Lecture 9 UE TBMC Cancer Biology PDF

MASTER 1 TBMC parcours Cancer Biology ANNEE 2024-2025 SANDRINE DABERNAT AND SAMUEL AMINTAS Shared signaling pathways in ontogenesis and oncogenesis Mechanisms of activation of proto-oncogenes Mechanism of inactivation of tumor suppressor genes Normal...

MASTER 1 TBMC parcours Cancer Biology ANNEE 2024-2025 SANDRINE DABERNAT AND SAMUEL AMINTAS Shared signaling pathways in ontogenesis and oncogenesis Mechanisms of activation of proto-oncogenes Mechanism of inactivation of tumor suppressor genes Normal G G Cells 1st event Mutation, Epigenetic (Germline* or Somatic) (*hereditary cancers or imprinting) Loss of function ( ) of one allele of a tumour suppressor gene (G) G G 2nd event (Somatic) Elimination Tumour G G G G G G G G or loss of function ( ) of the second allele of a Cells tumour suppressor gene (G) Monosomy Isodisomy Deletion Mitotic Mutation or Non-disjunction Recombination Methylation… Detection of loss of heterozygosity (LOH) From Dr. D Cappellen ACTIVITY 1  Wnt signalling network. PCP= planar cell polarity ligand-receptor complex signalosome dishevelled associated activator of morphogenesis Plasma membrane extension cycD1 c-Myc Daniel Routledge, Steffen Scholpp Development 2019 146: dev176073  Common features of WNT proteins Cleavage site NH2- Signal Peptide -COOH Family of secreted signaling molecules (19 in mammals) 21 invariant Cysteines Secreted but poorly soluble 1-4 N-linked glycosylations (except DWnt8) Lipid modifications: palmitic and palmitoleic acid (prenylation) MW between 36 and 50 KDa. From Dr. D Cappellen  Trafficking of Wnt outside of the cells. Wls=wntless 2 1 1’ Daniel Routledge, Steffen Scholpp Development 2019 146: dev176073  Facilitated diffusion of Wnt. Local diffusion (Chaperone-like) Lateral diffusion Daniel Routledge, Steffen Scholpp Development 2019 146: dev176073  Cytoneme-mediated Wnt transport Daniel Routledge, Steffen Scholpp Development 2019 146: dev176073 Gene gradient interplay for antero-posterior axis formation and liver development WNT antagonists Broken embryonic symmetry Perugorria et al. Nature Reviews Gastroenterology & Hepatology volume 16, pages121–136 (2019)  Gene gradient interplay for the intestinal crypt development and maintenance Du et al. PLOS Computational Biology | DOI:10.1371/journal.pcbi.1004285 ACTIVITY 2  Canonical Non canonical pathway pathway WNT2 FZD5 Normal/viable WNT3 BCAT APC1 WNT2B WNT 5B formation LRP6 VANGL2 A-P axis WNT5A WNT6B AXIN1 WNT8A WNT7A TCF3 WNT8B GSK3b patterning WNT1 WNT16 DVL2 FDZ1 D-V INVERSIN TCF4 FDZ2 CELSR1 development WNT9B FZD3 FDZ7 D-V Limb AXIN2 FDZ8 LEF1 CELSR3 WNT9A WNT11 development TCF1 Late fetal WNT4 WNT7B FDZ6 FZD4 After birth WNT10B FZD9 defect DVL1 LRP5 (Activity 2) Figure for activity oncogenesis ACTIVITY 4 A B MIN WT Migration Sanger sequence of the mAPC gene C Different germline mutations in APC influence polyps phenotype in mouse models  Step-wise tumorigenesis in colorectal cancer. ACF= aberrant crypt foci Master 1 Oncology Hedgehog pathway and cancer Amintas Samuel (PHU) Dabernat Sandrine (PU-PH) Hedgehog (HH) pathway ➔ Discover with the study of drosophila larvas (Larvas with KO of HH ligand gene looks like hedgehogs) Hedgehog (HH) pathway ➔ Key regulator of embryonic development ➔ Involved in embryonic cell differentiation ➔ Role in body segmentation and organogenesis ➔ Also involved in adult stem-cells regulation and homeostasis for tissue maintenance and regeneration Hedgehog (HH) ligands ➔ 3 HH homologous in mammalians › Desert Hedgehog › Indian Hedgehog › Sonic Hedgehog = SHH Phylogenetic relationship of hedgehog ligands (based on Ingham and McMahon, 2001). Hedgehog (HH) ligands biogenesis ➔ 45 kDa precursor with multiple post traductional modifications in ER u u u u Degradation by proteasome Hedgehog (HH) ligands secretion ➔ 4 different mecanisms of secretion Briscoe & al. ; Nature review 2013 Hedgehog (HH) ligands secretion ➔ Direct secretion Briscoe & al. ; Nature review 2013 Hedgehog (HH) ligands secretion ➔ Soluble oligomer Briscoe & al. ; Nature review 2013 Hedgehog (HH) ligands secretion ➔ Lipoprotein Briscoe & al. ; Nature review 2013 Hedgehog (HH) ligands secretion ➔ Exovesicle Briscoe & al. ; Nature review 2013 Hedgehog (HH) pathway Hedgehog (HH) pathway Ptch1 / SMO interaction « Sterol pump » Hedgehog (HH) pathway GLI activation SMO inactive u u u u u Ubiquitinylation and degradation Nucleus translocation and Briscoe & al. ; Nature review 2013 transcriptionnal repression GLI activation SMO active Nucleus translocation and transcriptionnal activation Briscoe & al. ; Nature review 2013 Hedgehog (HH) pathway and development ➔ Sonic HH is a major morphogene involved in embryogenesis ➔ Spatial and temporal gradients in tissue to guide cell during embryonic development Lee et al., Autism Res Treat, 2011  Hedgehog (HH) pathway and development ➔ Activity 5 To understand the role of Ptch1 in mediating Shh signaling in the developing cortex, we characterized the temporal and cell type- specific pattern of Ptch1 expression with heterozygous Ptch1-LacZ mutant mice, where a lacZ-neo fusion gene was inserted in the place of the start codon and portions of exon 1 and the entire exon 2 of the Ptch1 gene (LacZ gene induces expression of Beta-galacosidase enzyme) Q1 : Make a schematic representation of mutant mice Ptch1 gene 34  Hedgehog (HH) pathway and development Ptch1 gene ATG (start codon) Proximal 5’ promotor Exon 1 Exon 2 Exon 3 Exon n+ 3’ Cassette LacZ-neo 5’ LacZ Neo 3’ Modified Ptch1 gene ATG Proximal 5’ promotor LacZ Neo Exon 3 Exon n+ 3’ 35  Hedgehog (HH) pathway and development 36  Hedgehog (HH) pathway and development DAPI = nuclear DNA staining / P5 to P60 => Post-natal days / P60 : cortical development is over 37  Hedgehog (HH) pathway and development DAPI = nuclear DNA staining / P5 to P60 => Post-natal days / P60 : cortical development is over Q2 : How cell density vary in cortical layers during the first 60 days of mouse life? 38  Hedgehog (HH) pathway and development DAPI = nuclear DNA staining / P5 to P60 => Post-natal days / P60 : cortical development is over Cell density at P5 is higher in layers II/III and IV. On the contrary, these same layers present the lower cell density at P60 39  Hedgehog (HH) pathway and development Global By layer and by timepoint 40  Hedgehog (HH) pathway and development Global Q3 : How Pitch1 expression vary in cortical layers during the first 60 days By layer and of mouse life? by timepoint 41  Hedgehog (HH) pathway and development Global Pitch1 expression is significantly higher in the cortical layers at P60. At P5, layers IV and V mainly express Ptch1. The expression in By layer and homogenous between layers for P15 by timepoint and P28. At P60, Picth1 is mainly express by layers I/II/III and IV. 42  Hedgehog (HH) pathway and development S100B = astrocytes marker NeuN = Neuronal marker Olig2 = oligodendrocytes markers E: Proportion of Ptch1-LacZ-expressing cells with corresponding markers in P5–P60 cortex. Cell types switch of expression depending on the development timepoint => Ptch1 is mainly express by oligodendrocytes markers at P5, then by astrocytes between P15 and P28, and by neurons and astrocytes at P60 43  Hedgehog (HH) pathway and development We used an astrocyte-specific conditional mutant mouse line with conditional deletion (tamoxifen dependant) of Ptch1 (Ptch1cKO). We next compare gene expression by RNA-seq in WT and mutant astrocytes at P28 Cre/Lox system 44  Hedgehog (HH) pathway and development G: Volcano plot showing fold changes of gene expression in Ptch1cKO astrocytes. Red dots, p < 0.01, |log2FoldChange| > 1; orange dots, p < 0.01; remaining dots are gray. 45  Hedgehog (HH) pathway and development G: Volcano plot showing fold changes of gene expression in Ptch1cKO astrocytes. Red dots, p < 0.01, |log2FoldChange| > 1; orange dots, p < 0.01; remaining dots are gray. Q5 : Could you predict the consequences of Ptch1 conditional KO on SHH genes expression in astrocytes? What types of major cellular pathways would be impacted ? 