No description available.

The Drosophila Lipoprotein particles bear lipid-linked morphogens on their surface and are required for long-range signaling activity of Wingless and Hedgehog. They also bind a wide variety of gpi-linked proteins. Whether any of these proteins affect morphogen signaling is unknown. Here, I show that the gpi-linked heparan sulfate proteoglycan Dally is released from cell membranes and binds to lipoprotein particles both with and without its lipid anchor. Hedgehog signaling efficiency is reduced in Dally mutant discs, but can be rescued non-autonomously by expression of non-gpi-modified Dally. This Dally isoform colocalizes with Hedgehog, Patched and Lipophorin in endosomes and increases Hedgehog signaling efficiency without affecting Hedgehog distribution. These data show that Hedgehog signaling activity can be influenced by other Lipophorin-associated proteins, and suggest Lipoproteins provide a platform for regulation of morphogen signaling.

The Hedgehog (Hh) signaling pathway is vital for vertebrate embryogenesis and aberrant activation of the pathway can cause tumorigenesis in humans. In this study, we used a phosphatome RNAi screen for regulators of Hh signaling to identify a member of the Eyes Absent protein family, Eya1, as a positive regulator of Hh signal transduction. Eya1 is both a phosphatase and transcriptional regulator. Eya family members have been implicated in tumor biology, and Eya1 is highly expressed in a particular subtype of medulloblastoma (MB). Here we show that RNAi-mediated knock-down of Eya1, as well as knock-down of its co-factor, Six1, blocks Hh signaling as assessed by induction of Hh response genes. Utilizing small molecule agonists, RNAi, and protein over-expression methods, we place the influence of Eya1 and Six1 within the Hh signaling pathway downstream of Smoothened (Smo) and at or above the level of Suppressor of Fused (Sufu). Interestingly, Eya1 appears to be specifically required for Hh-responsive gene activation mediated by Gli transcriptional activators but not for Hh-mediated transcriptional de-repression mediated by the inhibition of Gli transcriptional repressors. Furthermore, we find that Eya1 and Six1 regulate the expression of Neuropilin1 (Nrp1) and Neuropilin2 (Nrp2), known positive regulators of Hh signaling, providing a mechanism by which Eya1 and Six1 exert their effects. Based on these data, we investigated a role of Eya1 in Hh signaling in vivo. We obtained Eya1-/- mice and focused our attention on the developing cerebellum, where Sonic Hedgehog (Shh) is a major factor promoting neural precursor proliferation. In the Eya1-/- cerebellum, we find a striking reduction in neural precursor proliferation. In addition, we surveyed several other locations where Shh and/or Eya1 are known to be important for development. These include the embryonic otic vesicle, neural tube, and lung. In the developing inner ear we find Eya1-/- mice display reduced Hh signaling in vivo and a genetic interaction between Eya1 and Hh signaling. In lung tissue, Eya1-/- mice have reduced levels of Nrp expression. Together, these data further our understanding of the Hh signaling pathway and provide evidence for a role of Eya1 in Hh signal transduction.

