CANCER AND THE BREAKDOWN OF GENE REGULATORY NETWORKS 1.GENE EXPRESSION AND TRANSCRIPTION 2.REGULATION OF GENE EXPRESSION 3.THE REGULATORY NETWORKS THAT PROTECT NORMAL CELLS AGAINST UNCONTROLLED PROLIFERATION 4.THE BREAKDOWN OF THESE NETWORKS ARE THE HALLMARKS OF CANCER OUTLINE

GENE EXPRESSION: GENE =SEGMENT OF DNA = BLUEPRINT FOR A SINGLE PROTEIN EACH CELL CONTAINS ALL GENES !!! WHEN A GENE IS EXPRESSED, THE PROTEIN IT CODES FOR IS SYNTHESIZED (AT RIBOSOMES) NOT ALL GENES ARE EXPRESSED IN ALL CELLS (OF A MULTICELLULAR ORGANISM) OR AT ALL TIMES Gene expression (EUCARYOTIC)

DIFFERENTIATION DIFFERENT CELL TYPES SYNTHESIZE DIFFERENT PROTEINS TYPICAL HUMAN CELL – ABOUT 10-20,000 EXPRESSED GENES FOR THE 2000 MOST ABUNDANT (>50,000 COPIES/CELL) FEW DIFFERENCES ARE FOUND (5 FOLD OR LESS) A FEW % ARE CELL TYPE SPECIFIC – BUT PROFILES DO DIFFER SIGNIFICANTLY A PARTICULAR CELL’S EXPRESSED GENES VARY WITH TIME IN RESPONSE TO EXTERNAL SIGNALS IN RESPONSE TO INTERNAL “CLOCKS” DEPENDING ON STATE – NORMAL, STRESSED, ABNORMAL HOW IS GENE EXPRESSION REGULATED??

TRANSCRIPTION Transcription M-RNA IS PROCESSED (SPLICED)

REGULATION AT DIFFERENT STEPS KINDS OF REGULATION mRNA IS PROCESSED (SPLICED) TRANSCRIPTIONAL CONTROL RNA PROCESSING CONTROL RNA TRANSPORT CONTROL TRANSLATIONAL CONTROL RNA DEGRADATION PROTEIN ACTIVITY PREVALENT (NO SYNTHESIS)

TRANSCRIPTION – CLOSER LOOK RNA POLYMERASE ATTACHES TO DNA, MOVES ALONG IT, OPENS DOUBLE HELIX, SYNTHESIZES mRNA. CONTROL OF EXPRESSION BY ASSISTING OR BLOCKING ATTACHMENT OF RNA POLYMERASE TO DNA. RNA POLYMERASE BINDS AT A SPECIFIC REGION, THE PROMOTER, AT THE START OF THE GENE, IF (1) AN ACTIVATOR IS ATTACHED AT AN ADJACENT SPECIFIC REGULATORY BINDING SEQUENCE (OPERATOR) AND (2) A REPRESSOR IS NOT ATTACHED TO ITS OWN OPERATOR TRANSCIPTION activator TRANSCRIPTION START

TRANSCRIPTION – CLOSER LOOK RNA POLYMERASE DOES NOT BIND AT THE PROMOTER, AT THE START OF THE GENE, IF A REPRESSOR IS ATTACHED TO IT’S OWN OPERATOR TRANSCIPTION repressor TRANSCRIPTION FACTORS

6-2,6-4

RECOGNITION OF BINDING MOTIFS IN DNA

RECOGNITION OF BINDING MOTIFS IN DNA 2 HOMEODOMAIN

TRYPTOPHAN REPRESSOR-SWITCH OPERON REGULATORY “NETWORK” Trp(P) repressor(P) Enzymes(G)

lac OPERON – E-COLI (PROCARYOTIC) OPERON – SET OF GENES PLACED ONE AFTER THE OTHER ON DNA, TAKING PART IN ONE PROCESS (BREAKDOWN OF lactose). E-COLI PREFERS glucose – WILL PROCESS lactose ONLY UNDER ( –glucose/+ lactose ) CONDITIONS. 4-SWITCH! Cyclic AMP CONCENTRATION WHEN +glucose allolactose CONCENTRATION WHEN +lactose ACTIVATOR: + (ACTIVE IF COMPLEX) --glucose/+lactose LAC OPERON KEEP! REPRESSOR: + (ACTIVE IF FREE)

