Placzek lectures

Shh in BCC history
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Terms in this set (51)
1998: aberrant activation of Shh pathway can lead to BCC
2000-05: Shh pathway is pathogenically relevant in more than 90% of BCCs
2003: Genentech/Curis - preclinical development of compounds that inhibit the Shh pathway
2007-09: Phase I and II studies performed in adult with locally advanced/metastatic BCCs that were refractory to standard therapy
2012: Erivedge - first FDA-licensed drug for patients with advanced BCC (described as the 'greatest advance for this disease')
-Act as link between blood and brain, i.e. their depletion is likely to affect core aspects of body control
-Central region --> connects to circulating plasma via fenestrated capillaries in the median eminence, and connects to CSF via the 3rd ventricle
-Many key nuclei, especially those controlling energy and stress balance - line close to 3rd ventricle in central hypothalamus --> specialised cells (tanycytes) line the ventral 2/3
-Throughout life adult mammals crucially depend on stem cell populations to maintain and repair their tissues to ensure life-long organ function
-Stem cells = capacity to extensively self-renew and give rise to one or more differentiated cell types - key to meeting changing demand for tissue replacement during normal lung homeostasis and regeneration after lung injury
Fgf10 signalling-Well-conserved signalling pathway governing epithelial-mesenchymal interactions during lung development as well as between different lung epithelial stem cells and their niches -Disruption leads to dysfunctional epithelial stem cell-niche unit, which may culminate in chronic lung diseases such as COPD, chronic asthma and idiopathic pulmonary fibrosis (IPF)Cyclopamine-1940-50: sheep-herders observed that normal adult sheep were giving birth to one-eyed (cyclopic) lambs -USDA found that this was due to them eating corn-lilies, which contain cyclopamine -1980s: researchers realised that Hh/Shh is conserved between invertebrates and vertebratesHh/Shh signalling pathway-1990s: Hh/Shh signalling pathway identified and characterised - show that it mediates its effects via the protein Smoothened -Shh-/- embryos have cyclopia and holoprosencephaly -Shh is not expressed in a segmental pattern - expressed along ventral midline and underlying axial mesoderm -Patched receptor (7TM) alleviates repression of Smoothened TM protein -Importance in brain, lungs, heart, digestive system components, liver and biliary system, pancreas and renal systemBCC, medulloblastoma, rhabdomyosarcoma and other human tumours-Demonstration that 'activating' Smoothened mutations can lead to BCC by making Smoothened completely refractory from inhibition by Patched (will always be activating) -Activating Smoothened mutation --> BCCs (abundant Gli protein) -Smoothened = proto-oncogene (activated) -Patched = tumour suppressor (inactivated)Cyclopamine directly antagonises Shh pathway-Shh signalling normally repressed as development proceeds -Failure to repress Shh will result in dysregulated cell proliferation - involved in many cancers -2000s: oncogenic mutations in Smoothened can be reversed by cyclopamine - completely antagonises Smoothened and causes TFs Gli1/2 to remain inactive (prevents expression of tumour-mediating genes)Cancer therapy-Smo and Ptch mediate cellular response to Hh secreted protein signal and oncogenic mutations affecting these proteins cause excess activity of Hh response pathway -Cyclopmine (teratogen) = inhibits Hh response -Synthetic derivatives with improved potency block activation of Hh response pathway and abnormal cell growth associated with both types of oncogenic mutation -Cyclopamine may act by influencing balance between active/inactive forms of SmoothenedErivedge-Genentech collaborate with Curis for preclinical development of compounds that inhibit the Hh pathway -Erivedge = substance that acts as cyclopamine-competitive antagonist of Smoothened -2007-09: Phase I and II studies performed in adult with locally advanced/metastatic BCC that were refractory to standard therapy 2011: Submission as FDA-approved drug under priority review programme 2012: First licensed medicine for patients with advanced BCCRetroviruses-Infect a host, in particular certain cells, then reverse-transcribe to form a dsDNA copy of themselves, which then integrates at random into host genome -As integrated DNA, retroviruses have 2 LTRs at each end -LTRs provide incredibly strong constitutively active promoters, responsible for driving transcription of viral DNA (to make new RNA, which can be packaged into new viral particles) -If virus integrates