Biology of metastasis

Swarnali Acharyya Nov 11, 2015

Dispersion&seedingDetec/onandremoval

ofprimarytumor

Treatments for primary breast cancer

89%survivalrateforlocalizedbreastcancers

Dispersion&seedingDetec/onandremoval

ofprimarytumor

Advances in breast cancer treatment

Over 90% survival rate for localized, well treated breast cancers

Be#erdetec)onandimaging

Newsurgicalop)ons

Targetedtherapy

Latency(monthstodecades)

Metastasisisresponsiblefor90%ofcancerdeaths

Overtmetastasis

hAp://seer.cancer.gov/staEacts/html/breast.html

Primary tumor

Metastasis

Cell-autonomous mechanisms: Genetic, epigenetic differences?

Howareprimarytumorsandmetastasisdifferent?

Non cell-autonomous mechanisms: Stromal influences

Metastasisaremoredifficulttotreatandareresistanttomostdrugs

Breast Cancer Genes

Shahetal.,Nature,2012

Mutations are highly concordant between primary tumor and metastasis Ref: Colon cancer, Brannon et al., Genome Biology, 2014

Breastcancersubtypes

Modified from: http://massgenomics.org/2012/09/a-comprehensive-atlas-of-breast-cancer-genomes.html

Luminal A slow growth and best prognosis

Luminal ca : ER positive Express luminal markers (ER alpha, cytokeratins 8/18)

Basal like triple negative cancers: worst prognosis Express basal cell markers (CK 5/6, 14, 17)

Basementmembrane

BasalorMyoepithelialcells

Luminalepithelialcells

HER2 positive breast cancer

Modified from Hongkong Breast Cancer Foundation

TargetHER2ProteinTrastuzumab

Signaling downstream of HER family

http://www.nature.com/nrclinonc/journal/v9/n1/fig_tab/nrclinonc.2011.177_F1.html

Reference: Ellis and Perou, Cancer Discovery, Jan 2013

Trends in breast cancer incidence and mortality

Metastasis: Differences in latencies in cancer types

Lung adenocarcinoma: Rapid metastasis to mutiple organs

Nature

Breast adenocarcinoma: Latency and metastatic speciation

. . . . . .

. . . . .

. . . . . .

WNT/TCF LEF1, HOXB9

EREG, COX2, FASCIN1, MMP1,

ANGPTL4, HBEGF, COX2,

FASCIN1, MMP1, ANGPTL4,

ST6GALNAC5

IL11, ADAMTS1,MMP1, TGFß, CTGF, PTHrP

miR335 miR126

Evolution of metastasis Step-wise or parallel?

Timelineofdevelopmentofmetastasisbasedonmathema)calmodeling

Luebeck,Nature,2010

ClonalEvolu)onofheterogeneousprimarytumorsgivingrisetometastasis

Alternative hypothesis: Parallel evolution of metastasis?

Bone marrow cancer cells genomically different from primary tumor mix -  Spread early and evolve

OR -  Different subpopulation

which might not give rise to overt metastasis?

Clinically- optimal to decide treatments based on DTCs or primary tumors?

Reference:Gray,CancerCell,2003

Phenotypically normal untransformed cells can persist in the lungs for months

Podsypanina et al., Science, 2008

Delayed oncogene induction still forms tumors

When normal mammary cells have reached the distant site in the absence of transformation at the primary site.

Seed or the soil: which matters in metastasis?

Ref: Fidler, Nat Rev Cancer, 2003

4millioncells/gofprimarytumorshed0.01%successful!

Brief history of metastasis research (Ref: Metastasis Research Society)

Metastatic cells spread through lymphatics or blood?

Blood (hematogenous spread)

Modified from www.iopscience.iop.org

Lymphatic spread

Modified from www.cancer.org

Lymphatic spread of breast cancer

•  Might depend on physical restrictions on invasive tumors. •  Easier to survive in passive, low shear flow. •  Lymphatics more leaky, lacking tight interendothelial junctions (higher chances of intravasation).

Lymphatics more accessible to cancer cells?

