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Invasion, metastasis and stem cells Prof. Robert Weinberg

Invasion, Metastasis and Stem Cells

Prof. Robert Weinberg

Whitehead Institute for Biomedical Research USA

1

The 1989 Vogelstein-Kinzler model of multi-step colon cancer pathogenesis

Tumorigenic pathways

SV40 ER

TGF- p19ARF MDM2 p53 ST

hTERT

hTERT Telomerase

RAS

How does this occur ­ additional genetic or epigenetic changes?

Growth factors RTK 17p

Chromosome: Alteration: Gene:

5q SMAD Loss

p15 / p16

12p PP2A

18q

Activation Loss Ras Loss Nutrient metabolism APC K RAS K-RAS p53 Telomere T l DCC? MAPK Cyclin D Apoptosis maintenance genomic AP-1 Rb instability Proliferative E2F DNA stimuli Loss of growth hypomethylation control

Other alterations

Normal epithelium

Hyperprolif. epithelium

Early adenoma

Intermediate adenoma

Late adenoma

Carcinoma

Metastasis

(Similar schemes can be drawn for tumors arising in a variety of other tissue sites)

2

An approach: propagate human mammary epithelial cells in distinct culture media in vitro

Lung metastases

Normal breast tissue

Standard medium Culture medium: MEGM

Special (novel) Culture medium: medium WIT These have a more luminal epithelial gene p g expression pattern

These have a more basal epithelial gene expression pattern Two distinct normal human mammary epithelial cell (MEC) populations

Primary cell culture BPE cells HME cells

Experimental transformation with identical introduced genes

BPLER Adenocarcinoma Metastatic

Adenocarcinoma

HMLER Squamous Ca Non-metastatic

Squamous cell carcinoma

Tan Ince

3

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1

Invasion, metastasis and stem cells Prof. Robert Weinberg

Conclusion: the nature of the normal cell-of-origin is a strong determinant of the phenotype of the tumorigenic cell, including its eventual tendency to metastasize

Chromosome: Alteration: Gene:

5q Loss APC

12p Activation K-RAS

18q Loss DCC?

17p Loss p53

DNA hypomethylation

Other alterations

Normal epithelium

Hyperprolif. epithelium

Early adenoma

Intermediate adenoma

Late adenoma

Carcinoma

Metastasis

4

Cell of origin determines tumor morphology and behavior

(Standard medium) (Special medium)

HMLER Ductal differentiation Invasion Stromal recruitment Metastasis Tumorigenicity

Tan Ince

BPLER Yes Yes Yes Yes

<102 cells needed to seed tumor

No Minimal Minimal No

106 cells needed to seed tumor

5

Similar hierarchical organization of cancer cells in a tumor

Tumor-initiating cell

This cell is qualified to seed a new metastasis

These cells cannot seed new metastasis a new tumor (because they lack self-renewal capability)

Figure 11.16b The Biology of Cancer (© Garland Science 2007)

6

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2

Invasion, metastasis and stem cells Prof. Robert Weinberg

Unanswered question:

Does the strong representation of tumor-initiating cells make BPLER cells growing within primary tumors far more competent to spawn macroscopic metastases??

Few stem cells that are qualified to seed macrometastases

HMLER ­

few metastases

BPLER ­

many metastases

Many stem cells that are qualified to seed macrometastases

s

7

How do cancer cells actually metastasize? The invasion-metastasis cascade:

Primary tumor Invasion Intravasation Transport

Colonization Metastasis Micrometastasis Extravasation

8

In fact, we can divide this cascade into two major phases:

The invasion-metastasis cascade

Physical dissemination Primary tumor Invasion Intravasation Transport

Metastasis

Adaptation to & proliferation in a foreign tissue

Colonization Micrometastasis Extravasation

9

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3

Invasion, metastasis and stem cells Prof. Robert Weinberg

The last step of the invasion ­ metastasis cascade

Colonization is likely to depend on the ability of disseminated micrometastatic cells to adapt to a novel/foreign tissue microenvironment

Colonization Micrometastasis Metastasis (macroscopic metastasis)

(a breast cancer micrometastasis in the bone marrow)

10

EMT = epithelial-mesenchymal transition

(Human) cytokeratinpositive cancer cells (therefore epithelial)

These cells have shut down epithelial marker expression and turned on mesenchymal marker expression instead, i.e., they have undergone an epithelial-mesenchymal p y transition (EMT)

