*pathological plasticity of epithelial cells *neuroendocrine differentiation (NED) *senescence associated secretory phenotype *cell cycle *epithelial-mesenchymal transition (EMT) *cancer stem cells (CSCs) *role of MDM2 *Transforming Growth Factor-b signal transduction *Growth/differentiation factor – 15 *signal transduction *role in cancer and damaged hematopoiesis * D:\Kaja\Manuscripts&Papers\NED&Cell Cycle_Zuzka\old\Pernicova_Figs_old\Links\CS_40x 1.tif Cancer is heterogeneous … …and not a single cell type disease. Pathological plasticity of prostate epithelial cells nNeuroendocrine transdiferentiation nEpithelial to mesenchymal transition •one of the leading causes of cancer related death in men worldwide •conventional treatment - hormonal therapy - luminal secretory cells are dependent on androgens - androgen deprivation therapy (hormonal therapy) – chirurgical castration or anti-androgen administration (Casodex (bicalutamide)) • patients - positive response at the beginning - tumor regression - after 1-2 years of treatment - tumor progression, metastasis - development of androgen independence Prostate cancer Sun et al., Am J Transl Res 2009, 1(2) Nelson et al., NEJM 2003, 349(4) Produced by NE cells Attar et al., Clin Cancer Res 2009, 15(10) Prostate neuroendocrine cells -Characteristic -uncertain origin -scattered in prostatic epithelium -dendrite-like protrusions -markers: NSE, TUBB3, CHGA -quiescent -do not express AR -secretion of various factors (bombesin, adrenomedulin, VEGF, serotonin, IL-6, IL-8, etc.) -Function -growth and differentiation regulation -modulates function of prostatic gland -regulation of homeostasis Taylor R A et al. Endocr Relat Cancer 2010;17:R273-R285 1.androgen deprivation therapy induces secretory, tumor-promoting senescent cells in prostate tumors; 2.role of MDM2 in EMT of benign and transformed cells; 3.CHK1 inhibition & DNA damaging drugs in prostate epithelial cells – preliminary screen 4.new methods & approaches: *isolation of normal mouse prostate stem cells *multicolor protocol for characterization and separation of human prostate cancer stem cells *new automatic cell cloning assay (ACCA) for determination of clonogenic capacity of cancer stem-like cells *1) androgen deprivation therapy induces secretory, tumor-promoting senescent cells in prostate tumors *We showed link between inhibition of androgen receptor signaling, down-regulation of S-phase kinase-associated protein 2, and the appearance of secretory, tumor-promoting senescent cells in prostate tumors. *We propose that androgen deprivation therapy may contribute to the development of androgen-independent prostate cancer through modulation of the tissue microenvironment by senescent cells. p53 dependent p53 independent EMT dependent MDM2 Cell migration TGF-b p14Arf Cancer transformation EMT MDMX Slug Twist Metastasis *a) isolation of normal mouse prostate stem cells * *b) multicolor protocol for characterization and separation of human prostate cancer stem cells Patient #123 Patient #24 *c) new automatic cell cloning assay (ACCA) for determination of clonogenic capacity of CSCs single cell/well up to 384 well plate re-culture after sorting (2D, 3D) analysis: CyQuant, ATP, xCelligence, microscopy Clonogenic capacity of CD44/CD133low vs. CD44/CD133high cells EMT in SCs/CSCs-like subpopulation DSC09145 DSC09145 Lin-/Sca1-CD49f- RNA isolation cDNA synthesis RealTime Ready Custom Panel RT-qPCR Lin-/Sca1+CD49fhigh/Trop2+ *Cdk inhibition induces neuroendocrine differentiation in prostate cancer cells siRNA CDKI *tools for molecular imaging *hydrogen peroxide sensor HyPer (Evrogen) for ratiometric detection of intracellular H2O2 level changes *HEK293 HyPer-dMito *HEK293 HyPer-cyto time (s) 100 mM H2O2 *tools for in vivo molecular imaging *Prostate, Breast, Melanoma, and Colon Carcinoma Models *for syngeneic immunocompetent strains C57Bl/6 or BALB/c *stable transfected with lentiviral luc vector *CT26 luc - mouse colon cancer *4T1 luc - mouse breast cancer *B16 F10 luc – mouse melanoma *TRAMP-C1 – mouse prostate cancer *RM-1 – mouse prostate cancer * * *tested compounds *CHK1 inhibitor SCH900779 *Ara-C, gemcitabine, HU *tested cell lines *non-tumorigenic – HPEpiC, BPH-1 *primary cancer – CAFTD-01, -03, LAPC-4 *metastatic cancer – LNCaP, PC3 *other – HeLa Fucci 8 *design *treatment with