03.29.12 - SLU PhD Admissions Seminar

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  • Figure 1: Backcross Scheme and Quantitative Trait Locus Mapping
    NZB and C58 mice were crossed to FVB then re-crossed with MMTV-PyMT-transgenic FVB (A). Primary tumor, pulmonary metastasis, and genotype data was obtained from transgene-positive mice developed tumors and was used to generate QTL maps for pulmonary metastasis (B).
  • Figure 3: Cadm1 Expression in Mammary Tumor and Lung
    The mRNA express of Cadm1 was measured by quantitative real time PCR in both mammary tumor (A) and normal lung tissue (B) of FVB and NZB. As depicted, inbred strains carrying the NZB chromosome 9 showed higher levels of Cadm1 mRNA in both tumor and the lung.
  • Figure 4: The Effects of Cadm1 Overexpression in vivo
    Mvt-1 and 6DT1 mouse mammary tumor cells stably expressing Cadm1 (A) were transplanted into mammary fat pads of syngeneic FVB mice. Data on primary tumor burden (B) and pulmonary surface metastases (C) was collected on 30 day. Pulmonary surface metastases were normalized by primary tumor mass to determine if Cadm1 overexpression showed an effect on metastasis independent of its tumor suppressive activity (C).
  • Figure 6: Pulmonary Metastases of Cadm1 Expressing Cells are Transgene Negative
    Matched primary tumor and lung sections were immunohistochemically stained with an antibody against the V5 epitope tag. Hematoxylin and eosin stained lung sections of lungs of mice transplanted with control (A) and Cadm1-V5 (B) expressing cells. Immunohistochemical staining of lung sections of control (C) and Cadm1-V5 (D). Immunohistochemical staining of the primary tumors produced by control (E) and Cadm1-V5 (F) expressing cells.
  • Figure 5: The Effects of Cadm1 Knockdown in vivo
    6DT1 cells expressing shRNA constructs targeting Cadm1 reducedCadm1 mRNA levels (A). Orthotopic tumor cell transplant data showing the effects of Cadm1 knockdown on primary tumor burden (B), pulmonary surface metastases (C), and tumor-normalized metastases (D).
  • Figure 8: Metastatic potential of Cadm1 Expressing Cells Rescued in Athymic Mice
  • Figure 7: Transwell Invasion and Migration Assay
  • 03.29.12 - SLU PhD Admissions Seminar

    1. 1. Cadm1, an Inherited Modifier of Metastasis that Suppresses Metastasis by Interacting with the Cell Mediated Immunity Kent Hunter Farhoud Faraji March 29, 2012
    2. 2. Agenda 1. Introduction 2. Genetic analysis reveals a locus on mouse Chr9 that is associated with metastasis 3. Validating Cadm1 as a metastasis-associated gene 4. Elucidating the mechanism of Cadm1-mediated metastasis suppression
    3. 3. Breast Cancer • Most common malignancy in women • In 2011: ~230,000 cases ~40,000 deaths (#2 killer after lung) • >90% of deaths are related to metastatic disease Cancer.org Hunter & Crawford. Cancer Res. 2006
    4. 4. Metastasis Biology Valastyan & Weinberg. Cell. 2011
    5. 5. Metastasis Biology Highly Complex Process •Numerous Gene Expression Programs •Tumor Phenotypic Transitions •Niche Remodeling •Intercellular Communication Where is potential for metastatic susceptibility encoded? Endpoint: Reaching and adapting to foreign environment Valastyan & Weinberg. Cell. 2011
    6. 6. The Genetic Background Definition: – Complement of genetic variation that distinguishes each of us as an individual More specifically: – Germline polymorphisms • SNP, Indel, CNV
    7. 7. Our Question Why do some breast cancer patients develop metastatic disease, whereas other patients, with seemingly similar tumors, don’t?