in-frame next to host gene, read-through can occur from strong viral promoter, so that now host gene is upregulated transcriptionallyProto-oncogenes/oncogenes-Proto-oncogene = act normally to control growth, but if hyper-activated develop into 'oncogenes' and cause cancer -Repressor = tumour suppressor gene -Activator = dominant oncogeneInt1-Homologous to Drosophila wingless gene (when mutated gave abnormal wings), and therefore re-named Wnt1 -Canonical Wnt pathway crucial to normal/abnormal growth -MMTV insertion leads to elevated levels of Wnt signalling, therefore aberrant growth/tumourigenesis -Wnt pathway genes are probably regulating cyclinsWnt pathway: absence of Wnt-Beta-catenin is phosphorylated by destruction complex -Destruction complex = Axin, scaffold protein that binds both APC, beta-catenin and 2 kinases (GSK3b and CKIa) -Beta-catenin is then phosphorylated by CKIa and GSK3b in this complex --> phosphorylated form is recognised by Slimb, which is associated with the complex as well, that causes beta-catenin to be ubiquitinated and degraded by proteasome -In absence of nuclear beta-catenin, TFs are bound to promoter of Wnt-responsive genes but associate with Groucho (transcriptional repressor) --> gene expression inhibitedWnt pathway: presence of Wnt-Wnt binds to Frizzled receptor -Dishevelled is recruited to Frizzled and is phosphorylated --> may bind Axin and is somehow involved in inactivating the destruction complex -Beta-catenin can now accumulate in the cytoplasm and translocate to the nucleus -Here it can displace Groucho from TCF DNA binding proteins and in combination with some additional transcriptional regulators it will activate target gene transcriptionAPC: shown to act as a tumour-Negative regulator of Wnt pathway -Found to show high correlation with human colon cancers (APC gene) -If both copies are knocked out in mouse --> early colon-like cancersWnt signalling controls cell growth-Activation of Wnt signalling can promote stem cell self-renewal -Uncontrolled self-renewal is hallmark of oncogenesis -Abnormal Wnt pathway activation in stem cells leads to their uncontrolled growth, and hence to earliest stages of cancerous phenotype -This type of evidence is coming particularly from evidence from colon cancers - cells in crypt are stem and progenitors, giving rise continuously in adult to new cells in villi -Elevated beta-catenin in early colon cancer (adenoma)Intestinal cells & cancer-Intestine lined with rapidly self-renewing epithelium -Organised into crypts (contain stem cells and transit-amplifying cells) and villi (protrusions covered with terminally differentiated cells) -Wnt pathway dominant force underlying proliferative activity of intestinal epithelium in both normal physiology and CRC (colorectal cancer)CRC cells-Proliferative crypt compartment harbours CRC cells, pluripotent and long-lived stem cells that give rise to all differentiated cell types of intestinal epitheliumLgr5-Orphan GPCR -First definitive adult intestinal stem cell marker -Lineage tracing experiments using inducible Cre knocked into Lgr5 locus generates ribbons of epithelial cells of all types and genetically marked by LacZ expression for up to 1 year after single Cre induction -From stem cells in crypts, a pool of transit-amplifying cells emanates - these cells will terminally differentiate into 1 of 4 principal epithelial cell lineages of intestineCancer stem cell concept-Proposes that tumours are generated and maintained by small, defined set of undifferentiated cells that are able to self-renew and differentiate into the bulk tumour population - these may be closely correlated to CBC (crypt basal columnar) cellsInbred mouse strains and genetically modified mice-To identify oncogenes/tumour suppressor genes -Identify signalling pathways that lead to specific tumour types -Predict which genes might give rise to cancers, if mutated -Correlate particular signalling pathways with tumour aggressionComponents of Wnt pathway implicated in human tumours/experimental cancer models-Wnt1 = oncogene activated by MMTV in murine breast cancer -APC = first isolated as tumour suppressor gene in human colon cancer -Recent example of link between cancer and Wnt signalling comes from identification of legless gene (Drosophila)Legless gene Human AXIN1 gene TCF1 Wnt5 SFRP family members-Homologue of Bcl-9 -Gene implicated in B cell malignancies -AXIN1 gene mutations reported in human hepatocellular carcinomas -TCF1 gene = can also act as tumour suppressor gene, as Tcf1 mutant mice develop adenomas in gut and mammary glands Wnt5 = can act to counteract signalling by other Wnts, appears to be able to act as tumour