Implications of hybrid epithelial/

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Implications of hybrid epithelial/

mesenchymal phenotype in metastasis:

Can theory help understand cancer biology?

Mohit Kumar Jolly

Department of Bioengineering and Center for Theoretical Biological Physics, Rice University

Philippine Genome Center June 28, 2016

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commons.wikimedia.org/wiki/File:Metastasis_sites_for_common_cancers.svg

Metastasis, the spread of cancer cells from one organ to another, claims

over 90% of all cancer deaths.

Metastasis: the Achilles’ heel in the ‘War against Cancer’

Past 50 years, great progress in:

1. Charting the genes/proteins involved in cancer 2. Listing the risk factors associated with cancer 3. Diagnosing cancer earlier

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Potential bottlenecks in halting metastasis

“Identifying all the genes and proteins in an organism is like listing all the parts of an airplane. While such a list provides a

catalog of the individual components, by itself it is not sufficient to understand the complexity underlying the

engineered object.”

Kitano, Science 2002

Can a systems biology

approach help defeat cancer?

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Systems Biology of Cancer

Goal: To develop a conceptual framework that can

a) Explain existing empirical experimental data

b) Predict cellular behavior and guide further experiments c) Help experimental biologists think more quantitatively

Without

1. Focusing on one specific subtype of cancer/ data from one lab 2. Inferring genetic networks from ‘omics’ (big-data) in an

automated manner

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How do cancer cells metastasize?

More than 80% cancers happen in epithelial organs, i.e.

cells do NOT move or invade.

Scheel and Weinberg, Semin Cancer Biol 2012

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EMT/MET: The engine of metastasis

Epithelial (E)

Tight cell-cell adhesion No migration, invasion

Mesenchymal (M) No cell-cell adhesion High migration, invasion

Mesenchymal-to-Epithelial Transition (MET)

Epithelial-to-Mesenchymal Transition (EMT)

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Are E and M the whole story? No!

Hybrid epithelial/mesenchymal (E/M) = Cell-cell adhesion (E) + Migration (M) =

Collective cell migration

Clusters of CTCs (Circulating Tumor Cells) are up to 50-times more metastatic than single CTCs

Aceto et al. Cell 2014 Liottta et al. Cancer Res 1976

Clusters identified in patients even before EMT was known!

Yu et al. Science 2013

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Hybrid E/M

Some cell-cell adhesion, some migration

Collective cell migration CTC clusters

Epithelial (E) Mesenchymal (M)

CTC clusters: the real ‘villains’ of metastasis

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Q1. How do cancer cells form these

clusters?

Q2. Can we find

targets to break these clusters?

Q3. Why do clusters form more

metastases?

Q1. How do cells attain 3 states – E, hybrid E/M, and M?

Q2. Can we destabilize the hybrid E/M state?

Q3. Are hybrid E/M cells more drug-resistant and/or more potent at initiating

tumors?

Tackling the real ‘villains’ of metastasis

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Q1. How do cells attain 3 states – E, hybrid E/M, and M?

(How do cancer cells form these clusters?)

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How do cells attain 3 phenotypes – E, M, E/M?

Lu*, Jolly* et al. PNAS 2013 Lu*, Jolly* et al. Cancer Res 2014

Core decision-making circuit for EMT

Transcriptional activation

Translational inhibition (microRNA) Transcriptional inhibition

• Each arrow/bar denotes a quantitative

relationship between input and output levels

• We developed a novel quantitative

framework to study translational regulation

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m₂₀₀ = g_m₂₀₀H^S(Z,l_Z,_m₂₀₀)H^S(S,l_S,_m₂₀₀)-m_ZY_m(m₂₀₀)-k_m₂₀₀m₂₀₀ m_Z =g_m_ZH^S(Z,l_Z,_m_Z)H^S(S,l_S,m_Z)-m_ZY_m(m₂₀₀)-k_m_Zm_Z Z =g_Zm_ZL(m₂₀₀)-k_ZZ

m₃₄ =g_m₃₄H^S(S,l_S_,_m

34)H^S(Z,l_Z,_m

34)-m_SY_m(m₃₄)-k_m₃₄m₃₄ m_S=g_m_SH^S(S,l_S_,m

S)H^S(I,l_I,_m

S)-m_SY_m(m₃₄)-k_m_Sm_S S=g_Sm_SL(m₃₄)-k_SS

Production Terms Production Terms Innate Degradation Terms

Innate Degradation Terms miRNA Degradation TermsmiRNA Degradation Terms miRNA Translation Inhibition miRNA Translation Inhibition

miRNA

miRNA mRNA

mRNA protein

protein

Quantitative framework for microRNA regulation

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Quantitative framework for microRNA regulation

