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 Remsik, et al, Carcinogenesis 2018 Dynamics of Trop-2 expression in vivo & in vitro Hypothesis: Trop-2 is dynamically regulated and reflects epithelial state of cells Remsik, et al, Carcinogenesis 2018 Regulation of Trop-2 expression by epigenetic and EMT machinery Hypothesis: ZEB1 & DNA methylation regulate Trop-2 Remsik, et al, Carcinogenesis 2018 Intratumoral heterogeneity of membrane Trop-2 expression better prognosis associated with epithelial phenotype Remsik, et al, Carcinogenesis 2018 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 – 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 (platinumbased, 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 Remsik et al, BJC, 2018 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 metastasisinitiating 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 ◼ Ximena Muresan, Tereza Suchanková ◼ Stanislav Drápela, Ondřej Vacek, Markéta Pícková, Barbora Kvokáčková, Karolína Kryštofová ◼ 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