Biology of Renal Cell Cancer. Disclosures

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1 Biology of Renal Cell Cancer Dr Joseph Ischia Uro-oncology Fellow No patents No honorariums Not on any boards Disclosures Essendon supporter 1

2 Renal Cell Cancer 2% of new cancers 30% have metastatic incurable disease at presentation No effective radio- or chemotherapy Need for a greater understanding of growth pathways and novel targeted therapies Clear cell 75% Papillary 15% Renal Cell Cancer Chromophobe 5% Oncytoma 5% Others rare (Duct of Bellini) 2

3 VHL and RCC VHL is a tumour suppressor gene Loss of function disease = autosomal recessive 50% of sporadic clear cell RCCs do not produce pvhl Deletion Mutation Hypermethylation No demonstrable VHL mutation??non-allelic mutations that affect VHL function HIF1α pathway- normal Normoxia -OH P HIF1α -OH Hypoxia VHLp Fe 2+ PHD PHD Fe 2+ VHLp P -OH HIF1α VHLp P HIF1α VEGF PDGF Proteasomal degradation of HIF1a Ub Ub Ub Ub P HIF1α HIF1β HIF1β TGFα HRE HIF1 target genes Cell nucleus 3

4 HIF1α pathway- mutated Normoxia -OH P HIF1α PHD Mutated VHLp P -OH HIF1α Fe 2+ Aberrant HIF1α activity P -OH HIF1α VEGF PDGF TGFα P -OH HIF1β HIF1α HIF1β HRE HIF1 target genes Cell nucleus Von-Hippel Lindau disease Hereditary cancer syndrome Urological Renal cysts Clear cell RCC Phaechromocytoma Non-urological Retinal and CNS haemangioblastomas Genetic mutation of one VHL gene on chromosome 3 VHL +/- Inherited or sporadic mutation Tumour develops with sporadic mutation of the remaining allele (now VHL -/-) 4

5 VHL and RCC >90% are due to 3p loss rather than mutations-?due to Loss of other tumour suppressor genes on 3p (eg PBRM1), or Technical failure to detect mutations VHL and RCC VHL inactivation alone is not sufficient to cause RCC E.g. VHL patients have hundreds of preneoplastic lesions (VHL -/-) but few RCCs Must be further chromosomal changes Eg loss of 14q and gain of 5q 5

6 HIFα There are three HIFα HIF1α- ubiquitous HIF2α- more restricted but most important HIF3α- unknown role HIF2α- preclinical data pvhl knockout mice- all activity through HIF2α Overexpression of HIF2α (not HIF1α) can overide pvhl HIF2α (not HIF1α) knockout stops tumour production in VHL -/- cells Axitinib Pazopanib HIF1α pathway 6

7 pvhl affected pathways HIFα VEGF, PDGF EGFR/TGFα Nuclear factor-κβ WNT c-met cyclin D IGF2, IGF1R Stromal Cell Derived Factor and C-X-C Chemokine Receptor Type 4 IL-6 RTK-Like Orphan Receptor 2 Aurora Kinase A Angiopoietin 4 Lactate Dehydrogenase A and Carbonic Anhydrase Why not HIFα antagonists? 7

8 Why not HIFα antagonists? Very poor activity of small molecule inhibitors of DNA-binding transcription factors (except steroid receptors) mtor inhibitors (temsirolimus, everolimus) Inhibit transcription and translation of HIF1α Mostly in inhibit mtor when it is in multiprotein complex TORC1, not TORC2 (which affects HIF2α) Summary of First-Line Phase III Data! Drug! Control! Study Design! Bevacizumab + IFN-α 1! Sunitinib 2,3,6! Pazopanib 6,7! Sorafenib 5! IFN-α! IFN-α! Placebo! Placebo! Temsirolimus 4! IFN-α! *Independent review. Investigator. Log-rank. Randomized 1:1, patients previously untreated (AVOREN/CALGB)! Randomized 1:1, patients previously untreated! Randomized 2:1, patients previously untreated or 1 prior cytokine! Randomized 1:1, patients previously treated with IL-2 or IFN! Randomized 1:1:1,! patients previously untreated who have poor prognosis! ORR! (%)! 31/26 vs 13! 39 vs 8*! 47 vs 12! 30 vs 3! 10 vs 2! PFS (months)! 10.2 vs 5.4!! (HR=0.63, P<0.0001)! 11 vs 5!! (HR=0.42; P<0.001)! 9.2 vs 4.2!! (HR=0.46; P<0.001)! 5.5 vs 2.8!! (HR=0.44; P<0.01)! 8.6 vs 4.8! 5.5 vs 3.1! OS (months)! 23 vs 21!! (HR=0.86, P=0.1291)! 26 vs 22!! (HR=0.82; P=0.051)! 23 vs 20.5!! HR=0.91 (p=. 224)! 19.3 vs 15.9!! (HR=0.77; P=0.02)! 10.9 vs 7.3!! (HR=0.73; P=0.008)! 1. Escudier. Lancet. 2007;370: Motzer. N Engl J Med. 2007;356: Figlin. J Clin Oncol. 2008;26 (suppl; abstr 5024). Hudes. N Engl J Med. 2007;356: Escudier. N Engl J Med. 2007;356: Prescribing Information Votrient (pazopanib) Sternberg. ASCO (abstr 5021). Rini- Cleveland Clinic 8