suppressor, as mice that are mutant for Wnt5A develop lymphoid malignancies SFRP family members = epigenetically activated in colon cancer - SFRPs may sequester and inactivate Wnt, loss of SFRP expression may contribute to Wnt activation in cancerRegenerative medicine-If we understand the molecular details that direct the differentiation of a particular cell type in vivo, we can recapitulate this, i.e. to direct the differentiation of a variety of 'stem' (pluripotent) cells -Through developmental biology approach, we know a lot about steps required for specification of particular cell typeJessell et al recapitulate development to make motor neurons from embryoid bodies-Embryoid body = mass of totipotent stem cells -When you grow embryoid bodies that have been pushed to neural fate in RA, the embryoid body-derived early neural cells change from 'anterior' neural identity to 'posterior' neural identity (A) Had already worked out pathway for motor neuron generation (B) Motor neurons derive from progenitor domain Nkx6.1 and Olig2, and weakly Pax6 (C) Addition of Shh at specific concentration to neutralised and RA-treated 'posteriorised' embryoid bodies, causes them to differentiate into motor neuron progenitor cellsCan these behave properly if you were to transplant them back into a host?-First get around problem of being able to get ESCs to differentiate purely along a motor neuron fate, and then be able to recognise the transplanted cells -This provides way of selecting for pure population of MNs (on FACS) -Can be used as a biological read-out for MNDTest(1) Find motor neuron-specific TF (HB9) (2) Subclone HB9 promoter upstream of GFP (3) Make transgenic mouse that now drives GFP in motor pools (4) Isolate ESCs from this HB9-eGFP transgenic mouse - treat them as described earlier (i.e. make embryoid bodies, neuralise, posteriorise, treat with Shh to get MNs) (5) All MNs will fluoresce green - transplanted MNs (recognised through HB9-driven expression of eGFP) integrate into host chick spinal cord and take normal axon trajectories associated with normal MNsShort-term goals(1) Development of drugs that are specific to MNs (2) Derivation of human ESCs and their differentiation into MNs (3) Use in studies to understand biology of MNDUse in drug screens(1) 5-day old human embryo (blastocyst) (2) Isolate inner cell mass, which contains ESCs (3) Culture ESCs in special conditions to grow different kinds of tissues (4) Get e.g. heart muscle cells, bone marrow cells, kidney cells (5) Inject into e.g. heartECS-derived directed MNs display wide variety of normal functional properties-E.g. ability to form synapses with muscle and make correct NT -Conclude that: (1) such routes will provide future basis for drug development - by enabling us to grow large amounts of specific cell types that can be screened in drug development/toxicity assays (2) there may be hope for this type of strategy in regenerative medicine approaches (3) much current work is trying to resolve whether you can get ESCs to integrate and function properly when transplanted into adultParkinson's Disease-Progressive disorder of CNS that occurs when DA-producing neurons are damaged, or die, in substantia nigra -DA = chemical messenger in brain responsible for coordinate functioning of body's muscles -Current therapies: L-dopa treatment (many problems) -Similar approach to that described for MNs to direct ESCs to differentiate into DA neurons is potentially more usefulGene regulatory pathway required for midbrain DA neuronal specification-Cell replacement therapies - elucidation of developmental pathways can facilitate generation of therapeutically important cell types from stem cells -Lmx1a and Msx1 function as determinants of midbrain DA neurons -Lmx1a is crucial and required to trigger DA cell differentiation -Early activity of Lmx1a is to induce expression of Msx1 -Msx1 complements Lmx1a by inducing proneural protein Ngn2 and neuronal differentiation -Expression of Lmx1a in ESCs results in robust generation of DA neurons with 'correct' midbrain identityDirect human pluripotent cell (iPS cell) to DA fate-Due to ethical/moreal problems associated with using ESCs, scientists are increasingly using iPS cells -'Reprogrammed' human skin cells from Parkinson's patients into ESC-like state -After removing reprogramming genes, scientists used iPS cells to create DA-producing neurons -To confirm that iPS cells had become DA-producing neurons, they stained the cells green for neuron-specific protein (class II beta-tubulin) and red for DA-producing neuron-specific enzyme (tyrosine hydroxylase)ESCs Vs iPSCs-Problem with ESCs = rejection -Program normal skin