2. Introduction to Cancer Apoptosis, a Developmental and Defense MechanismThe Structure and Function of IAP ProteinsSurvivin, a Crucial IAP Target in Cancer TherapyConclusion

3.  Normally cells utilize different death mechanisms, e.g.: Apoptosis, Autophagy, Necrosis to maintain homeostasis and physiological integrity of the organism, Cancer is typically a consequence of  Imbalance between cell death and proliferation in a way favorable to cell proliferation and survival

4. APOPTOSIS A pathway of cell death induced by a tightly regulated suicidal program, in which the cells destined to die activate enzymes that degrade cells own nuclear DNA and nuclear, cytoplasmic proteins. 4

5. Why should a cell commit suicide? 1. Programmed cell death is as needed for proper normal development as mitosis is. Examples: o The resorption of the tadpole tail in frog. o The formation of the fingers and toes of the fetus requires the removal, by apoptosis. o The sloughing off of the endometrium at the start of menstruation. o The formation of the proper connections (synapses) between neurons in the brain.

6. 2. Programmed cell death is needed to destroy cells that represent a threat to the integrity of the organism. Examples: o Cells infected with viruses o Cells of the immune system o Cells with DNA damage

8. Intrinsic pathway (damage): Mitochondria Cytochrome c release Pro-caspase 9 cleavage Pro-execution caspase (3) cleavage Caspase (3) cleavage of cellular proteins, nuclease activation, etc. Death BAX BAK BOK BCL-Xs BAD BID B IK BIM NIP3 BNIP3 BCL-2 BCL-XL BCL-W MCL1 BFL1 DIVA NR-13 Several viral proteins

9. Intracellular signals Oxidative damage from free radicals, Radiation, Virus infection, Nutrient deprivation, Pro-apoptotic Factors Damage to the mitochondrial membrane increasing permeability Entry of Cytochrome C into the cytoplasm Cytochrome C binds to Apaf-1 forming an apoptosome Apoptosome activates procaspase-9 to caspase-9 Caspase-9 cleaves and activates caspase-3 and caspase-7. This executioner caspases activate a cascade of proteolytic activity that leads to: Chromatin condensation, DNA fragmentation, Protein cleavage, Membrane permeability

10. Proapoptotic and anti apoptotic proteins containing BHdomains Balance shift from critical level in favor of proapoptotic proteins PROTEINS BIND TO MITOCHONDRIAL MEMBRANE AND DECREASE INTEGRITY MEMBRANE DEPOLARIZATION AND RELEASE OF MITOCHONDRAIL CONTENTS INTO CYTOPLASM

11.  The inhibitor apoptotic proteins family contain one to three baculovirus IAP repeat or BIR domains  cellular IAPs also possess new gene (RING) domain at their C- termini, and some also contain a caspase recruitment domain or CARD  X-IAPS attach the caspases and inhibit their function (controversial)

12.  142 amino acid residues  smallest IAP, with the unique characteristic of having a single BIR domain  Survivin levels and localization can be regulated by changes in transcription, physical association with chaperones, altering proteosomal degradation, and by other post-translational mechanisms such as phosphorylation and acetylation of key amino acid residues.

13.  Expressed from the fetal stage instead of adulthood  Plays role in normal cells (t-cells,hoemopoetic cells, vascular endothelial cells and Hepatic cells etc)  Most of the cancers have been found Survivin positive

14.  Nodal proteins(various cellular actions)  Mitotic regulator for many divisions  Chromosomal passenger complex and localize mitotic apparatus  INCEP+BOREALIN +Survivin=localization of enzymes=alignment+cell formation in mitosis

15.  DNA damage-induced activation of the checkpoint kinase 2 (CHK2) results in rapid release of Survivin from the mitochondria and consequently inhibition of cell death  DNA damage stimuli also stabilize p53, which in turn can repress the transcription of Survivin and help balance the degree to which activation of CHK2 promotes Survivin release and activation of caspases

17.  Survivin suppresses radiation-induced cytotoxicity  Survivin mRNA expression and radio sensitivity are inversely related  inverse relationship between Survivin expression and Radio sensitivity observed in colorectal cancer, glioblastoma, and melanoma, pancreatic cancer  Survivin inhibition= enhanced radiation and chemotherapy