gem, HU, Ara-C for 24h *2h treatment with CHK inhibitor, than media exchange *harvest 48h after treatment *readouts *CyQuant – concentration screen, synthetic lethality analysis *xCelligence – real-time analysis (selected concentrations) * Nature Methods - 5, 283 (2008) *Gemcitabine *in all tested cell lines very toxic – use lower concentrations *Ara-C + CHKI *BPH-1 – 5 + 0.25 mM synthetic effect *LNCaP – 5 + 2 mM synthetic effect *PC-3 – 5 + 2 mM synthetic effect *HU + CHKI *all tested cell lines 0.5 + 2 uM synthetic effect Experimental approaches and models •screening of cytotoxic concentrations of DNA damaging agents and inhibitors • –evaluation by CyQuant® proliferation assay – •monitoring of dynamics of cytotoxic effects of DNA damaging drugs • –xCELLigence system –live microscopic imaging • •evaluation of treatment in 3D conditions • –microscopic imaging –ATP bioluminiscence proliferation assay • TISSUE ORIGIN p53 STATUS BPH1 parental Prostate non – tumorigenic human inactivated BPH1 CAFTD03 Prostate tumorigenic human inactivated DU-145 brain metastais of prostate carcinoma human mt PC3 bone metastatis of prostatic adenocarcinoma human null HCT116 p53 +/+ colorectal carcinoma human +/+ HCT116 p53 -/- colorectal carcinoma human -/- HCT116 PTEN -/- colorectal carcinoma human +/+ MDCK kidney tissue non – tumorigenic dog wt B16-F10 skin melanoma mouse wt TRAMP C1 prostate adenocarcionma mouse wt 3219_Cover Olympus-ICC-Microscope Live microscopic imaging experiment set up BPH1par BPH1 c.03 HCT116 p53+/+ PC-3 DU-145 dmso OH209 SCH900776 GEMCITABINE GEMCITABINE + OH209 GEMCITABINE + SCH900776 cells seeding microscopic imaging 24 hod 48 hod 0 hod 50 hod 120 hod media exchange treatment by DNA- damaging agents treatment by Chk1 inhibitors Results Live microscopic imaging Cell lines indicated were seeded in microplate wells, treated and then monitored in real time using bright-field microscope and CCD camera. Images obtained at final time (120 hours) are compared for wells treated by gemcitabine (IC50 vaules for selected cell lines), by gemcitabine and SCH900776 (4 μM) and by SCH900776 (4 μM) only meritko_static PC3_gem_OH_static PC3_gem_static PC3 cell line PC3_gem PC3_gem_OH media exchange effect (inhibitor only) inhibitor only gemcitabine gemcitabine+ inhibitor meritko PC3_gem_static_end_34 PC3_gem_OH_static_end_47 meritko_static binres ANd9GcTL0opC7eTbERIqighMy75SCbSeqYo5X__rbYrW_PNBYOCN2hpfig •6 concentrations of DNA-damage drug (hydroxyurea) • •6 concentrations of 2 inhibitors of CHK1 (SCH900776, OH209) • •all combinations in quadruplicate and two biological repetition Running experiments - screening for synthetic lethality in panel of cell lines TYPE CELL LINE Colon SW480 SW620 HCT-116 p53+/+ HCT-116 p53 -/- HT29 Breast MCF10A MDA-MB-231 Sk-Br-3 Lung H441 A549 Ovarian A2780 A2780cis SKOV-3 Prostate BPH-1 BPH-1 CAFTD04 PC3 DU145 LNCaP LAPC-4 Pancreas MiaPaCa2 PANC-1 Kidney MDCK *Aleš Hampl (LF MU) – mGDF15 ICC (cryosections), CSCs, tissue engineering, SCID *Petr Vaňhara (LF MU) – GDF15 in dendritic cells, glioblastoma and ovarian cancer, lentiviral particles *Petr Beneš (PřF MU) – mGDF15 inducible plasmids *Kamil Paruch (PřF MU) – inhibitors *Stjepan Uldrijan (PřF MU) – MDM2 story *Lukáš Kubala (BFÚ) – EMT & ECM, tissue engineering * *Jiří Kohoutek (VÚVeL) – gdf15 knock-out colony managment *Michal Hofer (BFÚ)– hematopoiesis study *Jiří Pacherník (PřF MU) – GDF-15 in hypoxia, mES *Pavel Matula, Petr Matula (FI MU) – tube forming assay data analysis *Jiřina Procházková , Jan Vondráček (BFÚ) - GDF15 in cardiomyocytes, interaction of AhR and TGF-b *Miroslav Machala (VÚVeL) - interaction of AhR signaling with TGF-b *Jan Bouchal (UJP Olomouc) – EMT & ECM, prostate cancer clinical samples, CSCs, IHC * *Lukas Kenner (Ludwig Boltzman Institute, Vienna) - prostate cancer – mouse model, CSCs, *Giuseppe Valacchi (University of Ferrara) – redox signaling, autophagy * *Bakalářské *Úloha SNX9 v epiteliálně mesenchymálním přechodu u epiteliálních buněk prostaty *Diplomové *Změny v expresi proteinů MDM2 a MDMX v průběhu epiteliálně mesenchymálního přechodu *Epiteliálně mesenchymální přechod u normálních a nádorových kmenových buněk prostaty *Doktorské *Úloha Skp2 v cytokinetice nádorových kmenových buněk *Úloha epitelialně mesenchymálního přechodu v regulaci fenotypu nadorových kmenových buněk *