Magíster en Bioquímica área de Especialización en Bioquímica Clínica y memoria para optar al Título de Bioquímico / Autor no autoriza el acceso a texto completo de su documento hasta enero 2017 / El Carcinoma Basocelular Cutáneo (BCC), un tipo de cáncer de piel, es una de las neoplasias más frecuentes en el ser humano. Es causado por una desregulación de la vía Sonic Hedgehog/Gli (Shh/Gli), cuya señalización juega un rol importante en la proliferación y mantenimiento de nichos celulares troncales. Comúnmente el BCC se caracteriza por ser poco metastático, sin embargo es capaz de dañar significativamente el tejido y existen subtipos de gran agresividad y recurrencia. Por esto se hace necesario entender mejor esta patología y buscar nuevos blancos terapéuticos y/o marcadores tumorales que ayuden a mejorar la prognosis y tratamiento de esta enfermedad. Recientemente se reportó que la proteína Neogenina-1 (NEO1) es un blanco transcripcional de la vía Shh/Gli. NEO1 es un receptor transmembrana de Netrinas y RGM con funciones contexto y ligando dependientes. Se ha postulado que NEO1 pertenece a la familia de receptores de dependencia, entre otras funciones. La habilidad de estos receptores de gatillar apoptosis en ausencia de ligando se ha propuesto como un mecanismo de supresión tumoral, implicado tanto en proliferación como sobrevida de células tumorales. Diversos antecedentes indican que la expresión de NEO1 varía en distintos tipos de cáncer, relacionándose con su grado de proliferación, indiferenciación, agresividad y sobrevida. Estos antecedentes junto a un estudio piloto desarrollado en el laboratorio de la Dra. Palma que indica la presencia de NEO1 en muestras de BCC, permiten postular que: “Los niveles de expresión receptor de dependencia NEO1 y su(s) ligando(s) varían en la progresión del BCC ligado a la sobre-activación de la vía de señalización SHH/GLI”. Para comprobar esta hipótesis se analizó la expresión de mRNA de NEO1 y sus ligandos por PCR cuantitativo y por inmunotinciones en muestras de pacientes con BCC y muestras de piel sana correlacionándolos con la actividad de la vía SHH/GLI; se evaluó la relación entre los niveles de NEO1 y sus ligandos con la agresividad tumoral; y por último se analizaron las variaciones de este receptor, sus ligandos y reporteros la vía Shh/Gli en un modelo murino de BCC. Como principales resultados se observó la presencia de NEO1 y sus ligandos en piel sana y en BCC y se comprobó que los niveles de NEO1 y sus ligandos NTN1 y RGMA se correlacionan positivamente con los niveles de GLI1, un conocido reportero de la vía SHH/GLI. La inhibición de la vía SHH/GLI ex vivo produce una disminución de los niveles de mRNA de estos genes. Notablemente, los niveles de expresión de NEO1, RGMA y GLI1 son menores en las muestras correspondientes a subtipos más agresivos; en la progresión tumoral del BCC murino, dichos niveles disminuyen conforme avanza la enfermedad. El aumento de NEO1 y sus ligandos en las muestras de BCC indolentes podría guardar relación con su rol de receptor de dependencia, promoviendo la proliferación de las células tumorales. En tanto la disminución de NEO1 en subtipos agresivos, guarda relación a lo observado con otros blancos transcripcionales de la vía Shh/Gli y podría estar relacionado con