Not division…But growth

Cells 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 NPM ARF p68

Thinking about translation

The forgotten translation…top m RNA s Terminal Oligo Pyrimidine tracts: 5’-CTTTCTTCTC…

Raptor mLST8 mTOR Rictor mLST8 mTOR FKBP12 Actin cytoskeleton Ribosomal biogenesis Protein translation Rapamycin

PDK1 PTEN Mutations 5% LOH ~30% Akt Akt2 amplified < 5% S6K1 Amplified ~10% (17 q 23) PIK3CA Mutations in ~25% overall Gene amplification < 10% (~25%) (~45%) (<7%) ER + HER2 + ER + /HER2 + ER - /HER2 - ? m TOR NPM ? 4E-BP1 ? e If4e ? The growth pathway is subject to enormous mutation rates

PIK3CA mutations in ~25% of breast cancers (~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 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 RESIDENTSDRIFTERS NPM/B23 NCL/C23 Fibrillarin Cdc14B Nop5/Sik Bop1 R-proteins hnRNP’s TCOF1 NUMA1 Ki67 p19ARF Cdc14A TERT WRN ING1 PP1 SMN1 UBF Mdm2 MdmX Cyclin E p53 DKC1 PES1 BLM Mammals(~450) drosophilaYeast(~39)zebrafishxenopus

D. Trere (2000) Micron 31: UNIFORM AgNOR & SAPA SCORING METHOD

RIBOSOMAL GENES AND THE NUCLEOLUS FIVE rGENE CLUSTERS CHR. 13, 14, 15, 21, 22 OVER 400 TANDEM REPEATS ~47 KB REPEAT ELEMENT YIELDS 13KB TRANSCRIPT

Processing is everything

r rna modifications

Suppressing transcription…first important clue

Lots of steps to mess with…

Myc sends us another clue…

S6K eIF4E Protein Synthesis NPM Ribosomal Export rRNA Transcription CYTOPLASM NUCLEOLUS TIF1A RNA Pol I mTOR

Nuclear export nucleus cytosoplasm AAAAAAAA-3’ 5’ 60S 40S m TOR S6K NPM nucleolus Nucleophosmin acts in a late r RNA stage

Nucleophosmin is mutated in human cancers

nucleus cytosoplasm nucleolus 60S 40S 60S 40S AAAAAAAA-3’ 5’ PI3K S6K NPM r RNA Export Polysomes Protein synthesis This mutant is hyperactive in r RNA export

…The pathway can be addictive mTORC2 Rac1 NPM rRNA Export Rapamycin Neurofibromin K-Ras Polysomes Proteins None LY Rap Nf1+/+Nf1-/- Tubulin S6 P Neurofibromin controls the growth pathway

PI3K CELL SURVIVALCELL GROWTHCELL PROLIFERATION Nucleolar Integrity ARF/ p 53 Rapamycin ARF m TOR S6K NPM r RNA Export Polysomes Protein synthesis The pathway must have checks in place

THE INK4A-ARF LOCUS Fibrosarcomas Lymphomas Gliomas Melanomas Fibrosarcomas Lymphomas Gliomas 11 11 23 Ink4a / ARF Cyclin D-Cdk4Mdm2 Rb p 53 ?

Pol III 5S Pol I 5.8S18S 28S NUCLEOLUSNUCLEOPLASM 5S--- rp L5---NPM ARF 60S NUCLEAR EXPORT Synchronized ribosome export

Layers of potential targets… RTK Ras p85 p110 PDK1 AKT PTEN P P P P MDM2 IKK eNOS TSC1/2 GSK3  FOXO Myc mTOR Cyclin D p53 NF-  B p27kip1 Rheb cdk4 eIF4E NPM 28S 18S 5.8S 5S ARF S6K 4E-BP1 28S18S5.8S