    8. 8. Does the genetic background play a role in metastasis? MMTV-PyMT Transgenic mouse •Mammary tumors 100% penetrance (9wks) •Pulmonary metastasis >90% penetrance (100days) •FVB inbred background •Luminal-like, ER+ Credit: Robert Cardiff, UC Davis Transgenic Mouse Webpage http://www-mp.ucdavis.edu/tgmice/firststop.html
    9. 9. The Experiment: Does the genetic background play a role in metastasis? Mom Dad
    10. 10. The Observation: Constants: Oncogenic driver (PyMT) Paternal genotype (FVB) Variable: Maternal genotype
    11. 11. Altering the Maternal Genotype Results in a Non-binary Phenotype  Metastasis has a heritable component independent of the oncogenic driver  Phenotypic continuum implicates the interactions of two or more genes in the metastatic process Therefore: • Metastasis can be considered a complex trait • Genetic tools can be used to identify genomic elements involved in metastatic susceptibility
    12. 12. Our System Two approaches to investigate metastatic breast cancer 1. Identify candidate genes – associated with metastasis • Genetic screen 2. Validate – are candidate genes causative? • In vivo metastasis assay
    13. 13. Identification by Forward Genetic Screen 1) Outcross between inbred strains of varying metastatic propensity 2) Cross to FVB-PyMT 3) Determine phenotype and genotype 4) Which segments of genome segregate with metastasis?
    14. 14. Validation Mvt-1 and 6DT1 – murine mammary tumor cell lines •Driver: Myc •Pulmonary metastasis >90% penetrance (30 days) •Luminal-like, ER+ In vivo metastasis assay •Orthotopic graft of isogenic murine mammary tumor cells •Immune-competent
    15. 15. Agenda 1. Introduction 2. Genetic analysis reveals a locus on mouse Chr9 that is associated with metastasis 3. Validating Cadm1 as a metastasis-associated gene 4. Elucidating the mechanism of Cadm1-mediated metastasis suppression
    16. 16. Genetic Screen FVB: Metastasis-Prone C58 and NZB: Metastasis-Resistant
    17. 17. NZB/C58 + FVB Backcross Reveals QTL peak on Chromosome 9 x P NZB or C58 FVB/N-PyMT F1 x Measure: Primary Tumor Burden Pulmonary Metastases Genotype: Determine segments of genome segregating with metastasis N2 NZB Metastasis QTL C58 Metastasis QTL
    18. 18. Subcongenic Analysis Resolves Chr. 9 Susceptibility Locus to 49-67Mb Subcongenic Tumor Burden Subcongenic Metastasis * p < 0.05
    19. 19. Hundreds of genes on Chr 9 49-67Mb Filters • Haplotype structure: [C58=NZB] ≠ FVB • Differentially expressed between NZB and FVB Candidate list • Cadm1, Pias1, Zbtb16
    20. 20. Agenda 1. Introduction 2. Genetic analysis reveals a locus on mouse Chr9 that is associated with metastasis 3. Validating Cadm1 as a metastasis-associated gene 4. Elucidating the mechanism of Cadm1-mediated metastasis suppression
    21. 21. Exon Sequencing Reveals Synonymous SNP in Exon 2 524 530 540 550 « NZB (524) « FVB (520) Consensus (524) rs327213609 560 570
    22. 22. Cadm1 is Differentially Expressed in FVB and NZB Mice Normal Whole Lung Mammary Tumor p = 0.076 * (NZB Chr9) * p < 0.05
    23. 23. Level of Cadm1 Expression Predicts Survival in Patient Data Sets of Breast Cancer GOBO: ER-positive Tumors
    24. 24. Ectopic Expression of Cadm1 in Mvt1 and 6DT1 5 5 -V -V r r 1 1 to to m m c c ad ad Ve Ve C C -1 -1 T1 DT1 vt vt 6D 6 M M anti-Cadm1 anti-V5 anti-β-actin Overexpression is within physiological range.
