How do we get it? How does it kill us? Two distinct cancer research - - PowerPoint PPT Presentation

What is Cancer? How do we get it? How does it kill us?

Two distinct cancer research themes:

• 1. tumor onset

(how does it all start? Is it really just a question of bad luck?)

• 2. metastasis and

phenotypic plasticity

(what has personalized medicine to do with that?)

Mythbusting Cancer Research `For every complicated problem there is a solution that is simple, direct, understandable, and wrong. H.L. Mencken

Colorectal Cancer: the second deadliest

sequential clonal expansions (Darwin docet!)

Cancer: a multistep genetic process

2nd hit (gene B mutation) 1st hit (gene A mutation) 3rd hit (gene C mutation)

Cancer: a multistep genetic process

intestinal epithelial cell ACF adenoma

p53

• chr. 17p LOH

carcinoma

The Adenoma-Carcinoma Sequence in Colorectal Cancer: a genetic paradigm for tumor initiation, progression, and metastasis

APC K-RAS SMAD2/4

• chr. 18q LOH

What about the rate-limiting, initiating (APC) mutation event? Is it just a ‘random’ event (bad luck, shit happens), or do extrinsic (environmental) factors favor its occurrence?

Cancer: the role of “bad luck” vs. environmental risk factors

Cancer: the role of “bad luck” vs. environmental risk factors

Colorectal Cancer: the second deadliest, highest stem cell division rate (1012 over 60 yrs), and yet largely due to environmental factors.

Cancer: the role of “bad luck” vs. environmental risk factors

Why do we get colon cancer? 4 main etiological factors:

• 1. Age
• 2. Diet (life style)
• 3. Inflammation
• 4. Genetics

It’s all in the stem cell niche!

intestinal epithelial cell ACF adenoma

p53

• chr. 17p LOH

carcinoma

APC K-RAS SMAD2/4

• chr. 18q LOH

stem cell niche alterations

(i.e. expansion of cell targets for tumor initiation and progression)

metaplasia

(stem cell reprogramming)

Western-style diet Aging IBD Predisposition

Why do we get colon cancer? 4 main etiological factors:

• 1. Age
• 2. Diet (life style)
• 3. Inflammation
• 4. Genetics

It’s all in the stem cell niche!

How to model Western-Style diet-induced colon cancer in the mouse?

How to model Western-Style diet-induced colon cancer in the mouse?

NWD1: - 25% of C57BL/6J mice develop colon tumors (n= 1-2);

• Tumors arise at 1.5-2 years of age;
• Ratio carcinoma-to-adenoma of ~10%;

→ First and only model of dietary induced sporadic CRC NWD2: Increased Vit D3 and Ca2+ rescue the tumorigenic effect

0% 100% 200% 300% 400% 500% Fat Vit D3 Ca2+ Fiber Methyl donors % of control diet AIN76A NWD1 NWD2

Inflammatory Bowel Diseases: Crohn’s and Ulcerative Colitis

1 2 3 4 5 6 7 8 9 10

3% DSS H20

SACRIFICE

Lgr5-EGFP

1 2 3 4 5 6 7 8 9 10

H20

SACRIFICE

Lgr5-EGFP

Modelling inflammatory bowel disease by Dextran Sodium Sulfate (DSS)

The intestinal stem cell niche

cKit+ Paneth-like cells (in colon) LyscreERT2

(J. Van Es, H. Clevers)

cKitcreERT2

(D. Saur)

Lgr5EGFP-IRES-creERT2

(N. Barker, H. Clevers)

The intestinal stem cell niche

GFP

Control 3% DSS

GFP GFP/RFP GFP/RFP

GFP/YFP GFP/YFP GFP

Control diet NWD1 diet

GFP GFP Ki67 GFP Ki67

→ fewer Lgr5+ stem cells → Lgr5+ stem cell lineage tracing is inhibited

Both Western-style diet and inflammation suppress Lgr5+ stem cell function

→ Lgr5+ stem cells cease to proliferate

NWD1 inhibits proliferation, lineage tracing, and tumor-initiating capacity

• f Lgr5+ stem cells through the vitamin D receptor.

Western-style dietary factors and in particular vitamin D depletion inhibit Lgr5 stem cell function

The western-style NWD1 diet (low in vitamin D) represses the stem cell function of Lgr5+ cells and their capacity to underlie cell turnover in the small an large intestine. Are there other stem cells involved? Or are the dietary effects mediated by the niche?

‘Mini-Gut’ Organoid Assay

small intestine

• r colon

crypts 500 crypts/ well

Matrigel + growth factors

0h 5h 1day 5 days

AIN76A NWD1 NWD2 5x 20x 10x

20 40 60 80 100 120 140 160 180 200 AIN76A NWD1 NWD2 number of organoids

→ notwithstanding the fact that the Western-style diet suppresses stemness of Lgr5+ cells, mini-gut organoid forming capacity is increased. Hence, a distinct stem cell type is activated upon Western-style diet. Intestinal stemness is increased by Western-style diet

5 day old organoids

The intestinal stem cell niche

1000 Lgr5+ CBCs from mouse A 1000 Paneth cells from mouse B Plate out in matrigel for ‘mini-gut’ assay 30’ co-incubation at 37°C

• ptional: genetic (e.g. siRNA) or biochemical

modification of sorted lineages

• exp. mouse A

(e.g. control diet)

• exp. mouse B

(e.g. NWD1 diet)

Single cell digest of intestinal mucosa FACSort stem (Lgr5+) and niche (Paneth) cells separately

Functional analysis of stem vs. niche effects: the organoid reconstitution assay

Lgr5 cells Paneth cells

Western style diet

Lgr5+ stem cells inactivation

Homeostasis

Paneth cells provide niche support to Lgr5+ stem cells. +4 Lgr5+ Lyz+Paneth

Inflammation

Depletion of Lgr5+ stem cells

Regeneration

Paneth cells acquire stem cell properties and repopulate the intestinal epithelium.

