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Aggressive pituitary tumors are rare but difficult to manage, as there is no effective chemotherapy to restrict their growth and cause their shrinkage. Within these tumors, growth-promoting cascades, like the PI3K/mTOR pathway, appear to be activated. We tested the efficacy of two inhibitors of this pathway, NVP-BKM120 (Buparlisib; pan-PI3K) and NVP-BEZ235 (dual PI3K/mTOR), both in vitro on immortalized pituitary tumor cells (GH3) and on primary cell cultures of human pituitary tumors and in vivo on a rat model of prolactin (PRL) tumors (SMtTW3). In vitro, NVP-BEZ235 had a potent apoptotic and cytostatic effect that was characterized by decreased cyclin D/E and Cdk4/2 protein levels and subsequent accumulation of cells in G1. In vivo, the effect was transient, with a decrease in mitotic index and increase in apoptosis; long-term treatment had no significant inhibitory effect on tumor growth. In contrast, while NVP-BKM120 had little effect in vitro, it dramatically limited tumor growth in vivo. Increased Akt phosphorylation observed only in the NVP-BEZ235–treated tumors may explain the differential response to the two inhibitors. Primary cell cultures of human PRL pituitary tumors responded to NVP-BEZ235 with reduced cell viability and decreased hormone secretion, whereas NVP-BKM120 had little effect. Altogether, these results show a potential for PI3K inhibitors in the management of aggressive pituitary tumors. Mol Cancer Ther; 15(6); 1261–70. ©2016 AACR.

Tolerance of NVP-BKM120 treatment on rats. Growth curve (A) or glycemia (B) of rats before and during 4 weeks of NVP-BKM120 treatment 5 mg/kg/d, 5 days a week or control.

Primer pairs used for real-time quantitative RT-PCR.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly and aggressive cancer. Understanding mechanisms that drive preneoplastic pancreatic lesions is necessary to improve early diagnostic and therapeutic strategies. Mutations and inactivation of activin-like kinase (ALK4) have been demonstrated to favor PDAC onset. Surprisingly, little is known regarding the ligands that drive ALK4 signaling in pancreatic cancer or how this signaling pathway limits the initiation of neoplastic lesions. In this study, data mining and histologic analyses performed on human and mouse tumor tissues revealed that activin A is the major ALK4 ligand that drives PDAC initiation. Activin A, which is absent in normal acinar cells, was strongly induced during acinar-to-ductal metaplasia (ADM), which was promoted by pancreatitis or the activation of KrasG12D in mice. Activin A expression during ADM was associated with the cellular senescence program that is induced in precursor lesions. Blocking activin A signaling through the use of a soluble form of activin receptor IIB (sActRIIB-Fc) and ALK4 knockout in mice expressing KrasG12D resulted in reduced senescence associated with decreased expression of p21, reduced phosphorylation of H2A histone family member X (H2AX), and increased proliferation. Thus, this study indicates that activin A acts as a protective senescence-associated secretory phenotype factor produced by Kras-induced senescent cells during ADM, which limits the expansion and proliferation of pancreatic neoplastic lesions.Significance:This study identifies activin A to be a beneficial, senescence-secreted factor induced in pancreatic preneoplastic lesions, which limits their proliferation and ultimately slows progression into pancreatic cancers.

Supplemental Table 1. List of used Primary antibodies.

Supplementary Material & figure legends

Pituitary tumours are benign neoplasms that derive from hormone-producing cells of the pituitary gland. While medical treatments have emerged for most subtypes, gonadotroph tumours that express follicle-stimulating hormone (FSH) and/or luteinizing hormone still lack therapeutic options apart from surgery and radiotherapy. Activin ligands are physiological regulators of production and secretion of FSH by gonadotroph cells, but their role in gonadotroph tumourigenesis remains little explored. Using the LβT2 mouse gonadotroph cell line which produces FSH under activin stimulation, we first tested whether subcutaneous xenografts of LβT2 cells resulted in tumour formation in Rag2KO mice. Histological analysis confirmed the presence of LβT2 tumours with endothelial cells and macrophages in their microenvironment. FSH expression was found in a subset of clusters of LβT2 cells in the tumours. We subsequently addressed the consequences of targeting activin signalling via injection of a soluble activin decoy receptor (sActRIIB-Fc). sActRIIB-Fc treatment resulted in significantly decreased LβT2 tumour volume. Reduced Smad2 phosphorylation as well as inhibition of tumour-induced FSH production confirmed the efficient targeting of activin-downstream signalling in treated tumours. More interestingly, treated tumours showed significantly fewer endothelial cells associated with reduced Vegfa expression. In vitro treatment of LβT2 cells with sActRIIB-Fc had no effect on cell proliferation or apoptosis, but Vegfa expression was inhibited, pointing to a likely paracrine effect of LβT2 cells on endothelial cells through activin-mediated Vegfa regulation. Further in vitro and in vivo studies are now needed to pinpoint the exact roles of activin signalling in these processes prior to translating these observations to the clinic.

