Ariel Ruiz i Altaba

HEDGEHOG-GLI1 Signaling Regulates Human Glioma Growth, Cancer Stem Cell Self-Renewal, and Tumorigenicity

Cancer stem cells are rare tumor cells characterized by their ability to self-renew and to induce tumorigenesis. They are present in gliomas and may be responsible for the lethality of these incurable brain tumors. In the most aggressive and invasive type, glioblastoma multiforme (GBM), an average of about one year spans the period between detection and death [1]. The resistence of gliomas to current therapies may be related to the existence of cancer stem cells [2-6]. We find that human gliomas display a stemness signature and demonstrate that HEDGEHOG (HH)-GLI signaling regulates the expression of stemness genes in and the self-renewal of CD133(+) glioma cancer stem cells. HH-GLI signaling is also required for sustained glioma growth and survival. It displays additive and synergistic effects with temozolomide (TMZ), the current chemotherapeutic agent of choice. TMZ, however, does not block glioma stem cell self-renewal. Finally, interference of HH-GLI signaling with cyclopamine or through lentiviral-mediated silencing demonstrates that the tumorigenicity of human gliomas in mice requires an active pathway. Our results reveal the essential role of HH-GLI signaling in controlling the behavior of human glioma cancer stem cells and offer new therapeutic possibilities.

Gli and hedgehog in cancer: tumours, embryos and stem cells

Do tumours arise from stem cells, or are they derived from more differentiated cells that, for some reason, begin to recapitulate developmental programmes? Inappropriate activation of the Sonic hedgehog–Gli signalling pathway occurs in several types of tumour, including those of the brain and the skin. Studies in these and other systems suggest that inappropriate function of the Gli transcription factors in stem or precursor cells might lead to the onset of a tumorigenic programme and that these factors are prime targets for anticancer therapies.

Sonic hedgehog controls stem cell behavior in the postnatal and adult brain

Sonic hedgehog (Shh) signaling controls many aspects of ontogeny, orchestrating congruent growth and patterning. During brain development, Shh regulates early ventral patterning while later on it is critical for the regulation of precursor proliferation in the dorsal brain, namely in the neocortex, tectum and cerebellum. We have recently shown that Shh also controls the behavior of cells with stem cell properties in the mouse embryonic neocortex, and additional studies have implicated it in the control of cell proliferation in the adult ventral forebrain and in the hippocampus. However, it remains unclear whether it regulates adult stem cell lineages in an equivalent manner. Similarly, it is not known which cells respond to Shh signaling in stem cell niches. Here we demonstrate that Shh is required for cell proliferation in the mouse forebrains subventricular zone (SVZ) stem cell niche and for the production of new olfactory interneurons in vivo. We identify two populations of Gli1+ Shh signaling responding cells: GFAP+ SVZ stem cells and GFAP- precursors. Consistently, we show that Shh regulates the self-renewal of neurosphere-forming stem cells and that it modulates proliferation of SVZ lineages by acting as a mitogen in cooperation with epidermal growth factor (EGF). Together, our data demonstrate a critical and conserved role of Shh signaling in the regulation of stem cell lineages in the adult mammalian brain, highlight the subventricular stem cell astrocytes and their more abundant derived precursors as in vivo targets of Shh signaling, and demonstrate the requirement for Shh signaling in postnatal and adult neurogenesis.

Melanomas require HEDGEHOG-GLI signaling regulated by interactions between GLI1 and the RAS-MEK/AKT pathways

Melanoma is one of the most aggressive cancers, and its incidence is increasing. These tumors derive from the melanocyte lineage and remain incurable after metastasis. Here we report that SONIC HEDGEHOG (SHH)-GLI signaling is active in the matrix of human hair follicles, and that it is required for the normal proliferation of human melanocytes in culture. SHH-GLI signaling also regulates the proliferation and survival of human melanomas: the growth, recurrence, and metastasis of melanoma xenografts in mice are prevented by local or systemic interference of HH-GLI function. Moreover, we show that oncogenic RAS-induced melanomas in transgenic mice express Gli1 and require Hh-Gli signaling in vitro and in vivo. Finally, we provide evidence that endogenous RAS-MEK and AKT signaling regulate the nuclear localization and transcriptional activity of GLI1 in melanoma and other cancer cells. Our data uncover an unsuspected role of HH-GLI signaling in melanocytes and melanomas, demonstrate a role for this pathway in RAS-induced tumors, suggest a general integration of the RAS/AKT and HH-GLI pathways, and open a therapeutic approach for human melanomas.

Hedgehog–GLI signaling and the growth of the brain

The development of the vertebrate brain involves the creation of many cell types in precise locations and at precise times, followed by the formation of functional connections. To generate its cells in the correct numbers, the brain has to produce many precursors during a limited period. How this is achieved remains unclear, although several cytokines have been implicated in the proliferation of neural precursors. Understanding this process will provide profound insights, not only into the formation of the mammalian brain during ontogeny, but also into brain evolution. Here we review the role of the Sonic hedgehog–Gli pathway in brain development. Specifically, we discuss the role of this pathway in the cerebellar and cerebral cortices, and address the implications of these findings for morphological plasticity. We also highlight future directions of research that could help to clarify the mechanisms and consequences of Sonic hedgehog signalling in the brain.