Lineage tracing Lineage tracing

Regeneration

Niche reinforcement by Paneth cells, quiescent stem cell (+4) activation. +4 Lyz+Paneth

Overall, colon cancer risk factors such as diet and inflammation profoundly affect both niche and stem cells resulting in improved regeneration upon tissue injury. The recruitment of new stem cell types and the overall increased proliferation rate ultimately results in an increased mutation rate and cancer risk.

For every complicated problem there is a solution that is simple, direct, understandable, and wrong. H.L. Mencken

Two distinct cancer research themes:

• 1. tumor onset

(how does it all start? Is it really just a question of bad luck?)

• 2. metastasis and

phenotypic plasticity

(what has personalized medicine to do with that?)

20 30 40 50 60 70

Age (years) (epi)genetic mutation load

Tumor Evolution at the Primary Site (Darwin docet: sequential clonal expansion)

2nd hit 1st hit 3rd hit 2nd hit

x

3rd hit

x

EMT

surgery

Dissemination (CTCs & DTCs)

. . . . .

Tumorigenesis and Metastasis: Discontinous Evolution

. .

x

genetic hit (mutation) epigenetic hit (red: EMT; yellow: MET)

Distant Site Metastases

x x

. .

1st hit

x

Phenotypic Plasticity

The current ‘hype’ on personalized cancer therapy is misplaced and unjustified

‘a large majority of driver gene mutations are common to all metastases’ ‘the driver gene mutations that were not shared by all metastases are unlikely to have functional consequences’ 2002 2018

Tumor cells are capable of contributing to normal development and generate phenotypically normal chimeric organism with tumor cells present in most adult tissues

(mouse teratocarcinoma cells: Illmensee & Mintz, 1976; chicken embryonic cells infected with Rous sarcoma virus: Rous, 1979; Dolberg & Bissell, 1984; Stoker, Hatier, & Bissell, 1990).

Som

• me ea

earl rly (70’s and 80’s) exp xperimental evid viden ence th that t th thin ings gs are not

• t as sim

imple e as th that:

Mina Bissel, PhD:

phenotype is dominant over genotype!!

Notably, when cultured in vitro, these embryonic cells rapidly reveal pronounced tumorigenic features and undergo neoplastic transformation. Hence, the embryonic environment seems to attenuate oncogenesis possibly by inducing differentiation and suppressing cancer stemness.

Type, density and location of tumor-infiltrating immune cells in large cohorts of human colorectal cancers represent the strongest prognostic factors in terms of freedom from disease and overall survival at all stages of clinical disease (Galon et al., Science 2006).

Jerome Galone, PhD

The Overhyping of Precision Medicine

Science has a history of inflated promises when it comes to disease treatment. by Nathaniel Comfort, Dec. 16, 2016

www.theatlantic.com/health/archive/2016/12/the-peril-of-overhyping-precision-medicine/510326

Phenotypic Plasticity

The invasion-metastasis cascade: two main theories

….

“seed and soil” (single CTCs) vs. “collective cell migration”

Epithelial markers:

• E-cadherin
• Cytokeratin
• Desmoplakin
• Laminin

Mesenchymal markers:

• Vimentin
• Fibronectin
• N-cadherin
• 𝛃-SMA

Epithelial-to-mesenchymal transition: not a B&W process EMT transcription factors: ZEB1, ZEB2, Snail, Slug, Twist, FoxC2

Functional features:

• Tight cell-cell adhesion
• Non-motile
• Non-invasive

Functional features:

• No cell-cell adhesion
• Motile
• Invasive

Functional features:

• Weak cell-cell

adhesion

• Metastable

E/M markers:

??

Phenotypic Plasticity and Metastasis: conventional immortalized cell lines as experimental models

Common colon cancer cell lines encompass a CD44highEpCAMlow subpopulation with mesenchymal and highly motile and invasive features

Andrea Sacchetti

CD44highEpCAMlow colon cancer cells express

EMT markers and transcription factors (ZEB1)

CD44hiEpCAMlow =19% CD44hiEpCAMlow = 84%

EpCAM-FITC CD44-APC HCT116 - Sorted EpCAM low + 34 days

CD44highEpCAMlow cells have stem-like characteristics

CD44highEpCAMlow cells have high-metastatic capacity in vivo

HCT116 - Spleen injection

…and are resistant to chemotherapy

CD44hiEpCAMhigh CD44hiEpCAMlow

CD44hiEpCAMhigh CD44hiEpCAMlow

HCT116 – Top differentially expressed genes

EpCAM E-Cad ZEB1 VIM1 RSPO1

RNAseq analysis: functional and omics characterization

HCT116 and SW480: combined pathway analysis HCT116 and SW480: MDS analysis

CD44highEpCAMlow cells correlate with EMT signatures and the CMS4 subtype of colon cancer

HCT116 – chemotherapy

Post- treatment Pre- treatment Recurrence Post- treatment Pre- treatment Recurrence

SW480 – chemotherapy

CD44highEpCAMlow cells correlate with signatures of chemoresistance and have a worse clinical outcome

Diagnostic and prognostic CTC markers Targeted CTC therapy Future Plans: detection and targeting of single CTCs and CTC cluster in blood Single CTCs vs clusters Epithelial vs hybrid E/M vs mesenchymal

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