Targe&ng lactate transport in urothelial carcinoma

Transkript

1 Targe&ng lactate transport in urothelial carcinoma Todenhöfer T.,, Seiler R., Stewart C., Moskalev I., Gao J., Ladar S., Kamyabi A., Al Nakouzi N., Hayashi T., Choi S., Wang Y.Z., Daugaard M., Ooh H., HennenloNer J., Bedke J., Fazli L., Stenzl A., Black PC. Vancouver Prostate Centre, University of BriUsh Columbia, Vancouver, Canada Dept. of Urology, Eberhard- Karls University Tuebingen, Germany

2 Aerobe Glykolyse in Tumorzellen Porporato et al.

3 ONo Warburg (883-97)

4 LaktaNransport in Tumorzellen BSG = CD7 = EMMPRIN! Ankerprotein von MCT /MCT Parks et al., Nat Rev Can 3

5 Expressionsanalyse von MCT, MCT und CD7 in Gewebe von 6 Zystektomien MCT MCT CD7 * * Score 3 Score Score Score Tumor Urothel Primary tumor P- value: * <. <. * <. < Score 3 Score Score Benign Benign Primary tumor Score % of pa&ents.3. % of pa&ents % of pa&ents Score Score Score Benign Primary tumor Score

6 MCT und MCT sind mit dem basalen Subtyp des muskelinvasiven BC assoziiert Cancer Genome Atlas (TCGA) A B C RNA Expression (RNA Seq V) RNA Expression (RNA Seq V) RNA Expression (RNASeqV) RNA Expression (RNASeqV) I I * * II MCT * * III TCGA subtype * * II MCT * * * * * * III * * IV IV I I II II III III IV IV TCGA Subtype P- value: * <. <. * <. <. RNA Expression (RNASeqV) RNA Expression (RNASeqV) RNA Expression (RNASeqV) RNA Expression (RNASeqV) Luminale Marker 3 low MCT low MCT low MCT low MCT FOXA GATA3 KRT PPARG high MCT * high MCT high MCT * high MCT RNA Expression (RNASeqV) RNA Expression (RNASeqV) RNA Expression (RNASeqV) RNA Expression (RNASeqV). 6 Basale marker ).. low MCT low MCT low MCT low MCT KRT KRT6A KRT6B KRT6C * high MCT high MCT * high MCT * high MCT

7 Überexpression von MCT und MCT in basalen Karzinomen- Validierung in einer unabhängigen Kohorte normalized gene expression Basal MCT luminal normalized gene expression Basal MCT * luminal normalized gene expression.... Bas Basale Tumoren: - Expression von Stammzell- und EMT- Markern - Histologisch ol squamöse Differenzierung - Schlechte Prognose P- value: * <. <. * <. <.

8 MCT, MCT & CD7 Expression in Urothelkarzinom- Zelllinien MCT kda kda MCT kda CD7 kda Vinculin kda kda RT RTv6 UM- UC UM- UC UM- UCR UM- UC6 UM- UC UM- UC6 3JBV T UM- UC3 UM- UC3

9 Hemmung von MCT, MCT und CD7 durch RNA Interferenz (RNAi) MCT Protein MCT Protein CD7 MCT$ Protein MCT$ Vinculin$ T$.$$ T$siMCT$#$$ T$.$$ T$siMCT$#$$ T$siMCT$#$$ UC6$.$$ T$siMCT$#$$ UC6$siMCT$#$$ UC6$.$$ UC6$siMCT$#$$ UC6$siMCT$#$$ UC3$.$$ UC6$siMCT$#$$ UC3$siMCT$#$$ UC3$.$$ UC3$siMCT$#$$ UC3$siMCT$#$$ Vinculin$ UC3$siMCT$#$$ Rela;ve$expresion$.$.$ $.8$.6$.$.$ $ MCT T$ mrna UC6$ MCT MCT mrna UC3$ CD7 mrna T$.$ UC6$ UC3$.$.$.$.$.$.$.$.$ $.$ $ $ $ $.8$.8$.8$.8$.8$.6$.6$.6$.6$.6$.$.$.$.$.$.$.$.$.$ $.$ $ $ $ simct$#$ simct$#$ $ $ simct$#$ simct$#$ $ $ simct$#$ simct$#$ $ simct$#$ simct$#$ $ simct$#$ simct$#$ $ simct$#$ simct$#$ Rela;ve$expresion$ Rela;ve$expresion$$ Rela;ve$expresion$$ Rela;ve$expresion$ Rela;ve$expresion$