1AbstractSingle cell transcriptomics has recently seen a surge in popularity, leading to the need for data analysis pipelines that are reproducible, modular, and interoperable across different systems and institutions.To meet this demand, we introducescAN1.0, a processing pipeline for analyzing 10X single cell RNA sequencing data.scAN1.0is built using the Nextflow DSL2 and can be run on most computational systems. The modular design of Nextflow pipelines enables easy integration and evaluation of different blocks for specific analysis steps.We demonstrate the usefulness ofscAN1.0by showing its ability to examine the impact of the mapping step during the analysis of two datasets: (i) a 10X scRNAseq of a human pituitary gonadotroph tumor dataset and (ii) a murine 10X scRNAseq acquired on CD8 T cells during an immune response.

Folliculostellate cells are S100B-expressing cells with numerous functions in the normal anterior pituitary. These cells have also been identified in pituitary neuroendocrine tumours (PitNETs), where their precise role remains elusive. Here, we aimed to build a refined cartography of S100B-expressing cells to characterise their interpatient and intratumoural spatial distribution, and to start identifying their potential functions in PitNETs. High-throughput histological analysis of S100B-stained tumour sections of 54 PitNETs revealed a significant decrease in S100B + cells in PitNETs compared to the normal anterior pituitary. A Ki67 index ≥ 3, a mitosis count > 2/10 per high power fields, and a proliferative status, were all associated with fewer S100B + cells in gonadotroph tumours. Gonadotroph tumours also showed interpatient and intratumoural heterogeneity in the spatial distribution of S100B + cells. The existence of an intratumoural heterogeneity was further confirmed by the incorporation to our spatial analysis of additional markers: Ki67, FSH, LH, ERα and SSTR2. The tumour areas with fewer S100B + cells displayed a higher percentage of Ki67 + cells, whereas strong positive correlations were observed between S100B + , FSH + , and ERα + cells. Such spatial associations suggest that S100B + folliculostellate cells could play a role in gonadotroph tumorigenesis, and may contribute to the maintenance of tumour cells in a low proliferating, FSH + /ERα + differentiated state. Albeit, further in-depth functional studies are required to decipher the mechanisms underlying these spatial associations and to potentially identify a therapeutic use.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly and aggressive cancer. Understanding mechanisms that drive preneoplastic pancreatic lesions is necessary to improve early diagnostic and therapeutic strategies. Mutations and inactivation of activin-like kinase (ALK4) have been demonstrated to favor PDAC onset. Surprisingly, little is known regarding the ligands that drive ALK4 signaling in pancreatic cancer or how this signaling pathway limits the initiation of neoplastic lesions. In this study, data mining and histologic analyses performed on human and mouse tumor tissues revealed that activin A is the major ALK4 ligand that drives PDAC initiation. Activin A, which is absent in normal acinar cells, was strongly induced during acinar-to-ductal metaplasia (ADM), which was promoted by pancreatitis or the activation of KrasG12D in mice. Activin A expression during ADM was associated with the cellular senescence program that is induced in precursor lesions. Blocking activin A signaling through the use of a soluble form of activin receptor IIB (sActRIIB-Fc) and ALK4 knockout in mice expressing KrasG12D resulted in reduced senescence associated with decreased expression of p21, reduced phosphorylation of H2A histone family member X (H2AX), and increased proliferation. Thus, this study indicates that activin A acts as a protective senescence-associated secretory phenotype factor produced by Kras-induced senescent cells during ADM, which limits the expansion and proliferation of pancreatic neoplastic lesions. Significance: This study identifies activin A to be a beneficial, senescence-secreted factor induced in pancreatic preneoplastic lesions, which limits their proliferation and ultimately slows progression into pancreatic cancers.

