Cellular Signaling and Cancer
Plasticity Group ‐ CPG 
Dr.  Karel Souček
ksoucek@ibp.cz
Department of Cytokinetics               
Institute of Biophysics AS CR
Kralovopolska 135
612 65 Brno , Czech Republic
Tel:  + 420 541 517 166
St. Anne’s University Hospital Brno
International Clinical Research Center
Pekařská 53
656 91  Brno, Czech Republic
Tel:  + 420 543 181 111
Plasticity of cancer cells
• Cancer is heterogeneous and not single cell disease.
• Complex and dynamic, NOT static “ecosystem”.
• Diversity inside tumors is clinical problem limiting the efficacy of targeted 
therapies and compromising treatment outcomes.
Genomic instability
Microenviroment Therapy
Hallmarks of cancer
 90% of cancer related deaths are due to metastasis
 What kind of cells drives metastasis?
G Gundem et al. Nature, E1-E5 (2015) doi:10.1038/nature14347
Metastasis-to-metastasis seeding occurs either by a
linear or by a branching pattern of spread.
Hallmarks of metastasis‐initiating cells
Characterized by evolutionary advantageous traits 
that may originate in primary tumor and continue to 
evolve during dissemination & colonization:
– cellular plasticity
– metabolic reprogramming
– ability to enter/exit dormancy
– immune evasion
– co‐option of other tumor and stromal cells
Epithelial‐to‐mesenchymal transition (EMT)
 Reversible acquisition of migratory and invasive properties by epithelial 
cells
 Role in embryonic development, fibrosis, cancer
 Creates cells with stem‐like cells characteristics
 Both mesenchymal and epithelial phenotypes are required for efficient 
metastatic spread
 What is a role of EMT in prostate and breast 
cancer progression?
– Trop‐2 associates with epithelial phenotype of breast and prostate cancer cells
– EMT & metastatic signature of selected BCa subpopulations
 What is a role of cancer plasticity and 
heterogeneity in therapy resistance?
– Synthetic lethality as a concept for treatment drug resistant cancer
Questions
Trop‐2 associates with an epithelial phenotype 
in breast and prostate cancer cells
Trop‐2 marks epithelial subpopulation of 
BCa and PCa cell lines
Hypothesis: EMT is accompanied by changes in CSC‐like signature
Trop-2
• cell surface glycoprotein
• marks stem cells and progenitors
• role in stemness and multipotency maintenance mostly
unknown, e.g. in prostate activates basal cell program
• in some tissues, expression is epigenetically silenced in
differentiated adult cells
• described as oncogene and metastasis inductor
EpCAM vs. Trop-2
• both are commonly (over)expressed in adenoCa
• both are processed via RIP
• 67 % similarity
• 46 % promoter seq identity => quite unrelated
• EpCAM is known to be downregulated during EMT; but
what about Trop-2?
• EpCAM KO has lethal phenotype
unpublished
Dynamics of Trop‐2 expression 
in vivo & in vitro
Hypothesis: Trop‐2 is dynamically regulated and reflects epithelial state of cells
unpublished
Regulation of Trop‐2 expression 
by epigenetic and EMT machinery 
Hypothesis: ZEB1 & DNA methylation regulate Trop‐2
unpublished
Intratumoral heterogeneity of 
membrane Trop‐2 expression 
better prognosis
associated
with epithelial
phenotype
unpublished
Summary
 Trop‐2 associates with epithelial phenotype 
of breast and prostate cancer cells
 commonly accepted view of Trop‐2 as oncogene is too 
simplistic
Future plans
• To uncover molecular mechanisms by which Trop‐2 
contributes to cancer progression
– ZEB1 ChiP
– methylation status of human Trop‐2 promoter
– single cell qPCR in CTCs
EMT & metastatic signature of selected BCa subpopulations
Intratumoral heterogeneity and plasticity of cancer cells
IHC source: www.proteinatlas.org
EMT is commonly accepted source of plasticity, characterized 
by e.g. Cadherin‐switch and often accompanied by activation 
of stem‐like transcriptional programs
Motivation
TNBC is:
– Estrogen receptor (ER)‐negative
– Progesteron (PR)‐negative
– HER2 negative
• 15‐20% BCa
• Often in younger women, BRCA1 gene mutation
• Tends to be more aggressive, recur early and spread to 
other parts of body, poor prognosis
• treatment: surgery, radiation, chemotherapy (platinum‐
based, taxanes) = no targeted therapy available
‐ many intracellular markers known, but no reliable 
surface antigen enabling tracking of EMT available
Proteins associated with distinct cancer cell 
phenotypes
Surface antigens associated with distinct cancer 
cell phenotypes
next step:
11‐colour FACS protocol
for patient samples
Hypothesis: EMT‐ed cells have specific surface pattern
unpublished
Predicted 10‐molecule surface signature that associates 
with plasticity of epithelial cells.
Experimental strategy
source: www.proteinatlas.org, www.fda.gov
Heterogeneity examples
Mass cytometry
• measurement of over 40 
simultaneous cellular parameters 
at single‐cell resolution
Alternative … spectral FCM
Summary
 epithelial and stromal compartment of breast cancer 
tissue is composed of extremely heterogeneous mixture 
of cells
Future plans
• To identify cell surface signature which reflects cancer 
cell plasticity and mirrors enrichment in metastasis‐
initiating genes in patiens.
– sample collection (actually various subpopulations 
from 3 patients sorted), processing and analysis
– in vitro and in vivo tests for relevant markers (analysis 
of selected signaling pathways, migration, invasion)
Outlook
 Small‐molecule drugs and synthetic lethality 
 Plasticity of cancer cells and new targets for cancer therapy
 Modulation of tissue microenvironment, cell metabolism and 
drug efficiency
Team
 Lucia Binó, Ximena Muresan, Miroslav Huličiak
 Stanislav Drápela, Vojtěch Dvořák, Šárka Šimečková
 Radek Fedr
 Department of Cytokinetics, Institute of Biophysics AS
Cooperation
 Petr Beneš, Lucia Knopfová, Stjepan Uldrijan, Aleš Hampl, Petr Vaňhara, Josef Jaroš, 
Milan Ešner, Kamil Paruch, Lumír Krejčí, Jiří Damborský  ‐ Masaryk University
 Jan Bouchal, Gvantsa Kharaisvili ‐ UJP Olomouc
 Jiří Kohoutek ‐ Veterinary Research Institute, Brno
 Zoran Culig laboratory ‐ Medical University Innsbruck
 Gabri Van der Pluijm ‐ Leiden University Medical Centre
 Wytske van Weerden – Erasmus University, NL
 Michael Andäng, Karolinska Institutet
 Giuseppe Valachi, University of Ferrara