10 InhibiUon von MCT und CD7- Einfluss auf Wachstum von Urothelkarzinom- Zelllinien (T, UC6, UC3) OD 7 nm (fold change) 3 MCT RNAi T + simct# simct# OD 7 nm (fold change) 3 CD7 RNAi T sicd7# sicd7# OD 7 nm (fold change) UC6 simct# simct# OD 7 nm (fold change) UC6 * + sicd7# sicd7# UC3 OD 7 nm (fold change) 8 6 P- value: * <. <. * <. <. simct# simct# OD 7 nm (fold change) 8 6 UC3 sicd7# sicd7#

11 MCT RNA Interferenz hemmt Zellwachstum in MCT+ Zelllinien OD 7 nm (fold change) 3 T Normoxia * * + * 6 8 simct# simct# OD 7 nm (fold change) T Hypoxia + * * 6 8 simct# simct# OD 7 nm (fold change) UC6 Normoxia * * simct# simct# OD 7 nm (fold change) UC6 Hypoxia * * simct# simct# OD 7 nm (fold change) 8 6 UC3 Normoxia * simct# simct# OD 7 nm (fold change) 8 6 UC3 Hypoxia * + * simct# simct# P- value: * <. <. * <. <.

12 MCT Hemmung induziert Apoptose Annexin V FACS T UC6 UC3 Annexin V positive cells (fold change) 3 simct # simct # Annexin V positive cells (fold change) 3 simct # simct # Annexin V positive cells (fold change) 3 simct# simct# UC3 7AAD 7AAD 7AAD simct # simct # Annexin V Annexin V Annexin V P- value: * <. <. * <. <.

13 MCT Hemmung führt zu intrazellulärer LaktatakkumulaUon Lactate (nmol/ug Protein) T intracellular Lactate si Cntrl simct# simct# Lactate (nmol/ul/ug Protein) 3 3 T extracellular lactate * si Cntrl simct# simct# Lactate (nmol/ug Protein) UC6 intracellular Lactate si Cntrl simct# simct# Lactate (nmol/ul/ug Protein) UC6 extracellular lactate si Cntrl simct# * simct# P- value: * <. <. * <. <. UC3 intracellular Lactate UC3 extracellular lactate

14 UC3 MCT Hemmung führt zu reduzierter extrazellulärer AzidifikaUon Medium ohne Glukose O - Verbrauch Medium + Glukose Medium + Glukose+ Oligomycin Extrazelluläre Azidifika&on + (nicht glykoliusch) + + Oligomycin = Inhibitor der oxidauven Phosphorylierung! SUmulaUon Glykolyse T UC6 Glucose Oligomycin Glucose Oligomycin ECAR (mph/min) Phase simct# simct# ECAR (mph/min) 3 * * Phase simct# simct# P- value: * <. <. * <. <.

15 OD 7 nm (fold change) 3. Glukoseentzug neutralisiert wachstumshemmenden 3 Effekt einer.. MCT InhibiUon Time simct # simct # OD 7 nm (fold change). OD 7 nm (fold change) Time Time + * simct# # simct# # OD 7 nm (fold change) OD 7 nm (fold change) T Normoxia Time simct # simct # OD 7 nm (fold change) OD 7 nm (fold change) T Hypoxia UC6 Normoxia * Time Time simct simct# simct simct# OD 7 nm (fold change) 3 OD 7 nm (fold change)..... UC3 Normoxia UC6 Hypoxia Time simct simct# simct simct# OD 7 nm (fold change) Glukose "! Glykolyse "! LaktatprodukUon " OD 7 nm (fold change)..... UC6 Normoxia Time simct# simct# OD 7 nm (fold change) OD 7 nm (fold change) P- value: * <. <. * <. < UC3 Normoxia UC6 Hypoxia Time simct # simct# simct # simct# OD 7 nm (fold change) UC3 Hypoxia Time simct # simct #

16 MCT Hemmung induziert Synthese von Sauerstoffradikalen simct # simct # HDCFDA Fluorescence (Relative fold change) T simct # simct # HDCFDA Fluorescence (Relative fold change) UC6 3 simct # simct # HDCFDA Fluorescence (Relative fold change) UC simct # simct # P- value: * <. <. * <. <.