eventos tumorigénicos adicionales. Se concluye que NEO1 y sus ligandos se encuentran aumentados en BCC en correlación con la actividad de la vía SHH/GLI. NEO1 disminuye en casos agresivos de BCC lo que lo podría postular como un potencial marcador prognóstico tumoral / Basal Cell Carcinoma of the skin (BCC), a type of skin cancer, is one of the most common cancers in humans. It is mainly caused by a deregulation of Sonic Hedgehog/ Gli (Shh/Gli) pathway, which plays an important role in proliferation and maintenance of stem cell niches. Commonly, BCC is rarely metastatic, nevertheless it can cause large tissue damage and some aggressive subtypes with great recurrence are known. Therefore a deeper knowledge of this disease is required combined with the search for new therapeutic targets and/or tumor markers that might contribute to improve prognosis and treatment of this pathology. Recently it has been reported that Neogenin-1 (NEO1), a transmembrane receptor, is a transcriptional target of Shh/Gli pathway. NEO1 is a receptor of Netrin and RGM ligands and has both context and ligand dependent functions. NEO1 has been postulated as a death dependence receptor. In the absence of ligand interaction this kind of receptors can trigger apoptosis; process that has been proposed as a tumor suppression mechanism that could be altered in cancer promoting either proliferation or survival of tumor cells. NEO1 and ligand expression have been reported to vary in different types of cancer, in relation to the degree of tumor proliferation, differentiation, aggressiveness and survival. In addition, a pilot study developed in Dra. Palma’s laboratory shows presence of NEO1 in BCC samples. These findings lead us to propose that: “Neogenin-1 and ligand levels of expression vary in BCC tumor progression, relating with SHH/GLI pathway overactivation”. To test this hypothesis both NEO1 and ligand mRNA levels where quantified by qPCR and assessed by immunostaining on human BCC and healthy skin samples, correlating these results with SHH/GLI pathway activity; correlation between NEO1 and ligand levels with aggressiveness was assessed; and finally, variations in NEO1, ligand and SHH/GLI readouts levels were assessed in a BCC mouse model. As key results, presence of NEO1 and ligand was detected in healthy skin and in BCC. NEO1 and ligands (NTN1 and RGMA) mRNA levels presented a positive correlation with GLI1 levels, a classical SHH/GLI readout. Pharmacological inhibition of SHH/GLI pathway ex vivo decreased mRNA levels of these genes. Noteworthy, NEO1, RGMA and GLI1 levels where found to be significantly lower in aggressive subtypes of BCC and, in addition, a decrease their levels with tumor progression was observed. The increase of NEO1 and ligand in BCC samples could be related with its role as a death dependence receptor, promoting tumor cell proliferation. Meanwhile the decrease of NEO1 in aggressiveness could be related with additional tumorigenic events similar to what has been reported for other SHH/GLI transcriptional targets. In conclusion, NEO1 and it ligand are upregulated in BCC in relation to deregulation of the SHH/GLI pathway. NEO1 is decreased in aggressive BCC subtypes; therefore it is possible to evaluate NEO1 as a potential prognosis marker / Conicyt