fibroblasts from normal patient using combination of 4 TFs and use previous approach to direct them to midbrain DA neurons -Take iPS (from fibroblasts from normal person and person with Parkinson's) and direct them both to midbrain DA neurons -2 dishes of neurons: 1 healthy and 1 diseased --> ability to examine in detail what biological processes are going wrong in diseased patientParkin and pink1-/- will develop loss of DA and problems with motor activity - Good model for PD -Take humans with PD known to have mutation in parkin gene and make iPS cells from both patient groups --> showed that they had enough cells to start exploring mechanism of what was going wrong -Scientists in US successfully made human brain cells in lab that are an exact replica of genetically caused PD - means that they can now see exactly how mutations in the parkin gene cause disease in an estimated 1/10 patients with Parkinson's -Critical to study human neurons for PD because some animal models that lack parkin gene do not develop PDParkin mutation-Upregulation of monoamine oxidases (involved in oxidative stress - mitochondria) -Both Alzheimer's and PD are involved with elevated levels of MAO-B in brain -Normal MAO-B activity creates ROS, which directly damage cells -MAO-B levels increase with age, suggesting role in natural age related cognitive decline and increased likelihood of developing neurological diseases later in lifeTurn donated skin cells into brain tissue-Used skin samples from 4 volunteers: 2 healthy and 2 with PD caused by parkin gene mutation -Scientists showed that they can reverse the defect by putting a normal parkin gene into diseased neuronsNeurons derived from re-programmed fibroblasts functionally integrate into foetal brain and improve symptoms of rats with PD-Create patient-specific donor cells for transplantaon therapy, avoiding immunorejection, a major complication in current transplantation medicine -4 TFs Oct4, Sox2, Klf4 and c-Myc induce pluripotency in mouse fibroblasts -Show that iPS cells can be efficiently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture -Minimised risk of tumour formation from grafted cells by separating contaminating pluripotent cells and committed neural cells using fluorescence-activated cell sorting -Demonstrates therapeutic potential of directly reprogrammed fibroblasts of neural cell replacement in animal modelProcedure(1) Make iPS cells (2) Direct them in vitro to DAnergic fate (Nurr1/TH) using knowledge gained through animal models of development (3) Transplant into embryonic brain (4) In vivo, Nurr+TH+ cells are not just found in SN of midbrain (5) Transplanted cells actually end up integrating into various regions of brain, but including the SNPlasticity of somatic epigenome-Recent advances in somatic cell reprogramming have highlighted plasticity of somatic epigenome, particularly through demonstrations of direct lineage reprogramming of one somatic cell type to another by defined factors -However, not clear to what extent this type of reprogramming is able to generate fully functional differentiated cellsGWAS-Combination of plummeting of genomic sequencing coupled with massively improved computing power for meta-data analysis (analysis of very big data sets) -Now means that people can sequence thousands of genomes and get computers to do the crunching that says: 'do we see any association between any unusual bits of genetic material and the risk that those individuals will have a particular disease?'Largest meta-GWAS uncovers genetic links for PD-Revealed genetic risk underlying PD -After replication in 3361 cases and 4573 controls, they observed 2 strong association signals, 1 in gene encoding a-synuclein and another at MAPT locus -Most PD-associated SNPs fell into non-coding regions - might influence expression levels of one or more nearby genesResults-Measured mRNA expression as well as CpG DNA methylation in post-mortem brain tissue samples from healthy donors -By cross-referencing these data with genotype of each nearby PD-associated SNP, they identified 30 significant associations between variants and either mRNA levels or CpG methylation of nearby genes -In some cases, a single variant was associated with the expression and/or methylation of more than one gene (disease risk may result from complicated relationships between genes, even from a single polymorphism) -E.g. 1 PD-risk associated SNP on chromosome 7 - rs199347 - was associated with both decreased methylation of nearest gene, GPNMB, as well as increased expression of another nearby gene, NUPL2 -Therefore, this SNP could heighten PD risk by modulating expression of either or both of these genes