Peritumoral lymphatics matter more than intratumoral lymphatics

WongandHynes,CellCycle,2006

Potential pathways of reaching circulation from the primary tumor

WongandHynes,CellCycle,2006

Inprostatecancer,84%withlymphnodespreadhavehematogenousspread.

Drug resistance of metastases: yet another problem to solve

Sensi/ve Resistant

Kuczynskietal.,NatRevClinOncol.,2013

Challengestotargetdrugresistantclones

Latency(monthstodecades)

Overtmetastasis

Animal models to study the complex process of metastasis

Transgenic mouse models of breast cancer

Development of Patient derived xenografts

Kopetz et al., Clinical Cancer research, 2012

Fresh tumor implantation in immunocompromised mice

Tumor grafts in mice resemble the donor patient tumors by histology, genomics, growth

Sitesinpa)ents SitesinmiceMETASTASISSITES

Metastasis-associatedgeneexpression

ApproachI:Interrogatetheend-products

Kang et al Cancer Cell 2003 Minn et al Nature 2005 Tavazoie et al Nature 2008 Bos et al Nature 2009

BrM

LM

BoM

43genes

54genes

102genes

Pro

babi

lity

p < 0.0001

1.0

0.8

0.6

0.4

0.2

0 0 50 100 150

LMS+TβRS+

Lung metastasis-free survival (months)

LMS: 18-gene signature associated with lung relapse

EREG COX2 MMP1 ANGPTL4 FSCN1 NEDD9 ID1 CXCL1 TNC VCAM1 CXCR4 LTBP1 ROBO1 KRTHB1 MAN1A1 KYNU C10orf116 RARRES3

BreastCancerLungMetastasisSignature(LMS)

COX2: vascular permeability Epiregulin: cell motility Fascin1: invadopodia MMP1: collagenase ANGPTL4: endothelial disjunction

Tenascin-C

VCAM-1

Gupta et al Nature 2007 Padua et al Cell 2008 Tavazoie et al Nature 2008

Kim et al Cell 2009 Oskarsson et al JACS 2010

Tumor microenvironment interactions that support tumor progression

Quail and Joyce,, Nat Med, 2013

In transit

Role of platelets in the early hours of metastatic colonization

Reference Labelle et al., PNAS, 2014

At the distant site (lung)

Role of VCAM1 in establishing lung metastasis

Chen and Massague, Clinical Cancer Research, 2012

Fighting for survival

Role of extracellular matrix molecules in metastasis

Tenascin C at the invasive edge of tumors

Ref: Oskarsson et al., Nature Medicine, 2011

Depleting Tenascin-C in cancer cells reduces lung metastasis

Role of Tnc in outgrowth of micrometastasis

TNC interaction with cancer cells at the invasive front enhances NOTCH and WNT signaling

Enhances fitness of metastasis Initiating cells

Extended survival of breast cancer cells for decades in the bone

ModifiedfromhAp://www.myxgeva.com/breast-cancer-bone-metastases.html

The undetectable but present Latent metastasis

Zhangetal.,CancerCell,Jul7,2009

Role of Src signaling in the survival of bone metastasis

•  No role in homing to lungs or bone. •  If these cells reach, then Src signaling becomes critical for their

outgrowth.

Src enhances survival and outgrowth of latent metastatic cells in the bone marrow

Brain metastasis from breast cancer

Modifiedfromwww.braintumors.in

Plasmin- reactive brain stroma for protection of host

Valienteetal.,Cell,2014

Joyce JA and Pollard JW, Nat Rev Cancer, 2009

Tumor microenvironment components

Macrophage/microglia microenvironment targeting in brain tumors

Pyonteck et al., Nat Med, 2013

T regulatory cells are present in growing tumors and metastases

Poor prognosis in breast cancers

Treg cells ablated from established tumors can reduce metastatic growth

Macrophage

Tum

or fa

ctor

s Tumor

Dendritic Cells

Neutrophil Eosinophil Basophil

Modified from Wynn, Nat Immunology, 14(3), 197-99

Myeloid derived suppressor cells in cancer - Another group of poor prognosis immunosuppressive cells

CXCL1/2 mediates mammary tumor progression

sh Control

shCXCL1/2 0

100

200

300

400

0 20 40

Tum

or v

ol. (

mm

3 )

p<0.