oligodendrocytes migrate from the corpus callosum into the overlying cortex. The incidence of cilia did not change markedly across age groups, and did not vary consistently with the number of processes per cell, which was used as an indication of the maturation stage of OPCs and young OLs. The mean percent of Olig1 immunopositive (Olig1+) cells having cilia across ages was 33.1% + 16.5%, with all ages combined. In O4+ cells of these mice, 56.7 + 3.6% had primary cilia. If it is the case that adult OLs do not have cilia, the point in the lineage when primary cilia are lost is still unknown. Adult mice that had been injected with cyclopamine to block cilia-dependent Shh signaling were examined to determine whether the rate of generating new OPCs was influenced. In the CC of control mice, the numerical density of Olig1+/BrdU+ cells was 1.29 + 0.07/mm2 was reduced to 0.68 + 0.38/mm2 in the cyclopamine-injected group, and the numerical density of all BrdU+ cells (including both Olig1+ and Olig1- cells) of 4.55 + 1.50/mm2 in the control group was reduced to 3.14 + 1.27/mm2 in the cyclopamine-injected group. However, there were only 2 mice in each group and the differences were not statistically significant.

The Hedgehog protein (Hh) is a morphogen that is necessary for cell survival, growth and patterning in flies and mammals. In germline cells, alterations in the Hh signaling pathway can result in developmental disorders; in somatic cells, misregulation of the Hh signaling pathway can result in cancer. Most components of the signaling pathway were identified by genetic screens in Drosophila that were later found to be conserved in mammals. In the presence of the Hh signal, multiple Hh signaling components interact to mediate the induction of Hh target genes. In flies, Cubitus Interruptus (Ci) is the singular transcription factor of the pathway that is regulated by multiple upstream components of the pathway including Costal2 (Cos2). Cos2 is a scaffold protein that can both positively and negatively regulate Hh signaling by binding to Ci and various kinases such as Fused (Fu). We disrupted the binding of Cos2 to Fu using a physiological expressed genomic Costal2 transgene (gCosΔFu) and found that Fu must bind to Cos2 to promote efficient processing and activation of full-length Ci (Ci-155). Fu was thought to activate Ci-155 by phosphorylating Cos2 at sites S931 and S572, but we found that gCosS931A and gCosS572A did not reduce Ci activity in the fly wing disc. Instead, we hypothesize that Fu could directly phosphorylate Ci-155 or another unknown protein. To investigate if another protein was involved we developed a Hh sensitized genetic screen. We obtained multiple “hits” from the genetic screen but we did not find an obvious candidate that could be a substrate for Fu. Instead, we identified Mago Nashi and Srp54 which we found to be involved with the post-transcriptional regulation of ci RNA. We confirmed the existence of ci isoforms A and B and found that knockdown of Mago Nashi, resulted in an altered splicing pattern while knockdown of Srp54 reduced ci RNA levels. Mago Nashi inhibition and intronless Ci reduced Ci-155 protein levels, which suggests efficient splicing is necessary for normal Ci-155 levels. Furthermore, we found that reduced Ci-155 levels only affected Ci activity in sub-optimal Hh signaling conditions. In order to further dissect the mechanism Ci processing, activation and stabilization, we used physiologically expressed genomic Ci (gCi) and CRISPR Ci variants (crCi). First we examined Ci-S849A, which prevents Ci processing and we found that in the absence of processing, Ci-155 levels are uniformly high throughout the wing disc. Cos2 and PKA are necessary for Ci processing but we wanted to know if they had an additional role in Ci silencing We found that Cos2 but not PKA can silence and stabilize Ci-155 in the absence of processing. Activated Fu in the Ci-S849A wing disc highly activated and destabilized Ci-155, which was similar to Hh signaling at the AP Border. To test if Ci is the direct target of Fu, we are testing physiologically expressed Ci with point mutations and deletions that are near the Suppressor of Fused (Su(fu)) binding site to examine whether they are unresponsive to activated Fu. Su(fu) binds to Ci-155 to stabilize Ci- 155 levels and inhibit Ci activity, but the mechanism is not well understood. We developed Ci transgenes that have altered Su(fu) binding to determine if Su(fu) inhibits Ci by cytoplasmic anchoring, co-repressor recruitment, or by blocking a co-activator.

Breast and pancreatic cancers are among the major causes of cancer mortality in our society. There has been a significant decline in mortality from breast cancer over the last two decades, while pancreatic cancer has an exceptionally poor prognosis. Although these malignancies have very different clinical outcomes they share the common feature that metastatic disease is almost uniformly fatal. The existence of cancer stem cells has been postulated as a major factor in tumour recurrence after traditional chemo- or radio-therapy. Addressing this important clinical question requires a deeper understanding of the biology of normal and cancer stem cells and the signalling pathways involved in their regulation. The identity of the pancreatic stem cell remains elusive. However, using a murine model of haematopoietic stem cell (HSC) transplantation I have demonstrated for the first time transdifferentiation of these bone marrow derived cells into mature pancreatic acinar cells, where they appear to contribute to cell turnover ultimately forming acini and lobules. These data show that HSC have surprising developmental plasticity and provide insight into a potential stem cell niche in the pancreas. The Hedgehog, Wnt and Notch signalling pathways play a critical role in early development and in the maintenance and self-renewal of stem cells. There is also increasing evidence that dysregulation of these pathways contributes to the development of many malignancies. There is relatively little information regarding their role in breast cancer development and progression. I used immunohistochemistry for key proteins in these pathways, sonic hedgehog, beta-catenin and Notch 1 in three substantial series of human breast lesions and determined that abnormal expression of these proteins is an early event in the development in breast cancer, and is associated with particular breast cancer subtypes, Shh and beta-catenin expression is associated predominantly with the basal-like phenotype and Notch 1 with the HER2 amplified phenotype. Overexpression of Shh in particular confers a worse clinical outcome in invasive ductal carcinoma. Furthermore, increased levels of Shh in a 3D culture model of non-transformed mammary epithelial cells resulted in disorganisation of acini and the development of an abnormal discohesive phenotype. Finally the role of Shh was investigated in a mammary epithelial transplantation model, where overexpression of Shh resulted in the development of hyperplasia of the mammary ductal epithelium. Together these data confirm that the Hedgehog, Wnt and Notch developmental pathways are dysregulated in breast cancer and represent viable targets for further investigation of potential novel therapies in breast cancer.

