Jose Mario Gonzalez-Meljem

Stem cell senescence drives age-attenuated induction of pituitary tumours in mouse models of paediatric craniopharyngioma

Senescent cells may promote tumour progression through the activation of a senescence-associated secretory phenotype (SASP), whether these cells are capable of initiating tumourigenesis in vivo is not known. Expression of oncogenic β-catenin in Sox2+ young adult pituitary stem cells leads to formation of clusters of stem cells and induction of tumours resembling human adamantinomatous craniopharyngioma (ACP), derived from Sox2− cells in a paracrine manner. Here, we uncover the mechanisms underlying this paracrine tumourigenesis. We show that expression of oncogenic β-catenin in Hesx1+ embryonic precursors also results in stem cell clusters and paracrine tumours. We reveal that human and mouse clusters are analogous and share a common signature of senescence and SASP. Finally, we show that mice with reduced senescence and SASP responses exhibit decreased tumour-inducing potential. Together, we provide evidence that senescence and a stem cell-associated SASP drive cell transformation and tumour initiation in vivo in an age-dependent fashion.Senescent cells can promote tumour progression through the activation of a senescenceassociated secretory phenotype (SASP). Here, the authors show that SASP activation is associated with non-cell autonomous cell transformation and tumour initiation in an in vivo model of adamantinomatous craniopharyngioma.

Molecular Analyses Reveal Inflammatory Mediators in the Solid Component and Cyst Fluid of Human Adamantinomatous Craniopharyngioma.

Pediatric adamantinomatous craniopharyngioma (ACP) is a highly solid and cystic tumor, often causing substantial damage to critical neuroendocrine structures such as the hypothalamus, pituitary gland, and optic apparatus. Paracrine signaling mechanisms driving tumor behavior have been hypothesized, with IL-6R overexpression identified as a potential therapeutic target. To identify potential novel therapies, we characterized inflammatory and immunomodulatory factors in ACP cyst fluid and solid tumor components. Cytometric bead analysis revealed a highly pro-inflammatory cytokine pattern in fluid from ACP compared to fluids from another cystic pediatric brain tumor, pilocytic astrocytoma. Cytokines and chemokines with particularly elevated concentrations in ACPs were IL-6, CXCL1 (GRO), CXCL8 (IL-8) and the immunosuppressive cytokine IL-10. These data were concordant with solid tumor compartment transcriptomic data from a larger cohort of ACPs, other pediatric brain tumors and normal brain. The majority of receptors for these cytokines and chemokines were also over-expressed in ACPs. In addition to IL-10, the established immunosuppressive factor IDO-1 was overexpressed by ACPs at the mRNA and protein levels. These data indicate that ACP cyst fluids and solid tumor components are characterized by an inflammatory cytokine and chemokine expression pattern. Further study regarding selective cytokine blockade may inform novel therapeutic interventions.

MAPK pathway control of stem cell proliferation and differentiation in the embryonic pituitary provides insights into the pathogenesis of papillary craniopharyngioma

Despite the importance of the RAS-RAF-MAPK pathway in normal physiology and disease of numerous organs, its role during pituitary development and tumourigenesis remains largely unknown. Here, we show that the over-activation of the MAPK pathway, through conditional expression of the gain-of-function alleles BrafV600E and KrasG12D in the developing mouse pituitary, results in severe hyperplasia and abnormal morphogenesis of the gland by the end of gestation. Cell-lineage commitment and terminal differentiation are disrupted, leading to a significant reduction in numbers of most of the hormone-producing cells before birth, with the exception of corticotrophs. Of note, Sox2+ stem cells and clonogenic potential are drastically increased in the mutant pituitaries. Finally, we reveal that papillary craniopharyngioma (PCP), a benign human pituitary tumour harbouring BRAF p.V600E also contains Sox2+ cells with sustained proliferative capacity and disrupted pituitary differentiation. Together, our data demonstrate a crucial function of the MAPK pathway in controlling the balance between proliferation and differentiation of Sox2+ cells and suggest that persistent proliferative capacity of Sox2+ cells may underlie the pathogenesis of PCP. Highlighted Article: Constitutive activation of the MAPK/ERK pathway causes pituitary hyperplasia, abnormal morphogenesis, and abnormal endocrine cell specification due to the sustained proliferation of the Sox2+ stem cell compartment.

