LCCC Brown Bag Seminar 2009

Apoptosis, Cell Survival, and Cancer

Rebecca Riggins202.687.7451

rbr7@georgetown.edu

Overview

• What is apoptosis, and why is it important?

• What genes/pathways are involved?• How do we measure it?• How do cancer cells combat it?• How do we manipulate apoptosis and

cell survival in the treatment of cancer?• What are some alternatives to

apoptosis?

Definition of Apoptosis

• Also called “programmed cell death”• A specific program of events leading to

the death of a cell• Program is active in organisms from

yeast to humans• Important in removing cells that are

infected, growing inappropriately or otherwise damaged

• Essential in development

Apoptosis in Development

• Can be visible at the macroscopic and microscopic levels

http://transdeath.uni-graz.at/

PlantsFilamentousFungi

Dictyostelium Caenorhabditiselegans

Apoptosis in the Worm

• C. elegans is the best-studied model of developmental apoptosis

Lettre et al. Nature Reviews Molecular Cell Biology 7, 97–108 (February 2006) | doi:10.1038/nrm1836

Apoptosis in the Mammary Gland

• Cycle of cell growth and apoptosis during pregnancy and lactation

Ductal/Lobulo-Alveolar differentiation during four mammary gland stages

http://mammary.nih.gov/

Cellular Events in Apoptosis• Historically, apoptosis is identified by

morphology (appearance)• Chromatin condensation (pyknosis)• Nuclear fragmentation due to endonuclease

cleavage of DNA (karyorhexis)• Cell shrinkage• Ruffling of the plasma membrane• Key feature: cell membranes do not rupture…

they remain intact

←pyknosis

←karyorhexis

Wojcik, C. et al, Apoptosis. 1997;2(5):455-462.

Molecular Events in Apoptosis

• There are 2 major signal transduction pathways that control apoptosis

• Extrinsic pathway• Intrinsic pathway

Extrinsic = Outside the Cell

• Death Receptor Pathway

• Signals are received by receptors on the cell surface

• Other proteins are recruited to the cytoplasmic tails of these receptors

• These proteins activate the caspase enzyme family

Ashkenazi and Dixit, Science. 1998 Aug 28;281(5381):1305-1308.

PlasmaMembrane

Intrinsic = Inside the Cell

• Mitochondrial or Cell Stress Pathway

• Responds to DNA damage, other internal signals

• Mitochondrial membrane becomes permeable

• Cytochrome c is released, leading to activation of caspases

http://www.scq.ubc.ca/wp-content/uploads/2006/07/apoptosispathways.gif

Cross-Talk between the Pathways

Shajahan AN, Riggins RB, and Clarke R., 2008

Caspases

• A family of cysteine-dependent, aspartate-specific proteases

• Caspases hydrolyze peptide bonds on the carboxyl side of an aspartate residue – Eg.) DEVD↓….

Alnemri et al, Cell. 1996 Oct 18;87(2):171

Caspase Cooperation

Initiator Caspases

Effector CaspasesShajahan AN, Riggins RB, and Clarke R., 2008

What are the substrates of Effector Caspases?

• Cytoplasmic, structural proteins (actin)• Nuclear, structural proteins (lamin)• DNA repair proteins (PARP)• Kinases, other signaling molecules• Cell cycle proteins

Apoptotic Control of Cell Cycle Progression

http://www.cancerline.com/gUserFiles/stages_of_cell_cycle.gif

Apoptosis

Apoptosis in the Lab

• Visualization of DNA fragments by gel electrophoresis

Induce apoptosis

Isolate DNA and load onto agarose gel

Wojcik, C. et al, Apoptosis. 1997;2(5):455-462.

DNA Fragmentation, cont’d.

• Propidium iodide (P.I.) staining and fluorescence activated cell sorting (FACS)

• PI intercalates between DNA molecules, 1 dye per 4-5 base pairs

Induce apoptosis

Remove cells fromdish, stain with PI

MeasureFluorescence

Ex. 535 nm

Em. 617 nm

DNA Fragmentation Results

S

M

G1G2

control

0.95%

100 nM paclitaxel

7.5%

G1S

G2/M

- where a cell is in the cell cycle determines how much DNA is present

Cel

l C

ou

nt

subG1 peak

More Apoptosis in the Lab

Induce apoptosis

Isolate protein andload onto poly-acrylamide gel

• Western blot analysis…use of specific antibodies to detect full-length and cleaved caspase substrates

MW - +

Apoptosis

Apoptosis in Cancer: What goes wrong?