    25. 25. Cadm1 Expression Reduces Pulmonary Metastasis in vivo 6DT1 results demonstrate a metastasis-specific role for Cadm1 ** p < 0.01
    26. 26. Cadm1 Expression Reduces Pulmonary Metastasis in vivo
    27. 27. Metastases from Cadm1 Expressing Primary Tumors Do Not Express the Cadm1 Transgene Down-regulation of Cadm1 may be prerequisite for metastasis formation.
    28. 28. Stable Knockdown of Cadm1 by shRNA d 14 15 le b 1 1 m m m a ad ad cr S C C sh sh sh 1 1 1 DT 6DT DT 6 6 Cadm1 β-actin
    29. 29. Cadm1 Knockdown Promotes Pulmonary Metastasis Primary Tumor Burden 100 * Surface Metastasis Count Tumor Mass (g) 1.5 Pulmonary Metastases 1.0 0.5 0.0 T 6D cr 1S T 6D 14 NA hR 1s T 6D p = 0.07 ** 80 60 40 20 15 NA hR 1s 0 T1 6D r Sc T 6D N hR 1s 4 A1 T1 6D N shR 5 A1 Taken together, results confirm a causative role for Cadm1 in metastasis. Metastases Per Gram Tumor Tumor-Normalized Metastases 150 ** 100 * 50 0 T1 6D r Sc T 6D N hR 1s 4 A1 T 6D N hR 1s 5 A1 * p < 0.05 ** p < 0.01
    30. 30. Agenda 1. Introduction 2. Genetic analysis reveals a locus on mouse Chr9 that is associated with metastasis 3. Validating Cadm1 as a metastasis-associated gene 4. Elucidating the mechanism of Cadm1-mediated metastasis suppression
    31. 31. Cadm1 Initially Identified as the Gene Underlying Locus Frequently Deleted in Cancer • Observations: – 11q23 is deleted in NSCLC – Introduction 11q23.2 “completely suppresses tumor formation” • Cadm1 was identified as the responsible gene Murakami et al. PNAS. 1998 Kuramochi et al. Nat Genet. 2001
    32. 32. Loss of Cadm1 Associated with Poor Outcome in Numerous Solid Cancers Melanoma: – Loss/down-regulation (by promoter methylation) • Increased tumor stage • Significantly shorter disease-free survival Similar results in: -Neuroblastoma, Meningioma -Nasopharyngeal, Esophageal, Gastric, HCC -Pancreatic, Prostate, NSCLC -Ovarian, Cervical You et al. Melanoma Res. 2010 Murakami. Cancer Sci. 2005
    33. 33. Cadm1 is an adhesion molecule • Single-pass integral membrane protein • Homotypic cell-cell adhesion – Epithelial structure – adherens jn, hemidesmosome • Heterotypic cell-cell adhesion – Immunological synapse – Cell Differentiation • Synaptogenesis • Spermatogenesis • T-Lymphocyte Maturation Hagiyama et al. J Immunol. 2011 Wakayama et al. Anat Sci Int. 2009 Ito et al. Hepatology. 2007 Sakurai-Yageta et al. Biochem Biophys Res Commun. 2009
    34. 34. Could Cadm1 Suppress Metastasis by Regulating Motility or Invasion Properties? • 4.1 Binding Motif – Dal-1 – Tumor suppressor – Ezrin – Metastasis associated • PDZ Binding motif – Tiam1 – Metastasis associated • Rac GEF – involved in motility Busam et al. JBC. 2011 Wong et al. PNAS. 2007 Fujita et al. Am J Pathol. 2007
    35. 35. Summary of Cadm1 Effect on In Vitro Properties Cadm1 Expression: • No significant impact on tumor cell in vitro properties: – In vitro Growth – In vitro Motility – In vitro Invasion – 2D and 3D Morphology
    36. 36. Metastasis Biology •Effect of Cadm1 expression on metastasis might be distinct from early steps of metastatic cascade Valastyan & Weinberg. Cell. 2011