Invasive cell

EMT

Human vimentin-positive (therefore mesenchymal cells of human origin)

Signals Recruited mouse stroma from mouse stroma

K. Hartwell and T. Ince

11

BPLER tx human mammary epithelial cells in mouse host

Human vimentin (mesenchymal) Cytokeratins (epithelial)

Mouse stroma

Mouse stroma

K. Hartwell & T. Ince

Transformed human MECs (BPLERs)

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The screen versions of these slides have full details of copyright and acknowledgements

4

Invasion, metastasis and stem cells Prof. Robert Weinberg

(The EMT program also involves the acquisition of motility, invasiveness and increased resistance to apoptosis)

Conclusion: · The microenvironment of the primary tumor can contribute importantly to the phenotypic conversion occurring during an EMT, which involves an adaptation of cancer cells to contextual signals originating in the stroma

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The epithelial-mesenchymal transition (EMT) is a complex, multi-faceted program involving multiple changes in cell properties

Epithelial Mesenchymal

Cell-biological

Biochemical

Epithelial

Mesenchymal

EMT

14

EMTs play roles in various steps of embryogenesis

These cells have undergone an EMT

Mesenchymal

Epithelial

Courtesy of G Cherr

15

The screen versions of these slides have full details of copyright and acknowledgements

5

Invasion, metastasis and stem cells Prof. Robert Weinberg

EMT EMT

Emigration of melanocyte progenitors from the neural crest

These blue cells are leaving the top of the primitive spinal chord, are undergoing an EMT, and are migrating out to sites elsewhere in the body of the embryo

Courtesy of James Briscoe

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Twist transcription factor:

A transcription factor expressed in highly metastatic mouse breast cancer cells is also an inducer of mesoderm formation in Drosophila

Invagination

Epithelial

spatzle

Epithelial

Toll

(IL-1-like receptor) Mesenchymal Epithelial mesenchymal transition (EMT)

Dorsal (NFkB)

Migration

twist

snail

Mesoderm migration and specification

EMT plays a role in early embryogenesis, e.g., gastrulation

Mesenchymal

Adapted from Knust & Muller, 1998, Curr. Biol.; Arias AM 2001 Cell

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Twist is only one of a group of transcription factors that induce EMTs at various stages of embryogenesis

18

The screen versions of these slides have full details of copyright and acknowledgements

6

Invasion, metastasis and stem cells Prof. Robert Weinberg

Model: cancer cells appropriate early embryogenic programs in order to acquire traits of high-grade malignancy

(Human) cytokeratinpositive (therefore epithelial)

EMT

EMT

Human vimentin-positive (therefore mesenchymal)

Mesenchymal

Epithelial

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Twist acts pleiotropically to reprogram gene expression: epithelial markers shut down, mesenchymal are induced

Epithelial markers

Mesenchymal markers

J. Yang

20

Twist is essential for 4T1 tumors to metastasize from the mammary gland to the lung

Mouse #365

Mouse #368

Mouse #369

Shut down Twist: Lung mets down 85%

4T1 control-SiRNA

Mouse #352

Mouse #355

Mouse #358

Jing Yang

4T1 Twist-SiRNA

21

The screen versions of these slides have full details of copyright and acknowledgements

7

Invasion, metastasis and stem cells Prof. Robert Weinberg

How many steps of the invasion-metastasis cascade can the Twist transcription factor program?

Primary tumor Invasion Intravasation Transport

Colonization Metastasis Micrometastasis Extravasation

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If Twist expression can be induced in carcinoma cells by signals originating the tumor-associated stroma, then the ability to negotiate most of the steps of the invasion-metastasis cascade may be acquired without the need for additional mutations in the cancer cell genome

Primary Tumor Invasion Intravasation Transport

Metastasis

Colonization Micrometastasis Extravasation

23

Behavior of subcutaneously implanted, GFP-labeled, melanocytes that have been transformed with the same set of 5 pathway changes Bright field

A

Tumorigenic pathways

TGF- SMAD p15 / p16 Cyclin D Rb SV40 ER p19ARF MDM2 ST hTERT hTERT RAS Growth factors RTK Ras

GFP

B

M 10X OP M

p53 Apoptosis genomic instability

PP2A Telomerase Nutrient metabolism

Telomere maintenanceMAPK AP-1 Proliferative stimuli

E2F

Loss of growth control

OP

Lung

C

D

M OP

In contrast to transformation of a variety of epithelial cell BV 20X types, transformation of melanocytes generates numerous metastases