17 In- vivo Hemmung von MCT MCT Vinculin UC6 shcntrl UC6 shmct Tumor Volume (in mm 3 ) 3 Orthotopic Xenograft * 3 Days after injection * UC6 shcntrl (n=) UC6 shmct (n=) MCT IHC shcontrol MCT IHC shmct

18 Percent survival PrognosUsche Relevanz von Krebsspezifisches MCTs Überleben / CD7: Analyse von 6 MCT PaUenten = 3 nach Zystektomie Krebsspezifisches Überleben Log-Rank =.3 Wilcoxon =. MCT Expression Krebsspezifisches Überleben MCT < 3 Percent survival Log-Rank =.6 Wilcoxon =. MCT & CD7 Co- Expression Krebsspezifisches Überleben CD7 & MCT =3 Others Percent survival Log-Rank =.3 Wilcoxon =. Time (in months) MCT = 3 MCT < 3 Percent survival Log-Rank =.6 Wilcoxon =. Time (in months) CD7 & MCT =3 Others Time (in months) Time (in months) Gesamtüberleben Gesamtüberleben Percent survival Log-Rank =. Wilcoxon =. Gesamtüberleben MCT = 3 MCT < 3 Percent survival Log-Rank =. Wilcoxon =.9 Gesamtüberleben CD7 & MCT =3 Others urvival Time (in Log-Rank months) =. Wilcoxon =. MCT = 3 MCT < 3 urvival Log-Rank =. Time (in months) Wilcoxon =.9 CD7 & MCT =3 Others

19 PrognosUsche Relevanz nach Zystektomie (n=6) Tumorspezifisches Überleben Gesamtüberleben HR (9% CI) p-value HR (9% CI) p-value Age > vs..8 (.89-.)..6 (.-.6).3 <=Median pt > vs. <=.9 (.-3.8)..3 (.-3.86). pn vs. pn+.9 (.-3.)..93 (.-3.).6 R vs. R+.86 (.-3.3)..7 (.96-.9).6 M vs. M.3 (.86-.).9.7 (.7-3.6).8 MCT > vs. <=.67 (.-.89)..7 (.6-.86). Tumorspezifisches Überleben Gesamtüberleben HR (9% CI) p-value HR (9% CI) p-value Age > vs.. ( )..37 (.86-.7).7 <=Median pt > vs. <=.8 (.97-3.).6.9 (.7-3.6). pn vs. pn+.7 (.7-.89)..76 (.-.8). R vs. R+.73 ( ).6.6 ( ).8 M vs. M. (.8-.87).3.9 (.9-.).6 CD7 & MCT > vs. Rest.3 (.-3.3).7. (.-3.).

20 Zusammenfassung LaktaNransporter beim muskelinvasiven Blasenkarzinom überexprimiert KorrelaUon von MCT & mit basalem molekularem Subtyp Hemmung von MCT! LaktatakkumulaUon + AnsUeg von reakuven Sauerstoffspezies! Apoptose Reduziertes Tumorwachstum in vitro und in vivo durch MCT Hemmung Expression von MCT & CD7 mit schlechtem Outcome nach Zystektomie assoziiert! MCT Hemmung als potenueller Ansatzpunkt für die Behandlung des fortgeschrinenen muskelinvasiven BCs

21 Danke: Peter Black Roland Seiler Jian Gao Jörg HennenloNer Craig Stewart Alireza Kamyabi Simroop Ladar Shannon Awrey Stephen Choi Yuzhuo Wang Nader Al- Nakouzi Mads Daugaard Htoo Zarni Oo Ladan Fazli Jens Bedke Arnulf Stenzl Funding:

22 Antrag an die Notgemeinschal der Deutschen Wissenschal (9):