGB obtained highest accuracy for OS (74.5%), AB for IDH mutation (88%), MGMT methylation (71,7%), Ki-67 expression (86,6%), and EGFRvIII amplification (81,6%). CONCLUSIONS Best performing features shed light on possible correlations between MRI and tumor histology.

BACKGROUND The aim of this study was to report the first case of acute facial allograft transplantation (facial allograft transplantation) failure with allograft removal and autologous free-flap reconstruction. METHODS A 49-year-old female patient affected by neurofibromatosis type 1 with a massive neurofibroma infiltrating the whole left hemiface was planned for FAT for the left hemiface including the auricle, all skin and soft tissues from the temporal region, periorbital and nasal region, and up to the perioral area. The maxillary process of the zygomatic bone, left hemimaxilla, and hemimandible from contralateral parasyphysis to the incisura mandibulae were also included. RESULTS Total surgical time was 26 hours. There were 2 intraoperative arterial thromboses that were solved with new anastomoses and sufficient flap perfusion. On postoperative day 2, the allograft became pale with suspected arterial occlusion and the patient returned to the operative room for exploration no flow into the FAT was found. The allograft was removed and the recipient site reconstructed with a skin-grafted composite left latissimus dorsi-serratus anterior flap. CONCLUSIONS Hyperacute loss of FAT is a very dramatic event, and the activation of a backup surgical plan is crucial to save patient's life, give a reasonable temporary reconstruction, and return on the waiting-list for a second face transplantation.

Background A prostate cancer diagnosis is based on biopsy sampling that is an invasive, expensive procedure, and doesn’t accurately represent multifocal disease. Methods To establish a model using plasma miRs to distinguish Prostate cancer patients from non-cancer controls, we enrolled 600 patients histologically diagnosed as having or not prostate cancer at biopsy. Two hundred ninety patients were eligible for the analysis. Samples were randomly divided into discovery and validation cohorts. Results NGS-miR-expression profiling revealed a miRs signature able to distinguish prostate cancer from non-cancer plasma samples. Of 51 miRs selected in the discovery cohort, we successfully validated 5 miRs (4732-3p, 98-5p, let-7a-5p, 26b-5p, and 21-5p) deregulated in prostate cancer samples compared to controls (p ≤ 0.05). Multivariate and ROC analyses show miR-26b-5p as a strong predictor of PCa, with an AUC of 0.89 (CI = 0.83–0.95;p To distinguish between low and high-grade prostate cancer, we found that miR-4732-3p levels were significantly higher; instead, miR-26b-5p and miR-98-5p levels were lower in low-grade compared to the high-grade group (p ≤ 0.05). Combining miR-26b-5p and miR-4732-3p we have the highest diagnostic accuracy for high-grade prostate cancer patients, (AUC = 0.80; CI 0,69-0,873). Conclusions Noninvasive diagnostic tests may reduce the number of unnecessary prostate biopsies. The 2-miRs-diagnostic model (miR-26b-5p and miR-98-5p) and the 2-miRs-grade model (miR-26b-5p and miR-4732-3p) are promising minimally invasive tools in prostate cancer clinical management.

Isocitrate dehydrogenase (IDH) mutant and wildtype glioblastoma multiforme (GBM) often show overlapping features on magnetic resonance imaging (MRI), representing a diagnostic challenge. Deep learning showed promising results for IDH identification in mixed low/high grade glioma populations, however, a GBM-specific model is still lacking in the literature. Our aim was to develop a GBM-tailored deep-learning model for IDH prediction by applying convoluted neural networks (CNN) on multiparametric MRI. We selected 100 adult patients with pathologically demonstrated WHO grade IV gliomas and IDH testing. MRI sequences included: MPRAGE, T1, T2, FLAIR, rCBV and ADC. The model consisted of a 4-block 2D CNN, applied to each MRI sequence. Probability of IDH mutation was obtained from the last dense layer of a softmax activation function. Model performance was evaluated in the test cohort considering categorical cross-entropy loss (CCEL) and accuracy. Calculated performance was: rCBV (accuracy 83%, CCEL 0.64), T1 (accuracy 77%, CCEL 1.4), FLAIR (accuracy 77%, CCEL 1.98), T2 (accuracy 67%, CCEL 2.41), MPRAGE (accuracy 66%, CCEL 2.55). Lower performance was achieved on ADC maps. We present a GBM-specific deep-learning model for IDH mutation prediction, with a maximal accuracy of 83% on rCBV maps. Highest predictivity achieved on perfusion images possibly reflects the known link between IDH and neoangiogenesis through the hypoxia inducible factor.