M OP 40X

Liver

10X

E

F

Spleen

OP

M determinant

Once again: M Cell-of-origin is an important of eventual metastaticOP behavior40X 10X

M

G

H

LNs

Intestine

P. Gupta

C E C E

M

C E

10X

40X

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The screen versions of these slides have full details of copyright and acknowledgements

8

Invasion, metastasis and stem cells Prof. Robert Weinberg

The Slug TF plays a prominent role in the emigration of cells from the neural crest

These blue cells are leaving the top of the primitive spinal chord and undergoing an EMT and are migrating out to sites elsewhere in the body of the embryo Courtesy of James Briscoe

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A transcription factor - Slug - enables melanocyte emigration from primitive neural crest and is expressed in transformed melanocytes (1,000x higher in these cells than in breast cancer cells) 1200 1000 800 600 400 200 0

MCF7Ras

Slug expression

P. Gupta

Transformed melanocytes

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Slug suppression strongly inhibits (by 93%) experimental melanoma metastasis Primary tumor growth

Lung metasta area asis (arbitrary scale)

Lung metastasis

or Primary tumo mass (g)

Lung 1 Tumor

siControl siSlug3

Control

P. Gupta

siSlug

27

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9

Invasion, metastasis and stem cells Prof. Robert Weinberg

Expression of FOXC2 (a 3rd EMT-inducing TF) in MDCK (epithelial) cells induces migration & invasion (in vitro), and matrix metalloproteinase (MMP) secretion

Number of migrated cells m

Fold expression over control

180 160 140 120 100 80 60 40 20 0

400 300 200 100 0

MMP-2

MMP-9

14 12 10 8 6 4 2 0

FOXC2

FOXC2

Vector

Vector

Vector

FOXC2

Vector

FOXC2

Migration

Invasion

S.A. Mani

28

FOXC2 expression in human breast cancers: Survey for cells with high nuclear expression

Normal mammary gland

Negative ( ) (-)

Weakly cytoplasmic ( ) (-)

Strong cytoplasmic (+)

Andrea Richardson & S.A. Mani

Strong nuclear (+) Strong cytoplasmic/nuclear (+)

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FOXC2: association with basaloid breast cancers: ~15% of breast cancers /poor prognosis

(Triple receptor-negative: ER-, PR-, HER2/NeuNielson et al., (1996) Clinical cancer research 10:5367

30

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10

Invasion, metastasis and stem cells Prof. Robert Weinberg

High nuclear expression of FOXC2 in the highly aggressive human basal-like breast cancers (15% of br. ca. cases)

50 % of high nucle FOXC2 ear expression in tumors n

44.4

30

29.4 17.9

10

8.8 1.5

Basal HER2 Luminal ER-

2.4

ER+ Grade III

3.3

Grade I/II

S.A. Mani

31

EMT interactome: the EMT-inducing transcription factors intercommunicate

These EMT-inducing transcription factors act in collaborative cohorts to program the EMT; they often induce expression of one another

E-cadherin b-catenin

Twist SIP1

Roster of EMT-inducing TFs:

Twist1, 2 Snail Slug Goosecoid FOXC2 SIP1 ZEB2 (others?)

GSC

Snail

TGFb

Ras FOXC2 / Slug S.A. Mani

32

Speculation: different alliances of EMT-inducing transcription factors may operate to program high-grade malignancy in different types of carcinomas

33

The screen versions of these slides have full details of copyright and acknowledgements

11

Invasion, metastasis and stem cells Prof. Robert Weinberg

What is the nature of cells generated by an EMT?

Epithelial cells "Mesenchymal cells"

EMT

=

? ?

Fibroblasts

Epithelial cells forced thru EMT

TGF-b1 Vector Snail Twist

Normal mammary fibroblasts

· Conclusion: the EMT doesn't produce "mesenchymal cells" that resemble fibroblasts · Then what does it produce?

MFB1

MFB2

MFB3

E-cad CK5 CK14 FOXC2 GAPDH

Use gene expression patterns to distinguish the products of the EMT from fibroblasts

S.A. Mani

34

FOXC2 is expressed in cell nuclei of normal human mammary epithelium This EMT-inducing transcription factor is expressed in the nuclei of cells that resemble stem cells!

lumen

lumen Sites reminiscent of mammary epithelial stem cells

S.A. Mani & Andrea Richardson

35

What is the nature of cells generated by EMT?