• Cancer cells live because they “forget” how to die

• This can happen:– When key genes that induce apoptosis get

deleted, or turned off– When genes and pathways that induce cell

growth become amplified, or turned on

• Cell survival signaling directly counter-acts apoptotic signaling

Cancer’s “Drive to Survive”o growth factors and their receptors o all receptors are transmembrane proteinso 3 major features: o extracellular domain (ectodomain)o transmembrane regiono intracellular domaino where, when, and how they are expressed determines their biological function

Figure 5.10 The Biology of Cancer (© Garland Science 2007)

ErbB/HER Family

Epidermal Growth Factor Receptor (EGFR)

Seminars in Cancer Biology, Volume 14, Issue 4, August 2004, Pages 262-270

Growth Factor Receptor Action

Figure 5.15 The Biology of Cancer (© Garland Science 2007)

Cell Cycle Progression SURVIVAL

RASP

RTK

PGrb2SOS

PAkt

PI3K

p85

p110

RAS

Raf

MEK

P

P

ERK

GROWTH FACTOR

PIP3 PIP3

PDK1

PROLIFERATION

MDM2BAD

P

NF-ĸB

P

FKHR

P

CELL SURVIVAL

p70S6K

P

GSK3

P P

PROTEIN SYNTHESIS

Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).

Growth Factor Signaling is Complex!

Incidence of RAS mutations in human cancer

Lung (30% ki-ras)Breast

Brain

Colon (50% Ki-ras)

OvaryProstate

Pancreas (90% Ki-ras)

Bladder (10% Ha-ras)

Thyroid (50% ras)

Liver (30% N-ras)

Skin (14% N-ras)

Head & Neck

Leukemia (30% N-ras)

Kidney (10% Ha-ras)

A. Chan, Ras-MAPK Pathways. Sci. STKE 2005, tr5 (2005).

A. Chan, Ras-MAPK Pathways. Sci. STKE 2005, tr5 (2005).

Lung carcinoma K-Ras codon 12 (GGTgly) >AGTser

Pancreatic Carcinoma K-Ras codon 12 (GGTgly) >GTTval

Bladder Carcinoma H-Ras codon 12 (GGCgly) >GTCval

Melanoma N-Ras codon 61 (CAAgln)>CGAarg

Ras Mutations display Tumor Specificity

Apoptosis and Cell Survival in Cancer Therapy

• How do we stop inappropriate cell growth and survival, or re-start the process of apoptosis?

Drug Primary Target Mechanism(s) of Cell Death

Anthracyclines

Doxorubicin, Epirubicin DNA intercalation, topoisomerase II

BCL2 family regulation, NFκB inhibition, p53 activation

Alkylating Agents

Cisplatin, Cyclophosphamide

DNA crosslinking caspase activation, p53 activation, cytochrome c release

Antimetabolites

5-fluorouracil, Capecitabine

thymidylate synthase p53 activation, thymineless death

Microtubule Inhibitors

Docetaxel, Paclitaxel microtubule stabilization caspase activation, phosphorylation of BCL2 and BCL-X, JNK activation, CD95/FAS expression, autophagy

Vinblastine, Vincristine microtubule dissolution p53 activation, post-translational modification of BCL2 family members

Apoptosis and Cell Survival in Cancer Therapy

Shajahan AN, Riggins RB, and Clarke R., 2008

Drug Primary Target Mechanism(s) of Cell Death

Signal Transduction Inhibitors

Gefitinib EGFR kinase activity phosphorylation of BAD, downregulation of BCL2

Trastuzumab, CH401 HER2 extracellular domain inhibition of PI3K/Akt, phosphorylation of BAD, JNK activation

TRAIL TRAIL death receptor Death receptor/caspase 8 activation

Genasense BCL2 downregulation of BCL2

ABT-737, others BCL2 and BCL-X prevention of BCL2 and BCL-X interaction with pro-apoptotic BAX and BAK

Apoptosis and Cell Survival in Cancer Therapy

Shajahan AN, Riggins RB, and Clarke R., 2008

Some Final Thoughts• Is there more than apoptosis?

– Senescence– Mitotic Catastrophe– Necrosis– Autophagy

SENESCENCE• As cells divide, telomeres atchromosome ends become shorter• At some point, cells are unableto re-enter the cell cycle

MITOTIC CATASTROPHE• Cells die during mitosis (M

phase)• Caused by improper

chromosome segregation

NECROSIS• Chaotic death process in which

plasma membrane ruptures• Release of intracellular

contents leads to inflammation• Mechanism(s) still poorly

understood

Autophagy

• Autophagy, autophagocytosis = “self eating”

• Degradation of cellular components in a sequestering vacuole (autophagosome)

• This merges with the lysosome, where acidic hydrolases degrade its contents

Rubinsztein, Nat Rev Drug Disc. 2007 Apr; 6:304-312.

Autophagy vs. Apoptosis

Shajahan AN, Riggins RB, and Clarke R., 2008

Rubinsztein, Nat Rev Drug Disc. 2007 Apr; 6:304-312.

Mood stabilizer

Anti-malarial

Anti-estrogen

Regulating Autophagy Pathways

• If you would like to download this Powerpoint file for your reference, please visit:

http://openwetware.org/wiki/Riggins_Lab

• Under Resources, click Lectures then Summer Brown Bag

Thank You!