    37. 37. Does Cadm1 Expression Have an Effect on Late Events of the Metastatic Cascade? 21 days
    38. 38. Cadm1 Reduces Pulmonary Metastasis of Tail Vein Injected Tumor Cells Surface Metastasis Count Pulmonary Metastases 60 * * 40 20 0 -20 t-1 Mv cto Ve r t Mv adm -1 C or m1 ect ad V 1C T1 D DT 6 6 1 •Metastasis inhibitory effect of Cadm1 is not restricted to the early stages of metastasis. * p < 0.05
    39. 39. Cadm1 and Immunity NK cells NKT cells CD8+ T-cells Tumor Cell Extracellular ligand: – CRTAM - expressed on activated CTLs • increased secretion of IFNγ and IL-2 by activated CD8+ T-cells • Enhanced NK-cell cytotoxicity Galibert et al. J Biol Chem. 2005 Boles et al. Blood. 2005
    40. 40. Could Effects of Cadm1 on Metastasis be Mediated by the Immune System? Nude mouse – FoxN1 null – Athymic – Immunophenotype • Mature T-cells – Absent • B-cells – Present • NK-cells, APCs – Present and Functional
    41. 41. Cadm1 Expression Has No Effect on Metastasis or Tumorigenesis in Athymic Mice Immune Competent Host: Primary Tumor Burden Pulmonary Surface Metastases Metastasis Count 80 1.0 0.5 20 ad m 1 C 6D T1 Ve ct or 6D T1 C ad m 1 r Ve ct o ad m 1 C 6D T1 Ve ct or 6D T1 C ad m 1 M vt -1 Ve ct o M vt -1 40 0 r 0.0 60 M vt -1 Tumor Mass (g) 1.5 M vt -1 Athymic Host:
    42. 42. Hypothesis Cadm1 CRTAM Tumor Cell TCR MHCI CD8+ T-cell CD8 IFN-γ IL-2 1) CD8+ CTL-mediated tumor killing 2) NK-cell recruitment, activation, and tumor killing 3) IFN-γ-mediated tumor cytostaticity and/or killing Kuipers et al. Blood. 2011 Giangreco et al. J Immunol. 2012
    43. 43. Two Primary Questions: 1. Are CD8+ T-Cells Involved in Cadm1-Mediated Metastasis Suppression? 2. Are either IL-2 or IFN-γ involved?
    44. 44. 1) Are CD8+ T-Cells Involved in Cadm1-Mediated Metastasis Suppression? CD8+ Cell Depletion in Immune Competent Mice • Study in Progress • Design: _____IgG_____ ___anti-CD8___ Mvt1 Vector Mvt1 Cadm1 Mvt1 Vector Mvt1 Cadm1
    45. 45. 2) Do mice bearing Cadm1+ tumors show increased IFN-γ secreting lymphocytes? 30 days Fat Pad Injection Harvest: •Lung •Lymph Nodes Make Single Cell Suspension of Living Cells
    46. 46. 2) Do mice bearing Cadm1+ tumors show increased IFN-γ secreting lymphocytes? anti-IFN-γ anti-CD3
    47. 47. IFN-γ ELISPOT – Draining Lymph Nodes 6DT1 Vector 6DT1 Cadm1 Mvt1 Vector Mvt1 Cadm1 Tumor cells expressing Cadm1 may induce lymphocytes in draining lymph nodes to secrete IFN-γ secretion
    48. 48. Future Plans • If CD8+ T-cell depletion rescues metastatic phenotype: – Do CD8+ T-cells directly induce tumor cytotoxicity? • In vitro co-culture cytotoxicity assay by Cr51 release +/- Crtam blocking antibody +/- IFN-γ blocking antibody ELISA on supernatant for IL-2, IFN-γ • Is Crtam involved? – Cadm1 orthotopic transplant in Crtam KO mouse
    49. 49. Future Plans • If CD8+ T-cell depletion does not rescue metastatic phenotype: – CD4+ T-cell, NKT-cell depletion – Investigate role of stromal FoxN1 in Cadm1mediated metastasis suppression • Sort tumor stromal cells and check FoxN1 expression – BMDC, CAF, Endothelial cells, etc.