18.  expression of Survivin is substantially different in tumor cells compared to normal cells  Survivin has emerged as a potential early predictor of malignancies.  Survivin’s over-expression at the mRNA and proteins levels appears to correspond with higher malignant grades and reduced survival rates in different cancers  Studies suggest that the survival rate for Survivin negative patients is much more than Survivin negative patients  nuclear Survivin localization correlated with significantly lower survival rate than that of patients with low nuclear Survivin levels(acts as a biomarker)

19.  Survivin is not an enzyme nor it is a cell surface protein( so targeting it can be difficult studies are still being conducted)  Properties of drugs used: 1. function at the transcription level and inhibit the transcription of Survivin 2. inhibit Survivin at post-translational level include 3. vaccines that are based on cytotoxic activities of CD8+ T lymphocytes against specific Survivin epitopes  gene therapy methods including transfecting with dominant negative mutants

21.  Antisense oligonucleotides (AO) are short, single stranded RNA or DNA sequences that are complimentary to a specific RNA  It acts by hybridization to the target mRNA strand to suppress the expression of the particular gene  targeting Survivin triggered apoptosis in human melanoma cell lines hence leading to increased apoptosis

22.  Ribozymes are small RNA molecules that cleave target RNA by their endonucleolytic activity  transfection of human melanoma cell lines with ribozymes led to reduced expression of Survivin protein  SiRNAs are often more effective than other antisense approaches, rendering them better candidates for  clinical treatments Small interfering RNAs (siRNAs) are short, double-stranded RNAs that inhibit gene expression. triggered apoptosis  because lower concentrations of them is needed for each treatment resulting in less side effects compared to other techniques

24.  Delayed mitosis, misaligned chromosomes  decreased Survivin expression, enhanced cell death, reduced cell proliferation and inhibited tumor Growth  sensitivity of cancer cell lines to multiple treatments such as vincristine

25. Therapies:  HSP90inhibitors:interaction of Survivin with the molecular chaperone Hsp90 stabilizes the Survivin protein Shepherdin is a cell-permeable antagonist of the Hsp90-Survivin complex and acts through counteracting the binding of these two proteins  Cyclin-Dependent Kinase (CDK) Inhibitors:CDK inhibitors can counteract mitotic phosphorylation of Survivin on Thr34 and thereby lead to the destruction of Survivin and its function.it leads to increased apoptotic activities through different pathways.

26.  Promoter Inhibitors: imidazolium-based compound is the most important in this group of low molecular weight antagonists.  YM155 specifically inhibits Survivin gene expression by suppressing promoter activity and subsequent gene expression in cervical, colon, lung, and other cancer cells.  other low molecular weight antagonist of Survivin is terameprocol

27.  The recognition of specific tumor-associated antigens by T lymphocytes forms the basis for cancer immunotherapy.  These vaccines are able to induce tumor suppression in multiple cancers(first tested on animals)

28. 1. Dominant negative mutants:  one of the earliest strategies used to inhibit Survivin activity  studies reported that transfection with dominant-negative mutants of Survivin led to suppression of growth and increased apoptosis in gastric cancer cell lines and some other types of cancers in animal models. also increased sensitivity to other treatments.

29.  “genome editing” techniques, which are based on site-specific- directed nucleases, has raised enhanced feasibility of targeting the main causes of many diseases. The potential for therapeutic application of Zinc-finger nuclease (ZFNs),  transcription activator-like effector nucleases (TALENs) or clustered regulatory interspaced short  palindromic repeat (CRISPR/Cas-based) RNA-guided DNA endonucleases has enabled a paradigm-shift compared to conventional gene therapy methods

30.  Domains of the tools: 1. a designable and sequence-specific DNA-binding domain 2. a nuclease domain, which modifies the specific sequence of interest by leaving double-strand breaks behind

31. The differential expression of Survivin in malignant versus normal cells is a strong rationale for development of Survivin-based cancer therapeutics. Tumor-specific promoters like that of Survivin, may allow future studies to potentially direct the expression of other relevant apoptosis-inducing genes or apoptosis-inhibiting mutants in tumors. Approaches support the idea that the Survivin gene and promoter might have continued utility for developing novel agents to target and attack tumors of various types

32. REVIEW :Survivin as a Preferential Target for Cancer Therapy Mahsa Mobahat, Aru Narendran and Karl Riabowol Int. J. Mol. Sci. 2014, 15, 2494-2516; doi:10.3390/ijms15022494