The Hedgehog (Hh) signaling pathway has received attention regarding its important role in embryonic development, however the mechanism by which pathway regulators, such as Suppressor of fused (Sufu), Kinesin family member 7 (Kif7), and primary cilium, mediate Hh signaling transduction is not entirely understood. The work presented here examines the roles of Sufu and Kif7 in regulating Hh signaling in growth plate chondrocytes, as well as how they mediate parathyroid hormone-like hormone (Pthlh) signaling during chondrocyte development. I show here that Sufu and Kif7 are essential regulators of Indian hedgehog (Ihh) signaling. While Sufu negatively regulates Gli transcription factors, Kif7 functions both positively and negatively in chondrocytes. Kif7 plays a role in Sufu protein degradation and the exclusion of Sufu-Gli complexes from the primary cilium. Importantly, halving the dosage of Sufu restores normal Hh pathway activity and chondrocyte development in Kif7-null mice, demonstrating that the positive role of Kif7 is to restrict the inhibitory function of Sufu. Furthermore, Kif7 exerts inhibitory function on Gli transcriptional activity in chondrocytes when Sufu function is absent. Therefore, Kif7 regulates the activity of Gli transcription factors through both Sufu-dependent and Sufu-independent mechanisms. I show that Sufu is crucial for mediating the negative effect of Pthlh on Gli transcriptional activity and chondrocyte hypertrophic differentiation, whereas Kif7 and primary cilium are dispensable in this process. Although primary cilium is required for Hh ligand-mediated activation of Gli transcription, Pthlh negatively controls Gli transcriptional activity in a cilia-independent manner. The results of this work provide insight into how Hh signaling is regulated by Sufu and Kif7 in the context of primary cilium, but also suggest Sufu serves as an important link between Ihh and Pthlh signaling during growth plate chondrocyte development.

Aggressive fibromatosis is a benign fibroproliferative tumour that can occur as a sporadic lesion or a manifestation in patients with familial syndromes, such as familial adenomatous polyposis. Tumours are characterized by the stabilization of β-catenin and the activation of β-catenin-mediated transcription. Current treatment results are far from ideal, and recurrence rates are high. As a result, there remains a need for more effective therapeutic strategies. In this work, we demonstrate the effect of hedgehog signalling inhibition on aggressive fibromatosis tumour development and β-catenin modulation. We found that hedgehog inhibition decreased cell viability and proliferation as well as total β-catenin levels in human aggressive fibromatosis tumour cells in vitro. Furthermore, following hedgehog inhibition in Apc+/Apc1638N aggressive fibromatosis mouse model, the number and volume of the tumours formed was reduced. Together, this work suggests that hedgehog signalling inhibitor agents are potential candidates to effectively manage aggressive fibromatosis.

Aggressive fibromatosis is a benign fibroproliferative tumour that can occur as a sporadic lesion or a manifestation in patients with familial syndromes, such as familial adenomatous polyposis. Tumours are characterized by the stabilization of β-catenin and the activation of β-catenin-mediated transcription. Current treatment results are far from ideal, and recurrence rates are high. As a result, there remains a need for more effective therapeutic strategies. In this work, we demonstrate the effect of hedgehog signalling inhibition on aggressive fibromatosis tumour development and β-catenin modulation. We found that hedgehog inhibition decreased cell viability and proliferation as well as total β-catenin levels in human aggressive fibromatosis tumour cells in vitro. Furthermore, following hedgehog inhibition in Apc+/Apc1638N aggressive fibromatosis mouse model, the number and volume of the tumours formed was reduced. Together, this work suggests that hedgehog signalling inhibitor agents are potential candidates to effectively manage aggressive fibromatosis.