Monoclonal gammopathies (MG) encompass a variety of disorders related to clonal expansion and/or malignant transformation of B lymphocytes. Deposition of free immunoglobulin (Ig) components (light or heavy chains, LC/HC) within the kidney during MG may result over time in multiple types and degrees of injury, including acute kidney injury (AKI). AKI is generally a consequence of tubular obstruction by luminal aggregates of LC, a pattern known as “cast nephropathy”. Monoclonal Ig LC can also be found as intracellular crystals in glomerular podocytes or proximal tubular cells. Proliferative glomerulonephritis with monoclonal Ig deposits is another, less frequent form of kidney injury with a sizable impact on renal function. Hypercalcemia (in turn related to bone reabsorption triggered by proliferating plasmacytoid B cells) may lead to AKI via functional mechanisms. Pharmacologic treatment of MG may also result in additional renal injury due to local toxicity or the tumor lysis syndrome. The present review focuses on AKI complicating MG, evaluating predictors, risk factors, mechanisms of damage, prognosis, and options for treatment.

Novel DNA materials promise unpredictable perspectives for applications in cell biology. The realization of DNA-hydrogels built by a controlled association of DNA nanostars, whose binding can be tuned with minor changes in the nucleotide sequences, has been recently described. DNA hydrogels, with specific gelation properties that can be reassambled in desired culture media supplemented with drugs, RNA, DNA molecules and other bioactive compounds offer the opportunity to develop a novel nanomaterial for the delivery of single or multiple drugs in tumor tissues as an innovative and promising strategy. We provide here a comprehensive description of different, recently realized DNA- gels with the perspective of stimulating their biomedical application. Finally, we discuss the possibility to design sophisticated 3D tissue-like DNA-gels incorporating cell spheroids or single cells for the assembly of a novel kind of cellular matrix as a preclinical investigation for the implementation of tools for in vivo delivery of bioactive molecules.

In human prostate cancer (PCa), the neuroendocrine cells, expressing the prostate cancer stem cell (CSC) marker CD44, may be resistant to androgen ablation and promote tumor recurrence. During the study of heterogeneity of the highly aggressive neuroendocrine PCa cell lines PC3 and DU-145, we isolated and expanded in vitro a minor subpopulation of very small cells lacking CD44 (CD44neg). Unexpectedly, these sorted CD44neg cells rapidly and spontaneously converted to a stable CD44high phenotype specifically expressing the CD44v8-10 isoform which the sorted CD44high subpopulation failed to express. Surprisingly and potentially interesting, in these cells expression of CD44v8-10 was found to be induced in stem cell medium. CD44 variant isoforms are known to be more expressed in CSC and metastatic cells than CD44 standard isoform. In agreement, functional analysis of the two sorted and cultured subpopulations has shown that the CD44v8-10pos PC3 cells, resulting from the conversion of the CD44neg subpopulation, were more invasive in vitro and had a higher clonogenic potential than the sorted CD44high cells, in that they produced mainly holoclones, known to be enriched in stem-like cells. Of interest, the CD44v8-10 is more expressed in human PCa biopsies than in normal gland. The discovery of CD44v8-10pos cells with stem-like and invasive features, derived from a minoritarian CD44neg cell population in PCa, alerts on the high plasticity of stem-like markers and urges for prudency on the approaches to targeting the putative CSC.

Inadequate uterine receptivity is responsible for two-third of implanting failures. Aim of the study was to investigate the role of epithelial adherence and tight-junction molecules expressed by human endometrium in predicting womens' fertility outcome. A total of 76 consecutive women, including 24 fertile (G1), 40 primary infertile (G2), and 12 recurrent pregnancy loss (RPL, G3) women, who underwent diagnostic hysteroscopy plus endometrial biopsy between 2005 and 2016 at the Gynecology Division of Sant'Andrea Hospital, Sapienza University of Rome, in Italy, were retrospectively identified and included into the study. Endometrial biopsies were assessed for the immunohistochemical expression of beta-catenin (β-catenin), E-cadherin and K-cadherin biomarkers. Expression profiles were compared between the three groups of patients and were correlated with patients' fertility outcome. In infertile patients there was a significant lower endometrial expression of β-catenin (

