Cadm1, an Inherited Modifier of Metastasis that Suppresses Metastasis by Interacting with the Cell Mediated Immunity

Farhoud FarajiKent Hunter

March 29, 2012

Agenda

1. Introduction 2. Genetic analysis reveals a locus on mouse Chr9

that is associated with metastasis3. Validating Cadm1 as a metastasis-associated

gene4. Elucidating the mechanism of Cadm1-mediated

metastasis suppression

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.orgHunter & Crawford. Cancer Res. 2006

Metastasis Biology

Valastyan & Weinberg. Cell. 2011

Metastasis Biology

Valastyan & Weinberg. Cell. 2011

Highly Complex Process•Numerous Gene Expression Programs•Tumor Phenotypic Transitions•Niche Remodeling•Intercellular Communication

Endpoint: Reaching and adapting to foreign environment

Where is potential for metastatic susceptibility encoded?

The Genetic Background

Definition:– Complement of genetic variation that

distinguishes each of us as an individual

More specifically:– Germline polymorphisms

• SNP, Indel, CNV

Why do some breast cancer patients develop metastatic disease, whereas

other patients, with seemingly similar tumors, don’t?

Our Question

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

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

DadMom

Constants: Oncogenic driver (PyMT)Paternal genotype (FVB)

Variable: Maternal genotype

The Observation:

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

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

1) Outcross between inbred strains of varying metastatic propensity

2) Cross to FVB-PyMT3) Determine phenotype and genotype4) Which segments of genome segregate with metastasis?

Identification by Forward Genetic Screen

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

Validation

Agenda

1. Introduction 2. Genetic analysis reveals a locus on mouse Chr9

that is associated with metastasis3. Validating Cadm1 as a metastasis-associated

gene4. Elucidating the mechanism of Cadm1-mediated

metastasis suppression

FVB: Metastasis-Prone

C58 and NZB: Metastasis-Resistant

Genetic Screen

NZB Metastasis QTL C58 Metastasis QTL

x

FVB/N-PyMTNZB or C58

P

F1

x

N2

Measure: Primary Tumor Burden Pulmonary Metastases

Genotype: Determine segments of genome segregating with metastasis

NZB/C58 + FVB Backcross Reveals QTL peak on Chromosome 9

Subcongenic Analysis Resolves Chr. 9 Susceptibility Locus to 49-67Mb

* p < 0.05

Subcongenic Tumor Burden Subcongenic Metastasis

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

Agenda

1. Introduction 2. Genetic analysis reveals a locus on mouse Chr9 that

is associated with metastasis3. Validating Cadm1 as a metastasis-associated gene4. Elucidating the mechanism of Cadm1-mediated

metastasis suppression

Exon Sequencing Reveals Synonymous SNP in Exon 2

524 586530 540 550 560 570« NZB(524)

« FVB(520)

Consensus(524)

rs327213609

Cadm1 is Differentially Expressed in FVB and NZB Mice

Mammary Tumor Normal Whole Lung

p = 0.076 *

* p < 0.05(NZB Chr9)

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

GOBO: ER-positive Tumors

anti-V5

anti-Cadm1

anti-β-actin

6DT1 Vector

6DT1 Cadm1-V5

Mvt-1 Vecto

r

Mvt-1 Cadm1-V5

Ectopic Expression of Cadm1 in Mvt1 and 6DT1

Overexpression is within physiological range.

** p < 0.01

Cadm1 Expression Reduces Pulmonary Metastasis in vivo

6DT1 results demonstrate a metastasis-specific role for Cadm1

Cadm1 Expression Reduces Pulmonary Metastasis in vivo

Metastases from Cadm1 Expressing Primary Tumors Do Not Express the Cadm1 Transgene

Down-regulation of Cadm1 may be prerequisite for metastasis formation.

Stable Knockdown of Cadm1 by shRNA

Cadm1

β-actin

6DT1 shSc

rambled

6DT1 shCadm1 14

6DT1 shCadm1 15

Primary Tumor Burden

Tum

or M

ass

(g)

0.0

0.5

1.0

1.5

*

Tumor-Normalized Metastases

Met

asta

ses

Per

Gra

m T

umor

6DT1 Scr

6DT1 shRNA 14

6DT1 shRNA 150

50

100

150

* * *

Pulmonary Metastases

Sur

face

Met

asta

sis

Cou

nt

0

20

40

60

80

100

* * p = 0.07

Cadm1 Knockdown Promotes Pulmonary Metastasis

* p < 0.05

** p < 0.01

Taken together, results confirm a causative role for Cadm1 in metastasis.

Agenda

1. Introduction 2. Genetic analysis reveals a locus on mouse Chr9

that is associated with metastasis3. Validating Cadm1 as a metastasis-associated

gene4. Elucidating the mechanism of Cadm1-mediated

metastasis suppression

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. 1998Kuramochi et al. Nat Genet. 2001

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. 2010Murakami. Cancer Sci. 2005

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. 2009Ito et al. Hepatology. 2007

Sakurai-Yageta et al. Biochem Biophys Res Commun. 2009

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. 2011Wong et al. PNAS. 2007

Fujita et al. Am J Pathol. 2007

Cadm1 Expression: • No significant impact on tumor cell in vitro

properties:– In vitro Growth– In vitro Motility– In vitro Invasion– 2D and 3D Morphology

Summary of Cadm1 Effect onIn Vitro Properties

Metastasis Biology

Valastyan & Weinberg. Cell. 2011

•Effect of Cadm1 expression on metastasis might be distinct from early steps of metastatic cascade

21 days

Does Cadm1 Expression Have an Effect on Late Events of the Metastatic Cascade?