Stem cells and their role in pituitary tumorigenesis.

The presence of adult pituitary stem cells (PSCs) has been described in murine systems by comprehensive cellular profiling and genetic lineage tracing experiments. PSCs are thought to maintain multipotent capacity throughout life and give rise to all hormone-producing cell lineages, playing a role in pituitary gland homeostasis. Additionally, PSCs have been proposed to play a role in pituitary tumorigenesis, in both adenomas and adamantinomatous craniopharyngiomas. In this manuscript, we discuss the different approaches used to demonstrate the presence of PSCs in the murine adult pituitary, from marker analyses to genetic tracing. In addition, we review the published literature suggesting the existence of tumor stem cells in mouse and human pituitary tumors. Finally, we discuss the potential role of PSCs in pituitary tumorigenesis in the context of current models of carcinogenesis and present evidence showing that in contrast to pituitary adenoma, which follows a classical cancer stem cell paradigm, a novel mechanism has been revealed for paracrine, non-cell autonomous tumor initiation in adamantinomatous craniopharyngioma, a benign but clinically aggressive pediatric tumor.

Hypothalamic sonic hedgehog is required for cell specification and proliferation of LHX3/LHX4 pituitary embryonic precursors

Sonic hedgehog (SHH) is an essential morphogenetic signal that dictates cell fate decisions in several developing organs in mammals. In vitro data suggest that SHH is required to specify LHX3+/LHX4+ Rathkes pouch (RP) progenitor identity. However, in vivo studies have failed to reveal such a function, supporting instead a crucial role for SHH in promoting proliferation of these RP progenitors and for differentiation of pituitary cell types. Here, we have used a genetic approach to demonstrate that activation of the SHH pathway is necessary to induce LHX3+/LHX4+ RP identity in mouse embryos. First, we show that conditional deletion of Shh in the anterior hypothalamus results in a fully penetrant phenotype characterised by a complete arrest of RP development, with lack of Lhx3/Lhx4 expression in RP epithelium at 9.0 days post coitum (dpc) and total loss of pituitary tissue by 12.5 dpc. Conversely, overactivation of the SHH pathway by conditional deletion of Ptch1 in RP progenitors leads to severe hyperplasia and enlargement of the Sox2+ stem cell compartment by the end of gestation. Summary: Genetic approaches demonstrate that, during normal murine development, the SHH pathway is first required for normal specification of Rathkes pouch embryonic precursors and subsequently to control their proliferation.

Tumour compartment transcriptomics demonstrates the activation of inflammatory and odontogenic programmes in human adamantinomatous craniopharyngioma and identifies the MAPK/ERK pathway as a novel therapeutic target

Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. β-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP.

Paracrine roles of cellular senescence in promoting tumourigenesis

Senescent cells activate genetic programmes that irreversibly inhibit cellular proliferation, but also endow these cells with distinctive metabolic and signalling phenotypes. Although senescence has historically been considered a protective mechanism against tumourigenesis, the activities of senescent cells are increasingly being associated with age-related diseases, including cancer. An important feature of senescent cells is the secretion of a vast array of pro-inflammatory cytokines, chemokines, and growth factors collectively known as the senescence-associated secretory phenotype (SASP). Recent research has shown that SASP paracrine signalling can mediate several pro-tumourigenic effects, such as enhancing malignant phenotypes and promoting tumour initiation. In this review, we summarise the paracrine activities of senescent cells and their role in tumourigenesis through direct effects on growth and proliferation of tumour cells, tumour angiogenesis, invasion and metastasis, cellular reprogramming and emergence of tumour-initiating cells, and tumour interactions with the local immune environment. The evidence described here suggests cellular senescence acts as a double-edged sword in cancer pathogenesis, which demands further attention in order to support the use of senolytic or SASP-modulating compounds for cancer treatment.