Background Kidney cancer and clear cell renal carcinoma (ccRCC) are the 16th most common cause of death worldwide. ccRCC is often metastasized at diagnosis, and surgery remains the main treatment; therefore, early diagnosis and new therapeutic strategies are highly desirable. KAT inhibitor CPTH2 lowers histone H3 acetylation and induces apoptosis in colon cancer and cultured cerebellar granule neurons. In this study, we have evaluated the effects of CPTH2 on ccRCC 786-O cell line and analyzed drug targets expressed in ccRCC tumor tissues at different grade. Results CPTH2 decreases cell viability, adhesion, and invasiveness in ccRCC cell line 786-O. It shows preferential inhibition for KAT3B-p300 with hypoacetilating effects on histone H3 at specific H3-K18. Immunohistochemical analysis of 70 ccRCC tumor tissues compared with peritumoral normal epithelium showed a statistical significant reduction of p300/H3AcK18 paralleled by an increase of H3AcK14 in G1 grade and an opposed trend during tumor progression to worst grades. In this study, we demonstrate that these marks are CPTH2 targets and significative prognosticators of low-grade ccRCC tumor. Conclusions ccRCC is substantially insensitive to current therapies, and the efficacy of clinical treatment is dependent on the dissemination stage of the tumor. The present study shows that CPTH2 is able to induce apoptosis and decrease the invasiveness of a ccRCC cell line through the inhibition of KAT3B. In a tumor tissue analysis, we identified new prognosticator marks in grade G1 ccRCC tumors. Low KAT3B/H3AcK18 vs. high H3AcK14 were found in G1 while an opposed trend characterized tumor progression to worst grades. Our collected results suggest that CPTH2 reducing KAT3B and H3AcK18 can be considered a promising candidate for counteracting the progression of ccRCC tumors. Electronic supplementary material The online version of this article (10.1186/s13148-018-0473-4) contains supplementary material, which is available to authorized users.

Recently, a number of reports have described dominant C3 deposits in renal biopsies of patients with infection-related glomerulonephritis (GN). While acute post-infectious GN and membranoproliferative GN are commonly characterized by immune deposits containing C3 and/or C4, the absence of immunoglobulin (Ig) and/or immune complexes at light or electron microscopy is a rather unusual observation. Dominant C3 deposition is believed to result from the alternative pathway of complement activation via the C3bBb "tickover" convertase. The actual occurrence of C3 glomerulopathy could be underestimated, since infection-related GN often quickly subsides without the need for a renal biopsy. A more thorough understanding of the pathways that lead to complement assembly and deposition within the kidney is needed to support a new classification of complement-related lesions, including entities such as dense deposit disease, (atypical) hemolytic-uremic syndrome, dominant C1q, CFHR5, C4d, and C3 glomerulopathies. We will briefly review recent work in this area, focusing on GN with selective complement C3 deposits.

// Cosimo De Nunzio 1 , Simona Giglio 2 , Antonella Stoppacciaro 2 , Mauro Gacci 4 , Roberto Cirombella 2 , Emidio Luciani 2 , Andrea Tubaro 1 and Andrea Vecchione 2, 3 1 Urology Units, Department of Clinical and Molecular Medicine, Ospedale Sant’Andrea, Sapienza University, Rome, Italy 2 Surgical Pathology Units, Department of Clinical and Molecular Medicine, Ospedale Sant’Andrea, Sapienza University, Rome, Italy 3 Department of Cancer Biology and Genetics/CCC, The Ohio State University, Columbus, OH, USA 4 Department of Urology, Careggi Hospital, Firenze, Italy Correspondence to: Cosimo De Nunzio, email: cosimodenunzio@virgilio.it Keywords: autophagy, BPH, LUTS, inflammation, prostate Received: December 02, 2016 Accepted: January 11, 2017 Published: February 07, 2017 ABSTRACT Autophagy is a conserved evolutionary process that allows cells to maintain macromolecular synthesis and energy homeostasis during starvation and stressful conditions. We prospectively evaluated the relationship between autophagy and prostatic inflammation in a series of transurethral prostatic resection samples. Inflammatory infiltrates were defined according to the standardized classification of chronic prostatitis of the National Institute of Health. The inflammatory score (IS score) was calculated. High IS score was defined as ≥7. Each sample was stained for anti-LC3B and for anti-P62/SQSTM1 and scored. High p62 or LC3B percentage was defined as >25%, whereas low was defined as

Most frequent causes of androgenic manifestation are Cushing's syndrome, PCO, benign and malignant androgen-secreting non adrenal tumors and iatrogenic hirsutism. Hyperplasia or neoplasms of ectopic adrenocortical gland are rare. We report a case of a 63-year old female with hirsutism and alopecia. Laboratory data highlighted increased levels of androgens. Diagnostic imaging revealed normal morphology of adrenocortical gland and ovaries. In view of the clinical picture and suspected diagnosis of extra-adrenal cause, she underwent bilateral salpingo-oophorectomy. Histologic examination showed an ectopic adrenal gland with adenoma in the ovarian and peri-ovarian tissue. At six months of follow up, the patients has no sign of hyperandrogenism. In case of hyperandrogenism in postmenopausal women and in the absence of the adrenocortical gland abnormality, ovarian origin should be considered in the differential diagnosis.