Cadm1 Reduces Pulmonary Metastasis of Tail Vein Injected Tumor Cells

Pulmonary Metastases

Sur

face

Met

asta

sis

Cou

nt

Mvt-1 Vector

Mvt-1 Cadm1

6DT1 Vector

6DT1 Cadm1-20

0

20

40

60

* p < 0.05

* *

•Metastasis inhibitory effect of Cadm1 is not restricted to the early stages of metastasis.

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. 2005Boles et al. Blood. 2005

Cadm1 and Immunity

NK cellsNKT cells

CD8+ T-cellsTumor Cell

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

Cadm1 Expression Has No Effect on Metastasis or Tumorigenesis in Athymic Mice

ImmuneCompetentHost:

Primary Tumor Burden

Tu

mo

r M

ass

(g

)

Mvt

-1 V

ecto

r

Mvt

-1 C

adm

1

6DT1

Vecto

r

6DT1

Cadm

10.0

0.5

1.0

1.5

Pulmonary Surface Metastases

Met

asta

sis

Co

un

t

Mvt

-1 V

ecto

r

Mvt

-1 C

adm

1

6DT1

Vecto

r

6DT1

Cadm

10

20

40

60

80

AthymicHost:

Hypothesis

1) CD8+ CTL-mediated tumor killing2) NK-cell recruitment, activation, and tumor killing3) IFN-γ-mediated tumor cytostaticity and/or killing

Tumor Cell CD8+ T-cell

CRTAMCadm1

IFN-γIL-2

MHCITCR

CD8

Kuipers et al. Blood. 2011Giangreco et al. J Immunol. 2012

Two Primary Questions:

1. Are CD8+ T-Cells Involved in Cadm1-Mediated Metastasis Suppression?

2. Are either IL-2 or IFN-γ involved?

CD8+ Cell Depletion in Immune Competent Mice • Study in Progress• Design:

_____IgG_____ ___anti-CD8___ Mvt1 Mvt1 Mvt1 Mvt1

Vector Cadm1 Vector Cadm1

1) Are CD8+ T-Cells Involved inCadm1-Mediated Metastasis Suppression?

30 days

Fat Pad Injection

Harvest: •Lung•Lymph Nodes

Make Single Cell Suspension of Living Cells

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

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

anti-IFN-γ

anti-CD3

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

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 antibodyELISA on supernatant for IL-2, IFN-γ

• Is Crtam involved?– Cadm1 orthotopic transplant in Crtam KO mouse

Future Plans

• If CD8+ T-cell depletion does not rescue metastatic phenotype: – CD4+ T-cell, NKT-cell depletion– Investigate role of stromal FoxN1 in Cadm1-

mediated metastasis suppression• Sort tumor stromal cells and check FoxN1 expression

– BMDC, CAF, Endothelial cells, etc.

SummaryCadm1:1. Is an inherited modifier of metastatic risk in mice2.Is a metastasis suppressor in mice3.Metastasis suppression may involve host cell-

mediated immunity4.Expression levels in human tumor samples

predicts prognosis

ConclusionTumor-autonomous expression of Cadm1 impacts

tumor metastatic capability through non-tumor-autonomous mechanisms.

Cadm1 may sensitize tumor cells to immune surveillance and result in lymphocyte-mediated cytotoxicity.

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.

AcknowledgementsDr. Glenn Merlino• Dr. Chi-Ping Day• Dr. Raza Zaidi

Dr. Lalage Wakefield• Dr. Yuan Yang

Dr. Li Yang• Dr. Yanli Pang

Dr. Kent Hunter• Dr. Jude Alsarraj• Dr. Ling Bai• Dr. Natalie Goldberger• Dr. Luanne Lukes• Renard Walker• Dr. Scott Winter• Dr. Thos Geiger

Thanks for your attention

Questions?

Thanks for your attention

Questions?

Cadm1 localizes to cell-cell junctionsMvt1 Vector

Mvt1 Cadm1

6DT1 Vector

6DT1 Cadm1

No Significantly Impact on Cell Morphology or Actin Stress Fibers

Mvt1 Cadm1

Mvt1 Vector

6DT1 Cadm1

6DT1 Vector

No Significant Change in 3D Culture Growth Properties in

Mvt-1 Vector Mvt-1 Cadm1

6DT1 Vector 6DT1 Cadm1

No Significant Change in Motility in vitro

•Mvt1 Vector•Mvt1 Cadm1

•6DT1 Vector•6DT1 Cadm1

Tumor Cells

Matrigel-Coatedmembrane

Chemoattractant Rich Media

Chemoattractant Depleted Media

Transwell Migration and Invasion Assay

TranswellInsert

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

Cadm1 Expression Does Not Impact Tumor Cell Proliferation in vitro

•Mvt1 Vector•Mvt1 Cadm1

•6DT1 Vector•6DT1 Cadm1

IFN-γ ELISPOT – Lung

6DT1 Vector

6DT1 Cadm1

Mvt1 Vector

Mvt1 Cadm1

p=0.027 p=0.003

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

524 586530 540 550 560 570« NZB(524)

« FVB(520)

Consensus(524)

rs327213609

Exon Sequencing Reveals Synonymous SNP in Exon 2

IiExonIntron Intron

IiExon

PCR AmplifyTOPO Clone

Sanger Sequencing

IF CD8+ T-Cells play a major role, expect:

Mvt1 Mvt1 Mvt1 Mvt1 Vector Cadm1 Vector Cadm1 IgG anti-CD8

1) Are CD8+ T-Cells Involved inCadm1-Mediated Metastasis Suppression?

Met

asta

ses

Implications

Schreiber et al. Science. 2011

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. 2011Paulson et al. Br J Haematol. 2009

Nakahata et al. Leukemia. 2012

Chromosome 9 Metastasis QTL

NZB

C58