Purpose Pituitary neuroendocrine tumors (PitNETs) are frequent intracranial neoplasms that present heterogenic characteristics. Little is known about the immune cell network that exists in PitNETs and its contribution to their aggressive behavior. Methods Here we combined flow cytometry, t-SNE analysis, and histological approaches to define the immune landscape of surgically resected PitNETs. Xenografts of rodent pituitary tumor cells and resected PitNETs were performed in Rag2KO mice, in combination with in vitro analysis aimed at dissecting the role of pituitary tumor-cells in monocyte recruitment. Results We report that gonadotroph PitNETs present an increased CD68+ macrophage signature compared to somatotroph, lactotroph, and corticotroph PitNETs. Transcriptomic and histological characterizations confirmed gonadotroph infiltrating macrophages expressed CD163, MRC-1, ARG1, and CSF1R M2 macrophage markers. Use of growth hormone (GH)3/GH4 somatotroph and LβT2/αT3.1 gonadotroph cells drove THP1 macrophage migration through respective expression of CCL5 or CSF1. Although both LβT2 and GH3 cells recruited F4/80 macrophages following their engraftment in mice, only LβT2 gonadotroph cells showed a capacity for M2-like polarization. Similar observations were performed on patient-derived xenografts from somatotroph and gonadotroph tumors. Analysis of clinical data further demonstrated a significant correlation between the percentage of CD68+ and CD163+ infiltrating macrophages and the invasive character of gonadotroph tumors. Conclusions Gonadotroph tumor drive the recruitment of macrophages and their subsequent polarization to an M2-like phenotype. More importantly, the association between infiltrating CD68+/CD163+ macrophages and the invasiveness of gonadotroph tumors points to macrophage-targeted immunotherapies being a potent strategy to limit the progression of gonadotroph PitNETs.

Prolactinoma represents the most frequent hormone-secreting pituitary tumours. These tumours appear in a benign form, but some of them can reach an invasive and aggressive stage through an unknown mechanism. Discovering markers to identify prolactinoma proliferative and invading character is therefore crucial to develop new diagnostic/prognostic strategies. Interestingly, members of the TGFβ-Activin/BMP signalling pathways have emerged as important actors of pituitary development and adult function, but their role in prolactinomas remains to be precisely determined. Here, using a heterotopic allograft model derived from a rat prolactinoma, we report that the Activins orphan type I receptor ALK7 is ectopically expressed in prolactinomas-cells. Through immunohistological approaches, we further confirm that normal prolactin-producing cells lack ALK7-expression. Using a series of human tumour samples, we show that ALK7 expression in prolactinomas cells is evolutionary conserved between rat and human. More interestingly, our results highlight that tumours showing a robust expression of ALK7 present an increased proliferation as address by Ki67 expression and retrospective analysis of clinical data from 38 patients, presenting ALK7 as an appealing marker of prolactinoma aggressiveness. Beside this observation, our work pinpoints that the expression of prolactin is highly heterogeneous in prolactinoma cells. We further confirm the contribution of ALK7 in these observations and the existence of highly immunoreactive prolactin cells lacking ALK7 expression. Taken together, our observations suggest that Activin signalling mediated through ALK7 could therefore contribute to the hormonal heterogeneity and increased proliferation of prolactinomas.

Although Men1 is a well-known tumour suppressor gene, little is known about the functions of Menin, the protein it encodes for. Since few years, numerous publications support a major role of Menin in the control of epigenetics gene regulation. While Menin interaction with MLL complex favours transcriptional activation of target genes through H3K4me3 marks, Menin also represses gene expression via mechanisms involving the Polycomb repressing complex (PRC). Interestingly, Ezh2, the PRC-methyltransferase that catalyses H3K27me3 repressive marks and Menin have been shown to co-occupy a large number of promoters. However, lack of binding between Menin and Ezh2 suggests that another member of the PRC complex is mediating this indirect interaction. Having found that ActivinB - a TGFβ superfamily member encoded by the Inhbb gene - is upregulated in insulinoma tumours caused by Men1 invalidation, we hypothesize that Menin could directly participate in the epigenetic-repression of Inhbb gene expression. Using Animal model and cell lines, we report that loss of Menin is directly associated with ActivinB-induced expression both in vivo and in vitro. Our work further reveals that ActivinB expression is mediated through a direct modulation of H3K27me3 marks on the Inhbb locus in Menin-KO cell lines. More importantly, we show that Menin binds on the promoter of Inhbb gene where it favours the recruitment of Ezh2 via an indirect mechanism involving Akt-phosphorylation. Our data suggests therefore that Menin could take an important part to the Ezh2-epigenetic repressive landscape in many cells and tissues through its capacity to modulate Akt phosphorylation.