    50. 50. Summary Cadm1: 1.Is an inherited modifier of metastatic risk in mice 2.Is a metastasis suppressor in mice 3.Metastasis suppression may involve host cellmediated immunity 4.Expression levels in human tumor samples predicts prognosis
    51. 51. Conclusion Tumor-autonomous expression of Cadm1 impacts tumor metastatic capability through non-tumorautonomous mechanisms. Cadm1 may sensitize tumor cells to immune surveillance and result in lymphocyte-mediated cytotoxicity.
    52. 52. Implications The preponderance of data in the literature linking promoter hypermethylation of Cadm1 to advanced stage may indicate a critical role for loss of Cadm1 expression in cancer immunoediting.
    53. 53. Acknowledgements Dr. Kent Hunter • • • • • • • Dr. Jude Alsarraj Dr. Ling Bai Dr. Natalie Goldberger Dr. Luanne Lukes Renard Walker Dr. Scott Winter Dr. Thos Geiger Dr. Glenn Merlino • Dr. Chi-Ping Day • Dr. Raza Zaidi Dr. Lalage Wakefield • Dr. Yuan Yang Dr. Li Yang • Dr. Yanli Pang
    54. 54. Thanks for your attention Questions?
    55. 55. Thanks for your attention Questions?
    56. 56. Cadm1 localizes to cell-cell junctions Mvt1 Vector 6DT1 Vector Mvt1 Cadm1 6DT1 Cadm1
    57. 57. No Significantly Impact on Cell Morphology or Actin Stress Fibers Mvt1 Vector 6DT1 Vector Mvt1 Cadm1 6DT1 Cadm1
    58. 58. No Significant Change in 3D Culture Growth Properties in Mvt-1 Vector Mvt-1 Cadm1 6DT1 Vector 6DT1 Cadm1
    59. 59. No Significant Change in Motility in vitro •Mvt1 Vector •Mvt1 Cadm1 •6DT1 Vector •6DT1 Cadm1
    60. 60. Transwell Migration and Invasion Assay Transwell Insert Tumor Cells MatrigelCoated membrane Chemoattractant Depleted Media Chemoattractant Rich Media
    61. 61. No Significant Change in in vitro Migration or Invasion by Transwell Assay
    62. 62. Cadm1 Expression Does Not Impact Tumor Cell Proliferation in vitro •Mvt1 Vector •Mvt1 Cadm1 •6DT1 Vector •6DT1 Cadm1
    63. 63. IFN-γ ELISPOT – Lung p=0.027 p=0.003 6DT1 Vector 6DT1 Cadm1 Mvt1 Vector Mvt1 Cadm1
    64. 64. Level of Cadm1 Expression Predicts Survival in Patient Data Sets of Breast Cancer
    65. 65. Exon Sequencing Reveals Synonymous SNP in Exon 2 Intron Ii Exon « NZB (524) 524 530 Intron 540 550 PCR Amplify « FVB (520) TOPO Clone Ii Exon Consensus (524) Sanger Sequencing rs327213609
    66. 66. 1) Are CD8+ T-Cells Involved in Cadm1-Mediated Metastasis Suppression? Metastases IF CD8+ T-Cells play a major role, expect: Mvt1 Vector IgG Mvt1 Cadm1 Mvt1 Vector Mvt1 Cadm1 anti-CD8
    67. 67. Implications Schreiber et al. Science. 2011
    68. 68. Cadm1’s Divergent Role in Hematogenous Malignancies Myeloid & Lymphoblastic Leukemia: – High expression - better survival T-cell Leukemia and Lymphoma (ATLL, HTLV) – High expression – poor prognosis • Increased tumor infiltration • More aggressive tumors Kuipers et al. Blood. 2011 Paulson et al. Br J Haematol. 2009 Nakahata et al. Leukemia. 2012
    69. 69. Chromosome 9 Metastasis QTL NZB C58

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