9 – 21 lac 4-way switch

Decision-making by bacteria: choosing between two sugars can be tough… The first detailed map of a gene’s decision-making computation (Setty, Alon, 2003)

Eytan, the figure shows the rate of expression of the classic lactose operon of E. coli (z-axis), as a function of 100 combinations of its two regulating input signals. The first signal is IPTG which is related to the sugar lactose (y-axis). The second signal is cAMP, which is related to glucose starvation (x-axis) The operon is only expressed when there is lactose but no glucose (high IPTG And high cAMP). Previously this was thought to be a simple ‘AND gate’. We were surprised to find, using accurate GFP reporter measurments, that the gate is more intricate and has in fact four plateau levels and four thresholds, two for each signal. I think it’s the first time that an input function was mapped in such detail. We hope to use this experimental approach to map the input functions of virtually all genes In E. coli, to get a mapping of the logical decision making of the cell. (PS this is in press in PNAS- I can send you a copy if you wish)

EUCARYOTIC – MUCH MORE COMPLEX RNA POLYMERASE CANNOT INITIATE TRANSCRIPTION GENERAL TRANSCRIPTION FACTORS ASSEMBLE, FORM COMPLEX ON OPERATOR NEAR PROMOTER - ABUNDANT EUCARYOTIC 9 – 30,31 TBP – SUBUNIT OF TFIID

SPECIFIC REGULATORS OF TRANSCRIPTION (ENHANCERS) CAN ATTACH TO DNA MANY 1000 OF BP UPSTREAM, CAN EVEN BE PLACED DOWNSTREAM FROM START SITE VERY MINUTE AMOUNT PRESENT MAY NEED MORE THAN ONE TRANSCRIPTION FACTOR TO ACTIVATE GENE 9-36,9-45 control of human beta-globin proximity of GAL4 enhances 1000 fold the attachment of TFIIB to TFIID

REGULATORY NETWORKS TRANSCRIPTION FACTORS ARE PROTEINS THAT ACTIVATE OR REPRESS GENES’ TRANSCRIPTION INTO PROTEINS PROTEINS FORM COMPLEXES THAT INDUCE /TURN OFF /REGULATE A GENE’S TRANSCRIPTIONAL CAPACITY COMPLEX NETWORKS OF REGULATION OF GENE EXPRESSION EMERGE REGULATORY NETWORKS

HALLMARKS 1 CANCER IS CAUSED BY THE BREAKDOWN OF SEVERAL IMPORTANT NETWORKS,THAT GUARD AGAINST UNCONTROLLED PROLIFERATION HANAHAN & WEINBERG CELL 2000

NORMAL CELL STATES & CELL CYCLE Check Points (Internal and External signals) G1 –gap, decide whether to proliferate, wait or cross to non-dividing stage G0 S -- DNA Synthesis G2– gap, allow DNA repair M – Mitosis, cell division

NORMAL ENTRY TO/EXIT FROM CELL CYCLE Cell Division Proliferation Cell cycle arrest (G0) or Programmed Cell Death (Apoptosis) Limited replication, senescence, crisis Terminal differentiation Check Point (Internal and External signals) Growth Signals Induced Apoptosis too many divisions Anti Growth Signals

Cancer Cell-HALLMARKS Cell Division Proliferation Cell cycle arrest or Programmed Cell Death (Apoptosis) Limited replication, senescence, crisis Terminal differentiation Defective computation at check points, or failure to interpret signals or execute instructions: 1 Proliferation becomes independent of growth factors. 2 Loosing responses to cell cycle inhibitory signals. 3 Failure to apoptose when necessary. 4 Immortalization. series of random genetic accidents These are the main 4 HALLMARKS OF CANCER.

