Not division…But growth. Cells must grow PI3K GFR Akt Rheb Ras mTOR Tuberin/ Hamartin PTEN...

Not division… But growth

Cells must growCells must grow

PI3K

GFR

Akt

Rheb

Ras

mTOR

Tuberin/Hamartin

PTEN

Neurofibromin

Tuberous sclerosis complex

Neurofibromatosis type 1 (NF1)

Cowden Syndrome Lhermitte-Duclos

disease

S6KeIF4E

NPMARF p68

Thinking about translationThinking about translation

The forgotten translation…top The forgotten translation…top mmRNARNAss

Terminal Oligo Pyrimidine tracts: 5’-CTTTCTTCTC…

RaptormLST8

mTOR

RictormLST8

mTOR

FKBP12

Actin cytoskeleton

Ribosomal biogenesis

Protein translation

Rapamycin

PDK1

PTENMutations 5%LOH ~30%

Akt Akt2 amplified < 5%

S6K1 Amplified ~10% (17q23)

PIK3CA Mutations in ~25% overallGene amplification < 10%

(~25%)(~25%)(~45%)(<7%)

ER+

HER2+

ER+/HER2+

ER-/HER2-

?

mTOR

NPM ?

4E-BP1?

eIf4e?

The growth pathway is subject to enormous mutation ratesThe growth pathway is subject to enormous mutation rates

PIK3CA mutations in ~25% of breast cancers

(~25%)(~25%)(~45%)(<7%)

ER+

HER2+

ER+/HER2+

ER-/HER2-

~275 samples from ER+ patients undergoing Letrozole therapy

Developed sequencing pipeline for Exons 1,6,7,9,20 with GSC

Goal 1: Attack the genotypeGoal 1: Attack the genotype

Figure adapted from Cancer Res 65: 2554

Hypothesis: pik3ca mutations render tumors resistant to ER- and HER2-targeted therapies

CONSERVATION OF NUCLEOLAR STRUCTURE/FUNCTION CONSERVATION OF NUCLEOLAR STRUCTURE/FUNCTION

RESIDENTS DRIFTERS

NPM/B23NCL/C23

FibrillarinCdc14B

Nop5/SikBop1

R-proteinshnRNP’s

TCOF1NUMA1

Ki67p19ARF

Cdc14ATERTWRNING1PP1

SMN1UBF

Mdm2MdmX

Cyclin Ep53

DKC1PES1BLM

MammalsMammals(~450)(~450)

drosophiladrosophila

YeastYeast(~39)(~39)zebrafishzebrafish

xenopusxenopus

D. Trere (2000) D. Trere (2000) MicronMicron 3131:127-131:127-131

UNIFORM AgNOR & SAPA SCORING METHODUNIFORM AgNOR & SAPA SCORING METHOD

RIBOSOMAL GENES AND THE NUCLEOLUSRIBOSOMAL GENES AND THE NUCLEOLUS

FIVE rGENE CLUSTERSFIVE rGENE CLUSTERSCHR. 13, 14, 15, 21, 22CHR. 13, 14, 15, 21, 22OVER 400 TANDEM REPEATSOVER 400 TANDEM REPEATS~47 KB REPEAT ELEMENT~47 KB REPEAT ELEMENTYIELDS 13KB TRANSCRIPTYIELDS 13KB TRANSCRIPT

Processing is everythingProcessing is everything

rrrna modificationsrna modifications

Suppressing transcription…first important clueSuppressing transcription…first important clue

Lots of steps to mess with…Lots of steps to mess with…

Myc sends us another clue…Myc sends us another clue…

S6K

eIF4E

ProteinSynthesis

NPM

40

60 40

6040

60

RibosomalExport

rRNATranscription

CYTOPLASM

NUCLEOLUS

TIF1A

RNA Pol I

40

60

mTOR

Nuclear export

nucleus

cytosoplasm

AA

AA

AA

AA

-3’

5’60S 40S

mTOR

S6K

NPM

nucleolus

Nucleophosmin acts in a late Nucleophosmin acts in a late rrRNA stageRNA stage

Nucleophosmin is mutated in human cancersNucleophosmin is mutated in human cancers

nucleus

cytosoplasm

nucleolus

60S40S

60S40S

AA

AA

AA

AA

-3’

5’

PI3K

S6K

NPM

rRNA Export

Polysomes

Proteinsynthesis

This mutant is hyperactive in This mutant is hyperactive in rrRNA exportRNA export

…The pathway can be addictive

mTORC2

Rac1

NPM

rRNA Export

Rapamycin

Neurofibromin K-Ras

Polysomes

Proteins

None NoneLY LYRap Rap

Nf1+/+ Nf1-/-

Tubulin

S6P

Neurofibromin controls the growth pathwayNeurofibromin controls the growth pathway

PI3K

CELL SURVIVAL CELL GROWTH CELL PROLIFERATION

Nucleolar Integrity

ARF/p53

Rapamycin

ARF

mTOR

S6K

NPM

rRNA Export

Polysomes

Protein synthesis

The pathway must have checks in placeThe pathway must have checks in place

THE THE INK4A-ARFINK4A-ARF LOCUS LOCUS

FibrosarcomasLymphomas

Gliomas

Melanomas FibrosarcomasLymphomas

Gliomas

1 1 2 3

Ink4a/ARF

Cyclin D-Cdk4 Mdm2

Rb p53

?

Pol III

5S

Pol I

5.8S18S28S

NUCLEOLUS NUCLEOPLASM

5S---rpL5---NPM

ARF

60S

NUCLEAREXPORT

Synchronized ribosome exportSynchronized ribosome export

Layers of potential targets…Layers of potential targets…

RTK

Ras

p85p110

PDK1 AKT

PTENP P

PP

MDM2

IKK

eNOS

TSC1/2

GSK3

FOXO

Myc mTOR

Cyclin D

p53

NF-B

p27kip1

Rhebcdk4

eIF4ENPM

28S

18S

5.8S5S

ARF S6K 4E-BP1

28S 18S 5.8S