46  Hedgehog (HH) pathway and development G: Volcano plot showing fold changes of gene expression in H: Fold-changes of Ptch1, Smo and Hhip Ptch1cKO astrocytes. Red dots, p < 0.01, |log2FoldChange| > 1; expression in Ptch1cKO astrocytes. orange dots, p < 0.01; remaining dots are gray. L: Gene ontology analysis of differentially expressed genes between WT and Ptch1cKO cortical astrocytes. Ptch1 is a repressor of SHH pathway => its inhibition leads to the activation of the SHH pathway and upregulation of SMO expression Ptch1 KO leads to the disregulation of expression of multiples genes 47 involved in morphogenesis and developmental process Hedgehog (HH) pathway and cancer Gorlin syndrome Harmsen & al. , European journal of human genetics, 2009 Hedgehog (HH) pathway and cancer Normal cell PTCH PTCH WT WT PTCH function OK (100% of functional protein) Hedgehog (HH) pathway and cancer Normal Cell from Normal cell Gorlin patient PTCH PTCH PTCH PTCH WT WT WT mutation PTCH function OK Altered PTCH function (100% of functional protein) (50% of functional protein) Hedgehog (HH) pathway and cancer Normal Cell from Tumoral cell from Normal cell Gorlin patient Gorlin patient PTCH PTCH PTCH PTCH PTCH PTCH WT WT WT mutation mutation mutation PTCH function OK Altered PTCH function PTCH loss of function (100% of functional protein) (50% of functional protein) (Absence of functional protein) Hedgehog (HH) pathway and cancer Normal Cell from Tumoral cell from Normal cell Gorlin patient Gorlin patient PTCH PTCH PTCH PTCH PTCH WT WT WT mutation mutation PTCH LOH PTCH function OK Altered PTCH function PTCH loss of function (100% of functional protein) (50% of functional protein) (Absence of functional protein) Hedgehog (HH) pathway and cancer Normal Cell from Tumoral cell from Normal cell Gorlin patient Gorlin patient PTCH PTCH PTCH PTCH PTCH WT WT WT mutation mutation PTCH LOH PTCH function OK Altered PTCH function PTCH loss of function (100% of functional protein) (50% of functional protein) (Absence of functional protein) Hedgehog (HH) pathway and cancer ➔ The “cancer” roles of HH pathway components depending on their initial function ➔ Mutations of HH pathway effectors : ➔ HH pathway activator => proto-oncogene ➔ HH pathway inhibitor => tumor suppressor  Hedgehog (HH) pathway and cancer ➔ Little quizz : Proto-oncogene or suppressor tumor gene ? Sonic Hedgehog ? PTCH1 ? SMO ? GLI ? 55  Hedgehog (HH) pathway and cancer ➔ Little quizz : Proto-oncogene or suppressor tumor gene ? Sonic Hedgehog ? Oncogene PTCH1 ? SMO ? GLI ? 56  Hedgehog (HH) pathway and cancer ➔ Little quizz : Proto-oncogene or suppressor tumor gene ? Sonic Hedgehog ? Oncogene PTCH1 ?Tumor suppressor SMO ? GLI ? 57  Hedgehog (HH) pathway and cancer ➔ Little quizz : Proto-oncogene or suppressor tumor gene ? Sonic Hedgehog ? Oncogene PTCH1 ?Tumor suppressor SMO ? Oncogene GLI ? 58  Hedgehog (HH) pathway and cancer ➔ Little quizz : Proto-oncogene or suppressor tumor gene ? Sonic Hedgehog Oncogene PTCH1 Tumor suppressor SMO Oncogene GLI Oncogene 59 Hedgehog (HH) pathway and cancer Niyaz et al. ; Trans. Onc. 2019 Hedgehog (HH) pathway and cancer ➔ Mutations in HH pathway components : › Other activating mutations => SMO in Basal-cell carcinoma (10%) / SUFU and PTCH1 in medulloblastomas (30%) › But abnormal activation of HH is present in many different cancer types including, lung, liver, breast, prostate, stomach, colon and pancreas. Hedgehog (HH) pathway and cancer ➔ Dysregulated HH signaling lead to increased cellular proliferation and tumor growth ➔ 4 models for HH pathway activation in cancers: › Type I => Ligand independant model › Type II => Ligand dependant and autocrine activation model › Type III => Ligand dependant and paracrine activation model › Type IV => Aberrant activation of HH pathway in stem cells (autocrine or paracrine) Hedgehog (HH) pathway and cancer Niyaz et al. ; Trans. Onc. 2019 Hedgehog (HH) pathway and cancer