The aim of this retrospective study is to investigate the prevalence and pathological features of kidney inflammatory nephropathies diagnosed in Sant'Andrea Hospital, from January 2003 to April 2015. In this period, 246 kidney biopsies have been diagnosed in our Hospital. Excluding cases of kidney neoplasms and non-diagnostic samples, 195 cases were reviewed. Primary glomerulonephritis (GN) is the most common diagnosis. Among these, Membranous GN represents the majority of cases (20.4%), followed by IgA Nephropathy (12.7%). The higher prevalence of Membranous GN than IgA Nephropathy represents a difference between our study and national and international kidney biopsies registries. It can be considered a consequence of the average age of patients undergoing renal biopsy in our center (54,1 years). Patients with Diabetic Nephropathy are 1.5%. 10 out of 195 cases (5.1%) show end stage renal disease. This epidemiological study evaluates the prevalence of various kidney diseases in our database, the biopsy policy of SantAndrea Hospital and compares our results with national and international renal biopsies registries.

Background: Dysregulated innate immune responses play an important role in inflammatory bowel disease (IBD). NKG2D innate immunity receptor is a major sensor of tissue damage that, by recognizing multiple stress-induced, cell-associated ligands (MIC-A/B and ULBP1-5), potentiates the effector functions of “innate-like” (γ/δ TcR+, and natural killer receptor+ [NKR+]) T-cell populations. We analyzed the representivity, NKG2D/ligand expression pattern, and functional ability of the major innate immunity cell populations in pediatric IBD patients. Methods: We analyzed 41 Crohn's disease (CD) patients, 33 ulcerative colitis (UC) patients, and 51 age-matched non-IBD controls. The expression of NKG2D and its ligands, interferon-gamma (IFN-γ) production, and cytotoxic granule release were assessed by immunostaining and multiparameter cytofluorimetric analysis on circulating and mucosal mononuclear subsets; the inflammatory infiltrate was also characterized by immunohistochemistry. Results: The expression pattern of NKG2D receptor and its ligands on mucosal and circulating innate immunity populations is severely disturbed in IBD; NKG2D and ligands are upregulated on immune infiltrate in both CD and UC active lesions; receptor/ligand upregulation also occurs on circulating leukocyte populations, where it depends on both disease activity and type (UC vs. CD). Finally, the frequency and effector capability of peripheral blood “innate-like” T-cell populations are also altered in IBD patients. Conclusions: The circulating and mucosal innate immunity compartment is phenotypically and functionally altered in pediatric IBD; some alterations may represent a distinctive feature of the pediatric disease condition. The disturbance of NKG2D/ligand pathway may play a role in sustaining immune activation which leads to chronic inflammatory tissue damage. (Inflamm Bowel Dis 2012)

Purpose: The formation of G-quadruplex structures at telomeric DNA sequences blocks telomerase activity, offering an original strategy to design and develop new antitumor agents. The pentacyclic acridinium salt RHPS4 is one of the most effective and selective G4 ligands able to rapidly disrupt telomere architecture, resulting in apoptosis of cancer cells. Here, we studied the therapeutic index of RHPS4 and its integration with chemotherapeutics in preclinical model of solid tumors. Experimental Design: The antitumoral activity of RHPS4 was evaluated on human xenografts of different histotypes and compared with that of standard antineoplastic agents. Moreover, the effect of RHPS4/chemotherapeutics combinations on cell survival was studied and the most favorable combination was evaluated on tumor-bearing mice. Results: RHPS4 was active in vivo as single agent and showed a high therapeutic efficacy when compared with conventional drugs. Moreover, RHPS4 had antitumoral activity in human melanoma xenografts inherently resistant to chemotherapy and exhibited antimetastatic activity. RHPS4 also showed a strong synergistic interaction with camptothecins and this effect was strictly dependent on the drug sequence employed. Treatment of mice with irinotecan followed by RHPS4 was able to inhibit and delay tumor growth and to increase mice survival. Conclusions: Our data show that RHPS4 has a good pharmacodynamic profile and in combination therapy produces a strong antitumoral activity, identifying this drug as promising agent for clinical development.

