Apoptotic Cell Death : Opportunity of Oncological Application

Transcription

1 Session SS05 - Cell Death Mechanism Apoptotic Cell Death : Opportunity of Oncological Application Soo-Youl Kim, Ph.D. Division of Cancer Biology National Cancer Center, Korea kimsooyoul@gmail.com

2 What is Apoptosis? A Greek word meaning falling off as leaves from a tree

3 Apoptosis The term apoptosis was first used in a paper by Kerr, Wyllie, and Currie to describe a morphologically distinct form of cell death. Br J Cancer 26, , 1972 Dr. Horvitz found that programmed cell death occurs during the development of the nematode Caenorhabditis elegans (Horvitz, Cancer Res 59, 1701s 1706s,1999) 2002 Prof. H. Robert Horvitz The Nobel Prize in Physiology or Medicine In this organism 1090 somatic cells are generated in the formation of the adult worm, of which 131 of these cells undergo apoptosis or programmed cell death.

4 Morphology of Apoptosis Lymphocytes TEM A C B D (A) Large nuclei and scant cytoplasm / (B) Early during the chromatin condensation phase, arrow is a fragmented section of nucleus and the arrowhead indicates an apoptotic body/ (C) Extensive plasma membrane blebbing occurs followed by karyorrhexis and separation of cell fragments into apoptotic bodies during a process called budding / (D) These bodies are subsequently phagocytosed by macrophages, parenchymal cells, or neoplastic cells and degraded within phagolysosomes.

5 Apoptosis vs Necrosis It is mixed in the tumor tissues. Intracellular ATP Concentration: A Switch in the Decision Between Apoptosis and Necrosis J Exp Med. 185(8):1481-6, 1997

6 Apoptosis: Point of No Return to Life Mitochondria outer membrane permeabilization Most Confused Idea (1) apoptotic cells do not release their cellular constituents into the surrounding interstitial tissue; (2) they are quickly phagocytosed by surrounding cells thus likely preventing secondary necrosis; and, (3) the engulfing cells do not produce anti-inflammatory cytokines.

7 Major Pathways of Apoptosis Toxicologic Pathology, 35: , 2007 DISK: death inducing signaling complex MPT: mitochondrial perfusion transformation SET: nucleosome assembly protein

8 Intrinsic Apoptosis

9 Intrinsic Apoptosis The intrinsic signaling pathways that initiate apoptosis involve a diverse array of non receptor mediated stimuli that initiates mitochondrial events. Negative signals: the absence of certain growth factors, hormones and cytokines that can lead to failure of suppression of death programs, thereby triggering apoptosis. Positive signals: radiation, toxins, hypoxia, hyperthermia, viral infections, and free radicals.

10 Intrinsic Apoptosis mitochondrial permeability transition All of these stimuli cause changes in the inner mitochondrial membrane that results in an opening of the mitochondrial permeability transition pore, loss of the mitochondrial transmembrane potential and release of two main proapoptotic proteins including cytochrome c and Smac from the intermembrane space into the cytosol.

11 Intrinsic Apoptosis Mitochondria outer membrane permeabilization (MOMP) is considered the point of no return for apoptosis. The steps leading up to MOMP can be stopped in their tracks by inhibitor molecules, but once MOMP has been achieved, the cell will complete the death process. BAX (Green) forms ring like structure On the fragmented mitochondria

12 Intrinsic Apoptosis Cytochrome-C in the cell cytoplasm prompts the formation of the apoptosome with APAF1 that performs the final step to beginning cellular breakdown. The apoptosome turns procaspase-9 into caspase-9, which triggers a cascade of caspases activation. Inactive APAF1 Active apoptosome Cytochrome c

13 Intrinsic Pathway Proteins Cancer cells employ this molecules

14 Extrinsic Apoptosis

15 Extrinsic Apoptosis Extrinsic Pathways initiate apoptosis involved with transmembrane receptor-mediated Interactions such as FasL/FasR, TNF-α/TNFR1, Apo3L/DR3, Apo2L/DR4 and Apo2L/DR5. After signaling, a death-inducing signaling complex (DISC) is formed, resulting in the autocatalytic activation of procaspase-8. Once caspase-8 is activated, the execution phase of apoptosis is triggered.

16 cflip is an Inhibitor of Apoptosis Extrinsic Pathway Proteins Death receptor-mediated apoptosis can be inhibited by a protein called c-flip which will bind to FADD and caspase-8, rendering them ineffective.

17 TNF-Induced Apoptosis Dr. Lloyd J. Old, M.D., c TNFα, cachexin, or cachectin: discovered in 1975 Discovery of tumor necrosis factor (TNF), a key immune signaling molecule (cytokine) that, in addition to its promise for the treatment of cancer and other diseases, has provided a powerful research tool in biomedicine. Lloyd John Old

18 TNF inhibitor autoimmune and immune-mediated disorders such as rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, psoriasis, hidradenitis suppurativa and refractory asthma. The drugs inhibiting TNF include Remicade (infliximab), Enbrel (etanercept), Humira (adalimumab), Cimzia (certolizumab pegol) and Simponi (golimumab).

19 TRAIL and FAS Ligand Induced Apoptosis TNF-related apoptosis-inducing ligand (TRAIL) The FAS receptor (FasR), also known as apoptosis antigen 1 (APO-1 or APT), cluster of differentiation 95 (CD95) or tumor necrosis factor receptor superfamily member 6 (TNFRSF6)

20 Perforin/Granzyme Pathway

21 CTL Induced Apoptosis Cytotoxic T lymphocytes (CTLs) are able to kill target cells via the extrinsic pathway and the FasL/FasR interaction is the predominant method.

22 Perforin/Granzyme Pathway A novel pathway involves secretion of the transmembrane pore-forming molecule perforin with a subsequent exophytic release of cytoplasmic granules including granzymes A and B through the pore and into the target cell. Granzyme B can directly activate Caspase 3, BID, and ICAD which triggers apoptosis. Granzyme A can activates DNAse NM23-H1, which blocks the maintenance of chromatin structure integrity. ICAD: Inhibitor of Caspase Activated DNAse

23 Cancer PD-L1 Expression Avoids TCR Activated FasL/TRAIL by SHP2 T cell death Activation of cytotoxic T cells (Tc) is an antigen-specific process requiring the interaction of the TCR CD3 complex with a processed tumor antigen derived peptide bound to a MHC class I molecule. Tc activation Induces FASL and TRAIL to kill cancer cells. However, PD-1 activation by cancer PD-L1 stops Tc activation through SHP2 and triggers T cell death.

24 How to Cure Cancer with Apoptosis Inducers?

25 TNF-a Between Promotion and Suppression in Cancer Mouse bearing subcutaneous human tumour xenograft TNF treat Tnf / mouse Treatment with Coley s toxins A patient with round cell sarcoma of the jaw and abdominal metastases seen by coley in Photograph after 63 injections with coley s toxins; tumour had diminished to about half its original size. Wild-type mouse treated with the carcinogen DMBA and the tumour promoter TPA Coley s toxins : a mixture consisting of killed bacteria of species Streptococcus pyogenes and Serratia marcescens

26 Apoptosis Inducers

27 Oncogenic Apoptosis

28 Oncogenic Apoptosis Apoptosis can also cause unwanted effects that may even promote cancer. Understanding this may help maximizing anti-cancer apoptosis while minimizing pro-tumorigenic effects.

29 Enhancing Apoptosis MOMP: mitochondria outer membrane permeabilization SMAC: second mitochondria-derived activator of Caspases. XIAP: X-linked inhibitor of apoptosis protein (caspase inhibitor) CAD: caspase-activated Dnase TAM: tumour associated macrophage pink boxes: Possible enhancers of apoptosis blue boxes: inhibitors of unwanted effects Nat Rev Cancer Aug;16(8): celecoxib

30 Novel Approach to Apoptosis Induction Via p53 Stabilization

31 p53 is a Key Regulator in Cancer

32 Nature Reviews Cancer 9, (2009) Regulator of p53

33 Δ Δ p53 is the Major Target of Transglutaminase 2 TGase2 P TGase p Caki-1/p53 -/ IB: p53 p53 polymer BSA p53 monomer Coomassie p53 IP: IgG, IB: TGase2 IP: p53, IB: TGase2 INPUT: TGase2 INPUT: p Transactivation DNA binding domain Tetramerization Regulatory domain FASEB J Sep;27(9): K Q K Q

34 A New Regulator of p53 Transglutaminase 2 is Universally Increased in RCC By Microarray of TGase 2 using NCC 72 Cell Lines TGase 2 b-actin Oncogene. 30, , 2011 FASEB J Sep;27(9): RCC

35 mrna Expression (RNA Seq v2 RSEM) p53 Mutation is Only 4% in ccrcc TGase 2 is highly increased in ccrcc + high + low + high + low TGM2 (pvalue= ) p62/sqstm1 (p-value= ) TGM2 (p-value= ) Normal Tumor Instability of p53 in RCC is not associated with mutations because the COSMIC database showed only 4% mutation of p53 in clear cell RCC. Frequent mutations in clear cell renal cell carcinoma In COSMIC database Cell Death and Disease (2016) 7, e2163

36 Mechanism of TGase 2-p53 Regulation in RCC 1) Normal cells MDM 2 Proteasome degradation 2) RCC MDM 2 Nutlin-3 p53 TGase 2 KN383 Autophagy degradation p53 TGase 2 C C C N p53 N N TG2 LC3 binding p62 LC3II LC3II Phagophore p53-polymer Ca ++ Autophagosome Lysosome Autolysosome C p53 N C N N TG2 LC3 C UBA PB1 Ca ++ p62 C C CD CD p53 DBD N DBD N C TG2 Ca ++ C CD C DBD CD C DBD CD DBD Cell Death and Disease (2016) 7, e2163 FASEB J Sep;27(9): National Cancer Center, KOREA

37 GK921 CTL TGase 2 Inhibitor Reverses RCC via p53 Stabilization By collaboration with Prof. Gong Y.D H&E BrdU p53 J Cancer Res Clin Oncol. 140(5):757-67, 2014 National Cancer Center, KOREA

38 Cell Death and Disease (2016) 7, e2163 Tumor volume(mm 3 ) DNA damage + TGase 2 Inhibition = Synergy CAKI-1 Control GK921 (1 mm) Control Doxorubicin 1mg/kg 32 sitg2 Doxo (hr) TG2 p-p53(s15) GK921 2mg/kg Combination p53 p21 Doxorubicin (1 mm) GK921+Doxorubicin Bad Bax Puma β-actin P-p53 apoptosis 1000 ATM/ATR 500 DNA intercalation of Doxo Inhibits topo II p53 HDM2 TG2 GK week

39 Novel Approach to Apoptosis Induction Via ATP Depletion

40 Conventional Anti-Cancer Drug Development receptor tyrosine kinase EGFR, VEGFR, IGFR, PDGFR Rapamycin Ribavirin Pateamine A RAS Gefitinib, Erlotinib, Imatinib, Sutent Translation regulation mtor Cancer mtor produces survival signal molecules & nutrient uptake/catabolic Enzymes for proliferation Sorafenib RAF ME K ERK PI3 K AKT mto R Rapamycin, Temsirolimus Signaling Transcription regulation receptors Kinase inhibition transcription Translation Survival Proliferation Targeting Anabolism

41 ALDH Is Required for ATP Production in Cancer ALDH1L1 low Cancer Normal ALDH1L1 high (P-value = ) ALDH1L1 KN817 Oncotarget. 7: , 2016 Experimental & Molecular Medicine (2016) 48, e272 41

42 ALDH Inhibitor + Mito Complex I Inhibitor Xenograft mice model A KN ALDH1L1 low Cancer Normal ALDH1L1 high ALDH1L1 (P-value = ) ALDH1L1 Oncotarget. 7: , 2016

43 Summary : New Approach for Drug Dev Conventional anti-cancer drug development receptor tyrosine kinase EGFR, VEGFR, IGFR, PDGFR RAS Gefitinib, Erlotinib, Imatinib, Sutent ATP suppresses AMPK & activates mtor mtor Cancer mtor produces survival signal molecules & nutrient uptake/catabolic Enzymes for proliferation Sorafenib RAF MEK ERK PI3K AKT mtor Rapamycin, Temsirolimus ATP ATP Production via ETC ATP cnadh NADH transport Cytosolic NADH production increase via eg. ALDH transcription Translation Survival Proliferation Cell death Opportunity for anti-cancer drug development via regulation of oxidative cancer energy metabolism Cancer energy metabolism