Aggressive pituitary tumors are rare but difficult to manage, as there is no effective chemotherapy to restrict their growth and cause their shrinkage. Within these tumors, growth-promoting cascades, like the PI3K/mTOR pathway, appear to be activated. We tested the efficacy of two inhibitors of this pathway, NVP-BKM120 (Buparlisib; pan-PI3K) and NVP-BEZ235 (dual PI3K/mTOR), both in vitro on immortalized pituitary tumor cells (GH3) and on primary cell cultures of human pituitary tumors and in vivo on a rat model of prolactin (PRL) tumors (SMtTW3). In vitro, NVP-BEZ235 had a potent apoptotic and cytostatic effect that was characterized by decreased cyclin D/E and Cdk4/2 protein levels and subsequent accumulation of cells in G1. In vivo, the effect was transient, with a decrease in mitotic index and increase in apoptosis; long-term treatment had no significant inhibitory effect on tumor growth. In contrast, while NVP-BKM120 had little effect in vitro, it dramatically limited tumor growth in vivo. Increased Akt phosphorylation observed only in the NVP-BEZ235–treated tumors may explain the differential response to the two inhibitors. Primary cell cultures of human PRL pituitary tumors responded to NVP-BEZ235 with reduced cell viability and decreased hormone secretion, whereas NVP-BKM120 had little effect. Altogether, these results show a potential for PI3K inhibitors in the management of aggressive pituitary tumors. Mol Cancer Ther; 15(6); 1261–70. ©2016 AACR.

Our knowledge of multiple sclerosis (MS) neuropathology has benefited from a number of studies that provided an in-depth description of plaques and, more recently, diffuse alterations of the normal-appearing white or grey matter. However, there have been few studies focusing on the periplaque regions surrounding demyelinated plaques, notably in MS spinal cords. In this context, the present study aimed to analyze the molecular immunopathology of periplaque demyelinated lesions (PDLs) in the spinal cord of patients with a progressive form of MS. To achieve this goal, the neuropathological features of PDLs were analyzed in postmortem tissues derived from the cervical spinal cord of 21 patients with primary or secondary progressive MS. We found that PDLs covered unexpectedly large areas of incomplete demyelination and were characterized by the superimposition of pro- and anti-inflammatory molecular signatures. Accordingly, macrophages/microglia accumulated in PDLs but exhibited a poor phagocytic activity toward myelin debris. Interestingly, while genes of the oligodendrocyte lineage were consistently down-regulated in PDLs, astrocyte-related molecules such as aquaporin 4, connexin 43 and the glutamate transporter EAAT1, were significantly upregulated in PDLs at the mRNA and protein levels. Overall, our work indicates that in the spinal cord of patients with a progressive form of MS, a tissue remodeling process that is temporally remote from plaque development takes place in PDLs. We propose that in spinal cord PDLs, this process is supported by subtle alterations of astrocyte functions and by low-grade inflammatory events that drive a slowly progressive loss of myelin and a failure of remyelination.

Objective A link between diffuse axonal loss and diffuse inflammation has been established in the brain of patients with progressive multiple sclerosis (MS). In the present paper, we sought to determine whether such a link could be similarly demonstrated in the spinal cord of patients with progressive MS. Methods A neuropathological quantitative assessment of inflammation and axonal loss was performed in the cervical spinal cord of 18 patients with progressive MS and 5 control subjects. Results As previously reported, we found a mean 25% decrease of axonal density in the normal-appearing white matter (NAWM) of MS versus control spinal cords. T-cell perivascular infiltrates were rare, but a robust diffuse inflammation was observed in both the normal-appearing parenchyma and the meninges. The extent of diffuse axonal loss in the NAWM correlated with both the density of major histocompatibility complex (MHC) class II+ microglia in the NAWM and, surprisingly, the density of CD3+ T cells in the meninges. Interestingly, close interactions between T cells and MHC class II+ macrophages were observed in the meninges of spinal cords from MS patients. Interpretation Recent studies assigned a major role to meningeal B-cell follicles in the pathophysiology of secondary progressive MS. The present work also emphasizes the link between meningeal inflammation and parenchymal lesions and points to a specific role exerted by both meningeal T cells and activated microglia in diffuse axonal loss in the spinal cord. ANN NEUROL 2010

myo-Inositol-1-phosphatase from bovine brain was purified over 2000-fold. The native enzyme has a Mr of 59,000, and on SDS/polyacrylamide-gel electrophoresis the subunit Mr was 31,000. Thus the native enzyme is a dimer of two apparently identical subunits. The enzyme, purified to a specific activity of more than 300 units/mg of protein (1 unit of enzyme activity corresponds to the release of 1 mumol of Pi/h at 37 degrees C), catalysed the hydrolysis of a variety of phosphorylated compounds, the best one, in terms of V/Km, being D-myo-inositol 1-phosphate. Kinetic constants of compounds tested, including both isomers of glycerophosphate and two deoxy forms of beta-glycerophosphate, were measured. They show the importance of the two hydroxyl groups which are adjacent to the phosphate in myo-inositol 1-phosphate. With a wide variety of substrates Li+ was found to be an uncompetitive inhibitor whose Ki varied with substrate structure.