Functional telomeres are required for the replicability of cancer cells. The G-rich strand of telomeric DNA can fold into a 4-stranded structure known as the G-quadruplex (G4), whose stabilization alters telomere function limiting cancer cell growth. Therefore, the G4 ligand RHPS4 may possess antitumor activity. Here, we show that RHPS4 triggers a rapid and potent DNA damage response at telomeres in human transformed fibroblasts and melanoma cells, characterized by the formation of several telomeric foci containing phosphorylated DNA damage response factors gamma-H2AX, RAD17, and 53BP1. This was dependent on DNA repair enzyme ATR, correlated with delocalization of the protective telomeric DNA-binding protein POT1, and was antagonized by overexpression of POT1 or TRF2. In mice, RHPS4 exerted its antitumor effect on xenografts of human tumor cells of different histotype by telomere injury and tumor cell apoptosis. Tumor inhibition was accompanied by a strong DNA damage response, and tumors overexpressing POT1 or TRF2 were resistant to RHPS4 treatment. These data provide evidence that RHPS4 is a telomere damage inducer and that telomere disruption selectively triggered in malignant cells results in a high therapeutic index in mice. They also define a functional link between telomere damage and antitumor activity and reveal the key role of telomere-protective factors TRF2 and POT1 in response to this anti-telomere strategy.

This in vitro study evaluated the ability of prostate adenocarcinoma (ADC) cells to induce radiation-induced bystander effect (RIBE) exploring the factors that may be responsible and affect its intensity. The idea was to mimic a strong, clinically applicable RIBE that could lead to the development of innovative approaches in modern radiotherapy of prostate cancer, especially for those patients with hormone-refractory ADC in which radiotherapy might have a limited role.Two human prostate cancer cell lines of different differentiation, PC-3 and DU-145, have been irradiated using wide range of doses to obtain radiation-conditioned medium (RCM), which was used to treat the unirradiated cells and to evaluate the cytokines level. Using a trypan blue dye exclusion method, cell growth was assessed.Prostate ADC cells were able to induce RIBE; intensity depended on dose and cell differentiation. RIBE intensity of DU-145 was not correlated with the cytokines level, while for PC-3 Interleukin-6 (IL-6) correlates with strongest RIBE induced by 20 Gy.RIBE can be manipulated by modifying radiation dose and depends on cell differentiation status. IL-6 correlates with RIBE after exposure of PC-3 to a very high dose of radiation, thus indicates its possible involvement in bystander signaling.

Toll-like receptors (TLRs) are a family of highly conserved transmembrane proteins expressed in epithelial and immune cells that recognize pathogen associated molecular patterns. Besides their role in immune response against infections, numerous studies have shown an important role of different TLRs in cancer, indicating these receptors as potential targets for cancer therapy. We previously demonstrated that the activation of TLR3 by the synthetic double-stranded RNA analogue poly I:C induces apoptosis of androgen-sensitive prostate cancer (PCa) LNCaP cells and, much less efficiently, of the more aggressive PC3 cell line. Therefore, in this study we selected LNCaP cells to investigate the mechanism of TLR3-mediated apoptosis and the in vivo efficacy of poly I:C-based therapy. We show that interferon regulatory factor-3 (IRF-3) signalling plays an essential role in TLR3-mediated apoptosis in LNCaP cells through the activation of the intrinsic and extrinsic apoptotic pathways. Interestingly, hardly any apoptosis was induced by poly I:C in normal prostate epithelial cells RWPE-1. We also demonstrate for the first time the direct anticancer effect of poly I:C as a single therapeutic agent in a well-established human androgen-sensitive PCa xenograft model, by showing that tumour growth is highly impaired in poly I:C-treated immunodeficient mice. Immunohistochemical analysis of PCa xenografts highlights the antitumour role of poly I:C in vivo both on cancer cells and, indirectly, on endothelial cells. Notably, we show the presence of TLR3 and IRF-3 in both human normal and PCa clinical samples, potentially envisaging poly I:C-based therapy for PCa.

A 69-year-old woman presented a dropped head, caused by severe neck extensor weakness that had started two years before. She had also developed a mild degree of dysphagia, rhinolalia, eyelid ptosis and proximal limb weakness during the last months. EMG revealed myopathic changes. Muscle MRI detected fatty infiltration in the posterior neck muscles and tongue. Muscle biopsy revealed fiber size variations, sporadic rimmed vacuoles, small scattered angulated fibers and a patchy myofibrillar network. Genetic analysis revealed homozygous (GCN)11 expansions in the PABPN1 gene that were consistent with recessive oculopharyngeal muscular dystrophy (OPMD). There are a few reports of the recessive form, which has a later disease onset with milder symptoms and higher clinical variability than the typical dominantly inherited form. This patient, who is the first Italian and the eighth worldwide reported case of recessive OPMD, is also the first case of OPMD with dropped-head syndrome, which thus expands the clinical phenotype of recessive OPMD.