9 Tyrosine kinase receptors VEGF, PDGF TKIs- partially effective Use of VHL gene as biomarker is controversial Technical difficulties with identifying VHL mutations VHL +/+ tumors that are phenotypically pvhldefective Increasing side effects with increased TKR inhibition- may limit effectiveness Resistance to targeted therapies Rani, Atkins Lancet Oncology

10 Growth factor receptors in cancer VEGF PDGF EGF TGFα GRP Endothelin Chemokines Hormones cgmp/camp Tyrosine Kinase G-coupled protein Other Tumour growth Angiogenesis Metastasis Growth factor receptors in cancer VEGF PDGF EGF TGFα Tyrosine Kinase Inhibitors Tyrosine Kinase GRP Endothelin Chemokines G-coupled protein Hormones cgmp/camp Other Tumour growth Angiogenesis Metastasis 10

11 What is Gastrin-Releasing Peptide? 10 amino acid regulatory peptide Role in Metabolism Behaviour / itch sensation Cancer: renal, small cell lung, colon, pancreas, breast, prostate Angiogenesis Gastrin-Releasing Peptide (GRP) processing progrp Proteolytic cleavage GRP progrp G-protein coupled receptor Unknown receptor 11

12 GRP intracellular signalling HB-EGF GRP signalling EGFR ADAM Extracellular PLC PIP 2 G q DAG G 12 Src Rho Intracellular PI3K PKD PKC ROK PDK1 Akt mtor Raf MEK Focal adhesions PLA 2 Ins(1,4,5)P 3 p70 S6K p90 RSK ERK1/2 p42/p44 FAK Paxillin CAS Arichidonic acid release Ca 2+ mobilisation G 0 Cell nucleus S transition PGE 2 camp GRP intracellular signalling HB-EGF GRP signalling EGFR ADAM Extracellular PLC PIP 2 G q DAG G 12 Src Rho Intracellular PI3K PKD PKC ROK PDK1 Akt mtor Raf MEK Focal adhesions PLA 2 Ins(1,4,5)P 3 p70 S6K p90 RSK ERK1/2 p42/p44 FAK Paxillin CAS Arichidonic acid release Ca 2+ mobilisation G 0 Cell nucleus S transition PGE 2 camp 12

13 18 27 GRP processing GRP progrp GRP processing GRP progrp

14 18 27 GRP processing GRP progrp Initial PhD plan AIM Cure cancer PLAN

15 AIM Cure cancer Initial PhD plan PLAN 1. Start PhD AIM Cure cancer Initial PhD plan PLAN 1. Start PhD Cure cancer 15

16 AIM Cure cancer Initial PhD plan PLAN 1. Start PhD 2. Needed time to think 3. Cure cancer Aims 1. To study the expression of GRP, progrp and their receptors in normal kidney, RCC cell lines and resected human RCCs 2. To determine whether GRP and progrp are biologically active in RCC cell lines in vitro. 3. To test an RCC cell line in a xenograft mouse model to investigate the effects of GRP and progrp in vivo. 16

17 Expression of GRP and progrp in RCC cell lines RIA directed against 1. progrp carboxyl terminal of amidated GRP progrp Expression of GRP and progrp in RCC cell lines Origin RIA (fmol/10 6 cells) GRP18-27amide GRP1-10 ACHN Not detected 99 Caki-1 Not detected 170 Caki-2 Not detected

18 A C B Variable GRP expression in renal cell cancers A-C: clear cell A C B D Variable GRP expression in renal cell cancers A-C: clear cell D: papillary 18

19 Expression of GRP and progrp in RCC cell lines RIA directed against 1. progrp carboxyl terminal of amidated GRP progrp Expression of GRP and progrp in RCC cell lines RIA directed against 1. progrp carboxyl terminal of amidated GRP progrp E M E

20 Sandwich ELISA technique B B B B 1. Monoclonal Ab coated well 2. Sample addedantigen binds to Ab 3. Biotinylated secondary Ab, linked to streptavidin- HRP enzyme, binds to immobilized Ag 4. Substrate is added and converted to coloured product by enzyme Expression of GRP and progrp in RCC cell lines RIA directed against 1. progrp carboxyl terminal of amidated GRP progrp

21 Standard curves for progrp ELISA OD fmol/well Expression of GRP and progrp in RCC cell lines Origin RIA (fmol/10 6 cells) progrp ELISA (fmol/10 6 cells) GRP18-27amide GRP ACHN Not detected ± 8 83 ± ± 182 Caki-1 Not detected ± 2 Caki-2 Not detected ± ± ±

22 Expression of progrp in normal kidney and renal cancers ProGRP48-88 (fmol/g tissue) Kidney Normal kidney Matched renal cancer ND Expression of receptors for GRP and progrp 22

23 Renal cancer and BALB3T3 cell lines express binding sites for GRP Specifically bound 125 I-[Tyr4]bombesin (cpm) ACHN Caki-2 SK-01 SKRC-09 SKRC-17 SKRC-52 BALB3T3+hGRP-R Specific binding of 125 I-[Tyr 4 ]bombesin (10 3 cpm/pmol) to 4x10 4 renal cancer cells Renal cancer and BALB3T3 cell lines express binding sites for progrp Specifically bound 125 I-proGRP (cpm) I-proGRP I-[Tyr79]proGRP ACHN SK-01 SKRC-09 SKRC-17 SKRC-52 BALB3T3+hGRP-R BALB3T3 WT 23

24 A C B D Variable GRP- R expression in renal cell cancers A-D: clear cell Biological Activity 24

25 Proliferation by GRP in RCC cell lines ACHN Caki-1 Caki-2 Proliferation (% control) * * Proliferation (% control) * Proliferation (% control) * * 0 Control 1nM GRP 10nM GRP 100nM GRP 100nM GRP and 50nM GRP-R Antag 50nM GRP-R Antagonist 0 Control 10nM GRP 100nM GRP 100nM GRP and 50nM GRP-R Antag 50nM GRP-R Antagonist 10% FBS 0 Control 10nM GRP 100nM GRP 10nM GRP and 50nM GRP-R Antag 50nM GRP-R Antagonist * P<0.05 versus control Proliferation (% control) ProGRP fragments stimulate proliferation in ACHN cell line Control * 100nM progrp47-68 * 100nM progrp80-97 ACHN Caki-1 Caki-2 25

26 Boyden chamber migration assay Migration (% control) ** ** ACHN Caki-2 0 Control 100nM GRP 100nM progrp47-68 MAPK kinase activation by GRP in renal cancer cell lines phospho44 mapk phospho42 mapk total44 mapk Time (minutes) total42 mapk Average band density (% untreated cells) * * * ACHN Caki-2 GRP stimulates MAPK maximally at 5 mins 0 0 mins 2 mins 5 mins 10 mins 20 mins 60 mins * P<0.05 versus untreated cells 26

27 MAPK kinase activation by progrp in ACHN renal cancer cell line phospho44 mapk phospho42 mapk Time (minutes) total44 mapk total42 mapk Average band density (% untreated cells) * progrp stimulates MAPK maximally at 60+ mins 0 0 mins 2 mins 5 mins 10 mins 20 mins 60 mins * P<0.05 versus untreated cells MAPK phosphorylation Duration and intensity of response both important factors for coordinating cell proliferation, differentiation and motility* *Roux, P. P. and J. Blenis (2004). "ERK and p38 MAPK-Activated Protein Kinases: a Family of Protein Kinases with Diverse Biological Functions." Microbiol. Mol. Biol. Rev. 68(2): *Pouyssegur, J. and P. Lenormand (2003). "Fidelity and spatio-temporal control in MAP kinase (ERKs) signalling." Eur J Biochem 270(16):

28 Branching morphogenesis Begins mouse embryonic day 10 Corresponds to first appearance of GRP mrna Could GRP be involved? Branching morphogenesis 28

29 Branching morphogenesis No difference in number of branch points or tips for GRP, progrp47-68, GRP-R antagonist vs control Xenograft experiment Human renal cancer cell line: ACHN Xenografts- 6 week old SCID mice 5 treatment groups for 6 weeks Group 1 GRP Group 2 progrp47-68 Group 3 GRP-R antagonist Group 4 progrp47-68 and GRP-R antagonist Group 5 Control 20ug/day 20ug/day 20ug/day 20ug/day and 20ug/day Vehicle only 29

30 Xenograft tumour growth Tumour necrosis Gastrin-releasing peptide Control 30

31 Tumour necrosis Gastrin-releasing peptide Control Tumour necrosis Gastrin-releasing peptide Control 31

32 Tumour necrosis (%) Tumour necrosis * 0 Control GRP progrp47-68 GRP-R Antagonist progrp GRP-R Antag GRP group had increased viable tumour (p<0.05) Proliferative index Ki-67 Neoangiogenesis CD34 B There was no statistical difference between groups for Ki-67 or CD34 32

33 Summary A greater understanding of the the biology of RCC is driving new research and treatments GRP is an alternative growth pathway in RCC but comparatively far less significant than tyrosine kinases 33