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. 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. 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. Metastasis Biology
Valastyan & Weinberg. Cell. 2011

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. The Genetic Background
Definition:
– Complement of genetic variation that
distinguishes each of us as an individual
More specifically:
– Germline polymorphisms
• SNP, Indel, CNV

7. Our Question
Why do some breast cancer patients
develop metastatic disease, whereas
other patients, with seemingly
similar tumors, don’t?

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. The Experiment: Does the genetic background
play a role in metastasis?
Mom
Dad

10. The Observation:
Constants: Oncogenic driver (PyMT)
Paternal genotype (FVB)
Variable: Maternal genotype

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. 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. 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. 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. 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. Genetic Screen
FVB: Metastasis-Prone
C58 and NZB: Metastasis-Resistant

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. Subcongenic Analysis Resolves Chr. 9
Susceptibility Locus to 49-67Mb
Subcongenic Tumor Burden
Subcongenic Metastasis
* p < 0.05

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. 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. Exon Sequencing Reveals
Synonymous SNP in Exon 2
524
530
540
550
« NZB (524)
« FVB (520)
Consensus (524)
rs327213609
560
570

22. Cadm1 is Differentially Expressed
in FVB and NZB Mice
Normal Whole Lung
Mammary Tumor
p = 0.076
*
(NZB Chr9)
* p < 0.05

23. Level of Cadm1 Expression Predicts Survival in
Patient Data Sets of Breast Cancer
GOBO: ER-positive Tumors

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. Cadm1 Expression Reduces Pulmonary
Metastasis in vivo
6DT1 results demonstrate a
metastasis-specific role for Cadm1
** p < 0.01

26. Cadm1 Expression Reduces Pulmonary
Metastasis in vivo

27. Metastases from Cadm1 Expressing Primary
Tumors Do Not Express the Cadm1 Transgene
Down-regulation of Cadm1 may be prerequisite for metastasis formation.

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. 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. 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. 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. 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. 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. 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. 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. Metastasis Biology
•Effect of Cadm1 expression on metastasis might be
distinct from early steps of metastatic cascade
Valastyan & Weinberg. Cell. 2011

37. Does Cadm1 Expression Have an Effect on Late
Events of the Metastatic Cascade?
21 days

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. 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. 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. 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. 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. Two Primary Questions:
1. Are CD8+ T-Cells Involved in Cadm1-Mediated
Metastasis Suppression?
2. Are either IL-2 or IFN-γ involved?

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. 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. 2) Do mice bearing Cadm1+ tumors show
increased IFN-γ secreting lymphocytes?
anti-IFN-γ
anti-CD3

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. 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. 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. 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. 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. 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. 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. Thanks for your attention
Questions?

55. Thanks for your attention
Questions?

57. Cadm1 localizes to cell-cell junctions
Mvt1
Vector
6DT1
Vector
Mvt1
Cadm1
6DT1
Cadm1

58. No Significantly Impact on
Cell Morphology or Actin Stress Fibers
Mvt1
Vector
6DT1
Vector
Mvt1
Cadm1
6DT1
Cadm1

59. No Significant Change in
3D Culture Growth Properties in
Mvt-1 Vector
Mvt-1 Cadm1
6DT1 Vector
6DT1 Cadm1

60. No Significant Change in
Motility in vitro
•Mvt1 Vector
•Mvt1 Cadm1
•6DT1 Vector
•6DT1 Cadm1

61. Transwell Migration and Invasion Assay
Transwell
Insert
Tumor Cells
MatrigelCoated
membrane
Chemoattractant
Depleted Media
Chemoattractant
Rich Media

62. No Significant Change in in vitro
Migration or Invasion by Transwell Assay

63. Cadm1 Expression Does Not Impact
Tumor Cell Proliferation in vitro
•Mvt1 Vector
•Mvt1 Cadm1
•6DT1 Vector
•6DT1 Cadm1

64. IFN-γ ELISPOT – Lung
p=0.027
p=0.003
6DT1 Vector
6DT1 Cadm1
Mvt1 Vector
Mvt1 Cadm1

65. Level of Cadm1 Expression Predicts Survival in
Patient Data Sets of Breast Cancer

66. 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

67. 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

68. Implications
Schreiber et al.
Science. 2011

69. 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

70. Chromosome 9 Metastasis QTL
NZB
C58