Human colon cancer epithelial cells harbour active HEDGEHOG-GLI signalling that is essential for tumour growth, recurrence, metastasis and stem cell survival and expansion

Human colon cancers often start as benign adenomas through loss of APC, leading to enhanced βCATENIN (βCAT)/TCF function. These early lesions are efficiently managed but often progress to invasive carcinomas and incurable metastases through additional changes, the nature of which is unclear. We find that epithelial cells of human colon carcinomas (CCs) and their stem cells of all stages harbour an active HH‐GLI pathway. Unexpectedly, they acquire a high HEDGEHOG‐GLI (HH‐GLI) signature coincident with the development of metastases. We show that the growth of CC xenografts, their recurrence and metastases require HH‐GLI function, which induces a robust epithelial‐to‐mesenchymal transition (EMT). Moreover, using a novel tumour cell competition assay we show that the self‐renewal of CC stem cells in vivo relies on HH‐GLI activity. Our results indicate a key and essential role of the HH‐GLI1 pathway in promoting CC growth, stem cell self‐renewal and metastatic behavior in advanced cancers. Targeting HH‐GLI1, directly or indirectly, is thus predicted to decrease tumour bulk and eradicate CC stem cells and metastases.

Loss-of-function mutations in the human GLI2 gene are associated with pituitary anomalies and holoprosencephaly-like features

Diminished Sonic Hedgehog (Shh) signaling is associated with the most common forebrain defect in humans, holoprosencephaly (HPE), which includes cyclopia, a phenotype also seen in mice and other vertebrates with defective Shh signaling. The secreted protein Shh acts as a crucial factor that patterns the ventral forebrain and is required for the division of the primordial eye field and brain into two discrete halves. Gli2 is one of three vertebrate transcription factors implicated as obligatory mediators of Shh signal transduction. Here, we show that loss-of-function mutations in the human GLI2 gene are associated with a distinctive phenotype (within the HPE spectrum) whose primary features include defective anterior pituitary formation and pan-hypopituitarism, with or without overt forebrain cleavage abnormalities, and HPE-like midfacial hypoplasia. We also demonstrate that these mutations lack GLI2 activity. We report on a functional association between GLI2 and human disease and highlight the role of GLI2 in human head development.

Hedgehog-GLI signaling regulates the behavior of cells with stem cell properties in the developing neocortex

Stem cells are crucial for normal development and homeostasis, and their misbehavior may be related to the origin of cancer. Progress in these areas has been difficult because the mechanisms regulating stem cell lineages are not well understood. Here, we have investigated the role of the SHH-GLI pathway in the developing mouse neocortex. The results show that SHH signaling endogenously regulates the number of embryonic and postnatal mouse neocortical cells with stem cell properties, and controls precursor proliferation in a concentration-dependent manner in cooperation with EGF signaling. These findings identify a crucial mechanism for the regulation of the number of cells with stem cell properties that is unexpectedly conserved in different stem cell niches.

Gli proteins and Hedgehog signaling: development and cancer

A surprising number of apparently unrelated human diseases, including familial and sporadic cancers and a number of syndromes and malformations, seem to be associated with abnormal function of the Hedgehog (Hh) signaling pathway. Zinc-finger transcription factors of the Gli family play critical roles in the mediation and interpretation of Hh signals. Elucidating how Gli proteins work will enable us to further our knowledge of how cells proliferate, differentiate or survive in response to Hh signals, as well as to design rational therapies for these Hh-signaling related diseases (HSDs).

NANOG regulates glioma stem cells and is essential in vivo acting in a cross‐functional network with GLI1 and p53

A cohort of genes associated with embryonic stem (ES) cell behaviour, including NANOG, are expressed in a number of human cancers. They form an ES‐like signature we first described in glioblastoma multiforme (GBM), a highly invasive and incurable brain tumour. We have also shown that HEDGEHOG‐GLI (HH‐GLI) signalling is required for GBM growth, stem cell expansion and the expression of this (ES)‐like stemness signature. Here, we address the function of NANOG in human GBMs and its relationship with HH‐GLI activity. We find that NANOG modulates gliomasphere clonogenicity, CD133+ stem cell cell behavior and proliferation, and is regulated by HH‐GLI signalling. However, GLI1 also requires NANOG activity forming a positive loop, which is negatively controlled by p53 and vice versa. NANOG is essential for GBM tumourigenicity in orthotopic xenografts and it is epistatic to HH‐GLI activity. Our data establish NANOG as a novel HH‐GLI mediator essential for GBMs. We propose that this function is conserved and that tumour growth and stem cell behaviour rely on the status of a functional GLI1‐NANOG‐p53 network.