TGF-b1 Snail Twist Slug Epithelial cells SIP1 Goosecoid E2a si-E-cad EMT "Mesenchymal cells"

Stem cells

???

=

FOXC2

A speculation!

36

The screen versions of these slides have full details of copyright and acknowledgements

12

Invasion, metastasis and stem cells Prof. Robert Weinberg

CD24 & CD44 are useful markers to separate br. ca. stem cells from non-stem cells (al-Hajj et al., 2003) Bulk of tumor cells: Little if any ability to proliferate & seed new tumor

Separate cells on the basis of their cellsurface proteins

Figure 11.14a The Biology of Cancer (© Garland Science 2007)

37

Induction of EMT by TGF- 1 generates CD44hi and CD24lo HMLE human immortalized human MECs

CD44lo/CD24hi (position of non-stem cells)

CD44hi/CD24lo

(position of stem cells)

CD44

EMT

+TGF- 1

CD24

Day 0 Cobblestone epithelial morphology

S.A.Mani & W. Guo

Day 12 Fibroblastic mesenchymal morphology

5 ng/ml TGF-b1 induce EMT

38

Induction of EMT by Snail and Twist EMTinducing TFs also generates CD44hi CD24lo cells

HMLE HMLE-Snail HMLE-Twist

CD44hi/CD24lo (position of stem cells)

CD44lo/CD24hi (position of non stem cells) non-stem

CD44

CD24 Vector Morphological shift Snail Twist

S.A.Mani & W. Guo

EMT

39

The screen versions of these slides have full details of copyright and acknowledgements

13

Invasion, metastasis and stem cells Prof. Robert Weinberg

Naturally present CD44hi/CD24lo cells isolated from immortalized human mammary epithelial cells express mesenchymal markers

CD44hiCD24lo/CD44loCD24hi 100.0

Relative mRNA levels A

FOXC2

N-cad

SIP1

Twist

FN1

1.0 0.1 0.01

E-c cad

10.0

Slug

Non-stem cell Stem cell fraction fraction

"Stem cell" ÷ "non-stem cell"

(log10 scale)

Mesenchymal markers 0.001 Epithelial marker

EMT markers

(measured by RT-PCR)

The stem-like cells isolated from human reduction mammoplasty tissues express mesenchymal markers

CD44

Normal human mammary epithelial cells

CD24

Relative mRNA levels

1000 153.6 100 14.2 10 1 0.1 1.0 0.8 1.0 5.3 1.0 1.0

CD44hi24lo/CD44loCD24 hi

E-Cad

FOXC2

N-Cad

SIP1

EMT markers

S.A. Mani

Stem-like cells in primary human breast samples

(including normal and neoplastic cells exhibit mesenchymal traits)

CD44hiCD24lo CD44loCD24hi

Relative mRNA levels

Kornelia Polyak

Snail

Vim

40

41

42

The screen versions of these slides have full details of copyright and acknowledgements

14

Invasion, metastasis and stem cells Prof. Robert Weinberg

Activation of Twist-ER or Snail-ER for various time periods by tamoxifen:

Cells were exposed to active Twist in 2-dimensional culture for the indicated times and then introduced into 3-dimensional culture in the absence of further Twist exposure Mammospheres carry mammary epithelial stem cells # of mammospheres

W.Guo & S.A. Mani

Days treated with tamoxifen

43

EMT and cancer progression

1. Normal cells induced to undergo an EMT acquire stem-cell properties a. CD44hiCD24lo antigenic phenotype b. Ability to form mammospheres indefinitely 2. Same outcome with cancer cells 3. Epithelial cells in culture that are naturally CD44hiCD24lo show mesenchymal morphology; same is true of cells from reduction mammoplasty 4. Transient exposure to Snail or Twist causes descendants of exposed cells to form mammospheres indefinitely (as gauged by serial passage) Hence, induction of an EMT allows cancer cells a. To disseminate b. To become self-renewing

44

Most currently used chemotherapeutics kill non-CSCs preferentially

Putative effects of eliminating the non-CSCs therapeutically

Clinical response:

non-CSCs

Figure 16.30 The Biology of Cancer (© Garland Science 2007)

45

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15

Invasion, metastasis and stem cells Prof. Robert Weinberg

46

The screen versions of these slides have full details of copyright and acknowledgements

16

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