Biomedical Research in Aging

Biomedical research has been instrumental in identifying key molecular and cellular changes that occur throughout the aging process, also known as the Hallmarks of Aging. Notably, these are shared between humans and several other species that have served as models for the study of aging in the laboratory. In this chapter, we discuss current knowledge regarding the significance of hallmarks such as: decay of stem cell function, acquisition of genomic instability, DNA damage, telomere attrition, deregulated nutrient sensing, chronic inflammation and cellular senescence. We further describe current methodological issues, experimental techniques and best practices for the study of each hallmark across different in vivo and in vitro systems, while also pointing at their limitations. Finally, we provide future perspectives for the improvement of experimental designs in biomedical research of aging.

Senescence drives non-cell autonomous tumorigenesis in the pituitary gland

ABSTRACT Novel detrimental functions of senescent cells have been recently uncovered in the context of cancer development and progression, which they mainly exert through the secretion of several pro-tumorigenic factors. Here we discuss how cellular senescence and its secretory phenotype can be involved in the widely unexplored phenomenon of paracrine tumorigenesis.

Abstract 1804: Expression analysis of adamantinomatous craniopharyngioma suggests two subtypes associated with CTNNB1 mutational frequency and highlights potential therapeutic targets

We conducted RNA-sequencing analysis of Adamantinomatous Craniopharnygioma (ACP), including Laser capture micro-dissection (LCM) of individual cellular compartments, to characterise both disease heterogeneity and targetable deregulated molecular pathways. RNA sequencing was performed on 18 samples of ACP (17 pediatric, 1 adult) and six control samples (three fetal pituitaries, three non-functioning pituitary adenomas). LCM and RNA sequencing were performed on a subset of cases selecting nuclear β catenin accumulating clusters, and paired non-cluster tumour tissue (palisading epithelium). Mouse ACP models indicate that these clusters initiate tumorigenesis in a non-cell autonomous manner. Consensus clustering analysis revealed two subgroups of human ACP, one associated with a high CTNNB1 mutational frequency (>30%), a relatively high tumour content and fibrous reactive tissue (Group H) and another with a lower mutation frequency ( The meta-gene signature of Group H defined by non-negative matrix factorization included secreted factors, such as Sonic Hedgehog (SHH) and Fibroblast Growth Factors (FGFs) 3, 4 and 19, Keratins and Matrix Metalloproteinases and was enriched for genes related to odontogenesis. Weighted gene co-expression network analysis independently showed correlation of expression of such genes with mutational frequency. Glial markers and neural differentiation genes were up-regulated in Group L tumors. Differential expression showed up-regulation in ACPs of potential therapeutic targets including SHH (32x), EGFR (8.9x) and TNF (10x). Similarly, differential expression analysis of LCM dissected nuclear β catenin accumulating clusters identified up-regulation of SHH, FGFs and other secreted factors including as WNTs and BMPs. Analysis of the SHH pathway found that SHH is over expressed in clusters (9x) whilst its downstream targets Ptch1 (1.7x), Gli1 (2.1x), Gli3 (2.9x) are over expressed in palisading epithelium, indicating the presence of paracrine signaling. These findings were independently confirmed by in situ hybridisation in human samples and the mouse model. Clustering of murine expression data with those genes differentially expressed in the human clusters revealed murine and human clusters group together, suggesting they are similar structures functionally and biologically. Together, these results suggest that a subset of cells, carrying CTNNB1 mutations and showing nuclear β-catenin accumulation, express secreted pro-oncogenic factors and the response may be observed in neighboring cell types. Targeting such signalling offers an attractive therapeutic opportunity, for which the results suggest the mouse model is well placed for testing. The clinical significance of the two putative ACP subgroups remains to be determined. Citation Format: John R. Apps, Nital Jani, Gabriela Carreno, Jose M. Gonzalez-Meljem, Kyoko Tossell, Thomas J. Stone, Mark A. Ungless, Hywel J. Williams, Thomas S. Jacques, Juan-Pedro Martinez-Barbera. Expression analysis of adamantinomatous craniopharyngioma suggests two subtypes associated with CTNNB1 mutational frequency and highlights potential therapeutic targets. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1804.