Kim et al. J Cell Biol 2011; Goodall et al. Nat Cell Biol 2008; Lu*, Jolly* et al. PNAS 2013

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Integrator Existence of a Decision-making switch hybrid state

Lu*, Jolly* et al. PNAS 2013

Model predictions:

1. 3 phenotypes co-exist

2. (miR-200/ZEB) loop acts as a ‘decision- making’ three-way switch

E – (high miR-200, low ZEB) M – (low miR-200, high ZEB)

E/M – (medium miR-200, medium ZEB)

Design principles of EMT decision-making

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Increase in ZEB levels essential for cells to exit an

epithelial phenotype ZEB increase marks the

‘commitment point’ for EMT transition

ZEB1 levels have 3 ranges

• Anti-correlated with miR-200

• Hybrid E/M have intermediate levels

Sundarajan et al. Oncotarget 2015 Gregory et al. Mol Biol Cell 2011

Experimental validation of the predicted role of miR-200/ZEB

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Hybrid E/M phenotype a single-cell level

Grosse-Wilde et al. PLoS ONE 2015

Schliekelman et al. Cancer Res 2015

Andriani et al. Mol Oncol 2016

Jeevan et al. Anticancer Res 2016

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Q2. Can we destabilize the hybrid E/M phenotype?

(Can we find targets to break these clusters?)

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Let’s go back to the math!

Jolly et al. Oncotarget 2016

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H1975, T=0 H1975, T=2 months

≈

Green: VIM (Mesenchymal)

Red: CDH1 (Epithelial)

Hybrid E/M is a stable cell state

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H1975 H1299

T = 0 hour T = 12 hours

Disrupting such collective migration might be a new therapeutic strategy

Hybrid E/M cells move collectively

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Deleting OVOL2 or GRHL2 disrupts hybrid E/M phenotype

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Lung Cancer 982 patients

Breast Cancer 1117 patients

High GRHL2 correlates with poor patient survival

Adapted from Jolly et al. Oncotarget 2016

Hybrid E/M phenotype can be stable and its stability can aggravate tumor progression

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Q3. Are hybrid E/M cells more drug-resistant and/or more potent at initiating tumors?

(Why do clusters form more metastases?)

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The two major traits of cells responsible for tumor relapse (also referred to as ‘Cancer Stem Cells’ or CSCs):

??

The root of tumor relapse

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Three-way EMT/MET decision circuit

(Lu*, Jolly* et al. PNAS 2013; Lu*, Jolly* et al. Cancer Res 2014)

Stemness decision circuit

(Yang et al. Cancer Res 2010)

NF-κB

Jolly et al. J R Soc Interface 2014

OCT4

Stemness Window

Hybrid E/M cells can be more ‘stem-like’ than M cells Theory: Hybrid E/M cells can be more ‘stem-like’ than M

Niwa et al. Nat Genet 2000

Intermediate levels of OCT4 correspond to maximum stemness

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DTCs are CD44^hi CD24^hi

CD24+CD44+ (E/M cells) are drug-resistant and form much more tumors in

vitro and in vivo.

Goldman et al. Nat Comm 2015

Hybrid

Experiments confirm higher ‘stemness’ of hybrid E/M cells

Grosse-wilde et al. PLoS ONE 2015

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Summary

• Elucidated how tumor cells form stable clusters of Circulating Tumor Cells (CTCs) – the primary ‘villains’ of metastasis

• Identified potential targets that maintain these clusters and enhance their tumor forming ability – GRHL2

• Offered a potential mechanistic understanding of why clusters of CTCs form much more metastases

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Conclusion

Existing framework:

Hybrid E/M state is transient, i.e. cells cannot maintain it for a very long time

Proposed framework:

Hybrid E/M state is stable and can enable formation of CTC clusters

Tam and Weinberg, Nat Med 2013 Savagner, Curr Opin Dev Biol 2015

Jolly et al. Oncotarget 2016 Cheung and Ewald, Science 2016

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Clinical implications

Liquid biopsy

Quick

Easily obtainable Minimal invasive

Minimal pain

Focus on isolating clusters of Circulating Tumor Cells (CTCs)

• Categorize patients based on aggressiveness

• Suggest therapy options

• Monitor disease progression

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Herbert LevineJose’ N Onuchic

Acknowledgement

Eshel Ben-Jacob MC Farach-Carson

Mingyang Lu Dongya Jia Bin Huang Marcelo

Boareto A Dan Grigore Sendurai A Mani

Samir M Hanash Satyendra C

Tripathi

Aaron Goldman Shiladitya

Sengupta Kenneth J Pienta

Donald S Coffey Steven M Mooney

Jason Somarelli Samantha Shetler

Andrew Armstrong