1. SELF SUFFICIENCY IN GROWTH SIGNALS IN NORMAL CELLS, GROWTH FACTORS ARE RELEASED BY NEIGHBOR CELLS, BOUND BY GF RECEPTORS (USUALLY IN CELL MEMBRANE), WHICH GET MODIFIED AND INITIATE A CASCADE OF SIGNALING EVENTS. 1.Autonomous generation of growth factors 2.Receptor overexpression or alteration 3.Defective downstream processing MUTANT Ras SEND DOWNSTREAM GROWTH SIGNALS WITH NO STIMULUS FROM UPSTREAM What can go wrong?

2. IGNORING ANTI-GROWTH SIGNALS IN NORMAL CELLS, MOST ANTIGROWTH SIGNALS DIRECTING THE CELL TO G0 ARE CHANNELED THROUGH THE Rb (RETINOBLASTOMA) PROTEIN. TERMINAL DIFFERENTIATION IS INDUCED BY FORMATION OF Myc-Max COMPLEX.

3. EVADING APOPTOSIS THE APOPTOTIC MACHINERY RELIES ON SENSORS (THAT DETECT INTERNAL AND EXTERNAL SIGNALS) AND EFFECTORS, THAT INDUCE AND CARRY OUT THE DEATH SENTENCE. p53 IS A CENTRAL PLAYER IN APOPTOSIS.

4. IMMORTALIZATION A CELL CAN UNDERGO A LIMITED NUMBER OF DIVISIONS. THE “COUNTING DEVICE” IS A STRING OF SEVERAL 1000 REPEATS OF A 6-BP SEQUENCE ELEMENT AT THE END OF THE CHROMOSOMES (TELOMERS). IN EACH DIVISION 50 – 100 TELOMERIC BP ARE LOST. WHEN THEY RUN OUT, THE CHROMOSOME ENDS ARE UNPROTECTED AND FUSE, LEADING TO CRISIS AND DEATH OF THE CELL. CANCER CELLS ACQUIRE TELOMERE MAINTENANCE

Cancer Cell-HALLMARKS Cell Division Proliferation Cell cycle arrest or Programmed Cell Death (Apoptosis) Limited replication, senescence, crisis Terminal differentiation Defective computation at check points, or failure to interpret signals or execute instructions: 1 Proliferation becomes independent of growth factors. 2 Loosing responses to cell cycle inhibitory signals. 3 Failure to apoptose when necessary. 4 Immortalization. series of random genetic accidents These are the main 4 HALLMARKS OF CANCER.

TWO MORE: 5. Formation of new blood vessels in the tumor (Sustained Angiogenesis) (NEEDED TO ACHIEVE LARGE SIZE) 6. Acquirement of metastatic behavior (cancer cells spread to vital organs) (CAUSE OF 90% OF CANCER RELATED DEATHS)

Cancer results from anomalies in genes regulating cell growth: mutations, translocations Two classes of genes are involved: 1) - positive regulators promote cancer by hyperactivity (one allele is enough) 2) - negative regulators, promote cancer by loss of activity (both alleles must be mutated) Oncogenes Tumor Suppressor genes

AND/OR GENOMIC INSTABILITY: SCIENCE 2002

GENES ONCOGENES = GAS PEDAL TUMOR SUPPRESSORS = BRAKE PEDAL GENOME INTEGRITY GENES = MECHANIC

AIMS: UNDERSTAND THE GENETIC/MOLECULAR MECHANISMS THAT CAUSE CANCER. WHICH PATHWAYS BREAK DOWN IN VARIOUS CANCERS, AND HOW? IDENTIFY SUB-CLASSES OF EACH DISEASE. CLINICAL RELEVANCE: PERSONALIZED PREDICTIVE PREVENTIVE MEDICINE: PREDICT OUTCOME, DESIGN SUITABLE THERAPY DRUG DISCOVERY

MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia Armstrong et al, Nature Genetics 30, (2002)

PCA Leukemia 2 Korsmeyer ALL,AML,MLL 500 most separating genes

CANAANI GLEEVEC IN CML: