Who manipulates who in dysregulated mineralised tissue resorption? Dr Gurå Therese Bergkvist MRCVS
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1 Who manipulates who in dysregulated mineralised tissue resorption? Dr Gurå Therese Bergkvist MRCVS Senior Lecturer in Veterinary Anatomy & Clinical Research Associate of The Roslin Institute
2 Clinical diseases involving mineralised tissues Shared risk factors Shared environments Shared poor prognosis Shared molecular markers Feline oral squamous cell carcinomas Locally aggressive, commonly invade into bone. Highjacks the osteoclasts to invade? Feline tooth resorption Unknown cause, reactivation of tooth resorption by odontoclasts years after shedding of milk teeth
3 A. Feline tooth resorption A study of odontoclast dysregulation in feline tooth resorption
4 Feline tooth resorption Healthy teeth Early stage of TR in molar Multiple teeth affected, extensive tissue loss Final stage of TR Loss of dentition
5 Tooth shedding Replacement of milk teeth by permanent dentition Tooth root resorption of milk teeth Underlying adult tooth not resorbed n/curriculum/vm8054/vm80 54HP.htm Milk tooth being shed Gum line New tooth waiting to erupt How is this possible? Jaw bone
6 The cells that can resorb mineralised tissues like dentine, enamel, and bone: Odontoclast (in teeth) or Osteoclast (in bone)
7 The question How does the odontoclast know to resorb the milk teeth and not the adult teeth right next to it?
8 The answer Periodontal Ligament (PDL) cells & the cells of the microenvironment
9 The answer RANKL/RANK/OPG SYSTEM RANKL expressed from PDL of milk teeth >>permanent teeth RANKL RANKL-RANK interaction Stimulation of tooth resorption RANK Odontoclast OPG-RANKL interaction OPG is a decoy receptor so this interaction causes no activation. Produced by permanent teeth
10 It doesn t always work feline tooth resorption Dentine Odontoclast in molar with advanced stage TR sometimes odontoclasts become reactivated later in life
11 RNA seq study A = teeth from TR affected vs. TR free cats B = TR affected teeth vs. TR free teeth C = TR affected teeth and TR free teeth from same cat C showed 16,307 differentially expressed genes
12 RNA seq study results Analysis Abbreviation Name Gene selection Function MMP9 Matrix Metallopeptidase-9 Role in ECM degradation P2XR4 P2X purinergic receptor 4 Ligand-gated cation channel SPI.1 Spi-1 proto-oncogene Transcriptional activator, possible role in differentiation/activation of osteoclasts and odontoclasts CTSK Cathepsin-K Expressed by osteoclasts, role in ECM degradation RANKL Receptor Activator of Nuclear Factor -B Ligand Triggers osteoclast and odontoclast differentiation/activation OPG Osteoprotegerin Decoy receptor for RANKL
13 Fold changes normalised to TR - Quantitative qpcr MMP9 and P2XR4 were significantly upregulated 15 MMP9 paired comparison 10 5 TR1- TR1+ TR2- TR2+ TR3- TR3+ TR4- TR4+ TR5- TR5+ TR6-0 TR6+
14 H&E sections of healthy tooth & tooth resorption lesion
15 Immunohistochemistry MMP9 MMP9 positive odontoclasts in resorption pits Healthy TR +ve PDL Alveolar Bone Dentine TR +ve TR +ve
16 MMP9 expression increases rapidly at an early stage during osteoclast differentiation Bone marrow cells enriched for osteoclast precursors (myeloid origin) Terminally differentiate d osteoclasts
17 MMP9 inhibitor reduced osteoclast formation and resorption activity TRAP positive osteoclasts (% control) Medium Osteoclast formation Vehicle(DMSO) 5 ### ### 20 MMP9 inhibitor nm *** *** 1000 Resorption pits (% control) ** Medium Resorption activity Vehicle (DMSO) 5 *** 20 ### *** MMP9 inhibitor nm ### *** 1000 Number of osteoclasts formed was reduced Osteoclast resorption activity was reduced
18 MMP9 sirna reduced osteoclast formation but not resorption activity Number of osteoclasts formed was reduced Osteoclast resorption activity was reduced, but not compared to the scrambled control
19 A. Feline tooth resorption Summary Potential new therapeutic target further studies required Implications for osteoclast differentiation?
20 B. Feline Orals Squamous Cell Carcinomas Role of microenvironment in osteoclast activation
21 Feline Orals Squamous Cell Carcinomas (FOSCC)
22 FOSCC invasion is mediated by osteoclasts
23 Role of microenvironment in osteoclast activation??? ECM ph Tumour microenvironment Bone osteoblasts & osteocytes??? ECM fibroblasts ECM hypoxia RANKL? OPG IL 1β??
24 Proteomic analysis of bone invasive FOSCC cell line Silver stain Mass spectrophotometry revealed 168 different proteins (secreted, structural, enzymes) Identification of alternative/enhancing pathways to osteoclastogenesis SMG Conditioned medium protein precipitate Identification of possible cancer biomarkers
25 The effect of conditioned media on osteoclast formation and resorption A media only B media and conditioned media C CSF added D RANKL and CSF E RANKL, CSF & conditioned media
26 The effect of conditioned media on osteoclast formation and resorption The presence of conditioned media from the bone invasive FOSCC cell line causes: Increased osteoclast formation Increased resorption on dentin and mineralised plates Increased survival of osteoclasts in hypoxic conditions
27 Ex vivo mouse calvaria co-culture experiment BoneKEy Reports 5, Article number: 818 (2016) doi: /bonekey
28 Ex vivo mouse calvaria co-culture experiment Bone FOSCC cell line
29 B. FOSCCs, Microenvironment & Osteoclasts Summary FOSCCs release substances that enhance osteoclast formation, resorption and survival Proteomic analysis of CM from bone invasive and non-bone invasive tumours to narrow down potential candidates
30 C. FOSCC Development of targeted therapies
31 HNSCC & FOSCC Some common molecular markers: Epidermal Growth Factor Receptor (EGFR) c-src
32 Targeted therapies using RNA interference sirna validation c-src sirna EGFR sirna qpcr relative mrna levels Western Blot relative protein levels U: Untreated S: Scrambled C: Cocktail
33 Chicken Chorioallantoic Membrane (CAM) model Liu M, Scanlon CS, Banerjee R, Russo N, Inglehart RC, Willis AL, et al. Translational Oncology. 2013;6(3):
34 Development of targeted therapies Untreated Scrambled sirna treated H&E Cytokeratin
35 My research group Seungmee Lee Stratos Rokkos Monoar Pallab Mark Gray Nicola Mawson Abigail Mould Erika Abbondati Collaborations: David Argyle Group Lisa Pang Rhona Muirhead Donald Yool Colin Farquharson Group
36 Thank you for you attention
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