001

Days after injection

Tumor growth

0

20

40

Foci

num

ber/F

OV

shControl shCXCL1/2

p<0.0001

Lung

met

asta

sis shControl shCXCL1/2

Lung metastasis

shControl shCXCL1/2

Cle

aved

cas

pase

3

Apo

ptot

ic c

ells

/FO

V

0

10

20

30

shCon shCXCL1/2

* Apoptosis CXCR2

MMTV-PyMT MDA231-LM2

Granulocytic CD11b+Ly6G+ cells

CXCL1/2 recruit granulocytic CD11b+Ly6G+ cells in the tumor microenvironment

0 5 10 15

Unsorted

CD31+

CD11b+Gr1+

CD11b+Ly6C+

CD11b+Ly6G+

F4/80+

Cxcr2 mRNA levels (Relative units)

MDSC levels correlate with accelerated disease progression and poor survival in patients

- Breast Cancer (Diaz-Montero et al, 2009, Walter et al, 2012)

- Colorectal Cancer (Solito et al, 2011)

- Gastrointestinal Cancer (Gabitass et al., 2011)

- Lymphoma (Montero., 2012)

(Gabrilovich, Ostrand-Rosenberg and Bronte, 2012)

MDSC

Suppress Anti-tumor Immunity

Tumor Angiogenesis (Yang et al, 2004, Shojaei et al, 2007) Blood vessel remodelling and tumor cell extravasation (Yan et al, 2010)

0 20 40 60 80

100 120

0 20 40 60 Tum

or v

olum

e (m

m3 )

S100a9+/+bm S100a9-/- bm

p=0.

001

Days after injection

CXCL1/2 promotes tumor progression via myeloid cell S100A8/9

Met

asta

tic c

ells

/FO

V

S100a9-/- 0 2 4 6 8

10 12 14

p<0.0001

S100a9+/+

S100a9+/+ and S100a9-/- bone marrow reconstituted mice as recipients

Cancer cells to gland #4

Tumor growth Metastasis

Donor bone marrow

CXCL1/2

Breast primary

Metastasis Metasta/cstress

CXCL1/2

Cancer cell

CXCR2

CD11b+Gr1+MyeloidcellsSurvival

Survival

S100A8/9

S100A8/9

CXCL1/2 axis mediates tumor growth and metastasis

Invasionstress

Chemotherapy hyperactivates the CXCL1/2-S100A8/9 cycle

Neoadjuvant Rx samples

S10

0A8/

9 IH

C

scor

ing

Score 0 Score 1

Score 2 Score 3

S10

0A8/

9 sc

ore

Before chemo After chemo

p<0.0001

0

1 2

3 4

X

SB-265610

CXCR2 inhibitor enhances chemotherapy

Lung metastasis

Vehicle i-CXCR2 Two drugs

MD

A23

-LM

2 C

N34

-LM

1

AC chemo

Chemo

Met

asta

tic c

ells

/FO

V

CXCR2i +Chemo

0

4

8

12

16 MDA231-LM2

p=0.028

CN34-LM1

Chemo CXCR2i +Chemo

0

20

40

60

80 p=0.024 Days: 26

CXCR2 inhibitor

34 40 46 53 60

Chemo

0

Implant cancer cells

Primary tumor

Distant metastasis Brain

Bone

Vehicle chemo cxcr2i cxcr2i+chemo0

10000

20000

30000

40000Vehiclechemocxcr2icxcr2i+chemo

Nor

mal

ized

Pho

ton

Flux

Chemo Chemo+CXCR2i

Vehicle CXCR2i 0

10000

20000

30000

40000

Cytotoxic chemo, shortlived targeted therapy…..immunotherapy promise

hAp://www.revealtherapies.com/AllPages/Immunotherapy.html

Dispersion&seedingDetec/onandremoval

ofprimarytumor

Treatments for primary breast cancer

89%survivalrateforlocalizedbreastcancers