Type I => Ligand independant model Type I => Ligand independant model Niyaz et al. ; Trans. Onc. 2019 Hedgehog (HH) pathway and cancer Type II => Ligand Type II => Ligand dependant and autocrine activation model dependant and autocrine activation model Type Type II => II => Ligand Ligand dependant Type and depean and autocrine activation II => Ligand autocrine activation model model depndant and autocrine activation model Niyaz et al. ; Trans. Onc. 2019 Hedgehog (HH) pathway and cancer Type III => Ligand dependant and paracrine activation model Niyaz et al. ; Trans. Onc. 2019 Hedgehog (HH) pathway and cancer Type IV => Aberrant activation of HH pathway in stem cells (autocrine or paracrine) Niyaz et al. ; Trans. Onc. 2019 Hedgehog (HH) pathway and pancreatic cancer Hedgehog (HH) pathway and ➔ Downregulation of HH pancreatic signaling is critical cancer for the WT embryonic development of instestinal tract and pancreas Smo OE ➔ Constitutive Hedgehog Signaling Results in Abnormal Development of the Gut in xenopus embryo Smo WT Zhang j. & al. , Dev Biol. , 2001 overexpression Hedgehog (HH) pathway and pancreatic cancer ➔ In adult pancreas, HH signaling is limited to β cells ➔ Dysregulated HH signaling has been strongly implicated in the genesis of pancreatic ductal adenocarcinoma (PDAC) ➔ Overexpression of SHH ligand is already detected in PanINs (pancreatic intraepithelial neoplasia) and in PDAC with higher rates Thayer et al. , Nature, 2013 Hedgehog (HH) pathway and pancreatic cancer Hedgehog (HH) pathway and ➔ SHH cooperates with pancreatic cancer activate K-Ras to promote Pancreatic tumor development and acts to provide resistance to various chemotherapies Undiferiencied tumor J. P. Morton et al, PNAS, 2007 Hedgehog (HH) pathway and pancreatic cancer Hedgehog ➔ Activity 6 (HH) pathway and pancreatic cancer BrDU is a DNA intercalator Hedgehog (HH) pathway and pancreatic cancer Hedgehog ➔ Activity 6 (HH) pathway and pancreatic cancer BrDU is a DNA intercalator Cyclopamine : - Inhibit the DNA replication and slow down the cell-cyle Hedgehog (HH) pathway and pancreatic cancer Hedgehog ➔ Activity 6 (HH) pathway and pancreatic cancer BrDU is a DNA intercalator Cyclopamine : - Inhibit the DNA replication and slow down the cell-cyle - Increase apoptosis Hedgehog (HH) pathway and pancreatic cancer Hedehog ➔ Activity 6(HH) pathway and pancreatic cancer BrDU is a DNA intercalator Cyclopamine : - Inhibit the DNA replication and slow down the cell-cyle - Increase apoptosis - Decrease cell proliferation Hedgehog (HH) pathway and pancreatic cancer Hedgehog ➔ Activity 6 (HH) pathway and pancreatic cancer BrDU is a DNA intercalator Cyclopamine : - Inhibit the DNA replication and slow down the cell-cyle - Increase apoptosis - Decrease cell proliferation Cyclopamine inhibits a SHH pathway activator => SHH / SMO / GLI  HH Pathway : Therapeutics ➔ First compound identified as an HH inhibitor was cyclopamine, a natural alkaloid derived from Veratum californicum. › Block Smoothened (SMO) by binding to its heptahelical bundle, locking it in an inactive form 76  HH Pathway : Therapeutics ➔ First compound identified as an HH inhibitor was cyclopamine, a natural alkaloid derived from Veratum californicum. › Block Smoothened (SMO) by binding to its heptahelical bundle, locking it in an inactive form › Low bioavailability › Short half-life › Chemical instability Not a potent therapeutic 77  HH Pathway : Therapeutics ➔ First compound identified as an HH inhibitor was cyclopamine, a natural alkaloid derived from Veratum californicum. › Block Smoothened (SMO) by binding to its heptahelical bundle, locking it in an inactive form › Low bioavailability › Short half-life Not a potent therapeutic › Chemical instability ➔ Numerous SMO antagonists have been develloped based on cyclopamine structure but with higher potency and are now available ➔ Glasdegib : FDA approved in 2018 and Europe apporval in 2020 for treatment of acute myeloid leukemia 78  Thank you for your attention